Posterior capsular opacification (PCO), the most prevalent side effect of cataract surgery, occurs when residual lens epithelial cells (LECs) undergo fiber cell differentiation or epithelial-to-mesenchymal transition (EMT). Here, we used a murine cataract surgery model to investigate the role of the Zeb proteins, Smad interacting protein 1 (Sip1) and δ-crystallin enhancer-binding factor 1 (δEF1), during PCO.
Extracapsular extraction of lens fiber cells was performed on wild-type and Sip1 knockout mice. Protein expression patterns were assessed at multiple time points after surgery using confocal immunofluorescence. βB1-Crystallin mRNA levels were measured using quantitative RT-PCR. We used Transfac searches to identify δEF1 binding sites in the βB1-crystallin promoter and transfection analysis to test the ability of δEF1 to regulate βB1-crystallin expression.
δEF1, which, in other systems, can activate fibrotic genes (e.g., α-smooth muscle actin) and repress epithelial genes, upregulates by 48 hours after fiber cell removal. In culture, δEF1 repressed βB1-crystallin promoter activity, suggesting that it may also turn off lens gene expression following surgery, contributing to “fibrotic PCO” development. Sip1 also upregulates in LECs by 48 hours, but analysis of Sip1 knockout lenses demonstrated that Sip1 does not play a major role in EMT or fiber cell differentiation after surgery. However, Sip1 knockout LECs do express the ectodermal marker keratin 8, suggesting that Sip1 may limit the reprogramming of residual LECs to an embryonic state.
Zeb transcription factors likely play important, but distinct roles in PCO development after cataract surgery.
Zeb transcription factor expression upregulates following lens fiber cell removal in an animal model. The results presented correlate δEF1 expression with lens epithelial fibrosis while Sip1 upregulation appears to prevent dedifferentiation of lens cells to a precursor fate.
cataract surgery; epithelial-to-mesenchymal transition; lens regeneration; mouse disease model; posterior capsular opacification; Zeb
Posterior capsular opacification (PCO) is the major complication arising after cataract treatment. PCO occurs when the lens epithelial cells remaining following surgery (LCs) undergo a wound healing response producing a mixture of α-smooth muscle actin (α-SMA)-expressing myofibroblasts and lens fibre cells, which impair vision. Prior investigations have proposed that integrins play a central role in PCO and we found that, in a mouse fibre cell removal model of cataract surgery, expression of αV integrin and its interacting β-subunits β1, β5, β6, β8 are up-regulated concomitant with α-SMA in LCs following surgery. To test the hypothesis that αV integrins are functionally important in PCO pathogenesis, we created mice lacking the αV integrin subunit in all lens cells. Adult lenses lacking αV integrins are transparent and show no apparent morphological abnormalities when compared with control lenses. However, following surgical fibre cell removal, the LCs in control eyes increased cell proliferation, and up-regulated the expression of α-SMA, β1-integrin, fibronectin, tenascin-C and transforming growth factor beta (TGF-β)–induced protein within 48 hrs, while LCs lacking αV integrins exhibited much less cell proliferation and little to no up-regulation of any of the fibrotic markers tested. This effect appears to result from the known roles of αV integrins in latent TGF-β activation as αV integrin null lenses do not exhibit detectable SMAD-3 phosphorylation after surgery, while this occurs robustly in control lenses, consistent with the known roles for TGF-β in fibrotic PCO. These data suggest that therapeutics antagonizing αV integrin function could be used to prevent fibrotic PCO following cataract surgery.
lens; epithelial-to-mesenchymal transition; posterior capsular opacification; integrins; transforming growth factor beta; wound healing response; fibrosis; secondary cataract
We report analysis of the ocular lens phenotype of the recessive, larval lethal zebrafish mutant, lama1a69/a69. Previous work revealed that this mutant has a shortened body axis and eye defects including a defective hyaloid vasculature, focal corneal dysplasia, and loss of the crystalline lens. While these studies highlight the importance of laminin α1 in lens development, a detailed analysis of the lens defects seen in these mutants was not reported. In the present study, we analyze the lenticular anomalies seen in the lama1a69/a69 mutants and show that the lens defects result from the anterior extrusion of lens material from the eye secondary to structural defects in the lens capsule and developing corneal epithelium associated with basement membrane loss. Our analysis provides further insights into the role of the lens capsule and corneal basement membrane in the structural integrity of the developing eye.
