Molecular Genetic Analysis of the Mouse anorexia Mutation

by

Dennis Kim

A thesis submitted in conformity with the requirements

For the degree of Master of Science

Graduate Department of Molecular and Medical Genetics

University of Toronto

© Copyright by Dennis Kim 2008

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Abstract Molecular Genetics of the Mouse anorexia Mutation Dennis Kim

Master of Science 2008

Department of Molecular and Medical Genetics, University of Toronto

The serotonergic system regulates numerous behaviours and disruptions in this system

have been associated with disorders of mood and mind. Although molecular genetic

analysis has dissected many of the genes involved in the specification of the

serotonergic system, relatively little is known about the mechanisms that promote

axonal outgrowth from serotonin-producing neurons and how these projections are

directed to innervate and form synapses with their appropriate targets. The mouse

anorexia mutation causes hypersprouting of serotonergic projections in target fields and

has provided us the unique opportunity to examine the crucial events that lead to the

establishment of these complex serotonergic networks. Through positional cloning, I

have identified a candidate gene that is upregulated during a time in which innervation

and synaptogenesis of serotonergic neurons are maximal. I have assessed the

expression of this candidate gene in the brain and have found striking differences in the

pattern of expression between the normal and the mutant mouse. Furthermore, by

using transgenic methods, I have partially rescued several hallmark behavioural

phenotypes in the mutant mouse. Thus, this candidate almost certainly represents the

“Anorexia” gene.

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Table of Contents Content Page Abstract ii Table of Contents iii List of Figures v List of Tables vi List of Key Abbreviations vii Chapter 1: Introduction 1.1 The serotonergic system 1 1.2 Development of the serotonergic system 1 1.3 The mature mammalian serotonergic system 10 1.4 The mouse anorexia mutation 12 1.5 Aberrations in anx/anx hypothalamic circuitry 13 1.6 Positional cloning and candidate genes of the anx mutation 16 1.7 The Eph receptors and ephrins 17 1.8 The Trk receptors and neurotrophins 19 1.9 The receptor tyrosine kinase Tyro3 22 1.10 Summary 25 Chapter 2: Materials and Methods 2.1 Introduction 27 2.2 Meiotic recombination map 28 2.3 Candidate gene testing i. Brain-derived neurotrophic factor (BDNF) 29 ii. Glycine-amidinotransferase 29 2.4. Sequencing candidate genes 29

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Content Page 2.5 Generation of transgenic animals i. Mouse GFP-tagged normal and R7W-Tyro3 transgenes 30 ii. Human full-length Tyro3 transgene 32 iii. Identifying transgenic founder mice 33 2.6 Expression analysis of Tyro3 in normal and anx/anx mice by 33

RNA in situ hybridization 2.7 Weight and survival data from the progeny of transgenic 35 anx/+ heterozygote intercrosses

Chapter 3: Results 3.1 Introduction 36 3.2 Meiotic recombination mapping and candidate gene sequencing 38

identified a point mutation in the signal sequence of Tyro3 3.3 Tyro3 expression in normal P21 mouse brain 46 3.4 Tyro3 expression is altered in numerous brain regions in anx/anx mice 49 3.5 Generation and identification of transgenic mice 58 3.6 Transgenic anx/anx homozygotes have improved body weight at 61

P21 and live longer than their non-transgenic anx/anx littermates Chapter 4: Discussion 4.1 Signal sequence mutations 68 4.2 Expression analysis 69 4.3 Partial rescue in transgenic mice 71 4.4 Evidence suggestive that Tyro3 is causative of the anx mutation 74 4.5 Possible roles of Tyro3 75 Conclusion 77 References 78

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List of Figures Figure Page 1. Meiotic recombination mapping and sequencing of candidate genes reveal a 40 point mutation in the anorexia critical interval 2. Comparison of signal sequence predictions from SignalP analysis 44 3. Analysis of Tyro3 expression in the cerebral cortex in P21 normal and anx/anx 47 mice by RNA in situ hybridization 4. Analysis of Tyro3 expression in the hippocampus in P21 normal and anx/anx 50 mice by RNA in situ hybridization 5. Analysis of Tyro3 expression in the hypothalamus in P21 normal and anx/anx 52 mice by RNA in situ hybridization 6. Analysis of Tyro3 expression in the cerebellum in P21 normal and anx/anx 54 mice by RNA in situ hybridization 7. Analysis of Tyro3 expression in the brainstem in P21 normal mouse sagittal 56 sections by RNA in situ hybridization 8. Construction of the Tyro3 transgenes for rescue for mimic analyses 59 9. Analysis of bodyweight of transgenic anx/+ progeny at P21 62 10. Analysis of survival rates of transgenic and non-transgenic anx/anx progeny 66

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List of Tables Table Page 1. Genes within the anorexia critical interval 31 2. Percentage bodyweight difference in transgenic mouse lines 64

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List of Key Abbreviations 5-HT Serotonin anx anorexia mutation Arc Arcuate nucleus BDNF Brain-derived neurotrophic factor ME Median eminence P (e.g. P21) Post-natal day (21) PVN Paraventricular nucleus Vmh Ventromedial hypothalamus

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Chapter 1: Introduction

1.1 The serotonergic system

The serotonergic system is of crucial importance for normal functioning of the

developing and mature mammalian nervous system. Aberrations in the serotonergic

system can negatively affect physiological processes, such as sleep rhythms and

appetite regulation, and behaviours, such as anxiety and aggression. Subtle genetic

alterations can have wide-ranging consequences on proper wiring, firing, and

maintenance of serotonergic neurons and their targets. Variations in genes important for

serotonergic neurotransmission, such as the serotonin transporter (5HTT) and

tryptophan hydroxylase 2 (Tph2), are associated with psychiatric disorders, such as

phobic disorders (Furmark et al., 2004) and unipolar depressive disorder (Zhang et al.,

2006), respectively. Thus, improper development and/or maintenance of the

serotonergic system may contribute to disorders of mood and mind. Finally, there is

increasing evidence that serotonin itself plays a neuromodulatory role on neuronal

development along with its classical role as a neurotransmitter. All of these

observations highlight how crucial it is to have the correct amount of serotonin in the

developing nervous system at the right place and time.

1.2 Development of the serotonergic system

Serotonergic (5-HT) neurons are born in two main clusters along the midline of

the embryonic hindbrain, first appearing between embryonic (E) 10.5 -11.5 in the

mouse. Within one day of their birth, 5-HT neurons are capable of synthesizing

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serotonin and project axons toward their intended target areas in order to form

functional serotonergic networks, which, in the rat and mouse, do not fully mature until

after birth (Lidov and Molliver, 1982; Vitalis and Parnavelas, 2003). Once born, 5-HT

neurons migrate along the dorso-ventral axis and, at times, mediolaterally to eventually

form 9 cell groups collectively known as the raphe nuclei, labeled B1 to B9 from the

posterior to the anterior end. These nuclei are distinguished by their pre-migration

origins. The posterior groups, B1-B4, correspond to the raphe pallidus, the raphe

obscurus, the raphe magnus, and the raphe pontis, respectively, and arise from the

posterior hindbrain, while the anterior groups, B5-B9 collectively form the dorsal (B6/B7)

and median (B5/B8/B9) raphe nuclei. The boundaries of the raphe nuclei are diffuse

(Molliver, 1987) and only about one-third of the neurons within the raphe nuclei produce

5-HT (Descarries et al., 1982), thus characterizing the discrete locations of individual

nuclei is more difficult. There is also some debate as to whether the raphe nuclei

produced by the anterior cluster should indeed be considered as 5 distinct groups or

represent 2 larger functional groupings (reviewed in Cordes, 2005).

While our understanding of 5-HT neuron migration into the raphe nuclei is still

nebulous, great strides have been made towards understanding the signals that first

induce 5-HT neurons to be born and the transcriptional hierarchies important for their

specification. Experiments done on hindbrain explants suggest that Sonic hedgehog

(Shh) signaling from the notochord and floorplate in conjunction with FGF4 establish 5-

HT precursor identity (Ye et al., 1998). In line with these observations, the Shh-

downstream transcription factor, Gli2, has also been shown to play a role in 5-HT

induction as Gli2 null mutants exhibit a 50% reduction in 5-HTergic neurons in the

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mouse hindbrain (Matise et al., 1998). Additionally, FGF8, which is present in the

anterior hindbrain prior to exclusive production and secretion from the midbrain-

hindbrain organizing center (MHO), is required for the development of the anterior raphe

nuclei but not the posterior clusters (Ye et al., 1998). Displacement of the MHO either

rostral or caudal to its normal position causes a corresponding displacement of

serotonergic and dopaminergic cell clusters (Brodski et al., 2003), and transgenic mice

bearing a constitutively active form of Smoothened, a Shh receptor, produce 5-HT

precursors dorsal to their normal positions (Hynes et al., 2000). Shh signaling can

therefore establish both antero-posterior and dorsoventral patterning of monoaminergic

neurons in combination with other factors, but already during their earliest development

5-HT neurons in the anterior and posterior clusters are distinguished by their

dependence or independence on FGF8 signaling.

After early induction of 5-HT precursors, the concerted activity of several

transcription factors is required for subsequent position-appropriate differentiation of 5-

HT neurons. Initially, Nkx2.2, Nkx6.1, and Mash1 expression in hindbrain tissues

identify 5-HT precursors, which are followed by Mash1, Gata2, Gata3, Lmx1b, and Pet1

expression to establish 5-HT subtype selection (reviewed in Cordes, 2005). The

homeodomain transcription factor, Nkx2.2, which acts downstream of Shh, is required

for all but the dorsal raphe nuclei neurons to specify a 5-HTergic cell fate (Briscoe et al.,

1999). In the dorsal raphe nuclei, compensatory activity of Nkx2.9 is likely to direct the

5-HT neurochemical phenotype in the absence of Nkx2.2. Nkx2.2 also drives Nkx6.1

expression, which acts in cooperation to direct Gata2 and Gata3 expression required for

5-HT specification. Additionally, Nkx2.2 likely suppresses Phox2b expression, which,

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together with the down-regulation of Nkx2.9, marks the onset of 5-HT neuron

specification.

Differentiation of 5-HT precursors into terminal subtypes is directed by the

proneural helix-loop-helix transcription factor Ascl1/Mash1. Ascl1/Mash1 acts in two

ways; first, Ascl1/Mash1 downregulates Phox2b to generate post-mitotic 5-HT

precursors, leading to its second role in specifying 5-HT neurons (Pattyn et al., 2003).

The actions of Ascl1/Mash1 occur after the Nkx genes have exerted their effects to

induce 5-HT precursors. Ascl1/Mash1 null embryos lack later acting Ets-domain Pet1,

Lim homeodomain-containing Lmx1b, zinc finger Gata2 and Gata3 transcription factors

as well as 5-HT neurons altogether, even though expression of the earlier-acting Nkx

genes and Phox2b are unaltered. The zinc finger transcription factor Gata2 has been

shown to be necessary and sufficient to activate Lmx1b and Pet1 (Craven et al., 2004),

and is likely required for global 5-HT neuron development. Cultured Gata2-/- embryos at

E13 lack Pet1 and 5-HT neurons, even though expression of its companion zinc-finger

transcription factor Gata3 is unchanged. Gata3 acts in a cluster-specific role on

posterior 5-HT precursors to establish 5-HT specification. Gata3-/- mutants have an

80% reduction in neurons within these clusters, which likely acts in parallel with Lmx1b

and Pet1, both of which are unaffected in these mutants.

The Lim homeodomain transcription factor Lmx1b is required for 5-HT

specification through the Ets transcription factor, Pet1, which is lost in Lmx1b-/- embryos

(Ding et al., 2003, Cheng et al., 2003). Interestingly, ectopic 5-HT precursors in the

dorsal and ventral hindbrains of these mice suggest a role for Lmx1b in restricting the

cell migration of 5-HT neurons (Ding et al., 2003). Pet1 is exclusively required for the

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specification and differentiation of most hindbrain 5-HT neurons (Hendricks et al., 1999,

Hendricks et al., 2003). Loss of Pet1 causes a 70% loss of hindbrain 5-HT neurons due

to a failure to differentiate, and the remaining 5-HT neurons have reduced expression in

5-HTergic genes (Hendricks et al., 2003). However, the few Pet1-/- mice that do survive

until adulthood exhibit increased anxiety and aggression, in agreement with the notion

that proper 5-HTergic system development even during these early stages is necessary

for proper functioning in the adult and with the postulated role of 5-HT neurons in

modulating these behaviours.

Having established 5-HT precursor identity, factors that promote proliferation and

survival of 5-HTergic outgrowth are required to initiate the complex cascade leading

towards a proper 5-HT network. Some clues as to which molecules are involved in

these processes have been examined in culture and by using several mouse mutants

specific for a 5-HT axonal phenotype. Brain-derived neurotrophic factor (BDNF) has

been shown to promote the sprouting and survival of serotonergic neurons and their

projections. BDNF exposure of primary cultures of E14 rat rostral raphe nuclei induces

a near 2-fold increase in 5-HTergic neurons and importantly, a dramatic extension of

neurites (Rumajogee et al., 2002). Intracortical infusions of BDNF cause an increase in

axon density of 5-HTergic projections in the neocortex of adult rats, which also acts to

prevent chemically-induced lesioning of these fibers (Mamounas et al., 1995).

Conversely, mice with constitutively reduced levels of BDNF have a concomitant

decrease in 5-HT innervation of the hippocampus at 18 months (Luellen et al., 2007).

Mice heterozygous for a BDNF null allele exhibit increased aggressive behaviour,

hyperphagia, and weight gain; behaviours associated with 5-HT dysfunction, which is in

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part caused by the premature decline in forebrain 5-HT levels and 5-HTergic fiber

density (Lyons et al., 1999). The role of BDNF to promote neuron proliferation and

subsequent axon survival may also, in part, be regulated by the expression of pro-

apoptotic Bax and anti-apoptotic Bcl-2 genes. Damage to neural tissue due to cerebral

ischemia is reduced by pretreatment with BDNF which reduces the number of Bax-

positive neurons and increases the number of Bcl-2-positive neurons at the site of injury

(Schabitz et al., 2000). In double BDNF/Bax null mutant mice, cells of cranial sensory

neurons which are lost in BDNF null mice are completely rescued (Hellard et al., 2004).

However, these mutants are eventually overcome by the lethality of the BDNF null

phenotype due to a failure to innervate target tissues despite successful navigation to

their terminal fields. Whether 5-HT neurons cannot innervate their targets in these

animals has not been examined, but seems possible if not likely. Taken together, these

results suggest that BDNF acting on central neuronal populations for survival and

outgrowth of projections may converge with locally-acting BDNF required for in-growth

into target tissues. BDNF-sensitive neuronal populations, such as 5-HT neurons, may

therefore respond to such signals at both ends to provide trophic support and establish

functional synaptic connections.

Subsequent to adopting the 5-HT neuronal phenotype and expressing the

necessary genes thereafter, correct axonal guidance from the raphe nuclei to their

target fields is required to establish a functional 5-HT network. These complex

processes towards proper innervation are temporally and spatially regulated

(Parnavelas and Papadopalous, 1989), and though these processes are initiated during

gestation, complete 5-HTergic connectivity is not fully established until the third

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postnatal week as evidenced in the visual and primary somatosensory cortex in the rat

(Dori et al., 1996). While at times in immunostaining experiments, the wide-spread and

complex 5-HT projections may appear to be in a somewhat chaotic disarray, their

trajectories are far from haphazard, and axonal tracing experiments as well as chemical

lesioning have identified distinct areas innervated by specific raphe nuclei. The majority

of axonal projections from the dorsal and median raphe nuclei that constitute the

anterior group of 5-HT neurons ascend through the median forebrain bundle, but

diverge to innervate non-overlapping and complementary regions of the rat forebrain as

visualized by the anatomical tracer Phaseolus vulgaris-leucoagglutinin (Vertes, 1991,

Vertes et al., 1999). These axons also differ in their axonal morphologies. The axons

projecting from the dorsal raphe nuclei appear fine, and are beaded with tiny,

pleiomorphic varicosities unlike median raphe nuclei axons which have coarse

varicosities (Descarries et al., 1975, Maley et al., 1990). Furthermore, within these

anterior clusters, only dorsal raphe nuclei neurons are sensitive to treatment with such

psychotropic drugs as MDMA and PCA (Mamounas and Molliver, 1988; Molliver et al.,

1989). Furthermore, it has been shown that a small population of 5-HT neurons

projecting from B8 can branch out and innervate two separate areas (Kohler et al.,

1982, Kohler and Steinbusch, 1982). Hence, a single 5-HTergic axon can

simultaneously act on multiple and distinct areas of the brain, and this increases the

complexity of axon guidance for 5-HT neurons, since a single neuron must coordinate

the targeting of two different processes through territories that express distinct guidance

cues.

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There appear to be general axonal repulsive cues that affect both ascending and

descending axonal projections, including 5-HTergic projections, mediated by the Slit

proteins (Bagri et al., 2002). In Slit2 mutants, 5-HTergic fibers are displaced ventrally

through the diencephalon, while in Slit1;Slit2 double mutants, a number of 5-HTergic

fibers descend into the hypothalamus, and abnormally cross the midline in the basal

telencephalon. Thus, these repulsive cues act to maintain the dorsal position of these

pathways by inhibiting growth into ventral regions and into the midline, and to correctly

direct axons to target forebrain regions. However, the molecules involved to direct 5-

HTergic axonal growth and target specificity remain largely unknown. In a series of

explant and co-culture experiments, Petit et al. (2005) have begun to unravel some of

the properties of these guidance cues and repulsive molecules. Serotonergic axons

project from the dorsal raphe in a contact-dependent manner mediated by a GPI-linked

membrane protein. As these axons project into either ventral midbrain or striatal

explants, they adopt a preference for explants or membranes derived from the same

brain region by the activity of a membrane-bound molecule in these tissues, since this

preference is disrupted in the presence of high potassium chloride concentration.

