Ординатура / Офтальмология / Английские материалы / Development of the Ocular Lens_Lovicu, Lee Robinson_2004

34 Marilyn Fisher and Robert M. Grainger

since the antibody used by Karkinen-Ja¨askel¨ ainen¨ was apparently raised against the whole lens and was not specific to crystallins, it is possible that the positive immunoreactivity she observed did represent some response of the trunk ectoderm to factors provided by the OV but not necessarily lens crystallin production. Unfortunately, this apparently striking demonstration of the inductive power of the OV has proven difficult to confirm (C. Sullivan and R. Grainger, unpublished data). Thus, in light of the results described above, which raise questions about the necessity and sufficiency of the OV as an inductor, it is doubtful that during the course of normal development the OV of any vertebrate species is the primary inductor of lens development.

2.2.4. Role of Tissues Other Than the Optic Vesicle in Lens Induction

The early investigations focused on the properties of the OV as an inductor, but the results forced recognition of the fact that in any induction system the properties of the responding component must also be considered. It is clear from the studies already discussed that the ability of embryonic ectoderm to respond to induction by the OV varies in a temporal and spatial manner. In general, during the neurula stages, the head ectoderm is most responsive, and within the head ectoderm, that closest to the actual PLE retains its responsiveness the longest. That the PLE prior to contact with the OV already has some potential for lens differentiation was apparent from the development of free lenses in the absence of contact with the OV and inspired some to look for other potential lens-inducing factors. A number of early investigators addressed this problem (see discussion in Saha et al., 1989), but the most extensive contribution consisted of the studies of Jacobson (1958, 1966), who tested the lens-inducing capabilities of foregut endoderm and presumptive heart mesoderm, both of which he noted came to lie close beneath the PLE as a result of gastrulation movements (see Figs. 2.1B and C). He showed that these tissues had additive lens-inducing effects when recombined in vitro with PLE from early neurula stage amphibian embryos. However, since he tested PLE from neural plate stage embryos, tissue we now recognize as already biased (Grainger et al., 1997), he did not show that endoderm and mesoderm could act as primary inducers to elicit a lens response in young tissue that had not already been exposed to early lens-inducing signals. Mizuno (1972) tested the lens-inducing ability of early endomesoderm in chick embryos. After unexpectedly finding lens differentiation during his study of feather germ induction, he demonstrated that it was possible to induce readily recognizable lenses having elongated lens fibers in cephalic epiblast from primitive streak stage embryos by recombining it with cephalic hypoblast plus dermis from the back of 6.5-day-old embryos. While a variety of embryonic mesenchymes worked in this assay, dermis was most effective, and neither dermis nor hypoblast alone with epiblast was sufficient. In these cultures, there was no apparent neural differentiation, certainly not a recognizable OV. Using this same system, Mizuno (1973) further showed that epiblast taken from the trunk region or extraembryonically from the area opaca of H&H stage 4 embryos could form crystallin-positive lens vesicles when recombined with cephalic hypoblast plus dermis. Again, although a number of mesenchymes could substitute for back dermis, trunk hypoblast could not substitute for cephalic hypoblast. These results support the conclusion that, in chicks as well as amphibians, embryonic foregut endoderm is capable of contributing to the early stages of lens development.

The possibility that other anterior structures such as placodal ectoderm and early anterior neural plate might also have some lens-inducing potential has been raised. Several investigators noted that free lenses formed after optic rudiment ablation generally were more

advanced when they were in contact with the nasal pit (e.g., LeCron, 1907; Spemann, 1912; Jacobson, 1958; Reyer, 1962). The idea that early neural tissue, possibly the presumptive eye rudiment itself, might influence the adjacent ectoderm containing the PLE, arose from Nieuwkoop’s (1952, 1963) hypothesis that placodal ectoderm (including lens) is specified as an extension of neural induction by signals passing laterally through the plane of the ectoderm (see Figs. 2.1A and B). There was only questionable experimental evidence to support the idea that planar signals passing from neural to nonneural ectoderm might play a role in lens determination until Henry and Grainger (1990) tested it directly. They found that PLE from Nieuwkoop and Faber (N&F) stage 14 (Nieuwkoop and Faber, 1956) Xenopus laevis embryos cultured by itself could not form even rudimentary lens structures, but when cultured in combination with anterior neural plate of the same stage, crystallin-positive ectodermal thickenings formed. This result was enhanced when the PLE and anterior neural tissue were isolated and maintained as a continuous sheet, even when isolated as early as N&F stage 11.5–12. These results confirm that the neural plate is an early lens inducer. The importance of the anterior neural plate was further suggested by the demonstration that the ability to form free lenses was greatly reduced if anterior neural tissue was removed at N&F stage 14. Henry and Grainger (1990) found that free lenses formed in only 15% of cases following early ablation of the optic rudiment, compared with 94% of cases when the optic rudiment was ablated later (N&F stage 17–18).

