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

(Tomarev et al., 1994; Tomarev et al., 1992). Deletion (Tomarev et al., 1992) and sitedirected mutagenesis (Tomarev et al., 1994) experiments indicated that the consensus AP-1 and overlapping ARE sites are necessary for the activity of these squid crystallin promoters in the transfected cells. Since the S-crystallin promoters are active in lens cells and fibroblasts of vertebrates, the importance of these cis-regulatory elements binding ubiquitous factors for lens specificity in cephalopods remains open.

Although Pax6 has been implicated in the activity of numerous crystallin promoters in vertebrates (Cvekl and Piatigorsky, 1996), it is unclear whether Pax6 regulates the promoter activity of S-crystallin genes. A squid Pax6 has been cloned and can induce ectopic eyes in Drosophila, as can Pax6 from other species (Tomarev et al., 1997). Pax6 mRNA first appears in the region of the rudimentary eye well before the first appearance of S-crystallins in the lentigenic cells at day 17 of development (West et al., 1995). Pax6 mRNA was not detected in the lentigenic cells that give rise to the posterior lens segment, but it was found in the anterior part of the lens segment, where two S-crystallin genes (Lops 7 and 12) are expressed. Moreover, Pax6 mRNA was observed in the fold of tissue that is posterior to the eye and gives rise to the cornea (Arnold, 1984), although it was not followed in the developing cornea (Tomarev et al., 1997).

5.3.4.2. -Crystallin

The exon-intron structure of the scallop -crystallin gene is similar to that of the mammalian ALDH1/2 genes, with the exception that the scallop gene has an additional 5 untranslated exon (Carosa et al., 2000). In contrast to the coding sequences and gene structure, however, the scallop ALDH/ -crystallin promoter region resembles that of the mouse and chicken αA-crystallin genes rather than the mammalian ALDH genes (Carosa et al., 2002). Gel shift experiments demonstrated binding of Pax6 and CREB to the -crystallin promoter, and site-specific mutagenesis of the overlapping CREB/Jun and Pax6 sites abolished activity of the -crystallin promoter in transfected cells. It appears as if convergent evolutionary adaptations underlie the preferential expression of crystallin genes in the lens of vertebrates and invertebrates.

5.3.4.3. J-Crystallins

The promoter regions of the known J-crystallin genes of jellyfish have putative TATA boxes and several CAAT sequences (Piatigorsky et al., 1993). There is also a CAAT sequence in the promoter region of the SL20–1 gene of the squid (Tomarev et al., 1992). The promoter regions of the J-crystallin genes have a RARE half-site that can bind histidine-tagged jRXR, an RA receptor that was cloned from, Tripedalia cystophora, a species of jellyfish (Kostrouch et al., 1998). The possibility that RA receptors regulate the jellyfish J-crystallin genes deserves further study, since these transcription factors activate the promoters of many vertebrate crystallin genes (Gopal-Srivastava et al., 1998; Li et al., 1997; Tini et al., 1995; Tini et al., 1993; Tini et al., 1994).

Compelling evidence exists for the role of Pax family members in crystallin gene expression in jellyfish (Kozmik et al., 2003). Unlike vertebrates, jellyfish have a protein called PaxB comprising a structural and functional Pax2/5/8-like DNA binding paired domain and octapeptide, and a Pax6-like homeodomain. Transfection experiments showed that PaxB strongly activates the J3-crystallin and weakly activates the J1Aand J1B-crystallin promoters in transfection tests. J3-crystallin promoter activation was accompanied by and

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dependent upon PaxB binding to cognate sequences at positions −66 to −34. Interestingly, the J3-crystallin promoter was weakly activated by Pax2, but not by Pax6. In addition to the interesting implications these results have for the evolution of eyes in general, they provide additional support for a critical role of Pax proteins for crystallin gene expression throughout evolution.

5.3.4.4. Drosocrystallin

Both antibody and hybridization tests indicated that drosocrystallin synthesis is eye specific in Drosophila. Transgenesis experiments in Drosophila showed that a 441-bp 5 -flanking sequence of the drosocrystallin gene fused to the lacZ reporter gene is sufficient to reproduce the endogenous expression pattern of drosocrystallin (Janssens and Gehring, 1999). This includes gene expression in the ocellus as well as the compound eye. Intron sequences also appear to have some regulatory capacity, as judged by their ability to drive expression in the imaginal discs. The drosocrystallin promoter was inactive in transgenic mice, however, when tested with the luciferase reporter gene (Carosa and Piatigorsky, unpublished data). Nothing is known yet about the transcription factors that might regulate drosocrystallin during fly development. Possibilities such as Pax6 and prospero have been considered elsewhere (Tomarev and Piatigorsky, 1996).

