Axon guidance in the zebrafish visual system analysis of esrom mutant

List of FiguresFigure 1.1: Development of vertebrate nervous system Figure 1.2: Neurogenesis Figure 1.3: Netrin gradient in the spinal chord Figure 3.1: Diagram of the retino-tectal proj

AXON GUIDANCE IN THE ZEBRAFISH VISUAL

SYSTEM: ANALYSIS OF THE esrom MUTANT

JASMINE JOYCE D’SOUZA

( M.Sc University of Mumbai)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE

2005

feedback during annual work presentations Special thanks to the administrative staff fortheir help as well as Temasek Life Sciences Laboratory for their Funding.

I would like to thank all members of the Developmental Neurobiology group Specialthanks to Michael Hendricks and Sylvie Le Guyader My heartfelt thanks also go toSivan Subburaju, Song Choon and He Fang for their help in research work I would like

to Dr Alan Coulson form the Wellcome Trust Sanger Institute for his help is sequencingzebrafish genome

Special thanks to my family, especially my mother for her patience in waiting for myPh.D degree Thanks to all my friends

This thesis is dedicated to my father and mother

Table of Contents

1.3 Steps involved in the formation of neural network 5

1.7 Growth Cone Machinery 33

2.6 Pulse field gel electrophoresis and DNA fingerprinting 43

2.8 Construction of an eye cDNA library 44

2.19 Cloning of large genes 54

2.25 Preadsorbtion and antibody specificity 58

2.27 Generation of Dominant-Negative esrom construct 59

3.4 Esrom gene function is required in the eye 75

4.3 Genomic walk 92

Chapter V: Localization and molecular characterization of Esrom 110

5.2 Expression and localization of esrom 110

5.4 Esrom is involved in signal transduction 1175.5 Generating a Dominant-negative fragment 119

During development of the visual system, precise connections are formed between theeye and the brain Retinal ganglion cells (RGCs) are the only neural cells present in theretina that send connections to a specific region in the brain known as the optic tectum Aspatial correlation is maintained between the position of the RGCs within the retina andthe termination zone on the optic tectum This highly ordered connection gives rise to aretino-tectal map Little is known about the molecules that orchestrate the formation of aretino-tectal map In an effort to understand this process better, a large-scale mutant

screen was carried in zebrafish This has led to the isolation of a mutant called esrom.

This study involves the identification and characterization of the esrom gene Analysis of the zebrafish mutant esrom shows that it is required for axon fasciculation, targeting, branching and skin pigmentation In the visual system esrom is necessary for mapping

along the A-P and D-V axes, correct innervation of the pretectal targets and axon

bundling in the optic tract The gene function is required in the eye and not in the brain

for retinal ganglion cell (RGC) targeting The mapping of esrom revealed that there is a strong synteny between the esrom locus and human chromosome 13 q22.3 Using

positional cloning, esrom was established to encode a very large protein homologous to human PAM (Protein Associated with Myc)/ Drosophila Highwire/ C elegans Regulator

of Presynaptic Morphology 1 The cranofacial motoneurons in esrom mutants have expanded synapses at the neuromuscular junction like in Drosophila hiw mutants, and branch ectopically However RGC axon branching appears normal in size, unlike in hiw,

indicating that esrom has a different function in the vertebrate visual system This large

multidomain protein has an E3 ligase activity Esrom is ubiquitously expressed in theembryo unlike its invertebrate homologues HIW/RPM-1 In the RGCs it is required forproper activation of the p38 MAPK signal transduction pathway in response to

lysophosphatydic acid (LPA) Esrom is also known to regulate phosphorylated Tuberin, a

tumor suppressor within the growth cones These data suggest that esrom might act as a

molecular switch that integrates signal transduction events in axons.

List of Tables

Table 2: Clones obtained from eye cDNA library screening 94

Table 4: Morpholino1 (RCC1 morpholino) results 99Table 5: Morpholino2 (B-box morpholino) results 101Table 6: Standard morpholino control results 101

List of Figures

Figure 1.1: Development of vertebrate nervous system

Figure 1.2: Neurogenesis

Figure 1.3: Netrin gradient in the spinal chord

Figure 3.1: Diagram of the retino-tectal projection in 5-day-old zebrafish

Figure 3.2: Diagram showing the graded distribution Eph receptors and their ligands inthe visual system

Figure 3.3: The xanthophore phenotype of esrom

Figure 3.4: Xanthophore autofluorescence visualized under blue light illumination