While the PLE appears to increase steadily its lens-forming ability from the early neural plate stage onward, non-PLE head ectoderm has been shown by numerous investigators to lose its lens-forming responsiveness shortly after the stage when contact has been established between the PLE and the OV. While one could imagine a number of mechanisms by which this could happen, there is some evidence from experiments carried out by von Woellwarth (1961) that the neural crest might play an important role in restricting the region of lensforming responsiveness. He found that if he extirpated the anterior neural plate in newt embryos, no free lenses formed, but if he extirpated the posterior neural plate as well, eliminating the source of the head neural crest, free lenses did form. The neural crest is normally blocked from interacting with the PLE by virtue of its tight adherence to the underlying OV but would have access to the rest of the head ectoderm.

2.2.5. A Revised Model of Lens Determination

The persistence of interest in and experimentation on the problem of lens induction resulted in reevaluation and revision of the model of lens determination, which came to be viewed, not as a simple process elicited by interaction between head ectoderm and the optic vesicle, as envisioned by Spemann and Lewis, but a more protracted multistep process, as first suggested by Jacobson (1966). In Jacobson’s model, lens determination involves a series of inductive interactions beginning late in gastrulation as the PLE sequentially comes under the influence of endoderm, mesoderm, and finally the optic vesicle. Jacobson suggested that the influence of the separate inductors were qualitatively similar but that their relative strengths varied among species, explaining why free lenses form much more readily in some species than in others. Combining some of the elements of Jacobson’s model and some of Nieuwkoop’s ideas on the importance of signaling within the ectoderm during the induction of placodal structures (including the lens), Saha et al. (1989) proposed a new model of lens determination as a process with four distinguishable stages (see also Grainger, 1992, 1996). As the result of an additional decade of rigorous testing, the model has been further revised to recognize five important stages in lens determination: competence, bias, specification,

36 Marilyn Fisher and Robert M. Grainger

inhibition, and differentiation (Hirsch and Grainger, 2000). In the next sections, we review the evidence for the current understanding of the molecular bases of these stages and discuss likely sources of and candidates for lens-inducing signals.

2.3. Current Model of Lens Determination

The current model of lens determination is based on the vast body of amphibian lens induction studies done over the past century, including more recent studies from the last decade specifically designed to test and refine the elements of the model. We have reviewed lens induction studies of chick and mouse embryos to assess the general applicability of the principles of lens determination across vertebrate classes. There is a much smaller body of literature dealing with chick and mouse embryos than with amphibian embryos, owing to their being somewhat more challenging as experimental models. Because they need complex growth media, specialized atmospheric conditions, and elevated temperature, mouse embryos are particularly difficult to maintain in vitro, especially for periods longer than 1 or 2 days at a time, as would be required to observe the entire period of lens development from the earliest inductive interactions through the differentiation of lens fibers. Adding to the challenge of working with mammalian embryos are the dramatic size and shape changes these embryos undergo during gastrulation and early neurulation. Amphibian embryos are relatively large from the beginning and change very little in size between fertilization and lens placode formation. Mouse embryos, on the other hand, grow considerably in size during that period and undergo a dramatic change in configuration: during the gastrula stages, they are U-shaped, with the endoderm on the outer surface and the ectoderm on the inner surface, then they rotate along the body axis during the neurula stages to take on a more conventional configuration, with the ectoderm fully enclosing the embryo’s internal structures. For these reasons, we do not have the same depth of background information, especially for the earliest period of development, when, as we know, the first steps of lens determination are occurring in amphibian embryos. However, fate-mapping studies of various tissues from amphibian, chick, and mouse embryos show striking similarities in the relative positions of early rudiments in these organisms (see comparative fate maps of gastrula stage embryos in Lawson et al., 1991 and Quinlan et al., 1995 and of the prosencephalic neural plate in Inoue et al., 2000). So, despite the rather different sizes and configurations of these organisms during the blastula, gastrula, and early neural stages of development, it seems reasonable to postulate that, at least from the mid to late gastrula stages onward, the origins of presumptive retina and lens rudiments in the various vertebrate classes have a similar relationship to one another, as exemplified by the well-documented amphibian model. From the time the OV begins to grow out toward the overlying PLE through the maturation of the lens, the morphologic changes appear virtually identical across the vertebrate classes considered here. Furthermore, the data that are available from lens induction studies in chick and mouse embryos are generally consistent with the elements of the amphibian-based model, as noted earlier. To emphasize the common features that we believe are shared by these organisms, we have chosen to illustrate diagrammatically the steps in lens determination using the chick embryo (Figure 2.1).

