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Keywords:

  • induction;
  • lens;
  • maf;
  • Pax6;
  • transcription factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Classic and recent embryological studies
  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References

Since the pioneering work of the early 1900s, the lens has been used as a model system for the study of tissue development in vertebrates. A number of embryological transplantation experiments designed to elucidate the role of tissue interactions in the formation of the lens have led to the proposal of a stepwise determination model. This model has recently been refined through the identification of certain transcription factor genes, which exhibit distinct expression patterns and functional properties in the lens cell lineage. Otx2, Pax6, and Lens1 are induced by the adjacent anterior neural plate and expressed in predifferentiated lens ectoderm. Contact between the optic vesicle and lens ectoderm promotes expression of mafs, Soxs, and Prox1, which are responsible for the initiation of lens differentiation programs including crystallin expression, cell elongation, and cell cycle arrest. Further analysis of the expression and functional characteristics of these transcription factors will allow greater detail when describing the orchestration of genetic programs, which control tissue development from induction to maturation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Classic and recent embryological studies
  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References

An overview of lens development

During development, the lens originates from head ectoderm, maintaining a close relationship with the developing retinal primordium. Lineage analyses in chicken and Xenopus embryos have shown that, during neural plate stages, the presumptive lens area is located in lateral head ectoderm adjacent to the presumptive retina area of the anterior neural plate ( Fig. 1A–C; Couly & Le Douarin 1987, 1990; Eagleson et al. 1995 ; Zygar et al. 1998 ). After the anterior neural plate gives rise to the forebrain vesicle, the presumptive retina region protrudes from the forebrain toward the presumptive lens ectoderm (PLE) to form the optic vesicle ( Fig. 1D–F). When the developing optic vesicle reaches the PLE, the PLE cells begin to elongate and form the lens placode ( Fig. 1F,G; reviewed by Coulombre 1965; McAvoy 1980). The thickened lens placode invaginates toward the optic vesicle and subsequently pinches off from the ectoderm to form a hollow structure, the lens vesicle ( Fig. 1H–K). The cells composing the posterior wall of the lens vesicle dramatically elongate to fill the enclosed cavity while differentiating into primary lens fibers ( Fig. 1L). Cell cycle arrest and enucleation are associated with this differentiation process. The anterior cells in the lens vesicle become lens epithelial cells, a monolayer, which surrounds the lens fibers. The lens epithelial cells continuously proliferate at the equatorial region and differentiate into secondary lens fibers. Growth of the lens vesicle results from the continuous overlaying of new concentric layers of secondary lens fibers around the older layers. During the formation of the lens vesicle, the optic vesicle also changes its shape to form the optic cup. In subsequent stages, the inner and outer layers of the optic cup differentiate into the neural retina and retinal pigment epithelium, respectively.

image

Figure 1. Schematic representation of lens formation in vertebrates. The lens lineage is shown in blue, while the retina lineage is shown in orange. (a) Dorsal view of a chicken and (B) Xenopus embryo at the neural plate stage. (C–L) Cross-sections of a developing chicken embryo through lens- and retina-forming tissue. The development proceeds from (C) to (L). The dotted line in (a) indicates the plane of section for (C). (G–K) Close-ups showing lens vesicle formation. The rectangle in (F) indicates the region viewed in (G). (L) Close-up of a maturing lens. PLE, presumptive lens ectoderm; PR, presumptive retina; OV, optic vesicle; LP, lens placode; OC, optic cup; LV, lens vesicle; LF, lens fiber; LE, lens epithelium; NR, neural retina; PE, pigment epithelium.

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The differentiation of ectoderm into lens is accompanied by the specific activation of crystallin genes, which encode a group of structural proteins that confer transparency and reflectivity to the lens (reviewed by Piatigorsky 1981). Major crystallin proteins, which have been employed as molecular markers of lens tissue, are classified into four groups: α-, β-, δ-, and γ-crystallins. Of these groups, α- and β-crystallins are conserved throughout all major vertebrate species while the others show more variability. The γ-crystallins are found in fish, amphibians and mammals, whereas δ-crystallins are found in reptiles and birds. In the course of lens development, crystallin genes are expressed with distinct spatio-temporal specificity. In the chicken, δ-crystallins (α-crystallins in mammals) are expressed first in the lens placode, followed by expression of α-crystallins immediately after formation of the lens vesicle. Fiber differentiation in the lens vesicle is accompanied by activation of β-crystallins (and also γ-crystallins in mammals), with a concomitant steep increase in α- and β-crystallin expression.

