Vision Research 43 (2003) 899–912 www.elsevier.com/locate/visres
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Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
Received 3 July 2002; received in revised form 29 August 2002
Abstract
Knowledge of normal eye development is crucial for the development of retinal rescue strategies. I shall focus on two signalling pathways that affect retinal development. Fibroblast growth factors function in retinal cell proliferation, retinal ganglion cell axon guidance and target recognition, craniofacial patterning and lens induction. Hedgehog proteins are required for progression of the neurogenic wave, cell proliferation, photoreceptor differentiation, retinal ganglion cell axon growth and craniofacial patterning.
These signalling pathways have pleiotropic effects, can interact and have the potential to be used therapeutically. The zebrafish model organism may be well suited to studying how signalling pathways interact.
Ó 2002 Elsevier Science Ltd. All rights reserved.
Keywords: FGF; Hh; Retina; Development; Neuroprotection
1. Introduction
Blindness and visual impairment caused by retinal degeneration are found in 15 million people worldwide.
Much research is directed at understanding the course and mode of the degeneration and describing how the eye attempts to compensate, often inappropriately, during the degenerative process. Techniques are being developed for retinal cell rescue (aiding regeneration of the retina or optic nerve) and/or neuroprotection (protecting the eye from disease progression) and some are at clinical trial stage. Both understanding degeneration and devising rescue strategies require good animal models, of which there are several, but some diseases still lack animal equivalents (Chader, 2002). Even if some success is attained in combating some retinal degeneration disorders using mitogens, the same strategies are unlikely to cure all types of retinal degeneration. In addition, some retinal defects, such as glaucoma (high pressure within the eye that leads to optic disc damage and vision defects) and optic nerve hypoplasia (thinner optic nerve), may not benefit from such therapies. In these diseases, the primary defects are not a result of
*
Tel.: +44-7679-2928; fax: +44-20-7679-7349.
E-mail address: claire.russell@ucl.ac.uk
(C. Russell).
retinal degeneration, and therefore cannot be treated with mitogenic agents. It is therefore crucial that our understanding of retinal development is deepened, both so that new potential therapies might be tested and to provide new candidate genes that underlie eye defects.
In this review I shall focus on two classes of signalling molecules that have roles in retinal development, the
Fibroblast growth factor (Fgf) and Hedgehog (Hh) families. Their roles in proliferation, differentiation and axon growth and guidance suggest that they could have therapeutic value. In particular, I shall review the roles of Fgf8 and Shh in eye development. Fgf8 has not yet been assessed for its ability to rescue degenerating retina, but Shh failed to rescue a mouse retinal degeneration phenotype in vitro (Streichert, Birnbach, & Reh,
1999). Several recent studies have suggested that the signalling pathways downstream of these molecules can interact. In this light, and in the light of our own unpublished data, we suggest that both when studying development and considering therapies, we should not consider a single molecule or signalling pathway, but how two or more agents can interact.
Classical vertebrate model organisms have yielded a wealth of information. The zebrafish, Danio rerio , has more recently become established as a very useful model genetic organism. It is genetically tractable, the embryos are clear and amenable to embryonic manipulations,
0042-6989/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0042-6989(02)00416-9

900 C. Russell / Vision Research 43 (2003) 899–912
Fig. 1. Examples of zebrafish mutants that may be equivalent to human disease states. Coronal sections of 3-day-old embryos. In wildtype eyes (A) the three major laminae are present (ganglion cell layer (gcl), inner nuclear layer (inl) and photoreceptor layer (pcl)), as are the plexiform layers that separate the laminae and mainly contain neuronal projections (inner plexiform layer (inl) and outer plexiform layer (opl)). The pigmented epithelium
(pe) closely apposes the photoreceptor cell layer. In nagie oko (B), and oko meduzy retina (C), the layering is disorganised. Modified and reprinted from Malicki (2000), copyright (2000), with permission from Elsevier Science.
and transgenics can be made. The recent development of morpholino knockdown technology (Nasevicius & Ekker, 2000) has broadened the versatility of the zebrafish.
The morpholino is a modified antisense oligonucleotide that is usually specific for the 5 into the yolk of zebrafish embryos at the 1–4 cell stage, the morpholino prevents translation of the gene it is targeted to. As long as the zebrafish gene sequence is known (the Zebrafish Genome Project should be nearing completion by the end of 2002), gene function can be abrogated. In addition, several morpholinos can be used in concert, or in a mutant background, to assess how different genes act together (e.g., Bingham, Nasevicius,
Ekker, & Chandrasekhar, 2001). The zebrafish has therefore become a powerful genetic and developmental tool, and is amenable to the analysis of how signalling pathways can interact.
The eye of the zebrafish contains all the classes of cells seen in higher vertebrates (Fig. 1A) and specific markers are available (Malicki, 2000). As in other vertebrates, the production of the correct numbers of each cell type in the correct position within the eye is dependent on cell–cell signalling (Livesey & Cepko, 2001). Genetic screens for developmental, especially axon guidance, and degeneration phenotypes (Fig. 1B and C), have already been performed (Malicki, Pujic, Thisse, Thisse, &
Wei, 2002). Studies have also focussed on the regenerative capabilities of the zebrafish eye and optic nerve
(reviewed in Raymond & Hitchcock, 2000; Stenkamp &
Cameron, 2002), and the eye Õ s ability to continue growing throughout its lifespan (Marcus, Delaney, &
Easter, 1999).
2. Hedgehog function
0 of a gene. When injected
2.1. The Hedgehog signalling pathway
The Hedgehog (Hh) signalling pathway is involved in a huge variety of developmental processes in many organs including several examples of cell proliferation, cell survival, neurogenesis, patterning, induction and morphogenesis. These have been elegantly summarised in a recent review (Ingham & McMahon, 2001). Inappropriate regulation of Hh signalling is also the underlying factor in numerous tumours such as glioma, basal cell carcinoma and medulloblastoma (reviewed in Ming,
Roessler, & Muenke, 1998).
Hedgehog signalling proteins undergo post-translational processing to generate a potent secreted protein that can act at short or long range. Binding to the
Patched (Ptc) receptor on the receiving cell relieves the putative G-protein coupled receptor Smoothened (Smu) of inhibition. Smoothened then acts via an intracellular pathway in which the Gli proteins (or Ci in Drosophila ) act to either activate or repress target gene expression
(reviewed in Ingham & McMahon, 2001; Koebernick &
Pieler, 2002).
2.2. Hedgehog mutations and eye patterning
Mutations in the Hh signalling pathway can give rise to eye defects.
shh mutations in humans (Wallis &
Muenke, 2000) and mice (Chiang et al., 1996) result in holoprosencephaly (HPE). HPE is caused by a defect in development of the midline of the embryonic forebrain causing incomplete separation of the cerebral hemispheres and several craniofacial abnormalities including cyclopia (fused eyes) and ocular hypotelorism (eyes are closer together). Reduced Hh signalling from the ventral forebrain is likely to be the cause of this cyclopia. It has been shown in zebrafish that cells of the presumptive diencephalon, which also express Shh, must migrate forward to separate the single primordial eye field, and if this fails to occur, as in cyclops mutants, then cyclopia also occurs (Varga, Wegner, & Westerfield, 1999). It is not yet known whether a similar defect to that in zebrafish cyclops mutants underlies the most severe forms of human HPE. Mutations in Gli3 can cause hypertelorism (eyes further apart), as in Greig cephalopolysyndactyly (Wild et al., 1998).

C. Russell / Vision Research 43 (2003) 899–912
Loss of Shh has also been suggested as the cause of the dorsal–ventral patterning defects seen in the eyes of
BF-1 deficient mice. In these mice, the ventral optic stalk is missing and the retina is expanded (Huh, Hatini,
Marcus, Li, & Lai, 1999) suggesting a role for Shh in inducing ventral tissue in the optic cup. This is supported by overexpression of either shh or twhh in zebrafish, which ventralises the optic cup, giving an expanded pax2.1
domain at the expense of the pax6 domain, and subsequently an expanded optic stalk and reduced retina (Ekker et al., 1995; Macdonald et al.,
1995). Experiments to either block or overexpress Shh in the chick substantiate this data. It has been suggested that ventrally derived Shh and dorsally derived BMP4 may act antagonistically to maintain dorsal and ventral compartments of the optic primordium (Zhang & Yang,
2001a,b).
The zebrafish shh mutant, syu , however, does not display cyclopia (Schauerte et al., 1998), probably due to the presence of a second hh gene, twhh , which is similarly expressed. However, when both shh and twhh are reduced (Nasevicius & Ekker, 2000) there is late onset partial cyclopia, with the intraocular distance reducing with time. A similar phenotype is seen when the recently identified smoothened gene is mutated (Chen, Burgess, &
Hopkins, 2001; Varga et al., 2001).
smoothened ( smu ) embryos may therefore prove to be good models of human HPE.
