Harnessing the power of forward genetics – analysis of neuronal diversity and patterning in the zebrafish retina

REVIEW Harnessing the power of forward genetics – analysis of neuronal diversity and patterning in the zebrafish retina Jarema Malicki The seven major cell classes of the vertebrate retina are organized with remarkable precision into distinct layers.The appearance of this architecture during embryogenesis raises two questions of general importance.How do individual cell classes acquire their specialized structures and functions if they all originate from a morphologically uniform cell population? What mechanisms are responsible for the formation of such a complex and exact pattern? Recent advances present an opportunity to apply the tools of forward genetic analysis to identify mutations that affect these mechanisms in zebrafish. Molecular characterization will follow, providing insight into the basis of neuronal patterning in the vertebrate CNS. Trends Neurosci. (2000) 23, 531–541

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HE ZEBRAFISH (Danio rerio) has appeared in the limelight of scientific and occasionally nonscientific press over the past ten years. Several important characteristics have endeared this organism to developmental biologists and geneticists: small size, high fecundity, short lifecycle and ease of care. All of these had already been emphasized by the first proponent of the zebrafish genetic model, George Streisinger1, who also suggested that ‘large scale screening for mutants [in zebrafish] is possible because free-swimming seven-day old fish exhibit many behavioral and morphological traits of the parent’. It took a decade and a half for this prediction to come true. In 1996, two groups reported the completion of ‘large scale’ chemical mutagenesis screens that culminated in the isolation of nearly 2000 mutations that affect the first five days of zebrafish development2,3. These and several smaller experiments have led to the isolation of mutations affecting nearly every aspect of embryogenesis, from gastrulation to ear morphogenesis and retino–tectal pathfinding4–8. In order to facilitate the molecular characterization of mutant genes, substantial effort has been directed more recently towards development of mapping and cloning tools9–12. In parallel, transgenics and retroviral mutagenesis have enriched the repertoire of approaches available to manipulate gene function13,14. The tools of zebrafish genetics are now being used to characterize the genetic basis of neuronal development in one of the anatomically best-understood formations of the CNS: the retina.

Neurogenesis in zebrafish retina According to Ramón y Cajal the retina ‘is a genuine neural center, a sort of peripheral cerebral segment, whose thinness, transparency, and other qualities render it particularly favorable to histological analysis’. The vertebrate retina shares many of its qualities with other parts of the brain. During embryogenesis, as it evaginates out of the neural tube at the level of the diencephalon, its close 0166-2236/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

relationship to the forebrain is particularly obvious (Fig. 1b). The retinal evagination, continuous with the walls of the neural tube, is termed the ‘optic vesicle’ in most vertebrate species15–17. It comes into close contact with the surface epithelium where it is presumed to have a role in lens development. Subsequently, its distal portion invaginates to form a two-layered cup, referred to as the ‘optic cup’ (Fig. 1d,e). It is the inner layer of the optic cup that eventually gives rise to all neurons of the retina. This sequence of morphological transformations, with few exceptions (see legend to Fig. 1) also shapes the zebrafish eye18. The retina is easier to access than most brain regions and consists of relatively few well-characterized cell classes (Fig. 2). The laminar arrangement of these cells is more obvious than in other areas of the brain20. This is because laminae that consist mainly of cell perikarya are separated by layers of neuronal processes and because individual cell classes are characterized by distinct morphologies. This is particularly evident in the case of horizontal and photoreceptor cells, whose characteristic shapes are easy to recognize in very simple histological preparations (Fig. 2a). Isolation is another beneficial quality of the retina. In the course of early development, the connection between the retina and the diencephalon, known as the optic stalk, becomes progressively confined to a narrow shaft of cells. The resulting isolation limits cell migrations to and from other parts of the nervous system, and makes interpretation of developmental events considerably easier. Finally, in the central retina, neurogenesis is essentially complete by 60 h postfertilization (hpf). The cells that continue to be added at later stages of development do not change the overall architecture. The six major neuronal classes of the retina are organized into three major laminae: the ganglion cell layer, the inner nuclear layer and the photoreceptor cell layer, also known as the outer nuclear layer (Fig. 2a). Owing to its well characterized structure and accessibility, the fundamental questions PII: S0166-2236(00)01655-6

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Jarema Malicki is at the Dept of Ophthalmology, Harvard Medical School, Boston, MA 02134, USA. www. howelaboratory. harvard.edu/ malicki/htm

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Fig. 1. Early development of the zebrafish eye. The early CNS primordium in teleost fish, including the zebrafish, is a solid rod of tissue, termed ‘neural keel’, and not a hollow tube (neural tube) as in many other groups of vertebrates. (a) Eyes originate as a bilateral thickening of the anterior neural keel which first appears between 10 and 11 h postfertilization (hpf). Optic primordia lack lumen and hence are called ‘optic lobes’ and not ‘optic vesicles’ as in most vertebrates (arrowhead in all panels indicates midline). (b) By ~12 hpf, optic lobes (ol) are prominent on both sides of the brain. During the next few hours, optic lobes gradually rotate along their anteroposterior axes. As the result of this movement, their dorsal walls, fated to become neural retinae, turn away from the midline, while the ventral walls, fated to contribute to the pigmented epithelium, shift towards the brain. (c) A transverse section through the middle of the optic lobe at ca. 14.5 hpf. The rotation of the dorsal walls away from the midline has already begun. In parallel, starting posteriorly, optic lobes gradually detach from the neural keel. (d) At ~17 hpf, the posterior halves of the optic lobes are no longer in contact with the brain. Their anterior ends, however, still connect to the diencephalon through optic stalks (not shown). By this stage, the ventral walls of optic lobes (prpe) become markedly thinner as their cells gradually assume the flattened morphology of the pigmented epithelium. By contrast, the cells of dorsal walls (pnr) retain columnar morphology characteristic of the pseudostratified neuroepithelium. (e) By ~20 hpf, the dorsal surface of the eye primordium has invaginated forming the optic cup (oc). In parallel, the lens (le) has formed as a thickening of the overlying ectoderm. (f) At 24 hpf, the optic cup is well developed and the lens has detached from the overlying epidermis. All panels show transverse sections, oriented dorsal side up, approximately through the middle of the eye. Abbreviations: le, lens; oc, optic cup; ol, optic lobe; pnr, primordium of the neural retina; prpe, primordium of the retinal pigmented epithelium; rpe, retinal pigmented epithelium. Scale bar, 200 mm. Courtesy of Zac Pujic.