Classic and recent embryological studies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Classic and recent embryological studies
  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References

Early in this century, the close physical relationship between the development of the optic vesicle and the lens was examined by Spemann and Lewis in a famous set of experiments. Using a hot needle, Spemann destroyed the presumptive retina region of the anterior neural plates of frog (Rana fusca) embryos ( Spemann 1901). The resulting embryos failed to form not only optic vesicles but also lenses on the operated side, leading to his conclusion that stimuli from the optic vesicle are essential for lens formation. Independently, Lewis performed a more elaborate experiment in which he transplanted optic vesicles under flank ectoderm in Rana palustris embryos ( Lewis 1904). These embryos formed ectopic lenses associated with the transplanted optic vesicle; thus, he proposed that the optic vesicle was sufficient to convert multipotent ectoderm into lens. These studies are considered to be the first experimental documentation of embryonic induction, an important and widespread mechanism of epigenetic development.

Shortly after these initial studies, however, several other investigators reported evidence contradicting this understanding of lens induction (reviewed by Hamburger 1988). The first was Mencl, who found a double-headed salmon embryo in which two lenses had fully differentiated in the absence of optic cups ( Mencl 1903). King documented the formation of lens-like structures in R. palustris after carrying out a series of experiments in this species similar to those of Spemann ( King 1905). Spemann himself observed formation of lens-like structures when he performed a further series of retinal ablation experiments in Rana esculenta. Returning to R. fusca, he repeated his original experiments, but in no case observed the formation of lenses or lens-like structure in this species.

At the end of 1980s, lens induction, which had remained obscure, was systematically reinvestigated by Grainger and colleagues. They performed optic vesicle transplantation experiments using horseradish peroxidase as a lineage tracer to clearly distinguish donor tissues from host tissues ( Grainger et al. 1988 ). Using this technique, they revealed that the ectopically formed lenses are exclusively derived from contaminating donor cells carried along with the transplanted optic vesicles. These results were obtained using embryos from Xenopus as well as R. palustris, the first organism used by Lewis for such transplantation studies. Next, they carried out a further series of experiments in Xenopus to elucidate essential events in lens formation. The results led them to propose the currently favored stepwise determination model, which includes four phases: Competence, bias, specification, and differentiation ( Fig. 2; reviewed by Grainger 1992). Lens competence, the ability of the ectoderm to respond to putative lens-inducing signals, was examined by transplanting labeled ectoderm from different regions or stages into the presumptive lens area of neural plate stage hosts ( Henry & Grainger 1987). Competence first appears during mid-gastrula stages in all regions of animal cap ectoderm. As gastrulation proceeds, competence is rapidly confined, eventually to the region around the PLE upon reaching neural plate stages. The second phase, lens-forming bias, was elucidated via ablation experiments of the presumptive retina ( Henry & Grainger 1990). When the anterior portion of the neural plate containing the presumptive retina is surgically removed at early neural plate stages, the PLE cannot form the lens, as Spemann observed. However, when removed later, just prior to developing into the optic vesicle, the PLE usually forms the ‘free lens’. Based on these findings, Grainger proposed that the essential inductive events in lens formation occur during neural plate stages, at which time the competent PLE is strongly biased towards a lens fate by receiving ‘early inducing signals’ from the adjacent anterior neural plate. It is noted that the mesoderm underlying the PLE enhances this inductive effect. The third phase, lens specification, occurs around the time of optic vesicle contact with biased PLE during neural tube stages. In the stepwise model, the exact role of the optic vesicle remains elusive, but considering the rudimentary structure of the free lens and the fact that expression of the key regulators of lens differentiation commences directly after contact with the optic vesicle (described later), the optic vesicle seems to provide ‘late inducing signals’ to the PLE. These ‘late inducing signals’ are thought to fully activate the genetic programs of lens specification. The development of optic vesicle into optic cup accompanies the final phase, lens differentiation, in which the optic cup provides signals for the lens vesicle to stimulate terminal differentiation of lens epithelium into lens fibers.

image

Figure 2. Diagram showing a link between inductive interactions and expression of transcription factors during lens development. Developmental stages (at the top) and major inductive events between the retina- and lens-forming tissue (bold, dotted arrows) are illustrated according to the currently favored model ( Grainger 1992; Pittack et al. 1997 ). Signaling factors implicated in the respective inductive events are shown in yellow circles. The green and blue arrows represent the expression profile of transcription factors that were analyzed mainly at the messenger ribonucleic acid (mRNA) level in Xenopus, chicken, and/or mouse embryos. The transcription factors with green arrows are expressed in the presumptive lens ectoderm prior to contact with the optic vesicle, and those with blue arrows are expressed following contact. Only factors whose expression has been fairly well characterized are shown. In the final phase of lens differentiation, several fibroblast growth factors (FGF) may be involved (see text). References are described in the text.

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Transcription factors involved in specification of the presumptive lens field

  1. Top of page
  2. Abstract
  3. Introduction
  4. Classic and recent embryological studies
  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References

Lending support to the embryological studies that outlined the early events in specification of the PLE domain was the discovery of several transcription factors whose expression demarcates the PLE prior to optic vesicle contact (summarized in Fig. 2).

