A Nematode Neuron Identity Crisis Bruce Wightman Muhlenberg College

A Nematode Neuron Identity Crisis

Bruce WightmanMuhlenberg College

The nervous system is

comprised of many different

types of neurons

Homeodomain transcription factors regulate neuron identity in the

vertebrate spinal cord

The nematode C. elegans

• DNA Sequence known

• Rich genetics

• Simple nervous system

• Defined anatomy

The C. elegans nervous system

Axon anatomy can be examined by neuron-specific

promoter :: GFP fusions

Aequorea victoria

GFPNematodeneuron-specificpromoter

The AVK axons can be visualized with an flp-1::gfp transgene

AVKL

GFPflp-1

(Courtesy of C. Li)

The fax-1 gene is requiredfor AVK axon pathfinding

AVKL

Wild Type

AVKL

fax-1 (gm83)

fax-1 is also required for normallevels of flp-1::gfp expression

Wild-Type fax-1(gm83)

AVKLAVKR

AVKLAVKR

The flp-1 gene encodes a protein precursor for FMRFamide-like

peptide neurotransmitter

N C

SADPNFLRFSQPNFLRF

ASGDPNFLRFSDPNFLRF

AAADPNFLRFPNFLRF

AGSDPNFLRF Post-synapticneuron

Pre-synapticneuron

flp-1

FMRFamide is reduced in AVK neurons of fax-1 mutants

anti-FMRFamide immunostaining

fax-1 functions in both axon pathfinding and neuro-transmitter expression

FAX-1

FMRFamide

axon pathfinding

fax-1 encodes a protein that is a member of the nuclear hormone receptor family

N CDBD LBD

downstreamgene

NHR’s regulate transcriptionand bind lipophilic hormones

The FAX-1 DBD containstwo zinc- fingers

Zn2+

C C

CC

C C

CCZn2+

(J. Much)

FAX-1 is a member of a subclass of NHR’s that function in

nervous system development

Estrogen ReceptorRetinoic Acid Receptor

NHR-111TLX*

NHR-67

TLL*

FAX-1*

PNR*C. elegansDrosophilaH. sapiens

* functionsin nervoussystem

Mutations in human PNR cause defects in photoreceptor identity

Normal retina Enhanced S-conesyndrome retina

PNR is also known as NR2E3(J. Tanis)

fax-1 may regulate transcription of genes involved in speci-

fication of neuron identity

pathfindinggene

flp-1

Other neuron-specific gene

Examining FAX-1 expression using antibodies and immunofluroescence

staining

Anti-FAX-1 antibodies Immunofluorescencestaining

(N. Carmean)(B. Ebert)

fax-1 is expressed in 18 neurons including the AVKs

mid-stage embryo

adult

AVKL

AVKR

AVKAIY

RICDVA

Fax-1 Phenotypes:Unc, Che, Fab, Wandering

AVB AVAAVE MI

RICR AVBRAVARAVER

fax-1 may regulate transcription of genes involved in speci-

fication of AVK neuron identity

AVK path-finding gene

flp-1

Other AVK-specific gene

ncs-1::gfp expression in AVK requires fax-1

neuron-specific calcium sensor

wild-type ncs-1::gfp fax-1 (gm83) ncs-1::gfp

AVKL

AVKR

(Gomez, et al., 2001)

Summary of gene expression requirements

AVK

Requirefax-1

flp-1ncs-1

Cells:

What is the function of fax-1 in neurons other than the AVK’s?

DVA

AVKAIYRIC

AVBAVAAVEMI

The RIC neurons have normal anatomy and expression of tbh-1 in

fax-1 mutants

tyrosine -hydroxylase (for octopamine biosynthesis)

wild-type and fax-1 (gm83)

(M. Alkema and H.R. Horvitz, unpublished)

RIC

Expression of NMDA glutamate receptor subunits in AVA and AVE

requires fax-1

AVB

fax-1 (gm83) nmr-1::gfp

RIM

AVA/EAVB

RIM

AVG

AVBRIM

AVG

wild-type nmr-1::gfp

AVBRIMAVA

AVE

Both nmr-1 and nmr-2 show the same pattern

Expression of non-NMDA glutamate receptor subunits

does not require fax-1

AVA and AVE axons appear normal in fax-1 mutants:fax-1 not required for axon pathfinding in AVA/E

wild-type glr-1::gfp fax-1 (gm83) glr-1::gfp

AVA/EAVA/E

Summary of gene expression requirements

AVK AVA/E

Requirefax-1

flp-1ncs-1

nmr-1nmr-2

Cells:

Which genes may functionupstream of fax-1?