regarding the origins of neuronal patterning can be easier to address in the retina than in many other regions of the CNS. The when and where of vertebrate retinal neurogenesis have been characterized in considerable detail. These studies revealed several common features present in all or most of the species characterized so far21. Thus, the first cells to become postmitotic are ganglion cells. By contrast, bipolar cells and Müller glia consistently appear in the second half of neurogenesis. Cone photoreceptors and horizontal cells are generated simultaneously; in higher vertebrates, these two cell categories become postmitotic in the first half of neurogenesis, whereas in central retinae of the lower vertebrates, such as zebrafish, they appear towards the end (W. Nawrocki, PhD Thesis, University of Oregon, 1985)22–24. As a general rule, in the zebrafish retina cells of the ganglion cell layer become postmitotic first, followed by the inner nuclear layer and finally the photoreceptor cell layer 24. Neurogenesis also proceeds in an ordered fashion within individual cell layers. Ganglion cells of a small ventral region are the first to become postmitotic. From there, neurogenesis spreads dorsally across the ganglion cell layer (Fig. 3a–c). This spatial order of birth dates is later reflected in the appearance of differentiation markers. The zn-5 antibody, specific to ganglion cells in the retina, first labels a ventrally located group of cells27. A similar pattern is discernible in other layers. In the photoreceptor cell layer, for example, red opsin is first present in a few ventrally located cells known as the ‘ventral patch’25. From there it spreads into the nasal and then into the temporal and dorsal retina (Fig. 3d–h). 532

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In the plane of individual layers, cells are also arranged with remarkable precision (reviewed in Ref. 28). This is perhaps most obvious in the photoreceptor cell layer where five types of cones are arranged in rows (Fig. 3i,j). Cones of each row are arranged in the order: blue, red, green, UV, green, red, blue, etc. Neighboring rows of cones are separated by rod photoreceptors and are shifted with respect to each other so that blue cones of one row are next to UV cones of the neighboring rows (Fig. 3i)26. Individual photoreceptor cells are characterized by distinct spectral properties, morphologies and by the expression of specific opsin genes29. The genetic bases of diversity and spatial arrangement of retinal cells are poorly understood, even at the level of the most fundamental cellfate decisions that lead to the specification of major cell classes. Uncovering the details of specification and intricate architecture of all cell types is a far greater challenge. The zebrafish model offers an unprecedented opportunity to apply forward genetic approaches to gain insights into these processes.

Searching for mutations that affect retinal neurogenesis Genetic screens performed in zebrafish with the aim of isolating eye mutants have been impressive by scale or ingenuity, or both. The simplest one involved visual inspection of zebrafish larvae at 24, 48, 72 and 120 hpf. Approximately 500 000 embryos were screened over a period of a year and a half for alterations in eye size, shape or pigmentation, leading to the isolation of 49 mutants2,30. Screens that are based on morphological criteria, as the one described above, are limited to detecting gross morphological changes. Subtle differences in the number of neurons or in their organization would most probably remain undetected. In order to enhance the ability to detect subtle changes, one can visualize small cell populations using cell-type-specific labeling techniques. This approach has been used to study ganglion and interplexiform neurons in the eye, and primary motoneurons and neural crest cells outside the eye5,31–33. In the retina, to screen for mutations that affect retino–tectal projections, the axons of ganglion cells were labeled with lipophilic tracers. DiI and DiO were injected into separate quadrants of the eye and allowed to diffuse into the optic nerve5. An automated mounting and dyeinjection apparatus made it possible to turn this laborious screening procedure into a large-scale endeavor. Using these technical improvements, screening a single larva for retino–tectal defects took, on average, 1 min. In total, 125 000 larvae were screened, leading to the identification of 114 mutations that affect many aspects of retino–tectal pathfinding5,34,35.

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Fig. 2. Cells of the vertebrate retina form a distinct laminar pattern. Lens is indicated with an arrowhead. (a) Three major laminae, ganglion cell layer (gcl), inner nuclear layer (inl) and photoreceptor cell layer (pcl), also known as outer nuclear layer, are clearly visible in simple histological preparations. The neural retina is surrounded by a layer of darkly pigmented cells named retinal pigmented epithelium (rpe). Subsets of retinal cells can be visualized using molecular probes. (b) Ganglion cells form the innermost layer of the retina. As these are the only retinal neurons forming projections to the brain, they can be retrogradely labeled by DiI injections into the optic tectum. (c) A subset of amacrine cells stained with antibody to GABA occupies the innermost portion of the inner nuclear layer. (d) Müller glia span the entire thickness of the retina from the photoreceptor cell layer to the vitreal surface. Their morphology is visualized with anti-carbonic anhydrase antibody. (e) In addition to retrograde labeling, ganglion cells can be visualized with antibodies, in this case zn-5. (f) Rod opsin is a useful marker of rod photoreceptors. Here they are labeled by in situ hybridization with rod opsin probe (purple). (g) Similarly, red cones are specifically visualized with red opsin probe. (h) Interplexiform cells are the least numerous cell class in the retina. They are uniquely characterized by the expression of tyrosine hydroxylase. Here, they are stained with anti-tyrosine hydroxylase antibody (arrows). With the exception of (b), all panels show transverse sections through the retina at 3 (a), (e)–(g) and 5 (c), (d), (h) dpf. (b) shows a horizontal confocal section through the retina at 60 hpf. Scale bars, 100 mm. (h) Courtesy of Xiangyun Wei. (a)–(g) Reproduced, with permission, from Ref. 19.