Hence, target pathfinding of 5-HT projections is a multistep process in which broad

fields of 5-HTergic innervation are sequentially refined. When primary axons traverse

into target areas, they adopt a preference for innervating that specific target tissue and

divergent projections are inhibited from branching into and innervating alternate regions.

However, the exact molecules and pathways that allow 5-HT axons to acquire this

target-induced growth-inhibitory response are yet to be identified.

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The thalamocortical tract, a major ascending axonal pathway in the mammalian

brain, is modulated by 5-HT itself, and the overlapping expression of 5-HT1B and 5-HT1D

receptor transcripts with the axon guidance receptors Deleted in Colorectal Cancer

(DCC) and Unc5c interact to change attractive and repulsive cues in response to Netrin-

1 (Bonnin et al., 2007). The 5-HT1B and 5-HT1D are Gi/o-coupled receptors and upon

activation inhibit adenylate cyclase, thereby decreasing intracellular levels of cAMP

(Raymond et al., 2001). Using E14.5 day explant cultures, the attraction of posterior

dorsal thalamus axons to Netrin-1 is converted into a repulsive signal in the presence of

5-HT, which is dependent on the 5HT1B/1D receptor mechanism controlling camp

(Bonnin et al., 2007). The modulatory effects of 5-HT were also evident via in utero

electroporation of siRNAs to reduce the expression of 5-HT1B/1D at E12.5. Dorsal

thalamus axons expand ventrolaterally by E18.5, and in contrast, overexpression of

these 5-HT receptors has the exact opposite growth pattern, in which thalamic axons

are more restricted dorsomedially in the palladium. These results indicate that classic

neurotransmitters can play neuromodulatory roles, especially in the thalamocortical tract

which is shared by ascending 5-HT projections, and add a further complexity to 5-HT

neurodevelopment and its interrelation with other systems and overall brain

development.

Another poorly understood factor that affects 5-HT connectivity is feedback from

other neurotransmitter systems. This phenomenon is nicely exemplified by studies of

mice homozygous for the brindled-mottled mutation which affects copper metabolism

and thereby inhibits dopamine-beta-hydroxylase (DBH), which uses copper as a

cofactor to convert dopamine to norepinephrine (Martin et al., 1994). In brindled mottled

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homozygotes, 5-HT fibers exhibit heterotypic sprouting in the cerebral cortex. By P14,

axonal fiber density is 70% higher in the mutant than in control animals. Interestingly,

the brindled-mottled mutant highlights the often critical interaction of multiple systems

within the developing and mature nervous system. The only other known spontaneous

mouse mutant to directly affect 5-HT innervation and cause hypersprouting is the

anorexia mutation, which is the basis for the following study and will be discussed in

greater detail.

1.3 The mature mammalian serotonergic system

The mature 5-HTergic system is able to synthesize, release, and regulate 5-HT

between 5-HTergic terminals and target areas. This is accomplished by the coordinated

activity of at least 14 different 5-HT receptor subtypes, the 5-HT transporter (SERT),

and tryptophan hydroxylase 2 (Tph2), the 5-HT synthesizing enzyme, which is more

predominantly expressed in the brain than its companion isoform, Tph1 (Patel et al.,

2004, Sakowski et al., 2006). Several disorders of mood and mind have been traced to

disruptions in the 5-HTergic system, hence, model organisms that have altered 5-

HTergic systems may serve as models of human disease.

Convergent evidence from genetic analyses, animal models, and

pharmacological studies has elucidated the roles of some 5-HTergic genes. Targeted

inactivation of the 5-HT1A receptor in mice causes an increase in anxiety-related

behaviours (Heisler et al., 1998) as well as a decrease in cognitive ability (Sarnyai et al.,

2000). A polymorphism in the promoter region of the same gene in humans has been

positively linked to anxiety and major depression, potentially leading to suicidal

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behaviour (Hong et al., 2006, Kraus et al., 2007). 5-HT2C receptor null mutant mice are

cognitively deficient and exhibit hyperphagia-evoked weight gain (Heisler and Tecott,

1999). Similarly, 2 polymorphisms in the same gene in humans have been linked to

eating disorders, deficits in learning and memory, and the development of psychoses

(Massat et al., 2007, Hu et al., 2003). The serotonin transporter (SERT) and

monoamine oxidase A (MAOA) play roles in recycling and reducing the amount of 5-HT;

the first by uptake from the synaptic cleft into the presynaptic terminal, and the second

by deamination of 5-HT at the outer mitochondrial membrane. SERT null mice fail to

clear 5-HT and therefore, have excess levels of extracellular 5-HT (Holmes et al., 2003).

These mice show increased anxiety-related behaviours, reduced aggression, and

exaggerated stress responses. Interestingly, a human 5-HTT polymorphism has been

linked to anxiety and abnormal stress reactivity potentially leading to depression (Otte et

al., 2007, Munafo et al., 2005). This polymorphism is also linked to alcoholism (Feinn et

al., 2005) suggesting a role for 5-HTT in addictive behaviour, and eating disorders

(Monteleone et al., 2006, Matsushita et al., 2004). The MAOA knockout mouse displays

increased aggression (Popova et al., 2001) and abnormal stress reactivity (Seif and De

Maeyer, 1999, Popova et al., 2006). In humans, a polymorphism in MAOA has been

linked to antisocial behaviour, which can pathologically manifest as antisocial

personality disorder (Eisenberger et al., 2006). So far only single gene associations

have begun to be examined, but, given the substantial success of these limited efforts, it

is not difficult to imagine the vast number of potential direct or indirect interactions

between “serotonergic“ genes and the consequences of variations within them which

may contribute to the spectrum and complexity of psychiatric disorders. Identification of

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such genes and understanding their mechanisms of action will ultimately lead to tailored

and far more effective treatments for affected patients.

1.4 The mouse anorexia mutation

The mouse anorexia (anx) mutation displays a 5-HT-specific aberration in which

normal 5-HT terminal fields are hyper-innervated by 5-HT projections (Son et al., 1994).

At 3 weeks, in mice homozygous for the anx mutation, the density of 5-HT

immunoreactive fibers is greatly increased in the olfactory bulb, the cerebral cortex,

hippocampus, and cerebellum, whereas brain regions not normally innervated by 5-HT

projections remain virtually unaffected. Unlike the brindled mottled phenotype, in which

5-HT hyper-innervation occurs secondary to a DBH defect, anx/anx mice have normal

catecholaminergic innervation and cell body density as determined by tyrosine

hydroxylase immunostaining, and anx/anx mice preserve normal nuclear boundaries as

determined by Cresyl violet staining. A previously unreported phenotype has also been

found in our lab in anx/anx homozygotes and anx/+ heterozygotes at birth. Anx/anx

homozygotes lack any 5-HT in cortical areas suggesting a delay in 5-HT axon guidance,

while anx/+ heterozygotes have an intermediate level relative to normal littermates

(Huynh, unpublished). These data suggest that the effects of the anx mutation occur

during gestation and have a dosage-dependent effect.

The behavioural phenotype of anx/anx homozygotes also reflects the changes in

normal 5-HT innervation in the brains of these animals. Between P15- P18, anx/anx

mice develop body tremors, headweaving, abnormal gait and hyperactivity; behaviours

that can also be elicited by pharmacological stimulation of 5-HT receptors (Maltais et al.,

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1984). Additionally, treating P20 anx/anx mice with the 5-HT antagonist 5,7-

dihydroxytryptamine alleviates the severity of the behavioural phenotype. Hence, the

anx mutation appears to affect the 5-HT system directly.

1.5 Aberrations in anx/anx hypothalamic circuitry

Anx/anx mice are distinguishable from their normal and heterozygous littermates

by P9 by their reduced size and become emaciated in appearance until death by P22

(Maltais et al., 1984). From our observations and previous analyses, mutant mice at

P20 weigh only 30-50% of their unaffected littermates. These mutant mice were

therefore categorized as exhibiting “anorexia” due to their decreased food intake. At

P15, these mutant mice were devoid of fat and glycogen in their livers, in line with a

food intake abnormality. A hypothesis proposed by this group suggested that the

overstimulation of the 5-HT system in anx/anx mice inhibited the suckling response of

newborn pups, ultimately leading to the overall anorexic phenotype leading to death.

Metabolites of the 5-HT were known to be involved with the suckling response and

physiological control of food intake behaviour (Spear and Ristine, 1982), and 5-HT

agonists and antagonist had been shown to modulate this response (Caza and Spear,

1982). However, subsequent studies in our lab of the neural systems involved in

energy balance indicate that the anx mutation affects the circuitry regulating food intake

in the hypothalamus. Whether 5-HT overstimulation or disrupted neural circuitry is the

primary cause will be discussed in later sections.

The hypothalamus is divided into twelve nuclei which are individually sensitive to

numerous physiological cues to regulate homeostasis. The arcuate nucleus, located in

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the ventral region of the hypothalamus, sends projections dorsally and anteriorally to the

paraventricular nucleus (PVN), an area critical to food intake behaviour. The PVN

receives signals from two distinct cell groups from the arcuate nucleus. A subset of

arcuate nucleus neurons produce and secrete Neuropeptide Y (NPY) (Allen et al., 1983,

Chronwall et al., 1985, DeQuidt and Emson, 1986) and Agouti-Related Protein (AgRP)

(Shutter et al., 1997, Hahn et al., 1998), which, when released, stimulate food intake.

Another subset of arcuate nucleus neurons counteracts the effects of these orexigenic

signals. These neurons instead release Pro-Opiomelanocortin (POMC) (Bloch et al.,

1978, Bloom et al., 1978, Watson et al., 1978) and Cocaine- and amphetamine-

regulated transcript (CART) (Elias et al., 1998, Kristensen et al., 1998) deemed

anorexigenic in nature since they act to suppress food intake. Both cell populations

respond to peripheral signals including leptin from adipose tissue (Elias et al., 1999,

Elmquist et al., 1999), insulin and ghrelin (Lawrence et al., 2002, Seoane et al., 2003,

Scott et al., 2007). At low energy states, the release of NPY strongly stimulates feeding

behaviour (Stanley and Leibowitz, 1985). AgRP is co-expressed in the majority of NPY

neurons, which, when co-released, acts as an antagonist to the anorexigenic

melanocortin peptides (Fan et al., 1997, Ollmann et al. 1997). Thus, stimulation of

these neurons heightens food intake by suppressing hypothalamic inhibition of this

behaviour.

In the arcuate nucleus, POMC is further processed to generate

adrenocorticotropic hormone (ACTH) and α-melanocyte stimulating hormone (α -MSH),

which are increased in response to fasting (Brady et al., 1990). Pharmacological

stimulation of the melanocortin receptors to which these hormones bind can also

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suppress food intake (Fan et al., 1997, Grill et al., 1998). CART, which is produced by

these neurons, actively blocks feeding behaviour induced by NPY (Kristensen et al.,

1998, Lambert et al., 1998). However, regulation of food intake is also mediated

between these arcuate nucleus clusters as well. Not only are the projections from these

subsets of neurons linked and run parallel to each other (Zhang et al, 1994, Fuxe et al.,

1997, Broberger et al., 1997b, 1998, Csiffary et al., 1990), but POMC/CART neurons

can receive inputs from neighboring NPY neurons via NPY Y1 and Y5 receptors on their

cell surface (Broberger et al., 1997b, Fuxe et al., 1997), which, when activated,

stimulate food intake behaviour (Gerald et al., 1996, Stanley et al., 1992). Thus,

stimulation of NPY neurons can inhibit melanocortin release by acting on these

receptors enhancing food intake behaviour.

In the anx/anx hypothalamus, NPY is mislocalized to the cell body of arcuate

nucleus neurons as determined by NPY immunostaining (Broberger et al., 1997a,

1998). NPY and AgRP normally reside in the presynaptic terminal of arcuate nucleus

projections, leaving little NPY and AgRP in the cell body. Anx/anx arcuate nucleus

neurons exhibit increased NPY and AgRP immunoreactivity in the cell body, although

the transcription levels of the mRNAs for both peptides were normal as determined by

RNA in situ hybridization. Hence, in the anx/anx hypothalamus, 3 possibilities arise.

First, arcuate nucleus neurons fail to send axonal projections to terminal fields, in which

case the anx mutation would affect axonal outgrowth and/or maintenance of these

projections. Second, axonal projections extend normally to target areas, but axonal

transport mechanisms are disrupted leading to a sequestering of NPY and AgRP in the

cell body. Third, the anx/anx hypothalamic phenotype is secondary to the defect in 5-

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HT innervation. Although the hypothalamus is normally inundated with 5-HT fibers, a

very slight increase in 5-HT innervation has been previously reported in anx/anx mouse

brains (Son et al., 1994), in which case, the mechanism causing the 5-HT innervation

defect may nonetheless affect hypothalamic circuitry.

1.6 Positional cloning and candidate genes of the anx mutation

The anorexia mutation arose spontaneously in the F2 generation of a cross

between DW/J and an inbred strain derived from a cross between M. m. poschiavinus

and an inbred Swiss stock, and maps closely to pallid (pa) on chromosome 2 (Maltais et

al., 1984) in a relatively gene rich region. However, in looking for candidate genes

causative of the anx mutation, the most logical approach would be to examine genes

with known neurodevelopmental roles, and secreted or transmembrane proteins that

are similar to known axon guidance or neuronal maintenance molecules. In the case of

nervous system development, tyrosine kinase receptors, such as the Eph receptors and

Trk receptors, play particularly intriguing roles in axon guidance, neuronal cell migration,

and synaptogenesis. Two receptor tyrosine kinase genes, Tyro3 and Leukocyte

Tyrosine Kinase (Ltk), reside within the anx critical interval. Interestingly, the anx

mutation was also initially mapped near the BDNF gene, which is known to play a role in

5-HT neurite outgrowth and cell survival as described above, but, as I shall report in the

next chapter, we eliminated BDNF as a candidate gene via non-complementation and

further genetic mapping experiments.

17

1.7 The Eph receptors and ephrins

Among receptor tyrosine kinases (RTKs), the roles of the Eph receptor family in

axon guidance and synaptogenesis has been studied most extensively and, thus,

present a good starting point for understanding the possible roles of other RTKs in

modulating these complex neurobiological processes. The Eph receptor family is the

largest subfamily of RTKs and is comprised of 16 members classed as “A” or “B” based

on their binding affinities to 9 ephrin ligands, also classed as “A” or “B” (reviewed in

Goldshmit et al., 2006). Each receptor is characterized by a highly-conserved N-

terminal globular domain, a cysteine-rich motif, and two fibronectin type III repeats in the

extracellular region; the fibronectin type III repeats mediating receptor dimerization

(Lackmann et al., 1998). Two tyrosine residues located in the juxtamembrane region

are the major sites of receptor autophosphorylation and subsequent receptor signaling

(Bruckner and Klein, 1998, Holland, 1998). The intracellular kinase domain has been

posited to act on small GTPases and, thereby, affect cytoskeleton organization

(Nimnual et al., 1998), while a sterile alpha motif (SAM) domain formed by the last 60-

70 residues of the carboxy-terminus tail regulates cell-cell signal transduction (Schultz

et al., 1997, Stapleton et al., 1999). The inclusion of a post-synaptic density protein

zona occludens (PDZ)-binding domain is necessary to form Eph or ephrin complexes to

affect regulatory molecules and to direct these complexes to specific locations within the

cell (Kullander and Klein, 2002). To initiate a signal via Eph receptors, higher order

Eph-Ephrin complexes are an absolute necessity. These not only direct signals

downstream of the Eph receptor-expressing cell, but may also initiate reverse signaling

via clustered ephrins attached in the apposed cell membrane (Cowan and Henkemeyer,

18

2002; Davy and Soriano, 2005). Hence, a dual mechanism, in which initial binding of a

cluster of ephrins attached to one cell binds with a cluster of Eph receptors on the

apposed cell reinforcing cellular adhesion, which may be further reinforced or overcome

by intracellular changes affecting downstream processes (Klein, 2004).

The predominant of expression of most of the Eph receptors and ephrin ligands

in the nervous system is in line with their significant roles in neurodevelopment

(Flanagan and Vanderhaeghen, 1998; Nakamoto, 2000; Kullander and Klein, 2002,

Murai and Pasquale 2003). The Eph receptors and their associate ephrin ligands play a

role in the retinotectal mapping of visual circuits in the chick (Cheng et al., 1995,

Drescher et al., 1995, Nakamoto et al., 1996) and the mouse (Feldheim et al., 1998,

Feldheim et al., 2000), and direct axonal projections from motoneurons to their correct

targets in the hindlimb (Dottori et al., 1998; Kullander et al., 2001). The expression of

Eph B1, B2, B3, and A4 receptors and the B1, B2, and B3 ephrins is particularly high in

migratory regions in post-natal and adult mouse brains, and in regions of high plasticity

including the olfactory bulb, the hippocampus, and the cerebellum (Liebl et al., 2003).

EphB2 and EphB3 mutant mice are known for midline guidance defects of the tracts

forming the corpus callosum, and defects of the anterior commissure (Henkemeyer et

al., 1996, Orioli et al., 1996). EphA4, which can bind both A-class and B-class ephrins

(Gale et al., 1996), is required to establish corticospinal tract axons from the motor

cortex (Kullander et al., 2001). Additionally, a knock-in mouse generated to express a

constitutively active form of this receptor, EphA4EE, is deficient in forming

thalamocortical projections despite normal midline axon guidance and hindlimb

19

locomotion (Egea et al., 2005). These results highlight the complex nature of the Eph

receptors and ephrins and their roles in axon guidance and topographic mapping.