Briefly, the current view is that lens induction begins during mid to late gastrulation, when ectoderm acquires the ability (competence) to respond to the earliest lens-inducing signals that arise from the anterior neural plate (Figs. 2.1A and B). These early signals elicit within the entire head ectoderm a predisposition (bias) to form lens. Under the continued influence of the anterior neural plate as well as the underlying endomesoderm (Figs. 2.1B

and C), the head ectoderm becomes increasingly biased toward lens formation until reaching a threshold level that allows the PLE to begin differentiation even if isolated from further lens inductors. At this point the ectoderm is said to be “specified.” While a rather large area of head ectoderm initially acquires lens-forming bias, around the time of specification of the PLE there is a loss of lens-forming bias in head ectoderm outside of the PLE, possibly due to inhibition by the neural crest (Figs. 2.1D and E). Finally, under the influence of the OV, the PLE becomes fully determined and differentiates into a mature lens. In the following sections, we primarily discuss recent studies of lens induction that provide data relevant to individual components of the model while making reference to some earlier studies that were instrumental for developing the key concepts of the model.

2.3.1. Competence

Waddington (1932) was the first to use the term “competence” to describe the ability of a tissue to respond to a particular inducing stimulus, and he was the first to try to define experimentally the conditions necessary for establishing lens competence in amphibian embryonic ectoderm. Specifically, he hypothesized that competence for lens induction might depend on the prior induction of neural tissue or on some process controlled by mesoderm. He tested this idea by isolating early gastrula stage ectoderm (prior to the possibility of its being influenced by mesoderm or neural tissue) and culturing the ectoderm until control embryos reached the early neural plate stage. He then recombined the ectoderm isolates with presumptive retinal tissue from neural plate stage embryos and observed that in the cases where lenses developed there was no correlation between lens formation and neural or mesodermal induction. He concluded that apparently lens competence arose independently of any other specific inductive interactions, requiring only that the ectoderm take on the form of a thin-walled vesicle (not unlike the animal cap ectoderm of a mid to late gastrula).

More recently, Henry and Grainger (1987) and Servetnick and Grainger (1991) reexamined the issue of lens-forming competence. For this purpose, they defined competence operationally as the ability of a tissue to form a lens when exposed to the full range of normal inductive influences by being placed in the presumptive lens region of a young open neural plate stage embryo (for Xenopus, N&F stage 14). Using this assay system, they showed that there is a period from the mid to late gastrula stage (N&F stage 10.5–12) when animal cap ectoderm is competent to respond to lens-inducing signals and that the peak of lens responsiveness is at N&F stage 11–11.5. At these early stages, nearly all ectoderm shows some level of lens competence.

When competent ectoderm from mid gastrula stage embryos was transplanted over the outgrowing OV of an older neurula stage embryo (N&F stage 19), the lens-forming response was greatly diminished both qualitatively and quantitatively. On the other hand, neurula stage ectoderm (N&F stage 18) responded well to inductive influences of the OV. These results indicate that the OV is not a sufficient inductor of lens differentiation in tissues that have not been exposed to the full range of inductive signals occurring during gastrulation and early neurulation, prior to OV outgrowth. Henry and Grainger’s experiments also showed, just as Waddington had noted, that the sequential acquisition and loss of neural and then lens-forming competence by the ectoderm was tissue autonomous, occurring in isolated cultured ectodermal pieces on the same time scale as in the intact embryo. Although early investigators used the term “competent,” virtually all of the studies that addressed the regional ability of ectoderm to form a lens used the OV or the optic cup as the inductor and

