SIGNALLING PATHWAYS IN DROSOPHILA AND VERTEBRATE RETINAL DEVELOPMENT

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1 SIGNALLING PATHWAYS IN DROSOPHILA AND VERTEBRATE RETINAL DEVELOPMENT Justin P. Kumar The near-catholic conservation of paired box gene 6 (Pax6) and its supporting cast of retinal determination genes throughout the animal kingdom has sparked a scientific war over the evolutionary origins of the eye. The battle pits those who support a polyphyletic history for the eye against those who argue for a common ancestor for all seeing animals. Recent papers have shed light on how eyes in both vertebrates and invertebrates are patterned. New insights into the roles that signal-transduction cascades might have in determining the Drosophila melanogaster eye indicate that, like many developmental processes, eye specification is an inductive process. Department of Cell Biology, Emory University School of Medicine, 1648 Pierce Drive, Atlanta, Georgia 30033, USA. jkumar@cellbio.emory.edu At first glance, the eye of man is as different from that of the fruitfly as night is to day. This feeling has been reinforced by comparative studies of developing and adult retinas in both invertebrates and vertebrates across the animal kingdom (BOX 1). Despite these tremendous developmental and anatomical differences, numerous genes that affect eye development are surprisingly conserved across the animal kingdom (TABLE 1). In both vertebrates and fruitflies, these genes are expressed in a temporal and spatial pattern that is consistent with a function in eye development, and mutations in several of these genes result in retinal defects in both systems. This conservation of eye-specification factors has raised the intriguing question as to whether the mechanisms that control the specification of and pattern formation in the retina have also been conserved. Recent developments show that in the vertebrate retina, in a manner eerily reminiscent of the fruitfly eye, the secreted morphogen Hedgehog (Hh) initiates pattern formation by directing the propagation of a morphogenetic wave front across the optic disc 1. The preservation of eye-specification genes and patterning mechanisms indicates that these genes might be expressed and function solely in the developing retinas of both vertebrate and fly retinas. However, this is not the case; in both flies and vertebrates, these factors show dynamic spatial and temporal expression patterns during many stages of development. Furthermore, mutations in these genes affect not just the eye but also several other tissues. So, it is puzzling that these genes should be expressed in such dynamic patterns, while at the same time being evolutionarily conserved to produce eyes of drastically different forms and optical properties. Results from several laboratories now indicate that in the fruitfly, receptor tyrosine kinase (RTK) and Notch signalling pathways provide a spatial and temporal context for the specification of the eye 2 4. Some of these results have also challenged several established models for how and when the fly compound eye is specified. In this review, I discuss these results and the implications that arise from the discovery that a Hh-dependent morphogenetic wave exists in the vertebrate retina, just as it does in the fly, and from the demonstration that RTK and Notch signalling cascades direct the specification of the Drosophila melanogaster compound eye. Master control genes in the Drosophila eye The specification of the fruitfly compound eye is under the control of a set of seven nuclear factors (referred to hereinafter as eye-specification genes ): 846 NOVEMBER 2001 VOLUME 2

2 REVIEWS Box 1 Development and structure of the Drosophila and vertebrate retinas The Drosophila retina, which gives rise to the compound eye of the fly, is derived from a monolayer epithelium called the eye imaginal disc (shown in figure panel a as part of the eye antennal imaginal disc complex). During the final larval instar (the third larval stage), a wave of differentiation, which can be visualized by an indentation in the epithelium called the morphogenetic furrow, sweeps across the disc transforming an unpatterned and undifferentiated field of cells into a precise tiling of ~800 unit eyes, or ommatidia. The construction of an ommatidium involves a series of inductive events that result in the stereotyped recruitment of 20 cells: eight photoreceptors (R1 8), and 12 accessory cone and pigment cells (figure panel b). In the adult retina, the photoreceptor neurons make up the core of the ommatidium and project the rhabdomere a light-gathering organelle into the central lumen. Above this lumen lie four cone cells that secrete the overlying pseudocone and lens material. Surrounding the photoreceptors and cones are pigment cells that optically insulate each unit eye (figure panel b; seen in longitudinal section on the left and in cross-section at different positions on the right). The development of the vertebrate eye is first seen as a pair of bilateral depressions called optic pits in the developing forebrain (figure panel c). These pits eventually become pouches called optic vesicles (OV; step 1). As the overlying lens placode (LP) invaginates (step 2) to form the lens vesicle (LV; step 3) (and ultimately the lens (L); step 4), the underlying outer surface of the optic vesicle also invaginates to form the optic cup (OC) that now has two closely apposed layers; the inner layer becomes the neural retina (NR) and the outer layer forms the retinal pigmented epithelium (RPE). The overlying cornea is derived from the surface ectoderm. The developing neural retina (step 5) undergoes an ordered series of births and migrations of individual cell types, ultimately giving rise to an adult retina (figure panel d) that consists of two types of photoreceptor (rods and cones), horizontal, amacrine, bipolar and ganglion cells, and Muller glia (not shown on figure) that are organized into several layers. ON, optic nerve. (Panel b is reproduced with permission from REF. 103 (1993) Cold Spring Harbor Laboratory Press. Panel d is reproduced with permission from REF. 104 (1985) McGraw Hill.) a Morphogenetic furrow c 1 2 Ommatidium 3 RPE OC OV LP LV NR LP Point of furrow initiation Anterior 4 5 Neural retina RPE NR L Posterior Eye Cornea ON Lens Antenna b d Pigmented cell Cornea 10 mm Pseudocone Rod 3º pigment cell Rhabdomere Cone Cone cell R7 2º pigment cell 1º pigment cell R7 rhabdomere R4 R3 Cone cell process R5 R2 R6 R1 R7 R8 rhabdomere Horizontal Cone bipolar Amacrine R8 Ganglion cells Anterior R8 Cone cell foot Axons Equator NATURE REVIEWS GENETICS VOLUME 2 NOVEMBER