1.8 Trk receptors and neurotrophins

Another class of RTKs that have roles in neural development is the tropomyosin-

related kinase (Trk) receptors and their activating ligands, the neurotrophins. The

discovery of neurotrophins, in particular, nerve-growth factor (NGF), validated earlier

ablation and transplantation experiments in which a trophic factor acting locally at target

fields was necessary for survival and/or maintenance of neuronal projections (reviewed

in Levi-Montalcini, 1987). This also demonstrated the essential role of intercellular

communication in developmental processes. These molecules have numerous

functions acting not only as survival/trophic factors for neuronal projections as

discussed above for BDNF, but the interactions between Trk receptors and

neurotrophins have been shown to promote and direct axonal outgrowth (Tucker, 2002),

affect synaptic plasticity (Schinder and Poo, 2000), and mediate responses to injury

(Blesch et al., 1998). Furthermore, the Trk receptors and neurotrophins extend outside

of the nervous system with roles in vascularization (von Schack et al., 2001) and

oncogenesis (Descamps et al., 2001), highlighting the importance of these molecules in

both neural and non-neural tissues.

There are three Trk receptors which specifically bind four neurotrophins

(reviewed in Patapoutian and Reichardt, 2001). TrkA binds NGF, TrkB binds BDNF and

NT4, and TrkC binds NT3. The expression of these receptors and ligands in the

nervous system indicate roles in neuronal development. Each receptor is characterized

20

by a cleaved signal sequence followed by two cysteine clusters which flank three,

twenty-four-residue leucine-rich motifs. Adjacent to the second cysteine cluster, two

immunoglobulin-like domains complete the extracellular domain, which is then affixed to

the membrane and attached to an intracellular tyrosine kinase domain. Binding of a

neurotrophin to its specific Trk receptor occurs at the second Ig-like domain, although

the subsequent downstream events mediated by receptor binding are complicated by

several factors. The specificity of TrkA and TrkB to their proper neurotrophins is altered

in certain isoforms and by the promiscuity of NT3. Both receptors, when lacking an

insert by the juxtamembrane region, bind only their specific partners. However, when

the insert is included, TrkA and TrkB are able to bind NT3 (Clary and Reichardt, 1994,

Strohmaier et al., 1996). TrkB and TrkC also have isoforms lacking the intracellular

kinase domain, which may therefore act to suppress downstream signaling by

sequestering excess neurotrophin, although their exact role is unknown (Klein et al.

1990, Tsouflas et al., 1993). Additionally, the pan-neurotrophin receptor p75NTR causes

alterations in receptor binding behaviour of the Trk receptors. This receptor has low

binding affinity for each neurotrophin (Rodriguez-Tebar et al., 1991), however, p75NTR

activation enhances TrkA binding to NGF (Hempstead et al., 1991) and TrkB binding to

BDNF (Bibel et al., 1999) p75NTR is a distant member of the tumour necrosis factor

(TNF) receptor (Bothwell, 1995) and contains a cytoplasmic “death” domain (Liepinsh et

al., 1997). Ligand binding to p75NTR can induce neuronal death directly by initiating

apoptosis (Frade and Barde, 1998, Friedman, 2000). However, the activities of p75NTR

are necessary for neural development, since absence of this receptor causes

21

perturbations in axon growth in vitro and axon growth and target innervation in vivo (Lee

et al., 1994, Yamashita et al., 1999, Bentley and Lee, 2000).

In vitro experiments of all the neurotrophins indicate that signaling events

mediated by Trk receptor activation promote neurite outgrowth (reviewed in Patapoutian

and Reichardt, 2001). When subjected to gradients of neurotrophins, growth cones can

respond by redirecting their advancing trajectory. However, the reaction of neuronal

projections to neurotrophins as a chemoattractant or a chemorepellant is dependent on

the levels of cyclic nucleotides within those neurons (Song et al., 1997, Song and Poo,

1999). Inhibitors of the cAMP signaling cascade convert NGF- and BDNF-mediated

chemoattraction to chemorepulsion via their respective receptors. The chemoattraction

of TrkA activation via binding of NGF is lost in TrkA mutants unable to bind PI-3 kinase

(Ming et al., 1999). Interestingly, TrkC-NT3 mediated chemoattraction is converted to

chemorepulsion in the presence of cGMP signaling inhibitors (Song and Poo, 1999).

Thus, the intricate balance between chemoattraction and chemorepulsion may be

modulated by the presence of certain Trk receptor-neurotrophin signaling cascades and

further refined by intracellular events.

The activities of neurotrophins at target sites to promote innervation and

establish synaptic connections has been exemplified in studies in which excess

neurotrophin levels in normal targets lead to increased innervation and the presence of

neurotrophins in non-targets leads to ectopic innervation. NGF expression in pancreatic

islets driven by the insulin promoter causes dense sympathetic innervation of these

tissues which normally lack innervation (Edwards et al., 1989). Furthermore, when

driven by the keratin-14 promoter, similar innervation is induced in the epidermis

22

(Guidry et al., 1998). Additionally, ectopic expression of BDNF by the nestin promoter

stalls sensory axons from reaching the gustatory papillae and they are instead restricted

to the base of the tongue (Ringstedt et al., 1999). Thus, the central model of

neurotrophic factor action, in which targets of neuronal innervation secrete specific

amounts of survival factors which act to balance the size of a target tissue with the

number or amount of innervation, is repeatedly satisfied and reflects the careful neural

connections that are necessary to form functional networks in the brain.

1.9 The receptor tyrosine kinase Tyro3

Tyro3 is a member of the “TAM” family of receptor tyrosine kinases along with

Axl and Mer (reviewed in Hafizi and Dahlback, 2006). This receptor has adopted many

names since its identification including Rse (Mark et al., 1994), Sky (Ohashi et al., 1994)

Brt (Fujimoto and Yamamoto, 1994), Tif (Dai et al., 1994), Dtk (Crosier et al., 1994), and

Etk-2 (Biesecker et al, 1995) owing to its identification in different organisms and

different tissues. Of the TAM family members, Tyro3 is most extensively expressed in

the central nervous system, as shown in the mouse and rat (Lai et al., 1994; Ohashi et

al., 1994; Funakoshi et al., 2002; Prieto et al., 2000), but is also highly expressed in the

Sertoli cells of the testes. Additionally, Tyro3 has been shown to play a role in bone

resorption in osteoclasts (Nakamura et al., 1998), immune regulation (Lu and Lemke,

2001; Lemke and Lu, 2003), and platelet activation in response to injury (Dahlback and

Villoutreix, 2005), further underscoring the many potential roles of this receptor.

The extracellular domain in the mature protein contains a putative signal

sequence domain, and 2 immunoglobulin-like domains followed by 2 fibronectin type III

23

repeats, similar to neural-cell adhesion molecules (Lai et al., 1994). The

transmembrane receptor is complete with an intracellular tyrosine kinase domain,

which, upon activation by its ligands: growth-arrest specific gene 6 (Gas6) or Protein S,

initiates homophilic dimerization, cross-phosphorylation, and downstream signaling

through PI-3K/AKT or Ras/ERK signaling pathways (reviewed in Hafizi and Dahlback,

2006). Both ligands are composed of on N-terminal domain consisting of multiple post-

translationally modified γ–carboxyglutamic acid residues, which can interact with

negatively charged membrane phospholipids (Mann and Lawson, 1992). Following this,

four epidermal growth factor (EGF)-like repeats and a C-terminal globular sex hormone

binding globulin (SHBG)-like region consisting of two laminin G-like (LG) domains

complete each ligand (Tisi et al., 2000). An intervening loop region between the EGF-

like repeats and the SHBG-like region can be cleaved in Protein S but not Gas6 by

thrombin, ideal for its role in coagulation (Dahlback and Villoutreix, 2005). There is

some debate as to the exact binding partner of mouse and human Tyro3 based on

biochemical analysis with species-specific ligands. Human Tyro3 is preferentially bound

by human Gas6 and bovine Protein S over human Protein S, while mouse Tyro3

preferentially binds human protein S over human Gas6 (Godowski et al., 1995, Nyberg

et al., 1997, Stitt et al., 1995). These discrepancies between intraspecies ligand binding

affinities must be clarified to ascertain the roles of each ligand and receptor regarding

their effects in different tissues affecting different signaling pathways.

Like many other RTKs, interest in Tyro3 was drawn from its potential roles in

oncogenesis, but additional studies indicate that Tyro3 may have a role in mammalian

brain development. Published expression analyses have observed that Tyro3 is

24

expressed in the olfactory epithelium at E17.5 in the mouse, and is steadily upregulated

from birth to its maximum levels in the adult in layers 2/3, and 5 of the cerebral cortex, in

the CA1 region and at reduced levels in the CA3 region of the hippocampus, and in the

granule cells comprising the molecular layer of the cerebellum (Lai et al., 1994). In

comparing published expression analysis between the mouse and the rat, there are

some discrepancies as to the exact levels in certain regions of the brain. In the rat,

Tyro3 mRNA is reported to be expressed in layers 2, 3, and 6 of the cortex and at very

low but detectable levels in the dentate gyrus of the hippocampus (Prieto et al., 2000).

Despite these differences, Tyro3 activity in the brain appears to be important due to its

region-specific expression in the brain and its upregulation during a period of post-natal

development in which axonal projections and synaptogenesis are occurring at

maximum.

Early Northern blot analysis of RNA from numerous organs indicated high levels

of Tyro3 mRNA in the testes (Stitt et al., 1995). From birth to P18, Tyuro3 mRNA is

maintained at a high level in the testes and decreases slightly by P24 (Matsubara et al.,

1996). Tyro3 is expressed in the Sertoli cells of the testes which are responsible for

providing trophic support to the seminiferous tubules in concert with additional factors

secreted by surrounding Leydig cells. However, both the brain and testes appear

unaffected in a Tyro3 null mutant mouse line generated by insertion of a neo-cassette

replacing the second fibronectin type III domain (Lu, et al., 1999). These mice are

viable and fertile, and the only “neural” phenotype appears to be activity-induced

seizures that first occur around seven months. Additionally, double mutants of

Tyro3/Axl and Tyro3/Mer were also viable and fertile, with some aberrations in overall

25

health. However, Triple Tyro3/Axl/Mer mutants had numerous defects including cellular

degeneration in the neocortex, the hippocampus, and the cerebellum, and of the rods

and cones in the retina. Furthermore, the epithelium of the prostate, the parenchyma

of the liver and the walls of the blood vessels were also compromised, and the spleens

of these animals were enlarged though they were populated by apoptotic cells. In the

testes, the seminiferous tubules lacked mature sperm due to the progressive death of

differentiating germ cells. Tyro3, Axl, and Mer are all expressed in Sertoli cells, which

do not undergo cell death as Gata-1, a positive marker for Sertoli cells, was evident in

the testes of young adult triple mutant mice. The authors conclude that the coordinated

activity of these three receptors may be required for the necessary production of trophic

factors to support spermatogenesis. However, there are several caveats to the results

and discussions from these mouse mutants, especially regarding Tyro3. At the genetic

level, the neo cassette replacing the second fibronectin type III repeat was in-frame in

the final polypeptide. Hence, alternate splicing mechanisms may have indeed left some

of the functional aspects of the receptor intact. From the published Western blot, the

Tyro3 null mutants may actually be Tyro3 severe hypomorphs, since a very faint but

noticeable signal is visible in this mouse line.

3.4 Summary

From this brief summary it is clear that RTKs have important roles in

neurodevelopment. Aside from the more complex roles of BDNF and the interactions

between Eph receptors and their ephrin ligands, no other RTK signaling system has

been shown to definitively play a role in 5-HT axon guidance. In the next chapter, I will

26

present my studies which clearly show that the Tyro3 RTK plays an important,

previously unknown role in establishing and maintaining the 5-HT system.

27

Chapter 2: Materials and Methods

2.1 Introduction

In spite of the vast importance of this neurochemical, there are but few mutations

known to directly affect the development of the serotonergic system. We have further

characterized a 5-HT-specific developmental mouse mutation, known as anx, which is

specific for a defect in 5-HTergic innervation. This mutation causes a delay in 5-HT

axon guidance (Huynh, unpublished) which ultimately culminates in serotonergic

hypersprouting in terminal fields and death (Son et al., 1994). By characterizing the

neurobiological effects at this early stage in mammalian development, we can contribute

to the growing knowledge of molecules and pathways that mediate the complex

interactions required for brain development, and importantly, how these early events

play a role in the materialization of later, psychological disorders.

Forward genetic approaches and positional cloning allow for the identification of

novel genes and processes that might not otherwise have been implicated in a process

of interest – in this case, 5-HT system development. Here, I describe the positional

cloning of the anx gene which has selectively allowed us to provide strong evidence of a

receptor tyrosine kinase that has been previously shown to be expressed in nervous

tissue, but has not been shown to play a definite role in neurodevelopment. In

comparison to known receptor tyrosine kinases, our molecule of interest supports a role

in neurodevelopment.

The use of transgenic mouse models has provided a wealth of knowledge

regarding many different molecules and processes and how they are related to

28

numerous areas or research. In the following, I will also describe the results from the

use of transgenic mice to characterize the anx mutation, and discuss how our results

suggest certain roles which are disrupted by the anx mutation. I will also describe some

of the caveats of this approach to characterize anx, and additional research that may

elucidate some of the functional roles affected by the anx mutation.

2. 2 Meiotic recombination map

The anx mutation arose spontaneously in the F2 generation of a cross between

DW/J and an inbred strain derived from a cross between M. m. poschiavinus and an

inbred Swiss stock, and is maintained on a nonagouti (a) hybrid background referred to

as B6C3Fe a/a-anx/J. Heterozygous carriers of the mouse anorexia (anx) mutation

were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed onto

the Molossinus/Ei and C57Bl6/J strains. I identified simple sequence length

polymorphisms using the following markers: D2Mit207, D2Mit276, D2Mit484, D2Mit104,

D2Mit190, D2Mit397, D2Mit277, and D2Mit446 that distinguish anx/anx homozygotes

and anx/+ heterozygotes from normal mice on the C57Bl6/J, Molossinus, and anx

background strains. A polymorphism at D2Mit190 distinguished 21 anx/anx mice from

their unaffected littermates and was used thereafter to identify heterozygous carriers

and homozygous mutants. Carriers on the C57Bl6/J strain background produce a ~145

bp PCR product, distinguishable from normal animals that produce a PCR product of

123 base pairs using the D2Mit190 marker. Similar differences were present on the

Molossinus/Ei strain background. Progeny from heterozygous intercrosses on each

29

background were genotyped using the above list of markers surrounding D2Mit190

generating a meiotic recombination map.

2.3 Candidate gene testing

i. Brain-derived neurotrophic factor (BDNF)

To determine whether anx is an allele of the BDNF gene, we performed a

complementation test by intercrossing anx heterozygotes with BDNF+/- mice. BDNF+/-

mice were obtained from Jackson Laboratories and genotyped as described in

Henneberger et al., 2000. All progeny were assessed for phenotypes associated with

BDNF-/- mice and anx/anx homozygotes in the first 3 weeks after birth.

ii. Glycine-amidinotransferase (GatM)

To test for possible involvement of GatM mutations contributing to the anx

phenotype and to determine potential creatine metabolism defects, I performed a

nutritional rescue experiment. After intercrossing with anx heterozygote males and after

birthing their litters, anx/+ Dams were fed powdered-rodent chow diet containing 2%

creatine monohydrate (Prolab, Chatworth, California, USA) to supply creatine to

anx/anx and normal littermates via their milk. All progeny were assessed to determine if

creatine supplementation reduced or altered anx/anx phenotypes in the first 3 weeks

after birth.

2.4 Sequencing candidate genes

In the critical interval containing the anorexia mutation, the exons and at least 75

base pairs of flanking intronic sequence of candidate genes were amplified by

30

polymerase chain reaction (PCR) and sequenced to identify possible mutations. We

tested candidate genes with known or postulated neurodevelopmental roles and/or

encoding membrane-bound/secreted or secreted proteins first (listed in Table 1). A

cytosine to guanidine point mutation in the putative signal sequence of Tyro3 was

identified in anx homozygotes compared to normal littermates, resulting in an arginine to

tryptophan conversion at the seventh position of the translated protein. This mutation

will thus be referred to as R7W-Tyro3.

2.5 Generation of transgenic animals

i. Mouse GFP-tagged normal and R7W-Tyro3 transgenes

Whole brains from P21 anx/anx and normal littermates were homogenized in

TrizolTM (Invitrogen, Carlsbad, California, USA) and extracted according to

manufacturer’s instructions. cDNA was synthesized from polyA mRNA from normal and

anx/anx brains by using the Superscript First-Strand Synthesis RT System (Invitrogen).