Figure 3.5: Retino-tectal phenotype of esrom

Figure 3.6: Axon fasciculation defect of the optic nerve

Figure 3.7: Autonomy of esrom gene function

Figure 3.8: Retinal polarity

Figure 4.1: Crossing scheme for recombination based mapping of esrom

Figure 4.2: RAPD marker linkage

Figure 4.3: esrom maps to Linkage Group 9

Figure 4.4: Fine mapping of esrom

Figure 4.5: Genomic locus of esrom

Figure 4.6: cDNA library screening

Figure 4.7: Positional cloning of esrom

Figure 4.8: The esrom gene

Figure 4.9: Homologues of Esrom

Figure 5.1: RNA in-situ hybridization with esrom probe on whole mounts

Figure 5.2: Immunohistochemistry with PAM antibody

Figure 5.3: Esrom is an E3 ligase

Figure 5.4: LPA response

Figure 5.5: Generating a Dominant-Negative fragment

List of Abbreviations

Ab Antibody

AC Adenylyl cyclase

bHLH basic helix loop helix

BAC Bacterial artificial chromosome

BDNF Brain derived neurotrophic factor

BMP Bone morphogenetic protein

BSA Bulked segregant analysis

Ca2+ Calcium

CAM Cell Adhesion molecule

Chr chromosome

DBD DNA binding domain

DCC Deleted in colorectal cancer

DN Dominant-negative

DNA deoxyribo nucleic acid

ECM Extracellular matrix

EGF Epidermal growth factor

eIF elongation initiation factor

ENU N-ethyl N-nitrosourea

Eph Ephrin receptor

ER Endoplasmic reticulum

esr esrom

EST Expressed Sequence Tags

FAK Focal adhesion kinase

FGF Fibroblast growth factor

FN Fibronectin

GDNF Glial cell derived neurotrophic factorGEF Guanine nucleotide exchange factorGFP Green fluorescent protein

HHD Histone binding domain

PAC P1 artificial chromosome

PAM Protein Associated with Myc

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PFA Para formaldehyde

PKA Protein kinase A

PSA Polysialic acid

PTU Phenylthio urea

RAPD Randomly amplified polymorphic DNAsRGC Retinal ganglion cell

RING really interesting gene

RNA ribo nucleic acid

Robo Roundabout

RPTP Receptor protein tyrosine phosphatase

RTK Receptor tyrosine kinase

SAM Substrate adhesion molecule

Sema Semaphorin

Shh Sonic hedgehog

SSLP Simple sequence length polymorphism

SSCP Simple sequence conformation polymorphismSSR Simple sequence repeats

TAD Transactivating domain

TOR Target of rapamycin

Unc Uncoordinated

Ub Ubiquitin

YAC Yeast artificial chromosome

D'Souza J, Hendricks M, Le Guyader S, Subburaju S, Grunewald B, Scholich K,

Jesuthasan S Formation of the retinotectal projection requires Esrom, an ortholog of

PAM (protein associated with Myc) Development 2005 Jan;132(2):247-56

Chapter I: General introduction Introduction

Neurons are specialized cells that relay sensory information They have a unique cellularmorphology and are the longest cells in the body of a multicellular organism Neuronshave an extended shape with a long axon and dendrites In order to transmit sensorystimulus, neurons must connect with each other to form a network During developmentaxons and dendrites grow out, find their target and synapse with each other selectively

1.1 Vertebrate nervous system

The vertebrate nervous system is divided into the central nervous system and the

peripheral nervous system During embryogenesis the ectoderm is patterned to give rise

to a neuroectoderm which develops to form the brain, spinal cord and eye; components ofthe central nervous system (Sporle and Schughart, 1997) The neural plate on the dorsalside of the embryo buckles in at the midline to give rise to the neural tube (Fig 1.1) Thedorsal region of the neural tube forms the roof plate by closure of the dorsal tips of theneuroectoderm whereas the ventral region forms the floor plate The floor plate expressesSonic Hedgehog, which is a diffusible molecule The roof plate also expresses a

diffusible molecule BMP4 The concentration gradients of these two opposing

morphogens along the dorso-ventral axis within the neural tube control the gene

expression pattern of regulatory genes These include the homeodomain containingtranscription factors Pax3, Pax6, Pax7, Msx1 and Msx2 The Pax6, Pax3 and Pax7

expression in the caudal region of the neural tube subdivides the neural tube into distinct

Figure1.1: Development of vertebrate nervous system.The neural plate (purple) buckles

in to from the neural tube and the lateral edges of the neural folds come together and fuse

at the dorsal midline The notocord is an inductive tissue expressing sonic hedgehog thatpatterns the ventral neural axis The epidermal ectoderm (blue) fuses at the dorsal midlineand expresses a morphogen BMP4,7 The dorsal BMPs then induce the roof plate

structure to express BMP4 that patterns the dorsal neural axis (Adapted from:

Developmental Biology-5th Edition: Scott F Gilbert)

Figure 1.2: Neurogenesis Neural identity in the neural epithelium is attained by theaction of bHLH proteins and Notch signaling The conversion of a neural epitheliumconsisting exclusively of proliferating progenitor cells (gray) to one in which certain cellshave adopted a neuronal identity (green) by a process of lateral inhibition is shown Atthe onset of neuronal differentiation, the ventricular zone of the embryonic spinal cord issubdivided into dorsoventral domains that express different combinations of bHLHproteins, Notch ligands and Pax proteins The last diagram shows that subsets of neuronscan be distinguished by the expression of LIM homeodomain protein (Adapted from:TanabeY and Jessell TM., Science 274:1115-1123 )

tube Patterning along the anteroposterior axis establishes subdivisions of the neural tubethat prefigure the formation of midbrain, hindbrain and spinal cord (Lumsden and

Krumlauf, 1996) These subdivisions are conferred by the expression of Hox genes andretinoic acid signaling