3 Table 1 Selected factors involved in the specification and patterning of the retina Fly genes Protein Vertebrate gene(s) Loss-of-function Expression pattern phenotype twin of eyeless Homeodomain/ Small eye (Pax6) 107 Aniridia Anterior neural plate, lens placode (toy) and eyeless (ey) 9,12 paired domain and cornea sine oculis (so) and optix 7,10,13 Homeodomain/ Six family (Six3, Six6) 108,109 Bilateral anophthalmia Anterior neural plate, optic vesicle Six domain and stalk, neural retina and lens eyes absent (eya) (clift (cli) 6 ) Novel Eya1 Eya4 110,111 BOR syndrome Eya1 in lens placode; Eya3 in optic vesicle, lens vesicle and retina dachshund (dac) 8 Novel Dachshund homologue 1/2 Unknown Dach1 in optic vesicle, optic cup (Dach1, Dach2) 112,113 and retina; Dach2 in retina and surrounding mesenchyme eye gone (eyg) 55 Homeodomain Unknown Unknown Unknown hedgehog (hh) 68,114 Secreted morphogen Sonic hedgehog (Shh) 84,115, Cyclopia Shh and Twhh in neural retina and Tiggy winkle hedgehog retinal pigmented epithelium (Twhh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh) atonal (ato) 72,116 bhlh transcription Mouse atonal homologue 5 Loss of RGC Optic cup, RGC precursor factor (Math5) 78 EGF receptor (Egfr) 117,118 Receptor Waved2 119,120 Anterior segment Perioptic mesenchyme, eyelid tyrosine kinase dysgenesis, loss of epithelium, corneal epithelium anterior chamber, corneal scarring Notch (N) 52,121 Transmembrane Notch1 Notch4 Retrolentricular N1 in neural retina; N2 in lens receptor (N1 N4) hyperplasia, bilateral and retinal pigmented epithelium; microphthalmia N3 in lens and neural retina decapentaplegic (dpp) 125,126 TGF-β-secreted BMP family Block in lens induction, BMP4 in optic vesicle, lens morphogen (BMP4, BMP7) bilateral anophthalmia placode; BMP7 in surface ectoderm, lens placode, optic vesicle and stalk bhlh, basic helix loop helix; BMP, bone morphogenetic protein; BOR, branchiootorenal dysplasia; EGF, epidermal growth factor; Pax, paired box; RGC, retinal ganglion cell; Six, sine oculis homeobox; TGF-β, transforming growth factor-β. eyeless (ey), twin of eyeless (toy), sine oculis (so), eyes absent (eya), dachshund (dac), eye gone (eyg) and optix 5 13 (TABLE 1). Experiments both in vivo and in vitro indicate that these genes do not function as a linear biochemical or enzymatic pathway but rather exist in a complicated interwoven regulatory network (FIG. 1). Assays done in vitro show that these eye-specification genes interact with each other through direct transcriptional regulation and through the formation of biochemical complexes For example, Eyeless protein has been shown to bind directly to an eye-specific enhancer region that is located in the first intron of the so gene, and is also able to direct the expression of a reporter that has been fused to regulatory sequences located just upstream of the first exon of eya 15,16,18,19. In addition, the Eyes absent protein can form independent protein complexes with Sine oculis and Dachshund 14,20. The simplest explanation for such observations is that the seven eye-specification proteins form a loosely defined transcriptional unit that is required for the determination of the eye. Notwithstanding the attraction of such a simple model, our understanding of eye specification in Drosophila is complicated by the participation of additional factors, such as homothorax (hth), extradenticle (exd) and teashirt (tsh), and patterning genes, such as hedgehog (hh) and decapentaplegic (dpp), which in some cases function reiteratively in the eye-specification cascade Removal of any of the seven core eye-specification genes in the eye primordium results in a drastic reduction or deletion of the adult compound eye, whereas ectopic expression of these genes (except so) results in the induction of retinal development outside normal eye tissue 12,13, These twin characteristics led to the crowning of the factors as master control genes for the eye a term that was initially coined by Edward Lewis to describe the homeotic properties of the Bithorax and Antennapedia gene clusters (BXC and ANTC), famously the antenna-toleg fate transformation studies of Antennapedia mutations 29. The title of master control gene was further enshrined by the discovery of vertebrate homologues of eye-specification genes in the animals of all principal phyla (TABLE 1) and by the demonstration that ectopic eye tissue could be generated in flies, mice and Xenopus laevis by the expression of vertebrate homologues of the eye-specification genes 26 28, Furthermore, several human retinal disorders have been attributed to mutations in the human homologues of several fly eye-specification genes; for example, aniridia and bilateral anopthalmia are due to mutations in the human homologues of ey and so, respectively (TABLE 1). Despite these rather impressive qualities, there are still some doubts as to whether, in the fly, these genes truly can be called master regulators of eye specification. Every developmental mechanism might be expected to 848 NOVEMBER 2001 VOLUME 2

