Second International Symposium on Development of the EntericNervous System: Cells, Signals and Genes

UCL Institute of Child Health

London, UK

22–25 February 2009

These abstracts are published online as part of the February 2009 issue. Please visitwww3.interscience.wiley.com/journal/118498177/home to search and read the abstracts.

� 2009 Blackwell Publishing Ltd 223

Abstracts

Gene regulatory network underlying neural crest formation

m. bronner-fraser

Division of Biology, California Institute of Technology, Pasadena, CA, USA

The neural crest is a population of multipotent, migratory progenitor

cells that forms at the border of neural and non-neural ectoderm in

vertebrate embryos. These cells then migrate from the neural tube

along defined pathways, populate numerous sites and differentiate into

diverse cells types including melanocytes, sensory and autonomic

neurons, and mineralized matrices like bone and dentine. Data com-

piled from Xenopus, zebrafish, mouse and chick, suggest that a net-

work of interacting transcriptional regulators and downstream effector

genes confer properties like multipotency and migratory capacity to

nascent neural crest cells. These regulatory interactions can be divided

into distinct phases. The first involves inductive signals (e.g. Wnt,

BMP, FGF) that establish the neural plate border, by up-regulation of

border specifier genes like Msx1/2, Pax3/7, and Zic. These border genes

in turn up-regulate neural crest specifier genes like Slug/Snail, FoxD3

and the SoxE family. Finally, the neural crest specifiers turn on specific

downstream targets that render the neural crest migratory and multi-

potent. We are testing linkages in this hypothetical neural crest gene

regulatory network in chick and lamprey by systematically perturbing

a subset of the transcription factors involved in early neural crest

specification and examining the effect of these perturbations on likely

downstream genes in order to test the predicted interrelationships. In

addition, we are isolating cis-regulatory regions of genes in this puta-

tive neural crest regulatory network to identify neural crest enhancers,

determine additional inputs to the network and determine which

interactions are direct. The results suggest that a series of gene regu-

latory circuits are involved in the production of migratory neural crest

cells in the early vertebrate embryos and that many of these events

may be conserved to the base of vertebrates.

Characterization of the Stathmin SCG10 and its interactions with

Kinesin Binding Protein (KBP) in zebrafish

gm burzynski,* m alves,� e de graaf,� c hoogenraad,� bjl eggen,§

a brooks,� rmw hofstra� & i shepherd**Emory University, Altanta, GA, USA; �University Medical Center, Groningen,

the Netherlands; �Erasmus Medical Center, Rotterdam, the Netherlands; and

§University of Groningen, Groningen, the Netherlands

SCG10 (STMN2) is believed to be neuronal-specific stathmin that is

enriched in the growth cones of developing neurons and has a role in

neurite outgrowth. In all species so far examined SCG10 is expressed

in both the CNS and PNS. Recently we have shown that SCG10

interacts with the KBP (KIAA1279) protein. Mutations in kbp have

been shown to be responsible for human Goldberg-Shprintzen

(GOSHS) syndrome. Patients with this rare autosomal recessive

disorder manifest microcephaly, mental retardation, polymicrogyria,

facial dysmorphisms and Hirschsprung disease. The precise function of

KBP in nervous system development is currently not known. To begin

to understand the function of SCG10 we have determined the temporal

and spatial expression pattern of the two zebrafish SCG10 orthologues,

SCG10a and SCG10b, by RT-PCR and in situ hybridization. RT-PCR

shows that both transcripts are maternally deposited and are detectable

from 0 hpf through 5 days – the latest age we examined. In situ

hybridization analysis reveals that the pattern of expression of these

two genes is dynamic and spatially restricted but is nearly identical for

both homologues. SCG10a and b are principally restricted to the CNS

from 24–48 hpf. From 48 hpf onwards expression of both genes

becomes restricted to the anterior CNS and cranial ganglia. This pat-

tern of SCG10 expression at 48 hpf resembles the pattern of KBP gene

expression, which is limited to the anterior CNS from 48 hpf onwards.

Subsequently, both SCG10 genes are expressed additionally in the

developing gut from 72 hpf. To investigate the in vivo function of the

zebrafish SCG10 orthologues and their interactions with KBP, we

generated morpholinos (MOs) for all three genes. Morphant embryos

have a severe phenotype at relatively low MO concentration. It is

likely that there is redundancy between the two SCG10 genes as

double SCG10 morphants have a more severe phenotype when com-

pared to single morphants. Significantly, triple morphants injected

with sub-threshold doses of SCG10 and KBP MOs display a severe

phenotype, strongly suggesting an epistatic interaction between the

products of these genes. Our data suggests that SCG10 and KBP genes

are required for proper differentiation of pharyngeal arches, migration

of ENS precursors and later differentiation stages of the cranial ganglia.

Function of the RNA binding protein HERMES in gastrointestinal

morphology and motility

p de santa barbara, c rouleau, l le guen & c notarnicola

INSERM ERI25, �Muscle and Pathologies�, Montpellier, France

The motility of the digestive tract is ensured by the correct coordina-

tion of the autonomous enteric nervous system (ENS) and the visceral

smooth muscle cells (SMC). The ENS originates from neural crest cells

that migrate from the dorsal region of the neural tube and colonize the

whole gut to establish its innervation. The SMCs derive from the

splanchnopleural mesoderm that will form the undifferentiated vis-

ceral mesenchyme before their final differentiation. Motility disorders

in infants comprise a wide group of heterogeneous diseases. Hirsch-

sprung disease (HSCR) is a particular case due to an absence of the ENS

along certain lengths of the bowel. The SMC differentiation is also

often affected in patients with congenital gut malformations and

motility disorders such as Chronic Intestinal Pseudoobstruction (CIP).

However, few have investigated the status of SMC in physiopatho-

logical conditions. Our aim was to investigate the molecular mecha-

nisms that control the differentiation of the visceral mesenchyme into

SMCs in vertebrates. In order to identify these candidate pathways, we

developed and analyzed the gene expression profiles of undifferentiated

and differentiated avian stomach by microarray. We identified one new

candidate HERMES, a RNA binding protein and examined the function

of this factor during the development and the differentiation of the

SMC structure by performing exhaustive in vivo and in primary SMC/

ENS culture positive and negative approaches. We found that HERMES

control different aspects of the SMC differentiation and deregulation of

its expression and function alter the development of both SMC and

ENS systems. In addition, we analyzed the expression of homologous

HERMES gene in human physiopathological conditions. All these data

demonstrate the necessity of correct coordination between the

development/differentiation of the SMC and ENS and the importance

to focus on both motility effectors.

Development of the mucosal plexus of the human enteric nervous

system

m metzger,* as wallace,* kh schaefer,� aj burns* & n thapar**UCL Institute of Child Health, London, UK; and �Department of Biotechnology,

University of Applied Sciences, Kaiserslautern, Germany

Introduction: Knowledge regarding the structure and development of

the enteric nervous system (ENS) is crucial for the understanding of

gut function and its disorders. It is widely accepted that the ENS is

composed of two major plexus (myenteric and submucosal plexus)

arranged as concentric rings in the bowel wall. There is, however,

accumulating evidence from both animal studies, and our recent work

in humans, that nerve cells also reside within the post-natal intestinal

mucosa (mucosal plexus). The aim of this study was to examine the

development-dependent formation of an intrinsic mucosal plexus of

Neurogastroenterol Motil (2009)

� 2009 Blackwell Publishing Ltd i

the ENS in human gut. Material and methods: Human embryonic and

fetal gut tissue was obtained from the joint Medical Research Council

and the Wellcome Trust-funded Human Developmental Biology

Resource, and from the Department of Obstetrics, Clinical Faculty

Mannheim, University of Heidelberg under ethical approval. Human

post-natal tissue was obtained from patients of Great Ormond Street

Hospital, London, UK under ethical approval and with fully informed

consent. Immediately after preparation all tissues were fixed in 4%

paraformaldeyhde and processed for immunohistochemical analysis

using antibodies specific for enteric neurons and glia. Results: In

order to analyse the organization of the ENS in the mucosal plexus,

serial sections from human large intestine were studied by anti-Hu

(Anna-1), TuJ1, Sox10 and S100 immunohistochemistry. In addition,

expression of enteric neuronal subtype markers were investigated (VIP,

NOS). In contrast to the myenteric and submucosal plexus no neural

cells were observed within the mucosa of all analysed fetal gut stages.

Neural cell bodies were first detected in the newborn period and with

increasingly more prominent staining of individual cell bodies and

interconnected processes as post-natal age increased. The mucosal

plexus of large intestine most commonly contained isolated neural

cells at different topographical levels within the lamina propria and

around the mucosal crypts. On occasion small aggregates of cells akin

to true ganglia were observed. Conclusions: This study shows that

the ENS is not restricted to the myenteric and submucosal plexus, but

also exists as a mucosal plexus in post-natal gut. The development and

precise functional role of this plexus in post-natal human gut is

unclear, but it is likely that neural cells within the mucosa play

distinct and important roles perhaps in epithelial cross-talk and local

reflex activity of the gut. The regulatory mechanisms and importance

in mucosal functions need to be elucidated in detail in future studies.

Therapeutic potential of human enteric nervous system stem cells

derived from endoscopic gut mucosal biopsies

m metzger, c caldwell, aj barlow, aj burns & n thapar

UCL Institute of Child Health, London, UK

Introduction: There is increasing evidence that enteric nervous

system (ENS) stem cells may provide potential therapeutic tools for

disorders characterized by aganglionosis or defective ENS function.

Although full thickness human post-natal gut tissue has been used

successfully to generate such stem cells, its harvesting from surgical

resection poses significant practical limitations. Recent literature and

our own work supports the presence of a third distinct plexus of the

ENS within the intestinal mucosa. Such mucosa is routinely biopsied

during gastrointestinal endoscopy and provides a potential regenerat-

ing source of intestinal tissue. Objectives: The aim of this study was

to explore whether gut tissue obtained utilising minimally invasive

routine endoscopy techniques could be used to generate ENS stem

cells, which retain the potential to generate an ENS upon transplan-

tation into aganglionic gut. Methods: Post-natal human gut mucosal

tissue obtained from children undergoing gastrointestinal endoscopy

was used to generate cell cultures containing neurosphere like bodies

(NLBs), which have been shown to contain ENS stem cells. NLBs

generated from such mucosal biopsies were characterized by immu-

nostaining using a panel of established neural progenitor (Nestin) and

neural crest progenitor (p75 and Sox10) markers. The potential of

individual cells derived from NLBs to generate bi-potential colonies

was tested in clonogenic assays. The ability to generate mature neu-

ronal subtypes was examined by in-vitro differentiation of dissociated

NLBs and following their transplantation into models of aganglionic

gut. Results: Gut mucosal biopsies were obtained from 70 children

(aged 9 months 17 years). Immunostaining of the mucosal biopsies

showed they contained neuronal cells and cells positive for markers of

ENS stem cells (p75 and Sox10). On culturing of dissociated mucosal

biopsies characteristic NLBs were generated at all ages examined.

Post-natal mucosa-derived NLBs contained cells that were proliferat-

ing (Ki67), expressed ENS stem cell markers (p75, Sox10, Nestin), and

were capable of generating colonies containing neurons and glia in

clonogenic assays, and multiple ENS neuronal subtypes (CGRP, NOS,

Ser, VIP and ChAT). Upon transplantation into cultured recipient

aganglionic chick and human hindgut, cells from NLBs colonized re-

cipient gut, generated ganglia-like structures and enteric neurons and

glia, including mature neuronal subtypes. Conclusions: The results

show that ENS stem cells can be harvested from routine mucosal

biopsies of post-natal human gut using standard endoscopic proce-

dures. This represents a significant practical advance towards the

development of definitive cell replenishment therapies for aganglionic

gut disorders like Hirschsprung’s disease, or those characterized by

deficient or defective enteric neuronal function.

Interactions between L1cam and Sox10 during enteric nervous system

development

rb anderson & as wallace

Department of Anatomy & Cell Biology, University of Melbourne, Melbourne

Via, Australia

Introduction: Hirschsprung’s disease is a congenital disorder in which

there is an absence of enteric ganglia within the distal portion of the

gastrointestinal tract. There is increasing evidence to suggest that the

phenotypic variability and incomplete penetrance of Hirschsprung’s

disease is due to complex interactions between genes known to result

in Hirschsprung’s disease, such as Sox10, and unidentified modifier

genes. Human clinical and animal model studies have suggested that

the X-linked gene, L1CAM, may act as a modifier gene for the devel-

opment of Hirschsprung’s disease. Objectives: To examine whether

L1cam could interact with the transcription factor,

Sox10. Methods: To identify genetic interactions between L1cam and

Sox10, we used a two-locus complementation approach by crossing

female L1cam+/- mice with male Sox10+/- mice. To assay the effects of

any interaction, we examined whether the migration of enteric neural

crest-derived cells is altered in L1cam null mutant mice when com-

bined with a heterozygous mutation in Sox10. Results: We have

identified a significant genetic interaction between L1cam and Sox10.

In all ages examined, L1cam-/y; Sox10+/- mice showed a significant

delay in the colonization of the gut by enteric neural crest-derived

cells. To decipher the cellular basis of the enteric nervous system

(ENS) defect, we examined the rate of cell proliferation and cell death

of enteric neural crest-derived cells in the gut of single and double

mutant mice. The ENS defect observed in L1cam-/y; Sox10 +/- mice

does not appear to be due to changes in proliferation or cell

death. Conclusions: The results of this study show that the X-linked

gene, L1cam, can function as a modifier gene for the development of

Hirschsprung’s disease, and that the interactions between L1cam and

Sox10 are important for normal enteric nervous system development.

Development of organised motility patterns in the duodenum of the

mouse

jc bornstein,* rr roberts,*,� rm gwynne* & hm young�*Department of Physiology, University of Melbourne, Parkville, Vic, Australia;

and �Department of Anatomy and Cell Biology, University of Melbourne,

Parkville, Vic, Australia

Introduction: In adults, intestinal motility is regulated by interactions

between the enteric nervous system, pacemaker cells intrinsic to the

muscle layers, interstitial cells of Cajal (ICC), and the smooth muscle

itself. Both ICC and enteric neural circuits impose their own rhythms

on motility with their contributions differing between regions. In the

undistended mouse duodenum, most motor activity consists of small

circular muscle contractions that occur at ~50/min and propagate

anally. These may be due to pacemaker ICC. Distension triggers orally

propagating neurogenic motor patterns that manifest as low frequency

(1 every 2–5 min) waves of enhanced slow wave contractions. Anally

propagating contractile complexes (PCCs) are seen in mouse

colon. Objective: To determine when PCCs develop in relation to

enteric neurons and ICC. Methods: Spatiotemporal maps of intestinal

diameter were made from video recordings of embryonic and neonatal

mouse duodenum in vitro. This allows detailed analysis of spatial and

temporal relationships between contractions at neighbouring points.

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdii

We undertook electrophysiological analysis of smooth muscle and

immunohistochemical studies of the appearance of enteric neurons

and ICC. Results: At embryonic day 12.5 (E12.5), the duodenum was

quiescent. By E14.5 spontaneous contractions propagating orally and

anally away from point sources were seen. These were like the ripples

seen in neonatal mouse colon and were insensitive to the neural

blocker tetrodotoxin. At E14.5, Kit-immunoreactivity (marker for ICC)

labelled undifferentiated cells between the serosa and myenteric

plexus, but there were no morphological ICC. By E16.5, ripples were

prominent; also seen in duodenum of Ret-/- mice, which lack enteric

neurons. ICC had not differentiated. At E18.5, some organized neural

activity was seen; mucosal stimulation evoked junction potentials in

the muscle; and electrical slow wave activity was seen. ICC networks

were identified immunohistochemically. Two high frequency patterns

were identified; whether either or both were ripples or slow wave

contractions was unclear. Mature PCCs were not present in the duo-

denum at P0. Conclusions: Mature motility does not appear until

well after arrival and initial differentiation of enteric neurons. The first

organized motility is independent of neurons or ICC and may be

intrinsic to the smooth muscle itself.

Analysis of talpid3 mutant chicken embryos reveals defects in gut and

enteric nervous system development

aj burns,* l bradshaw,* lj freem,* mg davey� & n thapar**Neural Development Unit, UCL Institute of Child Health, London, UK; and

�Division of Genetics and Genomics, Roslin Institute, Midlothian, UK

Introduction: Talpid3 is a classical chicken mutant with a wide range

of phenotypic defects including paddle-shaped limbs, and face, skele-

ton and vascular abnormalities. Although these defects were originally

described over 40 years ago, it was only recently determined that tal-

pid3 encodes a novel gene product that is required for the function of

Gli repressor and activator transcription factors in the Hedgehog (Hh)

signalling pathway. Objective: Since the Hh signalling pathway has

been implicated in the development of the gastrointestinal tract and

enteric nervous system (ENS), our aim was to examine talpid3 mutant

embryos to gain further insight into the role of the Hh pathway in the

development of these tissues. Methods: Fertilised eggs from the tal-

pid3 flock were incubated for up to 8 days. The eggs were windowed in

order to determine stage of development and phenotype, then embryos

were removed, fixed in 4% PFA and processed for frozen sectioning and

immunohistochemistry. Antibody staining was performed to identify

smooth muscle (aSMA), neural crest cells (HNK-1), and early neuronal

cells (bIII Tubulin – TuJ1). Results: Approximately 20% of embryos

were identified as being homozygous talpid3 mutants, based on visual

inspection of the blood vessels and limb buds. Upon dissection, the gut

was normally patterned along its length (esophagus, stomach,

pre-umbilical intestine, post-umbilical intestine, hindgut) but was

significantly shorter in talpid3 embryos compared with stage-matched

controls. In talpid3 gut, although the distribution of HNK-1 and TuJ1-

positive cells was similar to controls, they were reduced in number. At

E6, the distribution of SMA was considerably altered in talpid3

mutants. In control esophagus, concentric rings of SMA encircled the

gut, whereas SMA staining was absent in mutants. Although weak

SMA immunoreactivity was present in the stomach of mutants, in the

pre- and post-umbilical intestine SMA was distributed across the

entire gut wall, and abutted the epithelium. In the hindgut of talpid3

mutants SMA immunoreactivity was patchy and positive cells did not

encircle the gut as in controls. Conclusion: Preliminary data suggest

that although neural crest cells are able to migrate and differentiate in

talpid3 gut, they are reduced in number compared with controls. In

addition, overall gut length is significantly reduced and SMA-positive

cells are not organised into presumptive circular muscle. These find-

ings suggest that perturbation of Hh signalling in the talpid3 chick

embryo results in malformation of the gastrointestinal tract, lack of

gut smooth muscle patterning, and altered ENS development.

Divergence of enteric neuronal and glial lineages from uncommitted

progenitors: roles of bone morphogenetic proteins, neuregulins,

neuropoietic cytokines and glial cell line derived neurotrophic factor

a chalazonitis,* f d�autreaux,� td pham,* ja kessler� & md gershon**Columbia University, New York, NY, USA; and �Ecole Normale Superieure,

Paris, France; and �Northwestern University, Feinberg Medical School, Chicago,

IL, USA

The enteric nervous system (ENS) develops from a multipotent pop-

ulation of neural crest-derived cells (ENCDC). Factors in the enteric

microenvironment that induce common neural/glial progenitors to

differentiate selectively as neurons or glia remain to be identified.

BMPs-2 and -4, which are expressed in the enteric microenvironment,

enhance neuronal differentiation at the expense of proliferating pre-

cursors; moreover, common enteric neural/glial progenitors are driven

by glial cell derived neurotrophic factor (GDNF) to develop as neurons.

Neuropoietic cytokines (LIF and CNTF; NPC) have also been found to

increase numbers of glia developing in vitro. Because mice that lack

ErbB3, the binding receptor for neuregulin-1 (NRG-1/GGF2) lack

enteric glia, enteric gliogenesis must be NRG-1-dependent. We tested

the hypotheses that NPC and/or BMP-2 and -4 enhance glial differ-

entiation directly or by increasing the responsiveness of precursors to

NRG-1/GGF2. To analyze the role of BMPs in enteric gliogenesis

in vivo, the BMP antagonist, noggin (NSE-noggin mice), or BMP4 (NSE-

BMP4 mice), were overexpressed in the ENS of transgenic mice.

Overexpression of noggin in the ENS decreased the density of enteric

glia and their ratio to neurons while overexpression of BMP4 increased

glial density and the glia/neuron ratio. In vitro, BMPs promoted ErB3

expression and glial differentiation of isolated ENCDC in a concen-

tration and stage-dependent manner. BMPs also induced nuclear

translocation of phosphorylated receptor-activated SMAD proteins in

cells that expressed the glial marker, GFAP. In situ, expression in the

fetal gut of ErB3 was detected as early as E12. Isolated crest-derived

cells selectively expressed ErB3 and its immunoreactivity in situ was

coincident with that of GFAP at E17. NRG-1/GGF2 expression (E14)

followed that of ErbB3 and both continued to adulthood. NRG-1/GGF2

induced glial proliferation and decreased GDNF-induced neurogenesis.

In contrast, GDNF enhanced neurogenesis at the expense of NRG-1/

GGF2-induced gliogenesis. In contrast to NRG-1/GGF2 and GDNF,

NPC increased the development both of neurons and glia. BMP- and

NPC-induced gliogenesis were not additive. Exposure of ENCDC to

BMPs decreased NRG-1/GGF2-stimulated glial proliferation but

increased NRG-1/GGF2 dependence. Observations support the

hypotheses that NPC promote survival/differentiation of common

neural/glial precursors, BMPs promote differentiation without speci-

fying lineage. In contrast, differentiation is biased toward a glial

lineage by NRG-1/GGF2 and toward a neuronal lineage by GDNF.

Supported by NIH grants DK58056 (AC) NS15547 (MDG) NS20013,

NS20778 (JAK).

Sox2 expression provides a means to identify and isolate ENS

progenitors

ta heanue & v pachnis

Division of Molecular Neurobiology, MRC-National Institute for Medical

Research, London, NW7 1AA, UK

The enteric nervous system (ENS) derives from neural crest cell pro-

genitors that emigrate from the neural tube and invade the developing

foregut and migrate in a rostral to caudal direction to completely col-

onize the developing gut. These progenitors proliferate to generate

sufficient cell numbers and then progressively differentiate into neu-

rons and glial cells and become organized into a series of intercon-

nected ganglia. While most progenitors undergo differentiation, cells

exhibiting the properties of enteric progenitor cells (EPCs) can be iso-

lated from mouse gut tissue from E11.5 until post-natal stages. Our

previous microarray screen, conducted to identify markers of the

mammalian enteric nervous system, identified the neural progenitor

and stem cell marker Sox2 to be expressed in the embryonic ENS. We

now show that SOX2 is expressed from embryonic to post-natal stages,

and is co-expressed with known enteric progenitor cell markers such

as SOX10 and NESTIN. We have developed a strategy that uses

selection on the basis of Sox2 expression as an alternative method for

Abstracts

� 2009 Blackwell Publishing Ltd iii

isolating EPCs. Cells derived from gut tissue from a transgenic mouse

line in which the bgal-neomycin resistance gene fusion protein has

been knocked into the Sox2 locus (Sox2-bgeo) can be cultured under

G418 selection, thus allowing substantial enrichment of neomycin

resistant SOX2 expressing cells within the population. Cells can be

selected in this way from both embryonic and adult tissues and can

proliferate extensively in culture over a period of months. The

expression profile of Sox2-bgeo selected cells is consistent with EPCs;

selected cells express the progenitor marker SOX10 and do not express

differentiation markers such as TuJ1 and GFAP. The functional prop-

erties of Sox2-bgeo selected cells have also been tested. Selected cells

transplanted into the ENS progenitor cell migratory pathway of mouse

and chick embryos were capable of migrating along appropriate routes.

Moreover, we demonstrate that when transplanted into an E11.5

mouse gut environment, Sox2-bgeo selected cells can migrate, differ-

entiate, and form apparently normal connections with endogenous

neurons, thereby exhibiting important properties of enteric progeni-

tors. The relative ease of maintaining a population of enteric

progenitors using this technique lends itself well for use in stem cell

replacement studies currently underway in the lab that aim to restore

enteric neurons to aganglionic regions in mouse models of Hirsch-

sprung’s disease.

Colonisation of the developing lungs by tangential migration of

foregut-derived vagal neural crest cells

lj freem, s escot, n thapar & aj burns

Neural Development Unit, UCL Institute of Child Health, London, UK

Introduction: Neural crest cells (NCC) are a transient population of

multipotent cells that migrate extensively throughout the embryo and

give rise to a variety of cell types including neurons of the enteric

nervous system and intrinsic neurons that innervate the airway

smooth muscle of the developing lungs. We have previously shown

that the NCC that initially colonize the lungs arise from a subset of

cells that migrates from the developing foregut into the lung

buds. Objectives: To analyse the spatiotemporal colonization of the

lungs by NCC and to determine the signalling cues that influence their

migration, proliferation and differentiation. Specific objectives are to:

(i) analyse the spatiotemporal migration of NCC from the foregut into

the lung buds in mouse and human embryos, (ii) analyse the expres-

sion of candidate signalling molecules in NCC and within the

lung mesenchyme, and (iii) test the chemoattractive and trophic

capabilities of candidate signalling molecules. Methods: ROSA26Y

FPStop;Wnt1Cre transgenic mice, in which all NCC express yellow

fluorescent protein (YFP), were used to analyse the spatiotemporal

development of NCC in the developing foregut and associated lungs.

The roles of candidate signalling molecules in NCC and lung buds

were analysed using immunohistochemistry on control and mutant

(Ret-/-, Gfra1-/-, Dom-/-) embryonic mouse and human lung tissues.

The effects of putative chemoattractants on the directed migration of

NCC within the lung were tested using an in vitro model of lung

colonisation/growth. Results: Studies of ROSA26YFPStop;Wnt1Cre

transgenic mice clearly described the tangential migration of YFP-la-

belled NCC from the developing foregut, beginning at E10.5, into the

lungs (E11.5–13.5). YFP-positive cells were closely associated with

the airway smooth muscle of the branching bronchi. No differences

in the extent of lung innervation by NCC-derived precursors were

evident in E14.5 Ret-/-, Gfra1-/-, Dom-/- mutant mice compared

with controls. Experiments using cultured lung explants exposed

to candidate molecules delivered within agarose beads demonstrated

that YFP-positive NCC were attracted towards GDNF, the RET

ligand. Conclusion: The determination of the mechanisms underly-

ing development of the intrinsic innervation of the lung would be

a significant advance and further our understanding of lung innerva-

tion in particular, and mechanisms of NCC development in general.

The project focuses attention on identification of potential neural

crest-related defects in the lung that could have significance in health

and disease before and after birth.

Mode of gut colonization by enteric neural crest-derived cells

h enomoto,* c nishiyama,* df newgreen� & hm young�*RIKEN Center for Developmental Biology, Kobe, Japan; �The Murdoch Childrens

Research Institute, Parkville, Via, Australia; and �Department of Anatomy & Cell

Biology, University of Melbourne, Melbourne,Via, Australia

Introduction: Recent time-lapse studies on GFP-labeled migrating

enteric neural crest-derived cells (ENCCs) have revealed dynamic

patterns of ENCC migration and suggested that distinct regions or

subsets of ENCCs contribute differentially to the formation of the

enteric nervous system. These studies also suggested a high level of

cell-cell interaction among ENCCs during migration. However, the

use of a single-wavelength fluorescence limits visualization of a given

cell population against other cell populations, and it is often difficult

to follow the behaviors and fate of ENCCs by this cell labeling tech-

nique. Objective and Methods: To more fully understand the mode of

gut colonization by ENCCs, we have generated Ednrb-Kik mice in

which all ENCCs are engineered to express the photo-convertible

fluorescent protein, humanized Kikume. Gut explants were dissected

from Ednrb-Kik embryos (embryonic day 12.5, E12.5) and time-lapse

imaging of ENCCs was performed using an inverted confocal

microscope. Kikume changes chromophore emission wavelength from

green to red upon UV exposure, enabling us to label a subset of ENCCs

at desired time points within the migrating network of ENCCs.