1.2 Neurogenesis

The differential maintenance of the homeodomain proteins along the dorso-ventral axis

of the neural tube activates the expression of proneural and neurogenic genes (Tanabeand Jessell, 1996) Proneural genes are transcription factors of the basic helix loop helix(bHLH) class and include neurogenin, Mash-1 (achaete-scute) and Math-1/atonal ( Fig.1.2) They are expressed in complementary, nonoverlapping domains along the dorso-ventral axis of the spinal cord Not all cells within the neural epithelium expressing theproneural genes become neurons The selection process involves lateral inhibition

mediated by neurogenic genes like the Notch receptor and its ligands Delta, Serrate andJagged The class of genes that act downstream of neurogenic genes to specify neuronalsubtypes remain unknown but the possibility that LIM homeodomain proteins regulate

neuronal subtype identity, has experimental evidince to support it islet-1 knockout mice

fails to develop spinal motor neurons Due to the action of specific transcription factorsthe commissural and association neurons differentiate dorsally, closer to the roof platewhereas the motor and ventral interneurons differentiate ventrally, closer to the floorplate Thus the cells in the neuroepithelial differentiate into neurons of different subtypesdepending on their location at different dorso-ventral positions

The peripheral nervous system is derived form the migrating neural crest cells on thedorsal side of the embryo and ectodermal placodes, thicked regions of epithelium Cells

in the dorsal side of the neural tube initially give rise to neural crest cells and

subsequently the roof plate cells at the dorsal midline The specification of neural crestcells has been shown to be influenced by Pax7 The olfactory and otic placodes can beinduced by mesoderm, endoderm and neuroectoderm, the trigeminal placode is induced

by dorsal neural tube, epibranchial placodes by pharyngeal endoderm (McCabe et al.,2004) The epibranchial, otic, and lateral line placodes arise from a common posteriorplacodal area characterized by Pax8 and Pax2 expression (Schlosser and Ahrens, 2004)

1.3 Steps involved in the formation of neural network

One of the earliest steps in the development of the central and peripheral nervous system

is the initiation of axon outgrowth from the newly born neurons Once differentiated,neurons send out long processes called axons and several short processes called

dendrites Axons travel long distances in order to find their right target to synapse Thegrowing axons continually reassess the environment to select correct pathways Whilenavigating, axons encounter many molecules that act as guideposts The tip of the axon iscalled the growth cone and acts as a sensor The growth cone is made of finger likeprotrusions called filopodia that contain actin cables and web like cytoplasmic veilscalled lamellipodia The growth cones steer the axons in the right orientation whilenavigating Formation of a neural network can be divided into the following steps (1)axon outgrowth (2) axon fasciculation (3) axon guidance (4) axon defasciculation(5)axon branching (6) synapse formation (7) axon pruning

Axon outgrowth is a process of initial sprouting of neurites followed by elongation Theprocess is influenced by several factors that are both adhesive and permissive for axongrowth Factors that regulate neurite outgrowth are extra cellular matrix molecules(ECM), cell adhesion molecules (CAM), neurotrophic factors, and several axon guidancemolecules The ECM include heparin binding growth associated molecule (HB-GAM),glycoproteins like laminin, tenascin, fibronectin, vitronectin, thrombospondin and

glycoaminoglycans like heparin sulfate and hyaluronate The CAMs include N-Cadherin,L1 family of molecules, NCAM, axonin/TAG-1 and contactin The neurotrophic factorsinclude nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cellderived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4)and the guidance molecules influencing axon outgrowth include semaphorins, netrins,ephrins and slits

1.4 Mechanism of axon guidance

About a century ago, Santiago Ramon y Cajal proposed that the target cells secretechemoattractive signals The directed growth of axons towards the target may be similar

to the process of chemotaxis of motile cells In 1963 Roger Sperry proposed the

chemoaffinity theory (Sperry, 1963) He hypothesized that during neural differentiationthe retinal ganglion cells and the tectal cells acquire at least two categories of

cytochemical labels, a functional label and a positional label As it is unlikely that thepositional labels are entirely unique because this would require enormous number ofdifferent molecules, Sperry posited a set of two orthogonal gradients in the retina that

used to match up with complementary gradients in the target field of the retinal

projection, the tectum For a long time, however, how complex neural networks areformed during development remained a puzzle

Many molecules have been discovered in the last decade that have been shown to

influence axon guidance both in-vitro and in-vivo Growth cones appear to be guided by

at least four different mechanisms: mediated attraction, chemoattraction, mediated repulsion and chemorepulsion (Tessier-Lavigne and Goodman, 1996)

contact-Guidance signals can either be permissive or attractive or inhibitory and repulsive.Diffusible molecules can generate a gradient of either a chemoattractant or

chemorepellent One of the first candidates identified for target-derived chemoattractantwas nerve growth factor NGF, which is expressed by the target cells of sympathetic andsensory axons NGF can attract regenerating sensory axons in culture and sympathethicaxons in vivo (Gundersen and Barrett, 1979; Gundersen, 1985; Tessier-Lavigne and

Placzek, 1991) In-vitro experiments conducted in which neurons were cultured together

with the target cells turned towards target cells Apart from long range chemoattractionexerted by diffusible attractants long range chemorepulsion by diffusible repellents hasalso been recently demonstrated Other guidance cues that have been cloned in the past

20 years include membrane associated proteins such as receptors and some ligands.These may be expressed in a spatially graded manner across a zone and may act aschemotrophic guidance cues in a manner similar to difusible chemotropic factors

To date four major classes of axon guidance molecules and their receptors have beendiscovered and their function and mechanism of guidance has been shown to be

conserved across species These include the netrins, ephrins, semaphorins and slits