4 MUSHROOM BODY The brain region of insects that might be implicated in complex types of behaviour, such as learning, social behaviour and spatial memory. NON AUTONOMOUS A cell non-autonomous trait is one in which genotypically mutant cells cause other cells (regardless of their genotype) to show a mutant phenotype. Optix Hh Dpp Notch Toy Ey Dac Egfr Tsh So + Eya MAPK RTK Eye specification Eyg Hth + Exd Figure 1 Genetic control of eye specification in Drosophila. A set of nuclear proteins, patterning pathways and signal-transduction cascades form a complicated regulatory network and are together required to specify the compound eye in Drosophila. The arrows in this diagram indicate the direction of the genetic, molecular and biochemical relationships. Dac, Dachshund; Dpp, Decapentaplegic; Egfr, Epidermal growth factor receptor; Exd, Extradenticle; Ey, Eyeless; Eya, Eyes absent; Eyg, Eye gone; Hh, Hedgehog; Hth, Homothorax; MAPK, Mitogenactivated protein kinase; RTK, receptor tyrosine kinase; So, Sine oculis; Toy, Twin of eyeless; Tsh, Teashirt. have a single master, but for Drosophila eye specification there are seven claimants. Second, unlike the homeotic transformations that accompanied the removal of genes in the ANTC and BXC, the adult fly eye is not replaced by another organ in loss-of-function mutants. In fact, the eye imaginal disc initially develops reasonably normally, but it later undergoes high levels of apoptosis 6 8,37. Third, the ability of the eye-specification genes to induce ectopic retinal development is limited to specific portions of just a few imaginal discs, and it has been indicated that the expression of the Hh and Dpp pathways are a prerequisite for retinal development in these discs 24. Fourth, the expression patterns of the fly eyespecification genes are not limited to the developing eye; each gene has a dynamic expression pattern and this is also true for the expression patterns of the vertebrate homologues. For example, in addition to expression in the fly eye imaginal disc, transcripts of the fly so gene have been localized to the Bolwig s organ (the larval eye), the optic lobes and a group of epidermal cells at the embryonic segmental boundaries 7,10,38. Likewise, the expression of one of the human homologues of so, SIX3, is not limited to the retina but is also found in the lens, hypothalamus and pituitary 39,40. In the fly, the loss of several eye-specification genes affects the development of other tissues; for example both ey and dac have been implicated in the development of MUSHROOM BODIES 41,42. Similarly, loss of mouse paired box gene 6 (Pax6) results in defects in nasal cavity formation as well as defects in lens development 43. And finally, it is known from other developmental contexts that there is interplay between master control genes and signal-transduction cascades. For example, it is known that signalling by the Ras pathway modulates the activity of proboscipedia (pb) and Ultrabithorax (Ubx) members of the ANTC and BXC 44. Because each eye-specification factor is a nuclear protein, it is possible that upstream signal-transduction cascades could exist to transmit instructive signals that are relevant to specify the eye. Signalling cascades in fly eye specification Recent articles from several laboratories might connect the eye-specification genes to the elusive master control gene for eye specification. In the first article, by Francis Hsiao and colleagues 2, the eye-specification protein Eyes absent (Eya) was identified as a direct target for phosphorylation by Mitogen-activated protein kinase (MAPK), which is encoded by the rolled (rl) locus in flies and is the most downstream cytoplasmic member of the Ras signalling cascade 2, The eya gene had been identified previously as a target of RTK signalling in a genetic screen for factors that interact with the epidermal growth factor receptor (Egfr) pathway antagonist, yan (also known as anterior open (aop)) 48. In both genetic and biochemical assays, the authors showed that the consensus MAPK phosphorylation sites in the Eya protein are indeed phosphorylated by MAPK. More interestingly, the removal of these sites reduces the ability of Eya to direct eye development in ectopic expression assays in vivo, indicating that during normal development eya lies directly downstream of a RTK signal-transduction cascade. If these results were extended to the eye-specification transcriptional unit, as I believe they can be, RTK signalling could be thought of as exerting a positive influence on the eye-specification gene set 2 (FIG. 1). These results further imply that the process of eye specification is a NON-AUTONOMOUS process. In a companion paper, J.P.K. and Kevin Moses 3 also suggest that the specification of the eye is a nonautonomous process. They have identified the Egfr and Notch signal-transduction cascades as potential upstream regulators of the expression and activity of genes required for eye specification in the developing fly retinal primordia. By testing the ability of many Egfr and Notch pathway members to affect eye development, complete homeotic transformations of the eye to antenna were obtained as a result of hyperactivating Egfr or downregulating Notch pathway signalling in the eye primordium (FIG. 2). In the newly transformed tissue, expression of several antennal markers is detected along with the nearly abolished transcription of eye-specification genes. These results imply that, genetically speaking, both pathways lie upstream of genes that specify the fate of both the fly eye and antenna. It remains to be determined whether these genetic interactions between the eye- and antennal-specification genes and the signal-transduction cascades occur at the transcriptional or protein level. NATURE REVIEWS GENETICS VOLUME 2 NOVEMBER

5 a b Eye Grk Spitz Pnt P1 Ras Raf MEK Vn Btl Egfr Htl MAPK Antenna Pnt P2 Egfr Notch Notch The reports by Hsiao et al. and Kumar and Moses seem to present contrasting roles for RTK signalling during fly eye specification. The genetic and molecular epistasis experiments described by Kumar and Moses indicate that the Egfr pathway might act in the antennal disc to prevent eye development, whereas Hsiao et al. present strong biochemical and genetic evidence for a positive influence on the developing eye primordium by RTK signalling. The reasons for the apparent dual and opposing roles for RTK signalling in eye specification might have roots in temporal and spatial developmental contexts. The conflicting role for RTK signalling is expected to take place in distinct tissues (in the eye in Hsiao et al. versus in the antennal discs in Kumar and Moses). Furthermore, these effects could take place at distinct times in development. Unlike Notch, the effects on eye specification by Egfr/RTK signalling have yet to be worked out in detail. Another difference between the Dl Antenna E(Spl)C Ser Su(H) Mam Figure 2 Egfr and Notch signalling control eye and antennal identity in Drosophila. a Hyperactivation of several components of the Epidermal growth factor receptor (Egfr) pathway in the eye primordium results in eye-to-antenna homeotic transformation, indicating that Egfr signalling might normally function in the developing