Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Large-scale analyses of gene expression in the cornea and lens

The basal layer of limbal epithelium between the cornea and the conjunctiva is enriched in stem cells that give rise to transient amplifying cells that migrate to the central corneal epithelium and become the basal epithelial cells. Laser capture microdissection followed by microarray analysis identified about 100 differentially expressed genes in limbal compared to corneal epithelial basal cells (Zhou et al., 2006). In another study, comparison of the rat limbal and central corneal transcripts by serial analysis of gene expression (SAGE) identified 759 transcripts specific for the limbus and 844 transcripts specific for the central cornea, with 2,292 transcripts present in both (Adachi et al., 2006). Comparison of PN9 and 6-week-old adult mouse corneas by SAGE demonstrated dynamic changes in gene expression during postnatal corneal maturation (Norman et al., 2004). Roughly one-third of the transcripts expressed in the cornea are present exclusively in the PN9 or mature corneas, and the remaining one-third are expressed at both stages. Abundantly expressed transcripts in the cornea are associated with

diverse functions such as metabolism, redox activities, and barrier integrity (Norman et al., 2004).

In the lens, younger and transcriptionally active differentiating cells envelop a core of mature fiber cells lacking nuclei and cytoplasmic organelles. Microarray comparison of gene expression profiles of young elongating and mature fiber cells captured by laser microdissection identified 65 differentially expressed genes important for lens cell differentiation (Ivanov et al., 2005). In humans, the expression of as many as 1,196 transcripts is elevated and the expression of 1,278 transcripts is decreased by twofold or more in lens epithelial cells compared to cortical fiber cells (Hawse et al., 2005). These gene expression changes correspond to distinct pathways and functions important for the formation of lens fiber cells.

Endogenous noncoding microRNAs (miRNAs) inhibit the translation and affect the stability of target mRNAs, and thus have a role in the regulation of development and differentiation (Kloosterman and Plasterk, 2006). Studies of miRNAs in the eye are just beginning. So far, several miRNAs expressed in a distinct tissueand cell-type-specific manner in different tissues of the eye have been detected using

miRNA microarrays (Ryan et al., 2006). MiRNA (mir)-184, one of the abundant miRNAs in cornea and lens, is expressed in the corneal epithelium and in the lens germinative zone epithelial cells. Mir-205 is widely expressed in the anterior segment epithelia and epidermis (Ryan et al., 2006). Specific functions of these miRNAs are not known yet.

Transcription factors involved in the development of cornea and lens

Studies employing transgenic mice and targeted deletion of genes helped accumulate useful information about the network of transcription factors influencing the development of cornea and lens (figure 57.2). Here we briefly review the contribution of different transcription factors to the development of cornea and lens.

Homeobox Transcription Factors Homeobox-contain- ing transcription factors have received preferential atten-

tion in studies on gene regulation during eye development, in view of their critical contribution. As indicated in the following discussion, in addition to their developmental roles, the homeobox transcription factors are also important regulators of crystallin gene expression.

Pax6. Pax6, a paired domain-homeobox transcription factor, is essential for the inductive interactions between the neuroectoderm and the surface ectoderm during early embryonic eye development (see figure 57.2) (Simpson and Price, 2002). Of historical interest, the importance of Pax6 for eye development was first noted in Drosophila, where its homologue is known as eyeless (Gehring, 2004). In humans, mutations in PAX6 result in severe eye defects (Graw, 2003; Hanson, 2003; Hanson et al., 1994). Homozygous Pax6 mutant mice develop only rudiments of the optic vesicle and die in the neonatal stage (Hogan et al., 1988). In heterozygotes, lens placode formation is delayed, resulting in a smaller lens with vacuolated fiber cells, frequently fused to

Figure 57.2 Transcription factors regulating the development of mouse lens and cornea. The network of transcription factors regulating the development of lens (red), or cornea (blue), or both (green) is shown, along with the embryonic age and the devel-

opmental stages of lens and cornea. Many of these transcription factors remain active in the mature lens and cornea. See color plate 66.

the cornea, resembling Peter’s anomaly (Collinson et al., 2001). In the mouse, Pax6 expression, first detected at E8 at the optic pit, head surface ectoderm, and neural ectoderm, is restricted to the lens placode, optic vesicle, and stalk at E9.5. After E13.5, Pax6 is expressed in the proliferating anterior epithelial cells of the lens vesicle and the surface ectoderm, which gives rise to cornea, conjunctiva, and eyelids. In the adult mouse, Pax6 is expressed in the lens epithelial cells, cornea, conjunctiva, iris, ciliary body, and retina (Koroma et al., 1997). Homeobox transcription factors Meis1 and Meis2, expressed in a pattern similar to Pax6, directly regulate Pax6 expression in presumptive lens ectoderm by binding the Pax6 lens placode enhancer (see figure 57.2) (Zhang et al., 2002).