1.5 Axon guidance molecules

1.5.1 Netrin

Netrins are a group of small secreted molecules related to laminin It was the first

vertebrate chemoattractive molecule to be discovered (Serafini et al., 1994) Marc

Tessier-Lavigne and his colleagues used the collagen culture method to prove the

existence of a secreted factor that attracted axons When a dorsal spinal cord explant wasplaced within a few hundred microns of the floor plate explant, commissural axons turnedand extended towards the floor plate from a distance These molecules were

biochemically isolated and named as Netrin-1 and Netrin-2 coined from the Sanskrit term

for ‘one who guides’ Netrins proved to be the vertebrate homologue of the C elegans

protein Unc-6, a laminin related molecule that is involved in axon guidance and cellmigration (Hedgecock et al., 1990) Following the isolation of the vertebrate netrin,

homologues in other species were soon identified including two Drosophila homologues

netrin A and netrinB (Harris et al., 1996) and zebrafish homologues netrin1, netrin-2, andnetrin-4 (Chisholm and Tessier-Lavigne, 1999) The role of Netrin has been

evolutionarily conserved in guiding axons towards the midline The N terminus contains

a signal peptide followed by domains of type V and VI B1 laminin chain, three predictedEGF repeats and a C terminal basic region which is the most variable between species

(Yin et al., 2000) In chick, netrin-1 is expressed in the floor plate cells and could formventral to dorsal gradient in a developing spinal cord (Kennedy et al., 1994; Serafini etal., 1994; Sim et al., 1999) In worms, Unc-6 is expressed in the ventral region and in

Drosophila Netrin-A is expressed along the midline of the CNS (Goodman, 1996).

The screen that isolated Unc-6 also isolated two more mutants Unc-5 and Unc-40 thateffected the dorso-ventral axon guidance Mutations in Unc-40 disrupt ventral migrations

of epidermal cells whereas mutations in Unc –5 disrupt the dorsal migrations of

epidermal cells (Hedgecock et al., 1990) Both molecules encode transmembrane proteinsthat have a structural feature of a receptor The response to Unc-6 gradient depends onwhich receptor is being expressed in the neurons (Tessier-Lavigne and Goodman, 1996).Neurons that express Unc-5 receptor extend their axons dorsally away from the ventralmidline while axons that express only Unc-40 move towards the ventral midline whichsecrets Unc-6 Unc–40 encodes a protein closely related to the immunoglobulin

superfamily that is similar to human DCC (Goodman, 1996) The DCC protein is atransmembrane protein containing four immunoglobulin domain and six type III

fibronectin repeats and a cytoplasmic tail with no known homologies to other proteins(Vielmetter et al., 1994) In addition, one other closely related protein, neogenin, hasbeen identified in the developing chick system (Vielmetter et al., 1997) The DCC proteinwas shown to function as Netrin receptor DCC blocking antibodies inhibit netrin

stimulated outgrowth of commissural axons in-vitro (Keino-Masu et al., 1996).

Moreover, DCC is expressed in the dorsal spinal cord region containing the commisuralneurons that are responsive to netrin The Neogenin protein has also been shown to bind

to Netrin-1 in vitro, suggesting that it too can function as Netrin receptor (Srinivasan et al., 2003) The Drosophila frazzled was proposed to encode a netrin receptor based on

the similarity of frazzled and netrin phenotype (Kolodziej et al., 1996) The cloning of thevertebrate homologues of Unc-5 that mediates repulsive activities of netrin has beenreported (Leonardo et al., 1997; Engelkamp, 2002) Members of this family of proteincontain two extracellular immunoglobulin-like and two thrombospondin type1 repeats

The possibility of netrin acting as a repellent in vertebrates has been studied The

trochlear motor axons originate near the floor plate and extend dorsally away from thefloor plate COS cells secreting recombinant Netrin-1 can repel trochlear motor axons

way from a distance in vitro suggesting that Netrins function as a chemorepellent to guide

the trochlear motor axons away from the floor plate (Colamarino and Tessier-Lavigne,1995) Thus in all species, netrins can function as both attractants and repellents, at bothshort range and long range with attraction mediated by DCC family proteins and

repulsion by Unc-5 family proteins (Fig 1.3)

Within the developing nervous system Netrin and its receptors are expressed in diversegroup of structures One particular example is the visual system The DCC receptor isexpressed in the retinal ganglion cell and netrin is expressed in the optic nerve head (Gad

et al., 2000) By analyzing both the DCC and netrin mutants netrin was shown to guideretinal ganglion cells to the optic nerve head (Deiner et al., 1997)

1.5.2 Ephrins

Ephrin- ligands and their receptors are known to act as axon repellents and promotegrowth cone collapse The Eph receptors belong to the largest subfamily of receptortyrosine kinases in vertebrates and can be divided into two subclasses (Eph-A and Eph-B)based on sequence specificity and ligand affinity (Flanagan and Vanderhaeghen, 1998).