Tyro3 cDNA was amplified with high-fidelity platinum Pfx (Invitrogen) yielding a 2.6kb

PCR product using primers containing attB sites flanking the open reading frame for

recombination into entry vectors using Gateway TechnologyTM (Invitrogen), and

truncating the 3’ stop codon for in-frame C-terminal fusion with green fluorescent protein

(GFP). Entry clones were generated by inserting attB-flanked PCR products into the

pDONR201 vector in the presence of BP clonase (Invitrogen) and transformed in DB3.1

cells. The sequence of normal and R7W-Tyro3 clones were verified with primers. To

allow for the expression of Tyro3 transgenes in endogenous Tyro3 expressing domains,

we generated the T3XPRSSN vector

31

Table 1. Genes within the anorexia Critical Interval

1. isovaleryl coenzyme A dehydrogenase 2. dermatan-4-sulfotransferase-1 3. gene model 631 4. RIKEN cDNA 4921503C21; CG6187-like 5. DNA repair protein RAD51 homolog 1 6. zinc finger, FYVE domain containing 19 7. PKC-dependent PP1 inhibitory protein subunit 14 8. Kunitz-type protease inhibitor 1 precursor (HGF activator inhibitor type 1) 9. ras homolog gene family, member V 10. Vacuolar protein sorting 18 11. Delta-like protein 4 precursor (Drosophila Delta homolog 4) 12. Calcium-binding protein p22 (CHP) (Calcineurin homologous protein) 13. nucleolar protein ANKT 14. Complex I intermediate-associated protein 30, mitochondrial precursor 15. inositol 1,4,5-trisphosphate 3-kinase A 16. RNA polymerase II associated protein 1 17. MAX-interacting protein; Max dimerization protein 5 18. mitogen activated protein kinase binding protein 1; JNK-binding protein 1 19. Similar to phospholipase A2, group IVB (Cytosolic) (Fragment) 20. EH-domain containing protein 4 (mPAST2) 21. phospholipase A2-like 22. Similar to phospholipase A2, group IVB 23. Vam6/Vps39-like protein (Vps39 protein) 24. Neutral alpha-glucosidase C (EC 3.2.1.-) 25. Calpain 3 (Calpain L3) (Calpain p94) (Calcium-activated neutral proteinase 3) 26. zinc finger protein 106 27. Synaptosomal-associated protein 23 (SNAP-23) 28. Motor domain of KIF16A 29. MKIAA1300 protein 30. Codanin 1 31. tau tubulin kinase 1; tau-tubulin kinase 32. ubiquitin protein ligase E3 component n-recognin 1 33. cyclin D-type binding-protein 1; maternal inhibitation of differentiation 34. Erythrocyte membrane protein band 4.2 (Erythrocyte protein 4.2) (P4.2) 35. transglutaminase 5 36. Gamma-tubulin complex component 4 (GCP-4) 37. transformation related protein 53 binding protein 1; murine p53-binding protein. 38. RIKEN cDNA B430315C20 gene; KIAA0377-like 39. Creatine kinase, ubiquitous mitochondrial precursor 40. Stereocilin precursor 41. sperm-associated cation channel 2 42. Protein disulfide isomerase A3 precursor (EC 5.3.4.1) (Disulfide isomerase ER-60) 43. RNA polymerase II elongation factor ELL3 44. Small EDRK-rich factor 2 (4F5rel) (h4F5rel) (Gastric cancer-related protein VRG107) 45. Huntingtin interacting protein K 46. microfibrillar-associated protein 1 47. RIKEN cDNA D130060C09 isoform 1 48. eukaryotic translation initiation factor 3, subunit 1 alpha; merged

32

consisting of the 9.5kb region upstream of exon 2c, sequence-specific attR

recombination sites for the insertion of Tyro3 cDNA, an in-frame C-terminal GFP, and

Sv40 IVS polyA. Because sequence comparison between the non-coding sequences of

human, mouse, dog, and rat Tyro3 showed the highest conservation in the regions

contained within the first 9.5kb upstream of exon 2c, we postulated that these regions

might be likely to direct Tyro3 and transgene expression in the endogenous expressing

domains. The translational start site for all known signal sequence containing forms of

Tyro3 is located in exon 2c. Normal Tyro3 and R7W-Tyro3 were inserted into the

T3XPRSSN vector in the presence of LR clonase (Invitrogen). Resulting normal and

R7W-Tyro3 expression constructs were digested with NruI (NEB, Ipswich,

Massachusetts, USA) creating a 14kb transgene product purified with GeneClean Kit

(Qbiogene, Irvine, California, USA), and used to generate transgenic animals by

standard pronuclear microinjection.

ii. Human full-length Tyro3 transgene

To generate a full-length, untagged version of Tyro3 which was distinguishable

from endogenous Tyro3, I purified human Tyro3 following EcoR1 (NEB) digestion of

cDNA-containing pUC SRα, which was directly inserted into the filled-in Sal1 (NEB)

sites of PDONR201 by blunt-end ligation. Insertion into T3XPRSSN, digestion and

subsequent purification was the same as outlined above.

I therefore generated the following T3XPRSSN transgenic constructs: mouse

Tyro3 with C-terminal GFP fusion, mouse R7W-Tyro3 with C-terminal GFP fusion, and

human-Tyro3 with an intact stop codon.

33

iii. Identifying transgenic founder mice

Positive transgenic carriers for the GFP-tagged normal and R7W-Tyro3

transgenes were identified by Southern blot analysis. Approximately 10µg of genomic

DNA was digested with EcoR1 (NEB), separated on a 1% agarose gel, depurinated in

0.1N HCl for 2 x 20 minutes, denatured for 1 hour, neutralized for 1 hour, and blotted in

10X SSC onto Biodyne B membrane (Pall Corporation, East Hills, New York, USA).

The membrane was probed with a 700 bp GFP probe labeled with P32 at 106 cpm/ml of

hybridization solution for 16 hours at 42OC. The membrane was washed as follows: 2 x

1 hour, 2XSSC, 0.1% SDS; 2 x 1 hour, 1XSSC, 0.1% SDS at 65OC. Positive transgenic

animals were identified by exposing the membrane to HyperfilmTM MP (Amersham

Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom) for 16-24 hours

at -80 OC. Positive transgenic animals for the full-length transgene were genotyped

using probes for the SV40 polyA IVS yielding a product size of 500 bp.

2.6 Expression analysis of Tyro3 in normal and anx/anx mice by RNA in situ

hybridization

Normal and anx/anx mice at P21 and normal mice at P14 were anaesthetized

with intraperitonial injection of Avertin (1.25% at 0.025ml/g) and cardially-perfused with

Ringer’s solution containing 50µg/ml heparin followed by 4% paraformaldehyde (PFA) in

diethyl-pyrocarbonate (DEPC)-treated 1xPBS. Brains were removed and post-fixed in

4% PFA overnight, rinsed 3 x 5 minutes in DEPC 1xPBS, washed 3 x 1 hour in DEPC

1xPBS, and placed in 30% sucrose in 1xPBS overnight. When no longer floating, the

34

brains were transferred into a 50:50 solution of 30% sucrose:optimal cutting

temperature (OCT) medium (Sakura Finetek, Torrance, California, USA) for 3 hours,

then washed 3 times in OCT for 1 hour each. All washes were performed at 4 OC.

Brains were embedded in OCT on dry ice and stored at -80OC. 14µm coronal sections

were taken using the Leica VT1000 cryostat onto Superfrost Plus microscope slides

(VWR, West Chester, Pennsylvania, USA).

Sense and antisense Tyro3 probes were generated by cloning a 588 bp region

coding for part of the intracellular kinase domain using HindIII (NEB) into pBluescript

KSII. To generate the sense probe, the Tyro3-containing vector was digested with

Xho1 (NEB) and in vitro transcribed with T3 RNA polymerase (Roche, Basel,

Switzerland). To generate the antisense probe, the vector was digested with EcoR1

(NEB) and in vitro transcribed with T7 RNA polymerase (Roche). For a positive control,

a probe for Kreisler was generated alongside Tyro3-specific probes by digesting 4CKS

with Nco1 (NEB) and in vitro transcribing with T7 RNA polymerase (Roche). All

riboprobes were DIG-tagged as specified in the manufacturer’s instructions.

RNA in situ hybridization was performed as outlined in Storm and Kingsley.

Staining was discontinued after 16-24 hours by 3 washes in 1x PBS and mounted with

either Permount (Daido Sangyo Co., Tokyo, Chiyoda-ku, Japan) or Cytoseal (Richard-

Allan Scientific, Kalamazoo, Michigan, USA) following standard dehydration and

rehydration into xylene.

35

2.7 Weight and survival data from the progeny of transgenic anx/+ heterozygote

intercrosses

Transgenic founders identified by Southern Blot analysis or by PCR genotyping

were crossed with anx/+ heterozygotes to generate transgenic anx/+ heterozygote

carriers. Litters from 5 mouse Tyro3 transgenic lines, 1 mouse R7W-Tyro3 mimic

transgenic line, and 3 human full-length Tyro3 transgenic lines were monitored regularly

from birth. Progeny from selected litters were weighed at P21. To measure the lifespan

of the offspring, animals with abnormal or anx-like behaviours were allowed to expire,

and the number of days survived was recorded.

36

Chapter 3: Results

3.1 Introduction

The anx mutation has provided a unique opportunity for studying a 5-HT-specific

aberration and allows for examining the relationship between axon guidance and

synaptogenesis between 5-HT projections and their terminal fields. Our data suggest

that among the known tyrosine kinases that play roles in cell proliferation, survival, and

migration, neurite/axonal outgrowth, and innervation to and synaptogenesis with central

targets, Tyro3, a tyrosine kinase of the TAM family of tyrosine kinase receptors, likely

plays a part in establishing these far-reaching circuits of the raphe nuclei. Tyro3 is

expressed at late embryonic stages in the mouse and its expression level increases in

the first 3 weeks after birth during a period of development in which axonal pathfinding

and synaptogenesis is maximal for 5-HT neurons. And, as Tyro3 has been found to be

predominantly expressed within specific regions of the mammalian brain, suggesting the

actions of Tyro3 may therefore be cell autonomous to 5-HT projections, we have found

Tyro3 expression in the hindbrain, where it has not been previously examined. Whether

this newfound expression coincides with the raphe nuclei has not been verified.

However, the activities of Tyro3 may indeed begin within the hindbrain or even 5-

HTergic neurons themselves.

Numerous lines of evidence have suggested to us that the point mutation

identified in the Tyro3 gene is indeed causative of the anx mutation. RNA in situ

analysis of Tyro3 mRNA at P21 is markedly reduced in known Tyro3-expressing

domains in the mammalian brain, and, perhaps even more profound than this, Tyro3

37

mRNA is absent in hypothalamic domains responsible for food intake behaviour, to

which the initial characterization of this mutation owes its name. Michael Huynh, a

previous graduate student in our lab, generated a TPH1-promoter driven PLAP reporter

system that solely labels Tph1-expressing axons and crossed these transgenic mice

onto an anx mouse line to generate transgenic anx/anx mice. At P15, these mice

exhibit much greater PLAP staining of several brain regions, and an altered pattern of

expression within the cortical layers. Within deeper cortical layers and at the most

superficial layer, dense PLAP staining is present in anx/anx mice relative to controls.

This pattern of PLAP expression was reversed in P0 transgenic mice in which anx/anx

mice lacked PLAP staining altogether and +/+ mice exhibited moderate staining.

Interestingly, heterozygous anx/+ mice exhibited an intermediate level of staining. This

research is the first to show a neonatal and heterozygous phenotype in anx mice which

has never been previously reported. Currently, Joanna Yu, a Ph. D. student in the lab,

has shown that anx/anx mice exhibit a platelet aggregation defect compared to normal

mice as determined by FACS sorting. Tyro3 is present in platelets and has a known

role in platelet aggregation, and a posited Tyro3 null mouse has been shown to have a

clotting defect. Hence, several lines of evidence strongly suggest that the point

mutation in Tyro3 is causative of the anx mutation.

To examine whether the point mutation found in Tyro3 is indeed causative of the

anx mutation, we generated several transgenic mouse lines to either rescue the anx

phenotype by insertion of a normal Tyro3 construct, or to mimic the anx phenotype by

insertion of a mutant Tyro3 construct. Two normal rescue Tyro3 constructs were

designed. One construct was made with mouse Tyro3 with an in-frame C-terminal GFP

38

tag. In doing so, these mouse lines could be used to trace transgene expression in

vibratome sections of fresh tissues by GFP fluorescence or by immunohistochemistry

using a GFP antibody. The other construct was made using human Tyro3, which is

virtually identical to the mouse Tyro3 gene. We labeled this construct “full-length” since

it contained an intact stop codon and would remove any potential interference of the

GFP tag, and importantly, allow us to perform future experiments that could selectively

knockdown the expression of this transgene. Using these transgenes, we have been

able to partially rescue two hallmarks of the anx phenotype by nearly doubling the body

weight and lifespan of anx/anx mice. We have also delayed the onset and severity of

the anx behavioural phenotype, though it has left us with the question of why either

rescue transgene could not overcome the endogenous mutation. Recently, Dr. Sabine

Cordes has examined cerebellar cytoarchitecture using immunofluorescent probes in

anx/anx brains transgenic for the mimic construct. In lieu of Purkinje cells normally

residing above the granule cell layer, large areas completely devoid of neurons are

present in transgenic anx/anx cerebella; an outcome suggestive of a gain-of-function

mutation in Tyro3. The consequences of this mutation and its effects are further

elaborated on in the discussion.

3.2 Meiotic recombination mapping and candidate gene sequencing identified a

point mutation in the signal sequence of Tyro3

To locate the anx mutation more precisely, we analyzed 335 progeny (670

meioses) from heterozygous intercrosses on the Molossinus/Ei (n = 224) and C57Bl6/J

(n = 111) strain backgrounds using simple sequence length polymorphisms (SSLPs),

39

and, thereby reduced the anx critical interval to a 3.2Mb region on chromosome 2

between markers D2Mit484 and D2Mit190 (Fig. 1A). During recombinational mapping,

we were able to eliminate two candidate genes within the critical interval. In the

complementation experiment in which BDNF+/- mice were crossed to anx/+

heterozygotes, compound BDNF/anx (n = 7) were normal and did not show anx

phenotypes at 3 weeks. Further recombinational mapping also positioned BDNF

outside of the anx critical interval. To test if GatM was causative of the anx phenotype,

we performed a nutritional rescue experiment in which anx/+ Dams were fed powdered-

rodent chow containing 2% creatine monohydrate after birthing. While normal and

heterozygous littermates were unaffected (n = 18), anx/anx homozygous mouse pups (n

= 5) receiving the nutritional supplement through the mother’s milk did not show

improvement from the anx phenotype and died by P21. Subsequently, we sequenced

the exons and flanking intronic regions of candidate genes having neurodevelopmental

roles or producing secreted or transmembrane proteins and identified a C to T point

mutation in the signal sequence of the receptor tyrosine kinase Tyro3 (Fig. 1C). In the

signal sequence-containing isoform of Tyro3, the mutation leads to a conversion from a

hydrophilic arginine to a hydrophobic tryptophan at the seventh amino acid position (Fig.

1D), henceforth referred to as R7W-Tyro3. The elimination of an NlaIV restriction site

by this point mutation was used in a PCR-based assay to genotype an additional 995

animals. All of the 176 affected progeny were homozygous for the point mutation,

whereas 819 unaffected animals were either heterozygous for the point mutation or

were normal (Fig.1E). We analyzed the predicted consequences of the R7W-Tyro3

mutation by using the Signal P program (www.cbs.dtu.dk/services/SignalP) a web-

42

Figure 1. Meiotic recombination mapping and sequencing of candidate genes

reveal a point mutation in the anorexia critical interval. (A) Meiotic mapping was

performed on a total of 335 progeny from anx/+ intercrosses. Progeny were analyzed

with the simple sequence length polymorphisms (SSLP) shown. Two recombinants

helped refine the critical interval to a 3.2Mb region between D2Mit484 and D2Mit190.

(B) A schematic diagram shows the positions of the SSLPs used for mapping and of

three relevant genes eliminated as candidates. A line diagram shows the approximate

positions of coding regions and Tyro3 within the anx critical interval. (C) Sequencing of

candidate genes within this region identified a point mutation in the signal sequence of

Tyro3, leading to an arginine-to-tryptophan conversion at the seventh position in the

translated protein, referred to as R7W-Tyro3. (D) The elimination of an NlaIV

restriction enzyme recognition site, which can be used to unambiguously genotype anx

animals, was further confirmed by analysis of 995 progeny from heterozygous anx/+

intercrosses. (E) Mice that exhibit the anx phenotype are homozygous for the point

mutation (n = 176), while mice that are heterozygous for R7W-Tyro3 (n = 584) or normal

(n = 235) do not show any anx-like phenotypes and appear normal.

43

based program to determine whether a given amino acid sequence has characteristics

of established protein signal sequences. The program does so by comparing an input

sequence to known characteristics of signal sequences from eukaryotic and prokaryotic

proteins. In all cases, three domains, known as the n-, h-, and c-region, are analyzed

and a potential signal sequence cleavage site is produced. In eukaryotes, the n-region

is only slightly arginine rich compared to prokaryotes, the h-region is short and very

hydrophobic, and the c-region lacks any amino acid sequence pattern. Analysis of the

first 45 amino acids of R7W-Tyro3 indicated that the observed amino acid change might

cause a slight deviation between the n-region and h-region of the signal sequence (Fig.

2). However, the R7W-Tyro3 conversion was not predicted to alter the status of the

translated polypeptide from that of a secretory molecule, nor was there a change in the

predicted cleavage site in the final protein product. These analyses suggested that the

signal sequence cleavage site should not be perturbed, but the mutation might affect

the localization, membrane retention, and/or potential post-translational modifications of

the extracellular domain which may be apparent in vivo. To more accurately determine

whether post-translational modifications were altered in the mutant protein, Joanna Yu,

a current Ph. D. candidate in our lab, performed an in vitro endoglycosidase H assay to

assess potential alterations to glycosylation of the extracellular domain. There was no

difference in the extent of glycosylation in the R7W-Tyro3 protein compared to normal

Tyro3. Additional analysis using GFP and RFP-tagged normal and R7W-Tyro3 that

were transfected in equal amounts in COS7 (Joanna Yu), Neuro2A (Joanna Yu and Dr.

Sabine Cordes), and HEK293 cells (Dr. Sabine Cordes) showed, at best, a very subtle

difference in the localization and apparent intensity of the mutant protein, in line with the

Figure 2.A. Normal Tyro3

B. R7W-Tyro3

44

45

Figure 2. Comparison of signal sequence predictions from SignalP analysis. The

first 45 amino acids of the normal Tyro3 and R7W-Tyro3 were examined using SignalP

to determine any aberrations resulting from the arginine-to-tryptophan conversion.

Comparison of the plots of the (A) normal and (B) mutant protein reveals an extremely

slight deviation between the n- and h-region in the mutant protein marked with an arrow.

However, the predicted cleavage site at amino acid 31 was virtually unaffected in the

mutant.