Results: We have made the following observations. (i) At least two

morphologically distinct cell populations are distinguishable within

the network of migrating ENCCs; multipolar immature neural crest-

like cells and unipolar neuron-like cells with a single neurite (the latter

migrated at the slower speed than the former), (ii) the wavefront of

migrating ENCCs is composed largely of immature neural crest-like

cells, (iii) although the meshwork formed by strands of ENCCs

maintains its basic structure over several hours, ENCCs continuously

change their neighbors within those strands, (iv) at least some of

the wavefront-derived ENCCs keep the �wavefront position� through-

out migration and (v) the ability of ENCCs to expand along the rostral-

caudal axis in the gut is highest in cell populations located at

the wavefront. Conclusions: The behavior of individual ENCCs sug-

gests a high degree of cell-cell and cell-matrix interactions. At E12.5

and thereafter, ENCCs located at the wavefront play a primary role in

colonizing the colon. Ednrb-Kik mice provide a valuable platform for

better understanding of dynamic and complex behaviors of ENCCs.

Regeneration in ileal anastomosis

s holland-cunz,* m chmelnik,* s weih,* m klotz� & kh schafer�*Department of Pediatric Surgery, University Hospital, Heidelberg, Germany; and

�Faculty of Science, Microsystem technique, Zweibruecken, Germany

Aim: This study wanted to show the regenerative capacity of the

bowel wall after interrupted and anastomosed small bowel Material

and Method: We performed laparotomy, ileal dissection and anasto-

mosis in 32 male Sprague Dawley rats. The anastomotic technique

varied between a single knot hand sutured anastomosis, the by our

team new developed CINPA method with non perforating single metal

clips and one case with a clued anastomosis, were we used a collagen

and fibrin layer. The anastomoses were resected after sacrificing the

animals at day 3 respectively 14 after the anastomosing procedure.

X-rays were performed for grading the stenosis rate of the anastomosed

region and stability tests, as bursting pressure and lengthening force

were realised.We calculated the morphological data and compared

between the different anastomotic techniques and the different sur-

vival times. Beside the nerval regeneration we observed changes in the

protein cluster and performed 2D DIGE separating of proteins. With

MALDI TOF the proteins were dedicated Results: Of these 32

anastomoses only 18 were applicable for a histological analyse. Out of

these 18 anastomoses we used 34 slides for the interpretation. There

was no increase of cell amount in between day three and 14 after

anastomosis. In the group of the sutured anastomoses there were more

nerve cells in the submucosal plexus and nearly equal relations in the

myenteric plexus. The area of the ganglions was a little bit enlarged in

the sutured group in comparison to the clipped ones. The glued

anastomosis showed similar results to the other ones.

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdiv

FGF16 (Fibroblast growth factor) und TRIP 13 (Thyreoid receptor

interactin protein) were isolated and dedicated by protein analysis and

showed regulative increase in dependence of postoperative state.

Discussion: Different methods of anastomosing the bowel result in

various scar forming. The influence of these mechanical forces on the

damaged bowel is nearly unknown with regard to the enteric nervous

system. If there is any functional consequence out of these observa-

tions is improbable, because the motility of the bowel seems only to be

deranged over a very short distance and not notable dependent of these

damaged nerves. To appreciate the differences in healing quality of

anastomoses it should be possible to determinate protein clusters that

are corresponding with the effectiveness of actual healing forces.

The clinical genetics of Hirschsprung’s disease

s lyonnet, as jannot, a tullio-pelet & j amiel

Department of Genetics and INSERM U-781, University Paris Descartes, Hopital

Necker-Enfants Malades, Paris, France

Hirschsprung disease (HSCR) is the most common congenital mal-

formation of the enteric nervous system. HSCR is characterized by the

congenital absence of intrinsic ganglion cells in the submucosal and

myenteric plexuses of the hindgut, leading to intestinal obstruction

and colonic distension in newborns. The length of aganglionosis is

variable and correlates with the severity of the disease, extending from

the rectum to the recto-sigmoid junction (short-segment, 80% of cases)

or beyond (long-segment, 20%). HSCR has an incidence of 1/5000 live

births, a circumstance which, together with the actual good surgical

prognosis, has allowed recruitment of quite a large series of patients

over long periods of time and also identification of familial cases.

Interestingly, in a significant number of cases (up to 30%), HSCR is

associated with other malformation or developmental anomaly,

including more complex neural crest defects, a fact that underscores

the great importance of a clinical and dysmorphological examination

of HSCR patients. Regarded as a single field neurocristopathy owing to

the embryonic origin of enteric neurons, HSCR is also a model of a

genetically complex inborn error of development. Despite the high

proportion of sporadic cases (80%), segregation analyses showed that

HSCR has a high heritability and is inherited as a sex-modified oligo-

genic trait with a variable and non-mendelian risk to siblings (3-22%

depending on the length of aganglionosis, the gender of the proband, and

the familiality). The major locus is clearly RET, probably involved in all

cases. In addition, a number of parametric and non-parametric linkage

analyses used to deconstruct the complex genetic multiplicative model

of inheritance underlying HSCR, suggest heterogeneous modifier loci

whose requirement might depend on the nature (coding or non-coding

sequence mutations), the �dose�, and the transmitting parent of mutant

alleles at the major locus RET, as well as possibly the genetic and ethnic

background. The purpose of the present talk will be to discuss these data

in a clinical setting and the interest for patients and families. In partic-

ular, this presentation will attempt to address some of the following

issues: (i) the clinical approach of syndromic HSCR, (ii) the links be-

tween HSCR, regarded as a developmental anomaly, and some inherited

predisposition to cancer, (iii) the clinical interest of genetic testing in

HSCR families, and (iv) the risk figures and genetic counselling issues in

HSCR families.

High-field magnetic resonance imaging: stem cell tracking and

phenotyping of transgenic mice

mf lythgoe

UCL Institute of Child Health, London, UK

In this seminar I will focus on two challenges in biomedical imaging

science (i) the tracking of stem cells to the sites of tissue damage and

(ii) non-invasive visualisation of abnormalities in embryo develop-

ment. Development of methods capable of labelling and tracking cells

in vivo would be of great benefit to the understanding of cell migration

under normal and pathological conditions. In a recent study, we

demonstrate that endogenous neuroblast migration cells can be la-

belled in vivo with an MRI contrast agent and that they can be visu-

alised using MRI, which indicates the feasibility of in vivo imaging of

cell migration using MRI. lMRI is now an emerging technique for

high-throughput phenotyping of transgenic mouse embryos, visualis-

ing abnormalities in development. The mouse is a key model for

research into human disease and is ideally suited to genetic study, as

the genome and techniques for manipulating its sequence are readily

available. One such technique is ENU mutagenesis, utilised by the

large-scale programmes underway to create mutant models for each

of the >25 000 genes to study their function. This increasing use of

genetically modified mice has highlighted the need for techniques to

rapidly characterise the new morphological phenotypes presented in

these models. lMRI may be an ideal high-throughput technique for the

imaging of multiple embryos.

Development of Neurons and the Role of Neural Activity in the

Developing Gut

mm hao,* re moore,* rb anderson,* jc bornstein,� ea jennings* &

hm young**Departments of Anatomy and Cell Biology, University of Melbourne, Mel-

bourne, Via, Australia ; and �Department of Physiology, University of Melbourne,

Melbourne, Via, Australia

Introduction: As they migrate through the developing gut, enteric

neural crest-derived cells (ENCCs) begin to differentiate into neu-

rons. In the developing CNS, neural activity has recently been

shown to affect the proliferation, differentiation and migration of

neighbouring neural precursor cells. Objectives: To examine the

timetable of neuronal differentiation in the developing gut and

establish whether neural activity affects the migration of

ENCCs. Methods: Immunohistochemistry was performed on

wholemount preparations of E10.5-E11.5 gut. Intact explants of mid-

plus hindgut from E11.5 RetTGM-GFP/+ mice, where all ENCCs

express GFP, were grown in culture with drugs that inhibit different

forms of neural activity: tetrodotoxin (TTX) to block neural action

potentials, nitro-L-arginine (NOLA) to inhibit nitric oxide produc-

tion, tetanus toxin to inhibit vesicle-dependent neurotransmitter

release, and clotrimazole to block IK channel activity. The distance

that ENCCs migrated was examined after 48 h. Whole cell patch-

clamp recordings were made from isolated neurons from the

embryonic gut of Ednrb-Kik mice, where all neurons express

the photo-convertible fluorescent protein, Kikume, to examine

the development of electrical properties. Results: As reported pre-

viously, around 15% of ENCCs in the gut from E10.5–E12.5 mice

expressed the pan-neuronal markers Hu, Tuj-1 and neurofilament-M,

and extended processes. Tuj-1+ and neurofilament-M+ nerve fibres

were present in close apposition to many of the ENCCs, including

those at the migratory wavefront. Commencing at E11.5, nitric

oxide synthase (NOS), calbindin and IK channel immunoreactivity

were present on a sub-population of ENCCs. In cultured explants of

embryonic gut, TTX, NOLA and clotrimazole did not affect ENCC

migration, but tetanus toxin significantly reduced ENCC migration

as reported previously1. In patch-clamp studies, action potentials

were recorded from isolated enteric neurons from E18.5

mice. Conclusions: Although neurons expressing pan-neuronal and

enteric neuron sub-type specific markers are in close association

with ENCCs while they are colonizing the gut, we found no evi-

dence that neuronal activity affects ENCC migration. Tetanus toxin

inhibited ENCC migration but it is unclear whether this is due to

inhibition of neurotransmitter release or inhibition of other mech-

anisms such as membrane re-cycling.

Reference:

1 Vohra BP et al. Differential gene expression and functional analysis

implicate novel mechanisms in enteric nervous system precursor

migration and neuritogenesis. Dev Biol 2006; 298: 259–71.

Abstracts

� 2009 Blackwell Publishing Ltd v

Vitamin A facilitates enteric nervous system precursor migration by

reducing PTEN accumulation

m fu,* y sato,* a lyons-warren,* bin zhang,� ma kane,� jl napoli� & ro

heuckeroth*,§,–*Department of Pediatrics, Washington University School of Medicine, St Louis,

MO, USA; �Department of Pathology and Immunology, Washington University

School of Medicine, St Louis, MO, USA; �Department of Nutritional Science and

Toxicology, University of California, Berkeley, CA, USA; §Department of

Developmental Biology, Washington University School of Medicine, St Louis,

MO, USA; and –HOPE Center for Neurological Disorders, Washington University

School of Medicine, St Louis, MO, USA

Aims: To test the hypothesis that retinoid signaling is important for

enteric nervous system precursor migration and to identify the molec-

ular machinery that responds to RA. Methods: We used several dif-

ferent culture techniques to investigate the effect or RA signaling on

ENS precursor migration including gut organ culture, slice culture, and

Boyden chamber assays. We also investigated the effect or RA in vivo

using mice deficient in serum retinol binding protein (Rbp4).

These animals are dependent on daily vitamin A ingestion to meet

tissue needs and can be rapidly depleted on circulating

retinoids. Results: Vitamin A depleted Rbp4 -/- mice have striking

distal bowel aganglionosis, but these mice have a normal appearing ENS

if they are maintained on Vitamin A containing food. Rbp4 -/- Ret +/-

double mutant mice have increased distal bowel aganglionosis even

when fed Vitamin A containing food substantiating an important role

for vitamin A as a non-genetic modifier of EN\S development. In vitro

studies demonstrate that RA signaling is critical for ENS precursor

migration and lamellipodia formation. This effect is attributable to

reductions in Pten protein levels in the most actively migrating cells.

Pten overexpression also reduces GDNF induced ENS precursor

migration in vitro consistent with the hypothesis that RA induced Pten

suppression is required for efficient ENS precursor migra-

tion. Conclusion: This study suggests the intriguing possibility is that

Vitamin A deficiency is a preventable cause of Hirschsprung’s disease.

Correlated expression of S100 and GFAP in enteric glia in rodent and

human gut

kh schafer,* s maas-omlor,* aj burns� & u rauch**University of Applied Sciences, Kaiserslautern, Germany; and �UCL, Institute of

Child Health, London, UK

Introduction: For years, enteric glia have played a �wallflower�-like

role, although their importance was already recognised by various

authors, including Michael Gershon, Georgio Gabella and Kristjan

Jesssen, who demonstrated that enteric glia contain the glial fibrillary

acidic protein GFAP, like reactive astrocytes in the central nervous

system. In recent years, the key role of enteric glia in supplying neu-

rotrophic support or responding to inflammation has become increas-

ingly obvious. However, there is still little known about the

distribution or the relationship between the two glial markers S100, as

a general marker, and GFAP, as a marker for reactive

glia. Objectives: To deliver a baseline for further studies on the role of

enteric glia in different diseases and species. To that end S100 and

GFAP immunoreactivity was assessed during development as well as

in various diseases or disease models such as the DSS (dextran sulfate

sodium) colitis. Methods: Tissue from all parts of the gstrointestinal

tract from esophagus to distal colon were collected from human and

rodent sources. In humans, tissue from the 7th week of gestation to old

age, and in rat from postnatal day 1 to adult was collected and

embedded in paraffin. Additional tissues were obtained from cases of

appendicitis, colon cancer, or from mice where a DSS colitis was

induced. 5 lm sections were cut and doublestained for S100 and

GFAP. Results: Both glial markers appear to be developmentally reg-

ulated independently from each other. In humans S100 is first seen in

samples from week 12 of gestation. Here it appears in the myenteric

plexus only and with a slight gradient from esophagus to the distal

colon. During development the S100 signal gets stronger and is fully

developed in the middle of the second trimester. In our sections GFAP

does not appear prenatally. After birth the GFAP signal is strongly

correlated to diseases. Its signal gets stronger when the tissue is

inflamed or also in colon cancer tissue. In the rat, both S100 and GFAP

do not appear before birth. At the first postnatal day S100 is only

present in the esophagus and stomach and gets stronger with the age of

the animal. At postnatal day (P)14 the signal is fully developed

throughout the gut, while GFAP reactivity cannot be seen all over the

full length of the gut before P28, although it appears partially from P7

onwards. In general, there seem to be glial cells in the muscle layer which

never show GFAP reactivity. Conclusion: There is evidence for at least

two distinct glial populations in the enteric glia which may react in

various amounts to external influences such as inflammation.

Neurospheres are not like neurospheres

kh schafer,* ma micci�,� & pj pasricha�*Universitiy of Applied Sciences, Kaiserslautern, Germany; �UTMB, Galverston,

USA; and �University Stanford, Stanford, CA, USA

Introduction: The transplantation of suitable neural stem cells seem

more and more to be a challenging alternative for the restoration of

gastrointestinal dysfunctions, such as nitrergic deficits in pylorus ste-

nosis, gastroparesis, achalasia or dysganglionosis. Individual attempts

were performed to open up new resources for auto- or heterologous

stem cell supply which can be used in clinical treatments. Potential

sources are the subventricular zone from fetal tissue or the enteric

nervous system itself. Depending on the site and time of isolation

of the neural stem cells, there might be a significant difference in

the yield of cells, as well as their potential to differentiate into

all cell types needed for the restoration of gastrointestinal func-

tion. Objectives: To demonstrate the problems which have to be faced

by using varying sources for neural stem cells, we compared the growth,

the needs and the differentiation of neurospheres derived from either

the central nervous system or the gastrointestinal tract from the same

animal. Methods: Stage pregnant mice at the embryonic age E13 were

sacrificed by an overdose of isoflurane. The embryos were immediately

removed and stored on ice. From each embryo the subventricular zone

as well as the whole gastrointestinal tract was removed. Both tissues

were processed identically. After incubation in a mixture of collagenase

and dispase, the tissues were dissociated, cell number counted and cells

plated at equivalent cell numbers in Neurobasal medium containing

the neurotrophic factors EGF (epidermal growth factor), FGF (fibroblast

growth factor) and GDNF (glial cell line derived neurotrophic factor).

The cultures were kept for several days until neurospheres could

be harvested in significant numbers. Neurosphere and sizes were

measured. After 5 days in culture, the neurospheres were harvested

and explanted in a three dimensional gel (extracellular

matrix). Results: There were significant differences concerning the

trophic supply, the growth and the differentiation of the two types of

neurospheres. While SVZ spheres could be cultured in EGF and FGF

alone, the ENS spheres needed additional GDNF for sufficient growth.

In general the SVZ spheres grew faster, bigger and more numerous.

Within the gel, the two types of neurospheres grew differentially. The

SVZ spheres differentiated slower, starting with a glial migration, fol-

lowed by a neurite outgrowth after three days in vitro, while the ENS

spheres started immediately with a very dense neurite network and

only few migrating glial cells. Conclusion: Before using individual

stem cell sources for clinical trıals, the quality of the isolated neural

stem cells have to be assessed of being usable in an appropriate way.

Interaction of the enteric nervous system and enterocytes in vitro is

increased by growth hormone treatment

l schwarz, d griesemer, u rauch, m klotz & kh schafer

Universitiy of Applied Sciences, Kaiserslautern, Germany

Introduction: Growth and regeneration of the mucous layer is a very

important prerequisite for the undisturbed function of the gastroi-

ntestinal tract. Lacking, insufficient or inaccurate regeneration and

proliferation might lead to severe disturbances of food resorption,

deficiencies or even malformations of the mucous layer. While in most

in vitro studies merely enterocyte cell lines were used to model the

mucosal barrier, the enormous influence of the intrinsic nervous sys-

tem, the enteric nervous system, upon the gastrointestinal regenera-

tion is mostly neglected. In this study the interaction of ENS and

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdvi

mucosal cells in vitro is investigated, especially under the influence of

growth hormone treatment. Objectives: The use of growth hormone

to increase the mucosal surface for a better resorption i.e. in short

bowel disease is still controversial. In the presented study the indirect

influence of GH upon mucosal proliferation via the enteric nervous

system is investigated. Methods: In vitro experiments were per-

formed using enterocytic cell lines alone and in coculture with isolated

myenteric plexus from the newborn rat. Both cell types were cultured

under the influence of growth hormone. Supernatants were collected

and fed to either one of the two cell types. Enterocyte proliferation was

measured using BrdU-ELISAs under or without the influence of neu-

ronal and glial secreted factors. Enterocyte cultures were fixed and

processed for scanning electron microscopy to evaluate the variation in

microvilli density. Results: The enterocyte proliferation in vitro

could significantly be increased by using supernatants from dissociated

myenteric plexus cultures, especially when treated with GH. These

supernatants where measured concerning neurotrophin content and

various neurotrophic factors such as NT-3 or CNTF could be detected.

Scanning electron evaluations of the enterocyte surfaces revealed

changes in cell size and microvilli density depending on the treat-

ment. Conclusion: The enteric nervous system and the enterocytes

do strongly influence each other. This influence might be used to

stimulate mucosal growth and regeneration by influencing the ENS

directly using various neurotrophic factors. The use of co-culture

models where several compounds of the gut wall such as mucosal

barrier and the ENS are represented might lead to a much better

understanding of the mucosa-nervous-system interaction.

Stage-specific control of neural crest stem cell proliferation

l sommer

University of Zurich, Zurich, Switzerland

During vertebrate development, neural crest (NC) cells emigrate from

the neural tube to generate a variety of neural and non-neural tissues.

Both migratory NC cells and their target structures contain so-called

neural crest-derived stem cells (NCSCs) that display multipotency and

self-renewal capacity. However, maintenance and proliferation of

these NCSC populations are differentially regulated. Indeed, Cdc42 or

Rac1 inactivation reduces self-renewal and proliferation of later stage

NCSCs, including those isolated from the enteric nervous system,

while self-renewal of early migratory NCSCs is Cdc42/Rac1-indepen-

dent. Accordingly, deletion of either Cdc42 or Rac1 in the NC results

in size reduction of multiple NC target structures because of increased

cell cycle exit. In contrast, NC cells emigrating from the neural tube

are not affected. This stage-specific requirement for small Rho GTP-

ases is due to changes in NCSCs that during development acquire

responsiveness to mitogenic EGF acting upstream of both Cdc42 and

Rac1. Thus, our data reveal distinct mechanisms for growth control of

NCSCs from different developmental stages.

Development and maintenance of interstitial cells of Cajal networks

in health and disease

sm ward, sj hwang, y bayguinov & km sanders

Department of Physiology and Cell Biology, University of Nevada, Reno, USA

Interstitial cells of Cajal (ICC) play critical roles in several GI motility

functions including: (i) generation of pacemaker activity (ii) propagation

of pacemaker slow waves (iii) mediation of excitatory and inhibitory

enteric motor inputs (iii) providing stretch-dependent responses within

the smooth muscle syncytium and (iv) guidance of vagal intramuscular

afferent nerves. It is well accepted that the development and mainte-

nance of ICC networks is a necessary pre-requisite for normal gut motor

function and their loss contributes to several GI dysmotilities. How the

development and maintenance of ICC networks is achieved within the

GI tract is quite controversial. It has been reported that ICC networks

develop from Kit(+) mesenchymal precursors between E15-E17.1

However, the importance of Kit signaling for the development of ICC

networks prior to birth is contentious.2,3 ICC may be replaced after

birth from Kit(low)CD44(+)CD34(+) Insr(+)Igf1r(+) progenitor cells.4

More recently it has also been demonstrated that Kit(+) ICC possess the

capability to undergo mitosis.5 Using a pulse-chase experimental

approach, tissues from animals of different ages were exposed to BrdU

and Edu for varying durations, to determine the capability of Kit(+) ICC

networks to undergo mitosis prior to and after birth. We further eval-

uated whether ICC undergo apoptosis and whether dividing ICC expand

networks to provide replacement of ICC in adult animals. We also

evaluated whether expansion of ICC networks during growth of the GI

tract occurs by cell division of mature Kit(+) ICC. In E17 tissues, nuclei

of Kit(+) ICC incorporated BrdU or EdU and this continued until at least

P30, suggesting that mature ICC within networks possess the capability

of cellular division. The number of ICC undergoing mitosis decreased

with age and dropped significantly after P10. We found little evidence

for apoptosis in Kit(+) ICC in GI tissues over the same time period, but

Kit immunoreactivity decreased and pacemaker activity ceased in tis-

sues following application of the pro-apoptotic agent camptothecin

(5lg ml-1; for 24 h). Acute application of camptothecin had no affect on

pacemaker activity. In conclusion Kit(+) ICC possess the capability to

undergo mitosis prior to and after birth and this process is likely to

ensure growth of ICC networks during development of the gut wall.

References:

1 Torihashi et al. Development of c-Kit-positive cells and the onset of

electrical rhythmicity in murine small intestine. Gastroenterology

1997; 112: 144–55.

2 Kluppel et al. Developmental origin and Kit-dependent development

of the interstitial cells of cajal in the mammalian small intestine.

Dev Dyn 1998, 211: 60–71.

3 Beckett et al. Kit signaling is essential for development and main-

tenance of interstitial cells of Cajal and electrical rhythmicity in the

embryonic gastrointestinal tract. Dev Dyn 2007, 236: 60–72.

4 Lorincz et al. Progenitors of interstitial cells of cajal in the postnatal

murine stomach. Gastroenterology 2008, 134: 1083–93.

5 Mei et al. An age-dependent proliferation is involved in the postnatal

development of interstitial cells of Cajal in the small intestine of

mice. Histochem Cell Biol 2008; Oct 3, Epub.

SOX10 genetic interactions: Waardenburg syndrome and mouse

double mutant studies

l stanchina,* y watanabe,* v baral,* m goossens,*,� v pingault*,� &

n bondurand**INSERM U955, IMRB, departement de genetique, Creteil, France; and �AP-HP,

Groupe Albert Chenevier-Henri Mondor, service de biochimie et genetique,

Creteil, France.

Study of the dominant megacolon mice and SOX10 involvement in

Waardenburg syndrome type 4 (WS4) first highlighted the crucial role

of this transcription factor for proper enteric nervous system and

melanocyte development. Recently, we described SOX10 deletions in

15% of patients presenting with WS2 (pigmentation defects without

HSCR). These results further documented the molecular complexity

and close relationship that link the different subtypes of WS, but still

leave 70% of WS2 and 25%–35% of WS4 with no molecular explana-

tion, and suggest that other genes might be involved, or that mutations

within the known genes still escape screening performed so far. Based

on these observations, we looked for point mutations and rearrange-

ments within the EDN3/EDNRB and MITF/SNAI2 genes in both WS2

and WS4 cases, and show that the molecular overlap between WS2 and

4 is mostly restricted to SOX10. We also considered the possibility that

mutations or deletions within the known regulatory sequences of

identified genes could explain some WS4 and WS2 cases. Results of

this ongoing work will be presented. In parallel, we pursued our effort

aiming at better apprehend SOX10 function during ENS development

and its interaction with other WS or HSCR genes. We recently focused

on ZEB2, a transcriptional repressor of the TGF-b/BMP signaling

pathway. Its alteration in human and mice causes severe defects of

neural crest development including ENS defects, but little is known on

its mechanism of action. Expression studies revealed that it is present

in SOX10+ enteric progenitor cells, and switches off during neuronal

differentiation in a manner reminiscent to what is observed for SOX10.

Based on these observations, we tested the hypothesis that interactions

between SOX10 and ZEB2 are essential to control ENS progenitor

maintenance and differentiation. To this end, we crossed Sox10 and

Zfhx1b knockout mice and compared the enteric phenotype of single

Abstracts

� 2009 Blackwell Publishing Ltd vii

and double heterozygotes. TuJ1 and Xgal immunostaining revealed a

more severe ENS phenotype in double mutants at all stages observed.

Experiments are ongoing to determine the cellular and molecular origin

of these observations.

Glutamate affects intestinal motility in zebrafish larvae

c olsson, c bergstrom & i wronski

Department of Zoology/Zoophysiology, University of Gothenburg, Goteborg,

Sweden

Glutamate is known to act as a neurotransmitter in the mammalian

gut. It is found in myenteric inter- and sensory nerons and in extrinsic

afferents and may affect gastrointestinal motility mainly by acting on

excitatory motorneurons. There are several receptor subclasses, many

of which are also present in the gut. Zebrafish (Danio rerio) larvae

provide an excellent model for in vivo studies on the development of

gut motility. Anally and orally propagating contraction waves are seen

from 3 days post fertilization (dpf), using video microscopy on the

intact transparent larvae (e.g. Holmberg et al 2003). Furthermore, the

effects of both inhibitory and excitatory transmitters have been

demonstrated using this method. In the present study, glutamate was

added to zebrafish larvae and the effect on propagating contraction

frequency was recorded. In addition, the effects of three different glu-

tamate receptor antagonists were examined: the NMDA-receptor

antagonist MK801, the AMPA-receptor antagonist CNQX and the

metabotropic receptor antagonist CPPG. The response to glutamate

itself was inconsistent. Most preparations did not show any effect on

contraction frequency, except a small increase in retrograde contrac-

tions at 7 dpf and a decrease at 9 dpf. In contrast, MK801 and CNQX

caused either a direct reduction in anterograde contraction frequency,

or a reduction was seen as a response to a subsequent glutamate

application at 4, 7 and 9 dpf. CPPG showed a reduction at 9 dpf only.

The effects on retrograde contractions were less consistent but fol-

lowed a similar pattern. The results indicate that glutamate can affect

gut motility very early in zebrafish development. The direct effect of

CNQX seen at 4 dpf further indicates that glutamate may be endoge-

nously released already at that stage. The results also indicate that

glutamate has both excitatory and inhibitory effects and this could be

the reason for the apparent lack of response to exogenous glutamate.

However, by blocking NMDA- and AMPA-receptors, an inhibitory

response to glutamate could be evoked. This suggests the involvement

of several receptor types in the glutamatergic control, but the exact

nature of the zebrafish receptors needs further investigations.