In general, Eph-A receptors show higher affinity for ephrin-As and Eph-B receptors showhigher affinity for ephrin-Bs, although some receptors have dual specificity The Ephreceptors are characterized by an extracellular region with a unique cystine-rich motifextending over at the N terminus followed by two fibronectin type III motifs The

receptors are closely related to other receptor tyrosine kinases with sequence identities ofapproximately 60-90% in the kinase domain and approximately 30-70% in the

extracellular domain Their ligands, the ephrins are also membrane bound All ligandsshare a conserved core sequence of about 125 amino acids, including four invariantcysteine residues probably corresponding to receptor binding domain This is followed bythe anchorage domain The ephrin A ligands (ephrin-A1 to ephrin-A5) are attached to thecell membranes by glycosylphosphatidylinositol (GPI) anchors whereas the ephrin-Bligands (ephrin-B1 to B3) posses a transmembrane domain

In contrast to the fourteen Eph receptors discovered in mammals, the genome of the

worm C elegans encodes a single Eph receptor, VAB-1 and four ephrins 1 to

EFN-4 (Chin-Sang et al., 1999; Chin-Sang et al., 2002) Drosophila has one Eph receptor,

DEK, suggesting that the divergence of Eph-A and Eph-B subclass is confined to

vertebrates (Scully et al., 1999; Bossing and Brand, 2002) Members of both subfamiliesshow widespread expression in the developing nervous system and other tissues involved

in a large number of different processes (Adams and Klein, 2000; Cowan and

Henkemeyer, 2002; Poliakov et al., 2004) The characterization of ephrin as a repellentmolecule comes from work done in the visual system (Flanagan and Vanderhaeghen,1998; O'Leary and Wilkinson, 1999) The retino-tectal projections from the RGCs

terminate orderly in the tectum in a manner that maintains their neighborhood

relationship In chick retina, the Eph-A receptors and ephrin-A ligands were found tohave graded distribution along the anterior posterior axis within the retino-tectal system,confirming Sperry’s chemoaffinity hypothesis In the1970’s, Bonhoeffer’s group

designed an elegant in-vitro assay called the stripe assay to directly test the growth

response of the retinal axons (Bonhoeffer and Huf, 1982; Walter et al., 1987) The retinalaxons were allowed to choose between alternating stripes of membrane carpets derivedform the anterior and posterior tectum The retinal axons were allowed to grow paralleltothe alternating tectal stripes The results of these experiment showed that the nasal

(anterior) axons grew equally well on both stripes while the temporal (posterior) axonsgrew preferentially on the anterior membrane Cleaving off the GPI linked moleculesusing phosphatidyl inositol specific phospholipase C (Walter et al., 1987; Roskies andO'Leary, 1994), reinstated the ability of the temporal axons to grow on posterior stripes,pointing out the repulsive property of the posterior tectum The transcripts and proteinsfor both ephrin-A2 and ephrin-A5 GPI linked ligands are distributed in an increasinganterior to posterior gradient across the tectum with ephrin-A2 being expressed as asmooth gradient and ephrin-A5 in a steeper gradient (Cheng et al., 1995; Drescher et al.,1995; Monschau et al., 1997) The Eph-A3 receptor is expressed at high levels in thetemporal retinal and low levels in the nasal retina All other Eph-A receptors are

uniformly expressed (Cheng et al., 1995; Nakamoto et al., 1996) Severe disturbances ofretino-tectal map formation are seen in ephrin-A2 -/- and ephrin-A5-/- double knockoutmice The projections are severely misguided along the anterior-posterior axis and to alesser extent along the dorso-ventral axis (Feldheim et al., 2000).

The Eph-B receptors and ligands are thought to establish the dorso-ventral connectivity

of the retina (Hindges et al., 2002; Mann et al., 2002; Mui et al., 2002) B2 and B3 receptors are expressed high in the ventral retina and low dorsally (Holash and

Eph-Pasquale, 1995; Connor et al., 1998) Their ligand ephrin-B1 is expressed high dorsallyand low ventrally (Braisted et al., 1997) In the retino-tectal system the Eph-Bs probablyact through an attractive mechanism since RGCs that express high Eph-B2 and Eph-B3map to the dorsal tectum which expresses high level of ephrin-B1 ligand (Barbera, 1975).This is supported by the experiment that shows ventral retinal cells adhering to substrates

of ephrin-B1 (Holash et al., 1997) Eph-B2; Eph-B3 deficient mice send ectopic arbors tothe lateral (ventral) tectum and exhibit increased frequency of guidance errors to the opticdisc (Hindges et al., 2002) There is growing evidence to show that the Eph-B receptorsand their ligands can signal bidirectionaly (Bruckner and Klein, 1998) The cytoplamicdomain of ephrin-Bs is highly conserved harboring five-conserved tyrosine residues and

a PDZ binding motif Upon receptor binding the C-terminus gets phosphorylated

potentially leading to signal transduction into the cell that expresses the ligand (reverse

signaling) In Xenopus the ephrin-B reverse signaling is important for establishing

dorso-ventral retino-tectal polarity (Mann et al., 2002) The dorsal retinal axons that expressephrin-B prefer to grow on clustered Eph-B stripes Moreover misexpressing full length

ephrin-B2 but not ephrin-B2∆C in the ventral retina causes the ventral axons to shiftmore ventrally on the tectum One of the molecule that regulates the transcription of Ephreceptors is c-Myc The c-Myc oncogene, is an extensively studied molecule and

microarray analysis show that c-Myc upregulates the expression of Eph-A2 and Eph-B2(Schuldiner and Benvenisty, 2001) The maintenance of gradients of these two moleculesacross the retina is quite crucial for establishing a retino-tectal topographic map

Although there is no direct evidence of c-Myc in axon guidance, it might indirectlyregulate molecules involved in axon guidance