46

bioinformatic analysis. However, due to the strength of our genetic analysis including

100% identification of anx/anx animals as being homozygous for the point mutation, and

the elimination of any mutations in other candidate genes, we explored the possibility

that the R7W-Tyro3 mutation was responsible for the anx phenotype further.

3.3 Tyro3 expression in normal P21 mouse brain

To test if Tyro3 was expressed in regions affected by the anx mutation, I

performed RNA in situ hybridization on sections from normal and anx/anx mouse brains

at P21 to determine if there were any differences in the level or location of Tyro3

expression. Previous analyses had reported Tyro3 expression in cortical layers 2/3 and

6 in the mouse, at high levels in the CA1 region of the hippocampus but low levels in the

adjacent CA3 region and negligible expression in the dentate gyrus, in the median

eminence of the hypothalamus, and in the granule cells and at low levels in the Purkinje

cells of the cerebellum. There have been no previous reports of Tyro3 expression in

other areas of the brain in the 3 week old mouse or rat.

In my analyses, Tyro3 was expressed throughout the cortex in what appear to be

large Pyramidal neurons. The highest expression was evident in cortical layers 2/3, and

layers 5 and 6. Tyro3 expression was barely detectable in layers 1 or 4 (Fig. 3A, C).

Within the hippocampus, Tyro3 expression was high in the CA1 region, the CA3 region

and the dentate gyrus. The strong expression of Tyro3 mRNA in the dentate gyrus is

not an artifact of increased cell density within this structure, as a sense probe against

Tyro3 yielded only minute signal strength above background in these neurons. The

pattern of expression in the CA1 and CA3 region appears as a uniform band that

48

Figure 3. Analysis of Tyro3 expression in the cerebral cortex in P21 normal and

anx/anx mice by RNA in situ hybridization. (A) Tyro3 expression in the brains of

P21 normal mice show widespread staining throughout the cortex. However, Tyro3

expression is layer-specific being mainly expressed in layers 2/3, 5 and 6. This is

particularly evident at higher magnification (C). (B) Conversely, anx/anx mice show

dramatically reduced levels of Tyro3 expression. Although the Tyro3 expression

appears to maintain its laminar expression, it appears to absent from layer 2 or shifted

into layer 3, as can be seen at higher magnification in (D).

49

stretches across the hippocampus in what are likely Pyramidal cells. At high

magnification, Tyro3 expression outlining the neurons of the dentate gyrus and into the

extension leading away from these neurons appears punctuate (Fig. 4A, C). In the

hypothalamus, Tyro3 was expressed in the median eminence as previously reported.

However, contrary to previous reports, I detected Tyro3 mRNA in regions important for

regulating energy balance: the arcuate nucleus, located dorsal and lateral to the median

eminence along the rostral-caudal axis, and in the large ventromedial nuclei of the

hypothalamus (Fig. 5A, C). In the cerebellum, Tyro3 is expressed at high levels in

granule cells and the Purkinje cells that separate the granule cell layer from the

molecular layer. At high magnification, Tyro3 forms a fine outline of the soma of

Purkinje cells. There are no previous reports of Tyro3 in other structures within the

central nervous system (Fig. 6A, C, E). However, in sagittal sections through the

brainstem of the adult mouse, Tyro3 is expressed in clusters which approximate the

location of neurons that form the raphe nuclei (Fig. 7).

Tyro3 expression in the arcuate nucleus, the hippocampus, and presumptive 5-

HT producing neurons is particularly notable as deficits in these neurons and these

regions have been observed in anx/anx mutants.

3.4 Tyro3 expression is altered in numerous brain regions in anx/anx mice

Next, I examined whether Tyro3 expression was affected in anx/anx mutants. At P21,

Tyro3 expression in anx/anx brains is altered in some regions. In the cortex, Tyro3

expression appears to maintain its laminar expression in layers 3, 5, and 6, but the

signal appears markedly reduced compared to normal cortices. Furthermore, Tyro3

51

Figure 4. Analysis of Tyro3 expression in the hippocampus in P21 normal and

anx/anx mice by RNA in situ hybridization. Tyro3 expression is present in the CA1

region, the CA3 region, and the dentate gyrus of P21 normal mice hippocampi. In

anx/anx hippocampi, Tyro3 expression is markedly reduced, but appears to remain high

in the dentate gyrus. In the CA1 region, the width of the band expression Tyro3 is

reduced compared to the normal Tyro3 expression pattern indicated by the boxed area

in (A) and (B) which is shown at higher magnification in (C) and (D), and the Tyro3

expressing region in the dentate gyrus appears condensed.

53

Figure 5. Analysis of Tyro3 expression in the hypothalamus in P21 normal and

anx/anx mice by RNA in situ hybridization. (A, B) In the hypothalamus of P21

normal brains, Tyro3 is expressed in the median eminence, the arcuate nucleus, and

the ventromedial hypothalamus. (C, D) In P21 anx/anx brains, Tyro3 expression is

absent in the arcuate nucleus and ventromedial hypothalamus, and is dramatically

reduced in the median eminence.

55

Figure 6. Analysis of Tyro3 expression in the cerebellum of P21 normal and

anx/anx mice by RNA in situ hybridization. (A) In the normal cerebellum, Tyro3 is

expressed in the granule cells; and can be seen to outline the soma of the large

Purkinje cells. In cerebellar white matter, fine Tyro3 expressing processes can be seen

at high magnification and appear beaded (marked with arrows in E).. In P21 anx/anx

brains, Tyro3 expression in granule cells is maintained, however, Tyro3 expression is

undetectable in Purkinje cells. Numerous Tyro3 positive cell bodies appear in the white

matter surrounding the cerebellum which are not present in normal brains, but Tyro3

expressing processes are not readily apparent.

57

Figure 7. Analysis of Tyro3 expression in the brainstem in P21 normal mouse

sagittal sections by RNA in situ hybridization. Tyro3 is expressed in the brainstem

of normal P21 mice in loose but noticeable clusters. (A) Tyro3 is expressed in neurons

just anterior and dorsal to the cerebellum. This is likely the dorsal raphe of the anterior

cluster of 5-HT neurons. (B-D) Tyro3 is also present in clusters located near the

ventral edge of the hindbrain.

58

expression in layer 2 is barely discernible from background. At high magnification,

Tyro3 expression forms a faint outline of individual neurons (Fig 3B, D). In the

hippocampus of anx/anx brains, Tyro3 expression is maintained in the CA1 region,

however, the signal strength is greatly reduced compared to that in normal brains. Also,

the thickness of the Tyro3 expressing band in this region is reduced. There is little to no

expression of Tyro3 in the CA3 region of mutant animals. In the dentate gyrus, Tyro3

expression is comparable to the signal strength in the CA1 region. However, within the

cell bodies of some neurons, additional large, spherical bodies are evident (Fig. 4B, D).

Tyro3 expression in the median eminence of the hypothalamus is intact in anx/anx

brains at P21. However, Tyro3 expression is absent in the arcuate and ventromedial

nuclei altogether (Fig. 5B, D). The cerebellum of anx/anx animals appears

morphologically normal, and at low magnification, Tyro3 expression in the granule cell

layer is intact. However, there is little to no Tyro3 expression in Purkinje cells lining the

granule cell layer. Surprisingly, cells expressing Tyro3 can be found in the most

superficial layer of the cerebellum (Fig. 6B, D, F).

3.5 Generation and identification of transgenic mice

Of all the transgenic mouse founders generated by pronuclear microinjection of

our constructs, five of six Rescue-GFP lines, two of three Rescue-Full Length, and one

of two Mimic-GFP lines were able to breed successfully. Mice were identified by

Southern Blot analysis using a GFP probe, and progeny were subsequently genotyped

in a PCR-based assay (Fig. 8).

60

Figure 8. Construction of the Tyro3 transgenes for rescue and mimic analyses.

(A) The Tyro3 transgenes were constructed using a 9.5kb BamH1-Not1 fragment just

upstream of the translational start site located in exon 2c (adapted from Biesecker et al.,

1995). The translational start site for all known signal sequence containing forms of

Tyro3 is located in exon 2c. The signal sequence of Tyro3 is indicated in black. (B)

The T3EXPRSSN vector was designed with sequence-specific attR sites to allow for the

insertion of Tyro3 or R7W-Tyro3 cDNA using the Gateway System (Invitrogen), two

selection agents, and a C-terminal GFP tag for future tracing experiments and a

stabilizing Sv40 IVS pA tail. (C) Positive clones for the Tyro3 transgenes were

digested with NruI yielding a 14kb transgene that was purified to generate transgenic

mice by pronuclear microinjection. Transgenic founders carrying the GFP-tagged Tyro3

(D) or R7W-Tyro3 (E) were identified by standard Southern blot analysis using a GFP

probe. (F) Transgenic founders for the Full-Length Tyro3 transgene were identified by

PCR using primers located in the Sv40 IVS pA region (F).

61

3.6 Transgenic anx/anx homozygotes have improved body weight at P21 and live

longer than their non-transgenic anx/anx littermates

Normal and anx/+ heterozygote mice transgenic for full-length human or GFP-

tagged mouse Tyro3 exhibited no obvious abnormal phenotypes, weight gain or loss or

impaired survival, and were indistinguishable from each other and their normal or

heterozygote littermates. However, anx/anx homozygote animals transgenic for either

the normal mouse or human Tyro3 transgene showed very mild or no anx-related

phenotypes at P21.

At P21, transgenic anx/anx animals had dramatic body weight increases

compared to their non-transgenic anx/anx littermates in all transgenic mouse lines (Fig

9). In GFP-tagged transgenic line Rescue-GFP#1, transgenic anx/anx mice (n = 15)

had an average increased body weight of 83.5% compared to their non-transgenic

anx/anx littermates (n = 5, p < 0.01, one-way ANOVA). Pooled body weight data of

transgenic anx/anx mice from the remaining GFP-tagged lines (n = 18) showed a 69.5%

increase in average body weight compared to their non-transgenic anx/anx littermates

(n = 5, p < 0.01, one-way ANOVA). In the Full-length Rescue line#1, transgenic

anx/anx mice (n = 4) weighed 92.8% more compared to their anx/anx littermates (n = 5,

p < 0.01, one-way ANOVA). When all transgenic anx/anx mice from all lines are

compared to all anx/anx littermates, there is an average body weight increase of 82.3%

at P21 (Table 2).

Normally, anx/anx mice exhibit anxiogenic behaviours including head weaving

and uncoordinated gait by P16-P17 and die by P21. In line Rescue-GFP#1, 23 anx/anx

transgenic mice showed little or no overt anx-like phenotypes at P21, and survived until

Rescue-GFP#1: Body Weight at P21

0

2

4

6

8

10

12

14

Genotype

Bo

dy

w

eig

ht (g

)

+/+ anx/+ anx/anx anx/anx, tg

Cumulate Body Weight Analysis: Transgenic Tyro3-GFP M ice at P21

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

Genotype

Bo

dy

w

eig

ht a

t P

21

(g

)

N o r m a l a n x / + a n x / a n x a n x / a n x ; t g

Body Weight of M ice from Full-Length Human Tyro3 at P21

0.00

2.00

4.00

6.00

8.00

10.00

12.00

Genotype

Bo

dy

W

eig

ht a

t P

21

(g

)

N o r m a l a n x / + a n x / a n x a n x / a n x ; t g

Body Weight of All Transgenic M ouse Lines at P21

0.00

2.00

4.00

6.00

8.00

10.00

12.00

G e n o t y p e

N o r m a l a n x / + a n x / a n x a n x / a n x ; t g

normal anx/+ anx/anx anx/anx;0

10.0

8.0

6.0

4.0

2.0

12.0

0

10.0

8.0

6.0

4.0

2.0

12.0Bo

dyw

eigh

t at P

21 (g

)

n = 7 37 4 15

0

10.0

8.0

6.0

4.0

2.0

12.0

Body

wei

ght a

t P21

(g)

0

10.0

8.0

6.0

4.0

2.0

12.0

Body

wei

ght a

t P21

(g)

normal anx/+ anx/anx anx/anx;

n = 32 64 5 33

normal anx/+ anx/anx anx/anx;

n = 7 12 5 4

normal anx/+ anx/anx anx/anx;

n = 39 76 10 37

Body

wei

ght a

t P21

(g)

0

5.0

4.0

3.0

2.0

1.0

6.0

Body

wei

ght a

t P21

(g)

Body Weight at P21: anx/anx vs. anx/anx;tg

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Bo

dy

we

igh

t a

t P

21

(g

)

anx/anx anx/anx anx/anxanx/anx;t

g

anx/anx;t

g

anx/anx;t

gRescue-GFP Full-Length #1 All Weight

Body Weight at P21: anx/anx vs. anx/anx;tg

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Bo

dy

we

igh

t a

t P

21

(g

)

anx/anx anx/anx anx/anxanx/anx;t

g

anx/anx;t

g

anx/anx;t

gRescue-GFP Full-Length #1 All Weight

Body Weight at P21: anx/anx vs. anx/anx;tg

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Bo

dy

we

igh

t a

t P

21

(g

)

anx/anx anx/anx anx/anxanx/anx;t

g

anx/anx;t

g

anx/anx;t

gRescue-GFP Full-Length #1 All Weight

Body Weight at P21: anx/anx vs. anx/anx;tg

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Bo

dy

we

igh

t a

t P

21

(g

)

anx/anx anx/anx anx/anxanx/anx;t

g

anx/anx;t

g

anx/anx;t

gRescue-GFP Full-Length #1 All Weight

Body Weight at P21: anx/anx vs. anx/anx;tg

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Bo

dy

we

igh

t a

t P

21

(g

)

anx/anx anx/anx anx/anxanx/anx;t

g

anx/anx;t

g

anx/anx;t

gRescue-GFP Full-Length #1 All Weight

non-Tg Tg non-Tg Tg non-Tg Tgn = 4 15 5 4 10 35

Rescue-GFP #1 Full-Length #1 Cumulative

A B

C D

E

Figure 9. Bodyweight of P21 Transgenic Mouse Lines

* *

* *

* * *

Tyro3-GFP#1 all Tyro3-GFP

Tyro3-FL#1 All Tyro3-Tg

* *

* *

62

63

Figure 9. Analysis of bodyweight of transgenic anx/+ progeny at P21. Transgenic

anx/anx progeny from all lines had improved bodyweight at P21. (A) In Rescue-GFP

#1, P21 transgenic anx/anx mice weighed significantly more than their anx/anx

littermates (p < 0.01). (B) When all mice transgenic for the GFP-tagged rescue

transgene are pooled, this trend is maintained (p < 0.01). (C) In Full-Length #1, P21

transgenic anx/anx also weighed significantly more than their anx/anx littermates (p <

0.01), which is shown when all transgenic mice for either the GFP-tagged or Full-

Length Tyro3 transgene are pooled (D, p < 0.01). (E) Transgenic anx/anx mice weigh

almost twice as much as their non-transgenic littermates (p < 0.01 for all groups).

92.8%6.001g ± 0.520(n = 4)

3.113g ± 0.268(n = 5)Full-Length #1

69.5%5.672g ± 0.800(n = 18)

Rescue-GFP#2, 4, 6

83.5%6.157g ± 1.176(n = 15)3.355g ± 0.580

(n = 5)

Rescue-GFP#1

% WeightIncreaseanx/anx; Tganx/anxMouse Line

Table 2. Percentage Bodyweight Difference in TransgenicMouse Lines

62

65

or past P35, unlike 9 of their anx/anx littermates that died by P21 (fig 10, p < 0.01, Χ2).

This is approximately a 66.7% increase in the lifespan of anx homozygotes. When the

lifespan of all other GFP-tagged mouse lines are combined, 19 transgenic anx/anx

animals survived at least until P35 compared to 7 anx/anx littermates that died by P21.

In two full-length human Tyro3 transgenic lines, ten transgenic anx/anx animals

exhibited the same lifespan increase compared to nine anx/anx littermates (p <

0.01, Χ2). Several transgenic anx/anx animals survived beyond P42, essentially

doubling their expected lifespan.

Figure 10. Survival of anx/anx Animals from Transgenic LinesN

umbe

r of M

ice

25

20

15

10

5

0

n = 9 0 0 23 8 0 0 19 4 0 0 5 5 0 0 5

Rescue-GFP Rescue-GFP Full-Length Full-Length #1 #2, 4, 5, 6 #1

#2

0

5

10

15

20

25

1 2 3 4 5 6 7 8anx/anx anx/anx; Tyro3-GFP

anx/anx anx/anx; Tyro3-GFP

anx/anx anx/anx; Tyro3-FL

anx/anx anx/anx; Tyro3-FL

Do not survive past P21

Survived past P35

66

67

Figure 10. Analysis of survival rates of transgenic and non-transgenic anx/anx

progeny. anx/anx homozygous mice are characterized by head weaving and

uncoordinated gait appearing between P15-P18 and die by P21. In all mouse lines

transgenic for either the GFP-tagged or Full-Length Tyro3 transgene, transgenic

anx/anx progeny show no or mild anx phenotypes at P21, which progressively increase

until death after P35. Many transgenic progeny survive up to and past P42, essentially

doubling the lifespan of anx/anx homozygous littermates.

68

Chapter 4: Discussion

4.1 Signal sequence mutations

Here, I have identified a point mutation in the signal sequence of the receptor

tyrosine kinase Tyro3 that is almost certainly causative of the anx mutation. A number

of mutations have previously been identified in the signal sequence of certain genes

that have affected post-translational processing and manifested in disease. A point

mutation causing an arginine to glycine conversion in the signal peptide of the human

factor X, known as XSanta Domingo inhibits cleavage by signal peptidase, even though the

mutant form is normally transported to the endoplasmic reticulum (Racchi et al., 1993).

The mutation also inhibits further post-translational processing of the mutant form,

which is retained in the endoplasmic reticulum, and is neither glycosylated nor secreted,

which normally occurs in the unaffected protein. This mutation is directly responsible for

bleeding diathesis in affected individuals.