Figure 2.2. (See color plate I.) Gene expression during lens determination. The dynamic expression in the PLE of several genes involved in lens determination is graphically represented in relation to the timing of the major phases of lens determination (shown across the top) and to key developmental stages (shown across the bottom). The various amphibian, chick, and mouse genes included are those for which there is the most complete expression information available for the developmental stages of interest. The genes are discussed individually (with references) throughout the text.

therefore were measuring not competence but a later stage in the lens-forming process, as discussed in the next section. There have been no studies of chick or mouse embryos to define a period of lens competence comparable to that established for Xenopus, although Servetnick et al. (1996) demonstrated a window of lens competence during gastrulation in axolotls.

2.3.1.1. Molecular Correlates of Competence

In Xenopus, animal cap ectoderm passes sequentially through periods of competence for mesoderm, neural, and lens induction. This sequence occurs even in animal cap tissue isolated and maintained in vitro (Grainger and Gurdon, 1989; Servetnick and Grainger, 1991), suggesting that these periods are somehow controlled by an autonomous timer (or timers) whose elements are at present unknown. While the molecular basis of lens competence has not yet been systematically investigated, some candidate regulatory genes are known to be expressed in animal cap ectoderm during the period of lens competence. One such candidate is Sox3, an HMG-box gene whose close relative Sox2 has been shown to increase the responsiveness of ectoderm to fibroblast growth factor (FGF) signaling during the neural competence period (Mizuseki et al., 1998). As illustrated in Figure 2.2, which shows gene expression in PLE during lens determination, Sox3 is expressed in animal cap ectoderm during the lens competence period (Zygar et al., 1998) and could similarly affect tissue responsiveness to lens-inducing signals. FGF has been suggested as a competence factor because of its role in enabling cells to respond to mesoderm induction (Cornell et al., 1995; Slack et al., 1996; Isaacs, 1997). Curran and Grainger (2000) showed that a shift from cytoplasmic to nuclear localization of activated MAP-kinase, an integral component of the FGF-signaling pathway, coincides with the onset of mesoderm competence. So, although one might hypothesize that competence is determined by the presence or functionality of receptors for inducing signals, it must be recognized that many components of the relevant signaling pathway could serve as target sites for modulating specific competencies.

2.3.2. Bias

A consistent observation in early studies of lens induction in amphibian and chick embryos was that the lens response elicited from the PLE by the OV improved as the age of the PLE used approached the stage at which close contact with the OV normally occurred. Grainger et al. (1997) documented this in studies with X. laevis. They showed that PLEs taken from progressively older embryos give increasingly better lens response when transplanted over the OV of N&F stage 18 hosts. Similarly, Enwright and Grainger (unpublished manuscript) found that in mice, just as in amphibians, there is a period during early neurulation when head ectoderm exhibits lens-forming bias. Head ectoderm taken from mouse embryos at several stages – from the open neural plate stage through the closed neural tube stage – shows increasing lens responsiveness when recombined in vitro with the OV from an embryo at embryonic day 9.5 (E9.5), the stage when tight apposition between these two tissues normally occurs. Experiments with chicks have also demonstrated a strong lensforming bias in head ectoderm from neurula stage embryos. In fact, according to Barabanov and Fedtsova (1982) and C. Sullivan and R. Grainger (unpublished data), chick embryonic head ectoderm is so strongly biased by the early neural plate stage that it is already capable of lens differentiation in the absence of interaction with the OV (i.e., it is already specified).