antenna to repress the expression of genes required for eye specification. Removal of specific Notch pathway elements also produces this homeotic transformation, indicating that Notch has a positive influence in eye specification. b The components of the Egfr and Notch pathways used in these experiments are shown in green and red boxes, respectively. Several components of each pathway (those not in boxes) seem to have no role in disc specification, indicating that pathway branchpoints could be crucial for developmental control of ubiquitously used pathways. The relationship between members that have been assigned roles in disc specification are linked by coloured arrows. Btl, breathless; Dl, Delta; E(Spl)C, Enhancer of Split complex; Grk, Gurken; Htl, Heartless; Mam, Mastermind; MAPK, Mitogen-activated protein kinase; MEK, MAP kinase kinase; Pnt, Pointed; Raf, MAP kinase kinase kinase; Ser, Serrate; Su(H), Suppressor of Hairless; Vn, Vein. functions of RTK signalling in this process is highlighted by the differential reliance on MAPK activity. The promotion of eye specification by RTK signalling is shown to occur through the phosphorylation of the Eya protein by MAPK, whereas the inhibition of eye development through Egfr signalling seems to be carried out through a MAPK-independent process. There is growing evidence that there are several possible branchpoints in the RTK/RAS/MAPK-signalling cascade. As one example, it has been recently shown that RAS, but not its downstream effector MEK (MAP kinase kinase), is required for cell movements in wound closure, indicating the possible involvement of a MAPK-independent mechanism in this process 49.If such branchpoints also exist in RTK signalling in the developing fly eye and antenna, it might account for the differences observed in the above experiments. The identification of the Notch pathway as a potential regulator of eye specification agrees with some aspects of an earlier seminal finding for this pathway in controlling appendage identity, and is consistent with reports of early roles for Notch in eye development 4,50,51. In a similar report to that by Kumar and Moses, Shoichiro Kurata et al. had expressed dominant-negative and constitutively active versions of the Notch receptor in the developing eye. Expression of a dominant-negative Notch receptor led to the elimination of the compound eye, whereas hyperactivating Notch signalling led to the formation of ectopic eyes in antennal-derived tissue 4. These results indicate a positive role for Notch in eye specification, as was suggested by Kumar and Moses. Kurata et al. go further and show that in an ey mutant background, increased Notch activity can promote antennal identity, as assayed by the homeotic transformation of retinal tissue into antennal segments. The observation that homeotic eye-to-antenna transformations (which have implications for the process of transdetermination; BOX 2) can occur either when Notch signalling is hyperactivated or downregulated complicates the interpretation of the role of Notch in eye specification 3,4. Notch has been suggested to have a permissive role in detemination events 52, so the genetic context in which the Notch pathway functions is certain to influence the developmental outcomes of its activity. The eye-to-antenna transformation seen when Notch activity is reduced occurred on an otherwise wild-type background, whereas the homeotic effects seen with increased Notch signalling occurred on a genetic background in which the eye-specification programme was severely compromised. The reaction of undifferentiated cells to the manipulations of signal-transduction cascades and tissue-specific regulators is an exciting and ongoing area of research. Timing of eye specification in Drosophila The suggestion that many signal-transduction cascades have inputs into the process of eye specification has prompted enquiries into the timing and source of the upstream signals. In Drosophila, it has been traditionally accepted that specification of the eye, as well 850 NOVEMBER 2001 VOLUME 2

6 Box 2 Transdetermination of imaginal disc identity in Drosophila a Disc transplantation in Drosophila Determination Wing b Disc transdetermination in Drosophila Wing disc Larva Transdetermination Antenna It has been shown in Drosophila that imaginal discs can be dissected, transplanted into host animals and cultured for long periods of time. If donor imaginal discs are transplanted into the abdomens of adult flies, the donor disc remains in a proliferative state and the disc can be recovered, thus establishing an in vivo culture system. However, if the donor disc is transplanted into donor larvae, then the disc will undergo metamorphosis along with the larva and will give rise to the correctly fated adult structure. Ernst Hadorn and colleagues took advantage of the fact that imaginal discs could be transplanted into a larval host and the fated adult derivative could be recovered after the host had matured into an adult, to map out the developmental capacity of each imaginal disc (figure panel a). Note that in this example, a transplanted wing disc will give rise to adult wing tissue. The wing disc is said to be determined to give rise to wing tissue. Amazingly, on rare occasions, the donor disc would undergo a change in fate and produce an adult structure that was different to its intended fate (figure panel b) through the process of transdetermination, a term coined to describe the alterations in organ fate that were observed in such imaginal disc transplantation. For instance, as shown in figure panel b, a wing disc could give rise to adult antennal tissue. A modified summary of several decades of transplantation experiments is shown in figure panel c, and indicates direction and relative frequency (proportional to the length of the arrows) of each c Transdetermining events in Drosophila transdetermining event. Naturally occurring homeotic Antenna Eye mutants by and large follow the directions of the disc transplantation experiments. Together these results Genital Wing Mesothorax indicate that the developmental capacity of each imaginal disc might be generally limited; in particular, the eye imaginal disc is quite restricted in its ability to Labial Leg Haltere produce anything but retinal tissue. By contrast, the recent