Vision-dependent behaviors provide another approach with which to screen for defects of the retina. Phototaxis, startle, optokinetic, optomotor and escape responses are all visual behaviors described in zebrafish36–38. The earliest two, the startle and optokinetic responses, are already well developed by three and a half days postfertilization (dpf)37. The optokinetic response is particularly useful because it is consistent, appears early and does not require a functional optic tectum. Its advantages were noted almost two decades ago by Clark and colleagues who also were the first to use it in a genetic screen (T. Clark, PhD thesis, University of Oregon, 1981) . In more recent years, at least six mutants have been isolated on the basis of behavioral criteria39. Some of them show obvious developmental defects, while others appear histologically normal but display defective electroretinograms. Two experimental tricks add flair to zebrafish genetic screens: the ability to generate haploid embryos easily and the ability to generate parthenogenetic diploid embryos (Box 1). Both procedures can be used to generate homo- or hemizygous mutant animals already in the F2 generation (second filial generation derived from mutagenized parents) of a genetic screen. Because in a regular diploid screen, only the F3 generation can be used to screen for recessive mutant phenotypes, these two procedures result in enormous savings of space, time and labor (Box 1). Although both are burdened with some disadvantages, they have been used successfully to isolate mutations in the development of several organs32,33,40. To search for mutations that affect dopaminergic neurons in the retina and brain, Guo

and colleagues have combined parthenogenesis with immunohistochemical detection of tyrosine-hydroxylase (TH)-positive cells. Their screen of ~700 F2 clutches of embryos, which was focused on a very small population of interplexiform neurons, has led to the isolation of two mutations that affect this cell type33. Chemical mutagenesis has been used in all zebrafish screens performed so far, almost without exception (Box 2). This can soon change. Retroviral vectors have recently been developed as an attractive alternative mutagenic agent. Although less effective than chemical mutagens, they provide the option of almost instantaneous cloning of mutant genes (Box 2). Several mutations generated using a retroviral vector have been described. One of them, not really finished (nrf ), specifically affects photoreceptor cells41 (Fig. 4i). Molecular characterization of this mutant locus has revealed that it is homologous to human nuclear respiratory factor 1 (NRF1)41. nrf is expressed through the developing CNS, including the retina, but its role in photoreceptor development is still ambiguous. As retroviral mutagenesis screens move forward, many more mutants should become available in the future14.

Mutants of cell specification and differentiation Although classical descriptions distinguish six major classes of neurons in the retina, careful morphological studies have subdivided most of them into many types20,44,45. Amacrine cells, one of the most diverse cell classes in the retina, can be subdivided into at least 22 morphologically distinct types in the rabbit46. This diversity of neurons originates from a TINS Vol. 23, No. 11, 2000

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Fig. 3. Neurogenesis proceeds in an ordered fashion in the plane of individual cell layers. (a) The first cells to become postmitotic in the ganglion cell layer are consistently located in a small region of the ventral retina. From there, neurogenesis spreads into central (b) and subsequently dorsal (c) ganglion cell layer. The general features of this pattern are also observed in other laminae (not shown). Neuronal differentiation also follows this spatiotemporal pattern. The first opsin-positive cells, for example, appear in a small ventrally located group of cells (d). From there, opsin expression spreads into other regions of the retina (e)–(h). Differentiated photoreceptor cells form a fairly precise pattern known as photoreceptor mosaic. (i) A schematic representation of the photoreceptor mosaic in zebrafish. UV-sensitive cones are indicated with dots. The short member of double cone pairs is darkly colored. (j) The same pattern visualized with the Fret43 antibody specific to double cones. Abbreviations: d, dorsal; n, nasal; t, temporal; v, ventral. Scale bar, 10 mm in (j). (a)–(c) Modified, with permission, from Ref. 24; (d)–(h) modified, with permission, from Ref. 25; and (i), (j) modified, with permission, from Ref. 26.

single population of neuroepithelial cells. Several questions should be asked about this process. What genetic mechanisms are responsible for the specification of major cell classes? What mechanisms guide their subdivision into distinct cell types? And, finally, what mechanisms lead to the formation of their unique morphological features such as the outer segments and synaptic terminals of photoreceptor cells? Genetic analysis has been extremely successful in addressing these questions in invertebrates. Could a similar approach be used in a vertebrate? The collection of zebrafish mutants isolated over the past five years suggests that this indeed could be the case. Mutations of nearly two dozen loci predominantly affect the early development of single retinal cell classes (Table 1). Photoreceptor cells, the hallmark of the retina, are characterized by an unusual morphology and the specialized function of detecting light. Although several factors are known to be involved in early photoreceptor development (reviewed in Refs 60–62), the understanding of this process is still rather fragmentary. 534

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Several loci in zebrafish could offer insights into the earliest steps of photoreceptor development. In the mikre oko retina (Fig. 4b), by the criteria of visual pigment expression, the full complement of photoreceptor cells does not appear to form30. Even the cells that do develop the elongated morphology characteristic of this cell class die by 5 dpf. Although somewhat later, similar developmental defects are present in brudas (Ref. 30; Fig. 4f). In this mutant, many cells in the photoreceptor cell layer also appear to die before or shortly after developing their typical elongated shape. Shortly after elaborating their characteristic oblong morphology, photoreceptor cells develop outer segments63. These rather unusual structures appear at the apical cell termini and consist of hundreds of parallel membrane folds that harbor opsins and other components of the phototransduction apparatus64. Although a few proteins, such as peripherin, ROM1 and opsins themselves are presumed to play a structural role in outer segments65,66, virtually nothing is known about the molecular events that initiate outer segment formation. The mutant elipsa could shed some light on this process. Its photoreceptor cells develop a characteristic elongated morphology as well as inner segments, but fail to differentiate outer segments30. Once thorough histological analysis is available, other zebrafish photoreceptor mutants, characterized by phenotypes similar to elipsa, for example, fleer, could also prove to impair outer segment formation specifically. Understanding the formation of another unusual morphological feature of photoreceptor cells, the synaptic terminus, can also benefit from studies of zebrafish mutants. Classical anatomists elegantly termed rod synaptic terminals, spherules and cone terminals, pedicles45. In zebrafish, between 65 and 75 hpf, postsynaptic processes of horizontal and bipolar cells invaginate as a single bundle into cone terminals and become surrounded by radially arranged presynaptic ribbons. The formation of this elaborate synaptic apparatus is disrupted in the retina of no optokinetic response c (nrc) (B. Allwardt, PhD thesis, Harvard University, 1999). In nrc, few postsynaptic processes partially invaginate into photoreceptor termini and ribbons appear to float in the cytoplasm instead of connecting to the presynaptic membrane. This defect can originate either in the photoreceptor pedicles or in the postsynaptic neurons. The answer to this problem has yet to be determined by generating genetically mosaic animals. Interestingly, this phenotype is specific to the outer retina, as it does not affect the ribbon synapses of the inner plexiform layer. Multiple photoreceptor types are present in the retinae of most vertebrates. Although distinct morphological, biochemical and functional properties characterize five types of zebrafish photoreceptor cells, the molecular basis of photoreceptor type identity is poorly understood. Visual pigments, together with several components of the phototransduction cascade, are the only known molecular correlates of photoreceptor diversity29,67. Rare insights into the process of photoreceptor type development can therefore come from studies of the pob (partial optokinetic response b) locus, which appears to be involved specifically in the survival of red cones47. Although initially present, the red cones of pob mutant animals are lost by 5 dpf, while blue opsin-synthesizing cells remain unaffected. The pob photoreceptor defect is not linked to the red