The unfolded protein response is a set of cell signaling pathways recently recognized to be activated in the lens during both normal development and endoplasmic reticulum stress induced by either unfolded proteins or oxidative damage. While mutations in the gene for connexin 50 are known to cause autosomal dominant cataracts, it has not been previously reported whether mutant connexins can activate the unfolded protein response in the lens. Mice homozygous for the S50P or G22R mutation of connexin 50 have reduced amounts of connexin 50 protein at the cell membrane, with some intracellular staining consistent with retention in the endoplasmic reticulum. Connexin 50 mutants have elevated levels of BiP expression in both lens epithelial and fiber cells from E15.5 with the most robust elevation detected in newborns. While this elevation decreases in magnitude postnatally, BiP expression is still abnormally high in adults, particularly in the perinuclear endoplasmic reticulum of cell nuclei that are inappropriately retained in adult homozygous mutant lenses. Xbp1 splicing was elevated in lenses from both connexin mutants studied, while Atf4 and Atf6 levels were not majorly affected. Overall, these data suggest that UPR may be a contributing factor to the phenotype of connexin 50 mutant lenses even though the relatively modest extent of the response suggests that it is unlikely to be a major driver of the pathology.
UPR; BiP; Cataract; Connexin 50
SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes, as highlighted by the pleiotropic defects observed in Mowat-Wilson Syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases, where it functionally links TGFβ signaling to the loss of epithelial cell characteristics and gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers, such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation, and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes. Furthermore, 34% of the genes with increased expression in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes with decreased expression in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts.
Sip1; Zeb2; lens development; ectodermal cell fate
Transforming Growth Factor-beta (TGF-β) has roles in embryonic development, the prevention of inappropriate inflammation and tumor suppression. However, TGF-β signaling also regulates pathological epithelial to-mesenchymal transition (EMT), inducing or progressing a number of diseases ranging from inflammatory disorders, to fibrosis and cancer. However, TGF-β signaling does not proceed linearly but rather induces a complex network of cascades that mutually influence each other and cross-talk with other pathways to successfully induce EMT. Particularly, there is substantial evidence for cross-talk between αV integrins and TGF-β during EMT, and anti-integrin therapeutics are under development as treatments for TGF-β-related disorders. However, TGF-β’s complex signaling network makes the development of therapeutics to block TGF-β mediated pathology challenging. Moreover, despite our current understanding of integrins and TGF-β function during EMT, the precise mechanism of their role during physiological versus pathological EMT is not fully understood. This review focuses on the circle of regulation between αV integrin and TGF-β signaling during TGF-β induced EMT, which pose as a significant driver to many known TGF-β-mediated disorders.
The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2 were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.
The lens capsule compartmentalizes the cells of the avascular lens from other ocular tissues. Small molecules required for lens cell metabolism, such as glucose, salts, and waste products, freely pass through the capsule. However, the lens capsule is selectively permeable to proteins such as growth hormones and substrate carriers which are required for proper lens growth and development. We used fluorescence recovery after photobleaching (FRAP) to characterize the diffusional behavior of various sized dextrans (3, 10, 40, 150, and 250kDa) and proteins endogenous to the lens environment (EGF, γD-crystallin, BSA, transferrin, ceruloplasmin, and IgG) within the capsules of whole living lenses. We found that proteins had dramatically different diffusion and partition coefficients as well as capsule matrix binding affinities than similar sized dextrans, but they had comparable permeabilities. We also found ionic interactions between proteins and the capsule matrix significantly influence permeability and binding affinity, while hydrophobic interactions had less of an effect. The removal of a single anionic residue from the surface of a protein, γD-crystallin [E107A], significantly altered its permeability and matrix binding affinity in the capsule. Our data indicated that permeabilities and binding affinities in the lens capsule varied between individual proteins and cannot be predicted by isoelectric points or molecular size alone.