recovery of eye-to-antenna transformations indicates that these developmental barriers might in fact be overcome and that imaginal discs are much more pluripotent than previously thought. The involvement of signal-transduction cascades, such as Hedgehog, transforming growth factor-β, receptor tyrosine kinases and Notch, provides a spatial and temporal context for disc specification. (Modified from REFS 105,106.) CLONAL ANALYSIS (also known as mosaic analysis). The process of following the progenitors derived from a single cell (a clone). Clonal analysis can be used to infer how many cells make up an anlage, when gene action takes place, and if lineage has a role in cell-fate determination. HOX GENE One of a group of linked regulatory genes involved in patterning the animal body axis during development. as that of the other appendages, occurs during embryogenesis. This is based on the expression patterns of several eye-specification genes, anatomical landmarks and on the CLONAL ANALYSIS of mutant markers 9,12, The factors that initiate eye imaginal disc development (those that lie upstream and initiate toy expression; FIG. 1) have yet to be identified, but several possible sources and types of cue can be considered. These upstream signals might be produced along the embryonic midline (which is a source of Spitz, an Egfr ligand) and secreted towards the regions in the embryo that will give rise to the imaginal discs. Alternatively, the decision to adopt an eye disc fate might be the result of a combinatorial code of HOX GENES. Other models include the correct combination of anteroposterior and dorsoventral signalling gradients or the correct combination of segmentationgene expression. An alternative model that has been proposed recently supports that the cells that give rise to the future eye are set aside during embryogenesis but that they might not be terminally committed to an eye fate until much later in development 3. This model is based in part on the expression patterns of the eyespecification genes. Collectively, several laboratories have shown that only three (ey, toy and eyg) of the seven factors are expressed together in the eye imaginal disc during embryogenesis 9,12,55. Because the available molecular, biochemical and genetic evidence indicates that these factors might function together as a transcriptional complex, one expectation might be that their expression patterns should coincide in the eye imaginal disc at the time of specification. It is not until the second larval instar that all eye-specification genes are restricted and co-expressed in the eye imaginal disc. At that time, the Notch receptor is also highly NATURE REVIEWS GENETICS VOLUME 2 NOVEMBER

7 a c Posterior Hh expression Morphogenetic furrow Hh-secreting photoreceptors Anterior d Midline b or Notch signalling during a crucial time window in the second larval instar stage of development a time that just precedes the initiation of the morphogenetic furrow (BOX 1). Similar timelines have been reported for the homeotic effects of removing Egfr from the fly wing and NOTUM 56. This now raises the possibility that in the fly, signal-transduction pathways are used late in development to temporally couple specification and pattern formation of organs. In this model, the Rubicon for eye specification might be crossed near the end of the second larval instar, when all the eye-specification genes are co-expressed along with the appropriate signal-transduction cascades and patterning elements. However, there are several limitations to this model; for instance, it is not clear whether the patterns of gene expression required for eye specification are regulated directly by either Notch or Egfr signalling. Furthermore, although the timing of the homeotic transformations occurs solely in the second larval instar stage, this does not rule out that the specification process begins during embryogenesis and continues through the later larval stages. The apparent stepwise addition of eye-specification genes to the eye imaginal disc could reflect different levels of differentiation or specification. Figure 3 Hedgehog is required for neurogenesis and for atonal expression in the fly eye. a Diagram of the Drosophila eye imaginal disc. Hedgehog (Hh) is expressed at the intersection of the posterior margin of the eye disc and the midline. The morphogenetic furrow initiates from this intersection and sweeps across the eye disc from posterior to anterior, leaving determined ommatidial founder photoreceptors (R8 cells; red) in its wake. R8 cells and the other photoreceptors that have been recruited into the growing ommatidium secrete Hh to cells anterior to the furrow, so pushing the furrow forward. b, c Imaginal eye discs stained with the proneural gene atonal (ato) (green). Developing photoreceptors are shown in red. b In wild-type eye discs, ato is expressed in a large swathe of cells ahead of and within the morphogenetic furrow (arrow). c When Hh signalling is removed using a hh conditional allele, ato expression is markedly reduced. d Ectopic expression of hh (green) ahead of the morphogenetic furrow leads to ectopic retinal development (shown in red). Note the circular pattern of ato expression that is just anterior to the endogenous ato pattern in the furrow (arrow). Anterior is to the left. (Images provided by Ulrike Heberlein.) (Panels b and c reproduced with permission from REF. 62 (1998) Academic Press. Panel d reproduced with permission from REF. 66 (1995) Macmillan Magazines Ltd.) NOTUM The dorsal or upper surface of any insect thoracic segment. enriched in the eye primordium, thus placing both the receptor and its potential downstream targets together in the eye imaginal disc 3. The coincident expression of the eye-specification genes and the Notch receptor, along with the timing of the eye-toantenna transformations, provides circumstantial evidence for a model in which the eye imaginal disc receives its final instructions to produce an eye late in larval life. By controlling the timing and duration of their overexpresssion experiments, Kumar and Moses showed that, surprisingly, the eye was transformed into an antenna in response to manipulations of Egfr One or many origins for the eye? In their seminal paper on the convergent evolution of the eye in different