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

enhancer” (EE), as it directs reporter gene expression to the ectoderm that gives rise to the lens, the lacrimal gland epithelium, and the corneal epithelium (Kammandel et al., 1999; Williams et al., 1998; Xu et al., 1999). Deletion of the EE by gene targeting results in mice with small eyes, small lenses, and a persistent lens stalk (Dimanlig et al., 2001). The presence of lenses in these animals, as well as a lower than normal level of Pax6 immunoreactivity in the lens placode, indicates that a second Pax6 enhancer must also have activity in the lens lineage (Dimanlig et al., 2001). Indeed, more recent analysis has identified the so-called SIMO element, a Pax6 enhancer that has activity in the lens lineage and is positioned 140 kb downstream (Kleinjan et al., 2001). SIMO was discovered through analysis of aniridia patients, in whom a translocation separated the SIMO element from the Pax6-coding region. When we combine (1) evidence that Pax6 is necessary and sufficient for lens induction with (2) analysis showing two distinct phases of Pax6 expression and

(3) evidence for the involvement of the EE and SIMO enhancers, we can propose a model (Fig. 11.2) describing some features of the genetic regulation of lens induction.

11.1.1.2. Meis1

The mammalian Meis family transcription factors are TALE-class homeodomain transcription factors (Burglin, 1997) and homologs of the homothorax gene of Drosophila melanogaster that has been implicated in eye development (Bessa et al., 2002). Recent analysis has implied that Meis1 and Meis2 have important roles in regulating lens induction in vertebrates (Zhang et al., 2002). Specifically, Meis-binding sites have been identified in the ectoderm enhancer of Pax6, and, furthermore, various experimental outcomes support the case that in vivo Meis transcription factors regulate Pax6 expression through this element. The experimental evidence includes (1) immunoidentification of Meis in a complex with EE probes in mobility shift assays, (2) transgenic mice showing that the activity of EE is dependent upon Meis-binding sites, (3) a genetic interaction between a Meis2 transgene and the Pax6Sey1-Neu allele, and (4) the demonstration that suppressor forms of Meis1 can down-regulate Pax6 expression when transiently expressed in the lens placode. Given the existence of two enhancers regulating Pax6 expression in the lens placode (Fig. 11.2), the latter experimental outcome requires that Meis transcription factors affect both the EE and SIMO elements. The identification of Meis-binding sites in the SIMO element is of great interest, but further experimentation will be required to precisely define the molecular genetics of this interaction. In particular, it will be very interesting to further analyze the eye phenotypes that arise in mice null or conditionally targeted for the Meis genes (Zhang et al., 2002). Since expression of the Meis genes is independent of Pax6, they are best incorporated into the model for genetic regulation of lens induction as an input upstream of both the EE and SIMO elements (Fig. 11.2).

11.1.1.3. FoxE3

FoxE3 is a forkhead family transcription factor that begins to be expressed in the mouse in a small region of the midbrain and the presumptive lens ectoderm at approximately E8.75 (Brownell et al., 2000). Analysis has indicated that FoxE3 is the gene mutated in the dysgenetic lens (dyl) mouse, which shows defective lens development (Blixt et al., 2000; Brownell et al., 2000). The phenotype apparent in the dyl mouse includes development of a small lens, probably due to reduced proliferation, and the lack of separation of lens vesicle and surface ectoderm (a Peters’ anomaly–like feature). It has been shown that in gene-targeted

11.1.3. FGF Receptor Signaling Plays a Role in Lens Induction and Cooperates with Bmp7

It is frequently the case that developmental events require the coordinated action of distinct signaling pathways. This and the observation that FGF signaling regulates lens polarization (section 11.2) provided the motivation to examine the question of whether FGF signaling is involved in lens induction. Expression of an inhibitory, dominant-negative form of FGFR1IIIc in the lens placode using the Pax6 ectoderm enhancer resulted in transgenic mice (designated Tfr7) with distinctive defects in lens and eye development (Faber et al., 2001). The Tfr7 mice showed a delay in the thickening and invagination of the lens placode, a persistent lens stalk, a small lens pit and vesicle, and ultimately microphthalmia. An examination of Pax6 levels showed that immunoreactivity in the lens placode was lower than in wild-type mice. This suggested that, like Bmp7, FGF receptor signaling was important for lens induction and was genetically upstream of the EE and SIMO regulatory elements (Fig. 11.3; Faber et al., 2001).