The role of Eph related receptors is diverse in other neuronal types including motorneurons which express the receptors only in a subset of neurons (O'Leary and Wilkinson,1999) They are also required for targeting the vomeronasal axons to the accessoryolfactory bulb (Knoll et al., 2001) Loss- of -function studies of Eph and ephrins havebeen limited to focused analysis of mice Defects in the formation of forebrain

commissures have been observed in mice lacking Eph-B2, Eph-B3 and Eph-A8

Patterning of the forebrain and hindbrain structures were disrupted in the presence ofdominant –negative Eph-A4 (O'Leary and Wilkinson, 1999) Nonetheless Eph and ephrinfunction is not restricted to the nervous system Ephrin-B ligands regulate the migration

of neural crest cells via repulsion (Smith et al., 1997; Santiago and Erickson, 2002).Ephrin-A1 is involved in tumor necrosis factor α induced angiogenesis and activation ofEph-B1 and Eph-A2 directs development of vascular system (Bruckner and Klein, 1998).Ephrins modulate cell adhesion and influence cell migration (Knoll and Drescher, 2002).Activated Eph-As and ephrin-As both modulate the integrin and MAPK signaling

pathways Activation of the Eph-A2 receptor by ephrin-A1 converts integrins into aninactive conformation leading to reduction in the cell adhesion and spreading Focaladhesion kinase plays a crucial role in this reaction as it quickly gets dephosphorylatedand inactivated and dissociates from the Eph-A receptor upon ligand binding Activation

of ephrin-A leads to increased cell adhesion and it requires β1 integrin and src familykinases On the other hand activation of Eph-A receptor leads to the inhibition of MAPkinase (ERK1, ERK2) pathway

1.5.3 Slits

Slits proteins are fairly large molecules structurally conserved across species Theycontain leucine-rich repeats, EGF like repeats and a laminin G domain (Rothberg et al.,

1990) Slit was first identified in Drosophila for its role in early pattern formation but

was later shown to be required for the proper development of the central nervous system

Commissural axons were disorganized at the midline in slit mutants (Kidd et al., 1999).

The role of Slit as a midline repellent was shown by a series of genetic experimentsconducted by Goodman's group The path to this discovery came from studies of a

transmembrane receptor called Roundabout (Robo) which is expressed by the axons thatnavigate the midline (Kidd et al., 1998) Longitudinal axons which express Robo never

cross the midline but in robo mutants these axons freely cross the midline suggesting that

there is a midline repellent cue sensed by Robo Similarly commisural axons which cross

the midline once do not recross the midline again but in robo mutants they recross the

midline Two lines of evidence have established that Slit is the repellent ligand for Robo

The first being trans-heterozygotes carrying one mutant copy of each robo and slit show a

phenotype similar to that of robo homozygous mutant (Kidd et al., 1999) Second, Slit

and Robo proteins have been shown to be binding partners in cell binding assay (Brose etal., 1999) Moreover ectopic Slit expression can cause misrouting of axons away for theectopic source (Battye et al., 1999)

The Robo proteins are phylogenetically conserved Ig superfamily members with theextracellular domain composed of Ig and FN III repeats and a large cytoplasmic domainlacking catalytically active domain but harboring a conserved CC motif required for Slitmediated chemorepulsion and silencing of netrin-mediated chemoattraction (Bashaw and

Goodman, 1999; Stein and Tessier-Lavigne, 2001) The Drosophila and C elegans

genome encodes for one Slit gene whereas the vertebrates have three Slits, Slit-1, Slit-2

and Slit-3 There is only one Robo receptor homologue in C elegans, Sax3,which is

implicated as a receptor for axon guidance, including the axons that navigate the midline

(Zallen et al., 1998) Drosophila has three Robo receptors Robo-1, Robo-2 and Robo-3.

In the case of vertebrates three Robo receptors have been identified so far These includeRobo-1, Robo-2 and Rig-1/ Robo-3 The Slit-Robo guidance mechanism seems to beevolutionary conserved in vertebrates (Brose et al., 1999) In mammals the Slit genes areexpressed in the ventral midline of the spinal chord and Robo-1 and -2 transcripts areexpressed in the region that includes the commissural neuronal cell bodies (Itoh et al.,1995; Brose et al., 1999; Piper et al., 2000; Long et al., 2004) In Slit triple mutants thecommissural axons project ventrally toward the floor plate but instead of forming tightlybundled commissures they are disorganized and appear defasciculated (Long et al.,2004) DiI labeling of the dorsal commisural axon showed that 72% of the axons stalled

at the floorplate (ventral midline) A few of them crossed the floor plate, made a turn andthen projected back to the ipsilteral side of the spinal chord, recrossing the floor plate.

The Robo protein is present on the cell surface of the commisural growth cones enabling

them to sense Slit In Drosophila, the concentration of Robo receptors on the commisural

axons is controlled by one key player, a molecule called Commissureless (Comm)

(Seeger et al., 1993; Georgiou and Tear, 2002) Prior to crossing the midline, Roboprotein levels are low on the surface of commissural axons Comm a type II

transmembrane protein removes the Robo protein from the cell surface by acting as anintracellular sorting receptor (Keleman et al., 2005) A LPSY motif in Comm

cytoplasmic region is required for Comm/Robo associations and serves to target Robo tolysosome degradation Comm requires the association with a ubiquitin ligase Dnedd-4which has been proposed to facilitate Robo ubiquitination leading to its subsequentdegradation (Myat et al., 2002) Once the commissural axons cross the midline Robo isupregulated, senses Slit and is repelled away from the midline No obvious homologues

of Drosophila Comm have been found in mammalian or C elegans gene data bases.

