Making the Connection: Retinal Axon Guidance in the Zebrafish

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seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp. 497–506 doi:10.1016/S1084–9521(02)00103-9, available online at http://www.idealibrary.com on

Making the connection: retinal axon guidance in the zebraﬁsh James Culverwell and Rolf O. Karlstrom∗

Genetic screens in zebrafish have identified a large number of for the formation of ‘neurospecificity’ that we still use mutations that affect neural connectivity in the developing today.1 In more modern terms, this model states that visual system. These mutants define genes essential for axons are guided to their appropriate targets by guid- accurate retinal axon guidance in the eye and brain and ance cues present in the growth environment. We now the characterization of these mutants is helping to define know that these environmental cues take the form of the cellular and molecular mechanisms that guide axons in molecules that help guide the motile tip of an axon, the vertebrate embryo. The combination of zebrafish genetic the growth cone, as it grows in the embryo. Guidance and embryological approaches promises to greatly increase molecules may attract or repel growthcones and may our understanding of how multiple guidance mechanisms be either diffusable or bound to the growth substrate.2 establish the complexneural interconnectivity of the vertebrate Most importantly, multiple guidance cues act on an in- brain. dividual growthcone to direct it to thecorrect target. The modern challenge is to identify the full suite of Key words: mutants / pathfinding / growth cone / tectum / guidance cues acting in the embryo, and to elucidate forebrain / genetic screens how growth cones integrate these cues to achieve the © 2002 Elsevier Science Ltd. All rights reserved. precise and reproducible neuronal connections that make a functioning brain. The zebrafish retinotectal projection, like that of the frog, forms as RGCs in the eye extend axons that con- nect to the optic tectum [Figure 1(A)]. In the eye, RGC axons grow in an organized way to the optic disc, where The retinotectal system in developmental they exit the eye and grow along the optic stalk/optic biology nerve to the optic chiasm. In lower vertebrates such as zebrafishand frogs, all retinal axons cross themidline The visual system in lower vertebrates has long been a where they enter the optic tract, resort, and grow dor- favorite model system for developmental biologists try- sally to the contralateral tectal lobe.3, 4 Upon reaching ing to understand how the brain gets wired. During the the tectum, each retinal axon terminates at a position past half century, classical embryological experiments that reflects the position of its cell body in the eye, in frogs have helped to define various aspects of the forming a topographic projection or sensory ‘map’ on assembly of the connections between retinal ganglion the tectum. Neurons from the dorsal eye send axons cells (RGCs) in the eye and their primary target in the to the ventral part of the tectum while ventral reti- brain, the optic tectum. By analyzing behaviors that re- nal neurons project dorsally. Nasal (anterior) retinal sulted from surgically induced defects, Roger Sperry neurons project to the posterior tectum and temporal and others were able to define the functionality and (posterior) neurons project anteriorly [Figure 1(A)]. formation of the retinotectal projection. This work re- The zebrafish model system, due to its experimen- sulted in a pivotal paper by Sperry where he summa- tal, optical, and genetic accessibility, is helping us rized the work and clarified the chemoaffinity model understand the mechanisms underlying the ‘neurospecificity’ that helps establish this neural connection. From the Department of Biology, University of Massachusetts, Amherst, We are now able to use the power of zebrafish genet- * MA 01003, USA. Corresponding author. ics to revisit many of the questions posed by Sperry in E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. the very same system. Genetic screens are revealing 1084–9521 / 02 / $– see front matter many of the defects seen by Sperry, but these defects