2.3. Hedgehogs and retinal proliferation and differentiation
In Drosophila , the photoreceptors differentiate in a wave that moves from posterior to anterior, beginning adjacent to the stalk of the eye imaginal disc. Expression of the proneural gene atonal moves anterior to the wave of differentiation and is required for neurogenesis in the Drosophila eye.
hh is expressed slightly posterior to atonal and diffuses ahead of atonal expression to promote atonal expression and therefore the forward movement of the wave of differentiation (Fig. 2A, and reviewed in Jarman, 2000; and Treisman & Heberlein,
1998). A similar mechanism has been found in zebrafish where shh is required to promote the wave front of retinal ganglion cell (RGC) differentiation and induce its own expression (Neumann & N usslein-Volhard, 2000).
Both shh and atonal (also known as ath5 , Masai,
Stemple, Okamoto, & Wilson, 2000) are first expressed in differentiating RGCs close to the optic stalk, and subsequently this expression spreads (Fig. 2B).
syu ( shh ) mutants have a delayed wave of differentiation, but a stronger effect can be obtained by treatment with cyclopamine, which blocks all Hh signalling and freezes the wave of differentiation. This indicates that twhh , which is also expressed in the RGCs, may also be involved in this process. Perturbations in the wave of
A.
Drosophila
A
B. Zebrafish
N
OS
OS
P
T cyclopamine
?
901
R8 photoreceptor or retinal ganglion cell hedgehog signalling direction of wave atonal expression
?
atonal expression not known
A anterior
P posterior
N nasal
T temporal
OS optic stalk
Fig. 2. Hhs and progression of the neurogenic wave in the eye. In
Drosophila (A), a wave of atonal expression precedes hh expression, and neurogenesis begins after atonal expression. Hh signalling is perceived in front of the wave and promotes atonal expression, causing it to spread into the undifferentiated epithelium. In the zebrafish (B), a similar wave propagated by shh exists. Unlike in Drosophila , expression of atonal is not down-regulated once neurogenesis begins. The wave of neurogenesis is blocked by cyclopamine, but differentiation still occurs in cells that have already been exposed to shh.
Possible disruption of atonal by cyclopamine has not yet been examined so atonal expression is not shown. Modified from Jarman (2000) and
Treisman and Heberlein (1998).
differentiation have so far only been demonstrated with a marker of differentiated RGCs. A concrete link between ath5 and differentiation of RGCs in the zebrafish retina is yet to be demonstrated (Fig. 2B). Conversely, ectopic shh can induce ectopic waves of shh expression, showing that shh is sufficient for induction of its own expression (Neumann & N usslein-Volhard, 2000). Similar mechanisms seem to exist in the chick, except that a further role of Shh to negatively regulate ganglion cell genesis behind the wave front was also uncovered
(Zhang & Yang, 2001a,b).
Hh signalling from the prechordal axial tissue may also be involved in inducing the initial expression of atonal and, by extrapolation, shh in the RGCs of the zebrafish eye (Masai et al., 2000).
atonal expression is absent, lamination is disrupted and neural differentiation is reduced in one-eyed pinhead ( oep ) eyes.
pax2.1
, which is normally expressed in the optic stalk, is also absent in oep , as is shh which is normally expressed in

902 the prechordal plate. When oep mutant embryos are injected with shh mRNA , pax2.1
expression is partially rescued so that it is expressed in a cluster of midline cells. Subsequently atonal is expressed in adjacent cells.
Similarly in wildtype embryos, injection of shh expands the pax2.1
-positive optic stalk, and atonal expression is initiated adjacent to the most distal pax2.1
expressing cells. Retinal differentiation in the pax2.1
mutant, noi , is normal (Macdonald et al., 1997) so pax2.1
itself is not required to induce atonal expression. Shh, however, can regulate the position of the optic stalk, which produces an unknown signal that is involved in inducing atonal expression. Therefore Shh indirectly regulates where
RGC differentiation begins.
Shh acts as a mitogen in the developing mammalian retina (Jensen & Wallace, 1997; Levine, Roelink, Turner,
& Reh, 1997) and brain (Dahmane et al., 2001). During retinal layer development in the mouse, Shh is expressed in the ganglion cell layer and inner nuclear layer, and Ihh is expressed in the inner nuclear layer, outer nuclear layer and pigmented epithelium. Perinatal mouse retinal cell cultures treated with Shh produce an increase in total cell numbers by proliferation, including rod photoreceptors, amacrine cells and M uller glial cells (Jensen & Wallace,
1997). In the zebrafish, shh and twhh are expressed in the retinal pigmented epithelium (RPE) in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina, and knockdown of shh and twhh expression slows or arrests rod and cone photoreceptor differentiation (Stenkamp, Frey, Prabhudesai, & Raymond, 2000). Therefore Hhs seems to be required for proliferation, generation and differentiation of many, if not all, cell classes in the eye.
Given the increasing volume of evidence that Hh signalling is involved in proliferation and differentiation of many cell types in the retina, it would seem that Hhs or their downstream intermediates would be good molecules to test for neuroprotective or rescue abilities in the retina.
2.4. Hedgehog functions in axon guidance
More recently, roles for Hh signalling in axon guidance have been reported. Mutagenesis screens in zebrafish have uncovered many mutations affecting the
C. Russell / Vision Research 43 (2003) 899–912 retinotectal projection (reviewed in Hutson & Chien,
2002). Amongst these mutants are the shh mutant ( sonic you or syu ), the smoothened mutant ( smu ), the gli2 mutant ( you-too or yot ), and detour ( dtr ), iguana ( igu ), and chameleon ( con ), which have all been classified as most probably having defects in Hh signalling due to similarity in phenotype, but the mutations have not yet been identified. Studies of our own show that syu , smu and con all have defects in RGC axon guidance within the eye
(Russell & Wilson, unpublished). In these mutants, most
RGC axons fasciculate at the optic nerve head but subsequently split into several fascicles, some of which take aberrant routes and fail to exit the eye (Fig. 3). This may be similar to human optic nerve hypoplasia, which is also often found in association with craniofacial defects, in both De Morsier Õ s and Kallman syndromes (Layman,
1999; Sener, 1996). On the other hand, con , dtr , igu , smu , syu and yot all have defects in midline crossing of the optic nerve, and subsequent defects in their retinotectal projection. The underlying mechanism by which Hh signalling is acting in retinotectal pathfinding is yet to be elucidated but some evidence from other organisms suggests several mechanisms.
Shh protein is found in RGC axons in vertebrates and
Drosophila . Experiments in Drosophila have shown that
Hh is transmitted along the retinal axons where it triggers neurogenesis in the brain, whilst also directing the assembly of the optic lamina to which they target (Huang & Kunes, 1996, 1998). In the rodent, RGC axons stimulate proliferation of astrocytes in the optic nerve.
This, and the level of Ptc expression (a readout of Hh signalling) can be reduced by treatment with anti-Shh antibodies (Wallace & Raff, 1999). Therefore Shh is probably the agent, or one of the agents, that causes astrocyte proliferation in the optic nerve. It is possible that a lack/reduction of astrocytes could contribute to the phenotypes caused by Hh pathway mutations. Significantly, in vitro experiments using Shh-N do not stimulate astrocyte proliferation, suggesting that the efficacy of Hh activity may be blocked by components of the culture medium or by a lack of cell contact. Another possibility is that Indian hedgehog (Ihh) rather than Shh stimulates astrocyte proliferation and can be blocked with the anti-Shh antibody. As Ihh is not expressed in the RGCs but in the optic stalk, both Ihh and Shh may
Fig. 3. The optic nerves in Hh pathway mutants display aberrant pathfinding. Ventral views of (A) wildtype, (B) syu , (C) con , and (D) smu embryos stained with anti-acetylated tubulin antibody to visualise the optic nerve. The mutant embryos all show, to a varying degree, split optic nerves
(arrowheads) and some defasciculation (asterisk).

C. Russell / Vision Research 43 (2003) 899–912 be required for stimulation, especially as Shh and Ihh have both redundant and unique properties (Ramalho-
Santos, Melton, & McMahon, 2000). In adult hamsters,
Shh has also been shown to be anterogradely transported in vivo from the retina, along the optic nerve, to the superior colliculus, possibly travelling in association with cholesterol-rich raft-like microdomains (Traiffort,
Moya, Faure, H assig, & Ruat, 2001).
Shh expressed at the optic chiasm border may have a role in guiding the optic nerve across the midline
(Trousse, Marti, Gruss, Torres, & Bovolenta, 2001). N-
Shh suppresses growth of RGC axons from chick retinal explants in vitro and can induce growth cone arrest. In established neurites in culture, addition of N-Shh reduces levels of cAMP, whereas forskolin, an activator of adenylate cyclase and an Hh antagonist, causes an increase in cAMP in the growth cone, and can negate the effects of N-Shh. This suggests that Shh has a repressive effect on RGC axon growth by regulating levels of cAMP, thereby constraining the pathway taken by the
RGC axons. In vivo retroviral-mediated ectopic shh along the chick visual pathway prevents axons from reaching the optic chiasm (high degree of viral infection), and misdirects a proportion of axons into the ipsilateral optic tract (Fig. 4). Staining with Pax2 and
Pax6 antibodies indicate no changes in eye patterning in the infected embryos, and the Tuj1 antibody shows no defects in retinal differentiation. In fact, axons expressing Tuj1 avoid the areas of high shh expression. This suggests that the phenotype is due to the action of Shh specifically on the extending retinal ganglion cell axons
(Trousse et al., 2001).