Otx2 expression in early presumptive lens ectoderm

One of the earliest genes to demarcate the PLE is Otx2, which encodes a bicoid-type homeodomain protein known to be essential for rostral head development (reviewed by Acampora & Simeone 1999). In addition to expression in anterior neural tissue and its underlying mesoderm from early gastrula stages, Otx2 is expressed by mid-neural plate stages in PLE as well as presumptive nasal ectoderm (PNE; Zygar et al. 1998). Otx2 expression in PLE subsequently disappears around the time of lens placode thickening. Tissue recombination experiments using lens- competent animal cap ectoderm and anterior neural plate tissue of Xenopus embryos suggest that expression in PLE is activated and/or maintained by putative lens-inducing signals provided by the anterior neural plate ( Zygar et al. 1998 ). Otx2 expression in both anterior neural tissue and PLE may suggest that these tissues share the same genetic machinery for development, at least during the initial stages of morphogenesis.

Pax6: A principal regulator of eye development

The Pax6 gene encodes a paired-type homeodomain protein whose expression occurs in PLE and PNE by late-neural plate stages, immediately following Otx2 activation in these tissues ( Zygar et al. 1998 ). Pax6 expression in cells of the lens lineage continues even after lens vesicle formation, but is restricted to the lens epithelium and nearly absent from lens fibers ( Grindley et al. 1995 ; Kamachi et al. 1998 ). Pax6 is also expressed in the presumptive retina region of the anterior neural plate, and subsequently in the optic vesicle and differentiating neural retina cells of the optic cup. Tissue recombination experiments performed on Xenopus embryos suggest that, like Otx2, expression of Pax6 in PLE depends on signals from the anterior neural plate ( Zygar et al. 1998 ).

The role of Pax6 in eye development seems to be fairly well conserved throughout the animal kingdom. The Drosophila homolog of Pax6, eyeless (ey), shows specific expression in the eye imaginal disc ( Quiring et al. 1994 ). Loss-of-function mutation of ey leads to the absence of eye structures, while targeted expression of this gene in other imaginal discs induces the formation of ectopic eye structures containing fully differentiated ommatidia ( Halder et al. 1995 ). Misexpression of Pax6 by injection of ribonucleic acid (RNA) into Xenopus embryos also leads to the formation of ectopic eyes with fully differentiated lenses and retinal tissues ( Chow et al. 1999 ). The mouse mutant of Pax6 is Small eye (Sey;Hill et al. 1991 ). In homozygous embryos of Sey, the lens placode fails to develop from PLE upon close apposition of the optic vesicle. Subsequently, the optic vesicle fails to develop into the optic cup, resulting in the absence of eyes. A mutation of Pax6 has also been identified in rats that exhibit a phenotype similar to that of Sey ( Matsuo et al. 1993 ). In tissue recombination experiments, PLE explants from + / + embryos form lenses even when cultured in combination with the rSey/rSey optic vesicles, whereas the rSey/rSey PLE explants never form lenses when cultured with the + / + optic vesicles ( Fujiwara et al. 1994 ). The results suggest that Pax6 activity in PLE but not in the optic vesicle is required for initiation of lens differentiation. These tissue recombination experiments and the overall course of Pax6 expression indicate involvement of this gene in maintenance of lens competence and/or induction of lens bias in PLE.

Lens1 maintains the state of lens ectoderm

Strong support for the model of PLE specification prior to optic vesicle contact comes from findings of the unique expression of Lens1 (Xlens1), whose product contains a fork head DNA-binding domain ( Kenyon et al. 1999 ). In contrast to Otx2 and Pax6, Lens1 is not expressed in the central nervous system and is only observed in PLE and PNE of Xenopus embryos by late-neural plate stages. Later, in the lens vesicle, Lens1 expression remains confined to the anterior lens epithelium. Misexpression of Pax6 in animal cap explants induces ectopic Lens1 expression, but Lens1 misexpression does not activate Pax6. In vivo, overexpression of Lens1 in Xenopus embryonic lens ectoderm causes the lens ectoderm to thicken and maintain gene expression characteristic of PLE. Interestingly, this ectoderm fails to differentiate into crystallin-expressing lens cells, suggesting that Lens1 acts to maintain the specified PLE in an undifferentiated state.

Transcription factors involved in lens development from presumptive lens ectoderm

  1. Top of page
  2. Abstract
  3. Introduction
  4. Classic and recent embryological studies
  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References

Contact of the optic vesicle with PLE is accompanied by activation of several transcription factor genes (summarized in Fig. 2), some of which were originally discovered as direct regulators of crystallin genes. They play overlapping but distinct roles in the promotion of various aspects of lens formation, such as crystallin expression, regional cell elongation and cell cycle arrest.