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Interestingly, the existence of a period during which a rather large region of ectoderm is predisposed toward a particular differentiation is not unique to the process of lens determination. In his studies on the determination of placode-derived tissues (lens, ear, and nasal tissues) in newts, Jacobson (1963a, 1963b, 1963c) described a broad strip of ectoderm adjacent to the neural plate that, during the early neural stages, was able to give rise to each of those structures when provided with region-specific inductive signals. That is, presumptive nasal ectoderm could give rise to the otic vesicle (and vice versa) when the ectoderm strip was transplanted with reversed anteroposterior (A-P) orientation. Indeed, sometimes when this “placodal strip” was transplanted with reversed A-P orientation, structures appropriate to both the origin and the new position differentiated adjacent to one another, such as the ear and the nose. Similarly, lenses could differentiate adjacent to the ear and the nose. Jacobson interpreted these results to indicate that determination of all three structures had begun already at the neural plate stage throughout the placodal zone and that the regional positioning of these structures normally depends on a combination of signals from neural, mesodermal, and endodermal tissues, which differ slightly in character along the A-P axis. These observations fit well with the broad area of lens-forming bias observed in frog, chick, and mouse embryos and are consistent with the observation of several investigators, mentioned earlier, that free lenses forming in contact with the nasal pit were more advanced than those forming in other locations.

2.3.2.1. Molecular Correlates of Bias

To understand the basis for the progressive change in lens-forming ability of head ectoderm, Zygar et al. (1998) looked for changes in the expression of some genes that seemed likely candidates to be involved in regulating lens formation. They found that changes in the expression patterns of three transcription factors, Otx2, Pax6, and Sox3, during the early stages of X. laevis development are consistent with the involvement of these genes during one or more of the early steps of lens determination. The dynamic expression patterns of these and additional genes from amphibian, chick, and mouse embryos are represented in Figure 2.2, illustrating that, to the extent that homologous genes have been examined across the vertebrate classes, there is remarkable similarity in both temporal and spatial expression patterns. Zygar et al. (1998) found that Sox3 is expressed during the competence period but down-regulated at the onset of the bias period and that Otx2 and Pax6 expression is upregulated during the bias phase. Furthermore, they showed that these genes were activated in lens-competent (N&F stage 11–11.5) but not postcompetent (N&F stage 12) ectoderm when transplanted to the PLE region but not when transplanted to other sites of N&F stage 14 hosts.

Another transcription factor that is first expressed in PLE during the bias period is Xlens1, a member of the forkhead family. Overexpression of Xlens1 in Xenopus embryos represses lens differentiation, suggesting that it might play some role in regulating the shift between proliferation and differentiation, as is further suggested by its later restriction to anterior lens epithelium and its loss from differentiating fiber cells (Kenyon et al., 1999).

A survey of the literature and our own unpublished observations suggest that during the bias period in mice (E8.5–9.5), just as in Xenopus, a number of potential regulatory genes show dynamic expression patterns. Figure 2.2 illustrates the temporal pattern of expression of several genes within the PLE during this period. In addition, several genes exhibit a dynamic spatial expression pattern within the larger context of the head ectoderm. Pax6, Otx2, and Sox2 (the mouse gene whose expression most closely resembles that of the

Xenopus Sox3), genes that are expressed broadly in head ectoderm at the open neural plate stage (E8.0), become restricted to PLE and/or presumptive nasal epithelium (PNE) by early E9.5 (Walther and Gruss, 1991; Simeone et al., 1993; Ang et al., 1994; Grindley et al., 1995; Collignon et al., 1996). Six3 is already expressed in the anterior neural plate at E8 but is expressed in the PLE, and PNE by E9.5 (Oliver et al., 1995). While there is no evidence to establish specific roles for these genes as “bias factors,” their expression patterns nonetheless serve as an indication that the head ectoderm is undergoing continuous change during this period.

One of the most intensely studied genes with regard to lens determination is Pax6, the paired-type homeobox gene homologous to the Drosophila eyeless gene (Quiring et al., 1994). Mutations in the Pax6 locus cause serious eye defects, including human aniridia (Ton et al., 1991) and the “small eye” phenotype in mice and rats (Hill et al., 1991; Fujiwara et al., 1994). Pax6 is expressed in PLE during the bias period before tight contact between the OV and PLE in amphibian, chick, and mammalian embryos (Li et al., 1994; Grindley et al., 1995; Zygar et al., 1998). Homozygous small eye mice and rats fail to form either lens or nasal placodes (Hogan et al., 1986; Fujiwara et al., 1994) and show defects in the expression of Sox2, Six3, and Otx2 in PLE and/or PNE. Furthermore, following up on the study of small eye rats by Fujiwara et al. (1994), J. Enwright and R. Grainger (unpublished data) found that head ectoderm from either homozygous or heterozygous small eye mouse embryos at E9.5 does not exhibit normal lens-forming bias, giving no or very poor lens response when recombined in culture with wild-type OVs (0–10% crystallin positive vs. 82% crystallin positive for wild-type recombinants).