Reference:

1 Holmberg et al. Ontogeny of intestinal motility in correlation to

neuronal development in zebrafish embryos and larvae. J Fish Biol

2003; 63: 318–31.

A full mutation spectrum in Hirschsprung disease: copy number

analysis

bq doan,* c stewart,� c kashuk,* sm arnold,* a chakravarti* & inter-

national hscr consortium*,�,§,–,**

*JHMI, Baltimore, MD USA; �Boston College, Boston, MA, USA; �INSERUM,

Paris, France §Istituto Gaslini, Italy; –U.Groningen, Netherlands; and

**H.U.Virgen del Rocıo, Spain

Introduction: Multiple genes with rare and common risk variants and

gene dosage sensitivity have been identified in Hirschsprung disease

(HSCR). Using genome-wide Affymetrix 500K SNP arrays, we initially

performed a family-based association analysis on 233 trios from the

International HSCR Consortium, and a novel set of genes were identified

on 7q: the semaphorins. Objective: Given that only one new set of

genes were identified and successfully replicated from our association

results, we subsequently performed a copy number analysis using SNP

intensity data to identify gene dosage sensitive regions. Methods: We

performed a copy number variant (CNV) analysis applying the ITALICS

copy number analysis tool to identify CNVs. ITALICS uses an aCGH

segmentation algorithm and controls for non-biological variation across

arrays. We also used the 270 HapMap samples to determine expected

levels of CNVs. Results: We identified a total of ~5000 CNVs (72 bp–

110 mb) in HSCR, with an average length per individual of 500 kb

(32SNPs : Nsp) or 270 kb (22SNPs : Sty). Multiple regions were identi-

fied that were biologically interesting. A de novo deletion of 1.4 mb on

6q25.1 was the most statistically significant variant, although present in

only one affected offspring. Corresponding genotype data revealed high

proportions of missing data, homozygous calls, and Mendelian errors

supporting the presence of a true deletion. Examining all CNVs within

this region, five more individuals with 1.3–62.9 kb deletions were

identified, as compared to only one 33 kb deletion in HapMap. This

suggests that although rare, the 6q25.1 region is potentially associated

with HSCR. Of great interest is the identification of 665 bp–112 kb

deletions (both de novo and segregating) in 29 individuals at 9q31, which

resides in the middle of a known linkage peak shown to be a genetic

modifier, and a 28 kb amplification in 11 individuals on 1q32.2, which

encompasses the PlexinA2 gene, the co-receptor for semaphorin3A.

Focusing on only recurrent de novo events reveals additional

variants of interest: a 128 kb region on 8p22 that occurs in four trios, and

a 203 kb amplification on 21q21.3 that occurs in three trios. The 21q21

region has also been previously shown to be a genetic modifier

locus. Conclusions: We have identified several gene dosage sensitive

regions, a few which reside in regions with previously known linkage

evidence, and a region that overlaps a gene identified as the co-receptor

for a gene recently identified from a genome-wide association study. To

minimize follow-up of false positives, we are reassessing these CNVs in

the context of rare and common disease variants identified from our

medical resequencing and genome wide association analysis on the

same study population. Prioritized regions are being experimentally

validated.

A zebrafish view of progressive fate restriction in neural crest

development

rn kelsh,* x yang,* ss lopes� & m nikaido**Department of Biology and Biochemistry, University of Bath, Claverton Down,

Bath, UK; and �Instituto Gulbenkian de Ciencia, Oeiras, Portugal

A fundamental issue in stem cell biology concerns the mechanisms

whereby cells adopt individual derivative fates. Neural crest cells con-

sist of multipotent progenitors for an astonishing diversity of different

cell-fates, including enteric neurons and glia, but also encompassing

multiple other peripheral neuronal and pigment cell types, as well as

varied skeletogenic fates. A major research objective in the field is to

uncover the mechanisms of fate specification from multipotent neural

crest (stem) cells. In vivo studies in the zebrafish model system strongly

suggest a progressive fate restriction model of neural crest cell devel-

opment, whereby progenitors become gradually restricted in the fates

that they can adopt. We will summarise briefly this evidence, before

describing our genetic and cell biological studies of the shady/leukocyte

tyrosine kinase (ltk) mutants (Lopes et al., 2008). Shady mutants lack

iridophores, a widespread pigment cell-type in cold-blooded vertebrates

and this results from gene function within the neural crest. Expression of

ltk at late stages [30 h post fertilisation (hpf) onwards] itself is an

excellent marker for differentiated iridophores and using this, together

with newly characterised markers for this cell-type, we show that dif-

ferentiation of iridophores fails in mutants. However, at c. 20 hpf ltk

expression is also absent, suggesting that ltk functions either in multi-

potent progenitors to mediate iridophore specification or very early in

iridoblasts to mediate their ongoing development.We show that in

mutants cell death is elevated, but only from later stages (c. 35 hpf on-

wards). We show that this phenotype parallels that in the well-charac-

terised sox10 mutants, in which fate specification of neural and pigment

cell fates is a common feature. Strikingly, early expression of ltk is

strongly increased in sox10 mutants. Importantly, this expression is

non-overlapping with that of very early neural crest cell markers like

snail2. We conclude that ltk expression may be a marker for a partially-

restricted neural crest progenitor, perhaps the hypothetical chromato-

blast; we propose that ltk function is critical for iridophore lineage

specification. We are now trying various approaches to test this

hypothesis and will present our progress to date.

Reference:

1 Lopes, SS, et al. Leucocyte tyrosine kinase functions in pigment cell

development. PLoS Genetics, 2008; 4: e1000026.

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdviii

Identification of mammalian neural crest enhancers through

transgenesis in Zebrafish

as mccallion,* wj pavan,� a chakravarti,* s johnson,� a antonellis,�jl huynh,* ze stine,* dm mcgaughey,* d gorkin,* mk prasad,*

es emison* & sk loftus�*McKusick–Nathans Institute of Genetic Medicine, The Johns Hopkins Univer-

sity School of Medicine, Baltimore, USA; �National Human Genome Research

Institute, National Institutes of Health, Bethesda, USA; and �Washington Uni-

versity, St Louis, USA

Introduction: Gene regulation is the framework on which cellular

diversity is built. The substantial cellular diversity that characterizes

embryonic development in vertebrates such as humans must therefore

require immense regulatory complexity. Although regulatory control

acts at many levels, we will focus on the roles played by cis-regulatory

elements (REs) in controlling the timing, location and levels of

neural crest transcripts. However, the biological relevance of non-coding

sequences cannot be inferred by examination of sequence alone.

Perhaps the most commonly used indicator of non-coding REs is

evolutionary sequence conservation. Although conservation can

uncover functionally constrained sequences, it cannot predict biological

function; regulatory function is also not confined to conserved se-

quences. At its simplest level, regulatory instructions are inscribed in

transcription factor binding sites (TFBS) within REs. Yet, while many

TFBS have been identified, TFBS combinations predictive of specific

regulatory control have not yet emerged for vertebrates. The absence of

such vocabularies serves as a significant impediment to correlations of

non-coding sequence variation and disease phenotypes. A role for such

non-coding variation in Hirschsprung Disease (HSCR) risk is now

well established; we will describe our efforts to identify HSCR

relevant REs and to assay the pathological significance of variants

therein. Objectives: We posit that motif combinations accounting for

tissue-specific regulatory control can be identified in REs of genes ex-

pressed in those cell types. We have set out to uncover NCREs at critical

NC genes, focusing on major HSCR and related loci. Our long-range goal

is to begin to identify TFBS combinations that can predict neural crest

(NC) REs – a first step in developing a regulatory lexicon. This data is

being integrated with ongoing human genetic efforts to evaluate the role

that non-coding variation may play in disease risk, in particular HSCR

risk. Methods: We have developed a highly efficient reporter transgene

system in zebrafish that can accurately evaluate the regulatory control of

mammalian sequences, enabling characterization of reporter expression

during development at a fraction of the cost in mice. We are actively

developing novel strategies to further increase the efficiency of

these assays and to enable assays of the in vivo functional consequences

of disease associated non-coding variation within identified

REs. Results: We have already identified many REs based on their

sequence conservation among vertebrates, although we have recently

demonstrated that they can also be embedded in non-conserved

sequence space. We are systematically evaluating the regulatory activity

of putative REs identified at all major HSCR loci. To date we have

identified ‡75 novel REs including numerous NCREs at genes including,

but not restricted to, RET, GFRA1, PHOX2B, SOX10, EDNRB

and ZFHX1B. Identified NCRE control ranges from discrete NC

subpopulations e.g. the enteric nervous system or melanocyte

populations, to pan neural crest control consistent with their varied and

often pleiotropic roles in development. We will describe our ongoing

efforts to determine the biological relevance of putative REs at these

and other genes; to build functional datasets large enough to begin the

search for regulatory vocabularies of specific NC populations and to

determine the pathological relevance of disease-associated identified

therein. Conclusions: The development of regulatory vocabularies

for specific NC cell lineages will potentially require the identification

ofhundreds of elements with overlapping regulatory control in

those cell types as well and those known to control expression

outside the NC. We have already cloned and analyzed in excess of

150 putative REs in vivo; we will discuss our progress in establishing

this functional sequence substrate and in illuminating pathological

variation therein.

Social controls of migration and proliferation of neural crest-derived

cells in the gut

df newgreen,* dc zhang,* b binder,� mj simpson,� ka landman,�h enomoto� & hm young§*The Murdoch Childrens Research Institute, Parkville, Via, Australia; �Depart-

ment of Mathematics and Statistics, University of Melbourne, Melbourne,

Melbourne, Via, Australia; �RIKEN Center for Developmental Biology, Kobe,

Japan; and §Department of Anatomy and Cell Biology, Melbourne, Via, Australia

Introduction: Enteric neural crest-derived cells (ENCC) colonize the

gut in a rostro-caudal wave. In Hirschsprung’s disease (HSCR), this

wave does not reach the distal gut, which remains aneuronal. Defects

in many genes predispose to HSCR, but a notable feature of HSCR

genes is the lack of penetrance. Objectives: To develop formal models

that replicate the colonization of the gut by ENCC, and illuminate the

HSCR phenotype. Methods: We developed avian and mouse models

via electroporation and transgenesis to enable the migration of ENCC

to be observed in 3D intestinal tissue by timelapse microscopy. We

also developed cellular automata (CA) models of ENCC behaviour

incorporating contact-influenced motility and proliferation as well as

simultaneous growth of the gut tissue. Results: Migrating ENCC did

not retain contacts with previous ENCC neighbours, and their trajec-

tories were unpredictable. Proliferation of ENCC was frequent at the

wavefront, but behind the wavefront proliferation was notable only

when the gut was also growing. Labelling few pre-migratory ENCC

with GFP via electroporation gave ENCC groups (probably clones) of

sizes and positions that varied widely between different individuals,

and which overlapped ENCC of different clonal origins. All these

conditions spontaneously emerged from CA models with few and

simple rules governing cell movement and cell proliferation. In CA

models the growth of the gut determined success or HSCR-like failure

of colonization. Interestingly, in CA simulations in which ENCC

invasion parameters were reduced, invasive outcomes (successful

colonization of the entire gut or a distal aneuronal zone) varied sto-

chastically between simulation runs with identical starting condi-

tions. Conclusions: We conclude that the ENCC behave as

individuals, yet they require interactions between the cells which

exerts a decisive overall control on migration outcomes. We propose

that population-scale directional invasion is driven by cell prolifera-

tion, crucially at the invasive front (�frontal expansion�). Growth of the

gut enables ENCC proliferation behind the front (�in-filling�) and

smearing of clonal boundaries. We suggest that events acting within

simple migration and proliferation rules but with stochastic compo-

nents at the cell level govern much of the colonisation process. In

addition, we raise the possibility that lack of penetrance of HSCR, as

observed even in highly inbred animal models, could also have a

stochastic element.

Netrin biosynthesized by enteric neurons mediates the attraction of

vagal sensory axons to the fetal gut

em ratcliffe,* a chalazonitis� & md gershon�*Department of Pediatrics, McMaster University, Hamilton, ON, Canada; and

�Department of Pathology & Cell Biology, Columbia University, New York, USA

Vagal sensory axons innervate the gut during fetal life. By applying DiI

to the nodose ganglia at a range of gestational ages in fetal mice, we

have found that vagal sensory axons descend to the stomach and small

bowel by E12 and E14 respectively, following the rostral-caudal

migration of crest-derived cells. DiI-labeling has furthermore revealed

that vagal sensory axons fail to innervate the aganglionic gut of mice

lacking the tyrosine kinase receptor, Ret. Previous work has shown

that netrin is expressed in the bowel wall and, by acting on its receptor

Deleted in Colorectal Cancer (DCC), mediates the guidance of vagal

sensory axons to the developing gut. We therefore tested the hypo-

thesis that developing enteric ganglia express netrin. Crest-derived and

non-crest-derived cells of E15 rat gut were separated by positive and

negative immunoselection with antibodies to p75NTR, a marker for

crest-derived cells in the fetal bowel. The p75NTR-immunoreactive

cells were then cultured for 6 days in media supplemented with the

Abstracts

� 2009 Blackwell Publishing Ltd ix

Ret ligand, glial cell derived neurotrophic factor (GDNF; 10 ng ml-1) to

promote neuronal differentiation. Transcripts encoding netrins-1 and -3

were found by RT-PCR in the cultured enteric neurons and in the non-

crest-derived cells, but not in the immunoselected crest-derived pre-

cursors. To test whether enteric neurons take up, as well as synthesize

netrin, the p75NTR-immunoreactive cells were cultured for 6 days in

3-dimensional collagen gels with stably transfected 293-EBNA cells

expressing c-Myc-tagged netrin-1. Controls included crest-derived cells

cultured alone or with parental 293-EBNA cells. In all three types of

culture, neurons (identified by their expression of PGP9.5) were found

to be netrin-immunoreactive. Cultures were immunostained with

antibodies to c-Myc to identify netrin secreted by the transfected

293-EBNA cells. Although the netrin-1-expressing 293-EBNA cells

were c-Myc-immunoreactive, the neurons that developed from the

crest-derived cells were not. Neurons thus did not take up secreted

netrin. To further investigate the role of netrin synthesized by enteric

ganglia, as opposed to from the enteric mesenchyme, protein was

extracted from the bowel of E14 Ret -/-, Ret +/- and Ret +/+ fetal mice.

Quantitative analysis of netrin-1 identified through Western blotting

revealed no significant difference in total amount of netrin-1 protein

per gut. We propose that the source of secreted netrin may play a more

important role than the aggregate amount of enteric netrin in guiding

vagal axons to their correction locations in the developing bowel.

Supported by AGA, CIHR and NS 15547/12969.

A functional approach to understand the role of the Kinesin Binding

Protein (KBP) in Goldberg-Shprintzen syndrome

mm alves,*,� as brooks,�,§ e de graaff,�,§ c hoogenraad,�,§ bjl eggen� &

rmw hofstra**Deptartment of Genetics University Medical Center Groningen, Groningen, The

Netherlands; �Deptartment of Developmental Genetics, Faculty of Mathematics

and Natural Sciences, University of Groningen, Haren, The Netherlands;

�Deparment of Clinical Genetics, Erasmus Medical Center, Rotterdam, The

Netherlands; and §Deparment of Neuroscience, Erasmus Medical Center,

Rotterdam, The Netherlands

Goldberg-Sphrintzen syndrome is a rare autosomal recessive disorder

characterized by polymicrogyria, mental retardation and Hirschsprung

disease. Recently, we showed that all patients suffering from this disease

have inactivating mutations in the KBP/KIAA1279 gene (1). However,

the function of its encoded protein is unknown. Considering that

mutations in KBP are associated with both enteric and central nervous

system defects, clarifying its function will lead to new insights about the

protein network associated with neuronal development. Based on its

primary sequence, KBP is predicted to have two tetratricopeptide repeats

(TPRs). Since the basic function of TPR motifs is to mediate protein-

protein interactions, a yeast two-hybrid screen was performed to identify

possible KBP interacting proteins. We obtained various possible inter-

actors, of which the two major categories were kinesin like proteins

involved in the transport of vesicles and organelles towards the plus end

of the microtubules, and microtubule destabilizing proteins implicated

in axonal growth. These interactions were further confirmed

by co-localization studies, co-immunoprecipitation assays and

expression studies in mice primary cortical neurons. A probable inter-

action with microtubules was also explored, both by co-localization and

in vitro binding assays. Based on the results obtained, we postulate that

KBP plays an important role in microtubule organization/stability

and may also be involved in vesicle transport. In conclusion, our

results bring new insights about Hirschsprung disease and polymicro-

gyria development pointing towards a microtubule associated problem

and present KBP as a new/major player involved in neuronal

development.

Reference:

1 Brooks AS et al. Homozygous nonsense mutations in KIAA1279 are

associated with malformations of the central and enteric nervous

systems. Am J Hum Genet 2005; 77 (1): 120–6.

The association of Bardet-Biedl Syndrome and Hirschsprung disease

highlights the role of the primary cilium in ENS development

j amiel,*,� l de pontual,*,1s thomas,*,1

na zaghloul,�,1dm mcgaughey,�

h dollfus,– c baumann,** ee davis,� a munnich,*,� h etchevers,*

m vekemans,*,� s lyonnet,*,� a mccallion,�,§ t attie-bitach*,� &

n katsanis�*Universite Paris Descartes, INSERM U-781, Paris, France; �Departement de

Genetique, Faculte de Medecine; AP-HP, Hopital Necker-Enfants-Malades, Paris,

France; �McKusick-Nathans Institute of Genetic Medicine Johns Hopkins

University School of Medicine, Baltimore, MD, USA; §Department of Molecular

and Comparative Pathobiology, Johns Hopkins University School of Medicine,

Baltimore, MD, USA; –Service de Genetique Medicale, Hopital de Haute-Pierre,

Strasbourg, France; and **Service de Genetique Medicale, Hopital Robert Debre,

Paris, France

Hirschsprung disease (HSCR) is a model neurocristopathy for under-

standing genetic diseases with a multigenic mode of inheritance.

Roughly 30% of the time, HSCR is associated with other congenital

anomalies as the result of chromosomal rearrangements (mostly Down

syndrome), monogenic Mendelian disorders of all modes of inheritance,

or other, unknown causes. In such associations, penetrance for the

HSCR trait is always incomplete (5–70%), suggesting additional

predisposing genetic factor(s). Bardet–Biedl syndrome (BBS, MIM

209900) is a genetically heterogeneous multisystemic disorder charac-

terized by postaxial polydactyly, progressive retinal dystrophy, obesity,

hypogonadism, renal dysfunction, variable learning difficulties and, in

about 5% of cases, HSCR. BBS proteins are involved in the assembly

and function of primary cilia or the function of basal bodies, affecting

both intraflagellar transport (IFT) and planar cell polarity (PCP). We

previously demonstrated the role of RET as a modifier gene for the

enteric phenotype in certain HSCR-predisposing syndromes (congenital

central hypoventilation, Down, and Bardet-Biedl syndromes) but not all

(Mowat-Wilson and Waardenburg type 4 due to SOX10 gene muta-

tions). This situation suggests that some HSCR cases are RET-depen-

dent and others, RET-independent. Surprisingly, the greatest RET

dependence was observed in the group of syndromic patients with both

BBS and HSCR, although functional interactions between RET and BBS

proteins are not known. We here report the co-segregation of mutations

at one BBS locus and at the RET locus for the HSCR trait to occur in BBS

familial and sporadic cases. We showed that human NCC possess a

primary cilium and express BBS genes as well as many components of

the PCP pathway. Genetic interactions between the BBS and RET

pathways were supported by zebrafish double Ret and Bbs4 morphants

in which enteric neurons were completely absent from the distal gut.

These data emphasize that the role of a primary cilium in NCC

migration and enteric nervous system development has been underes-

timated.

Semaphorin Family 3 Members Are Associated With Hirschsprung

Disease

s arnold,* m guy,* k kashuk,* y li,� g abecasis,� a chakravarti*

& international hscr consortium*,�*IGM, Johns Hopkins Univ Sch Med, Baltimore, MD, USA; and �Ctr for Stat Gen,

Department of Biostatistics, School of Public Health, University of Michigan, Ann

Arbor, MI, USA

Hirschsprung Disease (HSCR) is a complex disorder for which a

growing number of candidate genes and chromosomal regions hypo-

thetically modify the function of the major HSCR gene, the receptor

tyrosine kinase RET. HSCR varies in the length of colon affected by

aganglionosis, from the least severe and most common short segment

disease (S-HSCR) to the least common and most severe form, total

colonic agangionosis (TCA). The transmitted allele for a common RET

enhancer variant, RET+9.7, has greater penetrance with decreasing

severity of disease, while coding RET mutations are more common

with increasing severity of disease. In order to identify other common

variants that might contribute to the majority of HSCR cases, 220

1These authors contributed equally to the work.

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdx

S-HSCR trios were analyzed on the Affymetrix 500K SNP array plat-

form. Although RET displayed the highest degree of association by the

Transmission Disequilibrium Test (TDT), a significant cluster of SNPs

was identified in a region on chromosome 7 that fell downstream from

the axonal guidance protein SEMA3D and upstream from additional

SEMA family 3 members (3A, 3E, and 3C, in order of proximity). The

two SNPs with highest significance (SNP1, with P = 6.12 x 10-6 and

SNP2 with P = 5.15 x 10-6) were genotyped in an independent sample

of 420 HSCR trios that represented all segment lengths: both SNPs

maintained significance (P = 2.8 x 10-2 and 3.6 x 10-4, respectively).

Imputation, which added ~2 million SNPs to the analysis, strength-

ened the significance of the SEMA cluster and refined the location of

its peak, which included SNP2. The transmitted SNP2 allele displayed

greatest penetrance in males and in homozygous individuals, analo-

gous to the RET+9.7 enhancer variant. The four SEMA family three

members demonstrated very similar temporal and spatial patterns of

expression throughout the gut and were coexpressed with RET in these

tissues, supporting the possibility that one or all might modify RET

function in the developing enteric nervous system. Coding exons from

SEMA3D and SEMA3A, the two genes closest to the association peak,

were sequenced in 143 individuals with long segment disease or TCA

to identify deleterious mutations. While no novel variant was pre-

dicted to be deleterious by PolyPhen or SIFT analysis, the SEMA3D

variant S65P had a minor allele frequency that was five fold greater in

the HSCR population than in Caucasian controls (0.043 vs 0.009).

TCOF1 mutation affects the susceptibility to Hirschsprung’s Disease

aj barlow & pa trainor

Stowers Institute for Medical Research, Kansas City, MO 64110, USA

Hirschsprung’s disease (HSCR), or congenital aganglionic megacolon,

is a disorder of the digestive tract affecting 1 : 5000 live births. HSCR

is characterised by an absence of enteric ganglia from variable regions

of the colon. These ganglia comprise the enteric nervous system (ENS)

that is derived mainly from a migratory progenitor cell population

called the neural crest (NCC) which arises from the vagal level of the

neural tube at somites 1-7. At embryonic day (E) 9.0–9.5, ventrally

migrating NCC enter the foregut and over the next 4 days colonise the

entire length of the gut. The establishment of a proper functioning

ENS requires the coordination of NCC survival, migration, prolifera-

tion and differentiation. Data from animal models of HSCR reveal that

there is tight regulation of the ncc numbers that initially enter the

foregut and also that the maintenance of a sufficient pool of neural

crest progenitor cells is necessary during migration to ensure complete

colonisation (Barlow et al., 2003; 2008; Bondurand et al., 2006). Tcof1

encodes a nucleolar phosphoprotein known as treacle, that interacts

with upstream binding factor (UBF), and RNA polymerase I (Valdez

et al., 2004) to control ribosome biogenesis, a process vital for normal

cell growth and proliferation. Recently we demonstrated that Tcof1 is

essential for neural crest formation and proliferation during craniofa-

cial morphogenesis. Mutations in Tcof1 lead to a deficiency in the

number of migrating neural crest which is responsible for the cranio-

facial anomalies characteristic of Treacher Collins Syndrome (Dixon

et al., 2006). In addition to its role in governing cranial neural crest cell

development, we have investigated roles for Tcof1 both as an intrinsic

regulator and as a modifier of Hirschsprung’s disease. Using whole guts

isolated from E10.5 to 18.5 Tcof1± embryos immunostained with the

neuronal marker TuJ1, we observed a reduction in neural crest number

and delayed migration until E14.5. However, during later development,

the gut becomes completely colonised revealing that haploinsuffi-

ciency of Tcof1 alone is insufficient to cause HSCR. Interestingly, the

loss of Tcof1 was able to sensitize other mutant backgrounds to HSCR.

Pax3 heterozygote mice for example do not endogenously exhibit an

abnormal ENS phenotype, but do present HSCR at E18.5 when com-

bined with Tcof1 heterozygosity. Our data thus identifies Tcof1 as a

.new gene involved in HSCR susceptibility.

Effects of tissue age, presence of neurons and endothelin-3 on the

ability of enteric neuron precursors to colonize recipient gut:

Implications for cell-based therapies

aj bergner,* r hotta,* rb anderson,* df newgreen� & hm young**Department of Anatomy and Cell Biology, The University of Melbourne,

Victoria, Australia; and �Murdoch Children’s Research Institute, Royal

Children’s Hospital, Victoria, Australia

Introduction: There is currently great interest in the power of cell

therapy to replace diseased or absent enteric neurons in post-natal

humans. However, it is unclear whether neural crest stem/progenitor

cells will be able to colonize colon (i) in which the mesenchyme has

differentiated into distinct layers, (ii) that already contains enteric

neurons or (iii) that lacks a gene expressed by the gut mesenchyme,

such as endothelin-3 (Et-3). Objectives: To examine the effects of age,

presence of neurons and Et-3 on the ability of enteric neural crest-

derived cells (ENCCs) to colonize recipient gut. Methods: Co-cultures

were used to examine the ability of enteric ENCCs from E11.5 mouse

gut to colonize a variety of recipient hindguts. The area or the distance

colonized by donor ENCCs within the explants were compared. To

investigate whether premature neuronal differentiation affects the

ability of ENCCs migration, the proportion of ENCCs expressing

neuronal markers within the recipient gut was also deter-

mined. Results: ENCCs migrated in E14.5 and E16.5 aneural (lacking

enteric neurons) recipient colon in which the external muscle layers

had differentiated, but they did not migrate as far as they did in

younger colon. The reduced migratory ability of ENCCs in older

recipient gut did not appear to be due to premature neuronal differ-

entiation as the proportion of ENCCs expressing the neuronal marker,

Hu, was not significantly different in recipient gut of different ages.

ENCCs failed to enter most recipient E14.5 and E16.5 colon explants

containing enteric neurons, and when they did, they showed very

limited migration. Finally, ENCCs migrated a shorter distance in

recipient E11.5 Et-3-/- colon compared to wild-type recipient

colon. Conclusions: (1) Colonization of older aneural colon by intro-

duced cells is possible, but occurs more slowly than at earlier ages. (2)

A genetic defect in the colon mesenchyme slows but does not prevent

colonization by introduced cells. (3) Compared to colonization of

aneural colon, restitution of neuronal sub-populations by introduced

cells may be difficult to achieve as ENCCs failed to enter most

recipient explants containing enteric neurons.

