Genetic analysis of axon guidance and mapping in the zebrafish

A. Tolkovsky – Neurotrophic factors in action

given the quality of the work presented, there is probably not long to wait before the results are published in leading journals. References 1 Lin, L.F.H. et al. (1993) 1130–1132 2 Henderson, C.E. (1996) Neurobiol. 6, 64–70 3 Durbec, P. et al. (1996) 789–793 4 Trupp, M. et al. (1996) 785–789 5 Durbec, P.L. et al. (1996) 122, 349–358

Science 260, Curr. Opin. Nature 381, Nature 381, Development

6 Moore, M.W. et al. (1996) Nature 382, 76–79 7 Treanor, J.J.S. et al. (1996) Nature 382, 80–83 8 Jing, S.Q. et al. (1996) Cell 85, 1113–1124 9 Chao, M.V. (1994) J. Neurobiol. 25, 1373–1385 10 Greene, L.A. and Kaplan, D.R. (1995) Curr. Opin. Neurobiol. 5, 579–587 11 Dobrowsky, R.T. et al. (1994) Science 265, 1596–1599 12 Carter, B.D. et al. (1996) Science 272, 542–545 13 Lee, K.F. et al. (1994) Science 263, 1447–1449 14 Lee, K.F., Davies, A.M. and Jaenisch, R.

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(1994) Development 120, 1027–1033 15 Huber, L.J. and Chao, M.V. (1995) J. Neurosci. Res. 40, 557–563 16 Frade, J.M., Rodriguez-Tebar, A. and Barde, Y.A. (1996) Nature 383, 166–168 17 Davies, A.M., Minichiello, L. and Klein, R. (1995) EMBO J. 14, 4482–4489 18 Borasio, G.D. et al. (1993) J. Cell Biol. 121, 665–672 19 Nobes, C.D. and Tolkovsky, A.M. (1995) Eur. J. Neurosci. 7, 344–350 20 Vogel, K.S. et al. (1995) Cell 82, 733–742 21 Virdee, K. and Tolkovsky, A.M. (1995) Eur. J. Neurosci. 7, 2159–2169 22 Virdee, K. and Tolkovsky, A.M. (1996) J. Neurochem. 67, 1801–1805

TECHNIQUES Genetic analysis of axon guidance and mapping in the zebrafish Rolf O. Karlstrom, Torsten Trowe and Friedrich Bonhoeffer Systematic genetic screens have been powerful tools in identifying genes responsible for axon guidance in fruitflies and nematodes. This approach has now been extended to the study of axon guidance and the formation of topographic neuronal connections in the vertebrate brain. A systematic genetic screen was used to identify genes responsible for precise axon pathfinding and targeting in the retinotectal system of the zebrafish (Danio rerio). Over 30 genes were found that affect either: (1) retinal axon pathfinding to the contralateral tectal lobe; or (2) the topographic connection between the eye and the tectum. The zebrafish retinotectal mutants represent a new resource for the study of axon guidance in the vertebrate brain. Trends Neurosci. (1997) 20, 3–8

D

URING DEVELOPMENT axons grow precisely and reliably over long and complex pathways to reach their correct targets. Just as highway signs guide a driver towards a destination, molecules in the environment help guide the motile tip of an axon, the growth cone, along the correct pathway. These guidance molecules might attract or repel growing axons and might be diffusable or bound to the growth substrate1. Different axons can have different responses to the same guidance molecule2,3. Furthermore, gradients of guidance cues can help guide axons to their appropriate termination site4. It also seems that multiple guidance cues are responsible for accurate pathfinding of a single axon to its target. Given this complexity, it is a daunting task to identify the molecules that guide axons, and to elucidate how these molecules work together to achieve the precise and reproducible neuronal connections that make a functioning brain. A powerful approach to studying this problem is to use genetics to identify genes necessary for in vivo axon guidance. Mutations that result in defects in axonal projections define genes involved in establishing neuronal connectivity in the intact organism. By doing large-scale systematic screens, it is theoretically possible to identify all of the genes involved in a given Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0166 - 2236/97/$17.00

