Transgenic Lenses Research Articles

Microtubule‐associated protein tau (MAPT) in the lens of the eye in non‐transgenic and transgenic tau mutant (P301S) mice

AbstractBackgroundWe previously discovered that Aβ accumulates in the cortical/supranuclear region of the lens in people with Alzheimer’s Disease (AD) (Goldstein et al., 2003) and Down Syndrome (DS; (Moncaster et al., 2010). We also demonstrated Aβ in the Tg2576 APP Swedish mutation AD mouse model (Moncaster et al., 2022). Another protein that is involved in AD is Microtubule‐Associated Protein Tau (MAPT). Tau is expressed in the brain and becomes hyperphosphorylated in AD eventually forming neurofibrillary tangles. Tau has previously been reported to be expressed in the mouse lens (Bai et al., 2007; Zhao et al., 2013) but the results vary depending on the mouse model. Here we investigated whether tau protein was expressed in non‐transgenic mouse lenses and in a transgenic tau mutant (P301S) mouse model. We also studied whether cataracts may develop in this P301S tau mutant mouse model.MethodP301S tau mutant transgenic and non‐transgenic mice were bred and maintained at Boston University. Breeder mice were purchased from The Jackson Laboratory, Bar Harbor, ME. Male and Female transgenic and non‐transgenic mice were sacrificed throughout their lifespan at ages 3‐12 months. Mice were perfused with phosphate buffer saline, lenses were isolated and then imaged under two different sources of light using a D70 digital Nikon camera and a custom‐adapted Zeiss stereophotomicroscope. Lenses were then snap frozen and analyzed by Western Blotting using tau antibodies.ResultP301S transgenic and non‐transgenic mouse lenses all expressed tau during their lifespan when analyzed by Western blot. P301S transgenic lenses demonstrated an additional higher molecular weight band compared to the non‐transgenic mice. No cataract phenotype was observed in transgenic or non‐transgenic mouse lenses.ConclusionThere was no overt difference in lens phenotype between the P301S transgenic and non‐transgenic mice. However, the banding pattern observed by Western blot for P301S transgenic mice compared to non‐transgenic was different. Our data suggest the P301S tau mutation affects tau processing but does not result in a cataract phenotype. Based on our current and previous results, the data suggests that Aβ may play a more significant role than tau in lens pathology in AD.

Transcriptional profiling of single fiber cells in a transgenic paradigm of an inherited childhood cataract reveals absence of molecular heterogeneity

Our recent single-cell transcriptomic analysis has demonstrated that heterogeneous transcriptional activity attends molecular transition from the nascent to terminally differentiated fiber cells in the developing mouse lens. To understand the role of transcriptional heterogeneity in terminal differentiation and the functional phenotype (transparency) of this tissue, here we present a single-cell analysis of the developing lens, in a transgenic paradigm of an inherited pathology, known as the lamellar cataract. Cataracts hinder transmission of light into the eye. Lamellar cataract is the most prevalent bilateral childhood cataract. In this disease of early infancy, initially, the opacities remain confined to a few fiber cells, thus presenting an opportunity to investigate early molecular events that lead to cataractogenesis. We used a previously established paradigm that faithfully recapitulates this disease in transgenic mice. About 500 single fiber cells, manually isolated from a 2-day-old transgenic lens were interrogated individually for the expression of all known 17 crystallins and 78 other relevant genes using a Biomark HD (Fluidigm). We find that fiber cells from spatially and developmentally discrete regions of the transgenic (cataract) lens show remarkable absence of the heterogeneity of gene expression. Importantly, the molecular variability of cortical fiber cells, the hallmark of the WT lens, is absent in the transgenic cataract, suggesting absence of specific cell-type(s). Interestingly, we find a repetitive pattern of gene activity in progressive states of differentiation in the transgenic lens. This molecular dysfunction portends pathology much before the physical manifestations of the disease.

Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens

BackgroundDespite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous.ResultAlthough both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses.ConclusionsAlthough P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.

Transgenic lysyl oxidase homolog 1 overexpression in the mouse eye results in the formation and release of protein aggregates

Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular fibrillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken βB1-crystallin promoter was used to overexpress Loxl1 in the lenses of βB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. βB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20–50 nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of βB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2–5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients.

