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

44 Marilyn Fisher and Robert M. Grainger

2.3.5. Differentiation

The end point of determination is the irreversible commitment to overt lens differentiation. It now seems clear that this is the step where interaction with the closely apposed optic rudiment is crucial. The first steps in overt lens differentiation (early crystallin expression and placode and vesicle formation) can occur in the absence of the OV, as demonstrated both by the existence of free lenses in vivo and the small lenses that form in vitro from isolated specified PLE. However, such lenses do not attain the same degree of maturation as normal lenses, and in some cases free lenses degenerate in the absence of a sustained interaction with the optic cup. One might then hypothesize that there is an early phase of differentiation inextricably linked, by definition, to the event of tissue specification and a later phase encompassing the final maturation of the lens, including the formation of elongated lens fibers and the expression of the full range of lens-specific proteins. Recently, Offield et al. (2000) provided evidence that the onset of differentiation in specified ectoderm in vivo is not controlled by events intrinsic to lens ectoderm. Using a line of Xenopus tropicalis that carries a transgene construct linking γ -crystallin promoter elements with the coding sequence for green fluorescent protein (GFP), they were able to directly assay the onset of lens differentiation by montoring GFP expression in living animals and tissue explants. They found that specified lens ectoderm transplanted into young, neural plate stage hosts delayed GFP expression by about 4.5 hours (the time required for the hosts to reach the developmental stage of the donor tissue), and they also found that when specified lens ectoderm was explanted in vitro, GFP expression was delayed by around 16 hours relative to the control embryos. It is not yet known whether the signals in the embyro controlling the timing of onset of differentiation come from the optic vesicle or other adjacent tissues.

2.3.5.1. Molecular Correlates of Lens Differentiation

A number of transcriptional regulators and growth factors have already been implicated in the initiation and/or maintenance of lens differentiation. The timing of expression of many of these factors fits well with the idea that there are early and late phases to differentiation. For more detailed discussion of the regulation of crystallin expression and factors regulating lens fiber differentiation and maintenance, see chapters 5 and 11 in this volume.

As mentioned in a previous section, Pax6 is expressed in both the PLE and OV during the bias phase, and in homozogyous small eye mice the early phase of differentiation is blocked so that placodes do not form and other potential regulators or indicators of early differentiation (e.g., γ FCry, Sox2, Eya1, and Six3) fail to be expressed in the PLE around the time of placode formation (Oliver et al., 1995; Xu et al., 1997; Furuta and Hogan, 1998; M. Fisher and R. Grainger, unpublished manuscript). Clearly, Pax6 is important to the early phase of differentiation, and there is evidence from two sources that the block in small eye mutants involves a failure of retinoic acid (RA) signaling (discussed in the next section).

Another group of regulatory factors that appear to be involved at several levels of lens determination consist of Sox proteins, members of the Sry-related HMG domain family. At least three Sox proteins are believed to have roles in activating crystallin gene expression in chick and mouse embryos (Kamachi et al., 1998). As noted previously, in Xenopus, Sox3 is expressed during the lens competence period but is turned off at the onset of bias and reexpressed in PLE at the time of specification (see Fig. 2.2). PLE expression of its

mammalian counterpart, MSox2 (also expressed during gastrulation) is dependent on bone morphogenetic proteins (BMPs) 4 and 7 and Pax6 (Furuta and Hogan, 1998; Wawersik et al., 1999). Sox1 expression is required for lens fiber differentiation, and in fact Sox2 in both chick and mouse embryos is down-regulated in lenses when Sox1 is turned on (see Fig. 2.2; Nishiguchi et al., 1998; Kamachi et al., 1998).