L-Maf and other Mafs trigger lens differentiation

L-maf and other maf genes (mafB, c-maf, Nrl, mafK, mafF and mafG) encode proteins that carry a basic-leucine zipper (bZIP) motif for DNA binding and dimerization (reviewed by Blank & Andrews 1997). Of these maf family members, L-maf, mafB, c-maf, and Nrl together make up the large maf subfamily, because, in addition to the bZIP domain, their products contain a distinctive acidic domain that serves to activate transcription. The rest of the mafs encode proteins that lack this activation domain and thus are referred to as small mafs.

Recent studies show independent roles for large Maf proteins as vital regulators of cellular differentiation and morphogenesis in several distinct developmental pathways. The role of MafB in hindbrain development is demonstrated by mutants in mouse (kreisler) and zebrafish (valentino) that fail to exhibit segmental structures caudal to rhombomere3 ( Cordes & Barsh 1994; Moens et al. 1998 ). MafB also acts in the hematopoietic system to establish and maintain the myelomonocytic lineage ( Sieweke et al. 1996 ). c-Maf is emerging as an important player in lymphopoietic cell lineage decisions, where it controls Th-2-specific IL-4 expression ( Ho et al. 1996 ), while Nrl is dominantly expressed in post-mitotic retinal cells and is responsible for activation of the rhodopsin promoter ( Kumar et al. 1996 ).

The crucial role of large mafs in lens differentiation was revealed by studies of lens-specific transcription of crystallin genes. By searching for the identity of the chicken αA-crystallin gene enhancer-binding factor, Ogino and Yasuda identified a novel member of the large maf family ( Ogino & Yasuda 1998). Expression of this lens-specific maf gene, L-maf, shows extreme localization to lens cells and is initiated in PLE immediately after contact with the optic vesicle. Expression of δ-crystallin follows L-maf expression in the thickened lens placode. L-maf expression remains restricted to the invaginating lens placode and subsequently to the lens vesicle, where αA-crystallin is turned on. In the developing lens vesicle, L-maf is expressed in both lens epithelium and lens fibers. However, expression is predominantly in lens fibers facing the optic cup, implying that L-maf expression remains under the influence of signals from retinal tissue.

L-Maf binds and transactivates not only from the cis-regulatory element of the chicken αA-crystallin gene, but from other crystallin elements, including that of chicken βB1-, chicken δ-, mouse αA-, and mouse γF-crystallin ( Ogino & Yasuda 1998). These elements all contain sequences similar to MARE (Maf-responsive element; TGCTGAC(G)TCAGCA), a consensus binding sequence for Maf proteins determined in vitro ( Kataoka et al. 1994 ). MARE-like sequences seem to be widely conserved in a variety of crystallin genes throughout the animal kingdom ( Table 1), including ‘taxon-specific crystallins’, the genes for metabolic enzymes expressed in a lens-preferred manner in certain species (reviewed by Piatigorsky & Wistow 1989). For instance, the guinea-pig quinone reductase gene, which is referred to as ζ-crystallin because of its abundant expression in the lens of this species, has both a housekeeping and a lens-specific promotor. The latter contains a MARE-like sequence and is controlled by one or more Maf family proteins ( Sharon-Friling et al. 1998 ). Even in squid, MARE-like sequences are contained in the promoter region of the S-crystallin gene (SL11), a derivative of Glutathione S-Transferase P, whose gene product is a lens protein in this species. This squid promoter element still possesses lens-specific activity in primary cultures of chicken embryonic cells, but loses this activity when a mutation is introduced into its MARE-like sequence ( Tomarev et al. 1994 ). It is noteworthy that the gene products of both quinone reductase and the original Glutathione S-Transferase P are among the phase II detoxifying enzymes. Their expression in the liver is induced by small Mafs through anti-oxidant responsive elements, which are actually cis-elements resembling MAREs ( Itoh et al. 1997 ). These findings suggest the intriguing possibility of gene recruitment mechanisms in evolution. Perhaps, over time, minor mutations occurred in the cis- regulatory sequences of these anti-oxidant enzyme genes, allowing for the expression of their products in response to Maf proteins present in the lens. As oxidative insult can lead to cataract formation, these enzymes might be preferentially recruited in order to protect this tissue.