Molecular mechanism of Aquaporin 0-induced fiber cell to fiber cell adhesion in the eye lens

Cell-to-cell adhesion (CTCA), which is key for establishing lens transparency, is a critical function of Aquaporin 0 (AQP0). The aim of this investigation was to find out the possible mechanism by which AQP0 exerts CTCA between fiber cells, since there are two proposals currently, either an AQP0-AQP0 interaction or an AQP0-lipid interaction. We studied the mechanism of AQP0-induced CTCA in intact AQP0 and C-terminally cleaved AQP0 (CTC-AQP0). Assays showed CTCA between L-cells transfected with intact AQP0 or CTC-AQP0 and parental L-cells indicating AQP0-membrane interaction. Both forms of AQP0 significantly (P < 0.001) promoted adhesion to negatively charged l-α-phosphatidylserine lipid vesicles signifying AQP0-lipid interaction. AQP0-expressing L-cells also promoted adhesion of WT and AQP0-KO mouse lens fiber cell membrane vesicles (FCMVs) significantly (P < 0.001). However, when FCMVs of WT or AQP0-KO were plated over parental L-cells, only WT vesicles adhered significantly, corroborating AQP0-membrane interaction. After incubating with extracellular domain-specific AQP0 antibody, L-cells expressing intact AQP0 or CTC-AQP0 showed a significant reduction (P < 0.001) in the adhesion of AQP0-KO FCMVs indicating extracellular loop involvement in CTCA. WT FCMVs from outer cortex and inner cortex promoted adhesion to parental L-cells, without any statistically significant difference in adhesion efficiency (P > 0.05). Ultrastructure studies of WT, AQP0-KO and transgenic lenses showed AQP0 is critical for fiber CTCA and compaction. The data collected clearly demonstrate that the positively charged amino acids in the AQP0 extracellular loop domains interact with the negatively charged lipids in the plasma membrane to promote CTCA for compaction of fiber cells.

Expression of the HSF4 DNA binding domain-EGFP hybrid gene recreates early childhood lamellar cataract in transgenic mice.

The clinical management of cataracts in infancy involves surgical removal of the lens to ensure transmission of light to the retina, which is essential for normal neural development of the infant. This surgery, however, entails a lifelong follow-up and impaired vision. To our knowledge, no animal models recapitulate human lamellar opacities, the most prevalent form of early childhood cataracts. We present data on the recreation of the human lamellar cataract phenotype in transgenic mice. Mutations in the DNA binding domain (DBD) of the heat shock transcription factor 4 (HSF4) are known to be associated with early childhood autosomal dominant lamellar cataract. We used bacterial artificial chromosome (BAC) transgenesis to express a hybrid gene: Hsf4 (DBD)-enhanced green fluorescent protein (EGFP), by recombineering EGFP sequences into the DBD of the Hsf4 gene, to interfere with the DNA binding properties of Hsf4. We recapitulated the human lamellar cataract, in its temporal as well as spatial presentation, within the transgenic mouse lens. This phenotype was reproduced faithfully using four different BACs, indicating that EGFP can be used to target transcription factor function in transgenic mice. Molecular and cell biological examination of early postnatal transgenic lens reveals impairment of secondary fiber cell differentiation. Recreation of the human lamellar cataract phenotype in mice allows investigation of this human pathology at a level not possible previously and points to the relevance of fiber cell heterogeneity dictated by fiber cell-specific gene activity in the biogenesis of the lamellar cataract.

Design of an Amide N-Glycoside Derivative of β-Glucogallin: A Stable, Potent, and Specific Inhibitor of Aldose Reductase

β-Glucogallin (BGG), a major component of the Emblica officinalis medicinal plant, is a potent and selective inhibitor of aldose reductase (AKR1B1). New linkages (ether/triazole/amide) were introduced via high yielding, efficient syntheses to replace the labile ester, and an original two-step (90%) preparation of BGG was developed. Inhibition of AKR1B1was assessed in vitro and using transgenic lens organ cultures, which identified the amide linked glucoside (BGA) as a stable, potent, and selective therapeutic lead toward the treatment of diabetic eye disease.