497 J. Culverwell and R.O. Karlstrom

Figure 1. Mutations affecting the zebrafish retinotectal projection. (A) Schematic dorsal view of the 5-day wildtype retinotectal projection. RGC axons project out of the eye into the optic nerve, cross the midline of the diencephalon at the optic chiasm (arrow), enter the optic tract, and grow dorsally to the contralateral tectal lobes. Labeling of dorsal/nasal axons (green) and ventral/temporal (red) axons shows that RGC axons are topographically ordered in the optic nerve and tract and project topographically on the tectum. Fluorescent pictures show examples of mutant phenotypes found in the retinotectal screen. Arrows point to aberrant retinal axon projections. (B) In six mutants, axons occasionally make errors within the eye. (C) Ten mutants disrupt midline crossing, with axons projecting to the ipsilateral (incorrect) tectal lobe where they find their correct topographic position. Axons occasionally also cross the midline in these mutants (dotted lines). (D) Mutations in five genes lead to dramatic pathfinding errors in the forebrain. In different mutants, axons grow rostrally, ipsilaterally, or into the contralateral eye. (E) In any individual ast/robo-2 mutant, axons can make multiple pathfinding errors, including rostral and ipsilateral growth and recrossing of the midline. (F) In two mutants, few axons cross the midline. Those axons that reach the contralateral tectum often do not find their correct topographic position. (G) In three mutants, dorsal retinal axons fail to sort completely into the ventral optic tract. Despite this missorting, axons map correctly in the tectum. Occasional ipsilateral projections are also seen in these mutants. (H) Three mutations lead to defects in dorsal/ventral and/or anterior/posterior mapping. (I) Three mutations cause retinal axons to have expanded termination fields with corresponding mapping errors. Underlining indicates the gene affected in the mutant has been identified (see Table 1 for mutant name and gene name).

498 Making the connection –hindbrain tral forebrain. 6, 53 6, 53, 54, 76, 77 35, 64–66 5, 40, 41 6, 36, 37, 64, 68, 78 5, 11, 12 5, 31, 32 6, 32 5, 6, 46 5, 6, 45, 46, 53, 65 5, 16, 28, 67–69 5, 16, 24, 25, 70, 71 5, 11–13 9, 79–81 5, 6, 45, 46, 65,5, 72 16, 28, 73 5, 6, 42, 74 6 19, 53 10, 16, 45, 53, 67,5, 68 28 6 5, 16, 17, 28, 53, 67–69, 75, 82 5, 11–13, 32 5, 16, 28, 69, 75 currents, pig., touchresp. + HB Mot., spiral fibers in HB topography Circ., MHB, pronephros eye HB, NC, som. topography Fins, jaw topography Ear, fins, jaw, mot. eye Circ., curly, LFP, som. eye HB, NC, som. topography Ear, fins, jaw eye Circ., curly, fin, mot., som., Vfb eye HB, NC, OKR, som. + + + + + + + + + + Path.: FB Path.: FB Circ., ear, FB comm., heart, MHB Path.: FB None Path.: FB Path.: FB Cranial MN, curly, LFP Path.: FB Path.: FB Path.: FB Curly, circ., jaw, mid. lens, mot., som., vFB 1-laminin 1-laminin Unknown Topography Mot. ECM molecule Path.: FB Unknown Path.: FB UnknownUnknownUnknown Path.: FB Topography Path.: FB OKR Eye, FB comm., OKR γ Unknown Path.: FB Nodal related-2EXT-like gene Path.: FB Path.: FB curly, cyclopia, FP SmoothenedUnknown Path.: FB Path.: eye Circ., curly, cyclopia, mid. Lens, som. UnknownUnknownβ Topography Topography Pig. None UnknownUnknown Path.: FB Topography None Curly, FB comm., LFP UnknownUnknown Path.: FB Topography Na Curly, jaw, LFP, ect. lens, som. Mutant Gene Axon defects Other defects References Retinotectal axon guidance mutants at a glance (listed alphabetically) noi no isthmus pax2.1 nev nevermind Table 1. ace acerebellar fgf8 astbal astray bashful robo2 Notes: Abbreviations: circ., circulation; comm., commissure; ect., ectopic; FB, forebrain; HB, hindbrain; LFP, lateral floorplate; MHB, midbrain boundary; mot., motility; NC, notochord; OKR, optokinetic response; path., pathfinding; pig., pigment; resp., response; som., somite; and vFB, ven pic pinscher belbluboxboxer belladonna blumenkohl sly sleepy con chameleon cycdak cyclops dackel smuspc slow-muscle-omitted space cadet dtresrgnagup detour esrom gnarled grumpy gli1 syu sonic you sonic hedgehog umlwoeyot umleitung who-cares you-too gli2 igumao iguana macho