Table 1.  Conservation of MARE-like sequences in the promoter/enhancer regions of crystallin genes. Thumbnail image of

The striking activity of L-Maf to convert non-lens cells into lens cells was shown by ectopic expression experiments ( Ogino & Yasuda 1998). Misexpression of this transcription factor in cultured neural retina cells induces formation of lens-like structures (lentoids) in vitro. These lentoids are composed of large, elongated cells characteristic of terminally differentiated lens fibers and express every lens protein tested thus far, including every class of crystallins and a lens fiber- specific cytoskeletal protein, filensin. Misexpression of L-Maf in vivo was carried out by in ovo electroporation of chicken embryos, which also leads to ectopic lens cell differentiation in head ectoderm. This differentiation is especially prominent in the ectoderm surrounding the ventral side of the intrinsic lens ( Fig. 3A), implying a restricted distribution of competence to respond to L-Maf.

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Figure 3. Maf proteins may function in synergy with Sox proteins. (a) As described previously ( Ogino & Yasuda 1998), chicken embryos were electroporated with expression plasmids encoding L-Maf and GFP (green fluorescent protein) at stage 9, then stained with anti-δ-crystallin antibody after a 36 h incubation. The GFP signal in the left panel shows the electroporated ectodermal domain overlying the anterior end of telencephalon to ventral diencephalon. Ectopic lens cell differentiation is prominent in the region ventral to the intrinsic lens (indicated by the arrow in the right panel), but is not observed in the anterior region (arrowhead). (B) Schematic representation of putative binding sites for Maf and Sox proteins in δ- and γF-crystallin regulatory elements. The nucleotide sequences listed in Table 1 are shown. The nucleotides identical to the consensus binding sequence for Maf (MARE, see Table 1) or Sox (CT/ATTGT/AT/A, Pevny & Lovell-Badge 1997) are underlined.

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The basic DNA recognition domain of L-Maf shares remarkable similarity with that of c-Maf and MafB (90% amino acid identity), and c-Maf and MafB can also activate transcription through the cis-regulatory elements of crystallin genes when overexpressed (H. Ogino & K. Yasuda, unpubl. data, 1996). Indeed, they are expressed in the lens in various species, including chicken (H. Ogino et al. unpubl. data, 1996), mouse ( Kawauchi et al. 1999 ; Kim et al. 1999 ; Ring et al. 2000), rat ( Sakai et al. 1997 ), and zebrafish ( Moens et al. 1998 ). In the rodent lens, c-maf and mafB show an exclusive expression pattern: c-maf expression is initiated in the lens placode and subsequently restricted to lens fibers, whereas mafB is restricted to lens epithelium. The essential role of c-maf in lens development was examined by the generation of knockout mice ( Kawauchi et al. 1999 ; Kim et al. 1999 ; Ring et al. 2000). In these mice, the lens vesicle forms normally from the lens placode, but the resulting lens vesicle ceases development and remains as a small hollow structure. The posterior cells of this hollow structure fail to form elongated lens fibers and may continue to proliferate inappropriately. This phenotype partially overlaps with that of Sox1 and Prox1 knockout mice (see following text, also Nishiguchi et al. 1998 ; Wigle et al. 1999 ). Fiber-specific γ-crystallin expression (γA, γB, γC, γD, γE and γF) is completely abolished, and the expression of other classes of crystallin genes (α and β) unaffected in Sox1 or Prox1 knockout mice, is severely reduced. Expression of Sox1 and Prox1 is unaffected, however, as are the expression of Eya1 and Sox2. These results indicate the fundamental role of c-Maf in global control of crystallin expression and in promotion of lens fiber differentiation in the mouse.

Soxs in lens fiber differentiation

The Sox family encodes a group of Sry-related transcription factors that contain a high-mobility group (HMG) DNA-binding motif (reviewed by Pevny & Lovell-Badge 1997). The role of the Sox family members, Sox1, 2, and 3, as enhancer-binding factors of the chicken δ-crystallin gene was determined by Kamachi et al. 1998 . In the course of chicken lens development, Sox2 is expressed prior to optic vesicle contact in the ventral to lateral domain of head ectoderm, which includes the PLE. Upon contact with the optic vesicle, expression of Sox2 in PLE is locally enhanced, and expression of Sox3 in PLE is activated. Sox1 expression commences later, in the invaginating lens placode. Expression of these Sox genes continues over the course of lens vesicle development and, in the mature lens, their expression is stronger in lens fibers than in lens epithelium. In addition to lens tissue, these Sox genes are widely expressed in the central nervous system throughout development.