Transcriptomics approach of the human enteric nervous system

c besmond,* m clement-ziza,* jp jaıs,� s thomas,* h etchevers,* f joubert,�e sarfati,§ c de chaizematin§ & s lyonnet**Hospital Necker, Inserm U781, Paris, France; �School of Medecine, University

Paris-Descartes, Paris, France; �Pathology Department, Hospital Necker, Paris,

France; and §Surgery Department, Hospital Saint-Louis, Paris, France

Objectives: Our aim was to establish the transcriptomic repertoire of

the human enteric nervous system cells, i.e. neuronal and glial cells

from myenteric plexi, as well as of embryonic neural crest

cells. Methods: Starting from adult surgical colon samples, we per-

formed laser capture microdissection of individual neurons and pat-

ches of glial cells. Neural crest cells were recovered from cultured

neural tubes. As maintaining the highest RNA quality is critical when

performing expression studies on microarrays, we worked out an

alcoholic staining protocol and a heavier than air neutral gas, i.e. argon,

laser capture microscopy procedure. The goal was to prevent rehydra-

tion and subsequent activation of RNases in sample sections from

aqueous staining and air humidity during microdissection time. These

protocols allowed us to unambiguously identify the cells to be

captured and preserve RNA from degradation during up to 90 min of

microdissection time. RNA quantities recovered from these experi-

ments were estimated to range from 100 to 500 picograms. Same

quantities of RNA from the neural crest cells, neuronal and

glial cells were amplified using Nugen WT-amplification Pico

protocol. Results: Data were GC-RMA normalized and principal

component analysis showed that the data were grouped according to

the corresponding biological structures. After Range2 filtering

Abstracts

� 2009 Blackwell Publishing Ltd xi

(fdr<1%), 9866 probesets were selected and aggregated through non-

supervised classification into three sets of overexpressed probesets in

neural crest cells, glial cells and neurons. GO annotations for genes

overexpressed in neural crest cells featured transcription, translation,

replication, mitosis, embryonic development, neural tube formation,

morphogenesis, cardiac development and neural tube closure whereas

glial cells listed immunity, inflammation, cellular adhesion, extra-

cellular matrix, nervous system development and anion transport.

Annotations for neurons featured neurotransmitters metabolism,

synaptic transmission, axonal and dendritic growth and ionic trans-

port. As an example of superposing our differential analysis results to

Kegg networks, we saw that TGF-ß/BMP and WNT canonical path-

ways were activated in neural crest cells. This is consistent with the

fact that Smads, that we found overexpressed in neural crest cells,

transduce signals from the TGF-ß family and associate to transcription

factors to modulate expression of target genes. BMPs are essential to

development of neural crest cells because they influence their migra-

tion and WNT plays a role in induction and differentiation of these

cells.Conclusion: This transcriptomics study should allow us to

highlight the molecular specificities related to the cell types of the

enteric nervous system and putative candidate genes to neurocristop-

athies.

Stronger inhibitory neuromuscular transmission in the ileal

longitudinal muscle of neonatal guinea pigs

x bian

Michigan State University, East Lansing, MI, USA

Neurotransmission to smooth muscle is crucial for normal gut

motility but little is known about postnatal maturation of neuro-

muscular transmission. Postnatal development of neuromuscular

transmission in the gut is an important issue for understanding pae-

diatric motility disorders in the gastrointestinal tract. We investigated

excitatory and inhibitory neuromuscular transmission in vitro using

ileal nerve-muscle preparations from neonatal (£ 48 h postnatal) and

adult (~ 4 months postnatal) guinea pigs. Drugs and electrical field

stimulation (EFS, 20 V, 0.3 ms) were used to evoke cholinergic con-

tractions and non-adrenergic, non-cholinergic (NANC) relaxations in

longitudinal muscle. Immunohistochemistry was used to localise

substance P (SP) and nitric oxide synthase (NOS) in longitudinal

muscle with attached myenteric plexus (LMMP) preparations. In both

adult and neonatel tissues, nicotine (0.3–30 l mol L-1) contracted the

longitudinal muscle in a concentration dependent and tetrodotoxin

(TTX; 0.3 l mol L-1)-sensitive manner. Scopolamine (1 l mol L-1),

a cholinergic muscarinic receptor antagonist, largely inhibited the

nicotine-induced contractions in adult tissues and completely blocked

the nicotinic contractions in neonatal tissues. Neuronal fibers con-

taining SP were abundant in tertiary interganglionic strands in adults,

however, much fewer SP fibers were found in tertiary interganglionic

strands in neonates. In LMMP preparations in both adult and neonatal

animals, NOS immunoreactivity (IR) was present mainly in enteric

neurons in ganglia but very limited NOS-IR in neuronal intergangl-

ionic fibers. Quantitative studies demonstrated that the intensity of

NOS-IR in nerve fibers was higher in neonates than adults. In

the presence of the NOS inhibitor Nx-nitro-L-arginine (NLA;

100 l mol L-1), scopolamine (1 l mol L-1)-resistant nicotine-induced

contractions were revealed in neonatal tissues. NLA (100 l mol L-1)

also enhanced the nicotinic muscle contractions in neonates but not in

adults. The scopolamine (1 l mol L-1)-resistant nicotinic contractions

in adult tissues were enhnced by NLA (100 l mol L-1). Trains (5–25

Hz, 1 s) of EFS evoked frequency-dependent, NLA (100 l mol L-1) and

TTX (0.3 l mol L-1)-sensitive NANC relaxations. Frequency-response

curves in neonates were left-shifted compared to adults. Conclusions:

The neonatal gut is predominantly under the influence of inhibitory

neuromuscular transmission. It is likely that this inhibitory neuro-

transmission in the gut matures earlier than excitatory neurotrans-

mission. Delayed maturation of excitatory motor pathways may

contribute to paediatric motility disturbances.

Ret, caspases, cleavage and cell polarity: the complexity of being a

dependence receptor

jr cabrera, j bouzas, l giraud, s tauszig-delamasure & p mehlen

Laboratory Apoptosis Cancer and Development, CNRS UMR 5238, Center Leon

Berard, University of Lyon 1, Lyon, France

Introduction: Ret is a tyrosine kinase receptor activated by GDNF

Family Ligands (GFLs) that controls proliferation, differentiation and

migration of neural crest cells and derived cells. However we believe

Ret does not act only as a typical tyrosine kinase receptor but also as a

dependence receptor: this means that in the absence of trophic signal

Ret is cleaved by caspases, and this cleavage permits the release of

peptides with different biological activity from Ret full length recep-

tor. Objectives and Methods: Here we characterize a caspase-trun-

cated form of Ret detected in primary culture of neurons and in

transfected cell-lines. Results and Conclusions: We hypothesize

about a putative function of caspase-truncated form of Ret on cell

polarity during migration and axonal growth.

Migration of mouse sacral neural crest cells from their origin to the

hindgut

x wang,* lh bao,* m li,* aj burns� & wy chan**Department of Anatomy, Faculty of Medicine, The Chinese University of Hong

Kong, Hong Kong; and �Neural Development Unit, UCL Institute of Child

Health, London, UK

Enteric neurons and glia in the hindgut are derived from neural crest

cells (NCCs) from vagal and sacral regions. While it has been known

that vagal NCCs undergo extensive migration from the caudal hind-

brain to colonize the entire gastrointestinal tract, information on the

migratory pathways and behaviours of sacral NCCs is still scarce,

especially in rodents. In the present study, we sought to map the entire

migratory pathway of sacral NCCs from the caudal neural tube to the

hindgut in mouse embryos by using cell labelling, whole embryo

culture and immunohistochemical methods, and then examined their

migratory behaviours within the hindgut with time-lapse fluorescent

microscopy. Sacral NCCs caudal to somite 24 began their migration

from the dorsal neural tube at 27–28 somite stage (E9.5) and migrated

to regions around the dorsal aorta at 35–36 somite stage (E10.5). Most

of migrating NCCs expressed both Sox10 and p75. Double immuno-

fluorescence staining showed that these Sox10/p75 double positive

NCCs started to aggregate on two sides of the hindgut at E11.5 to form

pelvic ganglia. At E14.0, sacral NCCs entered the hindgut from pelvic

ganglia through the ventrolateral side of the hindgut at S2 and S3

levels. At this stage, sacral NCCs were closely associated with neuritis

extending from pelvic ganglia. By E14.5, sacral NCCs intermingled

with vagal NCCs migrating craniocaudally within the hindgut

and became morphologically indistinguishable from them. To

further examine their migratory behaviours within the hindgut, we

established an organ culture system, in which movements of green

fluorescent protein (GFP)-labelled sacral NCCs were observed with

time-lapse imaging. Consistent with the observations from immuno-

fluorescence staining, GFP-labelled sacral NCCs from pelvic ganglia

migrated into the hindgut via its ventrolateral side, while the dorsal

region of the distal hindgut seemed to be inaccessible to sacral NCCs.

Upon entering the hindgut, most of sacral NCCs dispersed within the

intestinal mesenchyme, in contrast to vagal NCCs which migrated

mostly in chains along cellular strands. Following arrival of vagal

NCCs to the caudal hindgut, sacral NCCs joined the cellular network

of the presumptive myenteric plexus formed by vagal NCCs. Our data

therefore showed that sacral NCCs were able to migrate from the

neural tube over long distances to enter the hindgut to contribute to

the enteric nervous system.

Acknowledgment: The work was supported by General Research

Funds from the Research Grants Council of the Hong Kong Special

Administrative Region, China (Project nos. CUHK4418/03M and

461808).

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxii

Beta1 integrins are required for the invasion of the caecum and

proximal hindgutby enteric neural crest cells

s dufour, ma breau,1 a dahmani, f broders-bondon & jp thiery2

UMR144 CNRS-Institut Curie, Paris, France

Integrins are major adhesive receptors for extracellular matrix with

various roles in development. We used the Ht-PA-Cre mouse strain to

target the b1 gene disruption in neural crest cell (NCC) derivatives. In

the mutant, b1-null enteric NCCs fail to colonize the gut completely,

leading to an aganglionosis of the descending colon. Moreover, mutant

enteric NCCs form abnormal aggregates in the gut wall, giving rise to a

severe alteration of the ganglia network organisation. Mice producing a

fluorescent protein in b1-null ENCC were generated using the

R26EYFP mouse strain with constitutive expression of the YFP-

encoding gene upon Cre-recombination at the R26REYFP locus. Live

imaging experiments showed that the migration defect occurs pri-

marily during the invasion of the caecum, where b1-null ENCC stop

their normal progression before invading the caecum and proximal

hindgut by keeping abnormaly aggregated. Whilst entering in the

caecum, the speed and the persistence of leading cells movements

were significantly reduced in the mutant. Unlike controls, the

migratory front in mutants was not organised in chains of cells in the

proximal hindgut. The caecum and the proximal hindgut express high

levels of fibronectin and tenascin-C, two well-known ligands of inte-

grins. In vitro, we showed that b1-null enteric neural crest cells

exhibited an enhanced response to the inhibitory effect of tenascin-C

on adhesion and migration, whereas they were unsensitive to the

stimulatory effect of fibronectin. These findings showed that b1 inte-

grins play crucial role at various steps of the enteric nervous system

development. They suggest that b1-integrins are required to overcome

the tenascin-C-mediated inhibition of migration within the caecum

and proximal hindgut and further fibronectin-dependent migration in

these regions.

Functional analysis of enteric nervous system progenitor cells

d edgar,* r lindley,*,� d hawcutt,� s theocharatos* & s kenny�*University of Liverpool School of Biomedical Sciences, Liverpool, UK; and

�Institute of Child Health, Royal Liverpool University Children’s Hospital, Alder

Hey, UK

Neural crest-derived progenitor cells of the enteric nervous system

(ENS) are a potential source of neurons that could be used in future

transplant therapies to help restore normal gut function in patients with

Hirschspung’s disease (congenital aganglionosis of the bowel). We and

others have previously developed techniques to isolate ENS progenitor

cells both from the embryonic mouse and neonatal human gut,

including samples obtained at surgery from Hirschsprung patients.

These cells may be maintained and amplified in culture as neuro-

spheres-aggregates of ENS stem cells and their differentiating neuronal

and glial cell progeny. Subsequently we have gone on to exploit a model

assay system using cultured explants of aganglionic embryonic mouse

gut to demonstrate that after justaposition of neurospheres on the gut

explants, cells migrate out of the neurosphere along the bowel wall and

differentiate into glia and neurons characteristic of the developing ENS.

Importantly, by observing the contractility and calcium fluxes that

develop in the differentiating smooth muscle of the gut explants, we

could show that while the endogenous contractility of the smooth

muscle requires the presence of interstitial cells of Cajal, it is inde-

pendent of the ENS. However, if these cultures are maintained so that

they reach term-equivalent stages of gut development, the neurosphere

transplants exert a regulatory role to coordinate calcium flux and

decrease the frequency of endogenous contractions in the distal colon,

resulting in slower but more powerful contractions. The characteristics

of these contractions are similar to those seen in normoganglionic colon

that has developed to term in vivo. Pharmacological analysis was used

to demonstrate that the regulatory role of the transplanted neurosphere-

derived cells is dependent on neuronal activity and is mediated largely

by nitrergic neurons. Current work is aimed at understanding the

mechanisms that regulate ENS stem cell proliferation and differentia-

tion in situ and when isolated from the neonatal gut. To this end we

have developed a lentiviral transduction technique to permanently label

with green fluorescent protein essentially all cells in cultured neuro-

spheres. This labelling allows us to follow the behaviour of ENS stem

cells and their progeny both after manipulation in culture and after

transplantation into bowel in vitro and in vivo.

Guts, germs, and the enteric nervous system: regulatory interactions

in the developing intestine

js eisen,* j kuhlman,* e mittge� & k guillemin�*Institute of Neuroscience, University of Oregon, Eugene, OR, USA; and

�Institute of Molecular Biology, University of Oregon, Eugene, OR, USA

Normal development and function of the intestinal tract requires

appropriate signalling amongst several different cell types, including

neural crest-derived neurons and glia of the enteric nervous system,

endoderm-derived cells of the gut epithelium, and mesenchyme-

derived muscle cells and interstitial cells of Cajal. The mesenchyme and

gut epithelium produce signals important for enteric nervous system

differentiation. A consortium of intestinal microbes, the gut microbiota,

is necessary for normal development of the gut epithelium, and thus may

have effects on differentiation of the enteric nervous system. One of our

goals is to understand the interactions among host cells and the micro-

biota, which regulate intestinal development and function, and what

happens when those interactions go awry. Here we describe several

mutations isolated in forward genetics screens in zebrafish that affect the

number and distribution of enteric nervous system neurons and how

changes inneuronnumberaffect intestinalmotility.Wealsodescribe the

role of the enteric nervous system in maintaining the integrity of the

intestinal microbial community and how changes in this community are

associated with an inflammatory response in which immune cells infil-

trate the intestine.

Regulation of Sox10 expression and function

m wegner

Institut fur Biochemie, Erlangen University, Erlangen, Germany

The HMG-box containing transcription factor Sox10 is essential for

enteric nervous system (ENS) development. Accordingly, Sox10

heterozygosity in human patients leads to Hirschsprung disease, and

deletion in the mouse causes complete ENS loss. During ENS devel-

opment, Sox10 is first expressed in vagal and enteric neural crest cells

where it is important for survival and maintenance of the undifferen-

tiated state. Later, Sox10 continues to be expressed in enteric glia

arguing that there may be an additional role for Sox10 at this later

stage. To understand the role of Sox10 in the developing ENS, it is

important to know how Sox10 expression is regulated and how its

activity is adjusted to different tasks during consecutive phases of ENS

development. My group has recently identified conserved enhancers

that drive Sox10 expression in different tissues and at different times of

development. These also include two enhancers that drive Sox10

expression in the developing ENS with very similar kinetics.

Additionally, we have obtained some evidence how Sox10 activity is

modulated in vivo and how its function can be adapted in glial cells to

serve stage-specific functions. In addition to functional interactions

with transcriptional activators that exhibit stage-specific expression,

we also detected crosstalks with transcriptional repressors including

proteins of the SoxD family. These results will be presented and dis-

cussed in light of their relevance for ENS development.

Congenital Short Bowel Syndrome

cs van der werf, jbgm verheij & rmw hofstra

Department of Genetics, University Medical Center Groningen, The Netherlands

Introduction: Congenital Short Bowel Syndrome is a rare gastrointes-

tinal disease. The main features patients share are inherited short

small intestines and malrotation. Biopsy specimen show an abnormal

1Present address: National Institute for Medical Research, Division of

Developmental Neurobiology, Mill Hill, London, UK.2Present address: IMCB A*STAR, Singapore.

Abstracts

� 2009 Blackwell Publishing Ltd xiii

appearance of the neuronal cells in the bowel wall in around 10% of

the patients described in literature. Sixty per cent of the cases are

familial. Because boys and girls are both affected and in 25% of

the cases the parents are consanguineous, an autosomal recessive

pattern of inheritance is suspected. Objectives: The aim of

our study was to identify the disease-mutation containing

gene. Methods: Homozygosity mapping was performed using 250K

SNP arrays from Affymatrix on a small consanguineous family with

two affected sibs, one of them was also diagnosed with Hirschsprung’s

disease. We searched for the longest homozygous shared haplotype, as

we hypothesised that this haplotype must contain the disease causing

gene. Using the NCBI database we determined which genes are present

in the shared region and subsequently the genes from this region were

sequenced. Moreover, DNA samples from three published CSBS pa-

tients were requested from the authors that reported these patients and

the data of the SNP arrays of these samples were used to confirm

linkage finding. Results: Homozygosity mapping on the consanguin-

eous family resulted in a longest shared homozygous region of 12 cM

located on chromosome 13. In the shared region 24 genes were located

and sequencing of 9 genes totally and 6 genes partially did not result in

the identification of a disease causing mutation. Finding a heterozy-

gous polymorphism in one of the genes in one of the patients helped us

to narrow down the shared homozygous region. We could not confirm

our linkage finding with the data of the SNP arrays performed on the

three additional families. Discussion and conclusion: We successfully

applied homozygosity mapping to a small family with CSBS and

identified the possible disease-mutation containing region on chro-

mosome 13. So far, sequencing did not result in the identification of

the mutated gene, however, it did make the shared homozygous region

slightly smaller. We could not confirm our linkage finding in the

additional families. Because these patients are from different parts of

the world, we do not expect that they will share the same disease-

mutation. Different mutations in the same gene or multiple genes

might be involved in this gastrointestinal disease.

RET promotor and genetic variation between the diverse ethnic groups

of South African Hirschsprung’s disease

sw moore & mg zaahl

University of Stellenbosch, Tygerberg, South Africa

Introduction: Reported associations between specific RET promoter

and intron 1 variations associated with other common variants and

haplotypes have been shown to interfere with RET function and

increasing the risk to HSCR pathogenesis. A paucity of information

exists about the nature of the promotor region in African populations

although some evidence of variation has been proposed. This study

focuses on specific RET mutations in the promotor region and other

related variations in a population sample of the diverse South African

population groups. Patients and methods: Ethical permission was

obtained and DNA extracted from whole blood samples in 30 patients

with histologically proven HSCR from the three main South African

ethnic groups. PCR products were screened for genetic variation of the

RET by direct sequencing analysis. Results: A sample of 30 HSCR pa-

tients from the three main ethnic groups [viz: Caucasian (10), mixed

(coloured) (10) Black African (10)] were studied. HSCR phenotypes did

not appear to differ between the ethnic groups. RET promotor variations

were detected in 13 patients (43%) situated at -1782A/G (11/30) -1697C/

G (3/30) -1479G/A (1/30) -1449G/A (1/30) -1285G/A (1/30) -1239C/T (2/

30) and -719T/C (8/30). Three patients had a combination of these vari-

ants. All these variants were identified in the heterozygous state except

for 1782A/G (5) and 719T/C (2) which were also identified in the

homozygous state. Four of these variants were identified only in the

Black African population. The intronic variants [SNP1 (rs 2506004) and

SNP2 (rs 2435357)] occurred at a high frequency in the South African

population. Conclusions: Potential disease-related RET mutations

identified in the promotor region in 43% of HSCR patients investigated.

The novel mutations identified in the promotor region adds to our

understanding of HSCR in African patients.

Regulation of proliferation of interstitial cells of Cajal

g farrugia

Mayo Clinic, Rochester, MN, USA

Normal gastrointestinal motility requires intact networks of inter-

stitial cells of Cajal (ICC). Loss of ICC is associated with several

motility disorders. Recent work strongly suggests that the number of

ICC at any given time point is not static. Rather, ICC networks

appear to be maintained by a dynamic, tightly regulated process,

involving, on one hand, growth factors, trophic factors and replen-

ishment from precursor cells and on the other, transdifferentiation

and pro-death factors. When the balance between cell loss and

replenishment is altered the result is a change in the number of

ICC. This presentation will focus on regulation of proliferation of

ICC. The stem cell factor/Kit pathway was the first pathway shown

to be critical in the regulation of ICC development and mainte-

nance. Loss of signaling is associated with decreased ICC numbers

and upregulation of the pathway associated with gastrointestinal

stromal tumors. Nitric oxide has been recently shown to be a

trophic factor with increased numbers of ICC in the presence of

nitric oxide and a decreased number of ICC when neuronally derived

nitric oxide or nNOS are absent. 5-HT has also emerged as an ICC

proliferation factor. ICC express 5HT2B receptors. Activation of the

5HT2B receptor in vitro in ICC cultures induces an increase in

numbers of proliferating ICC via a signaling cascade that involves

activation of PLC, IP3 receptor mediated intracellular calcium

release and activation of the calcium dependent PKCc. More recent

work has focused on determining if there is endogenous activation

of the ICC 5HT2B receptor and whether proliferation occurs in adult

ICC networks in vivo utilizing PKCc knockout mice and adult mice

with gene targeted deletion of the 5-HT2B receptor. The data suggest

that ICC proliferate in adult mouse ICC networks. In primary

culture, ICC derived from PKCc knockout mice do not show a

proliferative response to 5HT2B receptor agonists suggesting that

this PKC is required for the proliferative effect of activation of the

5HT2B receptor. Also, in the absence of the 5-HT2B receptor the

numbers of proliferating ICC in the myenteric plexus region of

whole mount preparations were significantly lower compared with

wild type controls. In conclusion, there appears to be ongoing

proliferation of ICC. This proliferation is regulated by a number of

growth and trophic factors. Alteration in these factors or in their

ligands result in abnormal ICC numbers and therefore modulation of

the pathways may serve to regulate ICC numbers.

Development redux: stimulated neurogenesis in the enteric nervous

system of adult mice

md gershon & m liu

Department of Pathology and Cell Biology, Columbia University, NY, USA

The enteric nervous system (ENS) develops from precursors that

migrate to the bowel from the neural crest. These precursors prolifer-

ate but cease dividing when they terminally differentiate as neurons

(glia remain capable of mitosis). Although a functioning ENS is

obligatorily present at birth to accommodate feeding, enteric neurons

continue to be born through P21. Growth of the bowel, however,

persists and enteric neurons increase in number. Evidence suggests

that the postnatal gut retains neuron-competent stem cells; moreover,

neuronal replacement, which occurs in the CNS, would seen to be

equally necessary in the ENS. Neurogenesis, however, has been diffi-

cult to demonstrate in adult ENS. Conceivably, stem cells might

normally, in adults, be quiescent and persist only in microenviron-

mental niches that are extraganglionic. These hypotheses were tested.

5-Bromo-2¢-deoxyuridine (BrdU) was continuously infused for 1 week

so as to label even infrequently dividing cells; BrdU infusion was fol-

lowed by 2 weeks to 6 months of chase to permit BrdU to be dissipated

in continually dividing cells and migration of BrdU-labelled neuro-

blasts to be detected. Under control conditions, no BrdU incorporation

into neurons was detected; however, if animals were simultaneously

treated with a 5-HT4 agonist (tegaserod or RS RS67506) or colitis was

induced with dextran sodium sulphate (DSS), then cells appeared in

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxiv

which BrdU was co-expressed with markers of mature (HuC/D) or

developing neurons (doublecortin, Sox10, Phox2b, and nestin). 5-HT4

agonists and inflammation stimulated proliferation, detected immu-

nocytochemically with antibodies to Ki67, in �germinal niches�between myenteric ganglia and the longitudinal muscle, where the

doubly labelled HuC/D+BrdU-immunoractive cells were initially

located. After extended periods of chase, BrdU+HuC/D-labelled neu-

rons approached, and by 4–6 months, entered myenteric ganglia. Nei-

ther tegaserod nor RS RS67506 caused HuC/D+BrdU-immunoractive

cells to become manifest in 5-HT4-/- mice; however, such cells were

seen during DSS-induced inflammation. Peripherin-immunoreactive

swellings, which lacked synaptic vesicle markers, were observed at the

ends of neurites in 5-HT4-/- mice and after DSS-induced inflamma-

tion. Electron microscopy confirmed that these swellings represented

nerve terminal degeneration and was accompanied by increased neu-

ronal autophagy. We suggest that enteric neurogenesis occurs in

retained, but extraganglionic �germinal niches� in the adult ENS.

Autophagy in neurites accompanies inflammation and may be linked

to the initiation of neurogenesis. 5-HT4 receptors appear to be neces-

sary for ENS maintenance. Supported by NIH NS12969 and NS15547.

Endothelial cells promote migration and proliferation of enteric neural

crest cells via ß1 integrin signaling

am goldstein,* o mwizerwa* & n nagy�*Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA;

and �Semmelweis University, Budapest, Hungary

Introduction: Enteric neural crest-derived cells (ENCCs) migrate along

the intestine and form an organized network of ganglia that comprises

the enteric nervous system (ENS). The signals directing the migration

of ENCCs, and their patterning into two concentric rings of ganglia,

are largely unknown. Objectives: The purpose of this study was

to identify cellular and molecular signals responsible for regulating

migration and patterning of ENCCs in the developing ENS.

Methods: Neurovascular development was examined during avian gut

development using immunohistochemistry and electron microscopy.

The role of the intestinal microvasculature in ENS formation was

tested in gut organ cultures grown on a variety of cellular and matrix

protein surfaces. ENCC migration, proliferation, and apoptosis were

analysed in these cultures. Results: Endothelial cells are present in

the gut prior to ENCC arrival and are patterned in concentric rings

predictive of the patterning of enteric ganglia. During ENS develop-

ment, ENCCs migrate adjacent to endothelial cells as they colonize

the gut. Inhibiting endothelial cell development using SU5416, a VEGF

receptor inhibitor, leads to intestinal aganglionosis, supporting an

essential role for the intestinal vasculature in ENCC migration. When

intestine is explanted onto cultured endothelial cells, ENCCs migrate

out of the gut and onto the surrounding cells, which promote both

migration and proliferation of ENCCs. This activity is inhibited in the

presence of a ß1 integrin function-blocking antibody. Laminin, which

is strongly expressed by the basement membrane of vessels, also serves

as an excellent substrate for ENCC migration, an activity inhibited by

the same antibody. Conclusions: These results support the existence

of important interactions between integrins on the ENCC surface and

the vascular basement membrane in regulating cell migration and

patterning in the developing ENS.

Sox10± mice as Hirschsprung model for cell replacement therapy with

embryonic stem cell-derived enteric neurons

a. kempe*, h. reichmann*, o. brustle� & g. gossrau**Department of Neurology, University of Dresden Medical Center, Dresden,

Germany; and �Institute of Reconstructive Neurobiology, University of Bonn

Medical Center, Bonn, Germany

The transcription factor Sox10 regulates early neural crest develop-

ment and specification of neural crest-derived lineages. Sox10± mice

reveal an aganglionosis in the intestines comparable to the human

Hirschsprung’s disease (HSCR), the most common developmental

disorder of the enteric nervous system. Because of limits associated

with the current surgical treatment of HSCR, we investigate whether

regenerative tissue repair of the gut using embryonic stem cell-derived

enteric neurons based on enrichment of Phox2b-expressing neural

precursors, represents an attractive alternative therapy for this disease.

Transplantation of ES cell-derived donor neurons in the intestines of

the Sox10 mouse mutants, a model of HSCR, indicates survival and

integration of the transplanted cells. Studies are ongoing to assess the

ability of these donor cells to restore a physiological motility pattern

following their transplantation into dysganglionotic intestines. The

in vitro differentiated putative enteric neurons can potentially serve as

donor cells for regenerative therapy in HSCR.

Genetic and non-genetic mechanisms of enteric nervous system

development

ro heuckeroth

Washington University School of Medicine, St. Louis, Mo, USA

Introduction: Hirschsprung disease is a complex multigenic disorder

with incomplete penetrance and variable expressivity. Many indi-

viduals with Hirschsprung disease also have associated anomalies.