Pax6 upregulates its own expression and influences eye development both directly, by controlling different genes encoding structural proteins required for eye formation, and indirectly, by controlling a number of genes encoding developmental transcription factors, including Six3, c-Maf, MafA/L-Maf, Prox1, Sox2, and FoxE3 (see figure 57.2) (Aota et al., 2003; Ashery-Padan et al., 2000; Brownell et al., 2000; Chauhan et al., 2002; Cvekl and Tamm, 2004; Cvekl et al., 2004; Duncan et al., 2000; Furuta and Hogan, 1998; Marquardt et al., 2001). Pax6 is autonomously required for formation of corneal epithelium, stroma, and endothelium between E10.5 and E16.5 (Collinson et al., 2003; Davis et al., 2003). The corneal epithelium in the heterozygous Pax6 (Small eye, Sey) mouse is thinner, with a reduced number of cell layers, despite increased cell proliferation. The levels of desmoglein, β-catenin, γ-catenin, and the intermediate filament keratin-12 are reduced in the Sey cornea, suggesting defective intercellular adhesion (Davis et al., 2003), an idea supported by a recent report showing that Pax6 heterozygote corneal epithelial cells have multiple glycoconjugate defects on the cell surface that restrict their ability to initiate migration in response to wound healing (Kucerova et al., 2006). Corneal epithelial cell proliferation is inhibited on overexpression of Pax6 (Ouyang et al., 2006). The distribution of neural crest–derived cells is abnormal in Sey mouse from early developmental stages to the adult, indicating that normal distribution and integration of neural crest–derived cells depend on a proper dosage of Pax6 (Kanakubo et al., 2006).

Pax6 is actively involved in the repair and maintenance of the adult corneal surface. During corneal wound healing, the amount of Pax6 increases at the migrating front of the resurfacing corneal epithelium, where it upregulates matrix metalloproteinase gelatinase B (gelB; MMP-9). Two Pax6binding sites exist within the gelB −522/+19 bp promoter fragment, which contains the necessary cis- elements for appropriate expression (Sivak et al., 2000). Pax6 paired domain controls the gelB promoter activity by interacting directly with one of these sites, and indirectly with the other

site, through cooperative interactions with AP2α. A reduced Pax6 dosage in heterozygous Sey mice results in a loss of gelB expression at the migrating epithelial front, which correlates with an increase in inflammation (Sivak et al., 2004).

Pax6 influences target gene expression both independently and in association with other transcription factors such as pRb, MafA, MitF, Sox2, and Sox3 in a cooperative manner (see figure 57.2). Sox2 and Sox3 interact with Pax6, leading to synergistic transcriptional activation (Aota et al., 2003). In the lens, both aA- and aB-crystallin genes are upregulated (Cvekl and Piatigorsky, 1996; Cvekl et al., 1994, 1995; Gopal-Srivastava et al., 1996; Haynes et al., 1996), and bB1-crystallin (Duncan et al., 1998) and g-crystallin (Yang et al., 2004) genes are downregulated by Pax6. Unlike the synergistic activation of aB-crystallin by Pax6 and c-Maf, Pax6 has no effect on c-Maf-mediated αA-crystallin promoter activation (Yang et al., 2004). Compound heterozygous mice with mutations in both Pax6 and Gli3, a zinc finger transcription factor gene expressed in the embryonic eye, develop more extensive abnormalities in the retina, iris, lens, and cornea than do single Gli3+/− or Pax6+/− mutants (Zaki et al., 2006). The requirement for a normal Gli3 gene dosage is greater in the absence of normal Pax6 gene dosage, suggesting that these two transcription factors cooperate during eye morphogenesis.

Six3. Six3, a Six domain containing homeobox transcription factor, is expressed in the anterior neural ectoderm at E7, developing retinal field at E8, and head ectoderm that forms the lens placode at E9 in the mouse (Lagutin et al., 2001; Oliver et al., 1995). After E9, Pax6 and Six3, expressed in an overlapping manner, regulate the expression of each other (see figure 57.2) (Goudreau et al., 2002). Conditional deletion of mouse Six3 in the presumptive lens ectoderm disrupts lens induction, resulting in the absence of the lens placode and lens in severe cases. Six3 influences eye development directly, by regulating the expression of structural and metabolic genes required for eye formation, and indirectly, by activating the expression of Pax6 in the lens preplacodal ectoderm (see figure 57.2) (Liu et al., 2006).