lens capsule; basement membrane; diffusion coefficient; permeability; binding affinity; partition coefficient; FRAP
Hyaluronan is an oligosaccharide found in the pericellular matrix of numerous cell types and hyaluronan induced signaling is known to facilitate fibrosis and cancer progression in some tissues. Hyaluronan is also commonly instilled into the eye during cataract surgery to protect the corneal endothelium from damage. Despite this, little is known about the distribution of hyaluronan or its receptors in the normal ocular lens. In this study, hyaluronan was found throughout the mouse lens, with apparently higher concentrations in the lens epithelium. CD44, a major cellular receptor for hyaluronan, is expressed predominately in mouse secondary lens fiber cells born from late embryogenesis into adulthood. Surgical removal of lens fiber cells from adult mice resulted in a robust upregulation of CD44 protein which preceded the upregulation of α-smooth muscle actin expression typically used as a marker of epithelial-mesenchyme transition in this model of lens epithelial cell fibrosis. Mice lacking the CD44 gene had morphologically normal lenses with a response to lens fiber cell removal similar to wildtype, although they exhibited an increase in cell associated hyaluronan. Overall, these data suggest that lens cells have a hyaluronan containing pericellular matrix whose structure is partially regulated by CD44. Further, these data indicate that CD44 upregulation in the lens epithelium may be an earlier marker of lens injury responses in the mouse lens than the upregulation of α-smooth muscle actin.
The lens capsule is a modified basement membrane that completely surrounds the ocular lens. It is known that this extracellular matrix is important for both the structure and biomechanics of the lens in addition to providing informational cues to maintain lens cell phenotype. This review covers the development and structure of the lens capsule, lens diseases associated with mutations in extracellular matrix genes and the role of the capsule in lens function including those proposed for visual accommodation, selective permeability to infectious agents, and cell signaling.
Accurate lens capsule thickness measurements are necessary for studies investigating mechanical characteristics of the capsule. Confocal Z-axis imaging was taken advantage of to measure the anterior lens capsule thickness of living intact lenses with minimal tissue manipulation. Measurements of the anterior capsule thickness is reported for the first time in young and old mice from four inbred strains, BALB/c, FVB/N, C57BL/6, and 129X1, and the outbred strain ICR. Our data demonstrates that the mouse anterior lens capsule continues to grow postnatally, as has been described with other mammals. It is also shown there is a significant difference in anterior lens capsule thickness between unrelated mouse strains, suggesting capsule thickness is a quantitative trait shared by strains with common ancestry. Measurements, taken from other regions of FVB/N capsules, revealed the anterior pole to be the thickest, followed by the equatorial region and posterior pole. In addition to mouse, anterior capsule measurements taken from intact cattle, rabbit, and rat lenses, as well as human capsulotomy specimens correlated with the over all size of the animal.
The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post coitum (dpc) mouse embryos however, by 13.5 dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5 dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5 dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around two weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.
Lens fiber cell differentiation is marked by the onset of βB1-crystallin expression and is controlled by the cooperative action of a set of transcription factors including Prox1, an atypical homeodomain protein. Previously, the authors reported that Prox1 directly interacts with the OL2 element found in the chicken βB1-crystallin basal promoter to activate the expression of this gene. Here they mapped the location of activating and repressing sequences of the full-length chicken βB1-crystallin promoter (−432/+30) in lens epithelial cells, annular pad cells, and intact lens and characterized Prox1-binding sites found in this region.
Transfection analysis and transgenic mice were used to characterize upstream regions of the chicken βB1-crystallin gene. DNaseI footprinting and chromatin immunoprecipitation was performed to identify Prox1-binding sites, and transfection analyses were used to characterize these sites functionally.
Sequences between −152 and −432 of the chicken βB1-crystallin promoter mediated either promoter activation or repression, depending on the stage of lens differentiation tested. Two new Prox1-binding sites were found in this region that bound Prox1 more avidly than the OL2 element. However, neither binding site conferred Prox1-mediated activation on a heterologous promoter; instead, each allowed Prox1 to repress promoter function.