This genetic complexity is a reflection of the elaborate biochemical

machinery required to facilitate ENS precursor migration through

the bowel, control cell proliferation and influence differentiation.

Even after ENS precursors colonize the entire bowel, specific

molecular signals guide neurite growth, control axon specification,

regulate neurotransmitter choice, and determine cell-cell interaction.

Defects in molecular signals controlling this developmental process

cause not only Hirschsprung disease, but also other intestinal

motility disorders. Objectives: To identify new ways to prevent

Hirschsprung disease and other intestinal motility disorders.

Methods: To identify mechanisms critical for ENS development and

for preventing human disease, we have systematically studied gene

expression patterns in the developing bowel, and are actively pur-

suing biochemical pathways controlled by known Hirschsprung

disease susceptibility genes. We are also focused on molecules crit-

ical for cell migration and neurite growth. Results: These studies

led to the observation that many of the regulatory molecules

required for ENS development are influenced by commonly used

medications or by specific micronutrients. We therefore now

hypothesize that non-genetic factors could critically influence dis-

ease severity and penetrance. To test this hypothesis, we have ini-

tiated studies to identify novel gene-environment interactions that

might influence ENS development. Conclusions: These approaches

have the potential to reduce the severity or the penetrance of

Hirschsprung disease and other intestinal motility disorders.

Goldberg–Shprintzen syndrome and microtubules

rmw hofstra,* m alves,* g burzynski,� e de graaf,� c hoogenraad,§

bjl eggen,– i shepherd� & a brooks�*Department of Genetics, University Medical Center Groningen, The Nether-

lands; �Department of Biology, Emory University, Altanta, GA, USA; �Depart-

ment of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands;

§Department of Neuroscience Erasmus Medical Center, Rotterdam, The Nether-

lands; and –Department of Developmental Genetics, University of Groningen,

The Netherlands

Introduction: The identification of genes involved in syndromic-Hir-

schsprung (HSCR) might give us new and unexpected knowledge on

the pathology of HSCR. We, therefore, focussed on such a syndromic

form of HSCR, Goldberg–Shprintzen syndrome (GOSHS). GOSHS is a

rare, autosomal recessive disorder characterized by polymicrogyria,

mental retardation and HSCR. Recently, we showed that Goldberg–

Shprintzen is caused by inactivating mutations in the KBP gene (pre-

viously called KIAA1279). Since then, we have identified several new

patients with more mutations in KBP, confirming our initial find-

ing Objectives: As both the enteric and the central nervous system

are involved in the disease, the KBP protein seems to play an important

role in neuronal development in general, although the precise function

of KBP is still largely unknown. This study aims at elucidating the

function of KBP. Methods: Based on its primary sequence, KBP is

predicted to have two tetratricopeptide repeats (TPRs). Since the basic

function of TPR motifs is to mediate protein-protein interactions, a

yeast two-hybrid screen was performed to identify possible proteins

interacting with KBP. Results: We determined various possible

Abstracts

� 2009 Blackwell Publishing Ltd xv

proteins, including five kinesin-like ones. These proteins are involved

in the transport of vesicles and organelles towards the plus end of the

microtubules. The most frequently found interactor was a stathmin

protein named SCG10, an important regulatory protein of microtubule

dynamics. Conformation of the two-hybrid data and further studies on

KBP’s functioning are being undertaken. All our data point towards

KBP being involved in microtubule organisation, and possibly in

vesicle transport. Conclusions: Our results show that indeed studying

syndromic HSCR does give new and unexpected knowledge on the

pathologyofGOSHS,andthustoHSCRandpolymicrogyriadevelopment,

with everything pointing towards a microtubule-associated problem.

Small molecule induction of neural crest stem cells from human

embryonic stem cell-derived neural progenitor cells

r hotta,* l pepdjonovic,� rb anderson,* d zhang,� aj bergner,* j leung,�a pebay,�,§ m dottori,�,§ hm young* & df newgreen�*Department of Anatomy and Cell Biology, The University of Melbourne,

Victoria, Australia; �Centre for Neuroscience, The University of Melbourne,

Victoria, Australia; �Murdoch Children’s Research Institute, Royal Children’s

Hospital, Victoria, Australia; and §Department of Pharmacology, The University

of Melbourne, Victoria, Australia

Introduction: Cell replacement therapy using human embryonic stem

cells (hESCs) holds great promise for treating diseases, including neu-

rocristopathies involving a deficit of neural crest (NC) cells such as

Hirschsprung’s disease. Objectives: To develop a robust system for

producing NC-like cells and enteric neurons from hESC-derived neural

progenitors. Methods: A hESC line, in which GFP is expressed by all

stem cells and their differentiated progeny, was used (Costa M et al.

2005). Neural progenitor cells were induced from hESCs using the

BMP inhibitor, noggin, as previously described (Reubinoff BE et al.

2001). To induce NC-like cells, hESC-derived neurospheres were cul-

tured on mouse embryonic fibroblasts (MEFs) for 24 hours and then

exposed to the ROCK1/2 inhibitor, Y27632. Co-cultures between the

neurospheres and explants of aneural embryonic mice gut, and in vivo

transplantation, were used to assess the ability of hESCs-derived

NC-like cells to migrate and differentiate. Results: A small number of

cells migrated from hESC-derived neurospheres grown on fibronectin

or MEFs, but only migratory cells exposed to MEFs expressed the NC

markers, p75 and Sox10. The number of p75+/Sox10+ cells migrating

from hESC-derived neurospheres grown on MEFS was dramatically

increased by exposure to Y27632. hESC-derived cells exposed to MEFs

and Y27632 migrated into explants of aneural embryonic mouse gut

and gave rise to NC-like cells expressing Sox10 or Hu. Some of the Hu+

neurons within the gut explants also expressed nitric oxide synthase,

which is expressed by sub-types of enteric neurons. Cells derived from

segments of hESC-derived neurospheres that had been exposed to

MEFs and Y27632 were implanted into quail embryos. GFP+ cells

migrated along NC pathways and differentiated into neural (Hu+) and

glial (S100b+) cells. Conclusions: We have developed a robust system of

producing NC-like cells from hESC-derived neural progenitors using the

smallmoleculeROCK1/2inhibitor,Y27632.Cellsderivedbythismethod

are able to migrate along NC pathways in avian embryos and within

murine bowel, and to differentiate into cells with neuronal markers.

The bHLH transcription factor Hand2 is necessary for specification and

differentiation of neurons in the enteric nervous system

mj howard, j lei, tj henderhot & p pugh

Medical University of Ohio, Toledo, OH, USA

The basic helix-loop-helix DNA binding protein Hand2 is expressed in

neural crest-derived precursors of enteric neurons and has been shown

to affect both neurogenesis and neurotransmitter specification in both

avian and murine embryos. Expression of Hand2 decreases with

increased developmental age in murine embryos prompting us to

determine the effect of loss of Hand2 following neural specification.

The objectives of this study were to more fully understand the role of

Hand2 in supporting differentiation of neurons, to determine whether

Hand2 has a role in gliogenesis, and to identify molecular mechanisms

of Hand2 function(s) in the developing enteric nervous system (ENS).

We selectively deleted Hand2 in neural precursor cells by crossing our

floxed Hand2 line of mice to mice in which Cre recombinase expres-

sion is under control of the Nestin promoter. Based on in-situ

hybridization, transcript encoding Hand2 is completely absent in the

gut but only partially deleted in the sympathetic chain ganglia. Loss of

Hand2 after neurons have been specified results in death at around

P20. Analysis of the ENS between P8 and P10 demonstrated that tar-

geted deletion of Hand2 in neural precursor cells resulted in a loss of

neurons with the proximal bowel being more severally affected than

the distal bowel. Differentiation of glial cells was also impacted by

deletion of Hand2. Interestingly, targeted deletion of Hand2 in neural

precursor cells results in patterning defects along the entire gastro-

intestinal tract with the distal bowel being more severely affected than

the proximal bowel. Our data indicate that Hand2 is essential to

support neurogenesis and gliogenesis. Proper patterning of the myen-

teric ganglia is dependent upon Hand2. Additionally, Hand2 has a

maintenance function since the number of enteric neurons decreases

with increasing developmental age. We conclude that Hand2 is a

multifunctional transcriptional regulator affecting specification,

differentiation and patterning in the ENS.

Density of nerve fibres changes in the colon of Hirschsprung disease

i jester,* m klotz,� s maas-omlor,� c hagl,� s holland-cunz§ &

kh schafer�*Department of General Surgery, Birmingham Children’s Hospital, Birmingham,

UK; �Department of Biotechnology, University of Applied Sciences Kaiserslau-

tern, Zweibrucken, Germany; �Department of Paediatric Surgery, University

Hospital Mannheim gGmbH, Medical Faculty of University Heidelberg, Mann-

heim, Germany; and §Department of Paediatric Surgery, University of Heidel-

berg, Heidelberg, Germany

Purpose: Various approaches are used to analyse ganglia in Hirsch-

sprung disease (HSCR). The direct relation between muscle tissue

and nerve fibre density was not addressed. Aim of the study was to

evaluate the nerve fibres density (NFD) in the circular muscle layer

in the ganglionic, transitional and aganglionic zone of HSCR.

Methods: Fifteen patients underwent pull-through surgery for HSCR.

The age of children at the time of pull-through surgery was

5.67 ± 5.97 months. Samples were taken along the longitudinal gut

axis and stained (PGP 9.5 (Ultraclone)). NFD were measured in three

regions from the myenteric plexus (mpl) towards the submucous

plexus (spl) individually. Additionally in three of these 15 patients

specimens were analysed for neurotrophic factor GDNF using ELISA.

Control specimens were obtained from four patients at surgical

resection of a colon descendens enterostomy not related to HSCR.

Results: NFD increased significantly (P < 0.01) 30% (mean) within the

circular muscle layer from the myenteric plexus (mpl) towards the

submucous layer (spl) in the ganglionic gut (1759/mm2 (mpl); (2638/

mm2 (spl)) and throughout the transitional zone (proximal: (2267/mm2

(mpl); (3084/mm2 (spl); distal: (1444/mm2 (mpl); (1900/mm2 (spl)).

Additionally nerve fibre density increased 20% between the ganglionic

bowel (2254/mm2) to the beginning of the transitional zone (2697/

mm2) (P < 0.02). In distal regions of the transitional zone (1742/mm2,

as well as in the aganglionic region (1601/mm2), there was a 40%

decrease (P < 0.05). With the increase of nerve fibre density, GDNF

concentration increased from the proximal resection margin

(44.3 ± 29.9 pg ml-1) to the proximal transition zone (62.6 ± 32.4 pg ml-

1) and decreased within the transition zone to the distal end of the gut

(46.7 ± 33.4 pg ml-1). In the control group no graduation of NFD within

the muscle layer was found. Conclusion: The findings demonstrate

an unknown pattern of NFD in the gut of HSCR. The clinical relevance

of the increased NFD and GDNF concentration of the transition zone

is unclear and might reflect a repair mechanism.

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxvi

A chemical screen in zebrafish for compounds impairing enteric

nervous system development

ji lake, o tusheva, bl graham & ro heuckeroth

Washington University in St Louis School of Medicine, St Louis, USA

Hirschsprung’s disease is a multigenic and partially penetrant disorder

of the ENS. While great strides have been made in determining the

roles of specific genes in the pathogenesis of the disease, less is known

about the effect of the environment on the developing ENS. We

propose that exposure to clinically used compounds may impair the

development of the ENS and affect disease penetrance in patients

with genetic risk. Such compounds may also provide tools and

suggest targets for further study of the development of the ENS.

Objectives: To screen chemicals in a high-throughput, in vivo system

and identify clinically relevant compounds that impair the develop-

ment of the enteric nervous system and test their roles as enviro-

mental contributors to disease penetrance. Methods: We screened the

Johns Hopkins Clinical Compound Library of 1520 compounds on

developing wild-type zebrafish embryos. Healthy embryos were

exposed to compounds in the library after the formation of the

embryonic gut and the extent of colonization of the distal gut was

measured by immunostaining for neuronal markers and micros-

copy. Results: We present several interesting compounds that impair

the development of the ENS, including sodium valproate. Valproate is an

anticonvulsant in wide clinical use, and is a known teratogen with well-

studied effects on the development of the CNS. Validation and charac-

terization of the results of the screen in zebrafish, in vitro, and murine

systems are ongoing.

Common regulatory mechanisms in enteric lymphoid and neuronal

organogenesis

h veiga-fernades,* a patel,* p pachnis,* m coles,* k foster,* v pachnis�& d kioussis**Division of Molecular Immunology, National Institute for Medical Research,

The Ridgeway, Mill Hill, London, NW7 1AA, UK; and �Division of Molecular

Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill,

London NW7 1AA, UK

Utilizing the GFP transgenic and knock-in mice has permitted an

analysis of cellular interactions, movement and function in lymphoid

organ development and function. The development of lymphoid organs

occurs during embryonic life as a result of interactions between lymph

node inducing cells and stromal cells. Utilizing human CD2-GFP

transgenic mice we have been able to analyse the molecular require-

ments for the development of the intestinal immune system. The

intestinal immune system consists of Peyers patches, cryptopatches,

intestinal lymphoid follicles and intraepithelial lymphocytes. The

development of Peyers patches follows the aggregation in the gut wall

of Peyers patch inducing cells during embryonic development.

Utilizing imaging technology we have been able to observe the role of

cell movement in the aggregation of Peyers patches. Utilizing a com-

bination of gene expression analyses of Peyers patch inducing cell

populations and genetic approaches we have studied molecules, such

as RET tyrosine kinase receptor and its ligands, which have an

important role in Peyers patch formation. Interestingly RET is a

molecule that is also essential for the mammalian enteric nervous

system (ENS) formation.

Differential proteomics of cultured enteric neural precursor cells

m klotz, u rauch & k-h schaefer

University of Applied Sciences Kaiserslautern/Zweibruecken, Germany

Introduction: The cultivation of isolated enteric plexus and neural

precursor cells obtained from it is important to understand the devel-

opment of the enteric nervous system and its ability for regeneration.

Monitoring the activation and differentiation of neural precursors

grown out of cultured myenteric plexus is a basic step towards using

them as a tool for the treatment of neuronal defects. Objectives: To

characterize the neural progenitors and their behaviour in culture, we

used differential proteomics to find the proteins regulated in neuro-

spheres collected after seven and ten days in culture compared to

isolated myenteric plexus. Methods: We used the 2-dimensional

fluorescence difference gel electrophoresis (DIGE) technique to search

for regulated proteins. Dissected and digested gut samples of newborn

rats were used to collect the myenteric plexus. These samples were

pooled and either used for the culture of which the neurospheres grew

out within 10 days, or directly frozen in liquid nitrogen for proteomics.

Seven and 10 days after their first appearance, neurospheres were

collected, pooled and frozen alike. Proteins of each pool were isolated,

labelled with fluorescent dye and then separated together in

2-dimensional electrophoresis. The overlay images of differentially

labelled myenteric plexus and neurospheres (day 7 or 10) were analysed

for regulated proteins by the Decyder software (GE) and proteins of

interest automatically picked out of the gel. Results: In the DIGE-gel

of plexus vs. neurospheres (day7) we found 55 differentially expressed

proteins out of over 1300 detected in total, plexus vs. neurospheres

(day10) showed 43 regulations. Several of these proteins could be

identified yet, among those ARHGAP17, which is down-regulated in

day 7 and day 10 neurospheres and Dihydrogenpyrimidine dehydro-

genase, which is down-regulated in day 7 and up-regulated in day 10

neurospheres compared to myenteric plexus. Conclusions: The dif-

ferential-proteomics approach is a promising way to characterize

neural progenitors in culture and tissue. It could help to understand

the effects of stimulation or environment on progenitors by providing

information of the reaction s of these cells on a protein level.

Watching Neural Crest Migration: How cells respond to embryonic

growth and microenvironmental signals to assemble the peripheral

nervous system

pm kulesa, m smith, r alexander, da stark, jc kasemeier & r mclennan

Stowers Institute for Medical Research, Kansas City, USA

Since the inception of intravital imaging, a fundamental problem has

been how the decisions of individual cells lead to stereotypical cell

migratory patterns in the vertebrate embryo. We combine our expertise

in the stem cell-like neural crest and high-resolution optical micros-

copy to study the mechanisms that regulate the programmed invasion

of the neural crest in the chick embryo. We show that there is a

coordination of molecular signals that regulate the attraction and

inhibition of neural crest behaviors to sculpt cells from the neural tube

towards specific peripheral targets. Using multicolour cell labelling

and multispectral imaging to accurately identify individual neural

crest cells, we correlate distinct cell morphometric characteristics

with position along a migratory route. Time-lapse analysis of neural

crest cell behaviours and photoactivation cell labelling of migratory

front and trailing cells reveal complex cell interactions that lead to

follow-the-leader behaviour and differences in local cell proliferation.

Our results suggest a model in which cell proliferation at the migratory

front drives neural crest expansion towards peripheral target sites, the

precise invasion of which is regulated by specific receptor-ligand

relationships.

Clonal deletion of RET using mosaic analysis with double markers

(MADM) in the enteric nervous system

r lasrado & v pachnis

National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7

1AA, UK

The enteric nervous system (ENS) is composed of a network of inter-

connected ganglia which reside within the gut wall and control its

motility, secretions and microcirculation. The ENS is one of the most

complex parts of the nervous system containing a vast number of neu-

rons whose axonal trajectory and connectivity is usually distinct from

that of its neighbours. The lack of obvious CNS-type �nuclei� composed

of a homogeneous neuronal population makes the tracing and study of

individual classes of neurons a daunting task. Therefore, the develop-

ment of tools that will allow the identification and study of individual

enteric neurons and their processes is fundamental for uncovering the

logic governing formation of enteric neuronal networks. Ret is a receptor

tyrosine kinase that is critical for the development of the mammalian

ENS. Despite extensive analysis of the developmental role of this sig-

nalling pathway, key questions relating to the in vivo role of Ret in

Abstracts

� 2009 Blackwell Publishing Ltd xvii

neuronal differentiation, connectivity and gangliogenesis remain

unanswered. Recently, we have demonstrated that, in addition to their

cell autonomous effects, mutations of Ret can also affect the early stages

of enteric neurogenesis in a non cell autonomous manner, raising the

possibility that the behaviour of individual neurons is influenced by

their immediate neighbours. To combine morphological and genetic

analysis of individual enteric neural crest cells/neurons, we have adop-

ted a novel approach called Mosaic Analysis with Double Markers

(MADM) which is based on Cre-loxP dependent inter-chromosomal

mitotic recombination. This approach requires the separate knock-in at

identical loci (such as Rosa26) on homologous chromosomes of two

reciprocally chimeric reporter genes (such as GFP and RFP), each con-

taining the N terminus of one marker and the C terminus of the other

interrupted by a loxP-containing intron. Cre-mediated inter-chromo-

somal recombination during mitosis results in two daughter cells

expressing one of the two fluorescent proteins upon G2-X segregation.

Introduction of a null mutation of Ret (which maps distal to the Rosa26

locus) allows the generation of genetic mosaics composed of wild-type

daughter cells labelled with one colour and homozygous mutant sibling

cells with another. We will be reporting on recent progress to study the

morphology, distribution and differentiation of enteric neurons in wild-

type and Ret-deficient MADM clones.

Condition Ptc1 knockout in vagal neural crest cells causes intestinal

hypoganglionosis

thc poon,* esw ngan,* fyl sit,* cc hui,� bj wainwright,� mh sham,§

pkh tam* & vch lui**Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong,

21 Sassoon Road, Pokfulam Hong Kong; �Program in Development & Stem Cell

Biology, The Hospital for Sick Children, University of Toronto, Toronto Medical

Discovery Towers, 101 College Street, Toronto, Ontario, Canada; �Institute for

Molecular Bioscience, The University of Queensland, Brisbane, Queensland,

Australia; and §Department of Biochemistry, LKS Faculty of Medicine, The

University of Hong Kong, 21 Sassoon Road, Pokfulam Hong Kong

Introduction: In vertebrates, vagal neural crest cells (NCC) colonize

the developing gut rostro-caudally giving rise to the neuron and glia

progenitors of the enteric nervous system (ENS). Hh-/- mice showed

patterning and colonization defects and reduction of neurons

(hypoganglionosis) of the ENS. However, the molecular mecha-

nisms by which Hh regulate ENS development are largely

unclear. Objecives: To investigate the cell autonomous functions of

Ptc1 on the vagal NCC/ENS progenitors. Methods: We have gener-

ated special mouse strains in which Ptc1 is deleted in vagal NCC by

Cre-loxP approach, and analysed the development of ENS progenitors

and ENS in embryos and adults. Mice with homozygous knockout of

Ptc1 in vagal NCC were generated by intercrossing of Ptc1flox (floxed

Ptc1) mice with two vagal NCC cre expressing mouse lines (Wnt1-cre

and b3-IIIa-cre mice). Results: In both of these conditional Ptc1

knockout mice, Ptc1-/- ENS progenitors colonized the intestine nor-

mally and displayed no patterning defect, suggesting that the coloni-

zation and patterning defects of ENS in Hh-/- mice was attributed to

the abnormally developed gut mesenchyme in Hh-/- mice. Instead, we

observed reduction of proliferative ENS progenitors in the mutant

embryonic guts, and reduction of enteric neurons in the mutant adult

intestines. Ptc2 was only weakly expressed by the ENS progenitors,

and that Ptc2 was not upregulated in Ptc1-/- ENS progenitors, indi-

cating that loss of Ptc1 activity in vagal NCC/ENS progenitors was not

compensated by an upregulation of Ptc2. Conclusions: Our data

indicated that a deletion of Ptc1 in vagal NCC resulted in a reduced

proliferation of the progenitors in embryonic gut giving rise to hypo-

ganglionosis in adult mice, suggesting a specific cell-autonomous

regulatory function of Ptc1 on the proliferation of ENS progeni-

tors. This work was supported by research grants from the Hong Kong

Research Grants Council (HKU7339/04M; HKU7528/06M) to VCH

Lui.

Contact inhibition of locomotion controls neural crest directional

migration

r mayor, c carmona-fontaine & hk matthews

Department of Cell and Developmental Biology, University College London,

London, UK

The Neural Crest cells have been called the �explorers of the embryos�because they migrate all over the embryo where they differentiate into

a variety of diverse kinds of cells. It has been found in recent years

that many of the factors that control neural crest migration work as

negative signals, such as semaphorins, ephrins, Slit/Robo, etc. How-

ever no positive signal that works as a chemoattractant for the neural

crest has been found so far. Here we propose a novel mechanism for

directional migration of neural crest cells, based on contact inhibition

of locomotion, where no chemoattractants are required. Contact

inhibition of locomotion was discovered by Abercrombie more than

50 years ago to describe the behaviour of fibroblast cells confronting

each other in vitro, where they retract their protrusions and change

direction upon contact. Its failure was suggested to contribute to

malignant invasion. However, the molecular basis of Contact inhibi-

tion of locomotion and whether it also occurs in vivo are still

unknown. Here we show that neural crest cells, a highly migratory

and multipotent embryonic cell population, whose behaviour has

been likened to malignant invasion, exhibit contact inhibition of

locomotion both in vivo and in vitro, and that this accounts for their

directional migration. When two migrating neural crest cells meet,

they stop, collapse their protrusions and change direction. By contrast,

when a neural crest cell meets another cell type, it fails to display

contact inhibition of locomotion; instead, it invades the other tissue,

like metastatic cancer cells. We show that inhibition of non-canonical

Wnt signalling abolishes both contact inhibition of locomotion and

the directionality of neural crest migration. Wnt signalling members

localize at the site of cell contact, leading to activation of RhoA in

this region. These results provide the first example of Contact

Inhibition of Locomotion in vivo, present an explanation for coherent

directional migration of group of cells and establish a novel role for

non-canonical Wnt signalling.

Abnormal enteric nervous system development in a Sox10N-GFP

mouse mutant

m zhang,* c leung,* vch lui,� pkh tam� & mh sham**Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of

Hong Kong, Pokfulam, Hong Kong SAR, China; and �Department of Surgery, Li Ka

Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong

SAR, China

Introduction: SOX10 mutations have been identified in human

Waardenburg–Hirschsprung (WS4) patients who displayed a varied

degree of intestinal aganglionosis. Studies on mouse mutants have

suggested that the Sox10 gene is implicated in modulating the prolif-

eration, maintenance, differentiation and migration of enteric neural

crest cells, but the mechanisms for regulating these processes remain

unclear. Objectives: To elucidate the roles of Sox10 in different

aspects of enteric nervous system development, in particular to visu-

alize the migration behaviour of abnormal enteric neural crest cells

and their derivatives, we have generated a novel mouse mutant

Sox10N-GFP in which the HMG DNA-binding domain and the

transactivation domain of Sox10 have been replaced by the EGFP

reporter. Methods: We analysed abnormal phenotypes of the Sox10N-

GFP mutants by immunohistochemical staining, NADPH-diaphorase

and acetylcholine esterase assays. We isolated and examined mutant

enteric neural crest cells by neurosphere cultures. We also visualized

the migration of the mutant enteric neural crest cells by time-lapse

imaging of gut explants in culture. Results: The heterozygous

Sox10N-GFP mutants displayed WS4 phenotypes including pigmen-

tation defects and megacolon. In Sox10N-GFP/+ mutants, the enteric

neural crest cells entered the midgut by E10.5, but migration of the

enteric neural crest cells was delayed by E12.5 and failed to populate

the full length of the gut at E14.5. Homozygous Sox10N-GFP/N-EGFP

mutants could survive till E13.5, at this stage enteric neurons could be

detected in the mutant oesophagus, but total aganglionosis was

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxviii

observed in the stomach and the gut. We have isolated the Sox10N-

GFP/+ mutant enteric neural crest cells and cultured them at clonal

density in neurosphere cultures. We are currently studying the differ-

entiation potential of the mutant neural crest cells and the abnormal

migration behaviour of Sox10N-GFP enteric neural crest cells by time-

lapse imaging.Conclusions: The Sox10N-GFP mutant serves as a no-

vel mouse model for Waardenburg–Shah Syndrome with abnormal

enteric nervous system development. This project was supported by a

research grant from the Research Grants Council of Hong Kong

(HKU7705/05M) to MHS.

Expansion and differentiation of neural progenitors derived from the

human adult enteric nervous system

m metzger, *,� pm bareiss, � t danker, � t skutella� & l just�*UCL Institute of Child Health, London, UK; �Institute of Anatomy, Tubingen,

Germany; and �Natural and Medical Sciences Institute, Tubingen, Germany

Objectives: A number of age-related gastrointestinal motility disor-

ders are caused by abnormalities of the enteric nervous system (ENS).

Stem or progenitor cell-based therapies may offer the potential of

replacing defective, damaged, or missing neural elements within the

bowel. Here, we describe a method suitable for the preparation of ENS

progenitor cells from human adult gut tissue and assess their trans-

plantation potential in vitro. Material and methods: Human enteric

spheroids from adult small and large gut were generated, expanded and

differentiated dependent on the applied cell culture conditions. For

implantation studies spheres were grafted into foetal slice cultures and

embryonic aganglionic hindgut explants of murine gut. Differentiating

enteric neural progenitors were characterized by BrdU labelling assay,

in situ hybridization, immunocytochemistry, quantitative RT-PCR

and electrophysiological studies. Results: We were able to generate

proliferating neurospheres from adult human gut tissues in vitro.

Spheroid derived cells could be differentiated into neuronal and glial

cells as demonstrated by the expression of a variety of neural markers

and clearly distinguishable sodium currents. Implantation experi-

ments into organotypic intestinal cultures showed the differentiation

capacity of neural progenitors in a three-dimensional environ-

ment. Conclusions: This study provides first evidence that adult

enteric neural progenitors persist in adult human gut tissue and that

they can be propagated in cell culture. Our findings open new possi-

bilities for enteric stem cell research and future development of

autologous cell-based therapies especially in the field of age-related

neurogastrointestinal disorders.