Prox1. Another well-conserved homeobox transcription factor influencing lens fiber cell elongation in the lens vesicle is Prox1 (see figure 57.2). Prox1 is expressed in the mouse at E9.5 in the lens placode, at E10.5 in the lens vesicle, and at E12.5 onward in the anterior epithelium, lens fiber cells, and surface ectoderm (Tomarev et al., 1998). Homozygous Prox1 null mice, which die around E14.5, show defective fiber cell elongation, abnormal cellular proliferation, downregulated expression of the cell cycle inhibitors Cdkn1b (p27KIP1) and Cdkn1c (p57KIP2), misexpression of E- cadherin, inappropriate apoptosis, and absence of gD- and gB-crystallin expression (Wigle and Oliver, 1999). As the

mutant lens cells fail to polarize and elongate properly, a hollow lens is formed. Prox1 binds and activates the mouse γD-crystallin promoter in vitro (Lengler et al., 2001).

POU homeodomain transcription factors. Bicoid-related POU homeodomain transcription factors Pitx2 and Pitx3 also regulate eye development (see figure 57.2). Deletion of Pitx2, normally expressed in the neural crest– and the mesodermderived precursors of the periocular mesenchyme, results in severe disruption of periocular mesenchyme structures and extrinsic defects in early optic nerve development. Studies with neural crest–specific Pitx2 null mice (Pitx2-ncko) indicate that Pitx2 is required in neural crest for specification of the corneal endothelium and stroma and the sclera (Evans and Gage, 2005). Central corneal thickness is reduced in heterozygous Pitx2 mutant mice (Asai-Coakwell et al., 2006). Overexpression of Pitx2a isoform in the mouse corneal mesenchyme and iris lead to corneal opacification, corneal hypertrophy, irido-corneal adhesions, and severely degenerated retina resembling glaucoma and Axenfeld-Rieger syndrome (Homberg et al., 2004). In humans, PITX2 or FOXC1 mutations account for up to 50% of the AxenfeldRieger malformations of the anterior segment (Graw, 2003).

Mutations in Pitx3, normally expressed from E10 in the thickening lens placode and later in the lens vesicle, are responsible for the aphakia mutant mice in which lens differentiation is affected after E11 (Semina et al., 1997, 2000). In humans, PITX3 mutations lead to posterior polar cataract and variable anterior segment mesenchymal dysgenesis (Addison et al., 2005; Semina et al., 1998).

Sip1. Sip1, a Smad-interacting zinc-finger homeodomain transcription factor, is expressed after lens placode induction in the lens epithelium and immature lens fibers of the bow region (Yoshimoto et al., 2005). Conditional deletion of Sip1 in the lens results in a small hollow lens connected to the surface ectoderm (Yoshimoto et al., 2005). Sip1 is required for lens fiber cell maturation, and γ-crystallin, a marker for mature fiber cells, is absent in the Sip1 null lenses (Yoshimoto et al., 2005). Sip1 activates Foxe3 expression in a Smadbinding domain–dependent manner in the lens (Yoshimoto et al., 2005).

Non-homeobox Transcription Factors Regulating

the Development of Lens and Cornea

High mobility group transcription factors Sox1 and Sox2. Sox transcription factors belonging to the high mobility group (HMG) family of DNA-binding proteins are involved in the regulation of diverse developmental processes. The targeted deletion of Sox1 in mice causes microphthalmia and cataract (Nishiguchi et al., 1998). Mutant lens fiber cells fail to elon-

gate and lack expression of γ-crystallins, as direct interaction of Sox1 protein with γ-crystallin promoters is required for their expression. Both Sox1 and Sox2 bind upstream lens enhancer elements in the mouse aB-crystallin gene (Ijichi et al., 2004). Transcription factors AP2, Pax6, and Prox1 upregulate Sox2 gene expression (see figure 57.2) (Lengler et al., 2001).

Hmgn1. The nucleosome-binding HMG protein Hmgn1, capable of altering the structure and activity of chromatin, affects the development of the corneal epithelium in mice (Birger et al., 2006). Hmgn1 null mice develop a thin, poorly stratified corneal epithelium depleted of suprabasal wing cells and a disorganized basement membrane. Epithelial cell–specific markers glutathione-S-transferase (GST)-α4 and -ω1 are reduced in Hmgn1 null corneas, while the components of adherens junctions—E-cadherin and α-, β- and γ-catenin—are upregulated (Birger et al., 2006).