Like the Sox family of proteins, members of the Maf family of basic leucine zipper transcription factors, including c-Maf, Mafβ, and L-Maf, also play roles in the early and late phases of lens differentiation by regulating expression of crystallins and other lens-specific proteins (Sakai et al. 1997; Yoshida et al., 1997; Ogino and Yasuda, 1998). Maf gene expression is up-regulated in specified ectoderm just prior to placode formation and early crystallin expression (Fig. 2.2; Ogino and Yasuda, 2000). Ectopic expression of L-Maf was shown to convert cultured neural retina cells into lens cells and to produce ectopic patches of δ-crystallin expression in the head ectoderm of chick embryos (Ogino and Yasuda, 1998). In homozygous c-Maf knockout mice, lens differentiation is blocked at the stage of fiber formation (Kawauchi et al., 1999; Kim et al., 1999).

A number of genes first expressed during the lens placode stage are implicated in the later differentiation and/or maintenance of either lens fiber cells or the anterior lens epithelium. Prox1, a homeobox gene homologous to Drosophila prospero, is first expressed in the placode stage in chick and mouse embryos and also appears to be important for the differentiation of lens fibers (Oliver et al., 1993; Tomarev et al., 1996; Wigle et al., 1999). Sw3–3, a putative basic-leucine zipper transcription factor expressed in chick lens placode and anterior lens epithelium, is suggested to have a role in lens differentiation (Wang and Adler, 1994). Similarly, FoxE3, a member of the forkhead family of transcription factors, is expressed in mouse lens placode and later is restricted to anterior lens epithelium. This factor maps to the murine dysgenetic lens locus, mutations in which result in the failure of lens vesicle separation from the corneal epithelium (Blixt et al., 2000; Brownell et al., 2000).

Thus, a number of factors are required for the onset of overt differentiation (i.e., placode and vesicle formation), and there is a second crucial point – that lens fiber differentiation requires a different set of factors. Interestingly, some of the factors required for later differentiation, such as Sox1 and c-Maf, are close relatives of factors required for early differentiation (i.e., Sox2/3 and L-Maf).

2.4. Inducing Signals

Presently we can only speculate as to the nature of the early lens-inducing signals that we now know must be arising first from the anterior neural plate and somewhat later from the foregut endoderm and head mesoderm. However, there is experimental evidence suggesting that RA, BMPs, and FGFs play important roles as signals during the stages of overt lens differentiation. Bavik et al. (1996) showed that RA deficiency beginning at 10–12 somite stages in mouse embryos leads to the failure of lens placode formation and optic vesicle invagination as well as to the interposition of mesenchyme between the OV and PLE – all features common to the small eye phenotype (Grindley et al., 1995). In addition, Enwright and Grainger (2000), using a transgenic mouse line carrying a RA response element (RARE)–β-galactosidase fusion construct, detected altered retinoid signaling in the heads of small eye mouse embryos. The RARE transgene expression pattern reveals that, during the development of wild-type embryos, RA-mediated gene activation is first apparent at late E8.5 in the OV and forebrain and by E8.75 in the PLE. By E9 the transgene is expressed in the OV, PLE, forebrain, PNE, and head mesenchyme (all structures that show

46 Marilyn Fisher and Robert M. Grainger

defects in small eye mice and that express Pax6 at this time). Enwright and Grainger also showed that RARE transgene expression was dramatically reduced in the PLE and PNE of small eye mice and that this was due to defects in the abilities of these tissues to respond to exogenously applied RA. Furthermore, the PNE and PLE did not produce RA, unlike their wild-type counterparts. These results suggest that, among other things, Pax6 acts within the head ectoderm to influence both response to and production of RA. In contrast to the PLE, the OV of small eye mutants appeared to have normal responsiveness to exogenous RA and only a mild reduction in RA production.