developmental pathway. This approach has been extremely successful in two invertebrate species, the fruitfly Drosophila melanogaster and the nematode worm Caenorhabditis elegans. An advantage of this approach lies in the unbiased nature of a genetic screen. Mutations can uncover novel genes or known genes that play unexpected roles. Mutated genes might control any aspect of guidance, from ligands and receptors outside the cell to second messengers and cytoskeletal proteins inside. Until recently, large-scale genetic screens have not been practical in vertebrates. A ‘reverse genetic’ approach, in which genes already cloned are disrupted or eliminated, has been employed in an attempt to understand the role of known proteins, such as adhesion molecules, in mouse development. Unfortunately, in most cases genetic knockouts of genes that might be expected to play a role in axon guidance have resulted in no obvious axon phenotype (for example, Refs 5,6). This could be due to the extreme subtlety of the phenotype, the fact that there are multiple proteins with overlapping guidance functions, or that the molecules function early in development and mice lacking the protein fail to survive to the stage of axon outgrowth. By employing forward genetics in zebrafish to study PII: S0166-2236(96)40005-4

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Rolf O. Karlstrom is at the Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 1st Avenue, New York, NY 10016, USA. Torsten Trowe and Friedrich Bonhoeffer are at the Max-PlanckInstitut für Entwicklungsbiologie, Spemannstr. 35/1, D-72076 Tübingen, Germany.

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coordinated movement (called ‘unc’ mutants). Several of these mutations affect the connectivity of neurons7,8. In particular, unc-5 (Ref. 9) and unc-6 (Ref. 10) affect growth-cone guidance directly. The unc-6 gene was cloned10, and encodes a protein homologous to vertebrate guidance molecules called netrins that attract spinal-cord commissural axons towards the midline. UNC-5 is a transmembrane receptor11 that interacts with the unc-6 gene product (UNC-6). Thus, UNC-5 and UNC-6 appear to be a receptor–ligand pair involved in dorsal–ventral axon guidance in nematodes. Recent genetic screens in Drosophila assayed the formation of several different axon pathways (for a review, see Ref. 12). Mutations were identified that affect the patterning of longitudinal and commissural axon pathways in the CNS (Ref. 13), neuromuscular specificity14, the formation of the visual system15, or the development of the PNS (Ref. 16). The mutations affect axon outgrowth and target recognition, as well as the formation of topographic projections in the brain15. The cloning of several of these genes has provided new and unexpected information about the mechanisms that help form specific neural connections. Mutations in the gene commissureless, for example, result in the absence of commissures13. commissureless encodes a novel transmembrane protein that seems to be transferred from midline glial cells to commissural axons as they cross the Drosophila midline17. As another example, mutations in the dreadlocks Fig. 1. Retinotectal projections in the wild-type zebrafish ( Danio rerio). (A) Schematic diagram of the retinotectal gene cause photoreceptor axons to projection in wild-type zebrafish as visualized during the screen. The left eye of each fish was injected with DiI (red) in make pathfinding errors on their the temporal–ventral quadrant and with DiO (green) in the nasal–dorsal quadrant, allowing the visualization of the way from the eye to the brain. The complete axon pathway from eye to contralateral tectal lobe. Axons leave the eye at the optic nerve head (papilla) and dreadlocks gene was found to enare sorted in the optic nerve according to position in the eye. After the chiasm, dorsal and ventral axons sort into vencode a protein containing SH2 and tral and dorsal branches of the optic tract, respectively. The axons grow dorsoposteriorly to the contralateral tectal lobe SH3 domains, which indicates a where they project topographically; temporal–ventral retinal neurons project to the anterior–dorsal tectum, and 18 nasal–dorsal retinal neurons project to the posterior–ventral tectum. (B) Confocal stereo pair showing the wild-type role in tyrosine kinase signaling . retinotectal projection. The fluorescently labeled axons are visible after they leave the eye at the papilla; they cannot It appears that dreadlocks is part of be seen within the eye because of the pigmented epithelium. Anterior to the left. Abbreviations: D, dorsal; N, nasal; signal-transduction mechanism that transmits guidance information T, temporal; V, ventral. Scale bars, 100 ␮m. within the growth cone. These inthe formation of relatively early forming axon tracts creasingly sophisticated genetic screens in invertebrate it has become possible to overcome this problem and species are proving to be powerful tools for furthering to analyse in vivo axon guidance systematically in a our understanding of axon guidance. vertebrate for the first time.