Winged helix-forkhead transcription factors. Human congenital primary aphakia, a rare developmental disorder characterized by microphthalmia, anterior segment dysgenesis, and in severe cases the complete absence of lens, is caused by null mutations in FOXE3, a winged helix-forkhead transcription factor (Valleix et al., 2006). In the mouse, mutations in Foxe3 result in the dysgenetic lens (dyl) mutant strain that has several defects in lens development and altered patterns of crystallin expression (Blixt et al., 2007; Brownell et al., 2000; Medina-Martinez et al., 2005). Foxe3 is initially expressed in the developing brain and the lens placode and later restricted to the anterior lens epithelium in a Pax6and Sip1-dependent manner, and is turned off on fiber cell differentiation (Yoshimoto et al., 2005). Targeted disruption of mouse Foxe3 results in an abnormal eye with a small, vacuolated lens. The anterior lens epithelium and the cornea do not separate, forming an unusual, multilayered tissue. These defects in lens development are accompanied by changes in the expression of DNase II-like acid DNase, Prox1, p57, and PDGF-α receptor (Blixt et al., 2007; Medina-Martinez et al., 2005).

Two other forkhead transcription factors, Foxc1 (Mf1) and Foxc2 (Mfh1), have nearly identical DNA binding domains, and largely overlapping expression patterns and functions in the developing eye (Hiemisch et al., 1998; Smith et al., 2000). Foxc1, expressed in the mesenchymal cells in the eye (Kidson et al., 1999; Kume et al., 1998), is restricted to the future trabecular meshwork by E16.5 (Kidson et al., 1999). Foxc1 null mice die at birth with multiple abnormalities, including severe anterior segment developmental defects, such as fused lens and cornea, a thickened corneal epithelium, disorganized stroma, and missing endothelium (Hong et al., 1999; Kidson et al., 1999; Kume et al., 1998). Foxc1 heterozygous mice are viable, with milder anterior

segment defects (Hong et al., 1999; Smith et al., 2000). Foxc1 and Foxc2 double heterozygous mice have malformations of the ciliary body not seen in either heterozygous mouse alone (Smith et al., 2000). In humans, FOXC1 mutations cause anterior segment dysgenesis and glaucoma (Honkanen et al., 2003; Mears et al., 1998; Mirzayans et al., 2000; Nishimura et al., 1998).

Heat shock factor 4. The heat shock factors (HSFs) belonging to the basic domain/leucine zipper family of transcription factors consist of three members in mammals (Hsf1, 2, and 4). HSFs upregulate the stress-inducible promoters by interacting with the heat shock responsive elements. In the embryonic mouse lens, Hsf1 and Hsf2 are expressed at high levels. Their levels decrease in the adult lens, where Hsf4 takes over (Somasundaram and Bhat, 2004). αB-Crystallin promoter is upregulated by Hsf4, but not by Hsf1 or Hsf2 (Somasundaram and Bhat, 2004). Hsf4 null mice show increased proliferation, premature, defective differentiation of the lens epithelial cells, and increased expression of growth factors FGF-1, FGF-4, and FGF-7, and develop early postnatal cataract with abnormal lens fiber cells containing inclusion-like structures (Fujimoto et al., 2004; Min et al., 2004). The human HSF4 locus is associated with autosomal recessive cataract (Smaoui et al., 2004) and autosomal dominant lamellar and Marner cataract (Bu et al., 2002).

Maf. Maf transcription factors belong to the basic domain/ leucine zipper family whose consensus target site, T-MARE, is an extended version of an AP1 site. Three members of the large Maf family, c-Maf, MafB, and Nrl, are expressed in the mouse lens (Kawauchi et al., 1999). The expression of c-Maf is earliest and most prominent in lens fiber cells and persists throughout lens development (see figure 57.2) (Kawauchi et al., 1999; Ring et al., 2000). Homozygous Maf mutant embryos and newborns show defective lens fiber cell differentiation, with severely impaired expression of crystallin genes. Posterior lens cells in Maf(lacZ) mutant mice do not elongate, do not express aA- and any of the b-crystallin genes, and display inappropriately high levels of DNA synthesis at E11.5 (Kawauchi et al., 1999; Kim et al., 1999; Ring et al., 2000). Maf interacts with Pax6 and Sox to synergistically upregulate the mouse αB-crystallin and γFcrystallin promoter activities, respectively (see figure 57.2) (Rajaram and Kerppola, 2004; Yang et al., 2004). Synergistic activation of these promoters by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract (Rajaram and Kerppola, 2004).