Each of the Sox proteins can bind to the same site on the δ-crystallin enhancer ( Kamachi et al. 1998 ). Overexpression of these Soxs increases the activity of this enhancer in lens cells but not in non-lens cells, indicating that Soxs require an additional factor(s) expressed in the lens to stimulate transcription. Both the δ-crystallin and γ-crystallin genes carry not only the Sox-binding sites but also Maf-binding sites in their regulatory elements ( Fig. 3B), hinting at an important synergistic effect of these two transcription factors in the control of expression. In fact, a base substitution in either the Maf-binding site or the Sox-binding site of the γF-crystallin promoter abolishes its activity ( Goring et al. 1993 ; Kamachi et al. 1995 ). This might reflect an intrinsic property of Sox proteins as architectural transcription factors. Binding of Sox proteins within the minor groove induces a dramatic bend in the DNA, which may facilitate the recruitment of other factors to adjacent sites in order to form an active transcriptional nucleoprotein complex ( Pevny & Lovell-Badge 1997). The idea that Soxs may in part provide competence to respond to Mafs is consistent with the observations made in L-maf misexpression experiments: The region of ventral head ectoderm showing remarkable potential to form ectopic lens cells corresponds to the Sox2-expressing domain ( Fig. 3A). Neural retina cells, which also efficiently transdifferentiate into lens cells in response to L-Maf, also express Sox genes.

Lens fiber formation is affected in Sox1 knockout mice in a similar manner to c-maf knockout mice ( Nishiguchi et al. 1998 ). Lens fiber formation is initiated normally upon withdrawal from the cell cycle, but is arrested prematurely before cell elongation. Expression of the fiber-specific γ-class of crystallin genes (γA, γB, γC, γD, γE, and γF) is severely reduced, while expression of α- and β-crystallins is unaffected.

Prox1 for cell cycle control of lens fiber differentiation

Prox1 is a homeobox gene originally identified as a vertebrate homolog of the Drosophila gene prospero ( Oliver et al. 1993 ). prospero is necessary in the development of the central nervous system in flies, where asymmetric distribution of transcript and protein from neuroblasts to their daughter cells, sibling neuroblasts and ganglion mother cells, is crucial for the formation of distinct cell lineages ( Hirata et al. 1995 ). prospero is also expressed in lens-secreting cone cells of the compound eye. In chicken and mouse embryos, Prox1 expression is prominent in the lens, gut, and rudimentary pancreas and liver ( Tomarev et al. 1996 ). Its expression in the lens cell lineage commences upon invagination of the thickened lens placode toward the optic vesicle. In the developing lens vesicle, its transcript accumulates in the bow region of the equator, the transitional zone where the daughter cells of actively dividing lens epithelial cells terminally differentiate into elongated, post-mitotic lens fibers, presumably under inductive influence from the adjacent ciliary margin of the optic cup.

Consistent with its expression, Prox1 knockout mice exhibit severe defects in lens fiber formation ( Wigle et al. 1999 ). Cell elongation is not observed, and instead, abnormal cellular proliferation is evident in the posterior region of the mutant lens vesicle. Fiber-specific expression of the cell cycle inhibitors p27KIP1 and p57KIP2, and some of the γ-crystallin genes (γB and γD) are abolished, whereas E-cadherin expression in the lens epithelium is expanded to the posterior cells. Expression of Sox1 and other crystallins is, however, still normal. Comparison of this phenotype with that of c-maf or Sox1 knockout mice suggests distinct but overlapping functions for these genes in lens fiber differentiation. c-maf is responsible for pan-crystallin regulation and cell elongation; Sox1 for expression of most of the γ-crystallins and cell elongation; and Prox1 for expression of a few of the γ-crystallins, cell cycle arrest and cell elongation ( Table 2).

Table 2.  Comparison of the lens fiber phenotype of Sox1, Prox1, and c-maf knockout mice
 WildSox1Prox1c-maf
Major phenotypestype–/––/––/–
  1. *A small number of the cells (less than 5%) still retain inappropriate mitotic activity.

  2. †The cell elongation commences, but terminates prematurely.

  3. ‡Only the expression of γB- and γD-crystallin is affected.

  4. **The expression is severely reduced but remained residuary.

Crystallins expression
α+++±**
β+++±**
γ+±
Cell cycle arrest++*+*
Cell elongation+±±

Other transcription factors involved in lens development

  1. Top of page
  2. Abstract
  3. Introduction
  4. Classic and recent embryological studies
  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References

In addition to those described earlier, several other transcription factor genes are implicated in lens development. Six3 is a vertebrate homolog of the Drosophila homeobox gene sine oculis (so), of which loss-of- function mutation results in a reduced eye or eyeless phenotype ( Oliver et al. 1995 ). In the vertebrate lens cell lineage, Six3 expression is initiated in the lens placode and subsequently restricted to the epithelium of the lens vesicle. Ectopic expression of this gene in medaka fish embryos resulted in formation of additional lenses in the area of the otic vesicle ( Oliver et al. 1996 ). In these embryos, expression of exogenous Six3 was mosaic and transient, and detected not in the ectopic lenses but in nearby cells, implying activation of a cell non-autonomous process by Six3 for lens formation.