5.4. Lessons from Transcriptional Control of Diverse Crystallin Genes: A Common Regulatory Mechanism?

The common denominator among diverse crystallin promoters and/or enhancers is the presence of clusters of binding sites for a small group of DNA-binding transcription factors, including Pax6, c-Maf, Sox1/2, and RARβ/RXRβ. As shown earlier, neither Pax-6, c-Maf, Sox1/2, nor RARβ/RXRβ is a tissue-specific factor. Thus, the emerging model to explain lens-specific expression is based on the combinatorial principle used also by many other genes (Chen, 1999). As the expression of these transcription factors is quantitatively and spatially controlled during development, they presumably interact dynamically on specific arrays of positively and negatively acting cis-elements in crystallin promoters and enhancers. Promoter and enhancer site occupancy then would determine the level of crystallin gene expression in the lens epithelium and lens fibers.

Strong evidence for the combinatorial mechanism is that the same factors play regulatory roles in the transcription of different crystallin genes. As shown in Figure 5.2, many crystallin promoters and enhancers have at least one Pax6 and MARE/CRE/AP-1 site. It was proposed earlier that the unique environment of the lens (e.g., the redox state of the lens) may be advantageous for the function of specific transcription factors, and from an evolutionary perspective, genes recruited as crystallins were those genes employing transcription factors that worked “optimally” in the lens (Piatigorsky, 1993; Piatigorsky and Zelenka, 1992). Collectively, the combinatorial code employing about a dozen known or yet unknown tissuerestricted transcription factors and gene recruitment based on the transcriptional control by those genes are not mutually exclusive models.

In addition, to achieve transcription in vivo, numerous multiprotein complexes (e.g., TFIID, TFIIA, and chromatin-remodeling complexes) and factors (e.g., TFIIB), as well as the RNA polymerase II holoenzyme, have to be recruited to the vicinity of the start site of transcription. The DNA-binding transcription factors (Pax6, c-Maf, Sox1/2, RARβ/RXRβ, and TFIID) have to gain access to their cognate sites by displacing the nucleosomes and

“opening” the chromatin for other transcription factors. Some DNA-binding factors may topologically alter the promoter and/or enhancer DNA through the mechanism of DNA bending and looping. Indeed, TFIID, Sox1/2/3, and possibly Pax6 can induce DNA bending (Connor et al., 1994; Gaston et al., 1992). In addition, some of the DNA-binding proteins (e.g., Pax6, c-Maf and RARβ/RXRβ) recruit transcriptional coactivators harboring the histone acetyltransferase (HAT) activity (e.g., p300 and CBP). HAT proteins modify histones by acetylating their tails and promote transcription of specific clusters of genes by remodeling the chromatin. It is noteworthy that lens fibers and nucleated erythrocytes derived from yolk sac are the only mouse embryonic cells expressing the histone H1o (Gjerset et al., 1982), and the prediction would be that there is a function for this histone in mouse embryonic lens. However, no abnormalities were found in histone H1o null lenses (Sirotkin et al., 1995).

While the research summarized in this chapter supports the global role of c-Maf, Pax6, RARβ/RXRβ, and Sox1/2, we do not know precisely how, where, and when all these factors interact in vivo with crystallin genes. The timing of the onset of the expression of crystallins, as well as the developmental control of the expression of those regulatory factors, is critical for understanding the complexity of crystallin-gene regulation (see Chapter 3). Pax6 is expressed in the head ectoderm/lens placode before the onset of αB- and αA-crystallins (Walther and Gruss, 1991), whereas c-Maf occurs later, at E10.5–11 (Ring et al., 2000). c-Maf is highly expressed in the differentiating lens fibers when the αA-crystallin gene and members of the β/γ -crystallin family of genes produce high amounts of transcripts. Later, c-Maf expression drops down in three-month-old rat lenses (Yoshida et al., 1997), but expression of some members of the β/γ -crystallin family is maintained after the cessation of c-Maf expression.