Frs2α enhances fibroblast growth factor-mediated survival and differentiation in lens development

Most growth factor receptor tyrosine kinases (RTKs) signal through similar intracellular pathways, but they often have divergent biological effects. Therefore, elucidating the mechanism of channeling the intracellular effect of RTK stimulation to facilitate specific biological responses represents a fundamental biological challenge. Lens epithelial cells express numerous RTKs with the ability to initiate the phosphorylation (activation) of Erk1/2 and PI3-K/Akt signaling. However, only Fgfr stimulation leads to lens fiber cell differentiation in the developing mammalian embryo. Additionally, within the lens, only Fgfrs activate the signal transduction molecule Frs2α. Loss of Frs2α in the lens significantly increases apoptosis and decreases phosphorylation of both Erk1/2 and Akt. Also, Frs2α deficiency decreases the expression of several proteins characteristic of lens fiber cell differentiation, including Prox1, p57(KIP2), aquaporin 0 and β-crystallins. Although not normally expressed in the lens, the RTK TrkC phosphorylates Frs2α in response to binding the ligand NT3. Transgenic lens epithelial cells expressing both TrkC and NT3 exhibit several features characteristic of lens fiber cells. These include elongation, increased Erk1/2 and Akt phosphorylation, and the expression of β-crystallins. All these characteristics of NT3-TrkC transgenic lens epithelial cells depend on Frs2α. Therefore, tyrosine phosphorylation of Frs2α mediates Fgfr-dependent lens cell survival and provides a mechanistic basis for the unique fiber-differentiating capacity of Fgfs on mammalian lens epithelial cells.

Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation

BackgroundBrahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4) and paired box gene 6 (Pax6)), chromatin structural proteins (for example, high-mobility group A1 (HMGA1)) and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R) in the Brg1 ATPase domain acts via a dominant-negative (dn) mechanism. Genetic studies have demonstrated that Brg1 is an essential gene for early (that is, prior implantation) mouse embryonic development. Brg1 also controls neural stem cell maintenance, terminal differentiation of multiple cell lineages and organs including the T-cells, glial cells and limbs.ResultsTo examine the roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific αA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (that is, denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in embryonic day 15.5 (E15.5) wild-type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous and Hsf4 homozygous lenses identified multiple genes coregulated by Brg1, Hsf4 and Pax6. DNase IIβ, a key enzyme required for lens fiber cell denucleation, was found to be downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation, for expression of DNase IIβ, for lens fiber cell denucleation and indirectly for retinal development.ConclusionsThese studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase IIβ as a potential direct target of SWI/SNF complexes. Brg1 is directly or indirectly involved in processes that degrade lens fiber cell chromatin. The presence of nuclei and other organelles generates scattered light incompatible with the optical requirements for the lens.

Lens Fiber Cell Differentiation and Denucleation Are Disrupted through Expression of the N-Terminal Nuclear Receptor Box ofNcoa6and Result in p53-dependent and p53-independent Apoptosis

Nuclear receptor coactivator 6 (NCOA6) is a multifunctional protein implicated in embryonic development, cell survival, and homeostasis. An 81-amino acid fragment, dnNCOA6, containing the N-terminal nuclear receptor box (LXXLL motif) of NCOA6, acts as a dominant-negative (dn) inhibitor of NCOA6. Here, we expressed dnNCOA6 in postmitotic transgenic mouse lens fiber cells. The transgenic lenses showed reduced growth; a wide spectrum of lens fiber cell differentiation defects, including reduced expression of gamma-crystallins; and cataract formation. Those lens fiber cells entered an alternate proapoptotic pathway, and the denucleation (karyolysis) process was stalled. Activation of caspase-3 at embryonic day (E)13.5 was followed by double-strand breaks (DSBs) formation monitored via a biomarker, gamma-H2AX. Intense terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) signals were found at E16.5. Thus, a window of approximately 72 h between these events suggested prolonged though incomplete apoptosis in the lens fiber cell compartment that preserved nuclei in its cells. Genetic experiments showed that the apoptotic-like processes in the transgenic lens were both p53-dependent and p53-independent. Lens-specific deletion of Ncoa6 also resulted in disrupted lens fiber cell differentiation. Our data demonstrate a cell-autonomous role of Ncoa6 in lens fiber cell differentiation and suggest novel insights into the process of lens fiber cell denucleation and apoptosis.

Activated Ras alters lens and corneal development through induction of distinct downstream targets

