Ординатура / Офтальмология / Английские материалы / Retinal Development_Sernagor, Eglen, Harris, Wong_2006

In summary, neural induction generates an early neural plate with an anterior neural fate bias. The presumptive neural plate is then patterned under the influence of caudalizing signals to generate different brain regions (fore-, midand hindbrain). Precise control of Wnt signalling is critical for patterning the forebrain. Disruptions of Wnt activity via components of the signalling cascades or the activity of Wnt inhibitors can have dramatic effects on eye formation. The evidence suggests that a gradient, or possibly distinct levels (low, intermediate and relatively high), of Wnt signalling modulated via both canonical and non-canonical signalling systems are required to pattern the telencephalon, eye field and diencephalon within the presumptive forebrain.

2.4 Eye field specification

Classical transplantation and fate-mapping experiments have determined the timing and location of eye field formation. A group of eye field transcription factors are synchronously expressed in the eye field during its specification. Each of these genes is required for normal eye formation and together they can induce the formation of additional eye fields and functional eyes. Although early models suggested a single signalling cascade, recent experiments point to a more complex model in which these genes act synergistically in a self-regulating feedback network to convert a region of the anterior neural plate into the eye field.

Transplantation experiments from the beginning of the last century demonstrated that the amphibian eye field is located in the neural plate. Removal of the anterior neural plate results in eyeless animals (Spemann, 1901; Lewis, 1907; Adelmann, 1929a). If this same region is transplanted to the belly wall, or simply grown in culture, a histologically normal eye will form (Adelmann, 1929b; Lopashov and Stroeva, 1964; Li et al., 1997).

Fate-mapping experiments have more precisely resolved the location and shape of the eye field. In amphibians and fish, the eye field is a single crescent of cells spanning the breadth of the early anterior neural plate (Figure 2.3 and Brun, 1981; Eagleson and Harris, 1990; Kimmel et al., 1990; Eagleson et al., 1995; Woo and Fraser, 1995). In contrast, at the earliest stages analysed, chick fate maps indicate the presence of two distinct eye fields separated by the embryonic midline (Fernandez-Garre et al., 2002). These differences could result from species-specific developmental processes. However, in amphibians, fish and mice the single eye field also resolves into two bilaterally symmetric eye primordia and several genes critical for eye field specification (see below) are expressed across the embryonic chick midline. These results suggest that a single eye field analogous to that observed in amphibians and fish may also exist in chick.

The location and timing of eye field specification in the anterior neural plate is synchronized with the coordinated expression of a group of eye field transcription factors or EFTFs (Table 2.1). The expression pattern of the Xenopus EFTFs ET, Rx, Pax6, Six3, Lhx2, tll and Optx2 overlap in the presumptive eye field during and immediately following its specification (Figure 2.3 and Zuber et al., 2003). The EFTFs of other species have a similar pattern

Table 2.1. The eye field transcription factors (EFTFs) (Xenopus homologues) including the origin of their abbreviated names and the transcription factor family to which they belong. Alternative names are shown in parentheses

Figure 2.3 Eye field transcription factors have overlapping expression patterns during eye field formation. Anterior, frontal views of neural plate-staged frog (Xenopus laevis), fish (zebrafish and medakafish), and mouse embryos show the expression domains of Rx, Pax6, Six3 and Optx2 homologues (Oliver et al., 1995; Mathers et al., 1997; Seo et al., 1998; Toy and Sundin, 1999; Inoue et al., 2000; Chuang and Raymond, 2002; Zuber et al., 2003; Bailey et al., 2004). Two Pax6 (Pax6a and Pax6b) and three Rx (Rx1, Rx2 and Rx3) homologues have been identified in fish. The expression pattern of mouse Rx (Rax) at embryonic day (E)8.25 was estimated from its published expression patterns at E7.5 and E8.5. The location of the presumptive eye field is indicated with a black dashed line. hpf, hours post-fertilization. For colour version, see Plate 1.

of coordinated expression. For example, at neural plate stages Pax6, Rx, Six3 and Optx2 are also observed in a single band of expression in the chick, zebrafish and mouse embryo (Figure 2.3 and Walther and Gruss, 1991; Li et al., 1994; Oliver et al., 1995; Mathers et al., 1997; Bovolenta et al., 1998; Toy et al., 1998; Ohuchi et al., 1999; Toy and Sundin, 1999; Chuang and Raymond, 2002).

Eye field transcription factors have been highly conserved through evolution and genetic evidence from multiple species demonstrates they are required for vertebrate eye formation.