phyla, Salvini Plawen and Mayr propose that the eye has evolved at least 40 and possibly 65 times during the past ~5 billion years 57.Their conclusion was based in part on the tremendous diversity in the structure of eyes throughout the animal kingdom and also on the types of eye found in extant phyla. For instance, in several cases, homologous eye types can be found in different taxa, whereas in others very different eye types can be found in a single phylum. In support of a convergent mechanism for eye development, it has been postulated that a camera, or simple eye, can evolve in a very short period of time (in evolutionary terms) 58. The diversity in eye types and optical properties, along with the differences in the use of lens proteins and the embryological origins of the eye, has been elegantly outlined in several recent discussions The recent discovery that the Pax6 gene and its accompanying cast can direct eye development in many taxa indicates that visual organs could have a monophyletic history after all. But it has been forcefully argued that the presence of a homologous structure or protein does not necessarily imply a homologous origin. As described above, several of the eye-specification genes are expressed and are involved in the development of non-eye tissue, but we do not say that the eye is homologous to the brain or to muscles. However, more recently, a common mechanism for propagating retinal patterning in both vertebrates and fruitfly eyes has been identified. This could re-ignite the debate and provide more evidence for the existence of a common ancestry for the eye. 852 NOVEMBER 2001 VOLUME 2

8 a c wt, 30 hpf wt, 52 hpf b d wt, 40 hpf syu, 52 hpf the elimination of ato expression and a subsequent block in both the initiation and progression of pattern formation an effect similar to that seen in ato loss-offunction mutants 62,64,65,72,76,77. Hh expression outside its normal confines is sufficient to activate the transcription of ato, thereby initiating ectopic retinal development 66 (FIG. 3). The acceleration in the completion of several genome projects has identified an avalanche of hh and ato homologues in vertebrate systems, several of which are expressed in the developing vertebrate retina Similar to their counterparts in the fruitfly, these genes are necessary for normal eye development. For instance, loss of Sonic hedgehog (Shh) and Mouse atonal homologue 5 (Math5), the mouse homologues of Drosophila hh and ato, leads to the production of a single fused optic vesicle and the near elimination of retinal ganglion cells (RGC), respectively The conservation of these two molecules in eye development indicates that the mechanisms for pattern formation, namely a morphogenetic wave front and a proneural selection method, are also preserved in both systems. Figure 4 Sonic hedgehog expression across the zebrafish retina. Side views of zebrafish retinas. Neurogenesis (indicated by staining for the Zn5 marker, red) and Sonic hedgehog Green fluorescent protein (Shh GFP, green) expression are shown at various hours post-fertilization (hpf). a c Shh expression occurs in a wave-like pattern across the wild-type (wt) zebrafish optic disc. Note that a subset of Zn5-positive cells are also positive for Shh GFP. d In mutants, such as sonic you (syu), in which normal zebrafish Shh signalling is disrupted, the initial Shh GFP is expressed in the retina but fails to expand through the retina. Note that neurogenesis is slightly retarded in the syu retina. Anterior is to the left and ventral is at the bottom. (Reproduced with permission from REF. 1 (2001) American Association for the Advancement of Science.) A conserved patterning mechanism The initiation and progression of the morphogenetic furrow across the fly eye imaginal disc is directed by the secreted morphogen Hh Before the onset of pattern formation, Hh is first expressed at the intersection of the midline and the posterior margin 64. During the final larval instar, the morphogenetic furrow initiates at this point and sweeps across the eye disc epithelium from posterior to anterior 69. A continuous supply of Hh is produced and secreted by newly born photoreceptors. Undifferentiated cells ahead of this advancing wave receive the diffusible Hh signal, undergo their final round of mitosis, enter the morphogenetic furrow and then go on to differentiate into photoreceptor neurons. This continuous cycle pushes the furrow across the retinal epithelium 70 (FIG. 3a). The first cells to differentiate behind the furrow are the R8 founder cells, the fates of which are directed by the proneural gene atonal (ato) a target of Hh signalling The ato gene is first expressed in a broad swathe of cells ahead of the advancing furrow. Expression of ato undergoes a series of successive refinements in and behind the furrow until it is expressed only in a single cell (the R8) in each ommatidium 72,74,75. Loss of Hh signalling in the developing fly eye results in A wave of Hedgehog signalling Mutations in the vertebrate Shh gene have been shown to result in the cyclopia phenotype in mice, zebrafish and humans, indicating a role for this pathway in eye development 84,85, Recent studies have shown that the production of a single optic vesicle is caused by a disruption of Shh at the embryonic midline rather than by a direct role in eye development; probably by altering the spatial distribution of the Pax2 and Pax6 homeodomain proteins 84,85. Now, a direct role for Hh signalling in vertebrate retinal development has been identified. A recent series of papers shows that the vertebrate retina does indeed use a Hh-dependent mechanism to propagate retinal development across the optic disc, and also a proneural mechanism for specifying the first neurons in the retina. Although the diverse cell types in the vertebrate retina are born, and migrate, to positions in a stereotyped sequence, the lack of an observable furrow in the vertebrate retina, like that seen in the fly eye, has dampened enthusiasm for a conservation of retinal patterning. Recently, Carl Neumann and Christianne Nuesslein-Volhard have favoured the view of common ancestry between insect and vertebrate eyes by showing that a wave of Shh patterns the zebrafish retina 1. Using reporter transgenes in which green fluorescent protein (GFP) is controlled by the Shh promoter (Shh GFP), the authors showed that a subset of first-born retinal ganglion cells express Shh (FIG. 4). They also showed that, as eye development continues, Shh expression expands to fill the neural retina, raising the possibility that this wave marks the movement of a cryptic furrow. They extended these results by driving expression of the Shh GFP transgene in zebrafish retinas that were genetically mutant for the endogenous Shh gene. In such experiments, Shh GFP was expressed at the point of normal neurogenic initiation but it failed to spread throughout the optic disc. As a possible consequence, the rate and extent of neurogenesis is retarded when NATURE REVIEWS GENETICS VOLUME 2 NOVEMBER