Shh is expressed in both Tuj1positive neurons and radial glia in the ventral hypothalamus before RGC axons reach the optic chiasm.
These are thought to be Ô guidepost cells Õ for the RGC axons (Mason & Sretevan, 1997). As the axons approach the hypothalamus, shh expression is downregulated at the position of the Tuj1-positive neurons, although the exact cells in which this occurs have not been demonstrated. Perhaps this downregulation allows axons to continue growing through the optic chiasm, whereas shh expression in the radial glia may constrain the pathway taken by the RGC axons. In accordance, the zebrafish pax2.1
mutant, noi , displays axon pathfinding defects first in the postoptic commisure (POC) and subsequently in the adjacent optic chiasm (Macdonald et al., 1997). Both the POC and the optic chiasm are less tightly fasciculated, with some axons extending rostrally instead of contralaterally. Some retinal ganglion cell axons also extend into the ipsilateral optic tract. This phenotype could now possibly be attributed to the expansion of shh expression in noi embryos, thereby repelling the RGC axons (Fig. 4). Conversely, the lack of post-optic and anterior commissures, and the optic chiasm, in zebrafish smu embryos (Chen et al.,
2001; Varga et al., 2001) suggests that reducing Hh
(A) Chick
(B)
Zebrafish vh poa control wildtype retinal ganglion cell axons eyes shh expression vh ventral hypothalamus poa pre-optic area noi
Fig. 4. Ectopic shh repels axons at the optic chiasm. Ectopic shh at the optic chiasm in retrovirus-infected chick embryos (A) or noi mutant zebrafish (B) causes retinal ganglion cell axons to avoid the optic chiasm. Axons either stall, or take the ipsilateral, rather than the contralateral route to the optic tectum. Modified from Trousse et al.
(2001) and Macdonald et al. (1997).
signalling may also disrupt RGC axon guidance. In smu mutants, it is not yet known if Hh expressed in the hypothalamus is required to attract RGC axons out of the eye towards the optic recess, or whether Hh expression within the RGCs themselves is important for RGC axon growth and pathfinding.
It is interesting to find that Shh is expressed both in
RGC axons, and at specific positions along their trajectory, and it will be exciting to find out how reciprocal signals (to and from the optic nerve) can be integrated.
Because of their variety and complexity of phenotypes, the zebrafish mutants may be extremely useful in further understanding the roles of Hh signalling in optic nerve pathfinding within the eye, across the midline and to the tectum.
To summarise, Hh signalling is important for retinal cell proliferation, retinal neurogenesis, and differentiation of various cell types in the eye. It is also important for optic nerve growth, guidance and targeting, and proliferation of astrocytes in the optic nerve.
3. Fgf function
3.1. Fgf signalling ectopic shh
903
The Fgf family of neurotrophic signalling proteins is made up of at least 23 ligands, some having several

904 C. Russell / Vision Research 43 (2003) 899–912 isoforms. There are four high affinity receptors, which undergo alternative splicing, and various low affinity heparan sulphate proteoglycans, which also bind the
Fgfs. Fgfs can certainly act over a distance of a few cell diameters within a tissue, but the full distance that Fgfs can act at is not yet known. Expression levels of the ETS domain proteins Erm and Pea3, however, are believed to give a readout of the levels of Fgf signalling, and closer examination of their distribution and regulation may answer this question (Raible & Brand, 2001). Expression studies demonstrate that at least 10 fgfs are expressed in the developing mammalian CNS, including fgf1 , fgf2 and fgf15 , which are expressed generally, and fgf8 and fgf17 , which are more tightly localised (Ford-Perriss,
Abud, & Murphy, 2001; Hicks, 1998; Tanihara, Inatani,
& Honda, 1997). Fgfs are involved in many aspects of development including gastrulation, neural induction and terminal differentiation, and each member of the family has its own specific roles in different tissues, regulated both by their receptor specificity and expression profiles (reviewed in Goldfarb, 1996; Hicks, 1998).
Fgfs are also upregulated in many tumours and are associated with craniofacial abnormalities (e.g., Fgf8,
Meyers, Lewandoski, & Martin, 1998). They have also been implicated in vasculogenesis and axon growth.
Fgfs have been cloned for many reasons and their expression patterns studied in a variety of tissues, but they have not all been examined closely for expression during eye development or in the adult retina. It has been reported, however, that fgf2 , 3 , 5 , 11 , 12 , 13 , and 15 are all expressed in the retina of various vertebrates (see within Ford-Perriss et al., 2001), and fgf1 and fgf2 in the murine lens (see within Govindarajan & Overbeek,
2001).
fgfr1 and 2 are expressed in the chick retina
(Tcheng, Fuhrmann, Hartmann, Courtois, & Jeanny,
1994), and fgfr1 , 3 and 4 in the Xenopus retina (Launay,
Fromentoux, Thery, Shi, & Boucaut, 1994).
fgf7 and fgf10 are expressed in the murine periocular mesenchymal cells (see within Govindarajan & Overbeek, 2001).
Due to the complexity of the expression patterns of the many Fgfs, I shall only describe the relevant expression patterns of those studied most recently and extensively for their roles in the eye.
Several fgfs and their receptors have now been cloned from the zebrafish. So far, three zebrafish fgfs are known to be expressed in the optic cup and/or eye. These are fgf3 , fgf8 and fgf17 .
fgf3 is most similar to Xenopus fgf3 but is also structurally analogous to mouse fgf3 (Keifer,
Str ahle, & Dickson, 1996). Zebrafish fgf8 is highly related to chick fgf8 , and slightly less so to the mouse and human homologues (Reifers et al., 1998). Zebrafish fgf17 , although more similar to zebrafish fgf8 than mouse fgf17 , is orthologous to mouse fgf17 because it maps to a region of synteny between mouse and zebrafish (Reifers, Adams, Mason, Schulte-Merker, & Brand,
2000). In fact zebrafish fgf17 is also very similar to mouse fgf18 , suggesting that fgf8 , fgf17 and fgf18 form their own subgroup. Zebrafish fgf3 , 8 and 17 are all expressed in the optic stalk from early stages (Reifers et al., 2000; Tsang, Friesel, Kudoh, & Dawid, 2002).
fgf8 is subsequently expressed in the choroid fissure and in the retina (Shanmugalingam et al., 2000) but the fgf3 and fgf17 expression patterns have not yet been fully described. Examination of fgf17 in the fgf8 mutant
(called acerebellar or ace ), and after fgf8 overexpression using implanted Fgf8-soaked beads, suggests that fgf17 is expressed downstream of fgf8 (Reifers et al., 2000).
Experiments are underway using morpholinos against fgf17 and fgf3 to ascertain their roles in eye development. Initial results suggest a role in cell proliferation and/or survival, as well as in choroid fissure development (Russell & Wilson, unpublished results). Zebrafish fgfr3 and fgfr4 are expressed in complementary domains in the eye, probably in the photoreceptor and RPE layers respectively, and around the lens (Sleptsova-
Friedrich et al., 2001). As more zebrafish fgfs and their receptors are cloned and Ô knocked down Õ , both singly and in combination, we should learn a great deal about the roles of Fgfs in the eye.
3.2. Fgfs in eye patterning and cell survival
Several Fgfs have been implicated in eye patterning, specifically in the distinction between RPE and neural retina. Chick Fgf2 (also known as bFgf) is expressed highly in the surface ectoderm overlying the optic vesicle and at lower levels in the presumptive neural retina and
RPE (Pittack, Grunwald, & Reh, 1997). If chick optic vesicles are cultured in the presence of Fgf1 (also known as aFgf) and Fgf2, the RPE does not differentiate but a double retina is formed instead. In contrast, if chick optic vesicles are cultured in FGF2 neutralising antibodies, neural differentiation is blocked but the RPE is normal. This suggests that Fgfs are required for neural retina differentiation (Pittack et al., 1997).
If the surface ectoderm of the optic vesicle, a rich source of Fgfs, is surgically removed in the chick, the optic vesicle later contains a mix of mingled neural and pigmented cells. Addition of Fgfs after surface ectoderm removal either by FGF-secreting fibroblasts or replication-incompetent retroviral expression vectors, results in segregated neural and pigmented epithelial domains, with the neural domain being located near the Fgf source (Hyer, Mima, & Mikawa, 1998). This indicates that Fgfs provide positional cues that organise the optic vesicle into neural retina and pigmented epithelium.
This can also be demonstrated by the expression of specific transcription factors such as Mitf and CHX10 .