Eya1, a member of Eya family, is also expressed in the lens placode and developing lens vesicle ( Xu et al. 1997 ). The Eya family was identified as vertebrate homologs of the Drosophila eyes absent gene (eya). Eya protein can act as a coactivator by directly interacting with the product of sine oculis, So, to stimulate transcription ( Pignoni et al. 1997 ) in vivo, co- misexpression of eya and so induces ectopic eyes in flies, as does misexpression of eyeless ( Halder et al. 1995 ). Eya1 is not expressed in the lens placodes of Small eye mutant mice, indicating that this gene acts downstream of Pax6 ( Xu et al. 1997 ). Eya protein also forms a complex with another nuclear protein, Dachshund, and these two factors act synergistically to induce the formation of ectopic eyes when co- misexpressed in flies ( Chen et al. 1997 ). The vertebrate homolog of the Drosophila dachshund gene, Dac, shows expression in various neuroectodermal and mesenchymal tissues ( Hammond et al. 1998 ). However, the importance of Dac in vertebrate eye development remains unclear.

Members of the POTX/Pitx family of bicoid-type homeobox genes show specific expression in lens cells. In Xenopus, Pitx1 expression originates in the PLE prior to optic vesicle contact and persists in the lens vesicle ( Hollemann & Pieler 1999). Another member of this family, Pitx3, is expressed in the lens placode of mice from the time of its formation onwards. A disruption in this gene is thought to be responsible for the aphakia mouse phenotype in which the lens placode appears to form normally but fails to invaginate ( Semina et al. 1997 ). In humans, Pitx3 is mutated in families with autosomal-dominant cataracts and anterior segment mesenchymal dysgenesis (ASMD; Semina et al. 1998 ). The other member of this family, Pitx2 (RIEG), is expressed in periocular mesenchyme of mouse embryos, but not in lens tissue. Human Pitx2 is responsible for Rieger syndrome, an autosomal-dominant disorder, which results in anomalies of the anterior chamber of the eye ( Semina et al. 1996 ).

Retinoid receptors, members of the superfamily of ligand-inducible transcription factors, are also involved in lens development. Transfection assays using lens cell cultures identified a retinoic acid responsive element (RARE) in the γF-crystallin promoter. The RAR and RXR bind to this responsive element and heterodimerize to activate transcription ( Tini et al. 1993 ). Although retinoid receptors are expressed in a variety of tissues, the transgenic indicator mouse, which carries a reporter gene driven by multimerized RARE sequences, shows specific reporter expression in the lens, suggesting a vital role for these receptors in this tissue ( Balkan et al. 1992 ). The fact that double mutant mice of RARα and RARβ develop impaired lenses containing abnormal fibers also implies the involvement of these nuclear receptors in lens development ( Lohnes et al. 1994 ).

AP-2 transcription factors, whose expression is responsive to retinoic acid, show a unique expression pattern during lens development ( West-Mays et al. 1999 ). Expression of AP-2α and AP-2β is evident early in surface ectoderm, and persists in the lens placode. Following lens vesicle formation, AP-2α expression localizes to the anterior region of the vesicle and subsequently persists in lens epithelial cells, whereas AP-2β expression disappears from the lens entirely. The phenotype of AP-2α knockout mice includes a persistent adhesion of the impaired lens to head surface ectoderm ( West-Mays et al. 1999 ). The mutant lens consists mainly of fiber cells, and does not contain cells that display the lens epithelial phenotype.

It is noteworthy that AP-2-binding sites are contained in the promoter regions of the cell adhesion molecule genes E-cadherin and N-cadherin ( Li et al. 1997 ; Batsche et al. 1998 ). E-cadherin is expressed in both surface ectoderm and lens epithelium, whereas N-cadherin is expressed in lens fibers ( Hatta & Takeichi 1986; Takeichi 1988). In certain epithelial cell lines, AP-2 activates transcription of the E-cadherin promoter in a RB- or c-Myc-dependent manner ( Batsche et al. 1998 ). AP-2-binding sites are also contained in the promoter regions of the lens-specific genes MIP and filensin ( Masaki et al. 1998 ; Ohtaka-Maruyama et al. 1998b ), although their expression in lens fibers is exclusive of AP-2α expression in lens epithelium. AP-2α gene products include both activator and repressor isoforms caused by alternative splicing and different usage of its promotors. Hence, it is suggested that repressor forms might be participating in the strict fiber specificity of MIP and filensin by repressing their expression in lens epithelium ( Ohtaka-Maruyama et al. 1998a ).