499 J. Culverwell and R.O. Karlstrom are the result of loss of gene function rather than sur- extremely useful in helping to explain how retinal gical manipulation. These mutants will thus provide a growth cones integrate cues from the ECM as they molecular explanation for well-described axon guid- grow out of the eye. ance and mapping phenomena. con and syu also disrupt retinal axon growthin the In a zebrafishretinotectal screen done in the1990s, eye and midline patterning.16 syu encodes the mid- Dr FriedrichBonhoeffer’slab identified over 30 reti- line secreted morphogen Sonic Hedgehog (Shh), nal projection mutants by labeling RGC axons with and con has been shown to disrupt Hedgehog (Hh) the lipophilic dyes DiI and DiO5–7 (Table 1). Errors signaling.10 Since Hhsignaling plays a major role in were seen at a variety of positions in the pathway be- patterning the ventral forebrain and optic vesicles,9, 17 tween the eye and tectum (Figure 1). These mutants these eye exit axon phenotypes might be due to early can be grouped into those affecting pathfinding in eye and optic stalk patterning defects. Alternatively, the eye (‘eye exit’ mutants), pathfinding across the since RGC differentiation depends on Hhsignaling, 18 forebrain to the tectum (‘pathfinding mutants’), and these pathfinding errors may reflect defects in the the formation of topographic connections on the tec- retinal cells themselves. Finally, space cadet (spc) mu- tum (‘topography mutants’) (reviewed in References tations result in a third, distinct, eye exit phenotype. 7, 8). The retinotectal mutants help to define choice In spc mutants, RGC axons often fail to leave the eye, points encountered by retinal axons as they grow to despite well-organized growthto theoptic disc. 19 their final target in the tectum. Many of these mutants, along withsome mutants found in related screens, have now been extensively characterized, and many Pathfinding in the forebrain of the affected genes have been identified. In this re- view we examine what has been learned about retinal Crossing the midline axon guidance from this ‘first generation’ of genetic screens. A large number of identified pathfinding mutations affect the ability of axons to grow toward and across the midline of the forebrain [Figure 1(C)]. In these Pathfinding within the eye: eye exit mutants mutant fish, retinal axons grow to the ipsilateral rather than the contralateral tectal lobe. Despite being on Several identified mutations interfere withtheability the wrong side of the brain, retinal axons find their of retinal axons to grow toward the optic disc and exit correct topographic target, confirming classic work the eye5, 9, 10 [Figure 1(B)]. In bashful (bal), grumpy which showed that guidance mechanisms within the (gup), and sleepy (sly) mutants retinal axon growth tectum can operate independently of mechanisms is disorganized in the eye, with axons wandering cir- guiding axons across the midline.20 Similar results cumferentially before reaching the optic nerve head. have been found in fly mutants with defective midline bal, gup, and sly share an array of axon phenotypes axon crossing,21 showing that the remarkable ability as well as similar defects in notochord and hind- of growthcones to make ‘appropriate’ connections brain development,11, 12 which suggested that these on the wrong side of the embryo after following aber- genes act in the same genetic pathway. This hypoth- rant pathways is evolutionarily conserved. In the fly esis was recently confirmed when gup and sly were commissureless (comm) mutant, axons completely fail shown to encode the β1 and γ subunits, respectively, to cross the midline,22 similar to the phenotype seen of the extracellular matrix (ECM)/axon guidance in the zebrafish midline mutants. Midline pathfind- molecule Laminin.13 bal may also encode a compo- ing is also defective in fly, worm, and mouse embryos nent of Laminin (D. Stemple, personal communica- mutant for the netrin guidance molecule (reviewed tion). RGCs