These data also implied that Bmp7 and FGF receptor signaling might cooperate in lens induction. To test this possibility, Bmp7 heterozygote and Tfr7 mice were crossed through two generations, and the resulting phenotype was examined in animals of compound genotype. Interestingly, the severity of the lens phenotype was dramatically increased in Tfr7/Tfr7,Bmp7+/− embryos compared with Bmp7+/− or Tfr7/Tfr7 embryos. This implied a genetic interaction between the two loci and suggested Bmp7, FGF receptor signaling cooperation (Faber et al., 2001). To test this possibility at stages of lens induction, an assessment of Pax6 levels was also performed in wild-type, Tfr7/Tfr7, and Bmp7+/−,Tfr7/Tfr7 embryos at E9.5. This showed that while Tfr7/Tfr7 embryos had lower levels than wild-type embryos, the lowest levels were apparent in Tfr7/Tfr7,Bmp7+/−embryos. This indicated a strong genetic interaction between the Bmp7 and Tfr7 loci and provided further evidence for cooperation between Bmp7 and FGF receptor signaling in the regulation of Pax6 expression (Fig. 11.3; Faber et al., 2001). The progressively lower level of FoxE3 expression in wildtype, Tfr7/Tfr7, and Tfr7/Tfr7,Bmp7+/− mice confirmed the proposed genetic relationship of FGF receptor and Bmp7 signaling to FoxE3 (Faber et al., 2001).

11.1.4. Bmp4 Involvement in Lens Induction and Development

Another member of the Bmp family, Bmp4, has been implicated in early development of the lens. Some Bmp4 null embryos survive until E10.5, and this is just sufficient to examine the consequences of Bmp4 absence for the first stages of eye development (Furuta and Hogan, 1998). Interestingly, absence of Bmp4 results in the lack of lens formation. Tissue explantation techniques have also been employed to extend the period in which analysis can be performed beyond E10.5 (Furuta and Hogan, 1998), and this strategy has been used to show that lens formation in Bmp4 null eye primordia could be rescued if the primordia were cultured in the presence of recombinant Bmp4 (Furuta and Hogan, 1998). Furthermore, lens formation from Bmp4 null presumptive lens ectoderm was rescued if the ectoderm was explanted adjacent to wild-type optic vesicle but not rescued if it was explanted only with Bmp4. These data have been interpreted to suggest that lens induction is a result of presumptive lens ectoderm responding to Bmp4 and at least one other signal derived from the optic vesicle (Furuta and Hogan, 1998). Alternatively, it is possible that Bmp4 has an essential role in the development of the optic vesicle and that in the absence of this factor optic vesicle–derived lens induction signals are not produced.

also not up-regulated in the usual way in Pax6Sey/Sey embryos, we can suggest that Sox2 is downstream of Pax6pre-placode and participates with Bmp4 in the proposed parallel pathway (Fig. 11.4). It is likely that currently unpublished work examining the requirement for different Bmp receptors in lens induction will help refine our understanding of Bmp4 involvement in lens induction.

11.1.5. Retinoic Acid

The study of retinoic acid (RA) involvement in any developmental process has some inherent difficulties because this signaling agent is not a gene product (one researcher recently stated that “we work on retinoic acid signaling, but we don’t like it”). Despite this, there is emerging evidence that RA and its related molecules have an important role to play in lens development.

RA receptors (RARs) reside in the nucleus and are ligand-inducible transcriptional regulators (Petkovich et al., 1987). RARs belong to a superfamily of nuclear receptors and are involved in a wide range of developmental processes. It now appears that there are two families of retinoid receptors. Members of the RAR family (RARs α, ß, and γ and their isoforms) are all activated by both all-trans RA and 9-cis RA. In contrast, members of the retinoid X receptor (RXR) family (RXRs α, ß, and γ ) are activated only by 9-cis RA. In addition to the ligand and its receptors, there are also cellular retinoic acid–binding proteins (CRABPs) that bind RA and can modulate the action of endogenous retinoids by sequestering RA, thus preventing it from activating the RARs (Perez-Castro et al., 1993).

A role for retinoid signaling in late lens development is supported by evidence that expression of some crystallin genes is regulated by retinoid receptors. For example, RARs and RXRs function additively with Pax6 to activate transcription of the mouse αB-crystallin gene (Gopal-Srivastava et al., 1998). Consistent with this is the observation that transgenic mice expressing a RA-binding protein at high levels in the lens show defects in the development of lens fiber cells. However, there is also strong evidence that RA signaling has an important role in lens induction.