Mitf is initially expressed throughout the undifferentiated mouse optic vesicle, and then becomes restricted to the presumptive RPE (Nguyen & Arnheiter, 2000). Fgf coated beads implanted near the presumptive RPE in

C. Russell / Vision Research 43 (2003) 899–912 cultured mouse optic vesicles results in downregulation of Mitf and development of neural retina instead of
RPE. Conversely, Mitf expression is retained in embryos where the surface ectoderm is removed, CHX10is downregulated and the epithelium becomes a pigmented monolayer with little retina. Application of Fgf inhibits this induction of RPE, suggesting that Fgf signals from the surface ectoderm to regulate gene expression and domain specification within the optic vesicle (Nguyen &
Arnheiter, 2000).
In the mouse, fgf9 is expressed in the distal region of the optic vesicle, which becomes the neural retina. If it is ectopically expressed in the proximal optic vesicle of transgenic mice, then neural differentiation also occurs in the presumptive RPE. This results in a duplicate mirror-image retina, where the polarity of the layers in the duplicate retina is reversed compared to the polarity of the layers of the original neural retina (Zhao et al.,
2001). In mouse embryos lacking fgf9 , the boundary between the RPE and neural retina is shifted so that cells of the RPE are found in the outer neural retina. This suggests that Fgf9 has a role in defining the boundary between neural retina and RPE in the optic vesicle, and that the RPE is not required for retinal growth and differentiation during embryonic stages. In addition, lens fiber cells were underdeveloped in the mouse fgf9 knockout, suggesting that Fgf9 may stimulate their differentiation (Zhao et al., 2001).
Similar results have been found using Fgf8-soaked beads applied to the temporal portion of chick eyes. The
RPE differentiates as neural retina, giving a mirror image to the endogenous neural retina. Fgf8 application also affects lens development, depending on the time of bead implantation. Generally lens differentiation occurs earlier than usual, and sometimes in ectopic positions
(Crossley, Martinez, Ohkubo, & Rubenstein, 2001;
Vogel-H opker et al., 2000). Unlike in fgf9 mutant mice, zebrafish mutant for fgf8 (called ace ) have normal eyes with no evidence of neural retina differentiating as RPE despite expression of fgf8 within the retina (Shanmugalingam et al., 2000), and the mouse fgf8 mutants were not examined closely for eye phenotypes (Meyers et al.,
1998) although fgf8 is expressed in the optic stalk and optic recess (Crossley & Martin, 1995). The similar results recovered from Fgf8 and Fgf9 overexpression suggest that they can activate the same developmental pathways when ectopically expressed, but the difference in their mutant phenotypes and expression patterns means that they probably have different endogenous roles.
Recently, morpholinos directed to splice sites within the zebrafish fgf8 pre-mRNA, have given stronger phenotypes than the ace mutation, or a morpholino directed against the fgf8 promoter (Araki & Brand, 2001). This could be due to increased gene knockdown, suggesting that the ace mutant is possibly not a null, and the pro-
905 moter-directed morpholino has only a weak phenotype.
In support of this, the splice-site directed morpholinos, which affect RNA levels, cause lower levels of fgf8 RNA in morpholino-injected embryos than those seen in ace embryos. Alternatively, the splice-blocking morpholinos may have additional non-specific effects. This seems a less likely explanation as two different splice-blocking morpholinos give the same phenotype. In splice-blocking morpholino embryos, apart from somite and tail defects, extensive necrosis is seen, which may also occur in the eye (Draper, Morcos, & Kimmel, 2001). This phenotype must be examined more carefully to determine if Fgf8 has a role in retinal cell survival.
Another zebrafish mutant, ( aussicht aus ), overexpresses fgf8 in the forebrain, optic stalk and retina
(Heisenberg, Brennan, & Wilson, 1999). Heterozygous aus embryos have a large optic stalk at early stages but a temporal outgrowth of retinal tissue later on, thought to be an outfolding of a normally layered retina. They also have delayed retinal differentiation and the optic nerve is defasciculated both within the eye and at the optic chiasm. In putative homozygous aus embryos, the ventral/ nasal part of the retina is reduced, the choroid fissure fails to close and the RPE is expanded out of the back of the eye (Heisenberg et al., 1999). The coloboma and optic nerve defasciculation phenotypes can probably be attributed to pax2.1
overexpression (Macdonald et al.,
1995), but several of the morphological phenotypes and the upregulation of pax2.1
were dependent on functional Fgf8. It is likely that overexpression of fgf8 causes the defects in eye patterning and it will be interesting to see what sort of protein the aus gene encodes, and its relationship to Fgf8.
3.3. Fgfs in proliferation and differentiation in the eye
Several techniques have been employed to block Fgf signalling in the developing eye. Application of a specific
FGFR inhibitor, SU5402, retards the wave of ganglion cell differentiation in the chick. Conversely, also in the chick, exogenous Fgf1 causes precocious development of RGCs in the peripheral retina, suggesting a role for
Fgfs in RGC differentiation, and progression of the wave of differentiation (McCabe, Gunther, & Reh,
1999). In contrast, in Xenopus , dominant negative XFD injected into dorsal animal blastomeres that give rise to retina causes loss of photoreceptor and amacrine cells and an increase in M uller glia cells, suggesting a role for
Fgfs in specifying cell fate (McFarlane, Zuber, & Holt,
1998). Expression of a dominant negative FGFR1 in the RPE of transgenic mice results in colobomas, impairment of eye growth and eye degeneration in homozygotes. In normal eyes the RPE cells extend apical finger-like cytoplasmic projections towards the photoreceptor outer segments (POS). In hemizygotes, the microvilli are present but do not come into tight contact

906 C. Russell / Vision Research 43 (2003) 899–912 with the POS, the choroid is thinner and the choroid vasculature is disrupted. Photoreceptors are then lost and retinal degeneration occurs. This is consistent with a role for Fgfs expressed in the RPE being required for photoreceptor survival, and suggests a role for Fgfs in choroidal angiogenesis (Rousseau et al., 2000). These apparently diverse effects caused by blocking FGFR function may be due to the species, inhibitor or method of delivery used. We do not know which of the Fgfs are responsible for these effects or if these methods really do block signalling through all FGFRs (as presumed) but the resulting phenotypes do demonstrate roles for Fgf signalling in RGC differentiation, retinal cell fate specification and photoreceptor survival.
Several experiments have been performed where Fgfs are overexpressed in the developing eye. Fgf2, which acts primarily to induce proliferation and survival in the
CNS (Hicks, 1998), has been studied most extensively.
Fgf2 is expressed in the embryonic and adult RPE (see within Hicks, 1998) and it is thought to be one of the signals from the RPE that stimulates retinal growth, organisation and differentiation in all layers but the
RPE. Overexpression of Fgf2 in Xenopus retinal precursors results in an increase in RGCs and a decrease in
M uller glia, and an increase of rod photoreceptors at the expense of cones (Patel & McFarlane, 2000). Similar experiments, where Fgf2 was added to embryonic rat retina explants containing uncommitted retinal precursors, accelerated the appearance of differentiated RGCs, whereas anti-Fgf2 antibodies delayed their appearance
(Zhao & Barnstable, 1996). These experiments show that Fgf2 can influence the timing of RGC differentiation, but not the numbers of RGCs produced from retinal precursors. In the same study, it was also found that although photoreceptor differentiation was not affected, rod photoreceptor rosette formation was inhibited, suggesting a role for Fgf2 in some properties of rods or adjacent M uller cells.
In addition to this effect, both Fgf1 and Fgf2 have been shown to have a proliferative effect on cultured rat retina (explants and monolayers) at E15–18 stages, which later declines (Lillien & Cepko, 1992). This indicates a change in the responsiveness to Fgf signals with time. Newborn rat retinal cells cultured as a monolayer with Fgf2 in the medium produce an increase in the number of photoreceptors although the total number of differentiated neurons and glia was not affected (Hicks &
Courtois, 1992). In this case, no increase in cell survival or proliferation was detected, suggesting that more uncommitted cells became photoreceptors. Equivalent cultures from postnatal day 3 rats showed a reduced stimulatory effect on photoreceptors, indicating that the effect of Fgf2 to promote differentiation depends on the state of the population of precursors in the retina. These overexpression studies indicate that Fgf2 has different effects depending on the stage at which it is applied. It can affect the proliferation of progenitors, the timing of
RGC differentiation, and shift the bias of differentiation.
fgf1 , fgf2 and fgf5 are all expressed in the embryonic chick RPE (Hicks, 1998) and both Fgf2 and Fgf1 can stimulate RPE to transdifferentiate into new neural retina in vivo (Park & Hollenberg, 1989, 1991) but fgf2 is expressed at higher levels in mature retina, suggesting a role in continued maintenance of retinal cells. A subsequent study showed that Fgf2 only stimulates transdifferentiation in sheets of chick embryonic RPE cultured in suspension, not in dissociated cultures that flatten and spread (Pittack, Jones, & Reh, 1991), suggesting that transdifferentiation depends upon a proper physical configuration of RPE cells. In vitro culture of chick embryonic presumptive RPE together with either
Fgf1 or Fgf2 affects all processes of transdifferentiation
(inhibition of RPE differentiation, increased proliferation and conversion to a retinal fate) but not equally
(Guillemot & Cepko, 1992). Fgf1 is more potent at inhibiting pigmentation and inducing retinal antigens than
Fgf2. Fgf1 also seems to have an independent effect on the differentiation of RGCs. Interestingly, conversion from an RPE cell to a neural retina cell does not require cell division (Guillemot & Cepko, 1992).