Development and physiology of enteric neurons in Drosophilai miguel-aliaga,* p cognigni,* s groenke� & ap bailey�*Department of Zoology, University of Cambridge, Cambridge, UK; �Institute of

Healthy Ageing, Department of Genetics, Evolution and Environment, UCL,

London, UK; and �MRC National Institute for Medical Research, London, UK

Invertebrate model systems such as Drosophila have greatly

contributed to our understanding of how somatic motor neurons

develop and interact with their target muscles. By contrast, the

power of Drosophila genetics has not yet been harnessed to inves-

tigate the development and physiology of visceral innervation. We

have investigated how and why the Drosophila digestive tract is

innnervated in both larvae and adult flies. In addition to the previ-

ously described innervation of the foregut and anterior midgut by

the stomatogastric nervous system,1 we have found peptidergic and

motor innervation of the hindgut by neurons with cell bodies

located in the central nervous system. These neuronal lineages

express Drosophila homologues of brain-gut peptides such as insu-

lin/IGF/relaxins. Developmental analysis reveals that several of

these fly visceral neurons are initially specified as pioneer neurons

that help form the longitudinal tracts of the embryonic central

nervous system. In the larva, they differentiate as visceral and

neurosecretory .2,3 Interestingly, some of the transcription factors

regulating their identity are similar to those involved in specifying

vertebrate visceral motor neurons. We are currently investigating the

mechanisms regulating targeting of visceral axons to the hindgut

muscles. In parallel, we have developed simple behavioural and

physiological assays to assess gut function in both wild-type flies

and mutants lacking specific peptides, or in which specific visceral

neurons have been genetically �silenced�. This allows us to deter-

mine the contribution of the peptidergic and motor innervation to

the functions of the digestive tract.

References:

1 Budnik et al. The Journal of Neuroscience 1989; 9: 2866–77.

2 Miguel-Aliaga et al. PLoS Biology 2008; 11; 6(3): e58.

3 Miguel-Aliaga and Thor Development 2004; 131(24): 6093–105.

Laminin, acetylcholinesterase and redundancy in ENS development

sw moore & ga johnson

University of Stellenbosch, Matieland, South Africa

Introduction: Laminin-111 and acetylcholinesterase (AChE) are both

involved in the promotion of cell adhesion and neurite outgrowth in

the developing ENS, a secondary function in AChE. We have

previously observed that AChE is able to bind laminin-

111 Objectives: In this study, we investigated the interaction sites of

AChE and Laminin. We also investigated the possibility that AChE

might be mimicking the function of other molecules during the

migration and differentiation stages of ENS develop-

ment. Methods: Preliminary localisation of the AChE molecule to

peripheral anionic site (PAS) region and the G4 domain of the laminin

alpha1 chain. Peptides representing various parts of these sequences

were synthesized and binding investigated by ELISA. Epitope analysis

of seven anti-AChE monoclonal antibodies blocking both cell adhe-

sion in neural cells and the AChE–laminin interaction was conducted

using a microarray of 500 peptides of varying lengths and degrees of

constraint. In silico docking of mouse AChE (1J06.pdb) and

mouse laminin (2JD4.pdb) was done on Hex 4.5. Bioinformatic anal-

ysis of structures was done on ProSite. Results: AChE was observed

on docking to bind to a discontinuous structure, 2718VRKRL, 2738YY,

2789YIKRK and 2817VGRK in the laminin alpha1 G4 domain. ELISAs

using synthetic peptides confirmed this. The major component of the

interaction site on AChE was found to be the sequence 90RELSED,

with additional contributions by 40PPV, 46R (linked to 94E by a salt

bridge) and 61DATTFQ. Epitope analysis of the antibodies showed

their major recognition site to be 40PPMGPRRFL; also cross-reaction

with Proline sequences 78PGFEGTE and 88PNRELSED. Antibodies

without 90RELSED motif recognition interfered with neither lami-

nin binding nor cell adhesion. The laminin site overlaps with the

heparin-binding site, and AChE competed with heparan sulfate

for laminin binding. This suggests possible functional redundancy

between AChE and HSPGs, [specifically, the syndecans, glypicans and

perlecan]. Search on molecules with 90RELSED and subsidiary motifs

yielded the receptor tyrosine kinase Mer, perlecan and the LDL

receptor. Conclusions: By defining sites for the interaction of laminin

and AChE, suggests an active role in developmental ENS cell adhe-

sion. The strong possibility of functional redundancy between AChE

and other molecules is also shown. Evidence suggests likely candi-

dates to include the syndecans, the glypicans, the receptor tyrosine

kinase Mer, the LDL receptor and, especially, perlecan.

Exploring aspects of chromosome 21 genes ITGB2 and SOD-1 in

Hirschsprungs disease

sw moore & mg zaahl

University of Stellenbosch, Matieland, South Africa

Introduction: Established associations between enteric nervous system

(ENS) pathology (e.g. Hirschsprungs disease (HSCR) and Chromosome

21 (e.g. Down’s syndrome, DS-HSCR) suggest a modifying link between

this region and the pathogenesis of an abnormal ENS. In addition to the

known susceptibility genes RET and EDNRB the role of the so-called

Down’s critical region at 21q22.2 remains uncertain. Potential chro-

mosome 21 candidate genes involved in ENS development flanking this

region include SOD1 and ITGB2 (CD18) which may have a modulatory

Abstracts

� 2009 Blackwell Publishing Ltd xix

effect on RET. Objectives: To investigate the genetic background of DS–

HSCR and the influence of the flanking genes SOD-1 and ITGB2 as

possible modifiers of the ENS in DS–HSCR. Methods: DNA extracted

from colonic tissue samples of 16 DS–HSCR [without associated

enterocolitis/cardiac lesions] and 20 sporadic HSCR patients was

screened for mutations in the RET, EDNRB,ITGB2 and SOD1 genes.

Results were compared with 60 control samples (20/ethnic group)

including unaffected Down’s syndrome patients. Polymerase chain

reaction (PCR) amplification was performed on the promoter and coding

region of the genes as specified, followed by heteroduplex single-strand

conformation polymorphism (HEX-SSCP) analysis. PCR products

demonstrating variation in the HEX-SSCP gels were subjected to auto-

mated DNA sequencing analysis Results: HEX-SSCP analysis revealed

RET variations in exons 2B (A45) 13 and 15 predominantly in DS HSCR.

An increased incidence of 561C/T in exon 2 of EDNRB was detected. The

ITGB2 gene demonstrated two known (V367, V441) as well as several

novel polymorphisms (-111T/C, 24G/T, 295G/A, 892A/G). In addition,

seven additional variants were identified in the 3�UTR of the gene. The

ITGB2-111T/C promoter polymorphism was statistically significantly

increased in HSCR (6/20) in the coloured population compared to con-

trols (1/20) (P < 0.05). A variant (D77N) was identified in one of 20 HSCR

patients and variants G567A, A572S, C575S, R586 was identified in four

of 13 DS–HSCR patients. In addition, aberrant HEX-SSCP banding pat-

terns were detected in the promoter region of SOD1 which included

three novel variants with one identified in exon 1 and the other in and

exon 3. Conclusion: In this study we demonstrate a possible interact-

ing chromosome 21 mechanisms involving ITGB2 and SOD genes in

HSCR pathogenesis The identification of several significant genetic

variants in this study indicates its probable role, as modifier loci, in

DS–HSCR phenotypes and possibly HSCR itself. The novel variants

described here, although present in low frequencies, may alter RET

expression.

Characterization and origin of a novel cell type in the avian enteric

nervous system

n nagy,* g csikos,* d molnar,* am goldstein� & i olah**Department of Human Morphology and Developmental Biology, Faculty of

Medicine, Semmelweis University, Budapest, Hungary; and �Department of

Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School,

Boston, MA, USA

The enteric nervous system (ENS) shares embryological, morphological,

neurochemical, and functional features with the CNS therefore, the

gastroenterologists and anatomists may be considered the ENS as the

�brain in the gut�. Both CNS and ENS are structured by neuronal

and supporting cells, namely glial and Schwann or satellite cells,

respectively. In the CNS the micro- or mesoglia contributes to innate

(phagocytosis) and acquire immunities. A cell with similar phenotype to

the microglia has not been identified in the peripheral nervous system

(PNS) including the ENS. Recently we have recognized a cell type in the

PNS, which share many features with the microglia of the CNS. For

collecting information of this type of cells in the ENS we have used

antibodies, which identify; neural crest derived cells, hemopoietic cells

including B and T lymphocytes, macrophages and dendritic cells. Anti-

MHC II antibody was also used to obtain preliminary information about

immune function. In the enteric and sensory ganglia the hemopoietic

marker CD45 recognized a highly ramified cell type. This cell type

express B cell, but not T cell, macrophage and dendritic cell markers. The

CD45+/Bu1b+/RCA (Ricinus communis agglutinin I) I+ phenotype

strongly supports their non-neural crest, but hemopoietic origin. The

presence of MHC class II antigen on their surface suggests their capa-

bility for antigen presentation. Their migratory capability and invasion

into the intestine were proved by chick-quail chimeras: quail hindgut

with nerve of Remak were isolated from 7 days old embryo and trans-

planted into the coelomic cavity of 3 days old chicken embryo. After

14 days of incubation the quail hindgut and nerve of Remak were tested

for chicken specific (CD45, Bu1, MHC II, RCA I) antigens, which re-

sulted in colonization of quail intestine and nerve of Remak by chicken

blood-borne cells, evidencing the immigration of hemopoietic cells into

the ENS. Our experiments indicate that there is a highly dendritic-like

cell in the PNS with identical phenotype of the microglia.

Development of the ENS in Ret51 animals: a mouse model for

understanding human Hirschsprung’s disease

d natarajan,* j dattani� & v pachnis**Division of Molecular Neurobiology, National Institute for Medical Research,

The Ridgeway, Mill Hill, London, UK; and �Department of Mathematics, Uni-

versity of Bath, Bath, UK

The gut motility disorder, Hirschsprung’s (HSCR) disease affects

1 : 4500 births and is characterized by failure of formation of the enteric

ganglia in the distal bowel. The enteric nervous system (ENS) is derived

from a relatively small number of (mostly) vagal neural crest cells which

invade the foregut and undergo extensive proliferation, migration and

differentiation into neurons and glia that make up a complex network of

interconnected ganglia distributed throughout the length of the gastro-

intestinal tract. Processes that disrupt normal development of the ENS

will often result in distal (colonic) aganglionosis. Several genes have

been identified as important regulators of ENS development and muta-

tions in these genes are often detected in patients with HSCR. Among

them, the gene encoding the receptor tyrosine kinase Ret is mutated in

approx. 50% of familial cases of HSCR, while changes in its activity are

thought to underlie all cases of this disorder. In mice, deletion of Ret

results in total intestinal aganglionosis, whereas failure to express the

Ret9 isoform can lead to colonic aganglionosis, a phenotype reminiscent

of the human condition (de Graaff et al, 2001) ). Here, we have used

monoisoformic Ret51 mice as an animal model for HSCR in order to

understand the pathogenesis of this condition, and assess the anatomical

and functional status of the ENS in the colonised (normoganglionic)

parts of the gut. To enable a more detailed analysis of enteric neuro-

genesis in mice homozygous for the Ret51 hypomorphic allele, we

introduced into this background the Rosa26YFPstop allele and the

Wnt1cre transgene, the combination of which marks all populations of

neural crest cells and their descendants, including those colonising the

gut. Our data so far indicate that normal activity of Ret is required for the

proliferation and migration of early neural crest cells. In addition, Ret

function is also required for the differentiation and axonogenesis of en-

teric neurons, as Ret51 homozygous animals have a smaller fraction of

neurons bearing significantly smaller processes. These findings suggest

that the functional abnormalities associated with HSCR are due to both

the lack of enteric ganglia in the colon and the presence of malfunc-

tioning neuronal networks in the ganglionated parts of the intestine.

This hypothesis is currently being tested by further analysis of the mu-

tant phenotype, including electrophysiological characterisation of con-

trol and Ret51 enteric neurons.

Forward Genetic Screen for Regulators of ENS Development

l niswander, t-h kim, y zhang & m hanson

HHMI and University of Colorado Denver, Denver, CO, USA

To gain insight into the complex process of enteric nervous system

development, we have used an unbiased approach of forward genetic

screening in mice to identify mutations that affect ENS development.

The screen was done at embryonic day 18.5 and we scored for the

presence of green or red material in the gut, in contrast to wildtype gut

that is yellow or light orange. We are currently working on three

mutants that derived from this screen. Our goal is to clone the genes

which when mutated cause ENS defects and to determine the mech-

anisms by which these genes act to regulate this critical embryonic

process. We are also characterizing the phenotype and using live

imaging to track the behaviours of the mutant cells to better under-

stand the cause of the ENS defects.

Ret/Gdnf signalling in vagal neural crest-derived neurons of the chick

embryo cloaca

am o�donnell & p puri

Children’s Research Centre, Oor Lady’s Childrens Hospital, Crumlin, Dublin,

Ireland

Introduction: The growth factor, �Glial cell line-Derived Neurotrophic

Factor� (GDNF), is involved in the development of enteric ganglia,

using the tyrosine kinase receptor �Rearranged during Transfection�(RET) to stimulate the proliferation and differentiation of neural crest-

derived precursor cells. To date, the presence of these signalling

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxx

molecules have not been studied in the developing cloaca, thus the aim

of this study was to investigate the distribution of RET and GDNF, and

analyse their co-localisation in vagal-derived neurons of the cloaca

using quail-chick chimera embryos. Materials and methods: Chicken

embryos were incubated until the 10–12 somite stage. The vagal neural

tube was microsurgically ablated in ovo and replaced with the vagal

neural tube from age-matched quail embryos. Quail-chick chimera

embryos were harvested at E12 and E14, fixed and embedded in paraffin

wax, and serially sectioned through the cloaca. Immunohistochemis-

try was performed using human natural killer-1 (HNK-1),

quail-cell-specific perinuclear (QCPN), GDNF and RET antibod-

ies. Results: HNK-1 labelled all ganglia in the myenteric and sub-

mucosal plexuses of the cloaca, while the quail-specific QCPN

antibody labelled all ganglia derived from the transplanted quail vagal

neural tube (Figure 1, A + B). RET and GDNF were found both

co-localised and expressed in separate ganglia in the cloaca (Figure 1,

C + D). The majority of QCPN-labelled vagal-derived neurons also

expressed RET and GDNF. Conclusion: Results show that GDNF and

RET signalling play a major role in ENS development in the chick

embryo cloaca. We have shown, for the first time, that the majority of

vagal neural crest-derived neurons co-express RET and GDNF, thus

highlighting the importance of these signalling factors in cloacal

development.

Effects of protein extracts from human HSD smooth muscle and

TGFß-family members on rat enteric nerve cell survival and

development in vitrou rauch,* m klotz,* e wink,� c hagl� & kh schafer**Department of Microsystems Technology/Biotechnology, University of Applied

Sciences, Zweibruecken, Germany; and �Department of Pediatric Surgery, Uni-

versity Hospital, Mannheim, Germany

The enteric nervous system (ENS) is a complex neuronal network which

is located within the muscle wall of the gastrointestinal tract (GIT).

Because of the importance of the microenvironment on cell survival,

development and function we studied the effects of gut smooth muscle

protein extracts from human Hirschsprung’s disease (HSD) patients on

isolated rat myenteric plexus cells. Due to the fact that sensory fibers

reaching from the gut to the CNS are also in contact with the same

microenvironment, we used DRG neurons to mimic that situation

in vitro. Furthermore we quantified the protein content and measured

the amount of GDNF and TGFbeta within the muscle wall. In parallel

we studied the effects of GDNF, Neurturin, Persephin, Artemin as well

as TGFbeta and bFGF alone and in combination on rat ENS plexus cells

in vitro. Most HSD-extracts induced an increase in neurite length and

neuron number and nearly half of them also an increase in the branching

pattern of isolated rat plexus neurons compared with control (defined

medium). Regarding the effects on DRG-fiber density, most extracts did

not induce a significant change. More than half of all extracts tested so

far induced significantly higher DRG cell numbers while the increase

induced by the other extracts was not statistical significant. The effects

of various neurotrophic factors on cell survival, neurite length and

neurite number per cell strongly depended on factors like age and origin

of cells (small intestine or colon) but especially on synergistic effects of

the factors. In this study we could clearly show that gut muscle extracts

from HSD patients did not have a negative effect on isolated rat plexus or

DRG neurons in vitro. This result is of particular importance, because it

shows that the aganglionic gut might provide a satisfactory micro-

environment for stem cell therapy in the future. Furthermore we could

show the importance of various neurotrophic factors, especially their

interaction, on rat ENS nerve cells in vitro.

MIND the gap: an astroglial perspective on intestinal barrier regulation

tc savidge

University of Texas Medical Branch, Galveston, TX, USA

The blood–brain barrier (BBB) is a specialized tissue-interface that

provides an important homeostatic and immunosurveillance role in

the central nervous system. Unlike most microvascular tissues that

readily promote paracellular passage of solutes and macromolecules,

the BBB is more analogous to polarized intestinal epithelia that restrict

such permeability in order to prevent disease-onset. Recent transgenic

ablation studies have demonstrated that BBB and intestinal tissues also

share a requirement for astroglial-regulated barrier-integrity. This

presentation highlights the emerging concept that astroglia regulate

barrier function at markedly different tissue-interfaces. It also explores

possible lessons that might be learnt by adopting epithelial model

paradigms of the BBB. For example, novel glial-derived S-nitrosylation

signals that regulate intestinal permeability in the digestive tract may

provide new mechanistic insights into barrier function at the BBB.

A better understanding of such universal mechanisms for barrier

regulation will facilitate novel therapeutic strategies targeting

permeability disorders at central nervous system and mucosal tissue

interfaces.

A novel Sox10-NGFP mouse model of Waardenburg–Hirschsprung

Syndrome

mh sham,* m zhang,* kh chu,* eym wong,* dmc chee,* mch cheung,*

vch lui� & pkh tam�Department of *Biochemistry, Li Ka Shing Faculty of Medicine, The University of

Hong Kong, Hong Kong SAR, China; and �Department of Surgery, Li Ka Shing

Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China

Introduction: Waardenburg–Hirschsprung (WS4) syndrome is charac-

terized by symptoms including sensorineural hearing loss, intestinal

aganglionosis, and pigmentation defects. Mutations of the SOX10 gene

have been identified in WS4 patients; molecular and mouse mutant

studies have suggested that the neural crest-derived abnormalities are

due to haploinsufficiency of Sox10. Objectives: The aim of this study

is to elucidate the roles of Sox10 during inner ear and enteric nervous

system development by mouse mutant analyses, in order to under-

stand the underlying pathogenic mechanisms for the sensorineural

hearing loss and intestinal obstruction displayed in WS4

patients. Methods: We have generated a novel mouse mutant Sox10-

NGFP in which the N-terminal domain of Sox10 is fused with the

EGFP gene. Using the Sox10-NGFP mutant, and in combination with

the Sox10-Dom, Sox9 or Sox2-GFP mutants, we examined the abnor-

mal development of the inner ear and ENS by morphological and

functional analyses. Results: By cell transfection and chick in-ovo

electroporation, we confirmed that Sox10-NGFP fusion proteins can

be stably expressed and have no transactivation functions. Heterozy-

gous Sox10-NGFP mutants displayed WS4 phenotypes including pig-

mentation defects, megacolon and mild hearing deficiency as

indicated by a slightly impaired auditory evolved brain response (ABR).

The Sox10-NGFP mutant embryos (E10.5) had reduced vestibular

ganglia with fewer neuronal and glial cells. The morphology of the

cochlear and vestibular structures of the Sox10-NGFP mutant

embryos was abnormal, particularly in homozygous mutants (E13.5),

though differentiation of sensory hair cells was not affected. By dye-

tracing, we showed that the innervation pattern of the spiral ganglion

was abnormal in the Sox10-NGFP mutant. Analysis of Sox9; Sox10

compound mutant embryos demonstrated that the inner ear defects

were more severe, indicating that the Group E Sox genes may have

distinct functions in the developing inner ear. On the other hand, in

Sox10-NGFP mutants, the colonization of enteric neural crest cells in

the gut was delayed at E12.5 and aganglionosis of the distal hindgut

was evident by E14.5. Conclusions: Our results on analysis of the

Sox10-NGFP mutant mice show that Sox10 has consistent roles in the

survival and differentiation of the enteric neural crest cells as well as

the progenitor cells in the vestibular ganglia, which would explain the

aganglionosis and hearing defects in WS4 patients.

Microarray–based identification of differentially expressed genes in the

intestines of Zebrafish ENS mutant lessen

i shepherd,* g burzynski,* l petrova,* j ngai� & j-m delalande**Department of Biology, Emory University, Atlanta, GA, USA; and �Department

of Molecular and Cell Biology, University of California, Berkeley, CA, USA

In order to gain insights into the molecular mechanisms that are

involved in the formation of the vertebrate enteric nervous system

Abstracts

� 2009 Blackwell Publishing Ltd xxi

(ENS) we have undertaken a microarray experiment to identify dif-

ferentially expressed genes in the intestine of the previously iden-

tified zebrafish ENS mutant lessen (lsn). The lsn mutant phenotype

is caused by a null mutation in MED24 (TRAP100), a subunit of the

Mediator co-transcriptional activator complex. lsn mutants have

fewer enteric neurons than wild type embryos due to the reduced

proliferation of ENS precursors. MED24 however is not expressed in

the ENS precursors but is expressed in the intestinal endoderm.

Based on these findings we hypothesized that the lsn mutant phe-

notype is caused by the reduced expression of endoderm derived

and/or endoderm regulated factors that are necessary for ENS pre-

cursor proliferation. To test this hypothesis we undertook a micro-

array experiment to identify differentially expressed genes in the

intestines of lsn mutant embryos as compared to wild type embryos

at a critical stage during zebrafish ENS development. In addition to

the microarray approach we also took a candidate gene strategy to

identify potential endoderm secreted ENS precursor mitogens.

Through the microarray experiment we have identified a large

number of genes that have reduced expression in the mutant. Down-

regulated genes include a number of previously identified ENS

associated transcription factors and components of several cell sig-

nalling pathways some of which had been previously implicated in

ENS development. We are currently characterizing the expression

and in vivo function of many of these genes using genetic and

pharmacological approaches. We will present describing these stud-

ies as well as more focused studies of investigating the function of

the Hedgehog and BMP signalling pathways in Zebrafish ENS

development.

Investigation of neural crest stem cell therapy for Hirschsprung’s

disease

n shimojima,* s shibata,� r hotta,* r nishikawa,* n nagoshi,�hj okano,� y morikawa* & h okano�*Keio University School of Medicine, Department of Pediatric Surgery, Tokyo,

Japan; �Keio University School of Medicine, Department of Physiology, Tokyo,

Japan; and �Keio University School of Medicine, Department of Orthopedic

Surgery, Tokyo, Japan

Introduction: Pathogenesis of Hirschsprung’s disease is thought that

migration arrest of neural crest cells results in a congenital absence of

ganglion cells in the distal segment of gut. The current only thera-

peutic choice is a surgical resection of the non-functioning

bowel. Length of the aganglionic (lack of neurons) segment vary and

patients with extensive lesion may need small bowel transplantation.

Therefore, novel therapeutic strategy, alternative to the surgery, is

highly expected. Objectives: The objective of this study is to inves-

tigate a potential of neural crest stem cells (NCSCs) transplantation as

a novel therapy for Hirschsprung’s disease. Methods: To make neural

crest derived cells distinct from other cells, special transgenic mice in

which all neural crest derived cells are enhanced green fluorescent

protein (EGFP) positive were used. Nagoshi N, et al. (2008). Embryonic

gut was used as NCSCs source. After the dissociation of gut, neural

crest derived cells were sorted as GFP positive cells. Sphere-forming

neural crest derived cells were identified after incubation with the

specific culture condition. Multilineage differentiation potency of the

sphere was assessed by immunocytochemistry for b-III tubulin,

smooth muscle actin (SMA), and glial fibrillary acidic protein (GFAP)

as neuron, smooth muscle, and glia markers, respectively. For the

transplantation experiment, NCSCs sphere was co-cultured with

recipient embryonic colon. Migration, localization, and differentiation

of transplanted NCSCs were analysed. Results: In the differentiation

assay, neural crest derived cells from mice differentiated into neuron,

smooth muscle, and glia. Transplanted NCSCs have migrated toward

the recipient gut at 16 h from the transplantation and the migration

progressed chronologically. Immunocytochemistry demonstrated the

existence of GFP and neuronal marker PGP9.5 double-positive cells in

migrating cells. Conclusions: We have isolated sphere-forming

NCSCs as EGFP-positive cells from mice embryonic gut. Transplanted

NCSCs successfully localized in the recipient gut and differentiated

into neurons. Although it is still preliminary data, we have started

experiments using human gut samples and similar spheres have been

formed. These data support the possibility of the future NCSCs

transplant therapy for Hirschsprung’s disease. Reference Nagoshi N,

et al. Ontogeny and multipotency of neural crest-derived stem cells in

mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem

Cell. 2008;10: 392–403.

The role of neuropilin-1 in neural crest cell invasion

r mclennan & pm kulesa

Stowers Institute for Medical Research, Kansas, KS, USA

During vertebrate development, neural crest cells (NCCs) delami-

nate from the neural tube and migrate in a stereotypical pattern to

specific destinations. In the cranial and post-otic regions, discrete

NCC migratory streams invade the branchial arches to form facial

structures and the gut to pattern the enteric nervous system, yet

signalling mechanisms that produce the migration pattern are still

unclear. Our aim is to explore the function of potential NCC

guidance factors and perform detailed analysis of NCC behaviours in

vivo. Here we investigate a putative NCC guidance cue, neuropilin-

1. Neuropilin-1 is a membrane co-receptor for ligands from the

Semaphorin and VEGF families. When a neuropilin-1 siRNA (Bron

et al, 2004) is used to knock down neuropilin-1 expression in ovo,

NCCs fail to fully invade the second branchial arch and the rostral

portion of branchial arch 3. Detailed cell morphometric and RT-PCR

expression analyses show non-invading neuropilin-1 siRNA

transfected NCCs exhibit a loss of cell polarity, shortened filopodia

and changes in expression of cell adhesion molecules. Motility

and directionality of these non-invading NCCs is rescued by trans-

plantation into the hindbrain (rhombomere 4) of younger host em-

bryos, however neuropilin-1 siRNA transfected NCCs fail to

contribute to cranial gangliogenesis and skeletal elements. Similar

experiments suggest that neuropilin-2 does not influence the initial

formation of the cranial NCC streams. We conclude that neuropilin-

1 is critical to NCC homing into the branchial arches by

maintaining an active motility state and responding to local

microenvironmental signals.

Genetic and genomic approaches to identify gene–gene interactions

that modify severity of aganglionosis in the sox10dom model of

Hirschsprung disease

em southard-smith, se owens, jt harris, lc walters, va cantrell,

km bradley, jr smith & dc airey

Vanderbilty University Medical Center, Nashville, USA

Hirschsprung disease (HSCR) arises from abnormal development of the

enteric nervous system (ENS) and presents as intestinal obstruction

secondary to the absence of enteric ganglia in a variable portion of the

distal intestine. Cumulative evidence suggests HSCR is the conse-

quence of multiple gene interactions that modulate the ability of

enteric neural crest – derived cells to populate the developing gut.