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Fly and worm genes affecting axon guidance

Using zebrafish genetics to identify guidance mechanisms in a vertebrate

In the past few years, genetic screens in D. melanogaster and C. elegans have provided new insights into the mechanisms of invertebrate axon guidance. Many genes necessary for proper axon pathfinding have been identified. In the nematode, a large number of mutations were identified because they cause un-

The convergence of biochemical approaches in vertebrates and genetic approaches in invertebrates has shown that some axon-guidance mechanisms are conserved to a remarkable degree through evolution (for example, Refs 10,19). At the same time, given the complexity of the vertebrate CNS, it seems likely that

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evolution has provided vertebrates with novel guidance mechanisms, or has used conserved guidance cues in novel ways. Thus, it is important to study axon guidance systematically in vertebrates as has been done in invertebrates. The refinement of the zebrafish (Danio rerio) as a genetic system20,21 made it possible to apply a systematic genetic approach to the study of axon guidance in a vertebrate, as was done recently in a large-scale screen in Tübingen, Germany22. In association with a screen for genes involved in early development undertaken in the Nüsslein-Volhard laboratory, larvae from mutagenized zebrafish families were screened for defects in axon projections between the eye and its main target in the brain, the optic tectum23–25. This retinotectal screen used an extremely specific assay designed to uncover genes involved in fine-scale mapping of retinal axons on the optic tectum. In addition to finding several genes involved in retinotectal topography, a large number of genes were found that affect axon guidance more generally.

Axon pathfinding in the retinotectal system of zebrafish

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Fig. 2. Examples of mutant phenotypes as found in the screen. For the retinotectal screen, whole mounted fish were viewed from the dorsal side under a fluorescent microscope the day after the dyes were injected. Anterior is to the left, putting the injected left eye at the bottom of the panel. In these photographs, fish were stained with DAPI (blue fluorescence) to outline the tectal neuropil, which is surrounded by DNA-containing cell bodies. Black spots are melanophores in the skin. (A) In a wild-type (wt) embryo the topographic projections on the contralateral tectal lobe are easily visualized. The optic nerve and ventral optic tract are ventral to this focal plane. (B) In astray (ast), retinal axons make pathfinding errors after reaching the midline. Instead of growing posteriorly and dorsally to the contralateral tectal lobe, axons often project anteriorly and sometimes recross the midline, as seen in this photograph. In other individuals, axons turn posteriorly but not dorsally, and grow ventral to the tectum (not shown). (C) In boxer (box), dorsal retinal axons fail to sort into the ventral brachium of the optic tract completely, as in the wild type (inset). Here the dorsal retinal axons are approximately evenly distributed between dorsal (arrowhead) and ventral (arrow) brachia. Upon reaching the tectum, mis-sorted dorsal axons traverse the tectum to reach their appropriate ventral target (not shown). (D) In who-cares (woe), dorsal retinal axons sort correctly in the optic tract, but grow inappropriately over the dorsal tectum. (E) Confocal image showing that some dorsal axons grow inappropriately over the dorsal tectum and terminate in the dorsal instead of ventral posterior tectum (star) in who-cares. Scale bar, 100 ␮m (A–D), 75 ␮m (E).

The retinotectal system of lower vertebrates is well characterized and provides a good system for studying both the process of axon pathfinding towards a target and the process of topographic connectivity within the target26,27. In the zebrafish, axons from retinal ganglion cells grow from the eye to the primary visual center in the brain, the optic tectum. Retinal axons follow a distinct pathway along the ventral and lateral surface of the diencephalon28,29. Axons from the two eyes cross each other at the ventral midline of the diencephalon to form the optic chiasm. After crossing the midline, retinal axons enter the optic tract and grow dorsally to the contralateral tectal lobe. Axons sort in the optic tract according to the position of their cell bodies in the eye, with axons from dorsal retinal ganglion cells forming the ventral branch of the optic tract, and ventral retinal axons forming the dorsal branch. Upon reaching the tectum, retinal axons terminate at a position in the tectum that reflects the position of the neuronal cell body in the eye, thus forming a so-called ‘topographic’ projection. Neurons from the dorsal eye send axons to the ventral part of the tectum while ventral retinal neurons project dorsally. Nasal (anterior) retinal neurons project to the posterior tectum and temporal neurons project anteriorly. The connections thus form an inverted map of the retina on the tectum (Fig. 1). The lipophilic tracer dyes DiI and DiO can be used to visualize this retinotectal projection in the zebrafish (Figs 1 and 2). To do this, the different colored dyes are injected into two positions in the eye where they