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Box 1. Some variations on chromosome mechanics in zebrafish genetic screens In a diploid organism, the phenotypes of recessive alleles obtained via mutagenesis are masked in the F1 generation (the first filial generation of mutagenized parents) by wild-type alleles. To analyze their phenotypes, one needs to bring mutant alleles to homozygosity. This is usually accomplished by outcrossing the F1 animals to a wild-type strain and subsequently incrossing the F2 generation (Fig. I, left). The mutant phenotypes are first detectable in 25% of the F3 generation embryos. Can this laborious and time-consuming process be shortened? Two approaches in zebrafish are frequently used to analyse recessive mutant phenotypes already in the F2 generation. The first one involves fertilizing eggs of F1 females with UV-irradiated, genetically inactive sperm (Fig. I, center). The resulting haploid F2 embryos lack the paternal contribution and if a female is a mutant heterozygote, 50% of its progeny will display the mutant phenotype. Since these mutant embryos contain only one copy of the genome, they are not mutant homozygotes but rather hemizygotes. Haploid development is relatively normal until 3 dpf, allowing for the identification of developmental mutations affecting most tissuesa. The second approach entails fertilization with UV-inactivated sperm followed by a high-pressure treatment to block the second meiotic division (Fig. I, right). At the time of spawning, zebrafish eggs are arrested in the prophase of the second meiotic division. When embryos are treated with high pressure shortly after fertilization, the second meiotic division does not occur and the resulting animals are diploidb. Parthenogenetic F2 embryos obtained via this treatment develop to adulthood. If an F1 female used in such an experiment carries a mutation, some of the resulting F2 embryos will be mutant homozygous. In the absence of crossing-over, the ratio of wild-type to mutant parthenogenetic embryos obtained from a heterozygous female would be one to one. Crossing-over events between a mutant locus and a centromere increase the number of heterozygotes at the expense of both wild-type and mutant homozygotes. If a mutant locus is located near a centromere, the frequency of crossing-over events is low, and nearly 50% of embryos are mutant. In the case of a telomeric locus, however, nearly all embryos are phenotypically wild-type heterozygotes. Thus, depending on the distance between a mutant locus and the centromere of its chromosome, the percentage of mutant embryos varies from 50% to less than 5% (Ref. c). Screening for mutant phenotypes in the F2 generation has a number of obvious advantages:

opsin gene – currently the only gene known to be expressed specifically in red cones. Although many other zebrafish loci appear to be involved in photoreceptor development (Table 1), their exact roles are difficult to evaluate in the absence of detailed descriptions of their phenotypes. In most mutants, photoreceptor cell loss is progressive and displays one of several patterns. In mikre oko and niezerka, for example, photoreceptor loss begins in the retinal periphery30. By contrast, brudas, elipsa, photoreceptors absent and fleer first lose centrally located photoreceptor cells30,55,56 (Fig. 4f). A third pattern is seen in krenty, sinusoida and discontinuous30. In these mutants, photoreceptor cells appear unaffected in some areas of the retina, while in others they are entirely absent30. Loss of photoreceptors commencing in the ventral region of the central retina can simply reflect the fact that these cells are the first to differentiate. The significance of the other patterns is open to speculation. It is important to note that, based on complementation tests (Box 3), almost all mutant loci involved in photoreceptor cell development are represented by single alleles. If these involve partial inactivation of gene

savings of time, labor, and most importantly, space required to raise many thousands of fish. References a Walker, C. (1999) Haploid screens and gamma-ray mutagenesis. Methods Cell Biol. 60, 43–70 b Streisinger, G. et al. (1981) Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature 291, 293–296 c Beattie, C.E. et al. (1999) Early pressure screens. Methods Cell Biol. 60, 71–86

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Fig. I. Three alternative breeding schemes used in zebrafish to detect recessive mutations. The parental generation (P) is subjected to a mutagenic regimen and crossed (outcrossed) to wild-type animals. The resulting F1 progeny is heterozygous for a hypothetical mutant allele (asterisk). It can be outcrossed to a wild-type strain to produce F2 generation (left). Subsequently, the F2 individuals can be crossed to each other (incrossed) to produce F3 generation. Alternatively, the F1 animals can be used to obtain haploid embryos (center) or parthenogenetic diploid embryos (right). Mutagenized chromosomes are in red. Boxes indicate homozygous or hemizygous mutant genotypes. Percentages of phenotypically mutant embryos in a clutch obtained by each of these methods are indicated in the figure below the method. Abbreviations: EP, early pressure; IVF, in vitro fertilization.

product, the isolation of other alleles that completely lack gene activity could reveal addition functions. It can thus emerge that some of these genes either act earlier than currently anticipated or that they are involved in additional aspects of development. The disproportionately large number of photoreceptor mutants compared with defects of other retinal cell populations most probably results from the large number and bulky size of photoreceptor cells relative to other cell categories. Because of these characteristics, the loss of photoreceptor cells affects eye size and is easy to detect in a morphological screen. In contrast to photoreceptor cells, interplexiform neurons are the least abundant retinal cell class45 (Fig. 2h). Because screening for defects in such a small cell population by morphological criteria is not possible, embryos were stained with an anti-TH antibody to detect mutations affecting interplexiform neurons33. Two mutants, foggy (fog) and motionless (mot), which lack TH immunoreactivity in the retina, have been isolated after screening of ~700 F2 parthenogenetic clutches. Given the lack of information about the fate of other neurons in these mutants, it is premature to speculate TINS Vol. 23, No. 11, 2000