Because of all these properties, Fgf2 has been widely tested as a neuroprotective agent and it can slow down the progression of an inherited disorder (Faktorovich,
Steinberg, Yasumura, Matthes, & LaVail, 1990). It is possible, however, that growth factors work better in combination, as when applied to rd mouse photoreceptors in culture (Ogilvie, Speck, & Lett, 2000). In the case of glaucoma and retinal ischemia, FGF-2 is significantly protective by 1 week post-ischemia in the rat (Unoki &
LaVail, 1994; Zhang, Takahashi, Lam, & Tso, 1994).
However, the usefulness of Fgfs, and many other neurotrophic factors, as neuroprotective agents in cases of retinal ischemia is somewhat limited because they are usually only effective when applied before ischemia.
3.4. Fgfs and axon guidance
Fgf receptors are known to modulate growth cone decisions mediated via cell adhesion molecules both in vitro (reviewed in Doherty, Williams, & Williams,
2000) and in vivo, in both Drosophila (Garc ııa-Alonso,
Romani, & Jim eenez, 2000) and transgenic mice (Saffell,
Williams, Mason, Walsh, & Doherty, 1997). Fgfs have a role in retinal axon extension (Perron & Bixby, 1999) via activation of ERK, which integrates several signals and is required for neurite outgrowth. More specifically,
Fgf2 can promote axonal sprouting and elongation at the inner limiting membrane of the chick retina, and this requires heparan sulphate, which is thought to bind Fgf and present it to cell-surface receptors (Chai & Morris,
1999).

C. Russell / Vision Research 43 (2003) 899–912
Fgf signalling is also required for target recognition by RGC axons. In Xenopus , Fgf2 (or bFgf) is found along the pathway taken by the optic nerve but not in the optic tectum. Conversely, FGFRs are found in the
RGC axons. Fgf2 stimulates neurite outgrowth from retinal cells in vitro but disrupts RGC axon targeting to the optic tectum in vivo, in an exposed brain preparation to which Fgf2 is applied. Further experiments indicate that Fgf2 is acting on the growth cone directly.
This suggests that Fgf2 is present in the optic tract to allow growth of the axons, but absent from the optic tectum to allow the axons to slow their growth, letting them recognise and synapse with their target (McFarlane, McNeill, & Holt, 1995). Conversely, inhibition of
FGFR activity in Xenopus RGC axons using in vivo transfection of dominant negative FGFR, causes axons to grow more slowly along the optic tract and bypass the optic tectum. This suggests that Fgf signalling is required for a normal rate of extension of the RGC axons, and is critical for recognition of the target (McFarlane,
Cornel, Amaya, & Holt, 1996).
Exogenous heparan sulphate can also cause RGC axons to bypass their target in Xenopus . Using heparan
907 sulphate side chains that preferentially bind Fgf1 or
Fgf2, it was found that binding Fgf2 specifically caused aberrant targeting. Early heparitinase treatment removes endogenous heparan sulphates at the beginning of optic tract formation, and this results in axon growth inhibition. This inhibition can be rescued by addition of
Fgf2 but the axons still fail to find their target. A later heparitinase treatment causes only the bypass phenotype. This suggests that Fgf2 requires heparan sulphates for normal rates of axon growth, and that heparan sulphates, either alone or together with another signal, are required for correct targeting (Walz et al., 1997).
The only fgf mutant so far found in zebrafish is ace , which probably encodes a truncated Fgf8 (Reifers et al.,
1998). In ace embryos, retinal axons misproject at the optic chiasm both ipsilaterally and rostrally to the telencephalon, and forms ectopic projections (Fig. 5).
Transplants of ace eyes into wildtype embryos rescues the projection phenotypes, indicating that it is Fgf8 at the midline, and not in the retina, that is required for correct axon pathfinding at the optic chiasm (Shanmugalingam et al., 2000). The eyes themselves are well patterned although an autonomous requirement for
Fig. 5. The optic nerve in ace embryos exhibits non-autonomous defects at the optic chiasm. Coronal sections of 2-day-old wildtype (A) and ace (B) eyes stained with anti-FRET43 (arrows). The layering in ace eyes looks normal. Transplants of eyes (shown with *): wildtype eyes to wildtype hosts
(C), ace eyes to wildtype hosts (D), ace eyes to ace hosts (E), and wt eyes to ace hosts (F). The optic nerve from an ace eye projects normally when placed in a wildtype brain, and the optic nerve from a wildtype eye projects abnormally in an ace brain. Abnormally projecting retinal ganglion cell axons do not cross the optic chiasm and project into the ipsilateral, rather than contralateral optic tectum, producing ectopic projections (arrowheads) and occasional defasciculation. Adapted and reprinted from Shanmugalingam et al. (2000), copyright (2000) with permission from The
Company of Biologists.

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4. Fgf and Hh interactions
C. Russell / Vision Research 43 (2003) 899–912
Fgf8 within the eye was found for the projection of dorsonasal RGC axons (Picker et al., 1999). In light of this, perhaps the mouse fgf8 alleles should be re-examined for projection phenotypes in the eye (Meyers et al.,
1998).
Fgf signalling is therefore important for patterning of the RPE and retina, differentiation and survival of various cell types, RGC neurite growth, and axon guidance and targeting. Fgfs may also be involved in lens development (not discussed). The endogenous roles of Fgfs in the eye must be examined further, both during development and adulthood, for us to deepen our understanding of how they function, through which signalling pathways, and in combination with which other signals.
brachyury expression (Brewster, Mullor, & Altaba,
2000). This suggests that Gli-expressing cells could integrate both Fgf and Hh signals. However, much more work is needed to elucidate the molecular basis of the interactions between these pathways. We are currently examining the eyes of double mutant and morpholino injected zebrafish embryos that have reduced activity of combinations of various Fgfs, Hhs and Glis to address possible interactions (Russell & Wilson, unpublished).
As I have discussed, Fgfs and Hhs have also been assigned similar roles in dorsoventral axis specification of the eye, and in neurogenic wave progression and differentiation in the retina. These are important considerations for neuroprotective therapies, so it is important to consider if Fgfs and Hhs may act together or complement each other, and if so, could they together have beneficial effects on retinal degenerations or improve retinal regeneration. In addition, their similar roles in axon growth, guidance and targeting suggest that they may have potential as therapeutic agents against optic nerve hypoplasia, retinal ischemia and glaucoma.
Hhs and Fgfs are closely expressed in many tissues of the developing embryo, including the telencephalon, optic vesicles and retina (Crossley et al., 2001). It has also been shown, specifically in the frontonasal process, that local retinoid signalling maintains local shh and fgf8 expression, thereby coordinating forebrain and facial morphogenesis. When retinoid signalling is transiently disrupted, forebrain tissue is absent and the eyes are fused (Schneider, Hu, Rubenstein, Maden, & Helms,
2001). Retinoic acid also controls expression of shh and fgf8 in the limb bud (Helms, Kim, Eichele, & Thaller,
1996; Stratford, Logan, Zile, & Maden, 1999).
A few examples of Hh and Fgf interactions are known, although only one example in the eye. In the
Medaka eye and mid-hindbrain boundary, injected shh induces spalt gene expression in the proximal optic vesicle, and this requires Fgf signalling, because dominant negative XFD co-injections block spalt induction
(Carl & Wittbrodt, 1999). Therefore, it is thought that
Fgf may specify a competence domain and Hh specifies the dorsoventral extent of spalt expression.
Perhaps in a similar manner, co-expression of Fgf8 and Shh in the vertebrate mid-hindbrain boundary and rostral forebrain creates induction sites for dopaminergic neurons. When Fgf4 is also present, hindbrain 5HT neurons are induced (Ye, Shimamura, Rubenstein, Hynes, & Rosenthal, 1998).
On the other hand, in the vertebrate limb, Fgf8 is required for the induction and maintenance of shh expression, via protein kinase C, which then leads to the upregulation of fgf4 (Johnson & Tabin, 1997; Lu,
Swindell, Sierralta, Eichele, & Thaller, 2001).
A possible link between the Fgf and Hh pathways is the Gli protein family. Gli proteins are effectors of Hh signalling, but often also antagonise Hh signalling, such as in ventral neuron specification (Litingtung & Chiang,
2000). In response to Fgf signalling during mesodermal development, Gli2 is expressed. Gli2 then induces
5. Zebrafish as a model organism for studying eye development, regeneration and rescue
Much of the recent progress in understanding Fgf and
Hh functions has come from studies in zebrafish. This is a relatively new model organism that can be exploited in many ways to increase our understanding of eye development and provide a tool for retinal rescue research.