While a number of genes have been identified whose expression within the PLE or surrounding tissues change during bias, most of the changes occur after the onset of bias and thus are likely not themselves responsible for, but rather contribute to, the increasing predisposition of the tissue toward lens formation. One might expect that a bias factor would be turned on and broadly expressed in the ectoderm during the mid to late gastrula stage (i.e., during or toward the end of the lens competence period). On the other hand, genes that come on in more restricted expression domains during the bias phase might be likely candidates for triggering a later stage of lens determination, such as specification or early differentiation. As mentioned above, XSox3 and MSox2 are both broadly expressed during the competence period. Similarly, Otx2 in both frog and mouse embryos and Pax6 in mouse embryos are broadly expressed in head ectoderm around the onset of bias, but it is not clear that their expression domains are coextensive with the area of head ectoderm that has demonstrated lens-forming bias. Other potential bias factors are members of the Eya and Dlx families, homologs of genes involved in Drosophila eye development and body patterning, respectively. Dlx3/dll2 (mouse, chick, zebrafish, Xenopus) is first expressed in ectoderm during gastrulation (zebrafish and Xenopus), before or near the time of onset of bias, and later expression excludes the PLE but includes both the otic and nasal placodes (mouse, chick, zebrafish, and Xenopus), suggesting a role in differentiation of those tissues but not the lens (Akimenko et al., 1994; Dirksen et al. 1994; Robinson and Mahon, 1994; Pera and Kessel, 1999). Eya1 in the mouse and frog (Xu et al., 1997b; Offield and Grainger, unpublished data) is expressed broadly around the onset of bias, at least coextensively with the placodal zone, and is later restricted to structures that include the lens and nasal placodes.

It is worth noting that PNE and PLE regions have overlapping but not identical molecular phenotypes, suggesting that they share certain features of determination. This is consistent with Jacobson’s (1963a, 1963b, 1963c) observations on the placodal zone of head ectoderm

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during the early lens bias period and with the effects of Pax6 mutation on both lens and nasal placode development (Grindley et al., 1995).

2.3.3. Inhibition

Since a relatively large area of ectoderm initially acquires lens-forming bias, one might hypothesize that there is a mechanism to ensure that lenses form only in that ectoderm that becomes properly positioned with respect to the developing eye rudiment. One could imagine that positive signals coming from the OV stabilize the lens-forming commitment in closely apposed ectoderm, while the lack of such positive signals in more distant ectoderm could contribute to destabilization and loss of lens-forming bias. Alternatively, or in addition to positive regulatory signals from the OV, there could be regulatory signals that come from some other source(s) and preferentially influence ectoderm not in contact with the OV, so as to alter its lens-forming bias and cause (or allow) it to differentiate along other pathways.

The progressive restriction of the ectodermal expression domains of a number of transcription factors (e.g., Otx2, MSox2, XSox3, and Pax6) likely to be important for lens formation bears testimony to the fact that the ectoderm is undergoing changes at the molecular level during the bias period, but it does not suggest any particular mechanism. As mentioned earlier, however, there is some experimental evidence that points to a potential inhibitory role for head neural crest. Von Woellwarth’s (1961) experiments demonstrated that free lenses were more likely to form if posterior neural tissue, including the source of head neural crest, was removed along with the retinal rudiment. Recently this hypothesis has been tested more directly in both mouse and chick embryos. J. Enwright and R. Grainger (unpublished data) found a strong inhibitory influence of head mesenchyme on the ability of specified head ectoderm from mouse embryos to form lenses in vitro (5% of early and 37% of late E9.5 head ectoderm plus head mesenchyme recombinants formed crystallin-positive lenses, compared with 37% of early head ectoderm and 71% of late E9.5 head ectoderm cultured alone). Similarly, C. Sullivan and R. Grainger (unpublished data) found that specified head ectoderm from outside the PLE region of H&H stage 10 chick embryos, when cultured alone, formed crystallin-positive lenses more than 90% of the time. In contrast, crystallin-positive lenses never formed when stage 10 specified head ectoderm was left attached to underlying mesenchyme, and they formed 30% of the time when the ectoderm was partially released from the head mesenchyme by brief trypsinization. Using the neural crest cell marker HNK-1, Sullivan and Grainger showed that, at H&H stage 10, neural crest is found throughout the head mesenchyme but not between the PLE and the OV.