9 a Hu, 30 hpf c Hu, 48 hpf + b Hu, 30 hpf lak + d Hu, 48 hpf lak Figure 5 Mutations in the zebrafish homologue of atonal inhibit the first wave of neurogenesis in the developing retina. a, c Wild-type retinas and b, d retinas mutant for lakritz (lak), the zebrafish atonal homologue (also known as ath) are shown here stained with Hu, a marker for post-mitotic neurons. a and b are retinas stained at 30 h postfertilization (hpf), when the neurogenic wave is beginning to form, whereas the retinas in c and d have been stained at 48 hpf, when the first wave of neurogenesis is mostly complete. a, c Hu staining can be seen in the nasal placode (arrowhead) and in the retina (arrows). b In the lak/ath mutants, Hu staining can still be seen in the nasal placode but d is absent in the retina during the first wave of neurogenesis. However, the first Hu-positive cells are seen in the retina. Anterior is to the left and dorsal is at the top. (Reproduced with permission from REF. 88 (2001) Cell Press.) assayed for the presence of the Zn5 neuronal marker (FIG. 4). Shh is also shown to be both necessary and sufficient for its own expression in the zebrafish eye, indicating that, as in the fly eye, Shh regulates retinal development in an autoregulatory fashion. Other Hh family members, such as tiggywinkle hedgehog (twhh), are implicated in patterning the retina, as the expanding wave of neurogenesis is much more severely inhibited when the retinas are treated with cyclopamine, a chemical that inhibits the function of all Hh proteins. The authors show that twhh is also expressed in the optic disc, confirming in part the partially redundant roles of the Hh proteins. Injections of a cocktail of shh and twhh antisense oligonucleotides in the zebrafish retinal pigmented epithelium (RPE) seem to inhibit rod photoreceptor and red cone differentiation, and adversely affect the overall size of the eye. Injections of either of the antisense oliognucleotides alone had similar effects, but the magnitude of the effects was reduced, again indicating a redundant role for twhh signalling in vertebrate eye development 80. Furthermore, reductions of zebrafish twhh also led to cyclopia phenotypes 93. Several laboratories have shown general roles for Hh in vertebrate eye development, but the published spatial and temporal expression patterns have never been entirely consistent with a role for Hh in initiating and patterning vertebrate retinal neurogenesis 79,80,83. This report by Neumann and Nuesslein-Volhard is the first concrete evidence that the retina is patterned by a mechanism that is conserved across evolution. That is not to say that there are not significant differences between the roles of Hh in the different optical systems. In the fly eye, Hh is required before the onset of neurogenesis for the initiation of the morphogenetic furrow 62,64.In zebrafish, it seems that Hh expression does not precede the onset of RGC development. Furthermore, in shh loss-of-function mutants or in retinas treated with cyclopamine, retinal neurogenesis is still able to initiate. By contrast, the early removal of hh from the fly eye completely blocks retinal development 62. Another significant difference is the apparent inability of Shh to induce its own expression (and drive retinal development) in regions of the optic disc that do not include the region of neurogenesis initiation. In flies, ectopic expression of hh ahead of the morphogenetic furrow can initiate and drive precocious retinal development 66. Despite these differences, the presence and wave-like expression pattern of shh in the developing vertebrate eye has significantly advanced the cause for a common ancestor for all seeing animals. Conserved proneural gene function Although each distinct cell type in both vertebrate and fly retinas is born in an invariant sequence, with the RGC and R8 cells being produced first, the identification of a common mechanism has, until recently, remained elusive. In the fruitfly eye, the fate of the R8 founder cell is dependent on the proneural gene ato, which encodes a basic helix loop helix (bhlh) nuclear protein 72,76,77.In ato mutants, the R8 precursors are never produced; and as the development of the ommatidium is an inductive process that requires cell cell interactions, this failure in R8 precursor development blocks the remaining steps of ommatidial assembly; subsequently, the eye fails to develop. Although there are several differences between the identity and origins of the R8 and RGC cells, they share the common feature that they are the first-born neurons in both the fly and vertebrate retinas, hinting that they might be evolutionarily related. Recent demonstrations that a homologue of Drosophila ato functions in RGC production provides further reasons for such speculation. Several vertebrate bhlh proteins with varying similarities to fly Atonal have been identified and, in fact, several are expressed in the developing retina. Their expression, however, does not pre-date the birth of the RGCs a requirement for a gene that is involved in RGC formation. Recently, the mouse Math5 gene has been cloned and shown to be the vertebrate family member most closely related to fly ato 78. Furthermore, Math5 is expressed in the mouse retina at embryonic stage (E)11, well before the onset of expression of the 854 NOVEMBER 2001 VOLUME 2