The homeodomain gene unc-42 may function upstream of fax-1

fax-1

UNC-42

AVK

• unc-42 is expressed in the AVK, AVA, AVE and other neurons

• unc-42 mutants have AVK path-finding defects that are similar to fax-1 mutant defects

(Wightman et al, 1997;Baran et al., 1999)

fax-1 is not expressed in some neurons of unc-42 mutants

Wild-type

unc-42 (e419)

FAX-1 is absent in AVB and two anterior neurons and reduced in AVK

DVA

AVKAIYRIC

AVBAVAAVEMI

AVA

AVERIC

AVK

wild-type FAX-1 unc-42(e419) FAX-1

AVA/E AVA/E

AVBRIC

AVKAVK

RIC

DVA

unc-42 is also required for flp-1::gfp expression

Percentage of AVK neurons

Express flp-1::gfp fluorescence

Wild-Type 100 fax-1 (gm83) 65 unc-42 (e419) 0 unc-42 (e419);

fax-1 (gm83) 0

(S. Mathieson)

Summary of gene expression requirements

AVK AVA/E

Requirefax-1

Requireunc-42

flp-1ncs-1

fax-1flp-1

nmr-1nmr-2

Cells:

Expression of ncs-1 also depends on unc-42

wild-type ncs-1::gfp unc-42(e419) ncs-1::gfp

AVKL

AVKR

Summary of gene expression requirements

AVK AVA/E

Requirefax-1

Requireunc-42

flp-1ncs-1

fax-1flp-1ncs-1

nmr-1nmr-2

Cells:

What about unc-42 and fax-1 in the AVA and AVE interneurons?

•Expression of non-NMDA glutamate receptor subunits in AVA/E requires unc-42•Expression of NMDA glutamate receptor subunits in AVA/E does NOT require unc-42

(Baran et al., 1999; Brockie et al., 2001)

wild-type unc-42(e419) fax-1(gm83)

nmr-1 + + -nmr-2 + + -glr-1 + - +glr-4 + - NDglr-5 + - +

Axon pathfinding by AVA and/or AVE appears defectivein unc-42 mutants

Summary of gene expression requirements

AVK AVA/E

Requirefax-1

Requireunc-42

flp-1ncs-1

fax-1flp-1ncs-1

nmr-1nmr-2

glr-1glr-4glr-5

Cells:

Models for the specification of neuron identity by unc-42 and fax-1

UNC-42

FAX-1

flp-1

Pathfindinggenes

ncs-1

AVK AVA/E

UNC-42 FAX-1

Pathfindinggenes

glr-1nmr-1

nmr-2glr-4

glr-5

Approaches to identification of FAX-1 downstream targets

(R. Haviland, R. Lombel, E. Murphy, K. Reinert, E. Smith, Y. Vidgop)

Other neuron-specific genes

Is expression of FAX-1 sufficient to change gene expression?

heat-shock promoter fax-1

(N. Bianco, T. Sundaresan)

anti-FAX-1 hs::fax-1; fax-1(gm83)

Participantsfax-1Sepi BazelNick BiancoNicole CarmeanSara CarrBryan EbertDavid GarbeRebecca HavilandChirag KalolaRebecca LombelJason MuchElissa MurphyGwen SarverDennis SladeEric SmithTilak SundaresanKristy Reinert Jessica TanisYelena Vidgop

unc-20Peter AlffJeff DotoBrook KohrtSon NguyenSuchi PandeyDoug PrechtelMatt Stein

Other genesDavid BrightbillAndrea Cerrone

Kelly KlampertSheila Mathieson

Mark Alkema, Cori Bargmann, Edouard DeCastro, Gian Garriga, Oliver Hobert, Christine Li, A.V. Maricq, Ruth Yu