express Integrins, receptors for Laminin, in Reference 23). So far, none of the zebrafish mid- and Laminin is known to play a role in retinal axon line crossing mutants have been shown to encode growthin theeye (reviewed in Reference 14). The these well-characterized guidance molecules. Instead, bal, gup, and sly phenotypes confirm that Laminin these mutations all affect the formation of the ventral plays a crucial role in fasciculation and directed axon forebrain, and thus these midline pathfinding defects growth toward the optic disc. These laminin subchain appear to be caused by mispatterning of the growth mutant phenotypes are surprisingly specific given the substrate. widespread roles played by Laminin in development All of the identified zebrafish midline mutations, (see Reference 15), and these mutants should be withthepossible exception of bel, may affect Hh

500 Making the connection signaling in the forebrain. The cyclops (cyc) mutation reduces ventral forebrain tissues (leading to cyclopia) and mild alleles of cyc have ipsilaterally projecting retinal projections. cyc encodes the TGF-β signaling molecule Nodal related-2 (Ndr-2),24, 25 which is needed for Hhsignaling in theventral neural tube 26 and for the formation of the ventral forebrain.27 As mentioned above, syu encodes Shh itself,10 while smu encodes the Hh receptor complex protein Smoothened.9 We recently showed that dtr and yot encode Gli1 and Gli2, respectively, transcription factors that interpret Hh signals in responding cells,17 (R. Karlstrom et al., unpublished results). The proteins encoded by con, igu, and uml have not been identified, but all of these mutants have phenotypes con- sistent withdefects in Hhsignaling, 10, 28 (O. Tyurina and R. Karlstrom, unpublished results). Our analysis shows that these mutations disrupt specific instructive (bothattractant and repellent) and permissive (cellular) guidance cues (M. Barresi and R. Karlstrom, unpublished results). In particular, it is known that the Hh signaling regulates netrin expression in the 29, 30 vertebrate ventral midline. Indeed, among other Figure 2. Retinal axon errors in belladonna. (A) In wildtype defects, we have seen that netrin expression is selec- embryos retinal axons (arrows) cross the midline to form tively reduced in thezebrafishHhpathwaymutants the optic chiasm (arrowhead) by 36 h of development. (B) (M. Barresi and R. Karlstrom, unpublished results). In bel mutants retinal axons (arrows) can grow up to, but Our analysis suggests that the midline mutants will not across, the midline (arrowhead). (C) By 38 h the chiasm be extremely valuable in characterizing the molec- is well formed (arrowhead) in wildtype embryos and retinal ular and cellular nature of the retinal axon growth axons have grown dorsally towards the tectum (arrows). (D) substrate in the forebrain. By 38 h, axons in bel mutants are no longer near the midline and grow dorsally along the ipsilateral diencephalon Making the wrong connection (arrows). (E) Schematic of wildtype retinal (red) and forebrain commissural (gray) axons at 38 h. (F) Schematic showing range of bel retinal axon defects and defects in forebrain Like the midline mutants described above, belladonna commissure formation (gray lines). Circled areas show re- (bel) mutants have ipsilateral retinotectal projections gions where retinal axons were observed in different bel that map to the correct position on the ipsilateral mutant embryos at early ages. By 48 h, all axons project ipsi- tectal lobe5, 31 [Figures 1(C) and 2]. Unlike the Hh laterally immediately after leaving the eye. (A–D) ZN-5 anti- pathway mutants, however, midline structures are gen- body labeling of retinal axons. All panels show ventral views, erally unaffected in bel.Thebel larvae have a reversed anterior up. response to visual stimuli,31, 32 indicating that retinal axons make functional connections on the wrong side of the brain. The bel phenotype thus reproduces Our lab examined retinal axon growthin bel mutants classic embryological experiments showing a reversed more carefully and we found that retinal axons can visual response in frogs