Functional inactivation of Pax6, Rx, Lhx2, tll, Six3 and Optx2 results in frogs, fish, rodents and/or humans with abnormal or no eyes (Hill et al., 1991; Mathers et al., 1997; Porter et al., 1997; Hollemann et al., 1998; Chow et al., 1999; Isaacs et al., 1999; Zuber et al., 1999; Wawersik and Maas, 2000; Yu et al., 2000; Loosli et al., 2001, 2003; Tucker et al., 2001; Carl et al., 2002; Li et al., 2002; Andreazzoli et al., 2003; Lagutin et al., 2003; Voronina et al., 2004). Eye field transcription factors are not only necessary for eye formation; in some contexts, they are also sufficient. Overexpression of Pax6, Six3, Rx and Optx2 can expand or induce eye tissues within the vertebrate nervous system (Oliver et al., 1996; Mathers et al., 1997; Andreazzoli et al., 1999; Chow et al., 1999; Loosli et al., 1999; Zuber et al., 1999, 2003; Bernier et al., 2000; Chuang and Raymond, 2001). Together these results demonstrate a critical role for EFTFs in early eye formation.

Many vertebrate EFTFs were originally identified as homologues of Drosophila genes required for fly eye formation. Pax6, for example, is a homologue of Drosophila eyeless (ey) (Quiring et al., 1994). Based on its remarkable evolutionary conservation, requirement for normal eye formation in multiple species and the ability of mammalian Pax6 orthologues to induce ectopic fly eyes, ey/Pax6 was hailed as a potential master regulator of eye formation (Halder et al., 1995; Callaerts et al., 1997). In initial models, ey was placed atop a hierarchy of genes that drove eye formation in the eye portion of the eye-antennal imaginal disc complex. However, a much more complex set of interactions soon emerged. The genes twin-of-eyeless (toy), sine oculis (so), optix, eyes absent (eya), dachshund (dac) and eye gone (eyg) are all required for fly eye formation. Individually or in combinations, these genes can also induce ectopic eyes, sometimes in the absence of ey. For instance, both toy and optix can induce ectopic eyes via an ey-independent mechanism (Czerny et al., 1999; Seimiya and Gehring, 2000; Punzo et al., 2004). In Drosophila, a model evolved in which toy, ey, so, optix, eya, dac and eyg behave as a network with hierarchical components as well as regulatory feedback loops including protein protein interactions (Chen et al., 1997; Pignoni et al., 1997; Kumar and Moses, 2001).

An analogous transcription factor network is at work during vertebrate eye formation. Pax6, Six3, Rx and Optx2 activate each other’s expression, while inactivation of each can reduce the expression of the others (Andreazzoli et al., 1999; Chow et al., 1999; Loosli et al., 1999; Zuber et al., 1999, 2003; Bernier et al., 2000; Chuang and Raymond, 2001; Lagutin et al., 2001, 2003; Wargelius et al., 2003). The evidence suggests that the network is conserved among species. For example, vertebrate Pax6 and Six3 and their fly homologues (toy, ey, so and optix) cross-regulate each other’s expression in flies, frogs, fish and mice (Pignoni et al., 1997; Halder et al., 1998; Seimiya and Gehring, 2000; Carl et al., 2002; Goudreau et al., 2002; Zuber et al., 2003). Eye field transcription factors can act synergistically and, as in the fly, functional interactions among the vertebrate EFTFs involve protein protein complexes and multiple levels of regulation (Zuber et al., 1999, 2003; Mikkola et al., 2001; Li et al., 2002; Stenman et al., 2003).

Based on their coordinated expression and the extensive analysis demonstrating the presence of a genetic network, Kumar and Moses proposed that Drosophila eye field specification might be driven by the coordinated expression of the fly EFTFs (Kumar and

in Rx−/−, Pax6−/−, Lhx2−/−, Six3−/−, Six6−/− and tll−/− mice are consistent with the hierarchical aspects of the frog model. In frog, Rx1 function is predicted to be required prior to Pax6 and Lhx2. Consistent with this order, Rx−/− mice lack optic sulci, vesicles and cups, while Pax6−/− and Lhx2−/− mice develop optic vesicles and even rudimentary optic cups (Grindley et al., 1995; Porter et al., 1997; Zhang et al., 2000). Rx−/− mice lack normal Pax6 expression in the optic primordia, while Rx1 expression is unaffected in the Pax6−/− mouse (Zhang et al., 2000). Neither Optx2 nor tll are required for the initial steps of eye field specification in Xenopus. Six6−/− (Optx2−/−) mice have normal (although small) eyes, while tll−/− mice do not develop retinal defects until three weeks after birth (Yu et al., 2000; Li et al., 2002). In mouse as well as frog, altering Optx2 levels have no effect on Pax6, Six3 or Rx expression (Li et al., 2002; Zuber et al., 2003).