Sp1/Krüppel-like transcription factors. Many Sp1/Krüppel-like transcription factors, members of the zinc finger family of DNA binding proteins, are expressed in the ocular surface and lens (see figure 57.2) (Chiambaretta et al., 2004; Norman

et al., 2004). Expression of Sp1 in the ectoderm and lens vesicle at E11 is much lower than that in the cornea from E15.5 to late stages (Nakamura et al., 2005). Sp1 levels in the cornea decline gradually following eyelid opening. Evidence for the involvement of Sp1 in the regulation of corneal gene expression comes from the finding that Sp1 activates involucrin gene expression in the differentiating corneal epithelium (Adhikary et al., 2005b).

Expression of Krüppel-like transcription factor Klf4, one of the most highly expressed transcription factors in the mouse cornea (Norman et al., 2004), is detectable in the ocular surface from around E10 and is sustained in the adult cornea (see figure 57.2). Conditional deletion of Klf4 in the surface ectoderm-derived structures of the eye leads to fragile corneal epithelium, swollen, vacuolated basal epithelial and endothelial cells, vacuolated lens, edematous stroma, and loss of conjunctival goblet cells (Swamynathan et al., 2007). Klf4 binds and activates keratin-12 and aquaporin-5 promoters in the corneal epithelium (Swamynathan et al., 2007). Klf4 cooperates with Oct3/4 and Sox2 and acts as a mediating factor that specifically binds to the proximal element to activate the Lefty1 core promoter in embryonic stem cells (Nakatake et al., 2006). Pluripotent stem cells could be derived from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under embryonic stem cell culture conditions (Takahashi and Yamanaka, 2006). As each of these factors is present in the corneal limbus, it is likely that they are involved in the maintenance of limbal stem cells, the source of epithelial cells in the mature cornea.

Another Krüppel-like factor, Klf6, is expressed in the lens pit at E10.5, in the ectoderm, mesenchyme, and the lens epithelium at E12.5, and in the lens and corneal epithelium and corneal stroma at E15.5 (Nakamura et al., 2004). Later on, the expression of Klf6 remains high in the cornea but decreases in the lens. In humans, expression of KLF6 is elevated in keratoconus, a progressive disease associated with thinning and scarring of the cornea (Chiambaretta et al., 2006). KLF6 binds and downregulates the a1-proteinase inhibitor (a1-PI) gene in corneal epithelial cells and may thereby be involved in keratoconus (Chiambaretta et al., 2006). Human KLF6 binds and activates keratin-12 promoter in cultured cells (Chiambaretta et al., 2002).

I-kB-z. I-κB-ζ, a regulator of the transcription factor NFκB, is expressed in the ocular surface epithelium, a part of the mucosal defense system (see figure 57.2) (Ueta et al., 2005). I-κB-ζ negatively regulates the pathological progression of ocular surface inflammation (Ueta et al., 2005). I-kB-z null mouse ocular surface shows chronic inflammation accompanied by loss of conjunctival goblet cells, indicating that I-κB-ζ is an integral part of the network of transcription factors required for ocular surface development (Ueta et al., 2005).

AP1. AP1, a group of dimeric complexes formed by the various Jun, Fos, Fra, and ATF proteins that regulate cell proliferation in response to various stimuli, is expressed in the lens and cornea (see figure 57.2) (Adhikary et al., 2005a; Ilagan et al., 1999; Norman et al., 2004; Okada et al., 2003; Shirai et al., 2004). AP1 activates mouse aA-crystallin expression through the conserved +25/+32 bp AP1 binding site (Ilagan et al., 1999). AP1 is necessary for expression of involucrin, a structural protein that is selectively expressed in differentiating corneal epithelial cells (Adhikary et al., 2005a, 2005b). In transgenic mice, removal of the AP1 site by truncation or point mutation results in a loss of involucrin expression, confirming the importance of AP1 for involucrin promoter activity during corneal epithelial cell differentiation.

AP2. The activating protein-2 (AP2) family of transcription factors, consisting of five different members, AP2α, AP2β, AP2γ, AP2δ, and AP2ε, stimulates proliferation and suppresses terminal differentiation in a cell-type-specific manner during embryonic development (Eckert et al., 2005). AP2α is expressed in the lens and corneal epithelium (see figure 57.2) (Ohtaka-Maruyama et al., 1998; West-Mays et al., 1999, 2003). Ap2a null embryos exhibit a range of phenotypes from a complete lack of eyes to defective lens attached to the overlying surface ectoderm (West-Mays et al., 1999). Conditional deletion of Ap2a in lens placode derivatives, including the corneal epithelium, results in a decrease in the expression of the cell-cell adhesion molecule E-cadherin, misexpression of laminin, entactin, and type IV collagen, and disruption of stromal collagen fibril organization, showing that AP2α is required for proper formation of the mouse cornea (Dwivedi et al., 2005; West-Mays et al., 2003). Pax6 and AP2α interact with each other and coordinate the expression of gelatinase-B (matrix metalloproteinase 9) and corneal epithelial repair (Sivak et al., 2004).