withsurgically induced ip- grow quite close to the midline before they turn ipsilat- silateral projections.20 Homozygous bel animals are erally (Figure 2(B)). This observation suggests that bel semiviable and exhibit abnormal swimming behav- disrupts axon guidance at or near the ventral midline. iors, further suggesting that the ipsilateral projections A careful analysis of gene expression in bel mutants re- are functional. The presence of (reversed) visual vealed subtle defects in forebrain patterning that may tracking, proper retinotectal mapping, and viability reflect the loss or misspecification of certain cell types of homozygous bel mutants all attest to the specificity at the midline where retinal axons normally cross (M. of the mutation for axon guidance across the midline Walkowicz and R. Karlstrom, unpublished results). of the ventral forebrain. Subtle forebrain defects, in combination withthe

501 J. Culverwell and R.O. Karlstrom observation that guidance errors are usually symmet- where axons do not grow.41 It thus appears that Slit rical across the midline, suggests that RGC guidance proteins may provide repellent cues that help confine defects result from defects in the growth substrate im- retinal axons to nonexpressing regions of the fore- mediately at the midline. The bel phenotype is remark- brain. The analysis of ast/robo2 function in zebrafish ably similar to achiasmatic disorders in humans and embryos reveals that vertebrates and invertebrates use dogs whichaffectdepthperception and otherbinoc- similar guidance mechanisms in different ways. In the ular functions.33, 34 The study of bel thus promises to Drosophila CNS, Slit acts as a midline barrier that is shed light on the molecular and developmental mech- crossed selectively after down-regulation of Robo re- anisms that may lead to these congenital disorders. ceptors in crossing axons. In the vertebrate forebrain, Slit seems to channel Robo-expressing RGCs into the Going haywire in the ventral forebrain appropriate region of the brain. There are multiple Robo receptors and Slit ligands, eachwithunique Several identified mutations lead to particularly dra- expression patterns, suggesting the Robo/Slit system matic axon defects, withretinal axons growing helps guide many different types of axons in different throughout the ventral forebrain [Figure 1(D)]. In ways. Analysis of Slit function in zebrafishwill help acerebellar (ace) mutants, retinal axons project ipsilater- elucidate how this one family of guidance molecules ally and rostrally in the telencephalon.35 The ace locus can play multiple roles in wiring the vertebrate brain. encodes FGF8, a secreted signaling molecule crucial esrom (esr) mutants have distinct pathfinding defects for normal forebrain patterning.35 Elegant eye trans- [Figure 1(F)]. In esr mutants few axons cross the mid- plantation experiments were used to create genetic line, and those that do reach the contralateral tectum mosaics and show that ace/fgf8 function is required fail to reachtheircorrect target field. 5, 6 esr mutants in the brain (not the eye) for normal retinal axon also have pigment defects, with reduced xanthophore guidance.35, 52 Ipsilateral and rostral axon growthis (yellow) pigmentation.42 The relationship between also seen in no isthmus (noi) mutants, withadditional defects in pigmentation and retinal axon defects is defects in axon fasciculation. Strikingly, retinal axons not understood, and the study of esr promises to link from the two eyes often intermingle in noi mutants, these apparently disparate phenomena. Interestingly, and they sometimes grow into the contralateral eye.36 albinism in mammals leads to defects in retinal pro- noi encodes pax2.137 which is expressed in glial cells jections that may be caused by misspecification of of the optic stalk, cells needed to organize axons in some RGCs.43 Thus a mechanistic link between pig- the optic nerve.36 ment formation and the formation of the retinotectal Retinal axons in bal, gup/β1-laminin, and sly/γ -lami- projection appears to be conserved across vertebrate nin mutants make numerous errors and can project species, including