In spite of the similarities outlined above, inconsistencies and potential differences still exist between the working models of vertebrate eye field specification. For instance, in frog ET activates Rx1 expression and both genes are proposed to play an early role in eye field specification. Although Xenopus ET is most homologous to Tbx3, Tbx3 null mice have no reported eye phenotype (Papaioannou, 2001; Davenport et al., 2003). Tbx3 is highly related to Tbx2 and both genes are members of the same subfamily of T-box containing genes (Papaioannou, 2001). The eyes of Tbx2 null mice do develop abnormally, however the expression of Xenopus Tbx2 is not detected in the early neural plate suggesting that it is not involved in initiating eye field specification (Takabatake et al., 2002; Harrelson et al., 2004). The fish Rx3 is required for normal eye formation. In Rx3 mutants, both Tbx2 and Tbx3 expression is lost in the retina, suggesting that Rx3 is genetically upstream of these genes (Mathers et al., 1997; Loosli et al., 2001, 2003). This directly contradicts the frog model in which Rx1 is downstream of ET (Tbx3) (Figure 2.4 and Zuber et al., 2003).

The discrepancies highlighted above may be a consequence of the different techniques used to identify functional interactions between gene products, for example, inductive analysis in frog and mutant analysis in mice. In addition, some genomes carry duplicate copies or highly homologous EFTFs. For example, distinct Rx homologues with similar expression patterns have been reported in Xenopus (2), medakafish (2) and zebrafish (3) suggesting functional redundancy and complicating the identification of true functional orthologues (Casarosa et al., 1997; Mathers et al., 1997; Chuang et al., 1999; Winkler et al., 2000; Loosli et al., 2001). Clearly, a significant amount of feedback and crossregulation is built into the system. Although there are clear advantages to the developing organism, it complicates analysis, making additional investigations necessary to more clearly define the genetic interactions necessary and required for vertebrate eye field specification.

The coordinated expression of the EFTFs is sufficient to generate ectopic eye fields and functional eyes. However, it is currently unclear how this coordinated expression is established. Recent evidence suggests that Wnt signalling, discussed in the previous section, directly controls expression of at least one EFTF. In the zebrafish anterior neural plate, Wnt11-expressing cells induce rx3 expression in neighbouring cells via a non-canonical Wnt signalling pathway (Cavodeassi et al., 2005). In Xenopus, non-canonical Wnt4 signalling

activates expression of EAF2 a component of the RNA polymerase II elongation factor complex. Wnt4 and EAF2 are both required for eye formation and EAF2 regulates Rx expression in vitro (Maurus et al., 2005). Wnt signalling is unlikely to induce every EFTF, however, since their expression patterns are not identical (Figure 2.3). Instead, distinct, as yet unidentified signalling systems coordinate EFTF expression.

In summary, transplantation and fate-mapping experiments have defined the location of the eye field. Eye field specification is synchronized with the coordinated expression of a group of EFTFs, most of which are required for normal eye development. When expressed individually, some EFTFs can induce ectopic, eye-like structures. The EFTFs form a selfregulating feedback network. Ectopic coexpression of these factors mimics the endogenous eye field, inducing ectopic, functional eyes. The signalling systems that coordinate their expression and the functional interactions among the EFTFs that are required for eye field specification and eye development remain largely unknown.

2.5 Separating the eye field into two eye primordia

All vertebrate embryos have a pair of eyes organized symmetrically across the body midline. As described above, the eye field forms as a single domain spanning the early anterior neural plate. In order to form two separate eyes, the single eye field must be split into two lateral eye primordia. Failure to do so results in cyclopic animals with one large midline eye. This is because the entire eye field is competent to form eye tissue. Midline as well as lateral anterior neural plate can form an eye (Adelmann, 1936).

The mechanism by which the eye field separates appears to be species specific. In amphibians, signals originating from the prechordal mesoderm underlying the anterior neural plate are responsible for separation of the eye field. Experiments in the 1930s by Mangold and Adelmann demonstrated that removal of the amphibian prechordal mesoderm results in cyclopic animals (Adelmann, 1930, 1934; Mangold, 1931).

Alterations in the expression patterns of EFTFs and fate-mapping experiments in Xenopus supports a mechanism in which prechordal mesoderm represses retinal fate in the midline of the anterior neural plate. Xenopus ET, Rx1, Pax6, Six3, Lhx2 and Optx2 are all expressed in the anterior neural plate. Their expression patterns are variable in size and extend to other regions of the neural plate, but all cover the eye field and each is expressed as a single band at stage 15 (Zuber et al., 2003). Without exception, the expression of each EFTF is repressed in the midline and resolves into the two eye primordia by stage 18 (Figure 2.5 and Casarosa et al., 1997; Hirsch and Harris, 1997; Li et al., 1997; Mathers et al., 1997; Zuber et al., 1999; Zhou et al., 2000; Viczian et al., 2006). If midline cells within the single Xenopus eye field are labelled with tracking dyes their progeny are found, not in the eyes, but at the midline in the ventral hypothalamus and optic stalk suggesting that they have been reprogrammed to ventral diencephalic fates (Figure 2.5 and Eagleson et al., 1995; Li et al., 1997). Furthermore, transplanted chick prechordal plate represses Pax6 expression in the anterior neural plate (Li et al., 1997). In the absence of the prechordal plate, Pax6 expression remains strong in the midline and chick embryos develop a single medial optic