Elf3. Ese-1/Elf3, an epithelium-specific transcription factor, is upregulated in differentiating mouse corneal epithelium and in immortalized human corneal epithelial cells (HCEs), and transactivates keratin-12 through Ets binding sites (Yoshida et al., 2000). Suppression of Ese-1/Elf3 by antisense RNA in HCE cells affects their differentiation, suggesting the involvement of Ese-1/Elf3 in differentiation of corneal epithelial cells (see figure 57.2).

Regulation of expression of specific genes in the lens and cornea

Crystallin Gene Expression in the Lens Differentiation of the lens is characterized by lens-preferred expression and accumulation of water-soluble crystallins, essential lens structural proteins required for light refraction and

transparency (Piatigorsky, 1998; Piatigorsky and Wistow, 1989). Much effort in the last two decades shows that the lens-preferred expression of crystallins is an outcome of synergistic interactions between developmentally regulated transcription factors such as Pax6, c-Maf, MafA/L-Maf, MafB, Nrl, Sox1, Sox2, Rarβ/Rxrβ, Prox1, Six3, and Hsf4, ubiquitously expressed factors such as AP1, Creb, pRb, and Usf, and chromatin remodeling proteins such as Asc-2 and Cbp/p300 (figure 57.3) (Cvekl and Piatigorsky, 1996; Cvekl and Tamm, 2004; Cvekl et al., 2004).

aA-Crystallin. Mouse αA-crystallin, a marker of lens fiber cell differentiation and one of the most abundant mouse lens crystallins, is expressed at high levels in the developing lens, beginning around E10.5, and continuing in the adult lens (Robinson and Overbeek, 1996; Wawrousek et al., 1990). The mouse αA-crystallin promoter contains a Maf responsive element (MARE) at −110/−98 bp position, and overlapping binding sites for αACryBP1/PrdII/Mbp-1 (Brady et al., 1995; Kantorow et al., 1993; Nakamura et al., 1990), Maf (Wawrousek et al., 1990; Yang and Cvekl, 2005), and Pax6 (Cvekl et al., 1995) at −88 to +46 bp position (see figure 57.3). Pax6 and c-Maf activate aA-crystallin moderately and strongly, respectively, in a nonsynergistic manner (Yang and Cvekl, 2005; Yang et al., 2004, 2006).

Three conserved distal control regions (DCR1, −7706/ −7492 bp; DCR2, −1900/−1670 bp; DCR3, +3650/ +3856 bp) regulate mouse αA-crystallin promoter activity (see figure 57.3) (Yang et al., 2004). DCR1, bound by Pax6, stimulates αA-crystallin promoter activity in primary lens explants. DCR1/DCR2/αA-crystallin promoter activity resembles the native αA-crystallin promoter activity in lens epithelium and fiber cells. In contrast, the DCR3/αAcrystallin promoter activity is restricted to late lens fibers (Yang et al., 2004, 2006). Pax6, c-Maf, and CREB recruit chromatin-remodeling enzymes such as Brg1, Snf2h, CBP, and/or p300, thereby leading to localized histone acetylation and upregulation of aA-crystallin gene expression (Chen et al., 2002; Yang et al., 2006).

aB-Crystallin. Expression of αB-crystallin, a member of the small heat shock protein (sHSP) family, begins in the lens placode at E10 and continues at high levels in the lens epithelium and the lens fibers, and in moderate levels in many tissues (Haynes et al., 1996; Iwaki et al., 1989). A skeletal muscle–preferred, orientation-dependent enhancer containing five distinct cis-acting regulatory elements (αBE-1, αBE-4 αBE-2, αBE-3, and MRF, an E box) is present at −427/−259 bp position (see figure 57.3) (Gopal-Srivastava and Piatigorsky, 1993; Gopal-Srivastava et al., 1995; Swamynathan and Piatigorsky, 2002). The −426/+44 bp, −339/+44 bp, and −164/+44 bp promoter fragments retain activity in the lens as well as corneal epithelium, indicating

Figure 57.3 Organization of different transcription factor binding sites on crystallin promoters (A) and enhancers (B). Note the use of similar transcription factor binding sites on diverse crystallin pro-

that the αB-crystallin enhancer is not required for basal promoter activity in the lens and cornea (Gopal-Srivastava et al., 2000). Another well-conserved lens-specific enhancer (−2656/−2267 bp) containing four putative Sox1and Sox2binding sites activates the αB-crystallin promoter, even though the HspB2 gene is located in between this enhancer and the αB-crystallin promoter (see figure 57.3) (Ijichi et al., 2004).