Although bioinformatic analysis did not predict an appreciable impact on Tyro3

processing and we did not observe dramatic changes in the gycosylation state of the

extracellular domain or localization of the mutant protein in cell culture, the absence of

noticeable changes assayed in vitro may not be able to uncover subtle changes

incurring dramatic effects in vivo. For example, Smad4 is a necessary component for

proper early mouse development and is shuttled constantly between the cytoplasm and

nucleus by the presence of a nuclear localization and nuclear export signal. When the

nuclear export signal is mutated, Smad4 has been shown to sequester in the nucleus of

embryonic stem cells and is therefore hypothesized to effect TGF-β signaling in these

69

cells and negatively affect embryonic development. However, mice engineered with

these mutations develop normally, indicating that Smad4 nucleocytoplasmic shuttling is

not essential for normal mouse embryonic development and that compensatory

mechanisms may exist (Biondi et al., 2007). Furthermore, the question remains as to

whether R7W-Tyro3 protein is indeed secreted in vivo, if it is stable, and if it’s tyrosine

kinase activity is retained.

4.2 Expression analysis

Consistent with previous results in the adult mouse brain, Tyro3 mRNA was

detected in layers 2/3, 5 and 6 of the cerebral cortex, in the CA1 region of the

hippocampus, in the median eminence of the hypothalamus, and in the granule cells of

the cerebellum (Lai et al., 1994, Schulz et al., 1995). However, in contrast to these

studies, I consistently detected Tyro3 mRNA at different expression levels in certain

areas and in other, previously unnoticed brain regions. These include the CA3 region

and dentate gyrus of the hippocampus, the arcuate nucleus and ventromedial nucleus

of the hippocampus, and the Purkinje cells of the cerebellum. These striking differences

can perhaps be attributed to several factors. In previous studies, radiolabeled Tyro3

cDNA riboprobes may not have had the necessary signal strength to be detected even

after extensive exposure to autoradiographic film in lower Tyro3 expressing brain

regions. This may be especially true for cerebellar Purkinje cells, in which the finer

Tyro3 expression outlining these neurons may be masked by the much stronger granule

cell expression. As well, the small region encompassing arcuate nucleus neurons in the

hypothalamus located at the dorsal edge of the brain may not produce an adequate

70

signal using radiolabeled probes that were consistently detected using DIG-labeled

riboprobes. Also, within the hypothalamus, the ventromedial nuclei are clustered very

closely to the midline of the brain. Sagittal sections that are not adjacent to the midline

may indeed “miss” this cluster of cells which are present in serial coronal sections

through the hypothalamus.

We detected Tyro3 mRNA consistently in the CA3 region and dentate gyrus in

the hippocampus at comparable levels to CA1, which would not be rectified by the

possible explanations given above for other brain regions. Why previous studies did not

detect as strong or virtually any signal in these regions remains a mystery. One

possibility is that Tyro3 expression specifically in the CA3 region and dentate gyrus is

reduced after P21. These previous studies only used “adult” mouse brains but did not

specify the exact age of the mice used. Another possibility is that our method of OCT

embedding our brains somehow preserves expression in these regions that are

negatively affected via paraffin embedding. However, this is very unlikely since other

areas expressing Tyro3 should also be affected in paraffin-embedded brains.

At P21, Tyro3 mRNA expression in anx/anx brains is altered in all Tyro3

expressing regions. In the cortex, Tyro3 expression is markedly reduced, and appears

virtually absent in layer 2. In the hippocampus, the thickness of the band expressing

Tyro3 in CA1 is reduced, and the neurons of the dentate gyrus are often marked with

internal regions of dark staining. The hypothalamus and cerebellum have a complete

absence of Tyro3 in the arcuate and ventromedial nuclei, and of the Purkinje cells,

respectively. In summary, Tyro3 is not only expressed in regions and neuronal

populations affected in anx mutant homozygotes, but in these animals, Tyro3

71

expression is markedly reduced. To date, no other gene with known or postulated roles

in neurodevelopment has been found to have such widespread change in its level of

pattern or expression in anx/anx animals. Thus Tyro3 expression in normal and mutant

animals is consistent with its possible role as the anx causative gene.

4.3 Partial rescue in transgenic mice

The presence of either GFP-tagged mouse or full-length human Tyro3 transgene

in anx/anx mice imparted partial rescue of the anx homozygous phenotype, reducing

the phenotype severity and increasing the overall health and bodyweight of these

animals at P21, and nearly doubling the lifespan of anx/anx homozygotes. The

question of whether this increase in weight is due to the reduced hyperactivity seen in

these animals, leading to less energy expenditure, or to somewhat corrected energy

and appetite regulation, is in part answered by NPY immunohistochemistry performed

by Dr. Sabine Cordes. In the hypothalamus of normal mice, axons from the arcuate

nucleus project anteriorly and dorsally to target areas including the paraventricular

nucleus and lateral hypothalamus, among other regions. Neuropeptide Y, which is

produced in the cell bodies of arcuate nucleus neurons, can be identified by the diffuse

staining ventral to this region where it is being transported in axons leading to the

presynaptic terminal. In anx/anx animals, Neuropeptide Y is found in the cell bodies of

arcuate nucleus neurons, with very little detectable staining in ventral regions (Cordes,

unpublished). Although Neuropeptide Y production does not appear affected, the

sequestering of the protein within arcuate nucleus neuron is the result of a failure of

proper cell body and possibly axon migration, thereby stunting the growth of these

72

projections (Cordes, unpublished). Interestingly, the hypothalamus of the transgenic

anx/anx mouse shows a phenotype intermediate between that of normal and anx/anx

mice. There appears to be a greater amount of NPY staining in the region ventral to the

arcuate nucleus, though not as great as in the normal brain, and there is less apparent

NPY staining in the arcuate nucleus neurons themselves compared to the anx/anx

hypothalamus. In P21 anx/anx mice, NPY-positive neurons are present in small

numbers in the arcuate nucleus, but some reside in the median eminence, a region

normally devoid of NPY-positive neurons. The transgenic anx/anx hypothalamus shows

NPY-positive neurons in mid-migration moving out of the median eminence towards the

arcuate nucleus. This intermediate phenotype at the neurobiological level appears to

reflect the intermediate effect of improved body weight in transgenic anx/anx animals.

The question as to the effect of the anx mutation on the absence of Tyro3 in key

areas of the hypothalamus involved in energy balance and appetite regulation gives rise

to a number of considerations. First, the failure of arcuate nucleus neurons to project

from the cell body suggests that Tyro3 is involved in axonal outgrowth and/or axonal

stability. Second, Tyro3 may also be responsible for stabilizing synapses between

incoming axons and target locations via cell adhesion or signaling at the terminal fields.

The inclusion of both Ig and FNIII domains in the extracellular region suggests that

Tyro3 expression in these cells may induce multiple consequences in order for axons to

project in the proper orientation and to secure proper synapses. Furthermore, Tyro3 is

known to mediate its downstream effects via homophilic interactions with other Tyro3

molecules. A disruption of the interactions of Tyro3 and the reduced expression within

the hypothalamus may ultimately result in the alterations observed in the hypothalamus.

73

The question remains as to why there is only partial rescue of the anx phenotype

in these transgenic mice. Initially, we had hypothesized that the C-terminal GFP tag

might disrupt the ability of Tyro3 receptors to signal normally by interfering with Tyro3

dimerization, or that GFP disallowed phosphorylation of tyrosine kinase targets.

Alternatively, GFP-mediated interactions between expressed transgenes may lead to

constitutive kinase activity causing additional disruptions in downstream activity.

Previous finding have shown that overexpression of Tyro3 causes ligand-independent

activation. However, full-length Tyro3 failed to fully rescue the anx phenotype and

these animals exhibited the same extent of partial rescue. We are currently

investigating several other possibilities. First, the transgenes may not contain all the

regulatory regions upstream of the 9.5kb genomic region cloned into the transgene or

perhaps within intronic regions which were removed between exons 3 to 18. Due to

this, normal Tyro3 may not be expressed in all the endogenous brain regions and/or at

physiologically relevant levels. Second, the anx mutation may cause dominant aversive

defects which cannot be completely overcome by the presence of a transgene. In this

case, the anx mutation might be a gain-of-function mutation that leads to a disruption of

normal brain development. Our most recent evidence from the cerebellum of mice

transgenic for the R7W-Tyro3 construct supports this hypothesis. As performed by Dr.

Sabine Cordes, immunohistochemical staining of Purkinje cells using an antibody

against calbindin showed that in the anx/anx cerebellum, Purkinje cells are present

though rounder and less uniformly-spaced than normal Purkinje cells, however, a large

number of Purkinje cells are missing in anx/anx cerebellum expressing R7W-Tyro3. In

the case that the anx mutation was a loss-of-function mutation, we would expect that

74

the presence of R7W-Tyro3 would have no effect on Purkinje cells in anx/anx mice.

These data suggest that the R7W-Tyro3 allele indeed has gain of function activity that is

deleterious to Purkinje cells and possibly, other neurons. These observations may

reflect a functional deficit in signaling through Tyro3-mediated pathways considering the

vast dendritic arborizations and inputs received by these cells, and thus neuronal

stimulation may also be a requirement for neuron survival.

4.4 Evidence suggestive that Tyro3 is causative of the anx mutation

In the anx/anx animal, we have identified a point mutation in Tyro3, a receptor

tyrosine kinase that was initially implicated for having roles in oncogenesis. The onset,

level, and pattern of Tyro3 expression within the nervous system are strongly correlated

to serotonergic innervation and targeting, and its role in neural development. Firstly,

Tyro3 expression in serotonergic target areas, namely the cortex, the hippocampus, the

hypothalamus, and the cerebellum, is upregulated just after birth during a period of time

when axonal targeting and synaptogenesis of serotonergic projections is maximal. It is

also these target areas that have extensive innervation due to aberrant hypersprouting

of serotonergic projections in the anx/anx animal. Furthermore, Tyro3 is expressed in

the brainstem similar to regions where the raphe nuclei reside. Secondly, mice that are

homozygous for this point mutation are the only animals that exhibit anx phenotype

characteristics, which has been shown in nearly 200 affected animals in 3 different

background strains. No other animal that is either heterozygous or lacking the mutation

altogether shows any abnormal behaviours associated with the anx phenotype. Thirdly,

Tyro3 expression in anx/anx brains is greatly affected in its level and pattern of

75

expression. In some areas, such as the cortex and hippocampus, there appears to be a

reduction of detectable Tyro3 mRNA as assayed by RNA in situ hybridization. In other

areas, such as the arcuate nucleus and ventromedial nucleus of the hypothalamus, and

of the Purkinje cells of the cerebellum, there is no detectable Tyro3 expression which is

clearly evident in normal brains. Fourthly, anx/anx animals carrying a normal Tyro3

transgene show partial rescue in bodyweight at P21 and of the failure to thrive

phenotype. These transgenic animals show attenuated anxiogenic behaviours, appear

healthier, and live almost twice as long as their non-transgenic anx/anx littermates.

Taken together, these data strongly suggest that the point mutation in Tyro3 is the

causative agent of the anorexia mutation and its associated phenotypes and

behaviours.

4.5 Possible roles of Tyro3

In anx/anx homozygotes, the reduction in Tyro3 mRNA may result from several

possibilities. The point mutation itself may lead to instability of the RNA transcript

thereby deteriorating prior to translation on its own or it may also be directed towards an

mRNA decay pathway. Tyro3 mRNA trafficking might also be disrupted, disallowing

transcripts to reach target areas prior to translation. However, if the anx mutation is

indeed a gain of function mutation in Tyro3, retrograde signaling from overactive Tyro3

may somehow downregulate Tyro3 mRNA transcription. In non-neural cells, the

intracellular kinase domain has been shown to immunoprecipitate with src, RanBPM,

and, in particular, the regulatory subunit p85 of phosphatidylinositol 3-kinase (PI3K) and

activation of Tyro3 leads to Akt phosphorylation. Akt phosphorylation has been shown

76

to affect neuronal survival, axon specification, axon outgrowth, neurite outgrowth, cell

polarity, neuroprotective effects due to injury and neurotoxicity. Interestingly, Akt

overexpression has been shown to inhibit cell differentiation in adult hippocampal neural

progenitor cells (Peltier et al., 2007). We are currently investigating whether Akt

phosphorylation is affected in anx/anx brains via Western Blot analysis and in R7W-

Tyro3 expressing cell lines.

At this point, our observations point to mislocalization of Tyro3 RNA and protein

as the likely primary cause of the anx phenotype, and, from preliminary Western

analyses, Akt phosphorylation and thus, Tyro3 signaling is only mildly impacted.

77

CONCLUSION

Partial rescue of the anx/anx phenotype by the presence of a normal Tyro3

transgene combined with disruptions in the mRNA expression in mutant brains and

100% linkage to a point mutation within the signal sequence lead us to believe that

Tyro3 is the anx causative gene. Our most recent data suggest that this is a gain-of-

function mutation and we are exploring whether RNA localization abnormalities

particularly evident in the cerebellum are a primary or secondary consequence of the

R7W-Tyro3 mutation and/or this mutation disrupts Tyro3 signaling. Continued

biochemical analysis to assess changes in the brain and in downstream signaling

pathways will elucidate the mechanisms underlying the neurobiological phenotypes

observed in the anx/anx mouse, and may ultimately provide greater insight into the

neurological pathologies of human affective disorders at the genetic, molecular, and

cellular level.

78

References

Allen, Y. S., Adrian, T. E., Allen, J. M., Tatemoto, K., Crow, T. J., Bloom, S. R.,

Polak, J. M. (1983). Neuropeptide Y distribution in the rat brain. Science 221, 877-879.

Bagri, A., Marin, O., Plump, A. S., Mak, J., Pleasure, S. J., Rubenstein, J. L.,

Tessier-Lavigne, M. (2002). Slit proteins prevent midline crossing and determine the

dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron 33,

153-155.

Bentley, C. A., Lee, K. F. (2000). p75 is important for axon growth and Schwann cell

migration during development. J Neurosci 20, 7706-7715.

Bibel, M., Hoppe, E., Barde, Y. A. (1999). Biochemical and functional interactions

between the neurotrophin receptors Trk and p75NTR. EMBO J 18, 616-622.

Biesecker, L. G., Giannola, D. M., Emerson, S. G. (1995). Identification of alternative

exons, including a novel exon, in the tyrosine kinase receptor gene Etk2/tyro3 that

explain differences in 5’ cDNA sequences. Oncogene 10, 2239-2242.

Biondi, C. A., Das, D., Howell, M., Islam, A., Bikoff, E. K., Hill, C. S., Robertson, E.

J. (2007). Mice develop normally in the absence of Smad4 nucleocytoplamic shuttling.

Biochem J 404, 235-245.

Blesch, A., Grill, R. J., Tuszynski, M. H. (1998). Neurotrophin gene therapy in CNS

models of trauma and degeneration. Prog Brain Res 117, 473-484.

Bloch, B., Bugnon, C., Fellmann, D., Lenys, D. (1978). Immunocytochemical

evidence that the same neurones in the human infundibular nucleus are stained with

anti-endorphins and antisera of other related peptides. Neurosci Lett 10, 147-152.

79

Bloom, S. R., Battenberg, E., Rossier, J., Ling, N., Guillemin, R. (1978). Neurones

containing beta-endorphin in rat brain exist separately from those containing

enkephalin: immunocytochemical studies. Proc Natl Acad Sci U S A 75, 1591-1595.

Bonnin, A., Torii, M., Wang, L., Rakic, P., Levitt, P. (2007). Serotonin modulates the

response of embryonic thalamocortical axons to netrin-1. Nat Neurosci 10, 588-597.

Bothwell, M. (1995). Functional interaction of neurotrophins and neurotrophin

receptors. Annu Rev Neurosci 18, 223-53.

Brady, L. S., Smith, M. A., Gold, P. W., Herkenham, M. (1990). Altered expression of

hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats.

Neuroendocrinology 52, 441-447.

Briscoe, J., Sussel, L., Serup, P., Hartigan-O'Connor, D., Jessell, T. M.,

Rubenstein, J. L., Ericson, J. (1999). Homeobox gene Nkx2.2 and specification of

neuronal identity by graded Sonic hedgehog signalling. Nature 398, 622-7.

Broberger, C., Johansen, J., Schalling, M., Hokfelt, T. (1997a). Hypothalamic

neurohistochemistry of the murine anorexia (anx/anx) mutation: altered processing of

neuropeptide Y in the arcuate nucleus. J Comp Neurol 387, 124-135.

Broberger, C., Landry, M., Wong, H., Walsh, J., Hokfelt, T. (1997b). Subtypes Y1

and Y2 of the neuropeptide Y receptor are respectively expressed in

proopiomelanocortin and neuropeptide Y-containing neurones of the rat hypothalamic

arcuate nucleus. Neuroendocrinology 66, 393-408.

Broberger, C., Johansen, J., Johansson, C., Schalling, M., Hokfelt, T. (1998). The

neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic,

and monosodium glutamate-treated mice. Proc Natl Acad Sci U S A 95, 15043-15048.

80

Brodski, C., Weisenhorn, D. M., Signore, M., Sillaber, I., Oesterheld, M., Broccoli,

V., Acampora, D., Simeone, A., Wurst, W. (2003). Location and size of dopaminergic

and serotonergic cell populations are controlled by the position of the midbrain-

hindbrain organizer. J Neurosci 23,4199-4207.

Bruckner, K., Klein, R. (1998). Signaling by Eph receptors and their ephrin ligands.

Curr Opin Neurobiol 8, 375-382.

Caza, P. A., Spear, L. P. (1982). Pharmacological manipulation of milk-induced

behaviors in three-day-old rat pups. Pharmacol Biochem Behav 16, 481-486.

Cheng, L., Chen, C. L., Luo, P., Tan, M., Qiu, M., Johnson, R., Ma, Q. (2003). Lmx1b,

Pet-1, and Nkx2.2 coordinately specify serotonergic neurotransmitter phenotype. J

Neurosci 23, 9961-9967.