each contact a small number of retinal ganglion cell bodies. The dyes diffuse throughout cell membranes to label axons along their entire length. In Figs 1 and 2, DiO (green) was injected into the dorsal–nasal quadrant and DiI (red) was injected into the ventral– temporal quadrant of the left eye of 5-day-old fish larvae. This labeling allows the visualization of the topographic arrangement of axon projections on the tectum in both the anterior–posterior and dorsal– ventral axes (Fig. 1 and Fig. 2A). As the zebrafish are largely transparent at this age, the labeled axons can be seen in whole mounted fish using a fluorescent microscope. To discover mutants affecting this projection, over 120 000 individual zebrafish larvae, representing nearly 3000 mutagenized genomes, were screened for defects in the retinotectal pathway. In order to assay such a large number of fish, a rapid and reliable method of labeling retinal ganglion cells was developed. Fixed 5-day-old fish were mounted carefully in agarose and injected sequentially with DiI and DiO using a vibrating tungsten wire. The injection apparatus and procedure are described in detail in Baier et al.23. On average, the screening procedure required approximately one minute of hands-on work per fish, including fixing, mounting, injecting and screening under a compound microscope. TINS Vol. 20, No. 1, 1997

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Fig. 3. Schematic of the pathfinding mutants. Genes are classified according to the position of pathfinding errors. The gene abbreviations and number of alleles found are shown for each type of pathfinding defect. The top two panels depict 48-hour embryos labeled with the ZN-5 antibody to show retinal ganglion cells and their axons. The remaining panels depict DiI and DiO labeling of retinal ganglion cell axons. Anterior is to the left. Gene names: ast, astray; bal, bashful; bel, belladonna; blw, blowout; box, boxer; con, chameleon; dak, dackel; dtr, detour; esr, esrom; gup, grumpy; igu, iguana; pic, pinscher; sly, sleepy; til, tilsit; tof, tofu; uml, umleitung; yot, you-too. Abbreviations: ON, optic nerve; OT, optic tract; RGCs, retinal ganglion cells. Adapted from Ref. 25.

Theoretically, a genetic screen of this size could achieve over 90% saturation of the genome23. Based on allele frequency (approximately 2.4 mutants per gene for the entire screen22), and the high number of single-allele loci (about half of the retinotectal mutants), it is clear the screen did not achieve this high level of saturation. Nonetheless, it seems safe to say that the retinotectal screen was on a scale sufficient to uncover mutations affecting most of the important guidance decisions made by a growth cone as it grows from the eye to its correct position in the tectum. In the zebrafish retinotectal screen, 112 mutant lines were identified in which labeled retinal axons deviated from the wild-type projection pattern. Errors were seen at a variety of positions in the pathway between the eye and tectum, and help define the pathway choice points encountered by a growth cone as it grows from eye to tectum. The mutants were classified based on the position of axon-guidance errors. Defects were classified generally as those affecting pathfinding to the tectum (‘pathfinding 6

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mutants’)25, the formation of topographic connections on the tectum (‘mapping mutants’)24, or development of the brain24. Figure 2 shows examples of some of these mutant phenotypes as seen during the screen. With complementation testing almost complete, these mutations define just over 30 genes. Below is a brief description of selected mutant phenotypes found in the screen.