BackgroundMammalian Ras genes regulate diverse cellular processes including proliferation and differentiation and are frequently mutated in human cancers. Tumor development in response to Ras activation varies between different tissues and the molecular basis for these variations are poorly understood. The murine lens and cornea have a common embryonic origin and arise from adjacent regions of the surface ectoderm. Activation of the fibroblast growth factor (FGF) signaling pathway induces the corneal epithelial cells to proliferate and the lens epithelial cells to exit the cell cycle. The molecular mechanisms that regulate the differential responses of these two related tissues have not been defined. We have generated transgenic mice that express a constitutively active version of human H-Ras in their lenses and corneas.ResultsRas transgenic lenses and corneal epithelial cells showed increased proliferation with concomitant increases in cyclin D1 and D2 expression. This initial increase in proliferation is sustained in the cornea but not in the lens epithelial cells. Coincidentally, cdk inhibitors p27Kip1 and p57Kip2 were upregulated in the Ras transgenic lenses but not in the corneas. Phospho-Erk1 and Erk2 levels were elevated in the lens but not in the cornea and Spry 1 and Spry 2, negative regulators of Ras-Raf-Erk signaling, were upregulated more in the corneal than in the lens epithelial cells. Both lens and corneal differentiation programs were sensitive to Ras activation. Ras transgenic embryos showed a distinctive alteration in the architecture of the lens pit. Ras activation, though sufficient for upregulation of Prox1, a transcription factor critical for cell cycle exit and initiation of fiber differentiation, is not sufficient for induction of terminal fiber differentiation. Expression of Keratin 12, a marker of corneal epithelial differentiation, was reduced in the Ras transgenic corneas.ConclusionsCollectively, these results suggest that Ras activation a) induces distinct sets of downstream targets in the lens and cornea resulting in distinct cellular responses and b) is sufficient for initiation but not completion of lens fiber differentiation.

Induction of Epithelial Mesenchimal Transition and Vasculogenesis in the Lenses of Dbl Oncogene Transgenic Mice

BackgroundThe Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. The numerous domains that are present in many Dbl family proteins suggest that they act to integrate multiple inputs in complicated signaling networks involving the Rho GTPases. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We generated transgenic mice introducing the cDNA of Dbl oncogene linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression in mouse lenses affected proliferation, migration and differentiation of lens epithelial cells.ResultsWe used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of 2 days, 2 weeks, and 6 weeks old transgenic mice. We observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT), such as down-regulation of epithelial cell markers and up-regulation of fibroblast markers. Genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses where vascularization can be readily observed.ConclusionOnco-Dbl expression in mouse lens correlated with modulation of genes involved in the regulation of EMT, apoptosis and vasculogenesis leading to disruption of the lens architecture, epithelial cell proliferation, and aberrant angiogenesis. We conclude that onco-Dbl has a potentially important, previously unreported, capacity to dramatically alter epithelial cell migration, replication, polarization and differentiation and to induce vascularization of an epithelial tissue.

Wnt signaling is required for organization of the lens fiber cell cytoskeleton and development of lens three-dimensional architecture

How an organ develops its characteristic shape is a major issue. This is particularly critical for the eye lens as its function depends on having appropriately ordered three-dimensional cellular architecture. Recent in vitro studies indicate that Wnt signaling plays key roles in regulating morphological events in FGF-induced fiber cell differentiation in the mammalian lens. To further investigate this the Wnt signaling antagonist, secreted frizzled-related protein 2 (Sfrp2), was overexpressed in lens fiber cells of transgenic mice. In these mice fiber cell elongation was attenuated and individual fibers exhibited irregular shapes and consequently did not align or pack regularly; microtubules, microfilaments and intermediate filaments were clearly disordered in these fibers. Furthermore, a striking feature of transgenic lenses was that fibers did not develop the convex curvature typically seen in normal lenses. This appears to be related to a lack of protrusive processes that are required for directed migratory activity at their apical and basal tips as well as for the formation of interlocking processes along their lateral margins. Components of the Wnt/Planar Cell Polarity (PCP) pathway were downregulated or inhibited. Taken together this supports a role for Wnt/PCP signaling in orchestrating the complex organization and dynamics of the fiber cell cytoskeleton.

Rho GDP dissociation inhibitor-mediated disruption of Rho GTPase activity impairs lens fiber cell migration, elongation and survival

To explore the role of the Rho GTPases in lens morphogenesis, we overexpressed bovine Rho GDP dissociation inhibitor (RhoGDIα), which serves as a negative regulator of Rho, Rac and Cdc42 GTPase activity, in a lens-specific manner in transgenic mice. This was achieved using a chimeric promoter of δ-crystallin enhancer and αA-crystallin, which is active at embryonic day 12. Several individual transgenic (Tg) lines were obtained, and exhibited ocular specific phenotype comprised of microphthalmic eyes with lens opacity. The overexpression of bovine RhoGDIα disrupted membrane translocation of Rho, Rac and Cdc42 GTPases in Tg lenses. Transgenic lenses also revealed abnormalities in the migration pattern, elongation and organization of lens fibers. These changes appeared to be associated with impaired organization of the actin cytoskeleton and cell–cell adhesions. At E14.5, the size of the RhoGDIα Tg lenses was larger compared to wild type (WT) and the central lens epithelium and differentiating fibers exhibited an abnormal increase of bromo-deoxy-uridine incorporation. Postnatal Tg eyes, however, were much smaller in size compared to WT eyes, revealing increased apoptosis in the disrupted lens fibers. Taken together, these data demonstrate a critical role for Rho GTPase-dependent signaling pathways in processes underlying morphogenesis, fiber cell migration, elongation and survival in the developing lens.