Cheng, H. J., Nakamoto, M., Bergemann, A. D., Flanagan, J. G. (1995).

Complementary gradients in expression and binding of ELF-1 and Mek4 in development

of the topographic retinotectal projection map. Cell 82, 371-381.

Chronwall, B. M., DiMaggio, D. A., Massar, V. J., Pickel, V. M., Ruggeiro, D. A.,

O’Donahue, T. L. (1985). The anatomy of neuropeptide Y-containing neurones in rat

brain. Neuroscience 15, 1159-1181.

Clary, D. O., Reichardt, L. F. (1994). An alternatively spliced form of the nerve growth

factor receptor TrkA confers an enhanced response to neurotrophin-3. Proc Natl Acad

Sci U S A 91, 11133-11137.

Cordes, S. P. (2005). Molecular genetics of the early development of hindbrain

serotonergic neurons. Clin Genet 68, 487-494.

81

Cowan, C. A., Henkemeyer, M. (2002). Ephrins in reverse, park and drive. Trends Cell

Biol 12 ,339-346.

Craven, S. E., Lim, K. C., Ye, W., Engel, J. K., de Sauvage, F., Rosenthal, A. (2004).

Gata2 specifies serotonergic neurons downstream of sonic hedgehog. Development

131, 1165-1173.

Crosier, K. E., Hall, L. R., Lewis, P. M., Morris, C. M., Wood, C. R., Morris, J. C.,

Crosier, P. S. (1994). Isolation and characterization of the human DTK receptor

tyrosine kinase. Growth Factors 11, 137-144.

Csiffary, A., Gorcs, T. J., Palkovits, M. (1990). Neuropeptide Y innervation of ACTH-

immunoreactive neurones in the arcuate nucleus of rats: a correlated light and electron

microscopic double immunolabeling study. Brain Res 506, 215-222.

Dahlback, B., Villoutreix, B. O. (2005). Regulation of blood coagulation by the protein

C anticoagulant pathway: novel insights into structure-function relationships and

molecular recognition. Arterioscler Thromb Vasc Biol 25, 1311-1320.

Dai, W., Pan, H., Hassanain, H., Gupta, S. L., Murphy, M. J. Jr. (1994). Molecular

cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and

testis. Oncogene 9, 975-979.

Davy, A., Soriano, P. (2005). Ephrin signaling in vivo: look both ways. Dev Dyn 232, 1-

10.

DeQuidt, M. E., Emson, P. C. (1986). Distribution of neuropeptide Y-like

immunoreactivity in the rat central nervous system. Immunohistochemical analysis.

Neuroscience 118, 545-618.

82

Descamps, S., Toillon, R. A., Adriaenssens, e., Pawlowski, V., Cool, S. M.,

Nurcombe, V., Le Bourhis, X., Boilly, B., Peyrat, J. P., Hondermarck, H. (2001).

Nerve growth factor stimulates proliferation and survival of human breast cancer cells

through two distinct signaling pathways. J Biol Chem 276, 17864-17870.

Descarries, L., Watkins, K. C., Garcia, S., Beaudet, A. (1982). The serotonin neurons

in nucleus raphe dorsalis of adult rat: a light and electron microscope radioautographic

study. J Comp Neurol 207, 239-254.

Descarries, L., Beaudet, A., Watkins, K. C. (1975). Serotonin nerve terminals in adult

rat neocortex. Brain Res 100, 563-588.

Ding, Y. Q., Marklund, U., Yuan, W, Yin, J., Wegman, L., Ericson, J., Deneris, E.,

Johnson, R. L., Chen, Z. F. (2003). Lmx1b is essential for the development of

serotonergic neurons. Nat Neurosci 6, 933-938.

Dori, I., Dinopoulos, A., Blue, M. E., Parnavelas, J. G. (1996). Regional differences in

the ontogeny of the serotonergic projection to the cerebral cortex. Exp Neurol 138, 1-14.

Dottori, M., Hartley, L., Galea, M., Paxinos, G., Polizzotto, M., Kilpatrick, T.,

Bartlett, P. F., Murphy, M., Kontgen, F., Boyd, A. W. (1998). Epha4 (Sek1) receptor

tyrosine kinase is required for the development of the corticospinal tract. Proc Natl Acad

Sci U S A 95, 13248-13253.

Drescher, U., Kremoser, C., Handwerker, C., Loschinger, J., Noda, M., Bonhoeffer,

F. (1995). In vitro guidance of retinal ganglion cell axons by RAGS, a 25kDA tectal

protein related to ligands for Eph receptor tyrosine kinases. Cell 82, 359-370.

83

Edwards, R. H., Rutter, W. J., Hanahan, D. (1989). Directed expression of NGF to

pancreatic beta cells in transgenic mice leads to selective hyperinnervation of the islets.

Cell 58, 161-170.

Egea, J., Nissen, U. V., Dufour, A., Sahin, M., Greer, P., Kullander, K., Mrsic-Flogel,

T. D., Greenberg, M. E., Kiehn, O., Vanderhaegen, P., Klein, R. (2005). Regulation of

EphA4 kinase activity is required for a subset of axon guidance decisions suggesting a

key role for receptor clustering in Eph function. Neuron 47, 515-28.

Eisenberger, N. I., Way, B. M., Taylor, S. E., Welch, W. T., Lieberman, M. D. (2007).

Understanding genetic risk for aggression: clues from the brain’s response to social

exclusion. Biol Psychiatry 61, 1100-8.

Elias, C. F., Lee, C., Kelly, J., Aschkenasi, C., Ahima, R. S., Couceyro, P. R., Kuhar,

M. J., Saper, C. B., Elmquist, J. K. (1998). Leptin activates hypothalamic CART

neurons projecting to the spinal cord. Neuron 21, 1375-1385.

Elias, C. F., Aschkenasi, C., Lee, C, Kelly, J., Ahima, R. S., Bjorbaek, C., Flier, J. S.,

Saper, C. B., Elmquist, J. K. (1999). Leptin differentially regulates NPY and POMC

neurons projecting to the lateral hypothalamic area. Neuron 23, 775-86.

Elmquist, J. K., Elias, C. F., Saper, C. B. (1999). From lesions to leptin: hypothalamic

control of food intake and body weight. Neuron 22, 221-232.

Fan, W., Boston, B. A., Kesterson, R. A., Hruby, V. J., Cone, R. D. (1997). Role of

melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385,

165-168.

84

Feinn, R., Nellissery, M., Kranzler, H. R. (2005). Meta-analysis of the association of a

functional serotonin transporter promoter polymorphism with alcohol dependence. Am J

Med Genet B Neuropsychiatr Genet 133, 79-84.

Feldheim, D. A., Vanderhaeghen, P., Hansen, M. J., Frisen, J., Lu, Q., Barbacid, M.,

Flanagan, J. G. (1998). Topographic guidance labels in a sensory projection to the

forebrain. Neuron 21, 1303-1313.

Feldheim, D. A., Kim, Y. I., Bergemann, A. D., Frisen, J., Barbacid, M., Flanagan, J.

G. (2000). Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in

multiple aspects of retinocollicular mapping. Neuron 25, 563-574.

Flanagan, J. G., Vanderhaeghen, P. (1998). The ephrins and Eph receptors in neural

development. Annu Rev Neurosci 21, 309-345.

Frade, J. M., Barde, Y. A. (1998). Nerve growth factor: two receptors, multiple

functions. Bioessays 20, 137-45.

Friedman, W. J. (2000). Neurotrophins induce death of hippocampal neurons via the

p75 receptor. J Neurosci 20, 6340-6.

Fujimoto, S., Yamamoto, T. (1994). brt, a mouse gene encoding a novel receptor-type

protein-tyrosine kinase, is preferentially expressed in the brain. Oncogene 238, 15-22.

Funakoshi, H., Yonemasu, T., Nakano, T., Matumoto, K., Nakamura, T. (2002).

Identification of Gas6, a putative ligand for Sky and Axl receptor tyrosine kinases, as a

novel neurotrophic factor for hippocampal neurons. J Neurosci Res 68, 150-160.

Furmark, T., Tillfors, M., Garpenstrand, H., Marteinsdottir, I., Langstrom, B.,

Oreland, L., and Fredrikson, M. (2004). Serotonin transporter polymorphism related to

85

amygdala excitability and symptom severity in patients with social phobia. Neurosci Lett

362, 189-192.

Fuxe, K., Tinner, B., Caberlotto, L., Bunnemann, B., Agnati. L. F. (1997). NPY Y1

receptor like immunoreactivity exists in a subpopulation of β-endorphin immunoreactive

nerve cells in the arcuate nucleus: a double immunolabelling analysis in the rat.

Neurosci Lett 225, 49-52.

Gale, N. W., Holland, S. J., Valenzuela, D. M., Flenniken, A., Pan, L., Ryan, T. E.,

Henkemeyer, M., Strebhardt, K., Hirai, H., Wilkinson, D. G., Pawson, T., Davis, S.,

Yancopoulos, G. D. (1996). Eph receptors and ligands comprise two major specificity

subclasses and are reciprocally compartmentalized during embryogenesis. Neuron 17,

9-19.

Gerald, C., Walker, M. W., Criscione, L., Gustafson, E. L., Batzi-Hartmann, C.,

Smith, K. E., Vaysse, P., Durkin, M. M., Laz, T. M., Linemeyer, D. L., Schaffhauser,

A. O., Whitebread, S., Hofbauer, K. G., Taber, R. I., Branchek, T. A., Weinshank, R.

L. (1996). A receptor subtype involved in neuropeptide-Y-induced food intake. Nature

382, 168-171.

Godowski, P. J., Mark, M. R., Chen, J., Sadick, M. D., Raab, H., Hammonds, R. G.

(1995). Reevaluation of the roles of protein S and Gas6 as ligands for the receptor

tyrosine kinase Rse/Tyro3. Cell 82, 355-358.

Goldshmit, Y., McLenachan, S., Turnley, A. (2006). Roles of Eph receptors and

ephrins in the normal and damaged adult CNS. Brian Res Rev 52, 327-345.

86

Grill, H. J., Ginsberg, A. B., Seeley, R. J., Kaplan, J. M. (1998). Brainstem application

of melanocortin receptor ligands produces long-lasting effects on feeding and body

weight. J Neurosci 18, 10128-10135.

Guidry, G., Landis, S. C., Davis, B. M., Albers, K. M. (1998). Overexpression of nerve

growth factor in epidermis disrupts the distribution and properties of sympathetic

innervation in footpads. J Comp Neurol 393, 231-243.

Hafizi, S., Dahlback, B. (2006). Signalling and functional diversity within the Axl

subfamily of receptor tyrosine kinases. Cytokine Growth Factor Rev 17, 295-304.

Hahn, T. M., Breininger, J. F., Baskin, D. G., Schwartz, M. W. (1998). Coexpression

of Agrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1, 271-272.

Heisler, L. K., Chu, H. M., Brennan, T. J., Danao, J. A., Bajwa, P., Parsons, L. H.,

Tecott, L. H. (1998). Elevated anxiety and antidepressant-like responses in serotonin 5-

HT1A receptor mutant mice. Proc Natl Acad Sci U S A 95, 15049-54.

Heisler, L. K., Tecott, L. H. (1999). Knockout Corner: Neurobehavioural consequences

of a serotonin 5-HT(2C) receptor gene mutation. Int J Neuropsychopharmacol 2, 67-69.

Hellard, D., Brosenitsch, T., Fritzsch, B., Katz, D. M. (2004). Cranial sensory neuron

development in the absence of brain-derived neurotrophic factor in BDNF/Bax double

null mice. Dev Biol 275, 34-43.

Hempstead, B. L., Martin-Zanca, D., Kaplan, D. R., Parada, L. F., Chao, M. V.

(1991). High-affinity NGF binding requires co-expression of the trk proto-oncogene and

the low affinity NGF receptor. Nature 350, 678-683.

87

Hendricks, T., Francis, N., Fyodorov, D., Deneris, E. S. (1999). The ETS domain

factor Pet-1 is an early and precise marker of central serotonin neurons and interacts

with a conserved element in serotonergic genes. J Neurosci 19, 10348-10356.

Henkemeyer, M., Orioli, D., Henderson, J. T., Saxton, T. M., Roder, J., Pawson, T.,

Klein R. (1996). Nuk controls pathfinding of commissural axons in the mammalian

central nervous system. Cell 86, 35-46.

Henneberger, C., Grantyn, R., Rothe, T. (2000). Rapid genotyping of newborn gene

mutant mice. J Neurosci Methods 100, 123-126.

Holland, S. J., Peles, E., Pawson, T., Schlessinger, J. (1998). Cell-contact-dependent

signaling in axon growth and guidance: Eph receptor tyrosine kinases and receptor

protein tyrosine phosphatase beta. Curr Opin Neurobiol 8, 117-127.

Holmes, A., Murphy, D. L., Crawley, J. N. (2003). Abnormal behavioral phenotypes of

serotonin transporter knockout mice: parallels with human anxiety and depression. Biol

Psychiatry 54, 953-9.

Hong, C. J., Chen, T. J., Yu, Y. W., Tsai, S. J. (2006). Response to fluoxetine and

serotonin 1a receptor (C-1019G) polymorphism in Taiwan Chinese major depressive

disorder. Pharmacogenomics J 6, 27-33.

Hu, X., Giotakis, O., Li, T., Karwautz, A., Treasure, J., Collier, D. A. (2003).

Association of the 5-HT2c gene with susceptibility and minimum body mass index in

anorexia nervosa. Neuroreport 14, 781-3.

Hynes, M., Ye, W., Wang, K., Stone, D., Murone, M., Sauvage, F., Rosenthal, A.

(2000). The seven-transmembrane receptor smoothened cell-autonomously induces

multiple ventral cell types. Nat Neurosci 3, 41-6.

88

Klein, R. (2004). Eph/ephrin signaling in morphogenesis, neural development and

plasticity. Curr Opin Cell Biol 16, 580-589.

Klein, R., Conway, D., Parada, L. F., Barbacid, M. (1990). The TrkB tyrosine protein

kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase

domain. Cell 61, 647-656.

Kohler, C., Chan-Palay, V., Steinbusch, H. (1982). The distribution and origin of

serotonin-containing fibers in the septal area: a combined immunohistochemical and

fluorescent retrograde tracing study in the rat. J Comp Neurol 209, 91-111.

Kohler, C., Steinbusch, H. (1982). Identification of serotonin and non-serotonin-

containing neurons of the mid-brain raphe projecting to the entorhinal area and the

hippocampal formation. A combined immunohistochemical and fluorescent retrograde

tracing study in the rat brain. Neuroscience 7, 951-975.

Kraus, M. R., Al-Taie, O., Schafer, A., Pfersdorff, M., Lesch, K. P., Scheurlen, M.

(2007). Serotonin-1A receptor gene HTR1A variation predicts interferon-induced

depression in chronic hepatitis C. Gastroenterology 132, 1279-86.

Kristensen, P., Judge, M. J., Thim, L., Ribel, U., Christjansen, K. N., Wulff, B. S.,

Clausen, J. T., Jensen, P. B., Madsen, O. D., Vrang, N., Larsen, P. J., Hastrup, S.

(1998). Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 393,

293-298.

Kullander, K., Mather, N. K., Diella, F., Dottori, M., Boyd, A. W., Klein, R. (2001).

Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon

tract formation in vivo. Neuron 29,73-84.

89

Kullander, K., Klein, R. (2002). Mechanisms and functions of Eph and ephrin signaling.

Nat Rev Mol Cell Biol 3, 475-486.

Lackmann, M., Oates, A. C., Dottori, M., Smith, F. M., Do, C., Power, M., Kravets,

L., Boyd, A. W. (1998). Distinct subdomains of the EphA3 receptor mediate ligand

binding and receptor dimerization. J Biol Chem 273, 13383-13392.

Lai, C., Gore, M., Lemke, G. (1994). Structure, expression, and activity of Tyro3, a

neural adhesion-related receptor tyrosine kinase. Oncogene 9, 2567-2578.

Lambert, P. D., Couceyro, P. R., McGirr, K. M., Dall Vechia, S. E., Smith, Y., Kuhar,

M. J. (1998) CART peptides in the central control of feeding and interactions with

neuropeptide Y. Synapse 29, 293-298.

Lawrence, C. B., Snape, A. C., Baudoin, F. M., Luckman, S. M. (2002). Acute central

ghrelin and GH secretagogues induce feeding and activate brain appetite centers.

Endocrinology 143, 155-62.

Lee, K. F., Bachman, K., Landis, S., Jaenisch, R. (1994). p75-deficient embryonic

dorsal root sensory and neonatal sympathetic neurons display a decreased sensitivity to

NGF. Development 120, 1027-33.

Lemke, G., Lu. Q. (2003). Macrophage regulation by Tyro 3 family receptors. Curr Opin

Immunol 15, 31-36.

Levi-Montalcini, R. (1987). The nerve growth factor 35 years later. Science 237, 1154-

1162.

Liebl, D. J., Morris, C. J., Henkemeyer, M., Parada, L. F. (2003). mRNA expression of

ephrins and Eph receptor tyrosine kinases in the neonatal and adult mouse central

nervous system. J Neurosci Res 71, 7-22.

90

Lidov, H. G., Molliver, M. E. (1982). An immunohistochemical study of serotonin

neuron development in the rat: ascending pathways and terminal fields. Brain Res Bull

8, 389-430.

Liepinsh, E., Ilag, L. L., Otting, G., Ibanez, C. F. (1997). NMR structure of the death

domain of the p75 neurotrophin receptor. EMBO J 16, 4999-5005.

Lu, Q., Gore, M., Zhang, Q., Camenisch, T., Boast, S., Casagranda, f., Lai, C.,

Skinner, M. K., Klein, R., Matsushima, G. K., Earp, H. S., Goff, S. P., Lemke, G.