When early mouse embryos are exposed to retinol-binding protein antisense oligonucleotides, RA signaling is effectively inhibited according to readout from a RA-sensitive lacZ reporter (Bavik et al., 1996). The inhibition of RA signaling results in defects in eye formation, specifically a failure of the lens placode to invaginate and form the lens pit (Bavik et al., 1996). This suggests that RA signaling has a critical early role in lens induction. Supporting this idea is the observation that the RA-responsive lacZ reporter is expressed in the neuroepithelium of the optic pit at E8.5 and in the lens lineage from E8.75 (Enwright and Grainger, 2000), at stages when lens induction signals are believed to be critical. Furthermore, Pax6Sey/Sey embryos, in which Pax6 activity is absent, show diminished expression of the RA-sensitive reporter in the lens placode (Enwright and Grainger, 2000), suggesting that by some means Pax6 regulates the activity of this pathway. It has thus been suggested (Enwright and Grainger, 2000) that some aspects of the Pax6 deficiency phenotypes are a result of diminished RA signaling. This notion is supported by the observation that RAR/RXR null mice have defects in lens development (Kastner et al., 1994) and that exposure of mouse embryos to 13-cis RA produces a Peters’ anomaly – like defect characteristic of Pax6 heterozygosity (Cook and Sulik, 1988).

Further implicating RA as an important lens induction signal is evidence that two RA responsive transcription factor genes are implicated in lens induction. The Meis

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transcription factors have already been mentioned in the context of Pax6 regulation, but in the limb it is clear that Meis1 and Meis2 are downstream of RA signaling and are critical proximal limb determinants (Capdevila et al., 1999; Mercader et al., 1999; Mercader et al., 2000). Given the possibility of conserved pathway function, it is worth investigating whether in lens induction RA signaling regulates the transcription of Meis genes. Similarly, the AP-2α gene is RA responsive and when mutated results in distinctive defects in early development of the eye, including the lens (Nottoli et al., 1998; West-Mays et al., 1999). With this information available, there is every opportunity to devise how RA regulates AP-2α and Meis transcription factors in the context of lens induction.

11.1.6. Other Elements of Lens Development Genetic Pathways

While there are still many genetic relationships that are not completely understood, several additional elements can be placed in the model of genetic regulation of early lens development. Six3 is a homeodomain transcription factor related to Drosophila sine oculis (Oliver et al., 1995). Analysis of Six3 expression in embryos with a conditional deletion of Pax6 in the lens placode (Ashery-Padan et al., 2000) indicates that Six3 lies genetically downstream of Pax6placode (Fig. 11.4). This is also true for Prox1 (Ashery-Padan et al., 2000), a homeobox transcription factor that is related to Drosophila prospero (Tomarev et al., 1998) and is required for lens fiber cell development (Wigle et al., 1999). Interestingly, the FoxE3dyl/dyl mice show an expansion of the Prox1 expression domain in the lens epithelium (Blixt et al., 2000), suggesting that FoxE3 plays a role in suppressing Prox1 at later stages of lens development (Fig. 11.4).

11.1.7. Future Challenges in the Analysis of Lens Induction Mechanisms

Several challenges face researchers interested in understanding lens induction. Despite the relatively complex genetic pathways that can be proposed to describe lens induction, we currently know remarkably little about how the various components orchestrate the cellcell interactions that are presumably crucial for development of the lens. For example, the Bmp7 gene is expressed in the presumptive lens ectoderm (Wawersik et al., 1999), and it is therefore tempting to suggest that it may signal to the presumptive retina. Thus far, however, there is no evidence for this type of signaling event. So too we currently have no direct evidence that an Fgf ligand is the mediator of an optic vesicle to presumptive lens signal in lens induction.

A further challenge will be to understand how the various signaling pathways involved in lens induction are integrated. There is strong evidence for involvement of Fgf and Bmp signaling pathways and circumstantial evidence for the involvement of RA signaling. Wnt signaling pathways have also been implicated. Modulation of Wnt signaling through misexpression of the Wnt receptor Xfz3 in Xenopus laevis results in the formation of lenses in the context of ectopic eyes (Rasmussen et al., 2001), and deletion of the Wnt signaling coreceptor Lrp6 results in a microphthalmia (Pinson et al., 2000) that appears to have its origin in the inductive phases of eye development (Stump et al., 2003). Thus, there are as many as four distinct pathways that must be integrated in a finely tuned way if the eye is to develop normally and reproducibly. An analysis of pathway integration will require all of the tools from the developmental geneticist’s toolkit.