Pathfinding mutants The zebrafish pathfinding mutants can be classified further according to the position of their axonal defects (Fig. 3)25. Pathfinding errors were seen in the eye, between the eye and the midline, and between the midline and the contralateral tectal lobe. In the largest class of pathfinding mutations retinal axons fail to cross the ventral midline of the brain. In these fish, retinal neurons connect to the ipsilateral rather than contralateral tectal lobe. In all of these mutants axons are able to find their correct topographic target, indicating that guidance mechanisms within the tectum can operate independently of mechanisms that guide axons to the tectum. Five of these genes (chameleon, detour, iguana, umleitung and you-too) affect differentiation of midline structures, as does the previously identified cyclops mutation30,31. These mutations highlight the central role the midline plays in guiding retinal axons. The careful analysis of the structures that are absent in these mutations, combined with experiments that rescue the axon-guidance defects by transplanting small numbers of wild-type cells into this region of mutant embryos, promise to define the specific midline cells that guide axons in the region of the chiasm. Embryos homozygous for the belladonna mutation also have ipsilateral retinotectal projections. Unlike the mutants mentioned above however, midline structures appear to differentiate normally, suggesting that the defects are more restricted to axon-guidance mechanisms. In fact, unlike mutants with general midline defects, homozygous belladonna fish are viable. Some belladonna homozygous fish show abnormal swimming behavior (circling) that suggests the retinal axons might form functional connections within the wrong (ipsilateral) tectal lobe32. While most axon pathways are unaffected in all of these mutations, the postoptic commissure, an early forming commissure in the same position as the optic nerve, fails to form in all of the mutants with ipsilateral retinotectal projections. Because these genes affect commissural guidance in the CNS, the ipsilateral mutant phenotypes are at least superficially similar to the Drosophila mutants with commissure defects, particularly the commissureless phenotype. The characterization of these mutants should further elucidate the role the midline plays in guiding axons further, including the cellular and molecular cues that guide axons towards and across the midline. Four genes affect the ability of axons to turn towards the contralateral tectal lobe after they reach the midline. In three of these genes, bashful, grumpy and sleepy, after axons cross the midline they often turn anteriorly and grow along the margin of the telencephalon. Axons in bashful also project ipsilaterally, or make pathfinding errors within the eye with axons occasionally taking extremely circuitous routes before exiting the eye25. These three genes also affect earlier

R.O. Karlstrom et al. – Zebrafish retinotectal mutants

developmental processes such as notochord differentiation and formation of the hindbrain33. Two alleles of bashful are viable (C.B. Chien, pers. commun.), while the remainder of these mutations are lethal. In contrast to bashful, astray homozygous fish show no obvious visible defects and all alleles seem to be viable. In this mutant, some retinal axons fail to turn dorsally after reaching the midline, and instead grow on the ventral surface of the brain in either an anterior or posterior direction (Fig. 2). astray is the only pathfinding gene that seems to affect only the retinal axons; other axon pathways, including the postoptic commissure, form normally.

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Fiber-sorting mutants Three genes affecting axon sorting in the optic tract were identified24,25(Fig. 2C). In the wild type, dorsal and ventral axons sort into different branches of the optic tract and arrive at different parts of the contralateral tectal lobe. In boxer, dackel and pinscher, not all dorsal axons sort into the ventral branch of the optic tract (Fig. 2C); instead, some dorsal retinal axons follow the inappropriate dorsal branch and arrive at the dorsal side of the tectum. These axons then traverse the tectum to find their correct ventral target, confirming embryological studies that demonstrate that retinal axons map correctly on the tectum independent of their point of entry34. These genes provide an entry point into the study of fiber sorting in a nerve bundle, a phenomenon that is poorly understood.

Mapping mutants Two genes (nevermind and who-cares) affect the mapping of retinal fibers along the dorsal–ventral axis (Fig. 4)24. In both mutants, nasodorsal axons terminate both ventrally and dorsally in the tectum. In nevermind, axons are already mis-sorted in the optic nerve and tract, while sorting appears normal in whocares. Two genes affect anterior–posterior mapping. Nasodorsal retinal axons in gnarled and macho begin to branch prematurely in the tectum instead of staying fasciculated until they reach their target site in the posterior tectum, as in the wild type. In gnarled, axons begin to branch in the anterior tectum, while in macho premature branching is more subtle and occurs in the posterior tectum. The mapping defects in nevermind, who-cares and gnarled are not symmetrical. Nasoventral axons map properly in the posterior dorsal tectum. These mapping mutant phenotypes are particularly intriguing when considered in regard to current models of map formation based on gradients of guidance cues (see Ref. 24). nevermind and who-cares also promise to give insights into dorsal–ventral mapping in the tectum, a phenomenon that, unlike anterior–posterior mapping35,36, has so far eluded experimental analysis.