Diffusible factors in lens development

  1. Top of page
  2. Abstract
  3. Introduction
  4. Classic and recent embryological studies
  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References

Recently, several diffusible factors have been identified as candidates for the putative signaling molecules, which mediate the inductive effect of retinal tissue. Bone morphogenetic protein-4 (BMP-4), a member of the tumor growth factor (TGF)-β family, is expressed strongly in the optic vesicle and weakly in the surrounding mesenchyme and head surface ectoderm. In Bmp-4 knockout mice, the lens placode fails to form from PLE upon contact with the optic vesicle ( Furuta & Hogan 1998). This phenotype is rescued by placing a BMP-4-carrying bead into the optic vesicle in explant cultures of mutant eye primordia. Substituting the optic vesicle with this bead in the explant, however, cannot rescue lens formation. The indication is that BMP-4 provides essential assistance for the optic vesicle to elicit lens-inducing activity by serving as one of multiple inducing molecules or/and by regulating downstream genes. Another member of this family, BMP-7, which is present in head surface ectoderm at the time of lens placode thickening, is also implicated in lens development ( Wawersik et al. 1999 ). Bmp-7 knockout mice display variable defects, including failure of lens placode formation or lens fiber differentiation. These embryos express neither Sox2 nor Pax6 in the region of PLE directly contacted by the optic vesicle.

Fibroblast growth factors (FGF) are implicated in the control of terminal differentiation of lens epithelial cells into lens fibers. In explant cultures, low concentrations of FGF-2 stimulate proliferation of lens epithelial cells, while high concentrations promote their differentiation into lens fibers ( McAvoy & Chamberlain 1989). Transgenic mice expressing different FGF (FGF-1, -4, -7, -8, and -9) under the control of the lens-specific αA-crystallin promoter show ectopic fiber differentiation in lens epithelia ( Robinson et al. 1995b ; Lovicu & Overbeek 1998). Of the fibroblast growth factor receptors (FGFR), FGFR3 shows unique expression in lens fibers ( Peters et al. 1993 ). To evaluate the necessity of FGF signaling, transgenic mice expressing kinase- deficient FGFR1 in the lens were generated ( Robinson et al. 1995a ). This truncated receptor is thought to act in a dominant negative fashion by heterodimerizing with endogenous FGFR. The resulting mice exhibited lens defects ranging from cataracts to severe microphthalmia, in which fiber differentiation was inhibited by ectopic apoptosis.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Classic and recent embryological studies
  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References

The currently favored model of lens development, based upon embryological manipulation studies, includes four phases: competence, bias, specification, and differentiation. This model is supported in part by the identification of transcription factors whose expression and/or function are tightly coupled to their respective phases (summarized in Fig. 2). Expression of Otx2, Pax6 and Lens1 in PLE seems to result in the acquisition of lens-forming bias. Activation of L-maf is considered to be a principal event in lens specification because of the extreme localization of its expression to the lens placode and its ability to convert non-lens cells into lens cells. Sox2/3 activation in the lens placode may also contribute to specification. c-maf, Sox1 and Prox1 are essential for differentiation into lens fibers, the final stage of lens development. However, genes involved in the first phase, lens competence, have not yet been identified.

The molecular nature of lens-inducing signals remains to be fully understood. None of the factors responsible for the bias-inducing activity of the anterior neural plate have yet been identified, and although BMP and FGF are implicated in late inductive events of specification and/or differentiation, they do not seem to be completely sufficient for the full inducing activity of the optic vesicle. An effective approach to this subject would be to analyze the expression mechanisms of transcription factors known to be involved in lens development. It is expected that the signals from the optic vesicle and the genetic cascade, which lead to lens-forming bias, are integrated at the L-maf and/or Sox2/3 promoter and result in the specification of lens fate. Additionally, analysis of the activation pathways of the early genes, Otx2, Pax6, and Lens1, should lead to the elucidation of the mechanisms of signaling from the anterior neural plate to the surrounding head ectoderm.

It is intriguing that overlapping expression of Otx2, Pax6, and Lens1 is observed in PNE, as well as in PLE, as both are located in head ectoderm surrounding the anterior neural plate and develop in parallel into placodal sensory structures. In fact, shared expression patterns of transcription factor genes is a common feature of the sensory primordia. For example, Six3 expression is observed in both lens and nasal placodes ( Oliver et al. 1995 ), while the distal-less gene family is expressed in nasal and otic placodes ( Papalopulu & Kintner 1993). Expression of Eya family genes and the PEA3 group genes of the ets family are shared by all three sensory placodes ( Chotteau-Lelievre et al. 1997 ; Xu et al. 1997 ). Analysis of the genetic programs that dictate expression of these transcription factors may lead to the elucidation of the fundamental mechanisms that govern the formation of vertebrate sensory tissues, the lens, nasal epithelium, and inner ear, all of which are believed to evolve from a common origin ( Jacobson 1966; Gans & Northcutt 1983).

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  5. Transcription factors involved in specification of the presumptive lens field
  6. Transcription factors involved in lens development from presumptive lens ectoderm
  7. Other transcription factors involved in lens development
  8. Diffusible factors in lens development
  9. Conclusions
  10. References
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