humans.33 ipsilaterally or rostrally into the telencephalon. Since Laminin is expressed along the retinal pathway in 38 other vertebrates, these errors may reflect a loss of Topography an attractive or permissive substrate that allows growth 39 across the midline and into the optic tract. Further Fiber sorting mutants analysis of growthcone behaviorsand laminin function in bal, gup/β1-laminin, and sly/γ -laminin is needed In boxer (box), dackel (dak), and pinscher (pic) mutants, to clarify the role of Laminin mediated cell–substrate dorsal axons do not all sort into the ventral branch of adhesion in retinal axon growth through the brain. the optic tract. Arriving at the incorrect, dorsal side of astray (ast) homozygous fish show no obvious vis- the tectum, these axons then traverse the tectum to ible defects and are viable. In this mutant, retinal find their correct ventral target [Figure 1(G)]. This axons make multiple errors, including ipsilateral observation confirms embryological studies which growth, rostral growth, and recrossing of the midline demonstrate that retinal axons map correctly on the [Figure 1(E)]. ast seems to affect only the retinal tectum independent of their point of entry.44 The axons; eye transplantation experiments show ast func- observation that these three mutants share retinal tion is needed in retinal axons rather than in the axon, jaw, and fin defects45, 46 suggests that box, dak, growthsubstrate. 40 ast encodes Robo-2, a receptor and pic may act in the same genetic pathway. In expressed on retinal growthcones thatbinds sur- work focusing on the jaw phenotype it was recently face molecules of the Slit family.40 slit2 and slit3 are reported that dak encodes a member of the exos- expressed in stripes across the forebrain in regions tosin (EXT) enzyme family which regulates heparan

502 Making the connection sulfate proteoglycan (HSPG) biosynthesis (H. Roehl RGC arbors are expanded to various degrees through- and C. Nüsslein-Volhard, personal communication). out the tectal neuropil. mao has another phenotype, Different HSPGs can bind a variety of axon guidance touch insensitivity, that sheds light on these retinal molecules (including Netrins, Slits, and Laminin), axon defects.53 mao RGCs and rohon-beard cells lack + guidance receptors (including DCC and Protein Ty- sodium currents, and blockage of Na channel activ- rosine Phosphatases47), and growthfactors (FGFs, ity with tetrodotoxin phenocopies the RGC projection TGF-βs) (reviewed in Reference 48). The recent defects seen in mao.54 Thus the expanded retinal pro- finding that heparan sulfate (HS) is essential for jections on the tectum seen in mao seems to be due Slit2/Robo-1 repulsive interactions49 supports the to the loss of activity dependent refinement of the model that HSPGs may act as cofactors that modu- retinotectal map. The further study of mao promises late ligand/receptor binding. HS has been directly to help us understand how neural activity refines implicated in retinal axon guidance in the frog,50 sensory maps in the brain. withspecific HS sequences required for retinal axon targeting.51 Since EXT enzymes are needed for the synthesis of numerous HSPGs, which can then in- Future connections in zebrafish teract witha wide range of axon guidance systems, determining the precise mechanisms by which EXT Given the long and productive history of research in proteins affect retinal axons may be a challenge. One other model systems, why should we study axon guid- hint may come from observations that cells at the bor- ance in the zebrafish? The answer lies in the unique der of the dorsal optic tract express a specific sulfation combination of genetic and experimental tools that enzyme, 6-O-sulfotransferase, which shows that this can be applied in zebrafishto examine axon guid- region may contain specific sulfated forms of HSPGs ance at boththecellular and molecular levels. Work needed for axon guidance in the tract.51 Thus the box, in Drosophila and Caenorhabditis elegans has shown the dak, and pic mutants are providing an important entry power of random mutagenesis screens in finding axon point into