Other factors that are likely to be involved in the early differentiation phase are BMP4 and BMP7, members of the transforming growth factor-β superfamily. Furuta and Hogan (1998) showed that, in mice, BMP4 is expressed in the OV but not the PLE and becomes further restricted to the distal tip of the OV underlying the PLE just before placode formation. They further showed that, in homozygous BMP4 knockout embryos that survive to E10.5, no lens placode forms, and Sox2 expression is not turned on in the PLE as it normally is, just prior to placode formation. In contrast, Pax6 and Six3 expression appears normal in the PLE of BMP4 mutant embryos. Lens formation in cultured eye rudiments from these mutants could be rescued by loading the OV with BMP4-coated beads, but the beads alone could not substitute for the OV. Furuta and Hogan also demonstrated that BMP4 was acting through the OV by showing that recombinants of wild-type OV and BMP4 mutant head ectoderm formed lenses at a high frequency whereas the reciprocal of mutant OV with wild-type head ectoderm formed lentoids at a low frequency. These results demonstrate that BMP4 acts within the OV to elicit the OV’s lens-inducing capabilities. Interestingly, they found that small eye mutants showed normal expression of BMP4 and its receptors, Alk3 and Alk6, suggesting that BMP4 and Pax6 are regulated and function independently with regard to lens determination.

BMP7 was first implicated in the late stages of lens differentiation (Dudley et al., 1995), but a more recent study by Wawersik et al. (1999) suggests that it is also involved in the early stages of differentiation. They showed that, on the C3H genetic background, a BMP7 knockout mutation blocked lens placode formation in homozygous embryos. They also demonstrated that both the BMP7 antagonist follistatin and a BMP7-neutralizing antibody could block lens-forming ability in cultured wild-type eye rudiments, reducing it to the same level as found in cultured eye rudiments from BMP7 mutants. Like Furuta and Hogan, they found that Sox2 was not expressed in the PLE of the mutants at E9.5, and in contrast to the BMP4 mutants, the BMP7 mutants also lost Pax6 expression from E9.5 PLE. Like BMP4, BMP7 was expressed normally in the PLE of small eye mutants.

Already mentioned earlier as potential candidates for competence factors, members of the FGF family are also implicated in lens fiber differentiation and maintenance. In chicks, FGF-8 is expressed in the distal optic vesicle when it contacts the PLE, and implantation of FGF-8–coated beads near the eye, soon after the OV contacts the PLE, leads to expansion of the lens field, as evidenced by the expanded domain of L-Maf expression (Vogel-Hopker¨

et al., 2000). The importance of FGF activity in fiber cell differentiation and maintenance is further underscored by a number of recent transgenic studies (see the review in chap. 11 of this volume). Targeted overexpression of FGFs to the developing lens of transgenic mice resulted in inappropriate differentiation of the lens epithelium (Robinson et al., 1995a, 1998; Lovicu and Overbeek, 1998). Furthermore, transgenic mice expressing dominantnegative FGF receptors specifically in the lens fibers, beginning at the earliest stages of overt differentiation, developed small lenses with disorganized fibers (Robinson et al., 1995b; Stolen and Griep, 2000).

2.5. Conclusions and Future Directions

After a century’s worth of investigative efforts directed toward understanding the seemingly simple phenomenon of induction of lens development by interaction with the OV, we have come to appreciate the complexity of this process. We have developed a model of lens determination that draws heavily on amphibian studies but appears to be generally applicable to other vertebrates based on the more limited information available from chick and mouse studies.

Our current model envisions a process that begins during gastrulation, when, through some intrinsic timing mechanism, ectoderm becomes competent to respond to signals from the anterior neural plate. These early signals impart a lens-forming bias to a large portion of the ectoderm. Additional signals, likely coming from mesoderm and/or endoderm, during the early neurula stages enhance the lens-forming bias in the ectoderm of the head until it reaches the point of specification, at which time the earliest stages of overt lens differentiation can occur, even if the ectoderm is removed from further inductive influences. At this point, the OV establishes an intimate relationship with a region of specified head ectoderm and provides the inductive signals necessary for final commitment and maturation of the lens. At this same point, head ectoderm not in close contact with the OV loses its lensforming bias, likely due at least in part to signals from the underlying migrating neural crest and ventral neural midline. Interference at any of these steps can disrupt lens determination. We do not yet know the nature of the several inductive signals that ultimately act on the PLE, although RA, BMPs, and FGFs have all been implicated in one or more phases of the process. Our present understanding of the timing and likely participants in interactions with the PLE should help to focus the search for the key inductive signals. Likewise, elucidating the relationships among the emerging array of transcription regulatory factors, many of which are also known to function in Drosophila eye development, will help to further clarify the complex process of lens determination.