Transgenic overexpression of connexin50 induces cataracts

To examine the effects of increased expression of Cx50 in the mouse lens, transgenic mice were generated using a DNA construct containing the human Cx50 coding region and a C-terminal FLAG epitope driven by the chicken βB1-crystallin promoter. Expression of this protein in paired Xenopus oocytes induced gap junctional currents of similar magnitude to wild type human Cx50. Three lines of transgenic mice expressing the transgenic protein were analyzed. Lenses from transgenic mice were smaller than those from non-transgenic littermates, and had cataracts that were already visible at postnatal day 1. Expression of the transgene resulted in a 3- to 13-fold increase in Cx50 protein levels above those of non-transgenic animals. Light microscopy revealed alterations in epithelial cell differentiation, fiber cell structure, interactions between fiber cells and areas of liquefaction. Scanning electron microscopy showed fiber cells of varying widths with bulging areas along single fibers. Anti-Cx50 and anti-FLAG immunoreactivities were detected at appositional membranes and in intracellular vesicles in transgenic lenses. N-cadherin, Cx46, ZO-1 and aquaporin 0 localized mainly at the plasma membrane, although some N-cadherin and aquaporin 0 was associated with the intracellular vesicles. The abundance and solubility/integrity of αA-, αB-, β- and γ-crystallin were unaffected. These results demonstrate that transgenic expression of Cx50 in mice leads to cataracts associated with formation of cytoplasmic vesicles containing Cx50 and decreased or slowed epithelial differentiation without major alterations in the distribution of other integral membrane or membrane-associated proteins or the integrity/solubility of crystallins.

Ras signaling is essential for lens cell proliferation and lens growth during development

The vertebrate ocular lens is a simple and continuously growing tissue. Growth factor-mediated receptor tyrosine kinases (RTKs) are believed to be required for lens cell proliferation, differentiation and survival. The signaling pathways downstream of the RTKs remain to be elucidated. Here, we demonstrate the important role of Ras in lens development by expressing a dominant-negative form of Ras (dn-Ras) in the lens of transgenic mice. We show that lens in the transgenic mice was smaller and lens growth was severely inhibited as compared to the wild-type lens. However, the lens shape, polarity and transparency appeared normal in the transgenic mice. Further analysis showed that cell proliferation is inhibited in the dn-Ras lens. For example, the percentage of 5-bromo-2′-deoxyuridine (BrdU)-labeled cells in epithelial layer was about 2- to 3-fold lower in the transgenic lens than in the wild-type lens, implying that Ras activity is required for normal cell proliferation during lens development. We also found a small number of apoptotic cells in both epithelial and fiber compartment of the transgenic lens, suggesting that Ras also plays a role in cell survival. Interestingly, although there was a delay in primary fiber cell differentiation, secondary fiber cell differentiation was not significantly affected in the transgenic mice. For example, the expression of β- and γ-crystallins, the marker proteins for fiber differentiation, was not changed in the transgenic mice. Biochemical analysis indicated that ERK activity, but not Akt activity, was significantly reduced in the dn-Ras transgenic lenses. Overall, our data imply that the RTK-Ras-ERK signaling pathway is essential for cell proliferation and, to a lesser extent, for cell survival, but not for crystallin gene expression during fiber differentiation. Thus, some of the fiber differentiation processes are likely mediated by RTK-dependent but Ras-independent pathways.

Regulation of αA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin

Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the alphaA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with alphaA-crystallin synthesis. A DCR1/alphaA-crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of alphaA-crystallin in lens epithelium and fibers. In contrast, the DCR3/alphaA/EGFP reporter was expressed only in 'late' lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the alphaA-crystallin promoter in lens chromatin and demonstrated that high levels of alphaA-crystallin expression correlate with increased binding of c-Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9-hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the alphaA-crystallin locus. Our data demonstrate a novel mechanism of Pax6, c-Maf and CREB function, through regulation of chromatin-remodeling enzymes, and suggest a multistage model for the activation of alphaA-crystallin during lens differentiation.