(1999). Tyro-3 family receptors are essential regulators of mammalian

spermatogenesis. Nature 398, 723-728.

Lu, Q., Lemke, G. (2001). Homeostatic regulation of the immune system by receptor

tyrosine kinases of the Tyro3 family. Science 293, 306-311.

Luellen, B. A., Bianco, L. E., Schneider, L. M., Andrews, A. M. (2007). Reduced

brain-derived neurotrophic factor is associated with a loss of serotonergic innervation in

the hippocampus of aging mice. Genes Brain Behav 6, 482-490.

Lyons, W. E., Mamounas, L. A., Ricaurte, G. A., Coppola, V., Reid, S. W., Bora, S.

H., Wihler, C., Koliatsos, V. E., Tessarolo, L. (1999). Brain-derived neurotrophic

factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain

serotonergic abnormalities. Proc Natl Acad Sci U S A 96, 15239-15244.

Maley, B. E., Engle, M. G., Humphreys, S., Vascik, D. A., Howes, K. A., Newton, B.

W., Elde, R. P. (1990). Monoamine synaptic structure and localization in the central

nervous system. J Electron Microsc Tech 15, 20-33.

Maltais, L. J., Lane, P. W., Beamer, W. G. (1984). Anorexia, a recessive mutation

causing starvation in preweanling mice. J Hered 75, 468-72.

91

Mamounas, L. A., Blue, M. E., Siuciak, J. A., Altar, C. A. (1995). Brain-derived

neurotrophic factor promotes the survival and sprouting of serotonergic axons in rat

brain. J Neurosci 15, 7929-7939.

Mamounas, L. A., Molliver, M. E. (1988). Evidence for dual serotonergic projections to

neocortex: axons from the dorsal and median raphe nuclei are differentially vulnerable

to the neurotoxin p-chloroamphetamine (PCA). Exp Neurol 102, 23-36.

Mann, K. G., Lawson, J. H. (1992). The role of the membrane in the expression of the

vitamin K-dependent enzymes. Arch Pathol Lab Med 116, 1330-6.

Mark, M. R., Scadden, D. T., Wang, Z., Gu, Q, Goddard, A., Godowski, P. J. (1994).

rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at

high levels in the brain. J Biol Chem 269, 10720-10728.

Martin, P., Ohno, M., Southerland, S. B., Mailman, R. B., Suzuki, K. (1994).

Heterotypic sprouting of serotonergic forebrain fibers in the brindled mottled mutant

mouse. Brain Res Dev Brain Res 77, 215-225.

Massat, I., Lerer, B., Souery, D., Blackwood, D., Muir, W., Kaneva, R., Nothen, M.

M., Oruc, L., Papadimitriou, G. N., Dikeos, D., Serretti, A., Bellivier, F., Golmard, J.

L., Milanova, V., Del-Favero, J., Van Broeckhoven, C., Mendlewicz, J. (2007).

HTR2C (cys23ser) polymorphism influences early onset in bipolar patients in a large

European multicenter association study. Mol Psychiatry 12, 797-8.

Matise, M. P., Epstein, D. J., Park, H. L., Platt, K. A., Joyner, A.L. (1998). Gli2 is

required for induction of floor plate and adjacent cells, but not most ventral neurons in

the mouse central nervous system. Development 125, 2759-2770.

92

Matsubara, N., Takahasi, Y, Nishina, Y, Mukouyama, Y., Yanagisawa, M.,

Watanabe, T., Nakano, T., Nomura, K., Arita, H., Nishimune, Y., Obinata, M.,

Matsui, Y. (1996). A receptor tyrosine kinase, Sky, and its ligand Gas 6 are expressed

in gonads and support primordial germ cell growth or survival in culture. Dev Biol 180,

499-510.

Matsushita, S., Suzuki, K., Murayama, M., Nishiguchi, N., Hishimoto, A., Takeda,

A., Shirakawa, O., Higuchi, S. (2004). Serotonin transporter regulatory region

polymorphism is associated with anorexia nervosa. Am J Genet B Neuropsychiatr

Genet 128, 114-117.

Ming, G., Song, H., Berninger, B., Inagaki, N., Tessier-Lavigne, M., Poo, M. (1999).

Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling

in nerve growth cone guidance. Neuron 23, 139-148.

Molliver, M. E. (1987). Serotonergic neuronal systems: what their anatomic

organization tells us about function. J Clin Psychopharmacol 7, 3S – 23S.

Molliver, M. E., Mamounas, L. A., Wilson, M. A. (1989). Effects of neurotoxic

amphetamines on serotonergic neurons: immunocytochemical studies. NIDA Res

Monogr 94, 270-305.

Monteleone, P., Tortorella, A., Castaldo, E., Maj, M. (2006). Associations of a

functional serotonin transporter gene polymorphism with binge eating disorder. Am J

Med Genet B Neuropsychiatr Genet 141, 7-9.Munafo, M. R., Clark, T. G., Roberts, K.

H., Johnstone, E.C. (2006). Neuroticism mediates the association of the serotonin

transporter gene with lifetime major depression. Neuropsychobiology 53, 1-8.

93

Murai, K. K., Pasquale, E. B. (2003). ‘Eph’ective signaling : forward, reverse and

crosstalk. J Cell Sci 116, 2823-2832.

Nakamoto, M., Cheng, H. J., Friedman, G. C., McLaughlin, T., Hansen, M. J., Yoon,

C. H., O’Leary, D. D., Flanagan, J. G. (1996). Topographically specific effects of ELF-1

on retinal axon guidance in vitro and retinal axon mapping in vivo. Cell 86, 755-766.

Nakamoto, M. (2000). Eph receptors and ephrins. Int J Biochem Cell Biol 32, 7-12.

Nakamura, Y. S., Hakeda, Y., Takakura, N., Kameda, T., Hamaguchi, I., Miyamoto,

T., Kakudo, S., Nakano, T., Kumegawa, M., Suda, T. (1998). Tyro 3 receptor tyrosine

kinase and its ligand, Gas6, stimulate the function of osteoclasts. Stem Cells 16, 229-

238.

Nimnual, A. S., Yatsula, B. A., Bar-Sagi, D. (1998). Coupling of Ras and Rac

guanosine triphosphatases through the Ras exchanger Sos. Science 279, 560-563.

Nyberg, P., He, X., Hardig, Y., Dahlback, B., Garcia de Frutos, P. (1997). Stimulation

of Sky tyrosine phosphorylation by bovine protein S-domains involved in the receptor-

ligand interaction. Eur J Biochem 246, 147-154.

Ohashi, K., Mizuno, K., Kuma, K., Miyata, T., Nakamura, T. (1994). Cloning of the

cDNA for a novel receptor tyrosine kinase, Sky, predominantly expressed in brain.

Oncogene 9, 699-705.

Ollmann, M. M., Wilson, B. D., Yang, Y. K., Kerns, J. A., Chen, Y., Gantz, L., Barsh,

G. S. (1997). Antagonism of central melanocortin receptors in vitro and in vivo by

agouti-related protein. Science 278, 135-137.

94

Orioli, D., Henkemeyer, M., Lemke, G., Klein, R., Pawson, T. (1996). Sek4 and Nuk

receptors cooperate in guidance of commissural axons and in palate formation. EMBO J

15, 6035-6049.

Otte, C., McCaffery, J., Ali, S., Whooley, M. A. (2007). Association of a serotonin

transporter polymorphism (5-HTTLPR) with depression, perceived stress, and

norepinephrine in patients with coronary disease: the Heart and Soul Study. Am J

Psychiatry 164, 1307-9.

Patapoutian, A., Reichardt, L. F. (2001). Trk receptors: mediators of neurotrophin

action. Curr Opin Neurobiol 11, 272-280.

Parnavelas, J. G., Papadopoulos, G. C. (1989). The monoaminergic innervation of the

cerebral cortex is not diffuse and nonspecific. Trends Neurosci 12, 315-319.

Patel, P. D., Pontrello, C., Burke, S. (2004). Robust and tissue-specific expression of

TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiatry 55, 428-433.

Pattyn, A., Vallstedt, A., Dias, J. M., Samad, O. A., Krumlauf, R., Rijli, F. M., Brunet,

J. F., Ericson, J. (2003). Coordinated temporal and spatial control of motor neuron and

serotonergic neuron generation from a common pool of CNS progenitors. Genes Dev

17, 729-37.

Peltier, J., O’Neill, A., Schaffer, D. V. (2007). PI3K/Akt and CREB regulate adult

neural hippocampal progenitor proliferation and differentiation. Dev Neurobiol 67, 1348-

1361.

Petit, A., Kennedy, T. E., Bagnard, D., Doucet, G. (2005). Membrane-associated

guidance cues direct the innervation of forebrain and midbrain by dorsal raphe-derived

sertonergic axons. Eur J Neurosci 22, 552-568.

95

Popova, N. K., Maslova, L. N., Morosova, E. A., Bulygina, V. V., Seif, I. (2006). MAO

A knockout attenuates adrenocortical response to various kinds of stress.

Psychoneuroendocrinology 31, 179-86.

Popova, N. K., Skrinskaya, Y. A., Amstislavskaya, T. G., Vishnivetskaya, G. B.,

Seif, I., de Meier, E. (2001). Behavioral characteristics of mice with genetic knockout of

monoamine oxidase type A. Neurosci Behav Physiol 31, 597-602.

Prieto, A. L., Weber, J. L., Lai, C. (2000). Expression of the receptor protein-tyrosine

kinases Tyro-3, Axl, and mer in the developing rat central nervous system. J Comp

Neurol 425, 295-314.

Racchi, M., Watzke, H. H., High, K. A., Lively, M. O. (1993). Human coagulation factor

X deficiency caused by a mutant signal peptidase as a cause for familial central

diabetes insipidus. J Clin Invest 91, 2565-2571.

Raymond, J. R., Mukhin, Y. V., Gelasco, A., Turner, J., Collinworth, G., Gettys, T.

W., Grewal, J. S., Garnoskaya, M. N. (2001). Multiplicity of mechanisms of serotonin

receptor signal transduction. Pharmacol Ther 92, 179-212.

Ringstedt, T., Ibanez, C. F., Nosrat, C. A. (1999). Role of brain-derived neurotrophic

factor in target invasion in the gustatory system. J Neurosci 19, 3507-3518.

Rodriguez-Tebar, A., Dechant, G., Barde, Y. A. (1991). Neurotrophins : structural

relatedness and receptor interactions. Philos Trans R Soc Lond B Biol Sci 331, 255-8.

Rumajogee, P., Verge, D., Hanoun, N., Brisorgueil, M. J., Hen, R., Lesch, K. P.,

Hamon, M., Miquel, M. C. (2004). Adaptation of the serotonergic neuronal phenotype in

the absence of 5-HT autoreceptors or the 5-HT transporter: involvement of BDNF and

cAMP. Eur J Neurosci 19, 937-944.

96

Sakowski, S. A., Geddes, T. J., Thomas, D. M., Levi, E., Hatfield, J. S., Kuhn, D. M.

(2006). Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as

revealed with monospecific antibodies. Brain Res 1085, 11-18.

Sarnyai, A., Sibille, E. L., Pavlides, C., Fenster, R. J., McEwen, B. S., Toth, M.

(2000). Impaired hippocampal-dependent learning and functional abnormalities in the

hippocampus in mice lacking serotonin(1A) receptors. Proc Natl Acad Sci U S A 97,

14731-6.

Schabitz, W. R., Sommer, C., Zoder, W., Kiessling, M., Schwaniger, M., Schwab, S.

(2000). Intravenous brain-derived neurotrophic factor reduces infarct size and

counterregulates Bax and Bcl-2 expression after temporary focal cerebral ischemia.

Stroke 31, 2212-2217.

Schultz, J., Ponting, C. P., Hofmann, K., Bork, P. (1997). SAM as a protein

interaction domain involved in developmental regulation. Protein Sci 6, 249-253.

Schulz, N. T., Paulhiac, C. I., Lee, L., Zhou, R. (1995). Isolation and expression

analysis of tyro3, a murine growth factor receptor tyrosine kinase preferentially

expressed in adult brain. Brain Res Mol Brain Res 28, 273-280.

Scott, V., McDade, D. M., Luckman, S. M. (2007). Rapid changes in the sensitivity of

arcuate nucleus neurons to central ghrelin in relation to feeding status. Physiol Behav

90, 180-185.

Seif, I., De Maeyer, E. (1999). Knockout Corner : Knouckout mice for monoamine

oxidase A. Int J Neuropsychopharmacol 2, 241-243.

97

Seoane, L. M., Lopez, M., Tovar, S., Casanueva, F. F., Senaris, R., Dieguez, C.

(2003). Agouti-related peptide, neuropeptide Y, and somatostatin-producing neurons

are targets for ghrelin actions in the rat hypothalamus. Endocrinology 144, 544-551.

Shinder, A. F., Poo, M. (2000). The neurotrophin hypothesis for synaptic plasticity.

Trends Neurosci 23, 639-645.

Shutter, J. R., Graham, M., Kinsey, A.C., Scully, S., Luthy, R., Stark, K. L. (1997).

Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in

obese and diabetic mutant mice. Genes Dev 11, 593-602.

Son, J. H., Baker, H., Park, D. H., Joh, T. H. (1994). Drastic and selective

hyperinnervation of central serotonergic neurons in a lethal neurodevelopmental mouse

mutant, Anorexia (anx). Brain Res Mol Brain Res 25, 129-134.

Song, H. J., Ming, G. L., Poo, M. M. (1997). cAMP-induced switching in turning

direction of nerve growth cones. Nature 388, 275-9.

Song, H. J., Poo, M. M. (1999). Signal transduction underlying growth cone guidance

by diffusible factors. Curr Opin Neurobiol 9, 355-363.

Spear, L. P., Ristine, L. A. (1982). Suckling behavior in neonatal rats:

psychopharmacological investigations. J Comp Physiol Psychol 96, 244-255.

Stanley, B. G. Leibowitz, S. F. (1985) Neuropeptide Y injected in the paraventricular

hypothalamus: a powerful stimulant of feeding behavior. Proc Natl Acad Sci U S A 82,

3940-3943.

Stanley, B. G., Magdalin, W., Seirafi, A., Nguyen, M. M., Leibowitz, S. F. (1992).

Evidence for neuropeptide Y mediation of eating produced by food deprivation and for a

variant of the Y1 receptor mediating this peptide’s effect. Peptides 13, 581-587.

98

Stapleton, D., Balan, I., Pawson, T., Sicheri, F. (1999). The crystal structure of an Eph

receptor SAM domain reveals a mechanism for modular dimerization. Nat Struct Biol 6,

44-49.

Stitt, T. N., Conn, G., Gore, M., Lai, C., Bruno, J., Radziejewski, C., Mattsson, K.,

Fisher, J., Gies, D. R., Jones, P. F., Masiakowski, P., Ryan, T. E., Tobkes, N. J.,

Chen, D. H., DiStefano, P. S., Long, G. L., Basilico, C., Goldfarb, M. P., Lemke, G.,

Glass, D. J., Yancopoulos, G. D. (1995). The anticoagulation factor protein S and its

relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 80,

661-670.

Strohmaier, C., Carter, B. D., Urfer, R., Barde, Y. A., Dechant, G. (1996). A splice

variant of the neurotrophin receptor trkB with increased specificity for brain-derived

neurotrophic factor. EMBO 15, 3332-3337.

Tisi, D., Talts, J. F., Timpl, R., Hohenester, E. (2000). Structure of the C-terminal

laminin G-like domain pair of the laminin alpha2 chain harbouring binding sites for

alpha-dystroglycan and heparin. EMBO J 19, 14323-1440.

Tsouflas, P., Soppet, E., Escandon, E., Tessarollo, L., Mendoza-Ramirez, J. L.,

Rosenthal, A., Nikolics, K., Parada, L. F. (1993). The rat trkC locus encodes multiple

neurogenic receptors that exhibit differential response to neurotrophin-3 in PC12 cells.

Neuron 10, 975-990.

Tucker, K. L. (2002). Neurotrophins and the control of axonal outgrowth. Panminerva

Med 44, 325-333.

Vertes, R. P., Fortin, W. J., Crane, A. M. (1999). Projections of the median raphe

nucleus in the rat. J Comp Neurol 407, 555-582.

99

Vertes, R. P. (1991). A PHA-L analysis of ascending projections of the dorsal raphe

nucleus in the rat. J Comp Neurol 313, 643-648.

Von Schack, D., Casademunt, E., Schweigretier, R., Meyer, M., Bibel, M., Dechant,

G. (2001). Complete ablation of the neurotrophin receptor p75NTR causes defects both

in the nervous and the vascular system. Nat Neurosci 4, 977-978.

Watson, S. J., Akil, H., Richard, C. W., Barchas, J. K. (1978). Evidence for two

separate opiate peptide neuronal systems. Nature 275, 226-228.

Yamashita, T., Tucker, K. L., Barde, Y. A. (1999). Neurotrophin binding to the p75

receptor modulates Rho activity and axonal outgrowth. Neuron 24, 585-593.

Ye, W., Shimamura, K., Rubenstein, G., Hynes, M. A., Rosenthal., A. (1998). FGF

and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural

plate. Cell 93, 755-766.

Zhang, X., Bao, L., Xu, Z.-Q., Kopp, J., Arvidsson, U., Elde, R., Hokfelt, T. (1994).

Localization of neuropeptide Y Y1 receptors in the rat nervous system with special

reference to somatic receptors on small dorsal root ganglion neurones. Proc Natl Acad

Sci U S A 91, 11738-11742.

Zhang, X., Beaulieu, J. M., Gainetdinov, R. R. and Caron, M. G. (2006). Functional

polymorphisms of the brain serotonin synthesizing enzyme tryptophan hyroxylase-2.

Cell Mol Life Sci 63, 6-11.