Two additional observations from mouse mutants are consistent with an inhibitory role for head neural crest. First, in both small eye mutants and Lhx2 knockout mice, the OV fails to achieve the tight contact with PLE that would normally preclude PLE interaction with neural crest, and no lens placodes form (Grindley et al., 1995; Porter et al., 1997). Second, J. Enwright and R. Gainger (unpublished data) found that head ectoderm from these mutants shows reduced level of lens-forming bias, perhaps partly because these tissues have more extensive interaction than normal with neural crest and/or because of deficiencies in the positive signals from the OV.

Further evidence that lens-forming bias in non-PLE is actively repressed comes from a zebrafish mutant (yot) with a defect in Gli2-mediated hedgehog signaling (Kondoh et al., 2000). In this mutant, Rathke’s pouch, which has been shown in chicks to have a strong bias toward lens differentiation, even transiently expressing high levels of δ-crystallin

(Barabanov, 1977; Fedtsova and Barabanov, 1978; Barabanov and Fedtsova, 1982), differentiates into normal-looking lenses while the adenohypophysis fails to form. These ectopic lenses have elongated fibers and express β-crystallin, but they degenerate by 120 hours of development, presumably due to the absence of necessary sustaining factors that would be supplied to a lens by the optic cup. A similar phenomenon is seen in the chick mutant talpid3. Ede and Kelly (1964) reported abnormalities, including the absence of Rathke’s pouch and the presence of ectopic lenses in its place. It has recently been shown that talpid3 mutants are unable to respond to sonic hedgehog signaling (Lewis et al., 1999). These results suggest that, during normal development, hedgehog signaling from the ventral midline of the CNS in the region of the diencephalon shifts the lens-forming bias of Rathke’s pouch toward adenohypophysis formation.

2.3.4. Specification

The PLE becomes increasingly biased toward lens formation throughout the neurula stage until around the time when the outgrowing OV establishes tight adherence with it. Around this time, the PLE has reached a level of determination that allows it to form a small, simple crystallin-positive lens even when removed from further inductive influences, as when explanted in vitro. At this point, the PLE is said to be “specified” to form a lens in the absence of other external cues. Specification is operationally distinct from determination, in that “specified” tissue might be induced to undergo alternative differentiation if placed in an appropriate environment whereas “determined” tissue is committed to follow a particular developmental course regardless of external cues. This point is clearly illustrated in chick embryos, where a large portion of epiblast is specified for lens formation even as the neural plate is forming (H&H stage 5, according to Barabanov and Fedtsova, 1982), at a stage when, in frog and mouse embryos, head ectoderm is only beginning to acquire lens-forming bias. Despite being able to form lens tissue in vitro at a very early stage (compared with other vertebrates), most of the epiblast ultimately will not differentiate as lens tissue if left in situ, where it will receive cues that will direct differentiation along several alternative pathways, including nasal, otic, and epidermis pathways. In amphibians and mice, PLE specification occurs at a very similar developmental stage, just before or right at the time when close contact with the OV is established (Karkinen-Ja¨askel¨ ainen,¨ 1978a; Henry and Grainger, 1990; J. Enwright and R. Grainger, unpublished manuscript).

So far, the relatively early specification of head ectoderm in chick embryos is the most striking difference we have found in the process of lens determination among the vertebrates studied, and it raises the point that although we can distinguish operationally between tissue bias and specification, we still do not know what is required at the molecular level for either condition. In amphibian and mouse systems, where the bias period lasts many hours, it is possible to demonstrate its progressive nature by testing ectoderm at several time points. Specification could represent attainment of a threshold level of some factor(s) activated during the bias phase (i.e., it could be a quantitative distinction rather than a qualitative one), or there may be distinct “specification factors” acting near the end of the bias phase. Early specification of chick head ectoderm does not necessarily signify a fundamental difference in the mechanism underlying lens determination. As far as can be judged from reviewing the literature, lens determination in chicks appears in all other regards to proceed similarly to that in mice and frogs. Because specification is defined operationally by the tissue’s ability to undergo at least rudimentary lens differentiation, we discuss the molecular correlates in the next section.