10 other ato homologues and before the birth of the first RGC that takes place at the late E12 early E13 stage of development. The spread of Math5 across the mouse retina coincides with and mimics the wave-like development of the RGC layer, indicating that, like the fly retina, the fate of the pioneering cells of the vertebrate retina might also require a proneural mechanism. A similar situation is observed in both the Xenopus and zebrafish retinas (FIG. 5): Xath5 and ath5 (the Xenopus and zebrafish homologues of ato, respectively) are expressed before the appearance of the first differentiated RGCs and, like the mouse retina, the expression of both orthologues fans out across the optic disc and prefigures RGC neurogenesis 81,82,88. Overexpression of Xath5 in tadpoles leads to the overproduction of RGC cells at the expense of other cell types in the retina, further promoting the idea that Ato directs retinal ganglion cell fate in both vertebrates and invertebrates 81. In the fruitfly, the spread of ato expression across the developing retinal epithelium is blocked when Hh signalling is eliminated in loss-of-function mutant clones or through the use of conditional, temperature-sensitive alleles 62,64,65. It is yet to be determined if the same relationship exists in the vertebrate retina. It is surprising that the timing of shh expression in zebrafish does not precede or correlate with the onset of ath5 transcription or with the appearance of the first differentiated RGCs 1. shh expression is first detected at 30 h post-fertilization, whereas the first retinal ganglion cells appear at 27 h, and ath5 expression is detected at 2 h before that event 88. In the vertebrate retina, the regulation of ath5 and thus RGC formation might depend on other members of the Hh family and could be considerably more complicated than their relationship in the fly eye. Loss of Math5 in the mouse retina markedly reduces the production of retinal ganglion cells 86,87. The loss of RGCs in Math5 knockout mice retinas is accompanied by an increase in the number of other retina cell types, including cone photoreceptors, which indicates that the loss of Math5 might lead to a cell fate switch. A similar gain in inner nuclear layer cell types at the expense of RGC cells is also observed in zebrafish retinas that are mutant for ath5 (REFS 78,86 88). The cell fate switch between RGC and other retinal cell types is not seen in Drosophila ato mutants 72. In the fly eye, there are three main classes of cell photoreceptors, cone cells and pigment cells giving a total of 20 cells per ommatidium 94.In ato mutants, the elimination of the R8 class of photoreceptor is not accompanied by a gain in any of the other cell types. The construction of the fly eye is a purely inductive process with the differentiation of each cell type being dependent on interactions with earlier born cell types 71,95,96. So, the entire fly eye is eliminated by programmed cell death if the initial specification of the R8 class of photoreceptor is not carried out correctly. Similarly, Egfr signalling is required for the specification and recruitment of the R2/R5 class of photoreceptor, which immediately follows and depends on the R8 founder cell. A block in R2/R5 specification by removal of Egfr signalling also halts any further cell-fate decisions in the developing ommatidium So, although there is some anecdotal, and now molecular, evidence that the R8 and RGC cells are evolutionary cousins, the mechanisms that induce the recruitment of the remaining cells might in fact be considerably different in the two systems. In the fruitfly, there is a single wave of differentiation in which the R8 class of photoreceptor develops first and acts like a nucleating factor for all remaining cell-fate decisions. By contrast, the vertebrate retina seems to undergo a series of successive rounds of birth, migration and differentiation in which each distinct cell type is born in a separate wave. These differences are in fact expected when one considers the marked differences in the structure of the diverse types of eye and should not be taken as evidence for the lack of a common ancestor. I believe that it is the similarities that provide the best clues to the origins of the eye. Concluding thoughts Although the list of commonly used molecules and pathways in insect and vertebrate eye development is continually growing, the great anatomical divide that separates different eye types is a difficult barrier to overcome in indicating a common ancestor. For this field to substantially move forward, those interested in such evolutionary questions will have to examine several issues that are still pending. These include why Pax6 and the other eye-specification genes were recruited for eye specification in the first place, and how these molecules, which are used in both flies and vertebrates, are used to produce such different types of eye. These questions are in many ways linked to the discussion of the type of eye possessed by the common ancestor of insects and vertebrates. I speculate that well before the split of insects and vertebrates, a common ancestor recruited Pax6 in subsets of cells to activate rhodopsin, as the ability to detect light would certainly be a considerable advantage. As evolutionary pressures placed a premium on the ability of organisms to avoid predators and seek prey, more complicated and sophisticated retinal and optical systems evolved. One can think of these evolutionary steps in this simple way. It is a step forward if you can detect light. It is even better if you can sense the approach of an object such as a predator. And it is still better if you can discriminate between a predator and a potential mate so that you can decide whether to run away or stick around. And so on The recruitment of several photoreceptors and the production of complicated eye structures has certainly involved the recruitment of established signalling and patterning cascades such as those controlled by Hh, transforming growth factor-β, RTKs and Notch. The diversity in optical properties and eye anatomy across the animal kingdom might be the result of environmental and adaptive pressures. The discovery that so many molecules and mechanisms are conserved across evolutionary lines should provide much solace and new-found inspiration to those who once thought it impossible to learn about one by looking at the other. It will now be considerably easier to look through the faceted lens of flies and into human eyes. NATURE REVIEWS GENETICS VOLUME 2 NOVEMBER