Figure 11.5. (See color plate XVI.) (A) Wholemount of the lens epithelium of a weanling rat, stained with hematoxylin. The equatorial region is shown. Anterior to the equator, the lens epithelial (epi) cells exist as a monolayer and show cobblestone-like packing. Below the equator, the cells are aligned in very distinct meridional rows. (B) The histological section of the weanling rat lens is stained with an antibody for ß-catenin to show cell boundaries. Cells above the equator are columnar in shape, whereas below the equator in the transitional zone (TZ) the cells are elongated. The arrows indicate equivalent regions known as meridional rows in wholemounts (A) and the transitional zone in sections (B). Together the wholemount and section show that the elongating fiber cells are highly aligned. Scale bar: 11 µm.

11.2. Lens Differentiation and Growth

The later stages of lens morphogenesis are characterized by the differentiation of two forms of lens cells from the lens vesicle. Differentiation of epithelial and fiber cells involves acquisition of distinctive morphological and molecular characteristics required for the function of cells in the two lens compartments. The epithelial cells are cuboidal and possess strong intercellular adhesion and communication properties. They are firmly attached to the lens capsule and form an epithelial sheet that covers the anterior surface of the fiber mass. The fibers are highly elongated cells. During differentiation, each cell develops a similar hexagonal shape, with four short sides and two long sides, and this results in the fibers assuming a highly ordered packing arrangement (see chap. 4 of this volume). This is readily evident in wholemount preparations that include the meridional rows (Fig. 11.5).

One of the best general markers for lens cell differentiation is the presence of crystallins. These proteins are abundant in the lens and progressively accumulate during morphogenesis and growth. In mammals, α-crystallin appears during formation of the lens pit, and as lens morphogenesis progresses, it is produced by all lens vesicle cells and their progeny, epithelial and fiber cells (McAvoy, 1978b). ß-Crystallin and γ -crystallin first appear in the posterior cells of the lens vesicle that form the primary fibers, but unlike α-crystallin, their expression remains restricted to the fiber cell compartment throughout life (Fig. 11.6; McAvoy, 1978a). Differences in crystallin composition are only one feature that distinguishes epithelial and fiber cells at the molecular level. Epithelial cells and fiber cells express their own distinctive repertoires of communication, adhesion, and cytoskeletal molecules, and these determine

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Figure 11.6. Typical distribution of crystallins in the neonatal rat lens. Immunohistochemistry carried out on serial sections shows α-crystallin (A) is detected in both lens epithelial (epi) and fiber cells. ß-crystallin (B) is first detected early in fiber elongation just below the lens equator (see Fig. 11.5), and γ -crystallin (C) is first detected in young fiber cells in the lens cortex. Neither ß- nor γ -crystallins are detected in the lens epithelium. Scale bar: 50 µm. (Adapted from McAvoy, 1978a.)

Figure 11.7. Section of an 11-day embryonic chicken lens which had been inverted and left in situ for five days. The polarity of the lens has become reversed. A new fiber mass has developed posteriorly from the original lens epithelium, and a new epithelial layer has formed anteriorly. Scale bar: 1 mm.

their different structures and functions (see chaps. 6 and 7 of this volume). How the two forms of lens cells differentiate to bring about lens polarity has been a major focus of research.

The importance of the ocular environment for determining and maintaining lens polarity was demonstrated in the classic lens inversion experiments of Coulombre and Coulombre (1963). They turned the chicken lens upside down so that the epithelial cells, which normally faced the aqueous and cornea, faced the vitreous and neural retina instead (Fig. 11.7). In this new environment, the epithelial cells elongated and differentiated into a new fiber mass, whereas a new epithelial sheet grew over the surface of the lens facing the cornea. Similar lens inversion experiments using mouse eyes confirmed that this

phenomenon also occurred in mammals (Yamamoto, 1976). These experiments showed that the vitreous environment promotes the differentiation of fiber cells and that the aqueous environment promotes epithelial differentiation and growth.

The ocular media have been shown to constitute a rich source of growth and regulatory factors (McAvoy and Chamberlain, 1990). The lens itself expresses members of major growth factor families and a variety of growth factor receptors and molecules involved in a range of signaling pathways. Studies over the last 20 years have mostly concentrated on identifying the factor(s) that controls fiber differentiation. The influence of members of major growth factor families has been investigated, and as a result of promising leads, members of the insulin/IGF, FGF, and TGFß growth factor families have received a lot of attention. This work will be evaluated in this chapter, and other factors that have been shown to influence the behavior of lens cells will also be reviewed.