Some of these advantages are discussed.
The ability of the zebrafish eye to continue growing throughout the fish Õ s lifespan has been poorly studied due to the slow rate of retinal growth compared to other fish such as the goldfish or trout. In fact, goldfish and trout have been used extensively to study retinal regeneration after retinal injury or insult (reviewed in Raymond & Hitchcock, 2000). However, several studies have focussed on the regenerative capabilities of the zebrafish optic nerve after axotomy. In particular, several molecules are re-expressed or newly expressed in the optic nerve and glia during this time, including the cell recognition molecules l1.1
, l1.2
and n-cam (Bernhardt,
Tongiorgi, Anzini, & Schachner, 1996), zfNLRR (Bormann, Roth, Andel, Ackermann, & Reinhard, 1999), b thymosin , the product of which binds actin monomers and modulates the actin cytoskeleton, and gelsolin , which encodes an actin-severing protein (Roth, Bormann, Wiederkehr, & Reinhard, 1999). Such studies could indeed lead to the discovery of agents that might aid optic nerve regeneration after retinal reattachment by surgery. Currently only 20–37% of patients regain reasonable sight after surgery, which leaves much room for improvement.

C. Russell / Vision Research 43 (2003) 899–912
Apart from the zebrafish mutants and knockdowns that I have already mentioned, there are many more that were recovered in numerous mutagenesis screens (Doerre & Malicki, 2002; Vihitelic & Hyde, 2002; and earlier screens reviewed in Malicki, 2000). Mutant phenotypes can be classified into those that have loss of layers, aberrant laminar pattern, growth retardation, small eyes associated with retinal degeneration, retinal degeneration associated with pigment defects, mis-positioning of the lens, loss of the proliferative zone, enlarged optic stalk/ventral eye, aberrant optic nerve pathfinding, and optic nerve defasciculation. Behavioural screens have also been performed in which mutants with an aberrant optokinetic response, optomotor response or visually mediated escape response were recovered (reviewed in
Baier, 2000).
The availability of a variety of degeneration phenotypes suggests that neuroprotective strategies could be tested in zebrafish mutants. Possible equivalents of human clinical conditions can be found in zebrafish
(Malicki, 2000). For example, Senior–Loken and Bardet–Biedl syndromes are forms of retinitis pigmentosa accompanied by renal abnormalities, the same combinations of defects being found in elipsa and fleer mutant zebrafish (Doerre & Malicki, 2002). Also glass onion
(Pujic & Malicki, 2001), nagie oko (Fig. 1B) (Wei &
Malicki, 2002), and oko meduzy (Fig. 1C) (Malicki &
Driever, 1999) may be equivalent to Walker–Warburg syndrome, muscle–eye–brain disease, and cerebro-ocular–muscular dystrophy. Although, these seem like they could provide good models for human diseases, the specificities of the pattern of degeneration and subsequent attempts to regenerate must be examined and compared to the human disease equivalent, especially as these may vary due to the regenerative capacity of the zebrafish eye, and the possible presence of some factors in the zebrafish eye which are absent in humans.
One frequent drawback of the zebrafish is that the long-term progression of an eye phenotype cannot be monitored because of pleiotropic requirements for the mutated gene, resulting in lethality. In these cases transplant technology, either of clones of cells or of whole eyes, can be used to investigate the phenotypic effects solely in the eye and whether or not a mutation acts cell autonomously (e.g., Picker et al., 1999; Pujic &
Malicki, 2001, and Fig. 5).
Another current drawback is the duration of the effectiveness of morpholinos. These are degraded over time and may not last for as long as you wish to examine the phenotype for. In the future, this hurdle might be overcome by mixing the morpholino with caged morpholino (Ando, Furuta, Tsien, & Okamoto, 2001), which is protected from degradation and can be uncaged at a later stage, allowing longer periods of morpholino action after injection into embryos. It must also be remembered that morpholinos can block translation of maternally deposited mRNAs, but not maternally deposited proteins.
Acknowledgements
909
Work in our group on eye development is supported by grants from the Wellcome Trust, BBSRC and European Community to Stephen Wilson.
References
Ando, H., Furuta, T., Tsien, R. Y., & Okamoto, H. (2001). Photomediated gene activation using caged RNA/DNA in zebrafish embryos.
Nature Genetics, 28 , 317–325.
Araki, I., & Brand, M. (2001). Morpholino induced knockdown of fgf8 efficiently phenocopies the acerebellar ( ace ) phenotype.
Genesis, 30 ,
157–159.
Baier, H. (2000). Zebrafish on the move: towards a behaviour-genetic analysis of vertebrate vision.
Current Opinion in Neurobiology, 10 ,
451–455.
Bernhardt, R. R., Tongiorgi, E., Anzini, P., & Schachner, M. (1996).
Increased expression of specific recognition molecules by retinal ganglion cells and by optic pathway glia accompanies the successful regeneration of retinal axons in adult zebrafish.
Journal Comparative Neurology, 376 , 253–264.
Bingham, S., Nasevicius, A., Ekker, S. C., & Chandrasekhar, A.
(2001).
Sonic hedgehog and tiggy-winkle hedgehog cooperatively induce zebrafish branchiomotor neurons.
Genesis, 30 , 170–174.
Bormann, P., Roth, L. W. A., Andel, D., Ackermann, M., &
Reinhard, E. (1999). ZfNLRR, a novel leucine-rich repeat protein is preferentially expressed during regeneration in zebrafish.
Molecular and Cellular Neuroscience, 13 , 167–179.
Brewster, R., Mullor, J. L., & Altaba, A. (2000). Gli2 functions in
FGF signalling during antero-posterior patterning.
Development,
127 , 4395–4405.
Carl, M., & Wittbrodt, J. (1999). Graded interference with FGF signalling reveals its dorsoventral asymmetry at the mid-hindbrain boundary.
Development, 126 , 5659–5667.
Chader, G. J. (2002). Animal models in research on retinal degenerations: past progress and future hope.
Vision Research, 42 , 393–
399.
Chai, L., & Morris, J. E. (1999). Heparan sulphate in the inner limiting membrane of embryonic chicken retina binds basic fibroblast growth factor to promote axonal outgrowth.
Experimental Neurology, 160 , 175–185.
Chen, W., Burgess, S., & Hopkins, N. (2001). Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity.
Development, 128 , 2385–2396.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L.,
Westphal, H., & Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking sonic hedgehog gene function.
Nature,
383 , 407–413.
Crossley, P. H., & Martin, G. R. (1995). The mouse fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo.
Development,
121 , 439–451.
Crossley, P. H., Martinez, S., Ohkubo, Y., & Rubenstein, J. L. R.
(2001). Coordinate expression of fgf8 , otx2 , bmp4 and shh in the rostral prosencephalon during development of the telencephalic and optic vesicles.
Neuroscience, 108 , 183–206.
Dahmane, N., Sanchez, P., Gitton, Y., Palma, V., Sun, T., Beyna, M.,
Weiner, H., & Ruiz i Altaba, A. (2001). The Sonic Hedgehog-Gli

910 pathway regulates dorsal brain growth and tumorigenesis.
Development, 128 , 5201–5212.
Doerre, G., & Malicki, J. (2002). Genetic analysis of photoreceptor cell development in the zebrafish retina.
Mechanisms of Development,
110 , 125–138.
Doherty, P., Williams, G., & Williams, E.-J. (2000). CAMs and axonal growth: a critical evaluation of the role of calcium and the MAPK cascade.
Molecular and Cellular Neuroscience, 16 , 283–295.
Draper, B. W., Morcos, P. A., & Kimmel, C. B. (2001). Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown.
Genesis, 30 , 154–156.
Ekker, S. C., Ungar, A. R., Greenstein, P., von Kessler, D. P., Porter,
J. A., Moon, R. T., & Beachy, P. A. (1995). Patterning activities of vertebrate hedgehog proteins in the developing eye and brain.
Current Biology, 5 , 944–955.
Faktorovich, E. G., Steinberg, R. H., Yasumura, D., Matthes, M. T.,
& LaVail, M. M. (1990). Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor.
Nature,
347 , 83–86.
Ford-Perriss, M., Abud, H., & Murphy, M. (2001). Fibroblast growth factors in the developing central nervous system.
Clinical and
Experimental Pharmacology and Physiology, 28 , 493–503.
Garc ııa-Alonso, L., Romani, S., & Jim eenez, F. (2000). The EGF and
FGF receptors mediate Neuroglian function to control growth cone decisions during sensory axon guidance in Drosophila .
Neuron, 28 , 742–752.
Goldfarb, M. (1996). Functions of Fibroblast Growth Factors in vertebrate development.
Cytokine and Growth factor Reviews, 7 ,
311–325.