the study of fiber sorting in a nerve bundle, guidance genes (see References 55, 56), and zebrafish a phenomenon that remains poorly understood. work brings this approach into a vertebrate species. This research bridges the gap between genetically ac- Mapping and termination mutants cessible but relatively simple invertebrate brains, and more complicated, yet less accessible, mammalian The retinotectal screen was designed to find muta- brains. Almost half of the mutations identified in the tions that affect sensory mapping. Mutants with both ‘first generation’ zebrafishscreens havenow been dorsal/ventral and anterior/posterior mapping de- identified, and all encode previously known proteins fects were isolated. nevermind (nev) and who-cares (woe) (see Table 1). We can only hope that as more labori- affect the mapping of retinal fibers along the dor- ous positional cloning projects are finished, some of sal/ventral axis6 [Figure 1(H)]. In bothmutants, na- the remaining mutants will be found to encode novel sodorsal axons terminate bothventrally and dorsally or surprising genes. Even now, the zebrafish retino- in the tectum instead of targeting only the ventral tectal mutants are revealing how molecular function region. In nev, axons missort in the optic nerve and is conserved across the animal kingdom. More im- tract, while sorting appears normal in woe. These dor- portantly, they are revealing new functions for evo- sal/ventral mapping mutants are of great interest, as lutionarily conserved molecules. This work will help very little is known about how the dorsal/ventral map direct research in higher vertebrates and is providing is generated. In ace/fgf8 mutants, which lack a mid- important models for human disease.57 brain/hindbrain boundary, mapping errors occur in Cell transplantation and ablation studies in ze- boththedorsal/ventral and anterior/posterior axes brafishhavecontributed enormously to our cellular of the tectum. Both dorsal/nasal and ventral/nasal understanding of axon guidance, and it is now rou- axons have expanded termination zones.52 tine to alter the growth environment in the zebrafish Other anterior/posterior mapping mutants include embryo by removing or adding cells. The ability to gnarled (gna), macho (mao), and blumenkohl (blu)6, 52 do these manipulations in different mutants will be [Figure 1(I)]. In gna mutants, nasodorsal retinal critical to help define the molecular mechanisms that axons begin to branchprematurely in theanterior mediate previously defined cellular interactions. As region of the tectum, with some axons terminating an example of this approach, cell transplantation ex- in this inappropriate region. In mao and blu mutants periments in the zebrafish spinal cord have shown

503 J. Culverwell and R.O. Karlstrom that specific muscle precursor cells (adaxial cells) are Acknowledgements needed for motor axon growthinto thesomites, 58 while ablations have shown that another set of muscle We thank our zebrafish colleagues who shared their cells (muscle pioneers) are not required for motor unpublished data and apologize for any omissions axon guidance59 (reviewed in Reference 60). In ad- owing to space constraints. Thanks to R. Murphey, dition, the generation of genetic mosaics has shown T. Godenschwege, M. Barresi, M. Walkowicz, P.Wilder, that ace/fgf8 function is required in the brain for nor- and B. Howlett for comments on the manuscript. R.O. mal retinal axon growth,52 while ast/robo-2 function Karlstrom is supported by the NIH (#RO1NS39994) is required in RGCs.40 These genetic/embryological and the Baystate/UMass Collaborative Biomedical experiments will continue to help define the molec- ResearchProgram. ular mechanisms that mediate interactions between growthcones and theenvironment. Coupled withgenetic and experimental accessibility References is the well-known optical clarity of the zebrafish em- 1. Sperry RW (1963) Chemoaffinity in the orderly growth of nerve bryo. Because zebrafishembryos develop so rapidly fiber patterns and connections. Proc Natl Acad Sci USA 50:703– and cells can be labeled and visualized in live embryos, 710 it is possible to