Recently, mammalian Rig-1/Robo-3 has been shown to function as an inhibitor of Slitresponsiveness in commissural axons prior to crossing the floor plate (Sabatier et al.,2004) Removal of Rig-1 results in the failure of commissural axons to cross the midlinebut unlike Comm, Rig-1 does not produce its effect by downregulating Robo receptors

Slit- Robo mediated guidance is also required for RGC pathfinding Slit 1 or Slit 2 singleknockout alone shows no pathfinding error probably due to functional redundancy

However, Slit1/2 double knockout mice display a variety of guidance defects, includingthe formation of ectopic chiasm (Plump et al., 2002) The phenotypes are similar to the

zebrafish astray (Robo 2) mutant (Hutson and Chien, 2002) Robo/Slit at the chiasm may

be acting in a different manner than at the midline It is not required to sort ipsilateral andcontralateral axons but ensures that the axons within the optic nerve remain tightly

fasciculated

Studies in vertebrate suggest that the Slit proteins play a distinct role in axon elongationand branching (Wang et al., 1999) When Dorsal Root Ganglion (DRG) were grown inthree-dimensional collagen matrix and treated with calf brain extract it promoted axonelongation and extensive branching Biochemical purification of the branch promotingfactor led to the identification of Slit-2 protein Only purified amino fragment Slit2-N,but not the full length slit molecule possesses this activity indicating that the cleavage ofSlit is important for the bioactivity It is not yet determined if Robo receptors mediate thisresponse

1.5.4 Semaphorins

Semaphorins belong to a large family of phylogenetically conserved secreted and

membrane bound proteins that have a characteristic ‘sema’ domain, a stretch of

approximately 500 amino acids containing 17 highly conserved cysteine residue (1999 ).Although this family of protein appears to be conserved they exhibit strong evolutionarydivergence at the mechanistic level Members of this family are capable of mediatingboth repulsive and attractive axon guidance during neural development (Raper, 2000)

There are more than 30 semaphorins identified and all share the conserved sema domain

at the N-terminal They can be classified into eight subfamilies depending on their

structural similarities and species of origin

The first semaphorin Sema 1a was identified in grasshopper (Kolodkin et al., 1992).Sema Ia is a transmembrane protein that is expressed on subsets of fasciculating axons inthe developing CNS Antibody perturbation experiments have shown that Sema Ia isinvolved in driving axons to fasciculation through repulsion presented in the surrounding

environment Other members were subsequently identified in Drosophila (Sema I and

Sema II) (Kolodkin et al., 1993) Loss-of-function mutations in sema II cause behaviouraldefects whereas ectopic expression of sema II in muscles which do not express it, inhibitscertain motoneuron (RP3) growth cone from forming normal terminal arborizations(Matthes et al., 1995) In an independent study a protein called collapsin was identified inchicken (Luo et al., 1993) This molecule had the ability to cause the collapse of sensory

growth cones in-vitro Collapsin appears to be homologous to Sema III Class I and class

II semaphorins are found in invertebrates (1999) Class III-VII semaphorins are found invertebrates and one final class of semaphorin is found in genomes of neurotrophic DNAviruses In vertebrates semaphorins in classes IV to VI are transmembrane proteins, ClassIII semaphorins are secreted and class VII semaphorin resemble class III semaphorin butare tethered to the cell surface by a phosphatidylinositol (GPI) linkage Many vertebrateSemas have been demonstrated to act as repellents to subsets of motor neurons in vitroassay Sema -3B, -3C, -3F have all shown to collapse or repel sympathetic growth cones(Adams et al., 1997) while Sema 3C has been shown to be an attractant for axons

extending from the cortical explants (Bagnard et al., 1998) Targeted deletion of Sema III

in mouse has provided evidence consistent with the role of Sema being required for axonguidance and fasciculation (Catalano et al., 1998; Ulupinar et al., 1999)

Two types of receptors have been implicated in mediating many semaphorin functions:plexins and neuropilins (Fujisawa and Kitsukawa, 1998) To date two neuropilins andnine plexins have been identified in the mammalian genome (Pasterkamp and Kolodkin,2003) Chimeric proteins in which the alkaline phosphate was fused to Sema 3A wereused to screen COS cells trasfected with plasmid expression libraries, which eventuallyled to the identification of a transmembrane protein, Neuropilin-1 (He and Tessier-Lavigne, 1997; Kolodkin et al., 1997) The most direct evidence that Neuropilin-1 isrequired for Sema 3A mediated response came from the observation of Neuropilin-1knockout mouse, in which the sensory ganglia were unaffected by Sema 3A (Kitsukawa

et al., 1997) A second Neuropilin-2 receptor was later identified (Giger et al., 2000).Neuropilin-1 binds with high affinity to Sema 3A but not Sema 3F, Sema 3F binds toNeuropilin-2 with higher affinity than Sema 3A while Sema 3C needs the cooperation ofboth Neuropilin-1 and Neuropilin-2 Dominant-negative Neurophilin-1 neutralizes Sema–3A and –3C signaling, but not Sema 3F (Raper, 2000) Neuropilins do not appear to be