11.2.1. Regulation of Fiber Differentiation

11.2.1.1. Insulin/IGF

The first factor reported to influence lens fiber differentiation was insulin. Piatigorsky (1973) showed that insulin stimulated explanted lens epithelial cells from six-day-old embryonic chicks to elongate. Beebe et al. (1980) reported that a factor from the vitreous, lentropin, stimulated chick lens epithelial explants to elongate and accumulate δ-crystallin, a crystallin that is markedly up-regulated during fiber differentiation in chicks. Subsequently, it was shown that lentropin is functionally and immunologically related to IGF-1 (Beebe et al., 1987). More recent studies have confirmed the ability of insulin/IGF-1 to induce chick lens epithelial explants to elongate and accumulate fiber differentiation markers, including CP49, a component of the fiber-specific beaded filaments (Le and Musil, 2001).

The influence of insulin and IGF-1 on the differentiation of epithelial explants from neonatal rats has also been studied. In contrast to their influence in chicks, insulin and IGF-1 each have only a slight stimulatory effect on the fiber differentiation response. However, in the presence of FGF, insulin or IGF can synergistically enhance fiber differentiation (Chamberlain et al., 1991). Further analysis of this phenomenon has shown that members of the insulin/IGF family of growth factors can maintain fiber differentiation once initiated by FGF (Klok et al., 1998; Leenders et al., 1997). Interestingly, an exposure of 15 minutes is sufficient to initiate this process, which IGF can then maintain. Members of the insulin/IGF growth factor family are present in the ocular media (Arnold et al., 1993), and because of their biological activity in vitro, they are likely to have a role in regulating the process of fiber differentiation in vivo. Their role may be less prominent in mammals than in chicks, as the fiber differentiation effects of insulin/IGFs (on their own) appear to be much less pronounced in the former. This is supported by studies with transgenic mice. When IGF-1 was overexpressed in the lens, from the αA-crystallin promoter there was no induction of premature differentiation in the lens epithelial compartment. Instead there was an apparent expansion of the germinative and transitional zones toward the posterior lens pole (Shirke et al., 2001), indicating a role for IGF-1 in regulating proliferation in vivo (see section 2.2.1).

11.2.1.2. FGF

Researchers over a number of years have developed a strong case that members of the FGF family of growth factors play a central role in regulating fiber differentiation (see

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Figure 11.8. Electron micrographs of explants cultured with FGF for four days. Transmission electron microscopy (A) and scanning electron microscopy (B) show parallel packing of flattened fiber cells, with interlocking processes at the lateral margins; for example, tonguelike flaps overlap complementary imprints in neighboring cells (arrows, B and inset). Scale bar: (A), 0.5 µm; (B), 1 µm; (B) inset, 0.4 µm. (Adapted from Lovicu and McAvoy, 1989.)

Chamberlain and McAvoy, 1997). This case was initially based on evidence from in vitro studies but has received support from a variety of in vivo models designed to test the hypothesis that FGF is a key regulator of this process.

In Vitro Studies. The development of a rat lens epithelial explant system was central to the identification of members of the FGF family as potential regulators of fiber differentiation. In this culture system, FGF induces lens epithelial cells to undergo morphological and molecular changes characteristic of fiber differentiation (Lovicu and McAvoy, 1989). The explants become multilayered as epithelial cells migrate and elongate into fibers. Structural specializations include loss of cytoplasmic organelles, formation of specialized cell-cell junctions, and denucleation (Fig. 11.8). These and other FGF-induced changes are characteristic of lens fiber differentiation in situ (Fig. 11.9).

A Role for FGF in Vivo. For FGF to be a regulator of fiber differentiation in vivo, it must be present in the lens environment, and lens cells must express the appropriate receptors in situ. Currently there are 23 known members of the FGF family (Bansal, 2002). FGF-1 and FGF-2 were the first FGFs to be identified, and their distribution in the eye has been studied extensively. For most of the more recent additions to the family, detailed distribution studies have not yet been carried out. Using immunohistochemical and biochemical methods, it has been shown that, in addition to being present in lens cells and the lens capsule, FGF-1 and FGF-2 are present in the ocular media that bathe the lens and tissues near the lens,