directly observe the processes of axon 2. Tessier-Lavigne M, Goodman CS (1996) The molecular biology guidance and synaptogenesis. In vivo observations of of axon guidance. Science 274:1123–1133 mutant growthcone behaviorcan uncover extremely 3. Stuermer CA (1988) Retinotopic organization of the developing retinotectal projection in the zebrafish embryo. J Neurosci subtle defects in axon guidance. As an example, the 8:4513–4530 analysis of growth cone behavior in the ast/robo-2 4. Burrill JD, Easter SSJ (1994) Development of the retinofugal mutant suggests that mutant growth cones differ projections in the embryonic and larval zebrafish. J Comp Neu- from wildtype growthcones in theirability to correct rol 346:583–600 pathfinding errors.41 This high resolution analysis 5. Karlstrom RO, Trowe T, Klostermann S, Baier H, Brand M, Crawford AD, Grunewald B, Haffter P, Hoffmann H, Meyer SU, of growthcone behaviorwill be an extremely pow- Muller BK, Richter S, van Eeden FJ, Nusslein-Volhard C, Bonho- erful way to uncover real-time aspects of axon guid- effer F (1996) Zebrafishmutations affecting retinotectal axon ance that might lead to significant defects in neural pathfinding. Development 123:427–438 connectivity. 6. Trowe T, Klostermann S, Baier H, Granato M, Crawford AD, Grunewald B, Hoffmann H, Karlstrom RO, Meyer SU, Muller We are now entering the ‘post-genome’ era for ze- BK, Richter S, Nusslein-Volhard C, Bonhoeffer F (1996) Muta- brafish. As sequencing of the zebrafish genome is tions disrupting the ordering and topographic mapping of ax- completed in the next year or two, and as genetic ons in the retinotectal projection of the zebrafish, Danio rerio. maps continue to improve, the identification of genes Development 123:439–450 7. Karlstrom RO, Trowe T, Bonhoeffer F (1997) Genetic analysis of important for axon guidance will become increas- axon guidance and mapping in the zebrafish. Trends Neurosci ingly straightforward. Second generation screens in 20:3–8 zebrafishwill employ new tricks borrowed from other 8. Hutson LD, Chien CB (2002) Wiring the zebrafish: axon guid- model systems (e.g. GFP expressing transgenic lines, ance and synaptogenesis. Curr Opin Neurobiol 12:87–92 insertional mutagenesis, transposon based mutagen- 9. Varga ZM, Amores A, Lewis KE, Yan YL, Postlethwait JH, Eisen JS, Westerfield M (2001) Zebrafishsmoothenedfunctions in esis, and Gal4 systems) and are already being devel- ventral neural tube specification and axon tract formation. De- oped to help identify new components of the axon velopment 128:3497–3509 guidance machinery. ‘Reverse genetic’ techniques 10. Schauerte HE, van Eeden FJ, Fricke C, Odenthal J, Strahle U, to identify gene function are also evolving quickly. Haffter P (1998) Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development New tools that benefit from a sequenced genome 125:2983–2993 include morpholino antisense oligonucleotides to 11. Stemple DL, Solnica-Krezel L, Zwartkruis F,Neuhauss SC, Schier knock down gene function (see Reference 61), laser AF, Malicki J, Stainier DY, AbdelilahS, Rangini Z, Mount- activation of the heat-shock promoter driven con- castle-Shah E, Driever W (1996) Mutations affecting develop- 62 ment of the notochord in zebrafish. Development 123:117–128 structs to misexpress genes in targeted cells, as well 12. Odenthal J, Haffter P, Vogelsang E, Brand M, van Eeden FJ, as targeting genetic lesions to known genes as is done Furutani-Seiki M, Granato M, Hammerschmidt M, Heisenberg in plants.63 The next few years hold great promise CP, Jiang YJ, Kane DA, KelshRN, Mullins MC, Warga RM, Al- for understanding axon guidance, and the zebrafish lende ML, Weinberg ES, Nusslein-Volhard C (1996) Mutations affecting the formation of the notochord in the zebrafish, Danio system will continue to play a central role in helping rerio. Development 123:103–115 us understand this process in the most complex of all 13. Parsons MJ, Pollard SM, Saude L, Feldman B, Coutinho P, Hirst interconnected systems, the vertebrate brain. EM, Stemple DL (2002) Zebrafishmutants identify an essen-

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