Generation of Lens Progenitor Cells and Lentoid Bodies from Pluripotent Stem Cells: Novel Tools for Human Lens Development and Ocular Disease Etiology.

In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids and specific ocular tissues from pluripotent stem cells has provided invaluable insights into the formation of lineage-committed progenitor cell populations, signal transduction pathways, and self-organization principles. This review provides a comprehensive summary of recent advances in generation of adenohypophyseal, olfactory, and lens placodes, lens progenitor cells and three-dimensional (3D) primitive lenses, "lentoid bodies", and "micro-lenses". These cells are produced alone or "community-grown" with other ocular tissues. Lentoid bodies/micro-lenses generated from human patients carrying mutations in crystallin genes demonstrate proof-of-principle that these cells are suitable for mechanistic studies of cataractogenesis. Taken together, current and emerging advanced in vitro differentiation methods pave the road to understand molecular mechanisms of cataract formation caused by the entire spectrum of mutations in DNA-binding regulatory genes, such as PAX6, SOX2, FOXE3, MAF, PITX3, and HSF4, individual crystallins, and other genes such as BFSP1, BFSP2, EPHA2, GJA3, GJA8, LIM2, MIP, and TDRD7 represented in human cataract patients.

Rinf Regulates Pluripotency Network Genes and Tet Enzymes in Embryonic Stem Cells.

The Retinoid inducible nuclear factor (Rinf), also known as CXXC5, is a nuclear protein, but its functions in the context of the chromatin are poorly defined. We find that in mouse embryonic stem cells (mESCs), Rinf binds to the chromatin and is enriched at promoters and enhancers of Tet1, Tet2, and pluripotency genes. The Rinf-bound regions show significant overlapping occupancy of pluripotency factors Nanog, Oct4, and Sox2, as well as Tet1 and Tet2. We found that Rinf forms a complex with Nanog, Oct4, Tet1, and Tet2 and facilitates their proper recruitment to regulatory regions of pluripotency and Tet genes in ESCs to positively regulate their transcription. Rinf deficiency in ESCs reduces expression of Rinf target genes, including several pluripotency factors and Tet enzymes, and causes aberrant differentiation. Together, our findings establish Rinf as a regulator of the pluripotency network genes and Tet enzymes in ESCs.

Profiling of chromatin accessibility and identification of general cis-regulatory mechanisms that control two ocular lens differentiation pathways.

BACKGROUND: Promoters and enhancers are cis-regulatory DNA sequences that control specificity and quantity of transcription. Both are rich on clusters of cis-acting sites that interact with sequence-specific DNA-binding transcription factors (TFs). At the level of chromatin, these regions display increased nuclease sensitivity, reduced nucleosome density, including nucleosome-free regions, and specific combinations of posttranslational modifications of core histone proteins. Together, "open" and "closed" chromatins represent transcriptionally active and repressed states of individual genes, respectively. Cellular differentiation is marked by changes in local chromatin structure. Lens morphogenesis, regulated by TF Pax6, includes differentiation of epithelial precursor cells into lens fibers in parallel with differentiation of epithelial precursors into the mature lens epithelium. RESULTS: Using ATAC-seq, we investigated dynamics of chromatin changes during mouse lens fibers and epithelium differentiation. Tissue-specific features of these processes are demonstrated via comparative studies of embryonic stem cells, forebrain, and liver chromatins. Unbiased analysis reveals cis-regulatory logic of lens differentiation through known (e.g., AP-1, Ets, Hsf4, Maf, and Pax6 sites) and novel (e.g., CTCF, Tead, and NF1) motifs. Twenty-six DNA-binding TFs, recognizing these cis-motifs, are markedly up-regulated in differentiating lens fibers. As specific examples, our ATAC-seq data uncovered both the regulatory regions and TF binding motifs in Foxe3, Prox1, and Mip loci that are consistent with previous, though incomplete, experimental data. A cross-examination of Pax6 binding with ATAC-seq data demonstrated that Pax6 bound to both open (H3K27ac and P300-enriched) and closed chromatin domains in lens and forebrain. CONCLUSIONS: Our study has generated the first lens chromatin accessibility maps that support a general model of stage-specific chromatin changes associated with transcriptional activities of batteries of genes required for lens fiber cell formation. Analysis of active (or open) promoters and enhancers reveals important cis-DNA motifs that establish the molecular foundation for temporally and spatially regulated gene expression in lens. Together, our data and models open new avenues for the field to conduct mechanistic studies of transcriptional control regions, reconstruction of gene regulatory networks that govern lens morphogenesis, and identification of cataract-causing mutations in noncoding sequences.

Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids.

Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium-retinal organoid-that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.

Bidirectional Analysis of Cryba4-Crybb1 Nascent Transcription and Nuclear Accumulation of Crybb3 mRNAs in Lens Fibers.

Purpose: Crystallin gene expression during lens fiber cell differentiation is tightly spatially and temporally regulated. A significant fraction of mammalian genes is transcribed from adjacent promoters in opposite directions ("bidirectional" promoters). It is not known whether two proximal genes located on the same allele are simultaneously transcribed. Methods: Mouse lens transcriptome was analyzed for paired genes whose transcriptional start sites are separated by less than 5 kbp to identify coexpressed bidirectional promoter gene pairs. To probe these transcriptional mechanisms, nascent transcription of Cryba4, Crybb1, and Crybb3 genes from gene-rich part of chromosome 5 was visualized by RNA fluorescent in situ hybridizations (RNA FISH) in individual lens fiber cell nuclei. Results: Genome-wide lens transcriptome analysis by RNA-seq revealed that the Cryba4-Crybb1 pair has the highest Pearson correlation coefficient between their steady-state mRNA levels. Analysis of Cryba4 and Crybb1 nascent transcription revealed frequent simultaneous expression of both genes from the same allele. Nascent Crybb3 transcript visualization in "early" but not "late" differentiating lens fibers show nuclear accumulation of the spliced Crybb3 transcripts that was not affected in abnormal lens fiber cell nuclei depleted of chromatin remodeling enzyme Snf2h (Smarca5). Conclusions: The current study shows for the first time that two highly expressed lens crystallin genes, Cryba4 and Crybb1, can be simultaneously transcribed from adjacent bidirectional promoters and do not show nuclear accumulation. In contrast, spliced Crybb3 mRNAs transiently accumulate in early lens fiber cell nuclei. The gene pairs coexpressed during lens development showed significant enrichment in human "cataract" phenotype.

Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers.

Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular compositions of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development and pathology in vitro. To compare protein compositions between the lens epithelium and fibers, we employed tandem mass spectrometry (2D-LC/MS) analysis of microdissected mouse P0.5 lenses. Functional classifications of the top 525 identified proteins into gene ontology categories by molecular processes and subcellular localizations, were adapted for the lens. Expression levels of both epithelial and fiber proteomes were compared with whole lens proteome and mRNA levels using E14.5, E16.5, E18.5, and P0.5 RNA-Seq data sets. During this developmental time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. As expected, crystallins showed a high correlation between their mRNA and protein levels. Comprehensive data analysis confirmed and/or predicted roles for transcription factors (TFs), RNA-binding proteins (e.g. Carhsp1), translational apparatus including ribosomal heterogeneity and initiation factors, microtubules, cytoskeletal [e.g. non-muscle myosin IIA heavy chain (Myh9) and ßB2-spectrin (Sptbn2)] and membrane proteins in lens formation and maturation. Our data highlighted many proteins with unknown functions in the lens that were preferentially enriched in epithelium or fibers, setting the stage for future studies to further dissect the roles of these proteins in fiber cell differentiation vs. epithelial cell maintenance. In conclusion, the present proteomic datasets represent the first mouse lens epithelium and fiber cell proteomes, establish comparative analyses of protein and RNA-Seq data, and characterize the major proteome remodeling required to form the mature lens fiber cells.

A comprehensive spatial-temporal transcriptomic analysis of differentiating nascent mouse lens epithelial and fiber cells.