Govindarajan, V., & Overbeek, P. A. (2001). Secreted FGFR3, but not
FGFR1, inhibits lens fiber differentiation.
Development, 128 , 1617–
1627.
Guillemot, F., & Cepko, C. L. (1992). Retinal fate and ganglion cell differentiation are potentiated by acidic FGF in an in vitro assay of early retinal development.
Development, 114 , 743–754.
Heisenberg, C.-P., Brennan, C., & Wilson, S. W. (1999). Zebrafish aussicht mutant embryos exhibit widespread overexpression of ace
( fgf8 ) and coincident defects in CNS development.
Development,
126 , 2129–2140.
Helms, J. A., Kim, C. H., Eichele, G., & Thaller, C. (1996). Retinoic acid signalling is required during early chick limb development.
Development, 122 , 1385–1394.
Hicks, D. (1998). Putative functions of fibroblast growth factors in retinal development, maturation and survival.
Seminars in Cell and
Developmental Biology, 9 , 263–269.
Hicks, D., & Courtois, Y. (1992). Fibroblast growth factor stimulates photoreceptor differentiation in vitro.
Journal of Neuroscience, 12 ,
2022–2033.
Hyer, J., Mima, T., & Mikawa, T. (1998). Fgf1 patterns the optic vesicle by directing the placement of the neural retina domain.
Development, 125 , 869–877.
Huang, Z., & Kunes, S. (1996). Hedgehog, transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the
Drosophila brain.
Cell, 86 , 411–422.
Huang, Z., & Kunes, S. (1998). Signals transmitted along retinal axons in Drosophila : hedgehog signal reception and the cell circuitry of lamina cartridge assembly.
Development, 125 , 3753–3764.
Huh, S., Hatini, V., Marcus, R. C., Li, S. C., & Lai, E. (1999). Dorsal– ventral patterning defects in the eye of BF-1-deficient mice associated with a restricted loss of shh expression.
Developmental
Biology, 211 , 53–63.
Hutson, L. D., & Chien, C.-B. (2002). Wiring the zebrafish: axon guidance and synaptogenesis.
Current Opinion in Neurobiology, 12 ,
87–92.
Ingham, P. W., & McMahon, A. P. (2001). Hedgehog signalling in animal development: paradigms and principles.
Genes and Development, 15 , 3059–3087.
C. Russell / Vision Research 43 (2003) 899–912
Jarman, A. P. (2000). Developmental genetics: vertebrates and insects see eye to eye.
Current Biology, 10 , R857–R859.
Jensen, A. M., & Wallace, V. A. (1997). Expression of Sonic hedgehog and its putative role as a precursor cell mitogen in the developing mouse retina.
Development, 124 , 363–371.
Johnson, R. L., & Tabin, C. J. (1997). Molecular models for vertebrate limb development.
Cell, 90 , 979–990.
Keifer, P., Str ahle, U., & Dickson, C. (1996). The zebrafish Fgf3 gene: cDNA sequence, transcript structure and genomic organisation.
Gene, 168 , 211–215.
Koebernick, K., & Pieler, T. (2002). Gli-type zinc finger proteins as bipotential transducers of Hedgehog signalling.
Differentiation, 70 ,
69–76.
Launay, C., Fromentoux, V., Thery, C., Shi, D. L., & Boucaut, J. C.
(1994). Comparative analysis of the tissue distribution of three fibroblast growth factor receptor mRNAs during amphibian morphogenesis.
Differentiation, 58 , 101–111.
Layman, L. C. (1999). Genetics of human hypogonadotropic hypogonadism.
American Journal Medical Genetics, 89 , 240–248.
Levine, E. M., Roelink, H., Turner, J., & Reh, T. A. (1997). Sonic
Hedgehog promotes rod photoreceptor differentiation in mammalian retinal cells in vitro.
Journal of Neuroscience, 17 , 6277–
6288.
Lillien, L., & Cepko, C. (1992). Control of proliferation in the retina: temporal changes in responsiveness to FGF and TGF a .
Development, 115 , 253–266.
Litingtung, Y., & Chiang, C. (2000). Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and
Gli3.
Nature Neuroscience, 3 , 979–985.
Livesey, F. J., & Cepko, C. L. (2001). Vertebrate neural cell fate determination: lessons from the retina.
Nature Reviews, 2 , 109–
118.
Lu, H.-C., Swindell, E. C., Sierralta, W. D., Eichele, G., & Thaller, C.
(2001). Evidence for a role of protein kinase C in FGF signal transduction in the developing chick limb bud.
Development, 128 ,
2451–2460.
Macdonald, R., Barth, K. A., Xu, Q., Holder, N., Mikkola, I., &
Wilson, S. W. (1995). Midline signalling is required for Pax gene regulation and patterning of the eyes.
Development, 121 , 3267–
3278.
Macdonald, R., Scholes, J., Str ahle, U., Brennan, C., Holder, N.,
Brand, M., & Wilson, S. W. (1997). The Pax protein Noi is required for commissural axon pathway formation in the rostral forebrain.
Development, 124 , 2397–2408.
Malicki, J. (2000). Harnessing the power of forward genetics––analysis of neuronal diversity and patterning in the zebrafish retina.
TINS,
23 , 531–541.
Malicki, J., & 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.
Malicki, J. J., Pujic, Z., Thisse, C., Thisse, B., & Wei, X. (2002).
Forward and reverse genetic approaches to the analysis of eye development in zebrafish.
Vision Research, 42 , 527–533.
Marcus, R. C., Delaney, C. L., & Easter, S. S., Jr. (1999). Neurogenesis in the visual system of embryonic and adult zebrafish ( Danio rerio ).
Visual Neuroscience, 16 , 417–424.
Masai, I., Stemple, D. L., Okamoto, H., & Wilson, S. W. (2000).
Midline signals regulate retinal neurogenesis in zebrafish.
Neuron,
27 , 251–263.
Mason, C. A., & Sretevan, D. W. (1997). Glia, neurons and axon pathfinding during optic chiasm development.
Current Opinion in
Neurobiology, 7 , 647–653.
McCabe, K. L., Gunther, E. C., & Reh, T. A. (1999). The development of the pattern of retinal ganglion cells in the chick retina: mechanisms that control differentiation.
Development, 126 , 5713–
5724.

C. Russell / Vision Research 43 (2003) 899–912
McFarlane, S., Cornel, E., Amaya, E., & Holt, C. E. (1996). Inhibition of FGF Receptor activity in retinal ganglion cell axons causes errors in target recognition.
Neuron, 17 , 245–254.
McFarlane, S., McNeill, L., & Holt, C. E. (1995). FGF signalling and target recognition in the developing Xenopus visual system.
Neuron,
15 , 1017–1028.
McFarlane, S., Zuber, M. E., & Holt, C. E. (1998). A role for the fibroblast growth factor receptor in cell fate decisions in the developing vertebrate retina.
Development, 125 , 3967–3975.
Meyers, E. N., Lewandoski, M., & Martin, G. R. (1998). An fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination.
Nature Genetics, 18 , 136–141.
Ming, J. E., Roessler, E., & Muenke, M. (1998). Human developmental disorders and the Sonic hedgehog pathway.
Molecular Medicine
Today, 4 , 343–349.
Nasevicius, A., & Ekker, S. C. (2000). Effective targeted gene
Ô knockdown Õ in zebrafish.
Nature Genetics, 26 , 216–220.
Neumann, C. J., & N usslein-Volhard, C. (2000). Patterning of the zebrafish retina by a wave of sonic hedgehog activity.
Science, 289 ,
2137–2139.
Nguyen, M., & Arnheiter, H. (2000). Signalling and transcriptional regulation in early mammalian eye development: a link between
FGF and MITF.
Development , 127, 3581–3591.
Ogilvie, J. M., Speck, J. D., & Lett, J. M. (2000). Growth factors in combination, but not individually, rescue rd mouse photoreceptors in organ culture.
Experimental Neurology, 161 , 676–685.
Park, C. M., & Hollenberg, M. J. (1989). Basic fibroblast growth factor induces retinal regeneration in vivo.
Developmental Biology, 134 ,
201–205.
Park, C. M., & Hollenberg, M. J. (1991). Induction of retinal regeneration in vivo by growth factors.
Developmental Biology,
148 , 322–333.
Patel, A., & McFarlane, S. (2000). Overexpression of FGF-2 alters cell fate specification in the developing retina of Xenopus laevis .
Developmental Biology, 222 , 170–180.
Perron, J. C., & Bixby, J. L. (1999). Distinct neurite outgrowth signalling pathways converge on ERK activation.
Molecular and
Cellular Neuroscience, 13 , 362–378.
Picker, A., Brennan, C., Reifers, F., Clarke, J. D. W., Holder, N., &
Brand, M. (1999). Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection.
Development, 126 , 2967–2978.
Pittack, C., Grunwald, G. B., & Reh, T. A. (1997). Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos.
Development, 124 , 805–816.