Similarly, Sox2 is initially expressed in the presumptive lens ectoderm from E8.5, and a high level of Sox2 is found during the formation of the lens pit; however, with the onset of Sox1 expression (at E11.5), Sox2 expression becomes down-regulated (Kamachi et al., 1998).

The RA-activated nuclear receptors RAR and RXR (present as α, β, and γ isoforms) form not only RAR/RXR heterodimers but also complexes with other nuclear receptors (e.g., T3 and ROR). However, the developmental expression patterns of RAR and RXR in the lens are not well known. Using reporter genes with single or multiple copies of RAREs, it was possible to visualize retinoid signaling in the developing mouse lens (Balkan et al., 1992; Enwright and Grainger, 2000; Rossant et al., 1991). Retinoid signaling is active in the lens placode (at E9.5) and persists during the lens vesicle stage but is lost in the lens by E12.5. Interestingly, retinoid signaling in the lens is dependent on Pax6 (Enwright and Grainger, 2000), and consequently some phenotypes observed in RXRα−/− and RARγ −/− mice are similar to Peters’ anomaly, a form of human ocular congenital defect due to mutations in the PAX6 gene (Hanson et al., 1994). An RXRα−/− phenotype resembles classic Peters’ anomaly; the lens remains attached to the surface ectoderm and rotates ventrally (Kastner et al., 1994). When double mutants were examined, an increased severity of eye malformations was observed (Lohnes et al., 1994; Mendelsohn et al., 1994). It is not known whether expression of the αB- and γ F-crystallins was affected in these mice. Thus, based on current data (summarized earlier), we propose here a model of coordinated crystallin gene expression as a sequence of events with at least four distinguishable phases.

First, as the head ectoderm is committed to become the lens placode, low-level expression of α-crystallins in the mouse (and δ-crystallin in the chicken) is initiated (“onset of crystallin

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transcription”), presumably as a result of transcriptional activation by Pax6 in combination with unknown factors (or perhaps Pax6 combined with Sox2/3 and RAR/RXR/ROR). As the lens placode becomes the lens pit and then the vesicle, these factors continue to cooperate to maintain crystallin expression in the presumptive lens epithelium. The repression of the β/γ -crystallin genes presumably occurs due to the absence of essential activators and to the presence of Pax6 acting as a repressor (Duncan et al., 1998). Then, when the cells in the posterior portion of the lens vesicle begin to elongate (“early embryonic fiber cell transcription”), c-Maf (or c-Maf and Sox1 together) becomes the prominent activator needed for high-level expression of the α-crystallins (or the β/γ -crystallins). While Pax6 protein expression is still detectable in secondary fiber cells (Duncan et al., 2000), its role in transcriptional control of α-/β-/γ -crystallins in fiber cells is not known. Similarly, RA receptors may terminate their engagement with αB-/γ F-/δ1-crystallin promoters after E12.5 (Enwright and Grainger, 2000).

The next developmental switch in crystallin expression occurs around birth in rodents, coincident with the initiation of βB2and γ S-crystallin expression and the loss of γ - crystallin expression in secondary lens fiber cells (“postnatal switching phase”). A similar switch occurs in the chicken lens around E15, at which time the transcriptional activity of the δ1-crystallin gene down-regulates sharply and there is a coincident up-regulation of β- crystallin expression (Hejtmancik et al., 1985). While these changes in crystallin expression result in the crystallin composition of the lens nucleus differing from that of the cortex (and presumably are essential for the formation of the refractive index gradient), the molecular basis of these changes is unknown.

Finally, another switch occurs in the juvenile/adult lens and causes a difference in the crystallin composition of the newly formed cortical fibers that will reside behind the iris and thus outside of the optical axis throughout life. While this postnatal change in crystallin expression has been poorly described as yet and has no clear function, it is apparent that the ratio of crystallin messages made by the lens changes with age. A marked example is that expression of all γ -crystallin genes except γ B-crystallin is lost from the adult rodent lens (Peek et al., 1990). Currently, the only hint of the molecular nature of this change is that c- Maf expression ceases in the lens after three months of age in the rat. We propose that changes in the relative expression levels of crystallin genes in lens fiber cells during development result from the changing levels and utilization of different transcription factors. It is possible that transcription factors with a broad expression pattern in the majority of cell types (e.g., CREB, CREM, AP-1, HSF, and USF) may participate in the maintained expression of crystallins in the postnatal lens without being essential for their developmentally regulated onset.