Although mammalian embryos are less well suited to the classical type of embryo manipulation that has proven so useful in defining critical tissue interactions during lens induction, they are proving quite valuable for their genetic manipulability. Naturally occurring and engineered mutations that affect lens determination provide a very useful resource for elucidating the molecular mechanisms that underlie each phase of lens determination. In this regard, the promise of X. tropicalis, the diploid, rapidly maturing relative of X. laevis, as a genetic system (Amaya et al., 1998; Offield et al., 2000) could give amphibians the clear edge, as far as experimental organisms go, by combining the best of both worlds – classical embryological manipulability combined with the potential for genetic analysis. In addition, new technologies for genetic analysis, such as DNA microarray analysis, are opening up the possibility of rapid identification of previously unrecognized genes that are involved in specific phases of lens determination.

addition, a combined misexpression of factors is often required (Bonini et al., 1997; Chen et al., 1997a; Pignoni et al., 1997; Shen and Mardon, 1997).

Remarkably, the vertebrate orthologs of these different factors have also been shown to be involved in eye development. Pax6, the vertebrate ortholog of ey, is required for eye development in most species so far studied (reviewed in Gehring and Ikeo, 1999; Wawersik et al., 2000), and experiments involving misexpression of Pax6 in Xenopus laevis have led to ectopic lens and eye formation (Altmann et al., 1997; Chow et al., 1999). Like in Drosophila, vertebrate Pax6 is therefore sufficient to initiate the chain of events leading to eye formation, indicating a widespread role for Pax6 as a key regulator of eye development. In vertebrates, the function of the ey-related toy gene is assumed to have been taken by Pax6, since an additional Pax6-like gene has only been identified in zebrafish (Nornes et al., 1998). Vertebrate orthologs for genes belonging to the remaining gene families have been isolated and shown, in some instances, to be expressed during eye formation (Borsani et al., 1999; Hammond et al., 1998; Heanue et al., 1999; Oliver et al., 1995a; Oliver et al., 1995b; Toy et al., 1998; Xu et al., 1997b) and to adopt gene regulatory strategies similar to those seen in Drosophila (Heanue et al., 1999; Loosli et al., 1999; Ohto et al., 1999; Zuber et al., 1999; reviewed in Relaix and Buckingham, 1999). Details concerning targeted mutations for the latter genes are, for the most part, either unavailable or inconclusive regarding their role in vertebrate eye development (Davis et al., 2001; Ozaki et al., 2001; Xu et al., 1999b). However, gain-of-function studies (Loosli et al., 1999; Oliver et al., 1996) and information gathered from cases of human mutations (Azuma et al., 2000; Gallardo et al., 1999; Wallis et al., 1999) indicate that these genes do participate in vertebrate eye formation.

3.3. Transcription Factors from Different Classes Are Involved in Lens Development

Recent studies have revealed additional transcription factors involved in eye and lens formation (see Fig. 3.2 and Table 3.1). These additional factors have been uncovered, for the most part, through reverse genetic approaches from existing Drosophila, mouse, or human mutations or on the basis of their capacity to bind to the regulatory sequences of lens-specific genes. These genes will be described according to the transcription factor classes to which they belong. Although members of most classes of transcription factors are expressed in the lens, transcription factors belonging to the homeodomain group are by far the must abundant and best studied regulatory factors involved in eye and lens formation and will therefore be described first.

3.3.1. Homeobox Genes

The homeodomain (Gehring et al., 1994) consists of a 60–amino acid core motif encoded by a 180–base pair sequence called the homeobox, which functions as a dimerization as

Figure 3.2. (facing page) Inductive phases and expression of transcription factors during lens development. Top: Schematic views on the different stages of eye development with respect to the lens inductive events. Middle: Links between retina lineage, secreted factors from the optic vesicle/retina, and lens lineage. Bottom: Expression timetable of transcription factors during lens development. C, cornea; LE, lens epithelial cells; LF, lens fiber cells; LP, lens placode; LV, lens vesicle; NR, neural retina; RPE, retinal pigmented epithelium; SE, surface ectoderm. For references, see corresponding chapters in the text. (After Ogino and Yasuda, 2000.)