The function of the upstream region of the chicken βB1-crystallin promoter changes depending on cellular context. These data suggest that Prox1 function as a transcriptional activator could be regulated at the DNA level based on the characteristics of the responsive elements.
β1-integrins are cell surface receptors that participate in sensing the cell’s external environment. We used the Cre-lox system to delete β1-integrin in all lens cells as the lens vesicle transitions into the lens. Adult mice lacking β1-integrin in the lens are microopthalmic due to apoptosis of the lens epithelium and neonatal disintegration of the lens fibers. The first morphological alterations in β1-integrin null lenses are seen at 16.5 dpc when the epithelium becomes disorganized and begins to upregulate the fiber cell markers β- and γ-crystallin, the transcription factors cMaf and Prox1 and down regulate Pax6 levels demonstrating that β1-integrin is essential to maintain the lens epithelial phenotype. Further, β1-integrin null lens epithelial cells upregulate the expression of α-smooth muscle actin and nuclear Smad4 and downregulate Smad6 suggesting that β1-integrin may brake TGFβ family signaling leading to epithelial-mesenchymal transitions in the lens. In contrast, β1-integrin null lens epithelial cells show increased E-cadherin immunoreactivity which supports the proposed role of β1-integrins in mediating complete EMT in response to TGFβ family members. Thus, β1-integrin is required to maintain the lens epithelial phenotype and block inappropriate activation of some aspects of the lens fiber cell differentiation program.
Prox1 is a transcription factor which can function either as a transcriptional activator, transcriptional repressor or a transcriptional corepressor. This paper seeks to better understand the role of protein–protein interactions in this multitude of functions.
We performed a yeast two-hybrid screen of an 11.5 day post coitum (dpc) mouse embryo cDNA library using the homeo-Prospero domain of Prox1 as bait. Computer modeling, cotransfection analysis and confocal immunolocalization were used to investigate the significance of one of the identified interactions.
Proliferating cell nuclear antigen (PCNA) was identified as a Prox1 interacting protein. Prox1 interactions with PCNA require the PCNA interacting protein motif (PIP box), located in the Prospero domain of Prox1. Computer modeling of this interaction identified the apparent geometry of this interface which maintains the accessibility of Prox1 to DNA. Prox1 activated the chicken βB1-crystallin promoter in cotransfection tests as previously reported, while PCNA squelched this transcriptional activation.
Since PCNA is expressed in the lens epithelium where Prox1 levels are low, while chicken βB1-crystallin expression activates in lens fibers where Prox1 expression is high and PCNA levels are low, these data suggest that Prox1-PCNA interactions may in part prevent the activation of βB1-crystallin expression in the lens epithelium.
βB2-crystallin is one of the most abundant proteins of the adult ocular lens of mammals although it is expressed at lower levels in several extralenticular locations. While mutations in βB2-crystallin are known to result in lens opacities, alterations in tissues besides the lens have not been previously investigated in these mutants. Since we found mice harboring the Crybb2Phil mutation bred poorly, here we assess the contribution of βB2-crystallin to mouse fertility and determine the expression pattern of βB2-crystallin in the testis.
The expression pattern of βB2-crystallin in the testis was analyzed by rt-PCR, western blotting, and immunohistochemistry. The fecundity of wildtype and Crybb2Phil mice was analyzed by quantitative fertility testing. The morphology of testes and ovaries was assessed by hematoxylin and eosin staining.
In the mouse testis, βB2-crystallin mRNA is found at low levels at birth, but its expression upregulates in this tissue as the testis is primed to initiate spermatogenesis. Western blotting detected βB2-crystallin protein in sperm obtained from mice, cattle, and humans while immunolocalization detected this protein in developing sperm from the spermatocyte stage onward. Male and female mice homozygous for a 12 nucleotide inframe deletion mutation in βB2-crystallin are subfertile when analyzed on a Swiss Webster derived background due to defects in egg and sperm production. However, mice harboring the same mutation on the C57Bl/6 genetic background did not exhibit any defects in reproductive function.
