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 ‘neuro- specificity’ 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 retino- tectal 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 Table 1. Retinotectal axon guidance mutants at a glance (listed alphabetically) Mutant Gene Axon defects Other defects References ace acerebellar fgf8 Path.: FB Circ., ear, FB comm., heart, MHB 35, 64–66 ast astray robo2 Path.: FB None 5, 40, 41 bal bashful ECM molecule Path.: FB + eye HB, NC, som. 5, 11, 12 bel belladonna Unknown Path.: FB Eye, FB comm., OKR 5, 31, 32 blu blumenkohl Unknown Topography OKR 6, 32 boxboxer Unknown Path.: FB + topography Ear, fins, jaw, mot. 5, 6, 45, 46, 53, 65 con chameleon Unknown Path.: FB + eye Circ., curly, LFP, som. 5, 16, 28, 67–69 cyc cyclops Nodal related-2 Path.: FB curly, cyclopia, FP 5, 16, 24, 25, 70, 71 dak dackel EXT-like gene Path.: FB + topography Ear, fins, jaw 5, 6, 45, 46, 65, 72 dtr detour gli1 Path.: FB Cranial MN, curly, LFP 5, 16, 28, 73 esr esrom Unknown Topography Pig. 5, 6, 42, 74 499 gna gnarled Unknown Topography None 6 gup grumpy β1-laminin Path.: FB + eye HB, NC, OKR, som. 5, 11–13, 32 igu iguana Unknown Path.: FB Curly, jaw, LFP, ect. lens, som. 5, 16, 28, 69, 75 + mao macho Unknown Topography Na currents, pig., touchresp. 6, 53, 54, 76, 77 nev nevermind Unknown Topography Mot. 6, 53 noi no isthmus pax2.1 Path.: FB + topography Circ., MHB, pronephros 6, 36, 37, 64, 68, 78 pic pinscher Unknown Path.: FB + topography Fins, jaw 5, 6, 46 sly sleepy γ 1-laminin Path.: FB + eye HB, NC, som. 5, 11–13 smu slow-muscle-omitted Smoothened Path.: FB Circ., curly, cyclopia, mid. Lens, som. 9, 79–81 spc space cadet Unknown Path.: eye + HB Mot., spiral fibers in HB 19, 53 syu sonic you sonic hedgehog Path.: FB + eye Circ., curly, fin, mot., som., Vfb 10, 16, 45, 53, 67, 68 uml umleitung Unknown Path.: FB Curly, FB comm., LFP 5, 28 woe who-cares Unknown Topography None 6 yot you-too gli2 Path.: FB Curly, circ., jaw, mid. lens, mot., som., vFB 5, 16, 17, 28, 53, 67–69, 75, 82 Notes: Abbreviations: circ., circulation; comm., commissure; ect., ectopic; FB, forebrain; HB, hindbrain; LFP, lateral floorplate; MHB, midbrain–hindbrain boundary; mot., motility; NC, notochord; OKR, optokinetic response; path., pathfinding; pig., pigment; resp., response; som., somite; and vFB, ventral forebrain. Making the connection 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.