Two well-conserved lens-specificity regions, LSR1 (−147/ −118 bp) and LSR2 (−78/−46 bp), are bound by Pax6, Maf, and RARs and are required for the lens activity of the mouse αB-crystallin promoter (see figure 57.3) (Gopal-Srivastava et al., 1996; Yang et al., 2004). The −164/+44 bp and −115/ +44 bp mouse αB-crystallin promoter activities are lens specific in transgenic mice, although the average activity is 30 times lower for the shorter fragment lacking LSR1. Sitespecific mutation of LSR1 eliminates both Pax6 binding and the −164/+44 bp promoter activity in transgenic mice (Gopal-Srivastava et al., 1996). Pax6-mediated activation of the αB-crystallin −162/+45 bp promoter is more robust in association with any of the Mafs and RARβ/RXRβ (Yang et al., 2004).

moters. DCR, distal control region. DNA fragments are not drawn to scale.

b- and g-Crystallins. β- and γ-crystallins are expressed in a lens fiber cell–specific manner. Several cis-elements required for lens-specific expression of the βB1-crystallin promoter have been identified by phylogenetic footprinting of the 5′ flanking sequence of the mouse, rat, human, and chicken bB1-crystallin genes (see figure 57.3) (Chen et al., 2001). The mouse bB1-crystallin −1493/+44 CAT transgene is expressed in the lens fiber cells (Chen et al., 2001). Transcription factor c-Maf directly activates many b-crystallin genes (see figure 57.3) (Ring et al., 2000).

Mutations in γ-crystallins lead to many dominant inherited cataracts in humans and mice (Garber et al., 1985; Graw, 1999). Sox1 binds a conserved element in the γ- crystallin promoter, and targeted deletion of Sox1 causes reduced g-crystallin expression, impaired fiber cell elongation, microphthalmia, and cataract (Nishiguchi et al., 1998). The heat shock factor HSF4 supports gF-crystallin expression in the lens fiber cells (Fujimoto et al., 2004). Pax6 represses the activation of the γF-crystallin promoter by large Mafs, Sox, and RARβ/RXRβ proteins in transiently transfected lens cells (Yang et al., 2004).

Gene Expression in the Cornea Unlike the lens, where a large body of work has focused on understanding the regulation of a handful of crystallin genes, in the cornea there is a limited amount of work, spread among different genes.

Keratocan. Keratocan (Ktcn), a keratan sulfate proteoglycan of the extracellular matrix, is first expressed at E13.5 in the periocular mesenchymal cells and after E14.5 is restricted to the stromal keratocytes (Liu et al., 1998). In adult transgenic mice with b-geo transgene driven by 3.2 kb 5′ flanking sequence, exon 1 and 0.4 kb of intron 1 of Ktcn, β-Gal activity is detected only in cornea. Spatiotemporal expression patterns of the −3.2 kb fragment transgene recapitulate that of endogenous Ktcn, suggesting that the 3.2 kb Ktcn upstream sequence contains the necessary cis-elements to regulate keratocan gene expression (Liu et al., 2000).

Keratins. Keratin-12 (Krt12), one of the more than 30 different keratins (intermediate filament components), each with a specific expression pattern in different epithelial cells, is expressed specifically in the stratified corneal epithelium (Tanifuji-Terai et al., 2006). Heterozygous mutations in KRT12 cause Meesmann corneal dystrophy, an autosomal dominant disorder affecting the human corneal epithelium. Krt12 null mice develop fragile corneal epithelium resembling Meesmann corneal dystrophy (Kao et al., 1996). During embryonic development, Krt12 expression is restricted to the suprabasal and/or superficial cells of the corneal epithelium from E15.5 to P10 in mice. After PN30, Krt12 expression is detected sporadically in the basal corneal epithelium, and the number of Krt12-positive basal cells increases as the mice grow older (Tanifuji-Terai et al., 2006). Pax6, KLF4, and KLF6 stimulate Krt12 promoter activity (Liu et al., 1999). Expression of Krt12 is delayed and downregulated in the Pax6+/− corneal epithelium, implying abnormal differentiation (Ramaesh et al., 2005).