Conclusions from the screen and future prospects The success of the zebrafish retinotectal screen has shown that it is possible to apply a large-scale genetic approach to the study of specific developmental processes in a vertebrate. The fact that individual genes can be mutated to give clear axon-guidance defects shows that some guidance systems are unique and do not have completely redundant or overlapping functions. At the same time, most of the zebrafish mutant phenotypes show a reduction in the number

brd (9)

Fig. 4. Schematic of sorting, mapping and other mutants. Genes are again classified according to phenotype with three-letter abbreviations and numbers of alleles indicated for each phenotype. In this diagram, tectal neuropils are shaded darker blue and are reduced or absent in some mutants. Gene names: blu, blumenkohl; box, boxer; brd, brain dead; dak, dackel; esr, esrom; gna, gnarled; mao, macho; nev, nevermind; noi, no-isthmus; pic, pinscher; tin, tiny neuropil; woe, who-cares. Adapted from Ref. 24.

of axons that arrive at the correct target, not a total elimination of accurate axon guidance. This suggests that multiple guidance cues are responsible for the extremely high-fidelity axon guidance seen in vivo. The prevalence of mutations with only partial axon defects might suggest the reason mouse knockouts have shown no clearly interpretable axon-guidance defects, namely other guidance mechanisms are sufficient to target axons more or less correctly. By taking a forward genetic approach, and by having a very sensitive assay, the zebrafish retinotectal screen uncovered mutations that affect axon guidance. A general finding of the screen is that most of the retinotectal mutants show other developmental defects. This indicates that molecular mechanisms used for axon guidance are also used in other developmental processes. It follows that genes absolutely necessary for early development were not found in the screen; affected embryos would not develop to a stage where axon guidance could be assayed. Further genetic screens designed to find conditional, temperaturesensitive mutations in zebrafish might provide a way to find more of the genes involved in axon guidance. In order to uncover the molecular mechanisms underlying the axon-guidance defects seen in the zebrafish mutants, it will be necessary to clone the affected genes. Cloning based on genetic map position has been successful in humans and mice (for example, Refs 37,38), and the generation of genetic maps in zebrafish39,40 makes this a viable approach for cloning TINS Vol. 20, No. 1, 1997

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the genes affected in the retinotectal mutants. Map position can be used immediately to identify candidate genes that map in the same region. One might expect that some of the pathfinding mutants, particularly the mutants with ipsilateral projections and midline defects, affect genes already known to be involved in midline formation and signaling, such as the hedgehog genes (see Ref. 41), or proteins mediating these signals42. Other candidates include axon-guidance molecules expressed at the midline such as the netrins43. Genes affected in the topographic mutants might include molecules known to play a role in retinotectal topography such as receptors of the Eph family or their ligands (for reviews, see Refs 44,45). Undoubtedly, unexpected and novel genes will be uncovered as well, expanding our mechanistic picture of axon guidance. Even before the mutated genes are cloned, the zebrafish mutants allow a unique analysis of axonguidance systems. Cell and tissue transplantation between mutant and wild-type individuals will determine the site of action of the mutations. These experiments will indicate whether the mutations disrupt guidance information in the environment, or affect the ability of the growth cone to read, interpret and respond to guidance cues. Closer analysis of the fibersorting and dorsal–ventral topographic mutants will give insights into these two previously uncharted phenomena. Finally, the creation of double mutants will determine if different pathfinding and targeting genes interact, and if they are part of a genetic hierarchy. Zebrafish genetics is an important new tool in the study of vertebrate development. The retinotectal mutants promise to shed light on novel and important molecular mechanisms of axon guidance. Other screens using in situ and antibody labeling can also be performed to find subtle defects in specific developmental processes46. These types of screens can be used in the future to find more genes involved in axon guidance and targeting, and can be applied to other developmental processes such as neurogenesis, brain regionalization and synapse formation. Behavioral screens are also possible47,48, and can be used to find subtle defects in complex neurodevelopmental processes. With the advent of large-scale zebrafish genetics, studies in vertebrate development are limited only by our imaginations.