conserved, they have not been found in C elegans genome, and repeated attempts have failed to isolate them in Drosophila Neuropilins are likely to require another receptor

component as they possess very short cytoplasmic tail with no known signaling motifs.The initial breakthrough in identifying the missing transducing component came from thefinding of a viral SemaA39R which utilizes VESPR as a functional receptor (Comeau et

al., 1998) now called as Plexin C1 Genetic and biochemical evidence in Drosophila

identified Plexin A as a functional receptor for Sema 1a, which is required for repulsiveguidance in motor axons (Winberg et al., 1998) Plexins form stable complexes withNeuropilins and Sema 3A has been shown to bind to Plexin A1/Neuropilin-1 complexwith roughly five fold greater affinity than to Neuropilin-1 alone (Takahashi et al., 1999)

plexin A3 mutant mice are deficient in class III secreted semaphorin repulsion

establishing that plexins are components of receptor complexes (Cheng et al., 2001;Yaron et al., 2005) It is interesting that plexins contain a divergent Sema domain at their

N terminus which, in the absence of ligands, silences Plexin A1 signaling through

intramolecular interaction (Takahashi and Strittmatter, 2001) Recently Sema 5A wasshown to function as a bifunctional guidance cue exherting both attractive and inhibitoryeffects on developing habenular neurons These effects are mediated by cell surfaceproteoglycans The trombospondin repeats of Sema 5A physically interact with

glycosoaminoglycan (GAG) portion of CSPGs (chondroitin sulfate proteoglycans) andHSPGs (heparan sulfate proteoglycans) Association with CSPGs results in inhibition ofaxon extensions while axonal HSPGs are required for Sema 5A mediated attraction(Kantor et al., 2004)

In addition to plexins and neuropilins, five other proteins have been implicated as

semaphorin receptors or components of the holoreceptor complex (Pasterkamp andKolodkin, 2003): first , off track (Otk), a protein similar to receptor tyrosine kinases but

which contains a catalytically inactive kinase domain associates with Drosophila

melanogaster PlexA to mediate Sema 1a repulsive function; second L1, a cell adhesion

molecule plays a role in repulsive responses to Sema 3A as a part of neuropilin/plexinreceptor complex; third, Met, a scatter factor/ hepatocyte growth factor receptor withintrinsic kinase activity is required to transduce Sema 4D mediated effects forming areceptor complex with PlexB1; fourth, CD72, a transmembrane protein belonging to the

Ca+2 dependent C type lectin superfamily acts as a Sema 4D receptor in the immunesystem and finally, Tim2, a member of the family of T cell immunoglobulin domain andmucin domain proteins, transduces Sema 4A mediated effects on T cell function

1.5.5 Other guidance molecules.

Recent studies have demonstrated that secreted morphogens such as bone morphogeneticprotein (BMPs), Sonic hedgehog, Fibroblast growth factor (FGF8) and Wnts also act asguidance molecules (Salinas, 2005)

The first axons to project are the pioneering axons They travel in an axon-free

environment during embryogenesis The later developing axons grow and elongate alongpre-existing axon tracts by selectively fasciculating with the pioneering axons Thepioneering axons thus lay a scaffold for axons that develop later Selective fasciculation

is one of the major determinants for proper connectivity The sorting of axons in the righttract is governed by many cell adhesion molecules (CAMs), substrate adhesion molecule(SAMs e.g integrins) and signaling molecules like netrin, semaphorin, ephrin, receptortyrosine kinases (RTKs) and phosphatases The CAMs fall in two classes, those thatbelong to the immunoglobulin family (NCAM, L1/NgCAM, neuroglian, DM-

GRASP/BEN/irreC, NrCAM, and TAG-1/axonin) and those that belong to the cadherinfamily (N-cadherin).

1.6 Cell adhesion molecules.

Molecules that belong to the immunoglobulin superfamily

1.6.1.1 NCAM: has five Ig and two fibnonectin domains in it extracellular region It is

abundantly expressed in the nervous system NCAM appears to ligate with itself

(homophilic binding) (Walsh and Doherty, 1997), and one of its most obvious functions

is in the self-association of nerve fibers to form fascicles It is expressed in three isoforms

NCAM-120, NCAM-140 and NCAM-180 (Goodman, 1996) In in-vitro experiments

neurite outgrowth is better on NCAM-140 lacking the VASE exon The adhesive

property of NCAM can be altered by the addition of a carbohydrate moiety polysialicacid (PSA) Removal of PSA leads to increased adhesion and decreased axon outgrowth

in-vitro When PSA is removed form chick motor neurons using an enzyme

endoneuraminidase N the axons fail to navigate and display abnormal trajectories

Analysis of NCAM null mice show subtle defect in guidance and connectivity (Cremer etal., 1994; Cremer et al., 1997) However, they show reduced spatial learning ability whentested in the Morris water maze test This suggests that NCAM may possibly play a role

in establishing synaptic plasticity The molecule Fasciclin found in invertebrates is

similar to vertebrate NCAM (Lin et al., 1994) It was first identified in a subset of

fasciculating axons in grasshopper embryos (Bastiani et al., 1987) In Drosophila loss of

function FasII mutations lead to the complete or partial defasciculation of the vMP2,