Corneal crystallins. An underlying assumption in many gene regulation–related studies is that gene expression is controlled mainly at the level of transcription. However, posttranscriptional regulation may play a significant role in the expression of abundant corneal crystallins aldehyde dehydrogenase IIIA1 (Aldh3a1) and transketolase (Tkt), which constitute roughly 50% and 10% of the water-soluble protein, respectively, and only about 1% each of the total mRNA in the adult cornea (Norman et al., 2004). Aldh3a1 expression in the mouse is at least 500-fold higher in the corneal epithelial cells than in other tissues (Kays and Piatigorsky, 1997). In transgenic mice, a 13 kb mouse Aldh3a1 promoter fragment is active selectively in tissues that express the endogenous Aldh3a1 gene; however, unlike the promoter activity of the endogenous wild-type gene, the

promoter activity of the 13 kb fragment in the transgene is higher in the stomach and bladder than in the cornea. By contrast, a 4.4 kb mouse Aldh3a1 promoter fragment drives transgene expression in transgenic mice specifically in the corneal epithelial cells and not in other tissues, indicating that cis-control elements for corneal promoter activity reside within this smaller DNA fragment (Kays and Piatigorsky, 1997). Current experiments indicate that Aldh3a1 promoter activity is controlled by transcription factors that also control lens crystallin genes (Pax6, Oct1) as well as additional transcription factors expressed highly in the cornea (KLF4, KLF5). In addition, a suppressor sequence resides in the first intron of the mouse Aldh3a1 gene, although it is not known at present how it might function in regulating endogenous gene expression (Davis et al., in press).

Another mouse corneal crystallin is Tkt, which is expressed at 30–50 times higher levels in the mature mouse cornea than in other tissues (Sax et al., 1996). Tkt mRNA levels increase six-fold in the mouse cornea in vivo within 1–2 days of eye opening. Both exposure to light and oxidative stresses appear to play a role in the up-regulation of Tkt gene expression after eye opening and during corneal maturation (Sax et al., 2000). The Tkt gene contains two transcription initiation sites separated by 630 bp, with a common initiator ATG codon (West-Mays et al., 1999). The distal transcription initiation site is used weakly in liver and the proximal GCrich transcription initiation site (within intron 1) lacking a TATA box is used in the liver and for high corneal expression. There is little knowledge yet of the molecular basis for the high expression of Tkt in the mouse cornea.

Concluding remarks

The studies summarized in this chapter indicate that the mouse lens and cornea utilize many similar transcription factors for their development and maintenance. One of the striking similarities in gene expression in these two tissues is that many (not all) of these developmental transcription factors are also employed to express their highly specific multifunctional proteins, the crystallins. In view of the similarities between lens and cornea in their (1) origin from ectodermal cells during embryogenesis, (2) utilization of common transcription factors during embryonic development and postnatal maturation, (3) accumulation of crystallins, and (4) optical function, it has been proposed that the cornea and lens be considered as a single unit called the refracton (Piatigorsky, 2001). The refracton concept is not meant to equate the needs for and extents of refraction served by the lens and/or cornea of different species, both of which may differ considerably among species (especially aquatic versus terrestrial species) or even within the same species at different developmental times (such as an aquatic larva and terrestrial vertebrate, i.e., a toad) or at different

behavioral times (such as an amphibious species in water or land, i.e., a frog or penguin). Rather, unifying the lens and cornea as the refracton underlines the numerous developmental and functional similarities of these two transparent eye structures, including especially the accumulation of taxon-specific, soluble proteins called collectively the lens or corneal crystallins, respectively. It is our hope that the refracton concept will stimulate cross-fertilization of ideas and knowledge concerning these refractive tissues of the eye by increasing dialogue and information exchange between basic scientists and clinicians presently specializing in the lens or the cornea.

We have witnessed substantial progress in our understanding of gene expression and its consequences for the development of the cornea and lens in the recent past. Many studies have utilized germ-line deletions or conditional inactivation of specific genes in the surface ectoderm–derived tissues of the eye. Improvements in our ability to delete or mutate specific genes in a spatiotemporally controlled manner will further aid this effort. We can look forward to the identification of additional roles for the transcription factors currently known to influence eye development, as well as to the discovery of novel transcription factors and new interactions among the transcription factors, in our quest to unravel the molecular basis, biological consequences, and medical implications of gene expression in the lens and cornea.

acknowledgments Work was supported by the intramural research program of the National Eye Institute, NIH. We are grateful to Dr. Janine Davis for a critical reading of the manuscript.

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