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Box 2. Mutagenesis in zebrafish The most potent mutagen currently used in zebrafish genetics is ENU (N-ethyl-N-nitrosourea). For this reason, the majority of genetic screens performed in zebrafish so far relied on this compounda,b. The main disadvantage of ENU is that to identify a mutant gene one has to embark on a laborious process of positional cloning. Although positional cloning is certain to become easier or even easy in the future, other mutagenesis approaches circumvent positional cloning entirely. In these approaches, DNA is both the target of mutagenesis and the mutagenic agent. Three types of DNA molecules have been used as mutagenic agents in genetic model systems: transgenes, retroviruses and transposons. Insertion of these molecules into a genome can disrupt the function of a nearby gene. It is easy to determine which gene is disrupted because the genomic fragments neighboring the insertion site can be isolated by standard methods of molecular biology. Retroviral- and transposon-mediated mutagenesis approaches have been the subject of pilot experiments in zebrafish. Of the two, retroviral mutagenesis is currently at a more advanced stage. To induce mutations, a retroviral construct is injected into the early zebrafish embryo. It integrates into the genome and sometimes disrupts the function of a nearby gene. The efficiency of this approach has been greatly improved over the past few years and is likely to increase even more in the future. A single injection results in ~25–30 integration eventsc. Slightly more than 1 in 100 retroviral

insertions results in a recessive mutant phenotype. According to current estimates, retroviral mutagenesis appears to be one tenth as efficient as ENU (Ref. c). It could thus become a useful alternative to chemical approaches. Transposon-mediated mutagenesis is another approach potentially applicable in zebrafish. Transposons are genetic elements capable of moving from one genomic site to another. The simplest of transposons require only one enzyme, the transposase, to catalyze a transposition event. It has been shown in zebrafish that exogenous transposable elements can be inserted into the genome and subsequently mobilized via transposase injections into embryosd,e. The efficiency of this process, however, needs to be improved to be useful in mutagenesis screens. References a Mullins, M.C. et al. (1994) Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate. Curr. Biol. 4, 189–202 b Solnica-Krezel, L. et al. (1994) Efficient recovery of ENU-induced mutations from the zebrafish germline. Genetics 136, 1–20 c Amsterdam, A. et al. (1999) A large-scale insertional mutagenesis screen in zebrafish. Genes Dev. 13, 2713–2724 d Raz, E. et al. (1998) Transposition of the nematode Caenorhabditis elegans Tc3 element in the zebrafish Danio rerio. Curr. Biol. 8, 82–88 e Ivics, Z. et al. (1999) Genetic applications of transposons and other repetitive elements in zebrafish. Methods Cell Biol. 60, 99–131

on the role of the fog and mot loci in retinal development. Their isolation demonstrates, however, the value of immunohistochemical screens in the genetic analysis of small cell populations. Before the onset of retinal neurogenesis, retinal progenitor cells have the potential to assume one of six neuronal cell fates and one glial fate. What genetic mechanisms assure that particular cell fates are chosen with the appropriate frequency? As all retinal neurons originate from a common pool of precursor cells, one would expect to find a class of mutant loci that result in the overproduction of one cell class at the expense of another. Such an outcome has been seen when cell-fate decisions of retinal progenitor cells are manipulated by transgene overexpression68,69. A defect of this type seems to indeed exist in the zebrafish mutant lakritz, originally isolated in a large scale mutagenesis screen, on the basis of defective pigmentation52. Its retinal phenotype has been discovered in a behavioral re-screen of previously isolated mutants on the basis of abnormal optokinetic response43. Further histological analysis revealed a loss of retinal ganglion cells accompanied by an expansion of the inner nuclear layer. Only 20% of the normal cell population is found in the lakritz ganglion cell layer at 4.5 dpf and the optic nerve is markedly thinner (Fig. 4h). At the same time, the number of interneurons of the inner nuclear layer appears larger43. This phenotype suggests that wild-type allele of the lakritz gene can have a role in biasing cell-fate choice towards ganglion cells, either as an intrinsic factor in progenitor cells or as an extrinsic factor in the environment.

Mutants of neuronal organization Although the cellular architecture of the retina has been described in detail20,44,45, the genetic underpinnings of its formation remain largely unexplored. Several mutants isolated in zebrafish display a disorganization of retinal neurons, offering access to the analysis of this problem. Both global mechanisms, as well as local patterning cues, appear to be affected in 536

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these mutants. In the group of loci that affects global patterning events, three, oko meduzy, glass onion and nagie oko, directly or indirectly regulate the positioning of all neuronal categories in the retina30 (Fig. 4c,d). The organization of retinal neurons in oko meduzy has been analyzed with cell-type-specific probes, leading to the conclusion that at least seven cell categories are distributed in an overtly abnormal pattern. Photoreceptor cells and ganglion cells, as well as inner nuclear layer (INL) interneurons and the Müller glia appear to be intermingled throughout the retina19. It is remarkable that the positions of all cell types are disorganized simultaneously. This unusually strong phenotype suggests that oko meduzy is of one of the ‘top dogs’ among the genes that orchestrate retinal organization. Additional insight into the role of oko meduzy is provided by the observation that its neuronal patterning phenotype is preceded by a disorganization of the retinal pseudostratified neuroepithelium. The apical termini of the retinal but not brain neuroepithelial cells are misaligned, and their centrosomes and adherens junctions localize to abnormal positions19. As the migration of postmitotic neurons in the developing retina occurs in the context of the neuroepithelial sheet, one is tempted to speculate that the neuronal patterning phenotype in this mutant can result from a disruption of positional cues present in the pseudostratified neuroepithelium. A thorough description of glass onion and nagie oko retinal defects is not yet available. On the basis of histological inspection, the phenotypes of these mutants in the retina are as severe as in oko meduzy, but outside the eye they are more pleiotropic30. All three loci appear to produce far stronger phenotypes in the retina than a handful of other genes shown to play a role in retinal lamination in other model organisms. NCAM (neural cell-adhesion molecule), integrin a6 and MARCKS (myristoylated, alanine-rich C kinase substrate) knockout mice, for example, show only a partial disorganization of neuronal laminae70–72. The

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Fig. 4. Examples of mutations affecting development of the zebrafish retina. (a),(g) In the wild-type retina, several classes of neurons can be distinguished in simple histological preparations at 3 days postfertilization (dpf) (a) as well as at 5 dpf (g). This is certainly true for photoreceptor cells (pcl) and ganglion cells (gcl). Additional cell categories can be distinguished upon closer inspection. (b) In mikre oko, many photoreceptor cells do not maintain elongated morphology and eventually die. This phenotype is usually more severe in the retinal periphery. (c) In nagie oko, the laminar pattern of neurons is almost entirely disorganized. (d) The phenotype of oko meduzy also involves a severe neuronal disorganization. (e) Although all neuronal laminae are present, the out of sight retina is markedly smaller when compared with the wild-type in (a). (f) In the brudas eye, photoreceptor cell loss is first obvious in the central retina and only later spreads into the periphery. At 3 dpf, some intact photoreceptors survive at the retinal margins (arrowheads). (h) lakritz is characterized by a specific loss of ganglion cells (arrow) and a severe reduction of the optic nerve (arrowhead) compared with wild-type retina at the same stage (g). (i) In contrast to brudas, the loss of photoreceptor cells in not really finished is most severe in the periphery. Some relatively intact photoreceptor cells persist in the central retina at least until 5 dpf. Sections were prepared from embryos collected at ~3 dpf (a)–(f) or ~5 dpf (g)–(i). Dorsal is up and midline is to the right in all panels [indicated with arrows in (c)]. Abbreviations: gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; le, lens; mz, marginal zone; on, optic nerve; opl, outer plexiform layer; pcl, photoreceptor cell layer; rpe, retinal pigmented epithelium. Scale bars, 100 mm in (a)–(f) and (g)–(i). (a) reproduced, with permission, from Ref. 42; (h) reproduced, with permission, from 43; and (i) reproduced, with permission, from Ref. 41.