Multiple HSCR susceptibility genes including RET, GDNF, EDNRB,

EDN3, and SOX10 have been identified through patient studies and

additional gene mutations are known to impact ENS development and

function based on studies in mouse models. However, little is known

of how interactions between genes impact the penetrance and severity

of aganglionosis. Few reports of gene-gene interaction effects specifi-

cally on aganglionosis have been published. Limited success identify-

ing relevant gene interactions stems in large part from the inherent

difficulties of studying aganglionosis phenotypes in mouse models

where enteric defects are typically only seen in homozygotes. Fortu-

itously, dominant negative forms of the NC transcription factor Sox10

(Sox10Dom) exhibit aganglionosis with variable penetrance and

severity in heterozygotes analogous to that seen in HSCR patients.

Analysis of Sox10Dom mice across multiple inbred strain backgrounds

demonstrates that a significant proportion of this phenotypic variation

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxxii

results from differences in genetic background. Consequently, the

Sox10Dom model facilitates identification of gene interactions that

impact ENS development and function. We have established extensive

genetic resources that incorporate the Sox10Dom allele with the aim

of mapping modifier genes, natural variants that interact with Sox10 to

alter aganglionosis. Genome wide studies have been combined with

focused analyses of candidate genes in linkage regions that are asso-

ciated with increased severity of Sox10Dom aganglionosis. Comple-

mentation testcrosses, immunohistochemical co-localization within

enteric progenitors and allele-specific expression studies have identi-

fied specific genes that interact with Sox10 to alter migration of enteric

progenitors into the developing intestine. Our results establish a

mechanistic basis for the effect of modifier genes on variation of

phenotype in HSCR disease.

Functional studies on Ret non-coding region mutations and the

involvement of the Nxf/Arnt2; Sim2/Arnt2 heterodimers in the

regulation of RET expression

y sribudiani, j osinga, k bos & rmw hofstra

Department of Genetics, University Medical Center of Groningen, Groningen,

The Netherlands

Ret coding mutations are considered the major cause of Hirschprung

(HSCR) disease. Up to 50% of familial cases and 15–20% of sporadic

cases proved to have such mutations. However, previous studies indi-

cated that non-coding Ret mutations, in particular SNP rs2506004 and/

or SNP rs2435357 both located in intron 1 of the Ret gene, are also

involved in HSCR susceptibility. It was hypothesized that the mutant

variants of those SNPs have an inhibiting effect on the expression of

RET. To test this hypothesis, we generated reporter constructs con-

taining the intronic sequence in which both non-coding Ret mutations

reside (separately or in combination) coupled to Luciferase. Moreover,

two different promoters were used (SV40 and RET). By using the SV40

constructs we have shown that in human and mouse neuroblastoma cell

lines the wild type variants either separately or in combination

enhanced Luciferase expression and that in contrast, both mutants

decreased Luciferase expression. Constructs with the RET promoter

however demonstrated that only the two RET mutant SNPs in combi-

nation are able to decrease the Luciferase expression. By using bioin-

formatics tools, we found that the sequences surrounding SNP

rs2506004 and six copies of similar sequences in the RET promoter are

the binding site for the Nxf/Arnt2 and Sim2/Arnt2 heterodimers.

Binding affinity was checked by Electrophoretic Mobility Shift Assay

(EMSA) and we have shown that Nxf/Arnt2 only binds to the wild type

SNP rs2506004 (-Cgtg-) and loses its binding affinity to the mutant SNP

(-Agtg-). Co-transfection of Nxf/Arnt2 with RET promoter constructs

into mouse neuroblastoma cell lines enhanced Luciferase expression

level by 40-fold compared to its control, proving that the Nxf/Arnt2

heterodimer acts as a transcription activator for RET. By contrast,

co-transfection of Sim2/Arnt2 with RET promoter constructs decreased

the luciferase expression compared to its control. Our data suggest that

SNPs rs2506004 and the six copies of similar sequences in the RET

promoter are likely to be involved in regulating RET expression and this

regulation is controlled by Nxf/Arnt2 and Sim2/Arnt2. This molecular

mechanism might also be a partial explanation for the common associ-

ation of HSCR with Down syndrome as Sim2 is located on chromosome

21.

Functional analysis of RET tyrosine kinase as a dependence receptor

m takahashi,* n asai* & f costantini�*Nagoya University Graduate School of Medicine, Nagoya, Japan; and �Columbia

University, New York, USA

RET encodes a receptor tyrosine kinase essential for the development

of the enteric nervous system and the kidney. Recently, RET was

reported to function as a �dependence receptor� which induces apop-

tosis in the absence of its ligand, GDNF. Without GDNF stimulation,

RET is cleaved by caspase-3 in vitro, thereby releasing a pro-apoptotic

fragment. Amino acids 707 and 1017 in RET are cleavage sites by

caspase-3, and both D707N and D1017N mutations failed to induce

cell death in vitro. We confirmed fragmentation of RET in cultured

human neuronal cells in a caspase-dependent manner, and wild-type

RET induced cell death. To determine the importance of RET pro-

apoptotic activity in vivo, we generated Ret knock-in mice in which

D707N mutation was introduced in the mouse Ret gene. D707

homozygous mutant mice showed hyperplasia of neural cells in the

enteric, sympathetic and parasympathetic nervous systems. However,

the neuron number in the distal half of the colon rather decreased. This

appeared to be due to migration defect of enteric neural crest cells

during embryogenesis. In addition, the adenopituitary lobe in the

pituitary gland was hyperplastic in D707N homozygous mice. These

findings suggest the important roles of RET pro-apoptotic activity for

normal tissue development. I discuss the significance of the results

observed in Ret D707N knock-in mice.

Using a lentiviral vector to label enteric nervous system progenitor

cells

s theocharatos,* s kenny,� r lindley*,� & d edgar**University of Liverpool, School of Biomedical Sciences, Liverpool, UK; and

�Institute of Child Health, Royal Liverpool University Children’s Hospital, Alder

Hey, UK

Understanding the enteric nervous system development is a critical step

in order to develop novel therapies for treating congenital disorders such

as Hirschsprung’s disease (aganglionosis of the bowel). Previous studies

have shown that neural crest-derived progenitor cells (NCCs) of the

enteric nervous system (ENS) migrate rostrocaudally along the bowel

during development and differentiate to form mature neurons and glial

cells. NCCs can be isolated from both embryonic mouse and neonatal

human bowel, and in culture under poorly adherent conditions they give

rise to floating aggregates of both stem cells and their progeny, known as

neurospheres. We have previously shown that transplantation of neur-

ospheres into aganglionic colon explants in vitro can restore the bowel

contractility as neurosphere-derived cells migrate into the explants and

differentiate into mature neurons and glial cells. However, little is

known about the mechanisms that control the migration and differen-

tiation of ENS precursors either in vivo or in vitro. Our current work

aims to track the fate of neurosphere-derived cells both within the

neurosphere and after transplantation into aganglionic bowel using an

EGFP expressing lentiviral vector. This construct utilizes the spleen

focus-forming virus (SFFV) promoter to enable constitutive expression of

EGFP in all cells after transduction. To do this, NCCs were isolated from

E11.5 mouse embryos and cultured in non-adherent conditions for at

least 20 days in order to form neurospheres. These neurospheres were

then dissociated and the resulting single cell population was transduced

by the lentivirus. Preliminary results showed that >85% of the neuro-

sphere cells were EGFP positive and when labelled neurospheres were

allowed to differentiate by attachment to adhesive tissue culture sub-

strates, axon-like fibers expressing EGFP extended from the neuro-

spheres, together with labelled cells. After culturing the transduced cells

for a minimum of 15 further days, EGFP positive neurospheres were

formed and were transplanted into E11.5 mouse colon explants. Single

labelled cells began to migrate from the neurosphere into the explant

during the first 24 h after transplantation. At later time points, EGFP

positive cells had migrated along the entire length of the explant. The

above lentiviral model enables us to transduce both dividing and non-

diving cells and it can be applied in both mouse and human NCCs.

Further steps will involve live cell imaging and immunostaining in order

to examine changes in the morphology and the identity of the EGFP

positive migratory cells.

Abstracts

� 2009 Blackwell Publishing Ltd xxiii

Bcl-xL-mediated rescue of enteric nervous system formation in a

mouse model for Hirschsprung disease

t uesaka & h enomoto

NDR, RIKEN Center for Developmental Biology, Kobe, Japan

The peripheral neurons that control the gut form a network known as

the enteric nervous system (ENS). Mutations in the RET gene are the

primary cause of Hirschsprung disease (HSCR), also known as con-

genital intestinal aganglionosis, in which enteric neurons fail to

develop in the distal colon. We previously demonstrated that condi-

tional ablation of Ret caused neuronal death in the colon and that mice

with diminished Ret expression show features of HSCR. In this mouse

model, aganglionosis was associated with impaired migration and

decreased survival of ENS precursors. (Journal of clinical investigation

2008; 118: 1890–8.). Since Bcl-xL can block enteric neuronal death

triggered by impaired RET signalling in vitro (Development 2007; 134:

2171–81.), we engineered Ret locus to allow elevated levels of Bcl-xL

expression in enteric neural crest-derived cells, and assessed the degree

of contribution of cell death to the emergence of aganglionosis in the

HSCR model mice. Now, we have demonstrated that Bcl-xL

overexpression reduced enteric neuronal loss in the colon by condi-

tional inactivation of Ret and rescue enteric neurons from colonic

aganglionosis in the HSCR model mice. These results strongly suggest

that cell death principally causes HSCR associated with Ret

mutations.

Neurochemical identification of enteric neurons in the larval and adult

intestine of the zebrafish (Danio rerio)

l uyttebroek,* f harrisson,* g hubens,* it shepherd,� j-p timmermans� &

l van nassauw*,�*Laboratory of Human Anatomy & Embryology, University of Antwerp,

Belgium; �Department of Biology, Emory University, Atlanta, GA, USA; and

�Laboratory of Cell Biology & Histology, University of Antwerp, Belgium

In the last decade, the zebrafish has emerged as a leading model

organism for the study of vertebrate developmental biology and has

begun to be used in studies of gastrointestinal congenital diseases.

While the general morphology and development of the enteric nervous

system (ENS) of the zebrafish are already known, specific details

regarding the physiological function and morphological characteristics

of enteric neurons is still incomplete. The aim of the present study is

to unravel the neurochemical coding of zebrafish enteric neurons,

revealing specific subpopulations. Using immunoenzymatic and mul-

tiple immunofluorescent staining methods on isolated intestines from

adult and larval zebrafish, we demonstrated and quantified the

expression of different neurochemical markers representing presump-

tive excitatory, inhibitory and sensory innervation. Three markers

[tyrosine hydroxylase, vasoactive intestinal peptide (VIP), and pituitary

adenylate cyclase-activating polypeptide (PACAP)] were only observed

in enteric nerve fibres, while other markers [calretinin (CR), calbindin

(CB), choline acetyltransferase (ChAT), serotonin (5HT) and neuronal

nitric oxide synthase (nNOS)] were also detected in neuronal cell

bodies. In all segments of the adult intestine, ±50% of the neurons

expressed CR, while ±40% expressed CB, ±40% ChAT and ±20%

nNOS. The proportion of 5HT(+) neurons significantly and progres-

sively decreased from the anterior part (±23%) to the posterior part

(±11%) of the gut. No colocalization was observed between 5HT and

CR or CB, while all CR(+) neurons expressed CB. ChAT colocalized

with CR and CB, but not with 5HT. VIP and PACAP were present from

72 hpf on in the mid- and posterior-gut. nNOS was also found from 72

hpf in these parts, while CR and CB were expressed in the midgut.

From 96 hpf on 5HT was also expressed in the ENS. The present results

indicate that inhibitory neurons are the first to differentiate in the

zebrafish ENS and that they play a significant role in the spontaneous

motor activity of the gut observed between hatching and the onset of

feeding. The results support also previous data that the ENS is well-

developed before the start of feeding. In the adult intestine, the results

are indicative of the presence of several subpopulations of enteric

neurons, and of the existence of regional differences.

Allelic influence of gain-of-function mutant KITK641E in the

development of KIT+ interstitial cells of Cajal in the mouse antrum

jm vanderwinden,* s ralea,* p hague,* p gromova* & b p rubin�*Universite Libre de Bruxelles, Brussels, Belgium; and �Lerner Research Institute

and Taussig Cancer Center, Cleveland, OH, USA

Introduction: Kit, a receptor tyrosine kinase, is essential for the

development of interstitial cells of Cajal (ICC) in the gut wall. Loss-

of-function Kit mutants cause defects in Kit expressing (Kit+) cells,

including ICC, while gain-of-function (oncogenic) mutants cause

either proliferation or defects in various Kit+ lineages. Kit alleles are

known to be co-dominant. Here we have investigated the influence of

the gain-of-function Kit mutant K641E1 in the development of the

muscularis propria in the postnatal mouse antrum. Material and

methods: Mice carrying wild type (WT), gain-of-function (K641E), loss-

of-function (Wv) and null (WZsGreen2) Kit alleles were interbred. In

viable genotypes, Kit+ ICC and smooth muscles were studied by

immunofluorescence for Kit and for alpha smooth muscle actin

immunoreactivity, respectively. Results: Only the WT Kit allele led

to normal development of the two KIT+ ICC populations (ICC-MP &

ICC-IM) and longitudinal muscle layer (LM) in the mouse antrum.

ICC-MP, myenteric ganglia and nerve fibers in the muscularis propria

were present in all genotypes. The gain-of-function allele K641E pro-

moted the proliferation of Kit+ ICC but in absence of WT allele, K641E

was unable to develop ICC-IM and it inhibited differentiation of the

LM. Discussion: This study highlights the different effects of Kit in

differentiation and in proliferation/survival processes. The gain-of-

function allele Kit K641E has a strong proliferative effect on Kit+ ICC,

while it impacts negatively the differentiation of Kit+ ICC-IM and of

LM in absence of WT. The signaling pathways involved and the

interactions between WT and mutant KIT are currently being inves-

tigated.

References:

1 Rubin BP et al. Cancer Research 2005; 65: 6631–39.

2 Wouters MM et al. Physiological genomics 2005; 22: 412–21.

Effects of partial loss of zebrafish intestinal smooth muscle on

migration and differentiation of enteric neural precursors

kn wallace & t akhtar

Biology Department, Clarkson University, Potsdam, NY, USA

Enteric neural precursors migrate and differentiate within intestinal

smooth muscle upon arrival in the digestive system. As a result,

changes in smooth muscle development may alter enteric neural pre-

cursor migration and differentiation. To this end, we disrupted intes-

tinal smooth muscle differentiation to determine if there are inductive

signals provided by this tissue as enteric neurons develop. FKBP9 was

previously shown to be required for differentiation of avian intestinal

smooth muscle. We identified the zebrafish homologue of fkbp9 and

characterized expression. zfkbp9 is expressed within the developing

anterior smooth muscle during the third day of embryogenesis when

smooth muscle is differentiating. To address the role of FKBP9 in

intestinal smooth muscle differentiation we used both FK506, a gen-

eral inhibitor of FKBPs, and a 5� morpholino to the gene. We find that

injection of either FK506 or the morpholino inhibits differentiation of

smooth muscle primarily in the anterior intestine. A morpholino to a

closely related gene, fkbp10, which is also expressed within developing

intestinal smooth muscle, does not result in inhibition of smooth

muscle differentiation. Disruption of intestinal smooth muscle

development by down-regulation of fkbp9 reveals a similar number of

enteric neurons in the anterior intestine with comparable axon pro-

jection. This suggests that either anterior smooth muscle does not

provide inductive signals to enteric neural precursors or remaining

smooth muscle produces sufficient signals. Alternatively, fkbp9 may

not affect early development of smooth muscle and signals for enteric

neuron differentiation may still be produced.

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxxiv

L1cam does not interact with Ret during enteric nervous system

development

as wallace, a bergner & rb anderson

Department of Anatomy and Cell Biology, University of Melbourne, Victoria,

Australia

Introduction: Hirschsprung’s disease is a congenital disorder affecting

1/5 000 live births in which there is an absence of enteric ganglia

within the distal gastrointestinal tract. The genetics of Hirschsprung’s

disease are highly complex and non-Mendelian with a male sex bias of

4 : 1. With only 50% of Hisrschsprung’s cases attributable to known

susceptibility genes there may be many unknown genes and gene–gene

interactions responsible for this condition. The X-linked gene L1CAM

has been implicated in several Hirschprung’s cases, making it an

excellent candidate as a modifier gene for the development of

Hirschsprung’s disease. Objective: To examine whether there is

genetic interaction between L1cam and Ret. Methods: We used a two-

locus complementation approach, crossing L1+/- female mice with

Ret+/- male mice. Using the pan-neuronal markers PGP9.5 and Tuj1

we assessed whether the migration of enteric neural crest cells and

their derivatives was altered in L1cam null mutant mice when com-

bined with a heterozygous mutation in Ret at E18.5 gut. Results: We

show that enteric neural crest-derived cell migration is not affected

in L1cam; Ret mutant mice at E18.5. Preliminary data also

suggests that neuronal cell density is not affected in these

animals. Conclusions: This study shows that L1cam does not interact

with Ret to alter neural crest-derived cell migration during the devel-

opment of the enteric nervous system. However, another study from

our laboratory has shown that L1cam can interact with the

transcription factor, Sox10, to disrupt enteric neural crest-derived cell

migration. Therefore, it is important to identify which Hisrschsprung’s

susceptibility genes interact with L1cam.

Permissive action of GDNF on HGF induced neurite growth from

enteric neuron precursor cells

h wang & ro heuckeroth

Washington University, St Louis, MO, USA

Introduction: Hepatocyte growth factor/scatter factor (HGF/SF) regu-

lates the development and function of neuronal precursors in the

central and sympathetic nervous system, but a role for HGF in the

enteric nervous system (ENS) has not been previously reported. Given

the complexity of the enteric nervous system and the number of dif-

ferent neuronal subtypes, the trophic factors currently known to sup-

port ENS development seem unlikely to adequately control neurite

growth. In particular, it may be necessary to selectively control neurite

growth in subsets of enteric neurons. Objectives: To determine if

HGF could influence ENS precursor development. Methods: E12.5

mouse ENS precursor cells were grown in defined medium in dispersed

cell culture or gut slice cultures in the presence or absence of HGF and

GDNF. Immunohistochemistry of the developing and mature ENS for

HGF and its c-met receptor were also performed. Results: Immuno-

histochemistry demonstrated the expression of c-met and HGF in

enteric neuronal precusor cells and in subsets of neurons of mature

ENS. Cell culture experiments demonstrated that although HGF alone

had no effect on neurite growth, HGF enhanced neurite growth when

ENS precursors were grown in media containing low levels of GDNF.

Similar effects of HGF on neurite outgrowth were also observed in

mid-gut slice culture experiments. Specifically, low levels of GDNF

that alone had no effect on neurite outgrowth from gut slices,

permissively increased neurite growth in the presence of HGF. HGF

receptor blocking antibody prevented HGF induced neurite

growth, consistent with a role for c-met and HGF in ENS develop-

ment. Conclusions: These data suggest that HGF signaling may sig-

nificantly influence subsets of ENS precursors and be important for

normal neurite network formation within the ENS.

Abnormalities of enteric nervous system structure and function in

proximal small intestine of the Ednrb model of Hirschrpung disease

kc williams,* g farrugia,� db polk* & em southard-smith**Vanderbilt University Medical Center, Nashville, TN, USA; and �Mayo Clinic,

Rochester, MN, USA

Background: Mutation of the gene that encodes for endothelin recep-

tor B can cause aganglionic megacolon, or Hirschsprung’s disease

(HSCR), in humans. Although surgical removal of the distal agangli-

onic segment rescues patients from life threatening intestinal

obstruction, up to 2/3 of HSCR survivors report ongoing gastrointes-

tinal sypmtoms suggestive of further motility abnormalities, such as

vomitting, abdominal pain, and constipation. Alterations of enteric

nervous system (ENS) proximal to the aganglionic segment of distal

colon may explain why many HSCR survivors continue to suffer

symptoms of gastrointestinal dysmotility. Previous studies of the

Endothelin Receptor B (Ednrb) mouse model of HSCR have demon-

strated that the structure of the ENS in the proximal small intestine

exhibits altered patterning in homozygous (Ednrb-/-) and heterozygous

(Ednrb-/+) mice as compared with wildtype littermates (Ednrb+/+).

Ednrb-/- mice develop aganglionic megacolon, while Ednrb-/+mice

develop ganglia in the distal colon and appear normal. Hypothesis: We

hypothesized that mutation of Ednrb alters the structure and function

of the ENS in the proximal small intestine even when agangliosis does

not develop in the distal colon. The Aim of this study was to evaluate

ENS morphology and function in the proximal small intestine of

Ednrb-/-, Ednrb-/+ and Ednrb+/+ littermates. Methods: Whole mount

preparations of the external duodenal muscle layers of 2-week-old mice

were studied. The volume of ganglia in the myenteric plexus region

was determined from 0.625 lm Zeiss apotome slices by 3D recon-

struction and volume rendering. Electrical field stimulation studies in

normal Krebs solution were utilized to evaluate the response of

smooth muscle to neurotransmitter release in duodenal seg-

ments. Results: The volume of the myenteric plexus was decreased in

homozygote and heterozygote littermates compared with wildtype

littermates. Contractile reponses induced by electrical field stimula-

tion were reduced in homozygote and heterozygote littermates as

compared with wildtype littermates. Conclusions: Our results indi-

cate that mutation of the Ednrb gene can alter proximal ENS structure

and function even when the mutation does not lead to aganglionosis in

the distal colon.

A sensitized mouse mutagenesis screen for modifiers of Sox10

neurocristopathies

wj pavan, de watkins-chow, i matera, k buac, d larson, sk loftus, l hou,

a incao, dl silver, c rivas, ec elliott & ll baxter

National Human Genome Research Institute, National Institutes of Health,

Bethesda, MD, USA

The neural crest is a multipotent cell population that arises during

mammalian development and gives rise to a variety of cell types

including cartilage, bone, melanocytes of the skin, and neurons and

glia of the peripheral nervous system. Disrupting the normal devel-

opment of these lineages can cause debilitating diseases, collectively

referred to as neurocristopathies, that present with a variety of phe-

notypes including deafness, blindness, cleft lip, congenital megacolon

and albinism. As genetic background is known to affect the severity of

neurocristophathies in both humans and mice, we have established an

enhancer screen to identify mutations that increase the phenotypic

severity of Sox10 haploinsufficient mice, a well-characterized mouse

model of human neurocristopathies. In analysis of 400 pedigrees, we

have identified four dominant modifiers of Sox10 neurocristopathies

(Mos1-4) and four recessive phenotypes affecting embryonic Sox10

expression (msp1-4). The causative mutations affect genes involved in

a variety of functions including hedgehog, neuregulin and semaphorin

signalling as well as ribosomal and RNA binding proteins. The phe-

notypes we have identified do not overlap previously known major

mouse spotting loci, thus demonstrating the feasibility of this screen

to provide a more detailed understanding of the critical genes regu-

lating mammalian neural crest development and to provide additional

disease models for human neurocristopathies.

Abstracts

� 2009 Blackwell Publishing Ltd xxv

Dynamic behaviour of immature enteric neurons

hm young,* mm hao,* rb anderson* & h enomoto�*Department of Anatomy & Cell Biology, University of Melbourne, 3010,

Australia; and �RIKEN Center for Developmental Biology, Kobe, Japan

Introduction: While they are migrating along the developing gut, a

sub-population of neural crest-derived cells starts to differentiate into

neurons. It is unknown whether neuronal differentiation halts their

migration. Objectives: To examine the dynamic behaviour of imma-

ture enteric neurons. Methods: Mice in which only immature enteric

neurons express GFP (TH-GFP mice) and mice in which all enteric

neural crest-derived cells express the photo-convertible fluorescent

protein, Kikume (Ednrb-Kik mice) were used. Neurons were imaged in

intact explants of embryonic gut. Results: Although neuronal migra-

tion is common in the developing CNS, it has been assumed that

neural crest cells cease migration at the onset of neural differentiation.

However, around 50% of immature neurons migrated during the

imaging period with an average speed of 15 lm h-. This is slower than

the speed at which the population of neural crest-derived cells

advances along the developing gut. Most migrating neurons migrated

caudally by extending a long leading process followed by translocation

of the cell body. In many migratory neurons, a swollen structure of

variable size would detach from the cell body and move along the

leading process in the direction in which the neuron was migrating.

The segment at the rear would subsequently translocate in the direc-

tion of the leading process and rejoin the swelling. This mode of

migration is different from that of non-neuronal enteric neural crest

cells and neural crest cells in other locations, which have multiple

transient processes that extend and retract rapidly. To determine if the

swollen structure along the leading process contains the centrosome,

antibodies to pericentrin were used. In all neurons examined, peri-

centrin staining was located exclusively in the cell body. Nerve fibres

are closely associated with the network of neural crest cells and their

processes. However, most nerve fibres project directly down the long

axis of the gut whereas neural crest cells migrate spirally around the

gut. Time-lapse imaging revealed that the leading processes of imma-

ture neurons sometimes initially extended in multiple directions

before growing in a caudal direction. Conclusions: Neuronal differ-

entiation seems to slow, but not halt, the caudal migration of neural

crest-derived cells along the gut. Axons follow more linear, longitu-

dinal pathways than the migrating crest cells.

Phactr4 regulates cytoskeletal dynamics in enteric nervous system cell

migration

y zhang, th kim & l niswander

Department of Pediatrics, Section of Developmental Biology, Howard Hughes

Medical Institute, University of Colorado at Denver and Health Sciences Center,

Aurora, CO, USA

Hirschsprung disease arises due to an embryonic defect in formation of

the enteric nervous system (ENS) leading to a reduction of ENS cells in

the gut and gastrointestinal blockage. Here we study the humdy mouse

mutant, with a disruption in the Phactr4 gene, and show there is an

embryonic gastrointestinal defect. Humdy mutant embryos have a

reduced number of ENS cells in the gut. This is due to an ENS

migration defect such that vagal neural crest cells do not migrate

normally. To visualize neural crest cell migration along the mouse gut,

heterozygous humdy mutant mice were crossed with pRet-GFP mice

in which the expression of GFP is under the control of Ret promoter. In

homozygous humdy mutant embryos, only a few cells migrate into the

stomach. Moreover, neural crest migration from the neural tube is also

aberrant. Previous study has shown that the humdy mutation

specifically disrupts interaction with Protein Phosphatase I (PP1).

Here, using neural crest (NC) cultures, inhibition of PP1 by

0.2 mmol L-1 okadaic acid (OA) does not show an obvious effect on cell

migration. This suggests there may be a redundancy of PP1 function in

the regulation of ENS progenitor cell migration.

HOXB5 synergizes TTF-1 in the transcription of human RET

jj zhu, tyy leon, esw ngan, pkh tam & vch lui

The University of Hong Kong, Hong Kong, China

Hirschsprung’s disease (HSCR) is an oligogenic disorder typified by its

complex pattern of inheritance with manifestation of incomplete

penetrance. The RET gene, which is crucial for the development of the

ENS, is reported to be the major gene in HSCR and the haploinsuffi-

ciency of the RET affects the disease penetrance. The other HSCR

genes codifying for transcription factors SOX10, PAX3, PHOX2B, TTF-

1 and HOXB5 have been shown to regulate RET expression. We have

previously reported that HOXB5 can induce RET transcription and

defects in the activation of RET by HOXB5 could lead to RET hap-

loinsufficiency. To investigate the contribution of HOXB5 in the reg-

ulation of RET expression and in the aetiology of HSCR, we sought to

(i) examine if HOXB5 cooperates with these factors in the transcription

of the human RET, and (ii) localize the regulatory element(s) respon-

sible for the HOXB5 induction. Using luciferase reporter assay with

the full length (3.7 kb) human RET promoter, HOXB5 showed a

threefold induction while TTF-1 displayed a 10-fold induction from the

RET promoter. In contrast, we observed a 19-fold induction when

HOXB5 and TTF-1 were co-transfected. However, such synergistic

interaction was not observed when HOXB5 was co-transfected with

either PAX3, PHOX2B or SOX10. We have also localized the regulatory

elements in the human RET promoter responsible for the HOXB5

induction. Taken together all these suggest that HOXB5 may act as a

transcriptional co-activator and synergizes TTF-1 to induce the RET

transcription. HOXB5 may function as a HSCR modifier locus, in

which defects in the activation of RET by HOXB5 co-operating with

TTF-1 lead to RET haploinsufficiency and HSCR.