5.5. Current Questions

Early crystallin promoter studies were bolstered by the discovery that a short fragment of 5 -flanking sequence from the mouse αA-crystallin gene drove lens-specific expression in transfected cells (Chepelinsky et al., 1985) and transgenic mice (Overbeek et al., 1985). The strict evolutionary conservation of the mechanisms controlling lens-specific expression was then confirmed by the finding that chicken α-, β-, and δ-crystallin promoters also drove lens-specific expression in transgenic mice. In 1994–8, detailed study of the transcriptional regulation of αA- and δ1-crystallin made Pax6, Sox1/2/3, L-Maf/c-Maf/Nrl, and RAR/RXR leading candidates as transcription factors regulating the lens-specific expression of crystallin genes. Subsequently, binding sites for these transcription factors have been found in

diverse crystallin promoters driving expression of both ubiquitous and taxon-specific crystallins. In 1998–2000, direct in vivo experiments confirmed that Sox1 and c-Maf are critical regulatory genes for the expression of many crystallin genes in the mouse lens.

While the minimal promoter necessary for lens-specific expression has been identified for many crystallins, in most, the sequences that control their precise temporal and spatial activity pattern during development are not known. Further, even when the known promoter sequences appear to recapitulate the developmental expression pattern of their genes, in only one case (chicken βB1-crystallin) have these sequences been shown to be sufficient to raise transgene expression to a level approaching that of an endogenous crystallin gene in the lens (Duncan et al., 1996b; Taube et al., 2002). Thus, at this time, the molecular mechanisms that allow a single copy gene to be expressed at levels approaching 50% of total cellular mRNA (chicken δ1-crystallin in embryonic lens fiber cells; Hejtmancik et al., 1985) are completely unknown. It can be hoped that the sequencing of the human, mouse, rat, and zebrafish genomes will allow for phylogenetic footprinting of the “ubiquitous” crystallin genes that may predict functionally conserved regulatory regions.

How the precise balance of transcription factors work together in the context of the complex, overlapping elements found in most crystallin minimal lens-specific promoter regions remains a key research area in crystallin gene expression. While c-Maf and Sox1 are expressed in lens fibers, other factors (e.g., Pax6) involved in crystallin gene expression are expressed primarily or preferentially in the lens epithelium. The investigation of this question should give great insight into both the mechanisms controlling tissue-specific gene expression during development and the biochemistry of the individual factors. It should be noted that, at this time, we cannot exclude the possibility that a truly lens specific transcriptional activator or coactivator exists.

There is accumulating evidence that growth factor–receptor (e.g., FGF family members and IGF-1; see chap. 11) and cell surface receptor-extracellular matrix interactions (e.g., integrin and lens capsule; see chap. 10) are involved in the regulation of lens fiber cell differentiation. In both cases, ligand binding to the receptor initiates a signal transduction cascade that changes the phosphorylation states of downstream signaling molecules. This signaling often changes the phosphorylation state of transcription factors, causing them to either translocate into the nucleus or change in function within the nucleus. Thus, integrin or growth factor signaling could cause a change in crystallin gene expression by influencing the function of preexisting transcription factors. Alternatively, this signaling could induce the expression of factors important for crystallin expression. While little direct evidence is available yet to support either possibility, it has been established that treatment of lens epithelial explants with bFGF (FGF-2) can induce crystallin gene expression (McAvoy et al., 1991). In addition, a recent study demonstrated that FGF-2 concentrations capable of promoting fiber cell differentiation in rat lens explants also activate a construct consisting of the mouse αA-crystallin –366 to +44 promoter fragment (Ueda et al., 2000). A better understanding of the downstream targets of signal transduction cascades in the lens, combined with information on how signal transduction influences factors known to be involved in crystallin transcription, is essential for a unified picture of the mechanisms regulating crystallin gene expression.

While most work on crystallin gene transcription has focused on the function of specific transcription factors, it is clear from studies in other systems that crystallin gene regulation is likely to be much more complex in vivo at the level of the endogenous gene. To address the role of chromatin in the transcriptional control of the mouse αA-crystallin promoter, two promoter fragments having 5 ends at positions −1809 and −366 were introduced into

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stably transfected lens epithelial cells (Sax et al., 1996). The results showed substantial promoter activity of the −1809 to +46 fragment in stably transfected cells, while the same promoter fragment was less active in transient transfections. Thus, the role that chromatin organization and remodeling plays in crystallin gene expression is an important subject for future studies involving the identification of potential locus control regions (LCRs).