HES1 (hairy and enhancer of split homolog 1) phenotype, by contrast, is associated with massive cell death that could account for most of its patterning defects73. The extreme severity of defects in oko meduzy, nagie oko and glass onion suggest that these mutants expose previously unknown fundamental mechanisms involved in the formation of retinal architecture.

Milder defects of neuronal organization, perhaps affecting local patterning events, are observed in other mutants. In bashful, ganglion cells do not form a uniform layer along the vitreal surface of the retina, and in some regions are missing entirely. Their axonal projections make pathfinding errors both within the eye and in the brain34. The chameleon mutations affect TINS Vol. 23, No. 11, 2000

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TABLE 1. Mutations of retinal neurogenesis in zebrafish Name

Eye phenotype

Other phenotypes

Mutations affecting differentiation of specific cell types lakritz (lak) GC loss, supernumerary cells in INL (4.5 dpf a) No background adaptation foggy (fog) IPC loss (3 dpf) Blood circulation disrupted, pericardial edema motionless (mot) IPC loss (4 dpf a) Reduced touch response, apoptosis in telencephalon and lens, blood circulation disrupted partial optokinetic PRC loss specific to red cones (5 dpf, No OKR in red light, abnormal ERG response b (pob) normal at 3 dpf) crocodile (cro) PRC loss (6 dpf a) Reduced or absent touch response flathead (fla) PRC loss (6 dpf a) Abnormal craniofacial skeleton, small eyes and brain, reduced pectoral fins fade out (fad) PRC loss (6 dpf a) and RPE defect Reduced chromatophore pigmentation fading vision (fdv) PRC loss (6 dpf a) and RPE defect Reduced chromatophore pigmentation ivory (ivy) PRC loss (6 dpf a) and RPE defect Reduced chromatophore pigmentation, brain degeneration sunbleached (sbl) PRC loss (6 dpf a) and RPE defect Delayed pigmentation, abnormal craniofacial skeleton elipsa (eli) PRC loss starting in central retina (3 dpf) Pronephric cysts, body axis curved brudas (bru) PRC loss starting in central retina (3 dpf) Pigmentation darker, by 3 dpf lack of touch response Not named PRC loss starting in central retina (6 dpf a) Pronephric cysts, body axis curved mikre oko (mok) PRC loss starting in peripheral retina (3 dpf) Not all individuals develop swim bladders niezerka (nie) PRC loss starting in peripheral retina (3 dpf) Brain slightly smaller, no swim bladder fleer (flr) PRC loss starting in central retina (3 dpf) Pronephric cysts, body axis curved photoreceptors PRC loss starting in central retina (4 dpf) None reported absent (pca) discontinuous (dis) PRC loss, patchy (3 dpf) Smaller brain, no swim bladder krenty (krt) PRC loss, patchy (3 dpf) Smaller brain, no swim bladder sinusoida (sid) PRC loss, patchy (3 dpf) Smaller brain, no swim bladder not really PRC loss (3 dpf) 60% of individuals do not develop swim bladders finished (nrf) no optokinetic PRC synaptic termini abnormal (6 dpf a) No OKR in white light, abnormal ERG response c (nrc) Mutations affecting neuronal patterning oko meduzy (ome) RPE (30 hpf) and RN defective, subsequently all cellular laminae disorganized nagie oko (nok) RPE (30 hpf) and RN defective, subsequently all cellular laminae disorganized glass onion (glo) RPE (24 hpf) and RN defective, subsequently all cellular laminae disorganized blowout (blw)

Central retina ruptured (5 dpfa)

bashful (bal)

GC disorganized (48 hpf)

young (yng)

Cell differentiation delayed, patterning defective in some layers (60 hpf) GC projections misrouted in the retina (48 hpf)

chameleon (con)

Other mutations heart and soul Reduced eye size and degeneration (48 hpf), (has) RPE defect (36 hpf) out of sight (out) Reduced eye size (36 hpf) pandora (pan) Ventral retina underdeveloped (48 dpf)

Somewhat abnormal brain shape, blood circulation reduced Abnormal brain shape, blood circulation reduced, body axis curved Abnormal brain shape accompanied by degeneration between 12 and 24 hpf, blood circulation reduced, tail morphology abnormal Retinotectal projections navigate to ipsilateral tectum Retinotectal projection defectively routed at multiple sites along its navigation path Cardiac edema, dorsally flexed tail

Alleles

Refs

th241c m806 m807

19,43 33 33

a1

47

tw212d ta53c, tf21c, th5b, ty76a, tu255e tc7b, tp94c, tm63c th236a tm271a, tp30

43,48 43,49–51

to4a m649, tp49d m148 tz288b m632 m743 m477 a2

43,50–53 30 30 43,54 30 30 55 56

m704 m699 m604 hi399a

30 30 30 41

a14

39,b

m98, m289, m298, m320 m227, m520

19,30,57

m117

30

tc294z

34

43,51,52 43,51,52 43,51,52

30,57

tp82, tp86, tm220, 34 tr259b etc. (15 total) a8, a50 58

Retinotectal projections navigate to ipsilateral tectum, curved body axis, neural tube

tf18b, th6d, tm15, tu214, ty60

34

Abnormal brain shape, blood circulation reduced, body axis curved Not all individuals develop swim bladders Abnormal brain, ear, and tail morphology, pigmentation delayed or absent

m129, m567, 30,57 m781 m233, m360, m390 30 m313 6,30,59

Several mutant categories are not included in this table: neuronal degenerations predominantly affecting retinal margin or multiple cell layers; defects of retino-tectal pathfinding outside the eye; defects of optokinetic response not associated with histological abnormalities. As many mutations were not complementation tested to each other, future studies might reveal that mutations currently listed as representing separate loci are in fact allelic. Numbers in parentheses indicate at what developmental stage a phenotype has been reported to be first discernible. a Indicates that a timecourse of phenotype appearance has not yet been reported. A phenotype could thus first appear significantly (more than 24 h) earlier than indicated. No information about earlier stages of development is, however, available. Abbreviations: dpf, days postfertilization; ERG, electroretinogram; GC, ganglion cells; hpf, hours postfertilization; INL, inner nuclear layer; IPC, interplexiform cells; OKR, optokinetic response; RN, retinal neuroepithelium; RPE, pigmented epithelium; PRC, photoreceptor cells. b B. Allwardt, PhD thesis, Harvard University, 1999.