Selected references 1 Goodman, C.S. (1996) Annu. Rev. Neurosci. 19, 341–377 2 Colamarino, S.A. and Tessier-Lavigne, M. (1995) Cell 81, 621–629 3 Monschau, B. et al. EMBO J. (in press) 4 Walter, J., Müller, B. and Bonhoeffer, F. (1990) Journal de Physiologie 84, 104–110 5 Tomasiewicz, H. et al. (1993) Neuron 11, 1163–1174 6 Magyar, J.P. et al. (1994) J. Cell Biol. 127, 835–845 7 McIntire, S.L. et al. (1992) Neuron 8, 301–322 8 Wadsworth, W.G. and Hedgecock, E.M. (1992) Curr. Opin. Neurobiol. 2, 36– 41 9 Hamelin, M. et al. (1993) Nature 364, 327–330 10 Ishii, N. et al. (1992) Neuron 9, 873–881 11 Leung-Hagesteijn, C. et al. (1992) Cell 71, 289–299 12 Seeger, M.A. (1993) Curr. Opin. Neurobiol. 4, 56–62 13 Seeger, M. et al. (1993) Neuron 10, 409–426 14 van Vactor, D.V. et al. (1993) Cell 73, 1137–1153 15 Martin, K.A. et al. (1995) Cell 14, 229–240 16 Kolodziej, P.A., Jan, L.Y. and Jan, Y.N. (1995) Neuron 15, 273–286 17 Tear, G. et al. (1996) Neuron 16, 301–514 18 Garrity, P.A. et al. (1996) Cell 85, 639–650 19 Serafini, T. et al. (1994) Cell 78, 409– 424 20 Mullins, M.C. et al. (1994) Curr. Biol. 4, 189–202 21 Grunwald, D.J. and Streisinger, G. (1992) Genet. Res. 59, 103–116 22 Haffter, P. et al. (1996) Development 123, 1–36 23 Baier, H. et al. (1996) Development 123, 415–425 24 Trowe, T. et al. (1996) Development 123, 439–450 25 Karlstrom, R.O. et al. (1996) Development 123, 427–438 26 Holt, C.E. and Harris, W.A. (1993) J. Neurobiol. 24, 1400–1422 27 Chien, C-B. and Harris, W.A. (1994) Curr. Top. Dev. Biol. 29, 135–169 28 Burrill, J.D. and Easter, S.S.J. (1995) J. Neurosci. 15, 2935–2947 29 Stuermer, C.A. (1988) J. Neurosci. 8, 4513–4530 30 Hatta, K. et al. (1991) Science 350, 339–341 31 Bernhardt, R.R., Nguyen, N. and Kuwada, J.Y. (1992) Neuron 8, 869–882 32 Easter, S.S.J. and Schmidt, J.T. (1977) J. Neurophysiol. 40, 1245–1253 33 Odenthal, J. et al. (1996) Development 123, 103–115 34 Yoon, M.G. (1975) J. Physiol. 252, 137–158 35 Drescher, U. et al. (1995) Cell 82, 359–370 36 Walter, J., Henke-Fahle, S. and Bonhoeffer, F. (1987) Development 101, 909–913 37 Kingsley, D.M. et al. (1992) Cell 71, 399–410 38 Riordan, J.R. et al. (1989) Science 245, 1066–1073 39 Postlethwait, J.H. et al. (1994) Science 264, 699–703 40 Knapik, E.W. et al. (1996) Development 123, 451–460 41 Ingham, P.W. (1995) Curr. Opin. Genet. Dev. 5, 492–498 42 Hammerschmidt, M., Bitgood, M.J. and McMahon, A.P. (1996) Genes Dev. 10, 647–658 43 Kennedy, T.E. et al. (1994) Cell 78, 425–435 44 Pandey, A., Lindberg, R.A. and Dixit, V.M. (1995) Curr. Biol. 5, 986–989 45 Brambilla, R. and Klein, R. (1995) Mol. Cell. Neurosci. 6, 487–495 46 Henion, P.D. et al. (1996) Dev. Genet. 11, 11–17 47 Granato, M. et al. (1996) Development 123, 399–413 48 Brockerhoff, S.E. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10545–10549

TINS Editorial Policy Trends in Neurosciences is the leading neuroscience review journal (Impact Factor 19.972; SCI Journals Citation Reports® 1995), publishing timely and wide-ranging feature articles that enable neuroscientists to keep up to date in and around their own research fields. Reviews form the foundations of each issue and offer concise and authoritative summaries of important areas of neuroscientific research; Meeting Reports and Research News articles describe recent developments within a particular field; Perspectives discuss areas of historical, social and more oblique interest – offering interesting insights into current research as it applies to neuroscience; Viewpoints include more controversial or synthetic ideas. The Debate section provides a series of alternative viewpoints on a currently hotly debated topic. Perspectives on Disease discuss how basic neurobiological studies can be applied to neurological disease; Techniques offers readers a guide to new methods and their use. TINS articles are specially commissioned by the Editor, in consultation with the Advisory Editorial Board. All submissions are subject to peer and editorial review – commissioning does not guarantee publication.

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