54 Guy Goudreau, Nicole Baumer,¨ and Peter Gruss

well as a sequence-specific DNA-binding element. Additional motifs are often found in homeodomain proteins, and these have also been shown to participate in protein-protein interactions and in DNA binding. Of the more than 25 classes of homeodomain identified to this date, the members from 7 classes of homeodomain proteins have been shown to participate in lens development.

3.3.1.1. Pax6

Pax genes were isolated on the basis of the presence of a 384–base pair sequence called the paired box, which shares sequence similarities to the Drosophila paired gene (Noll, 1993). The resulting 128-amino-acid-long protein structure, the paired domain, acts as a sequence-specific DNA-binding domain (Wilson et al., 1995). Members of the Pax gene family have been subdivided primarily according to the presence or absence of a pairedtype homeobox, which may be partial or complete, or according to whether they encode a conserved octapeptide (Noll, 1993; Walther et al., 1991). The latter motif corresponds to the consensus sequence (H/Y)S(I/V)(N/S)G(I/L)LG (Noll, 1993) and is related to the core (eh1) region of the engrailed repressor domain (Eberhard et al., 2000). Pax genes have been isolated from several vertebrate and invertebrate species, are widely expressed during embryogenesis, and are involved in developmental processes and in tumorigenesis (Barr, 1999; Dahl et al., 1997; Mansouri et al., 1999; Nutt et al., 2001). In vertebrates, nine members of the Pax gene family have been identified. Of these nine Pax genes, only Pax6 is expressed in the lens (Walther and Gruss, 1991). In addition to its well-described function during eye development (Gehring and Ikeo, 1999; Mathers and Jamrich, 2000; Wawersik et al., 2000) in vertebrate species, Pax6 is also involved in the development of the central nervous system (Bishop et al., 2000), nose (Quinn et al., 1996), pituitary gland (Kioussi et al., 1999), and pancreas (Dohrmann et al., 2000). These different requirements for Pax6 function are indicative of the multiple functions that Pax6 is likely to carry out during development.

Structurally, human and murine Pax6 proteins are 422 amino acids in length and contain a paired domain and a paired-type homeodomain but do not contain an octapeptide (Callaerts et al., 1997). Pax6 encodes a C-terminal proline-serine-threonine (PST)–rich domain, which is thought to function as a transactivating domain. Both the homeodomain (Sheng et al., 1997a; Sheng et al., 1997b) and the paired domain (Czerny and Busslinger, 1995; Epstein et al., 1994a) of Pax6 appear to function independently as well as in cooperation to bind specific DNA sequences. Pax6 can also bind DNA either through the N-terminal or C-terminal portion of the paired domain, increasing the number of possible DNA-binding sites (Epstein et al., 1994b; Xu et al., 1999a). Additionally, a splice variant (Pax6(5a)) has been identified in several vertebrate Pax6 genes, and it results in a 14–amino acid insertion in the N-terminal portion of the paired domain (Epstein et al., 1994b; Kozmik et al., 1997). In vitro experiments have indicated that this insertion disrupts the ability of the paired domain to bind DNA, since the extended paired domain only recognizes DNA through its C-terminal portion, therefore limiting its DNA-binding capacity (Duncan et al., 2000; Epstein et al., 1994b; Kozmik et al., 1997).

Pax6 Expression during Lens Formation. All Pax6 genes so far studied have been found to be expressed in the developing eye (Gehring and Ikeo, 1999), and detailed analysis of Pax6 expression has been done in the developing ocular structures of several species (Callaerts et al., 1997). In the developing mouse eye (Walther and Gruss, 1991), Pax6 mRNA is first detected at embryonic day 8.0–8.5 (E8.0–8.5) on the head surface