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Box 3. Glossary Complementation test A standard genetic test that allows one to determine whether two independently obtained recessive mutations affect the same gene. Organisms carrying mutations in question are crossed to each other. Mutations are assumed to affect the same gene if the resulting progeny is phenotypically mutant. Expressed sequence tag (EST) A stretch of nucleotide sequence obtained by a partial analysis of a cDNA clone randomly selected from a cDNA library. ESTs serve both as a source of candidate genes and genetic markers in gene mapping and cloning. GAL4-UAS system A set of two transgenes that allows one to express a gene of interest in a spatially and temporally restricted manner. One of the transgenes expresses a potent transcriptional activator, GAL4, in a desired place and time. The other transgene contains the gene of interest under the control of a GAL4-inducible promoter. To avoid lethality that can be associated with the expression of the gene of interest, the two transgenes are maintained in separate lines and assembled together in a single organism by crossing. Inducible promoter A gene regulatory element that can be activated at a desired timepoint. Some of the most commonly used inducible promoters are derived from heat-shock genes and can be activated by increasing temperature. Large-insert genomic library A library containing large genomic DNA fragments sometimes exceeding one million base pairs in length. Such libraries are indispensable in positional cloning experiments (see below).

even later aspects of ganglion cell development. Although ganglion cell bodies are positioned properly, their projections frequently grow abnormally along the equatorial region of the retina and fail to leave the eye34. Numerous additional mutants have been shown to affect retinotectal pathfinding outside the eye (reviewed in Ref. 74). As axonal navigation is a broad topic in itself, a discussion of these phenotypes extends beyond the scope of this article.

Zebrafish mutants versus human disease Disorders of vision in the human retina most often involve photoreceptor and ganglion cells. Retinitis pigmentosa, cone and cone–rod dystrophies involve the loss of photoreceptor cells, whereas glaucoma is characterized by the loss of ganglion neurons75,76. The abundance of photoreceptor defects in zebrafish is thus fortunate. Can zebrafish mutants be used as models of human pathological conditions? A rather striking similarity between certain zebrafish photoreceptor phenotypes and syndromic forms of retinitis pigmentosa suggests a positive answer to this question. For example, Senior–Loken and Bardet–Biedl syndromes are forms of retinitis pigmentosa associated with renal abnormalities77,78. The same combination of anomalies is induced by the elipsa and fleer mutations, suggesting that these zebrafish defects and human disorders could involve defects in homologous loci30,55. Clinical correlates can also be found for the neuronal patterning loci glass onion, nagie oko and oko meduzy. A combination of eye and brain defects characteristic of these mutants is present in a group of phenotypically related human conditions: Walker–Warburg syndrome, muscle–eye–brain disease and cerebro–oculo–muscular dystrophy79. Given these similarities, it is safe to predict that the

Positional cloning Cloning of a mutant gene based primarily on its genetic map position. It involves mapping of a mutant gene, cloning of the genomic region containing this gene, and the identification of a mutant transcript. Radiation hybrid panel An array of cell lines, usually rodent, each containing a set of exogenous chromosome fragments randomly derived from another organism, zebrafish for example. Two zebrafish genes that are physically located close to each other will tend to be present in the same cell lines. This provides the basis for the use of such panels in gene mapping. Sequence polymorphism A sequence known to vary between wild-type individuals of the same species. Such sequences of known genomic position are used to map mutant genes. Short DNA sequence repeats, such as (CA dinucleotide)n, are known to vary in length frequently and are called ‘simple sequence length polymorphisms’ (SSLPs). Temperature-sensitive allele A mutant allele encoding a protein product inactive at higher temperatures presumably due to heat-induced changes in amino acid chain conformation. Such alleles have been isolated for many types of proteins and can be used to inactivate protein function in a desired window of time. Transposable element A genetic element capable of changing its location in the genome. Several classes of transposable elements have been identified based on nucleotide sequence similarities. Among others, Tc-1/ mariner family transposons are present in the zebrafish genome.

molecular characterization of zebrafish mutations will most probably contribute to a better understanding of human disorders.

Where genetics meets genomics Is there a need for additional mutagenesis screens in zebrafish? Certainly, yes. Although past experiments have been impressive in scale, even larger endeavors are currently in progress. The most compelling reason to continue searching for mutants is the observation that among the loci identified so far, few were isolated multiple times (Table 1), implying that only a fraction of genes that control retinal development have been identified30,39. Several technological innovations will improve the efficiency of future mutagenesis screens. Chemical mutagenesis will most probably continue to be a major approach although an attractive alternative method using retroviral vectors as a mutagenic agent is already available80 (Box 2). Until very recently, retroviral mutagenesis protocols were at the stage of pilot experiments, and the number of retrovirusinduced mutant alleles is still rather low. A large-scale retroviral mutagenesis screen is, however, currently in progress. The magnitude of this endeavor is impressive to say the least. The plan is to test 80 000–150 000 insertions for recessive phenotypic effects14. As pilot experiments indicate that 1 in 70 to 100 retroviral insertions is mutagenic, the expected outcome of this screen is the identification of hundreds of mutant alleles. The major advantage of retroviral mutagenesis is that the mutant genes will be available for immediate cloning. Another technology that can become available in the future is transposon-mediated mutagenesis. Mariner/Tc-1 family transposable elements TINS Vol. 23, No. 11, 2000