A Transgenic Mouse Model for Human Autosomal Dominant Cataract

To characterize lenses from transgenic mice designed to express mutant and wild-type alphaA-crystallin subunits. A series of transgenic mouse strains was created to express mutant (R116C) and wild-type human alphaA-crystallin in fiber cells of the lens. Dissected lenses were phenotypically scored for the presence and extent of opacities, fiber cell morphology, and posterior suture morphology. Gene transcripts derived from integrated transgenes were detected by reverse transcriptase-PCR. Distribution of expressed transgenic protein was determined by immunohistochemical staining of lens tissue sections. The abundance of endogenous and transgenic lens proteins was estimated by quantitative Western blot analysis. Expression of R116C mutant alphaA-crystallin subunits resulted in posterior cortical cataracts and abnormalities associated with the posterior suture. The severity of lens abnormalities did not increase between the ages of 9 and 30 weeks. With respect to opacities and morphologic abnormalities, lenses from transgenic mice that express wild-type human alphaA-crystallin subunits were indistinguishable from age-matched nontransgenic control mice. Similar phenotypes were observed in different independent lines of R116C transgenic mice that differed by at least two orders of magnitude in the expression level of the mutant transgenic protein. The results show that lens opacities and posterior sutural defects occur when mutant R116C alphaA-crystallin subunits are expressed on the background of wild-type endogenous mouse alpha-crystallins. Low levels of R116C alphaA-crystallin subunits are sufficient to induce lens opacities and sutural defects.

Transgenic Mice Expressing Dominant-negative Osmotic-response Element-binding Protein (OREBP) in Lens Exhibit Fiber Cell Elongation Defect Associated with Increased DNA Breaks

Osmotic-response element-binding protein (OREBP), also known as TonEBP or NFAT5, is thought to be responsible for the induction of osmolyte-accumulating genes when cells are under hypertonic stress. Recent studies suggest that OREBP also plays a role in water reabsorption in the kidney, T-cell proliferation, and embryonic development. We developed transgenic mice that express the dominant-negative OREBP (OREBPdn) specifically in the lens because our earlier studies showed that it is particularly sensitive to osmotic stress. The transgenic mice developed nuclear cataract soon after birth, suggesting defects in lens development. The developing transgenic lenses showed incomplete elongation of fiber cells and formation of vacuoles. This is accompanied by evidence of DNA strand breaks, activation of p53, and induction of checkpoint kinase, suggesting that the developing fiber cells lacking OREBP are in a similar physiological state as cells experiencing hypertonic stress. These results indicate that OREBP-mediated accumulation of osmolytes is essential during elongation of the lens fiber cells.

Ectopic Pax6 expression disturbs lens fiber cell differentiation.

Pax6 is a transcription factor necessary for the specification and subsequent formation of the ocular lens. It is expressed in all lens cells at early stages of development. After lens formation, Pax6 expression is maintained in the lens epithelium, whereas its level abruptly decreases in differentiated fiber cells. This study is to test the hypothesis that normal fiber cell differentiation would be perturbed by sustained Pax6 expression. Transgenic mice expressing the canonical form of mouse Pax6 were created under the control of a modified mouse alphaA-crystallin promoter. The phenotypic changes in the transgenic lens were analyzed by light and electron microscopy. The effect of ectopic Pax6 expression on the lens fiber cells was investigated by in situ hybridization, immunohistochemical staining, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and two-dimensional (2-D) gel electrophoresis. Transgenic mice from seven different lines all had cataracts with severity that correlated with the transgene expression level in lens fiber cells. In severely affected lines, a lumen was present between the apical surfaces of the epithelial and fiber cells, suggesting that secondary fiber cell elongation is incomplete. Electron microscopy analysis showed that the ball-and-socket interdigitations between neighboring fiber cells were underdeveloped or attenuated in the transgenic lens. Most interesting, elevated levels of Pax6 in fiber cells reduced the protein levels of transcription factor cMaf, which is known to be essential in fiber cell differentiation. Furthermore, the total amount of lens proteins was 60% less than normal in the Pax6 transgenic lens. Among the crystallins examined, the relative ratio of intact betaB1-crystallin protein to total lens protein was significantly reduced. Real-time reverse transcriptase PCR showed that the ratio of betaB1-crystallin transcript levels to total mRNA levels were reduced by 87%. The data demonstrate that high levels of Pax6 expression disrupt normal fiber cell differentiation and maturation.