Finally, to understand both the evolution of lens function and crystallin gene expression, it is necessary to address the mechanisms by which crystallin genes have been recruited for high-level lens expression. Identification of common regulatory genes implicated in crystallin gene regulation may be used to explain some molecular events implicated in crystallin gene recruitment. We can experimentally test this hypothesis using transgenic “knock-in experiments.” Three models may be particularly attractive in this respect. First, it is possible to knock the ZPE region from the guinea pig ζ -crystallin into the homologous mouse gene. Second, it is possible to knock LSR1/LSR2 from the mouse αB-crystallin promoter into the promoters of small heat-shock proteins, hsp27 and 29. Finally, chicken δ-crystallin/ASL lens-specific enhancer can be knocked in the mouse ASL gene locus.

5.6. Conclusion

During the past 20 years, our understanding of both the function of crystallin proteins and the control of their expression during lens development has increased greatly. While many unanswered questions remain, the rate of current progress in these fields suggests that many advances will be made in the next decade.

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continually adding new fiber cells at its equator, an area important for outward current flow, it is interesting to speculate that establishing and maintaining spatial differences in membrane transport proteins is an integral part of lens differentiation. To support this contention, the structural, functional, and molecular evidence that indicates the existence of spatial differences in membrane transport proteins between epithelial cells, newly differentiating fiber cells, and inner fiber cells is reviewed.

6.3. Membrane Conductances Vary between Lens Regions

Impedance analysis and ion substitution experiments have tentatively localized the majority of the K+ conductance to cells near the surface of the lens and the Na+ and Cl− conductances to the inner fiber cells (Mathias et al., 1979). It has been calculated that the specific membrane resistance of the inner fiber cells is very high, indicating that under normal conditions the conductance of individual fiber cells is low. However, because the lens is made up of many fiber cells, their conductance generates a relatively large circulating ion flux. Further, because the surface and inner lens regions are electrically coupled via gap junction channels, this difference in electromotive potential generates the radially circulating current that powers the internal circulation while at the same time ensuring that the inner fiber cells have a negative membrane potential.

To determine the identity and spatial localization of these conductances, the patch clamp technique was initially used. More recently, molecular cloning techniques have been employed. Although we consider the membrane properties of three distinct cell types – epithelial cells, differentiating fiber cells, and mature fiber cells – most of the data accumulated concern the epithelial cells, as they have proven to be the most amenable to patch clamp analysis (Rae and Cooper, 1990). Attempts to patch clamp the inner fiber cells have been hampered by difficulties in obtaining a viable preparation of elongated fiber cells. This is of course not surprising, as these cells lack the K+ channels and Na+ pumps necessary for generating a negative membrane potential. Thus, once isolated from the surface cells, the inner fiber cells have no means of generating a membrane potential, and instead they depolarize. Their depolarization initiates a cascade of events that includes ion influx, cell swelling, and the Ca2+-dependent activation of intracellular proteases, which ultimately leads to fiber cell globulization (Wang et al., 1997; Bhatnagar et al., 1997). To prevent this globulization, patch clamping of isolated elongated fiber cells needs to be performed in Ca2+-free Ringers solutions (Eckert et al., 1998).

6.3.1. Potassium Conductance

The patch clamping of isolated epithelial cells has shown that the membrane properties of these cells are, as predicted, dominated by K+ conductances. In subsequent studies, the molecular identity of the channel proteins underlying these conductances was revealed. This work has predominately been done by Rae and collegues (see Table 6.1 for references). They identified at least three major K+ channel types: an inwardly rectifying K+ channel, a delayed outwardly rectifying K+ channel, and a large conductance Ca2+-activated K+ channel (Maxi-K). The relative expression of these different channel types varies in lenses from different species. An inwardly rectifying K+ channel dominates the conductance properties of epithelial cells in rat, mouse, and rabbit lenses, while in the human, bovine, pig, frog, and chick lenses a delayed outwardly rectifying K+ channel dominates (Table 6.1). It is believed that these two K+ channel types are responsible for setting the membrane potential