Pittack, C., Jones, M., & Reh, T. A. (1991). Basic fibroblast growth factor induces retinal pigment epithelium to generate neural retina in vitro.
Development, 113 , 577–588.
Pujic, Z., & Malicki, J. (2001). Mutation of the zebrafish glass onion locus causes early cell-nonautonomous loss of neuroepithelial integrity followed by severe neuronal patterning defects in the retina.
Developmental Biology, 234 , 454–469.
Raible, F., & Brand, M. (2001). Tight transcriptional control of the
ETS domain factors Erm and Pea3 by Fgf signalling during early zebrafish development.
Mechanisms of Development, 107 , 105–117.
Ramalho-Santos, M., Melton, D. A., & McMahon, A. P. (2000).
Hedgehog signals regulate multiple aspects of gastrointestinal development.
Development, 127 , 2763–2772.
Raymond, P. A., & Hitchcock, P. F. (2000). How the neural retina regenerates.
Results and Problems in Cell Differentiation, 31 , 197–
218.
Reifers, F., Adams, J., Mason, I. J., Schulte-Merker, S., & Brand, M.
(2000). Overlapping and distinct functions provided by fgf17 , a new zebrafish member of the Fgf8/17/18 subgroup of Fgfs.
Mechanisms of Development, 99 , 39–49.
Reifers, F., Bohli, H., Walsh, E. C., Crossley, P. H., Stainier, D. Y., &
Brand, M. (1998).
fgf8 is mutated in zebrafish acerebellar ( ace )
911 mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis.
Development, 125 ,
2381–2395.
Roth, L. W. A., Bormann, P., Wiederkehr, C., & Reinhard, E. (1999).
b -thymosin, a modulator of the actin cytoskeleton is increased in regenerating retinal ganglion cells.
European Journal of Neuroscience, 11 , 3488–3498.
Rousseau, B., Dubayle, D., Sennlaub, F., Jeanny, J.-C., Costet, P.,
Bikfalvi, A., & Javerzat, S. (2000). Neural and angiogenic defects in eyes of transgenic mice expressing a dominant-negative FGF receptor in the pigmented cells.
Experimental Eye Research, 71 ,
395–404.
Saffell, J. L., Williams, E. J., Mason, I. J., Walsh, F. S., & Doherty, P.
(1997). Expression of a dominant negative FGF Receptor inhibits axonal growth and FGF Receptor phosphorylation stimulated by
CAMs.
Neuron, 18 , 231–242.
Schauerte, H. E., van Eeden, F. J., Fricke, C., Odenthal, J., Str ahle,
U., & Haffter, P. (1998). Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish.
Development,
125 , 2983–2993.
Schneider, R. A., Hu, D., Rubenstein, J. L. R., Maden, M., & Helms,
J. A. (2001). Local retinoid signalling coordinates forebrain and facial morphogenesis by maintaining FGF8 and SHH.
Development, 128 , 2755–2767.
Sener, R. N. (1996). Septo-optic dysplasia associated with cerebral cortical dysplasia (cortico-septo-optic dysplasia).
Journal of Neuroradiology, 23 , 245–247.
Shanmugalingam, S., Houart, C., Picker, A., Reifers, F., Macdonald,
R., Barth, A., Griffin, K., Brand, M., & Wilson, S. W. (2000). Ace/
Fgf8 is required for forebrain commissure formation and patterning of the telencephalon.
Development, 127 , 2549–2561.
Sleptsova-Friedrich, I., Li, Y., Emelyanov, A., Ekker, M., Korzh, V.,
& Ge, R. (2001).
fgfr3 and regionalization of anterior neural tube in zebrafish.
Mechanisms of Development, 102 , 213–217.
Stenkamp, D. L., & Cameron, D. A. (2002). Cellular pattern formation in the retina: retinal regeneration as a model system.
Molecular Vision, 8 , 280–293.
Stenkamp, D. L., Frey, R. A., Prabhudesai, S. N., & Raymond, P. A.
(2000). Function for Hedgehog genes in zebrafish retinal development.
Developmental Biology, 220 , 238–252.
Stratford, T., Logan, C., Zile, M., & Maden, M. (1999). Abnormal anteroposterior and dorsoventral patterning of the limb bud in the absence of retinoids.
Mechanisms of Development, 81 , 115–125.
Streichert, L. C., Birnbach, C. D., & Reh, T. A. (1999). A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of Retinitis Pigmentosa.
Journal of
Neurobiology, 39 , 475–490.
Tanihara, H., Inatani, M., & Honda, Y. (1997). Growth factors and their receptors in the retina and pigment epithelium.
Progress in
Retinal and Eye Research, 16 , 271–301.
Tcheng, M., Fuhrmann, G., Hartmann, M.-P., Courtois, Y., &
Jeanny, J.-C. (1994). Spatial and temporal expression patterns of
Fgf receptor genes type 1 and type 2 in the developing chick retina.
Experimental Eye Research, 58 , 351–358.
Traiffort, E., Moya, K. L., Faure, H., H assig, R., & Ruat, M. (2001).
High expression and anterograde axonal transport of aminoterminal Sonic hedgehog in the adult hamster brain.
European Journal of
Neuroscience, 14 , 839–850.
Treisman, J. E., & Heberlein, U. (1998). Eye development in
Drosophila : formation of the eye field and control of differentiation.
Current Topics in Developmental Biology, 39 , 119–158.
Trousse, F., Marti, E., Gruss, P., Torres, M., & Bovolenta, P. (2001).
Control of retinal ganglion cell axon growth: a new role for Sonic hedgehog.
Development, 128 , 3927–3936.
Tsang, M., Friesel, R., Kudoh, T., & Dawid, I. B. (2002). Identification of Sef, a novel modulator of FGF signalling.
Nature Cell
Biology, 4 , 165–169.

912 C. Russell / Vision Research 43 (2003) 899–912
Unoki, K., & LaVail, M. M. (1994). Protection of the rat retina from ischemic injury by brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor.
Investigative Opthalmology and Visual Science, 35 , 907–915.
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y. L., Postlethwait, J.
H., Eisen, J. S., & Westerfield, M. (2001). Zebrafish smoothened functions in ventral neural tube specification and axon tract formation.
Development, 128 , 3497–3509.
Varga, Z. M., Wegner, J., & Westerfield, M. (1999). Anterior movement of ventral diencephalic precursors separates the primordial eye field in the neural plate and requires cyclops .
Development,
126 , 5533–5546.
Vihitelic, T. S., & Hyde, D. R. (2002). Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.
Vision
Research, 42 , 535–540.
Vogel-H opker, A., Momose, T., Rohrer, H., Yasuda, K., Ishihara, L.,
& Rapaport, D. H. (2000). Multiple functions of fibroblast growth factor-8 (FGF-8) in chick eye development.
Mechanisms of
Development, 94 , 25–36.
Wallace, V. A., & Raff, M. C. (1999). A role for Sonic hedgehog in axon-to-astrocyte signalling in the rodent optic nerve.
Development, 126 , 2901–2909.
Wallis, D., & Muenke, M. (2000). Mutations in holoprosencephaly.
Human Mutation, 16 , 99–108.
Walz, A., McFarlane, S., Brickman, Y. G., Nurcombe, V., Bartlett, P.
F., & Holt, C. E. (1997). Essential role of heparan sulphates in axon navigation and targeting in the developing visual system.
Development, 124 , 2421–2430.
Wei, X., & Malicki, J. (2002).
nagie oko , encoding an MAGUK-family protein, is essential for cellular patterning of the retina.
Nature
Genetics, 31 , 150–157.
Wild, A., Kalff-Suske, M., Vortkamp, A., Bornholdt, D., Konig, R., &
Grzeschik, K. H. (1998). Point mutations in human Gli3 cause
Greig syndrome.
Human Molecular Genetics, 6 , 1979–1984.
Ye, W., Shimamura, K., Rubenstein, J. L. R., Hynes, M. A., &
Rosenthal, A. (1998). FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate.
Cell, 93 , 755–
766.
Zhang, X.-M., & Yang, X.-J. (2001a). Regulation of retinal ganglion cell production by Sonic hedgehog.
Development, 128 , 943–957.
Zhang, X.-M., & Yang, X.-J. (2001b). Temporal and spatial effects of
Sonic hedgehog signalling in chick eye morphogenesis.
Developmental Biology, 233 , 271–290.
Zhang, C., Takahashi, K., Lam, T. T., & Tso, M. O. M. (1994). Effects of basic fibroblast growth factor on retinal ischemia.
Investigative
Opthalmology and Visual Science, 35 , 3163–3168.
Zhao, S., & Barnstable, C. J. (1996). Differential effects of bFGF on development of the rat retina.
Brain Research, 723 , 169–176.
Zhao, S., Hung, F.-C., Colvin, J. S., White, A., Dai, W., Lovicu, F. J.,
Ornitz, D. M., & Overbeek, P. A. (2001). Patterning the optic neuroepithelium by FGF signalling and Ras activation.
Development, 128 , 5051–5060.