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(Box 3) have been successfully mobilized in the zebrafish genome and can potentially be useful as mutagenic agents81. Again in this case, cloning of mutant genes isolated using this approach will be straightforward. In addition to advances in mutagenesis approaches, the development of zebrafish transgenics, new cell-type-specific molecular probes, and additional behavioral testing techniques will broaden the scope of mutagenesis screens even further. Transgenics alone could be used to improve the efficiency of mutagenesis screens in at least two ways. First, reporter transgenes driven by cell-type-specific promoters can be used to visualize specific cell populations selectively, which obviates the need for antibody or in situ staining. This is usually necessary when a targeted cell population is small and not accessible to visual inspection. Second, transgenes can be used to create sensitized genetic backgrounds. These new mutagenesis and screening tools will assure that future genetic screens in zebrafish will access an even broader spectrum of loci. Given the flood of mutants generated by mutagenesis screens, a major goal for the coming years is the molecular characterization of the hundreds of chemically induced mutant alleles. The molecular identity of these mutant genes can be determined using positional or candidate cloning. The rapid development of genomics is greatly improving the efficiency of both approaches. Many essential genomics resources are already well developed in zebrafish and continue to improve. These include genome maps, large-insert genomic libraries, radiation hybrid panels and expressed sequence tag (EST) databanks (Box 3). EST cloning and mapping projects are especially valuable for mutant gene identification because they contribute both genomic markers and candidate loci. A convincing indicator of progress in zebrafish genomics is the number of available SSLP (simple sequence length polymorphism) map markers. Since the publication of the first zebrafish SSLP-based genomic map, their number has grown over tenfold and continues to increase9,82. Given all recent advances, it is reasonable to assume that positional cloning in zebrafish will soon become a fairly standard approach. As in other model systems, the ultimate aim of zebrafish genomics is to obtain the full sequence of the entire genome. Collecting ever increasing numbers of mutants via progressively larger and more sophisticated mutagenesis screens cannot be the only goal of zebrafish genetics, even if it is followed by effective cloning procedures. Many elegant studies in invertebrates have shown that genes and their developmental functions cannot be assigned to each other by a simple one to one relationship. A single gene can play a role at multiple stages of development; it can function in parallel in several tissues; or it can be involved both in developmental and housekeeping functions in a single cell83. To make the situation even more complicated, the role of a gene can be obscured by a functional redundancy with other factors. Invertebrate geneticists have developed an impressive arsenal of tools to deal with the complexities of gene function. To dissect the individual functional aspects of a gene or to circumvent the problem of redundancy, gene activity can be manipulated (increased or decreased) in a specific tissue or in a specific window of time. This is 540

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accomplished through the use of temperature-sensitive alleles, inducible promoters (such as the heatshock promoter), the Gal4-UAS expression system (Box 3), or through the analysis of mutant phenotypes in clones of homozygous mutant cells84,85. Are such tools available in zebrafish? Zebrafish genetics has been steadily catching up with the technological gadgetry of other systems. Expression of transgenes from tissue-specific and heat-inducible promoters has been accomplished13,86. These experiments have been taken a step further to engineer a Gal4-UAS expression system87. In addition, the expression of reporter genes has been achieved from large (.100 kb) transgenes allowing for faithful reproduction of native expression patterns88. Much still needs to be done. Further work on inducible promoters is an obvious necessity. A targeted recombination method that would allow for the generation of knockout phenotypes is also a very worthwhile, although challenging, goal. The zebrafish is the only vertebrate model system where the classical forward genetic approach of mutagenesis screen can be carried out on a large scale, to isolate thousands of recessive mutations. An ability to perform mutagenesis screens, combined with the increasingly sophisticated tools of genomics and gene function analysis, creates an exceptionally powerful analytical approach to dissect the molecular basis of neuronal diversity and patterning in the vertebrate retina. Selected references 1 Streisinger, G. et al. (1981) Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature 291, 293–296 2 Driever, W. et al. (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37–46 3 Haffter, P. et al. (1996) The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1–36 4 Solnica-Krezel, L. et al. (1996) Mutations affecting cell fates and cellular rearrangements during gastrulation in zebrafish. Development 123, 67–80 5 Baier, H. et al. (1996) Genetic dissection of the retinotectal projection. Development 123, 415–425 6 Malicki, J. et al. (1996) Mutations affecting development of the zebrafish ear. Development 123, 275–283 7 Whitfield, T.T. et al. (1996) Mutations affecting development of the zebrafish inner ear and lateral line. Development 123, 241–254 8 Mullins, M.C. et al. (1996) Genes establishing dorsoventral pattern formation in the zebrafish embryo: the ventral specifying genes. Development 123, 81–93 9 Shimoda, N. et al. (1999) Zebrafish genetic map with 2000 microsatellite markers. Genomics 58, 219–232 10 Hukriede, N.A. et al. (1999) Radiation hybrid mapping of the zebrafish genome. Proc. Natl. Acad. Sci. U. S. A. 96, 9745–9750 11 Geisler, R. et al. (1999) A radiation hybrid map of the zebrafish genome. Nat. Genet. 23, 86–89 12 Zhong, T.P. et al. (1998) Zebrafish genomic library in yeast artificial chromosomes. Genomics 48, 136–138 13 Long, Q. et al. (1997) GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development 124, 4105–4111 14 Amsterdam, A. et al. (1999) A large-scale insertional mutagenesis screen in zebrafish. Genes Dev. 13, 2713–2724 15 Schook, P. (1980) Morphogenetic movements during the early development of the chick eye. A light microscopic and spatial reconstructive study. Acta. Morphol. Neerl. Scand. 18, 1–30 16 Pei, Y.F. and Rhodin, J.A. (1970) The prenatal development of the mouse eye. Anat. Rec. 168, 105–125 17 Grant, P. et al. (1980) Ontogeny of the retina and optic nerve in Xenopus laevis. I. Stages in the early development of the retina. J. Comp. Neurol. 189, 593–613 18 Schmitt, E. and Dowling, J. (1994) Early eye morphogenesis in the Zebrafish, Brachydanio rerio. J. Comp. Neurol. 344, 532–542 19 Malicki, J. and Driever, W. (1999) oko meduzy mutations affect neuronal patterning in the zebrafish retina and reveal cell–cell interactions of the retinal neuroepithelial sheet. Development 126, 1235–1246 20 Ramón y Cajal, S. (1893) La retine des vertebres. La Cellule 9, 17–257

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Acknowledgements The author is grateful for insightful comments from Connie Cepko, Bill Harris, Pamela Yelick, Francesca Pignoni, Bill Sewell, Clint Makino, Geoffrey Doerre and Zac Pujic. The author’s research is supported by grants from Research to Prevent Blindness, March of Dimes Birth Defects Foundation and the National Eye Institute.

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