βB2-crystallin is expressed in developing and mature sperm and mice of both sexes harboring the Philly mutation in the βB2-crystallin gene are subfertile when analyzed on a Swiss Webster genetic background. While these data are suggestive of a role for βB2-crystallin in fertility, definitive determination of this will await the creation of a βB2-crystallin null mouse.
Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.
Astrocytes; βA3/A1crystallin; Eye development; Ganglion cells; Lens; Retina; In situ hybridization
Sip1; Smad Interacting Protein 1; ZEB2; Development; Epithelial Mesenchymal transition; Mowat-Wilson Syndrome; Hirshsprung Disease
Inflammation and angiogenesis are integral parts of wound healing. However, excessive and persistent wound-induced inflammation and angiogenesis in an avascular tissue such as the cornea may be associated with scarring and visual impairment. Junctional adhesion molecule A (Jam-A) is a tight junction protein that regulates leukocyte transmigration as well as fibroblast growth factor-2 (FGF-2)-induced angiogenesis. However its function in wound-induced inflammation and angiogenesis is still unknown. In this study, we report spontaneous corneal opacity in Jam-A deficient mice associated with inflammation, angiogenesis and the presence of myofibroblasts. Since wounds and/or corneal infections cause corneal opacities, we tested the role of Jam-A in wound-induced inflammation, angiogenesis and scarring by subjecting Jam-A deficient mice to full thickness corneal wounding. Analysis of these wounds demonstrated increased inflammation, angiogenesis, and increased number of myofibroblasts thereby indicating that Jam-A regulates the wound-healing response by controlling wound-induced inflammation, angiogenesis and scarring in the cornea. These effects were not due to inflammation alone since the inflammation-induced wound-healing response in Jam-A deficient mice was similar to wild type mice. In order to determine the molecular mechanism associated with the observed aberrant corneal wound healing in Jam-A deficient mice, we assessed the expression of the components of vascular endothelial growth factor A (VEGF-A)/vascular endothelial growth factor receptor- 2(VEGFR-2) signaling pathway. Interestingly, we observed increased levels of VEGF-A mRNA in Jam-A deficient eyes. We also observed nuclear localization of phosphorylated SMAD3 (pSMAD3) indicative of TGFβ pathway activation in the Jam-A deficient eyes. Furthermore the increased wound-induced corneal inflammation, angiogenesis, and scarring in Jam-A deficient mice was attenuated by treatment with DC101, an anti-vascular endothelial growth factor receptor-2 (VEGFR-2) antibody. Our results suggest that in the absence of Jam-A, the VEGF-A/VEGFR-2 pathway is upregulated, thereby augmenting wound induced corneal inflammation, angiogenesis, and myofibroblast accumulation leading to scarring.
Recent studies demonstrated a number of links between chromatin structure, gene expression, extracellular signaling and cellular differentiation during lens development. Lens progenitor cells originate from a pool of common progenitor cells, the pre-placodal region (PPR) which is formed due to a complex exchange of extracellular signals between the neural plate, naïve ectoderm and mesendoderm. A specific commitment to the lens program over alternate choices such as the formation of olfactory epithelium or the anterior pituitary is manifested by the formation of a thickened surface ectoderm, the lens placode. Mouse lens progenitor cells are characterized by the expression of a complement of lens lineage-specific transcription factors including Pax6, Six3 and Sox2, controlled by FGF and BMP signaling, followed later by c-Maf, Mab21like1, Prox1 and FoxE3. Proliferation of lens progenitors together with their morphogenetic movements results in the formation of the lens vesicle. This transient structure, comprised of lens precursor cells, is polarized with its anterior cells retaining their epithelial morphology and proliferative capacity, whereas the posterior lens precursor cells initiate terminal differentiation forming the primary lens fibers. Lens differentiation is marked by expression and accumulation of crystallins and other structural proteins. The transcriptional control of crystallin genes is characterized by the reiterative use of transcription factors required for the establishment of lens precursors in combination with more ubiquitously expressed factors (e.g. AP-1, AP-2α, CREB and USF) and recruitment of histone acetyltransferases (HATs) CBP and p300, and chromatin remodeling complexes SWI/SNF and ISWI. These studies have poised the study of lens development at the forefront of efforts to understand the connections between development, cell signaling, gene transcription and chromatin remodeling.
development, lens differentiation; histone acetylation and methylation; Pax6; c-Maf; chromatin remodeling; transcriptional regulation
Pax6 is a transcription factor that is required for induction, growth, and maintenance of the lens; however, few direct target genes of Pax6 are known.