In fish, mutants have been invaluable in identifying the signalling systems regulating eye field separation. cyclops (cyc) is a Nodal-related member of the transforming growth factor-β family of secreted signalling molecules (Feldman et al., 1998; Rebagliati et al., 1998; Sampath et al., 1998). In cyc mutants posterior diencephalic precursors express opl instead of foxb1.2 and fail to move anteriorly resulting in cyclopic embryos lacking ventral forebrain (Hatta et al., 1991; Varga et al., 1999). Zebrafish mutations in Nodals (cyc and squint), Nodal cofactors (one-eyed pinhead; oep) and Nodal signal transducing transcription factors (schmalspur) all result in cyclopia (Hammerschmidt et al., 1996; Feldman et al., 1998; Pogoda et al., 2000). Activation of the Nodal signalling cascade (by overexpression of Nodal receptor ActRIIB or the transcription factor Smad2) rescues the cyc phenotype (Gritsman et al., 1999).

In mice, null mutations effecting Nodal signalling are early embryonic lethal, however genetic combinations of heterozygous Nodal (Nodal+/−) with heterozygous Nodal receptor or Smad2 mutations cause cyclopia (Nomura and Li, 1998; Song et al., 1999). Interestingly, ablation studies show that in order to induce cyclopia in wild-type fish embryos, it is necessary to remove prechordal plate (which also migrates anteriorly during gastrulation) as well as medial ventral diencephalic precursors (Varga et al., 1999). cyc and oep are both expressed in the prechordal mesoderm, have prechordal plate defects and are cyclopic (Hammerschmidt et al., 1996; Feldman et al., 1998; Sampath et al., 1998). These results suggest that the mechanisms driving fish and frog eye field separation are perhaps not quite so dissimilar as they initially appear and that Nodal signalling in the prechordal mesoderm is a general requirement for vertebrate eye field separation.

Nodal signalling modulates its effects on the eye field, at least in part, through the hedgehog (Hh) family of secreted morphogens. The transcriptional regulator of the Nodal signalling pathway, Smad2, directly modulates the Sonic hedgehog (Shh) promoter in zebrafish and chick neural tissue (Muller et al., 2000). Sonic hedgehog expression is absent from the neuroectoderm of early stage cyc mutants (Krauss et al., 1993). Smad2 overexpression induces Shh expression and rescues the cyc eye phenotype in fish (Muller et al., 2000). A conserved requirement for Hh in vertebrates is also demonstrated by the fact that humans, mice and fish lacking functional Hh signalling are cyclopic (Chiang et al., 1996; Roessler et al., 1996; Nasevicius and Ekker, 2000). Hedgehog overexpression expands the ventral forebrain (including optic stalk) at the expense of the retina. In fish, frogs and mice, ectopic Hh represses the EFTF Pax6 while activating the expression of the optic stalk marker Pax2 and other ventral forebrain markers (Barth and Wilson, 1995; Ekker et al., 1995; Macdonald et al., 1995; Shimamura et al., 1995; Shimamura and Rubenstein, 1997; Perron et al., 2003).

In summary, separation of the single eye field into the two eye primordia is required for normal eye formation. Fate-mapping studies suggest that at least two different mechanisms are used – physical displacement of eye field cells and/or reprogramming of midline eye field cells to ventral forebrain fates. In spite of these distinct mechanisms, misexpression and genetic analysis demonstrate that eye field separation is controlled through the Nodal and Hh signalling systems in vertebrates.

The embryonic location of the vertebrate eye field was first determined over a century ago. It has only been in the last decade, however, that the genes required for eye field and eye formation have been identified. Consequently, many unanswered questions remain regarding how the eye field is specified within the anterior neural plate.

A current model of vertebrate eye field formation, based on experiments in frogs, suggests that the combined expression of a group of EFTFs is sufficient to specify the eye field in the anterior neural plate. Is this mechanism conserved among vertebrate species? Are all these genes required for eye field formation? What are the upstream regulators that coordinate their expression? What are the functional interactions among the EFTFs necessary for eye field specification? Do the EFTFs act coordinately to regulate later aspects of eye formation? How are the dorso-ventral and proximo-distal axes of the eye field established?

The answers to these questions, and others, will provide a better understanding of how the cells of the early anterior neural plate are specified to form the eye field and then generate all the cells of the mature retina.

Acknowledgements

Thanks to Andrea Viczian for constructing the chapter figures and table. The authors acknowledge support from Research to Prevent Blindness through a Career Development Award to MZ.

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