Temporal patterning of gastroesophageal innervation is abnormal in

rat embryos and fetuses with experimental diaphragmatic hernia

f pederiva, r aras lopez, l martinez & ja tovar

Department of Pediatric Surgery and Research Laboratory, Hospital Universitario

La Paz, Madrid, Spain

Background: Gastroesophageal reflux (GER) has been often described in

babies with congenital diaphragmatic hernia (CDH). Since deficient

innervation of the esophagus has been demonstrated in babies with

CDH, we tested the hypothesis that the temporal patterning of gastro-

esophageal innervation might be abnormal in rat embryos and fetuses

with experimental CDH. Material and methods: Pregnant rats were

given either nitrofen or vehicle on E9.5. Embryos were recovered on E15,

E18 and E21 and 10 control and nitrofen/CDH pups were studied. E15

and E18 esophagi were stained for anti-protein gene product 9.5 (PGP 9.5)

antibody and the intrinsic neural network was examined under confocal

microscopy. The intramural ganglia of E18 and E21 whole mount prep-

aration of stomachs stained for AChE were counted and measured.

Transverse sections of esophagi were immunostained for antineurotro-

phin receptor (p75NTR) and anti-PGP 9.5 antibodies. The relative sur-

face occupied by neural structures over the smooth muscle surface was

measured in the esophagus. Comparison between groups was made with

non-parametric tests. Results: Esophageal neural structures were

underdeveloped on E15 and E18 in nitrofen exposed pups. The relative

neural/muscle surface was decreased in the esophagus of CDH embryos

on E15 and E18 at all levels and tended to normalize on E21. The neural

network of the stomach was similar in control and nitrofen-exposed

embryos on E15 but on both E18 and E21 the number and the

mean surface of ganglia were significantly smaller in CDH

fetuses. Conclusions: Neural structures of the esophagus are sparser

and the ganglia of the stomach are fewer and smaller in rat fetuses with

CDH. These findings confirm the value of this model for further inves-

tigating the mechanisms of upper g.i. dysmotility in patients with

CDH.

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxxvi

How many genes for Hirschsprung diease?

a chakravarti

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University,

Baltimore, MD USA

Despite the diversity of genetic models for the inheritance of Hir-

schsprung disease (HSCR) it is clear that it is multifactorial with a

host of genes affecting its pathophysiology. Three features are evi-

dent: (1) The necessity of RET mutations: I shall describe studies

from the International HSCR consortium that demonstrates the RET

effect in all forms of HSCR, the variation in this effect based on

gender, familiality and segment length of aganglionosis and the

complemetary genetic properties of rare coding versus common non-

coding (enhancer) mutations. (2) The existence of many common

polymorphisms in HSCR: recent studies from Paul Tam’s group and

the International Consortium suggests that a common non-coding

variant in Neuregulin 1 (NRG1) and within the Semaphorin 3 cluster

(likely SEMA 3A) affect HSCR. Since statistical power for detecting

these effects were low this suggests many other such undetected

polymorphic factors. (3) Structural abnormalities and copy number

variants impact HSCR: I shall describe new studies from our group

and the International Consortium that show novel dosage abnor-

malities in HSCR including those for the SEMA3A co-receptor

PlexinA2. From a variety of genetic experiments over 70 genes can be

implicated in HSCR and even more discovered. However, most of

them have not been directly tested structurally or functionally. It is

important to create an extensive resource of samples from patients

and their families to test genes, unravel pathophysiology and provide

improved genetic testing to individuals.

Genome-wide association study identifies NRG1 as a susceptibility

locus for Hirschsprung’s disease

pk h tam,* csm tang,� esw ngan,* vch lui,* y chen,* mt so,* tyy leon,*

xp miao,*,� cky shum,* fq liu,* my yeung,� zw yuan,§ wh guo,– l liu,�xb sun,** lm huang,�� jf tou,�� yq song,§§ d chan,§§ kmc cheung,––

kky wong,* ss cherny,�,*** pc sham�,*** & mm garcia-barcelo**Department of Surgery, The University of Hong Kong, Hong Kong, China;

�Department of Psychiatry, The University of Hong Kong, Hong Kong, China;

�Department of Surgery, Shenzhen Children’s Hospital, Shenzhen, China;

§Department of Paediatric Surgery, Shengjing Hospital, China Medical Univer-

sity, Shenyang, China; –Department of Surgery, Beijing Children’s Hospital,

Beijing, China; **Department of Pediatric Surgery, Shandong Medical University,

Shandong, China; ��Department of Surgery, Beijing University, Beijing, China;

��Department of Surgery, Zhejiang Children’s Hospital, Zhejiang, China;

§§Department of Biochemistry, The University of Hong Kong, Hong Kong, China;

––Department of Orthopaedics and Traumatology of the Li Ka Shing Faculty of

Medicine, The University of Hong Kong, Hong Kong, China; and ***Genome

Research Centre. The University of Hong Kong, Hong Kong, China

Background and objectives: Hirschsprung’s disease (HSCR), or

aganglionic megacolon, is a congenital disorder characterized by the

absence of enteric ganglia in variable portions of the distal intestine.

RET is a well-established susceptibility locus, although existing

evidence strongly suggests additional loci contributing to sporadic

HSCR. To identify these additional genetic loci, we carried out

a genome-wide association study using the Affymetrix 500K marker

set. Methods: We genotyped 4 93 840 single-nucleotide polymor-

phisms (SNPs) in 200 Chinese subjects with sporadic HSCR and 306

ethnically matched control subjects. The SNPs most associated with

HSCR were genotyped in an independent set of 190 HSCR and 510

control subjects. Results: Aside from SNPs in RET, the strongest

overall associations were found for two SNPs located in intron 1 of the

neuregulin1 gene (NRG1) on 8p12, with rs16879552 and rs7835688

yielding odds ratios of 1.68 [CI 95%:(1.40, 2.00), P = 1.80 · 10–8] and

1.98 [CI 95%:(1.59, 2.47), P = 1.12 · 10–9], respectively, for the

heterozygous risk genotypes under an additive model. There was also a

significant interaction between RET and NRG1 (P = 0.0095),

increasing the odds ratio 2.3-fold to 19.53 for the RET rs2435357 risk

genotype (TT) in the presence of the NRG1 rs7835688 hetero-

zygote. Conclusions: Our highly significant association findings are

backed-up by the important role of NRG1 as regulator of the devel-

opment of the enteric ganglia precursors. The identification of NRG1

as a new HSCR susceptibility locus not only opens new fields of

investigation into the mechanisms underlying the HSCR pathology,

but also the mechanisms by which a discrete number of loci interact

with each other to cause disease.

A Sox10-Histone2BVenus BAC transgene enables imaging and

isolation of multipotent enteric neural crest-derived progenitors

jc corpening, va cantrell, s byers, kk deal & em southard-smith

Departments of Medicine and Cell & Developmental Biology, Vanderbilt

University, Nashville, TN, USA

Sox10 is a neural crest (NC) transcription factor required for develop-

ment of enteric ganglia and other NC derivatives. Current methods of

visualizing Sox10+ lineages during NC development rely either on

antibodies or histochemical reporters. These approaches restrict anal-

ysis to fixed tissues and are problematic due to cross-reactivity

between Sox gene family members. To enable dynamic imaging and

isolation of viable enteric progenitors (EPs) that express Sox10, we

generated mouse transgenic lines that drive expression of a His-

tone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. The

Sox10-H2BVenus transgene construct relied on modification of a BAC

spanning a 218 kb interval at the Sox10 locus that has been shown to

contain elements necessary to recapitulate Sox10 expression in vivo.

This strategy does not alter the endogenous Sox10 locus and thus

facilitates studies of normal NC-derived progenitors in enteric nervous

system development. The H2BVenus moiety also allows clear dis-

crimination of individual cells as a consequence of nuclear–localized

fluorescence. Expression of our Sox10-H2BVenus BAC transgene

exhibits appropriate temporal and spatial patterns of Sox10 expression.

H2B reporter expression illuminates migration of individual Sox10+

progenitors as they leave the neural tube, traffic through vagal and

sacral pathways and populate the intestine. Immunohistochemical

co-localization of transgene expression with lineage markers demon-

strates appropriate cell-type specific restriction during development

and reveals maintenance of Sox10-H2BVenus in mature enteric glial

cells. H2BVenus+ EPs are readily isolated by flow cytometry and

exhibit the ability to give rise to multi-potent colonies in clonal

cultures. These EPs also readily form neurospheres and are capable of

self-renewal in vitro. Interestingly, differential levels of reporter

expression are evident by both confocal microscopy and flow cytom-

etry due to down regulation of the transgene among distinct popula-

tions as lineage divergence of enteric populations occurs. Analysis of

gene expression in purified populations of enteric Sox10-H2BVenus+

cells identifies transcription of multiple stem cell genes, some of

which have previously not been recognized in enteric lineages. The

ability to image, isolate and compare expression between NC lineages

based on Sox10-H2BVenus trangene expression opens multiple options

for investigating distinct lineages and the effects of mutant alleles

during NC development.

Neural stem cell transplantation in the enteric nervous system:

roadmaps and roadblocks

pj pasricha

Stanford University, Stanford, CA, USA

The use of neural stem cells for the restoration of function in the

aganglionic gut is becoming increasingly feasible with several experi-

mental studies showing that CNS- or ENS-derived neural stem cells

(NSC) can be successfully transplanted into the gut and are capable of

modulating neuromuscular activity. However, in order for the promise

of cell replacement therapy to become a clinical reality several

important questions remain to be answered, including: (i) does func-

tional restoration require faithful recreation of myenteric ganglia and

related structures? (ii) what is the ideal stem cell source for trans-

plantation? (iii) what is the most appropriate route of stem cell

administration? (iv) what is the best approach (including in vitro

preparation and post-transplantation manipulation) to achieve an

appropriate, functional, and long-lasting integration of transplanted

stem cells into the host tissue? (v) what should the first clinical targets

be? This review will suggest possible pathways to clinical trials as well

as highlight the many gaps in our knowledge.

Abstracts

� 2009 Blackwell Publishing Ltd xxvii

Role of Rac GTPases in the ENS network formation and patterning

v sasselli, s bogni & v pachnis

National Institute for Medical Research, Mill Hill, London, UK

The functional development of the enteric nervous system (ENS)

requires newly generated neurons and their progenitors to migrate to

their appropriate sites, extend neurites and dendrites to suitable loca-

tions and, finally, establish synaptic connections with the appropriate

targets. Very little is known about the molecular mechanism under-

lying these processes, however recent evidence points towards a

potential role of Rho GTPases as key components in ENS patterning

and circuitry formation. Our current study addresses the in vivo role

and possible genetic interactions of two members of the Rho GTPase

family, Rac1 and Rac3, in enteric neurogenesis. Taking advantage of

the Cre/loxP recombination system and a Rac1 conditional inactiva-

tion mouse strain (Rac1flox/flox), we generated a Sox10Cre; Rac1flox/

flox; R26StopYFP mouse line, where Rac1 gene is specifically ablated

in the neural crest population, while Cre recombinase activity is

monitored by YFP fluorescence. Secondly, we generated double

Rac1Rac3 mutant animals by crossing the Sox10Cre; Rac1flox/flox;

R26StopYFP mouse line to a constitutive Rac3 KO strain (Rac3-/-).

Rac3-/- mice are fertile, survive to adulthood and do not show any

apparent ENS phenotype. In this analysis we therefore compared single

Rac1 mutants and double Rac1Rac3 mutants for possible genetic

interaction between the two loci. Rac1 and Rac3 are expressed in

enteric neural crest cells (ENCCs) during development. Genetic abla-

tion of Rac1 results in a delayed migration of ENCCs detectable from

embryonic day (E) 11.5 and maintained at least until E15.5, when Rac1

deficiency causes embryonic lethality. This delay in migration could

not be attributed to impaired proliferation or survival of progenitor

cells. Nevertheless, Rac1 mutant ENCCs exhibited a clear deficit in

migration using in vitro assays, and shorter neurite length both in vitro

and in vivo. Interestingly, Rac1Rac3 mutants showed no additional

migratory defects compared to single Rac1 mutants, but axons of

enteric neurons had an abnormal projection pattern at E11.5 and E12.5.

At these stages longitudinally oriented tracts become obvious, espe-

cially at the migratory wavefront. In Rac1Rac3 mutant guts, single

processes, or bundles of them, seem to have lost their normal longi-

tudinal and caudal projection pattern and they now project either

circumferentially or randomly. Ongoing analysis is addressing the

migratory behaviour and morphological features of ENCCs and enteric

neurons in Rac1 and Rac1Rac3 mutants, which might help to dissect

the specific role of the Rac subfamily of Rho GTPases on enteric

neurogenesis.

Enteric nervous system: too complex to understand?

v pachnis

National Institute for Medical Research, Mill Hill, London, UK

The enteric nervous system (ENS) is one of the most complex parts of

the nervous system in vertebrates. The complexity of the ENS is due to

the vast number of neurons and glial cells it contains, the large number

of distinct neuronal subtypes and the difficulty to predict the position

or axonal projection of a given neuron within the enteric ganglia. As

the mechanisms underlying the development of the ENS become

clearer, a new challenge emerges, namely the understanding of the

logic governing the formation of a highly complex and integrated

neuronal network from a relatively small number of undifferentiated

neural crest progenitors. I will report on studies which address the

dynamic regulation of enteric neurogenesis and gliogenesis at prenatal

and postnatal stages and discuss experiments that identify genetic

regulators of axonal pathfinding during ENS development.

Temporal regulation of neurogenesis in the enteric nervous system

catia laranjeira,* nicoletta kessaris� & vassilis pachnis**Division of Molecular Neurobiology, MRC National Institute for Medical

Research, London, UK; and �The Wolfson Institute, UCL, London, UK

The enteric nervous system (ENS) is a complex network of neurons

and glia within the gut wall which originate from neural crest cells.

Recently, a number of cell culture studies have shown that self-

renewing multipotential progenitors of the ENS can be isolated from

the gut of foetal mice and rats as well as from newborn and adult

animals. Despite these studies, the identity of the multilineage pro-

genitor of the ENS and the regulation of its neurogenic potential in

vivo, are currently unknown. Sox10 is an HMG-containing transcrip-

tional regulator which is expressed in progenitors of the ENS and

in enteric glia but is absent from enteric neurons. To establish whether

Sox10 is expressed in multipotential progenitors of the ENS in vivo, we

have combined the Sox10-Cre transgene with the R26ReYFP reporter

allele to lineally mark the progeny of Sox10-expressing cells. Our

analysis shows that in adult animals both the Sox10- neurons and

Sox10+ glia cells are derived from a common pool of Sox10-expressing

progenitors. To examine the temporal regulation of the neurogenic

potential of Sox10+ ENS progenitors, we generated the Sox10iCreERT2

transgenic line, in which expression of a tamoxifen inducible Cre

recombinase is under the control of the Sox10 promoter. Analysis of

Sox10iCreERT2; R26ReYFP double transgenics exposed to tamoxifen

at different time points showed that the neurogenic potential of Sox10-

expressing progenitor cells decreases progressively during embryogen-

esis and is undetectable at some point between P30 and P84. These

findings raise the question of the origin of multilineage ENS progeni-

tors isolated from cultures of postneurogenic gut. To address the

possibility that such progenitors originate from Sox10-expressing glial

cells, we cultured dissociated myenteric plexus of Sox10iCreERT2,

R26ReYFP double transgenics exposed to tamoxifen at p84. In such

cultures, glial cells proliferate, and at least a subset of them can give

rise to nNos+, VIP+ and NPY+ neurons. Similar results were obtained

using the hGFAPCreERT2; R26ReYFP transgenic mice. Taken

together, our data suggest that, although Sox10-expressing cells in the

ENS of adult animals loose their neurogenic capacity in vivo, they can,

under certain conditions be activated to generate self renewing,

multipotential progenitors.

Ultrasound-guided grafting of ENS progenitor cells into murine

embryonic gut in vivok sandgren & v pachnis

National Institute for Medical Research, Division of Molecular Neurobiology, The

Ridgeway, Mill Hill, London, UK

Hirschsprung’s Disease (HSCR) is a congenital disorder in which

enteric neurones are absent from varying lengths of the distal bowel.

The defect causes tonic contraction of the affected bowel segment

resulting in severe intestinal obstruction. Current treatment is surgi-

cal resection of the aganglionic segment. Transplantation of ENS or

other neural progenitors into aganglionic or abnormally innervated

bowel to restore ENS function has been suggested by several groups.

Aim of this study is to examine the feasibility of ENS progenitor cell

(EPC) transplantation as a means of rescuing the neuronal deficit in

HSCR. Microinjection of EPCs into isolated embryonic guts or into

whole embryos kept in organotypic cultures in vitro is a valuable tool

to study the ability of grafted cells to survive, proliferate, migrate, and

differentiate and also their capacity to colonize both wild-type and

aganglionic gut. In the present study, we developed a new method for

specifically targeting the gut in mouse embryos in vivo using an

ultrasound-based injection system. Reasons for establishing this sys-

tem are that we want to follow the fate of grafted EPCs within

embryonic gut in vivo providing a �niche� for neural crest stem cells.

Further, when considering postnatal grafting many important muta-

tions that mimic HSCR will die within a few days to a couple of weeks

after birth. Therefore transplantation into embryonic gut in vivo in

mutants with congenital aganglionosis prolongs the time window for

evaluating the potential of grafted EPCs to colonize aganglionic gut. In

this study EPCs were isolated from embryonic guts of the double

transgenic TgWnt1Cre/R26YFPStop mice and grafted into embryonic wild-

type intestine using ultrasound backscatter bio-microscopy. The

transplanted guts were harvested after 2–8 days, and evaluation was by

immunostaining for green fluorescent protein (GFP) and the neuronal

marker TUJ1. The preparations were analysed using epifluorescence

and confocal microscopy. Murine embryonic gut could be visualized

and EPCs grafted from embryonic stage (E) 10.5 onwards. Grafted cells

could be detected in approx. 50–70% of the transplanted guts as shown

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxxviii

by immunostaining for GFP. The overall survival of the embryos

varied between 73% and 84%. The successful outcome of such

experiments will provide information regarding molecular and cellular

mechanisms that control the migration and differentiation of neural

crest cells and thereby improve our understanding of diseases affecting

the ENS, and help in the search for new treatment modalities.

Effects of genetic background on ENS development in the Sox10Dom

model of Hirschsprung disease

lc walters, va cantrell & em southard-smith

Departments of Medicine and Cell & Developmental Biology, Vanderbilt

University, Nashville, TN, USA

Abnormalities in development of enteric neural progenitors (ENPs) can

lead to aganglionosis in a variable portion of the distal intestine,

causing Hirschsprung disease (HSCR). Cumulative evidence suggests

that variation in HSCR is the consequence of gene interactions that

modulate the ability of ENPs to populate the developing gut. Sox10 is

an essential gene for enteric ganglion development. Sox10Dom mice on

a mixed genetic background exhibit variable aganglionosis. We have

established congenic lines of Sox10Dom mice on distinct inbred genetic

backgrounds, C57BL/6J (B6) and C3HeB/FeJ (C3Fe). These lines differ

in penetrance and extent of aganglionosis. To define the impact of

genetic background on processes during enteric nervous system

ontogeny, we assayed these congenic lines for differences in migration,

lineage potential and proliferative capacity of ENPs. We analyzed ENP

migration in E12.5 guts by whole-mount immunohistochemistry. Both

strains of Sox10Dom mice displayed deficits in migration and decreased

density of enteric NC. However, the phenotype of B6 Sox10Dom

embryos was more pronounced with approximately half the cell

density and one third the migration as their wild-type littermates.

Differences in developmental potential were assayed by isolating

enteric NC stem cells (eNCSC) from Sox10Dom congenic lines by flow

cytometry. eNCSC were purified on the basis of p75/HNK-1

immunolabeling and cultured at clonal density. Immunohistochem-

istry was applied to identify cell lineages within the resulting colonies.

We observed significant differences in lineage potential, capacity for

multipotency, and total number of viable colonies between the strains

and genotypic classes. In particular, eNCSC from B6 guts have a

greater potential to develop a glial fate, while those from C3Fe guts are

more inclined toward a neuronal fate. We analysed proliferative

capacity by immunohistochemistry on dissociated E12.5 ENPs. The

Sox10Dom mutation had no effect on proliferation in either strain,

regardless of the gut region analysed. Our analysis indicates that the

deficiencies of enteric NC development leading to aganglionosis are

not strictly due to migration defects. The significant differences in

developmental potential between the Sox10Dom congenic lines suggest

that abnormalities in lineage determination events within ENPs can

contribute significantly to aganglionosis. The study of the Sox10Dom

congenic lines is valuable for understanding mechanisms that

contribute to variation in HSCR disease phenotype.

Enteric nervous system developmental potential of vagal and sacral

neural crest cells

aj burns

Neural Development Unit, UCL Institute of Child Health, London, UK

Neural crest cells (NCC) are a transient population of multipotent

cells that migrate extensively throughout the embryo and give rise to a

wide variety of cell types including the neurons and glial cells that

comprise the enteric nervous system (ENS), the intrinsic innervation

of the gastrointestinal tract. The ENS is specifically formed from vagal

and sacral neural crest-derived precursors. Although vagal NCC

(adjacent to somites 1–7) give rise to most of the ENS along the entire

gut, sub-regions of vagal crest have intrinsic differences in their ability

to colonize the gut, as evidenced by neural crest ablation and hetero-

topic transplantation experiments. In addition, our recent studies have

demonstrated that a subpopulation of these �enteric� vagal NCC leaves

the gut and migrates into the developing lungs where they form

intrinsic neural ganglia that innervate airway smooth muscle. Sacral

NCC, which normally contribute neural cells to the postumbilical gut

only, are much less invasive of the gut than vagal NCC, a property

likely due to their lower levels of RET expression. The aim of my

laboratory is to better understand the molecular basis of the differences

in developmental potential of vagal and sacral-derived ENS precursors

using quail-chick chimeric grafting, gene electroporation, DNA

microarray analysis and proteomics approaches. Data suggest that

intrinsic differences, such as varying levels of gene expression, differ-

ent capacity for proliferation, and cell death within the precursor

population, may account for the variation in ENS developmental po-

tential of different NCC.

ENS stem cells: practical problems, practical solutions

n thapar

Gastroenterology and Neural Development Units, UCL Institute of Child Health,

30 Guilford Street, London, UK

Many congenital and acquired ENS disorders are characterized by

severe gut dysmotility. This translates to a life-time of recurrent epi-

sodes of intestinal obstruction and management of complications,

which mainly relate to sepsis and aspects of parenteral nutrition on

which many patients are reliant for their survival. Even Hirschsprung’s

disease, with its long established surgical therapy, carries a question-

able long-term prognosis irrespective of the length of gut agangliono-

sis. Therefore, not surprisingly, the last few years has seen tremendous

progress in the field of ENS stem cell biology and therapeutics. Our

group and many others have reported successes in the isolation of ENS

stem cells including from human postnatal gut and their transplanta-

tion into models of aganglionic gut with emerging evidence of func-

tional rescue. Yet there is no doubting the many enormous challenges

of the task in hand, such as scaling up to human gut, identification of

the optimal source of stem cells and their harvesting from tissue,

overcoming problems of defective allogeneic stem cells, unwelcome

environments of recipient gut and limited colonization capacity of

transplanted cells, and whether true functional rescue can be effected.

More fundamental questions include what diseases should be consid-

ered for ENS stem cell transplantation? Perhaps Hirschsprung’s disease

is not the best target for ENS stem cell treatments. The outcome of

short segment disease is arguably adequate and the prospect of colo-

nizing the gut in long-segment disease perhaps unrealistic. Further-

more, in addition to aganglionosis, Hirschsprung’s gut is characterized

by significant hypertrophy of presumed extrinsic innervation; can the

functional effects of this ever be overcome? Clearly many of these

issues need addressing before cell therapy for ENS disorders can be

applied in the clinical arena. Maybe all is not so bleak. The structurally

�hypoganglionic� gut seen with aging is not associated with functional

failure giving hope that restitution of a complete, normal ENS is per-

haps not needed. Gene therapy is routinely used in clinical therapies

and the genetic rescue of defective ENS stem cells derived from murine

models of Hirschsprung’s disease has been shown to be possible. Tis-

sue transplantation and its immunological management is well

established and unlikely to be a major obstacle. Our recent work

addressing practicalities of harvesting cells for therapy has shown that

minimally invasive procedures such as endoscopy can be used to iso-

late ENS stem cells from a regenerating source of intestinal tissue and

ultimately deliver them back into gut. Transplantation of such cells

into models of aganglionic gut has demonstrated they are capable of

colonizing gut and generating components of the ENS.

Abstracts

� 2009 Blackwell Publishing Ltd xxix

Hirschsprung’s disease associated enterocolitis is related to defects

in innate immunity

n alexander,*,� s eaton,* m haston,� as wallace,� vv smith,–

aj burns,§ j salomon,�,** n shah,� nj klein,� a pierro* & n thapar�*Academic Unit of Paediatric Surgery, UCL Institute of Child Health, London,

UK; �Infectious Disease and Microbiology Unit, UCL Institute of Child Health,

London, UK; �Gastroenterology Unit, UCL Institute of Child Health, London,

UK; §Neural Development Unit, UCL Institute of Child Health, London, UK;

–Histopathology Department, Great Ormond Street Hospital, London, UK; and

**current address: Hopital Necker-Enfants Malades, 75015 Paris, France

Background and aims: Patients with Hirschsprung’s disease (HSCR)

frequently suffer HSCR-associated enterocolitis (HAEC) from very

early in life. The etiology and nature of HAEC remain unclear and it is

most commonly diagnosed on clinical criteria including abdominal

distension, diarrhoea (±blood), vomiting and fever. There is evidence of

an overlap between the genetics of ENS development and gut associ-

ated immunity.1 The aims of this study were to evaluate the preva-

lence of HAEC and whether it is associated with defects in innate

immunity. Methods: The study consisted of a retrospective review of

gut histology of HSCR patients presenting over a 15-year period and a

prospective study examining the relationship between the diagnosis

and clinical spectrum of HAEC and innate immunity in HSCR

patients. No genotyping of the HSCR patients was carried

out. Results: Gut inflammation, present from early life, was evident

in more than 30% of all HSCR patients. HSCR patients had signifi-

cantly reduced monocyte MHC class II expression compared with

controls and those with clinical HAEC had a plasma mannose binding

lectin (MBL) deficiency in addition to the MHC class II defect. The

MBL deficiency was strongly correlated to recognized mutations in the

MBL gene. Conclusions: It is likely that HSCR is associated with

inherent immunodysregulation, resulting in a spectrum of clinical

disease states. These come under the umbrella label of HAEC and

range from local gut inflammation to more generalized clinical man-

ifestations of immune deficiency and its complications. Although the

exact aetiology of HAEC is unclear the novel finding of immunopa-

ralysis provides evidence of an underlying immune disorder. Impor-

tantly, additional defects in innate immunity, namely MBL deficiency

appear to predispose individuals to develop systemic and potentially

more severe clinical disease. It is unknown whether the immune de-

fects relate to abnormalities in gut-associated immunity.

Reference:

1 Veiga-Fernandes H, Coles MC, Foster KE, Patel A, Williams A,

Natarajan D, Barlow A, Pachnis V, Kioussis D. Tyrosine kinase

receptor RET is a key regulator of Peyer’s patch organogenesis.

Nature 2007; 446(7135):547–51.

Abstracts Neurogastroenterology and Motility

� 2009 Blackwell Publishing Ltdxxx