In this report, we describe the results of a cDNA microarray analysis of lens transcripts from transgenic mice over-expressing Pax6 in lens fibre cells in order to narrow the field of potential direct Pax6 target genes. This study revealed that the transcript levels were significantly altered for 508 of the 9700 genes analysed, including five genes encoding the cell adhesion molecules β1-integrin, JAM1, L1 CAM, NCAM-140 and neogenin. Notably, comparisons between the genes differentially expressed in Pax6 heterozygous and Pax6 over-expressing lenses identified 13 common genes, including paralemmin, GDIβ, ATF1, Hrp12 and Brg1. Immunohistochemistry and Western blotting demonstrated that Brg1 is expressed in the embryonic and neonatal (2-week-old) but not in 14-week adult lenses, and confirmed altered expression in transgenic lenses over-expressing Pax6. Furthermore, EMSA demonstrated that the BRG1 promoter contains Pax6 binding sites, further supporting the proposition that it is directly regulated by Pax6.
These results provide a list of genes with possible roles in lens biology and cataracts that are directly or indirectly regulated by Pax6.
Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6.
The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR.
In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens.
Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation.
HMGN proteins promote chromatin unfolding, enhance access to nucleosomes, and modulate transcription from chromatin templates. It is not known whether they act indiscriminately as general modulators of transcription or whether they regulate specific gene expression. Here, we investigated the role of HMGN3, a recently discovered HMGN family member, in transcription in vivo. We created cell lines overexpressing HMGN3a or its splice variant, HMGN3b, and analyzed their gene expression profiles using microarrays and reverse transcriptase PCR. We found that ectopic expression of HMGN3a alters the expression of approximately 0.8% of genes. Both HMGN3a and HMGN3b upregulate the expression of the glycine transporter 1 gene (Glyt1). Glyt1 encodes a membrane transporter that regulates the glycine concentration in synaptic junctions. Both GLYT1 and HMGN3 are highly expressed in glia cells and the eye, and we show that both proteins are coexpressed in the retina. Chromatin immunoprecipitation assays showed that HMGN3 protein is recruited to a region of the Glyt1 gene encompassing the Glyt1a transcriptional start site. These results suggest that HMGN3 regulates Glyt1 expression and demonstrate that members of the HMGN family can regulate the transcription of specific genes.
It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the αB-, αA-, δ1-, and ζ-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and β-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the βB1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full-length mouse Pax-6 coding sequences into primary embryonic chicken lens epithelial cells or fibroblasts repressed the activity of this promoter by as much as 90%. Pax-6 constructs lacking the C-terminal activation domain repressed βB1-crystallin promoter activity as effectively as the full-length protein, but the PD alone or Pax-6 (5a), a splice variant with an altered PD affecting its DNA binding specificity, did not. DNase footprinting analysis revealed that truncated Pax-6 (PD+HD) binds to three regions (−183 to −152, −120 to −48, and −30 to +1) of the βB1-crystallin promoter. Earlier experiments showed that the βB1-crystallin promoter sequence from −120 to −48 contains a cis element (PL2 at −90 to −76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. In the present study, we show by electrophoretic mobility shift assay and cotransfection that Pax-6 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminated binding by Pax-6. Taken together, our data indicate that Pax-6 (via its PD and HD) represses the βB1-crystallin promoter by direct interaction with the PL2 element. We thus suggest that the relatively high concentration of Pax-6 contributes to the absence of βB1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens fiber cells during development allow activation of this gene.