Elucidation of both the molecular composition and organization of the ocular lens is a prerequisite to understand its development, function, pathology, regenerative capacity, as well as to model lens development and disease using in vitro differentiation of pluripotent stem cells. Lens is comprised of the anterior lens epithelium and posterior lens fibers, which form the bulk of the lens. Lens fibers differentiate from lens epithelial cells through cell cycle exit-coupled differentiation that includes cellular elongation, accumulation of crystallins, cytoskeleton and membrane remodeling, and degradation of organelles within the central region of the lens. Here, we profiled spatiotemporal expression dynamics of both mRNAs and non-coding RNAs from microdissected mouse nascent lens epithelium and lens fibers at four developmental time points (embryonic [E] day 14.5, E16.5, E18.5, and P0.5) by RNA-seq. During this critical time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. Throughout this developmental window, 3544 and 3518 genes show consistently and significantly greater expression in the nascent lens epithelium and fibers, respectively. Comprehensive data analysis confirmed major roles of FGF-MAPK, Wnt/ß-catenin, PI3K/AKT, TGF-ß, and BMP signaling pathways and revealed significant novel contributions of mTOR, EIF2, EIF4, and p70S6K signaling in lens formation. Unbiased motif analysis within promoter regions of these genes with consistent expression changes between epithelium and fiber cells revealed an enrichment for both established (e.g. E2Fs, Etv5, Hsf4, c-Maf, MafG, MafK, N-Myc, and Pax6) transcription factors and a number of novel regulators of lens formation, such as Arntl2, Dmrta2, Stat5a, Stat5b, and Tulp3. In conclusion, the present RNA-seq data serves as a comprehensive reference resource for deciphering molecular principles of normal mammalian lens differentiation, mapping a full spectrum of signaling pathways and DNA-binding transcription factors operating in both lens compartments, and predicting novel pathways required to establish lens transparency.

Evolutionary Origins of Pax6 Control of Crystallin Genes.

The birth of novel genes, including their cell-specific transcriptional control, is a major source of evolutionary innovation. The lens-preferred proteins, crystallins (vertebrates: α- and ß/γ-crystallins), provide a gateway to study eye evolution. Diversity of crystallins was thought to originate from convergent evolution through multiple, independent formation of Pax6/PaxB-binding sites within the promoters of genes able to act as crystallins. Here, we propose that αB-crystallin arose from a duplication of small heat shock protein (Hspb1-like) gene accompanied by Pax6-site and heat shock element (HSE) formation, followed by another duplication to generate the αA-crystallin gene in which HSE was converted into another Pax6-binding site. The founding ß/γ-crystallin gene arose from the ancestral Hspb1-like gene promoter inserted into a Ca2+-binding protein coding region, early in the cephalochordate/tunicate lineage. Likewise, an ancestral aldehyde dehydrogenase (Aldh) gene, through multiple gene duplications, expanded into a multigene family, with specific genes expressed in invertebrate lenses (Ω-crystallin/Aldh1a9) and both vertebrate lenses (η-crystallin/Aldh1a7 and Aldh3a1) and corneas (Aldh3a1). Collectively, the present data reconstruct the evolution of diverse crystallin gene families.

N-myc regulates growth and fiber cell differentiation in lens development.

Myc proto-oncogenes regulate diverse cellular processes during development, but their roles during morphogenesis of specific tissues are not fully understood. We found that c-myc regulates cell proliferation in mouse lens development and previous genome-wide studies suggested functional roles for N-myc in developing lens. Here, we examined the role of N-myc in mouse lens development. Genetic inactivation of N-myc in the surface ectoderm or lens vesicle impaired eye and lens growth, while "late" inactivation in lens fibers had no effect. Unexpectedly, defective growth of N-myc-deficient lenses was not associated with alterations in lens progenitor cell proliferation or survival. Notably, N-myc-deficient lens exhibited a delay in degradation of DNA in terminally differentiating lens fiber cells. RNA-sequencing analysis of N-myc-deficient lenses identified a cohort of down-regulated genes associated with fiber cell differentiation that included DNaseIIß. Further, an integrated analysis of differentially expressed genes in N-myc-deficient lens using normal lens expression patterns of iSyTE, N-myc-binding motif analysis and molecular interaction data from the String database led to the derivation of an N-myc-based gene regulatory network in the lens. Finally, analysis of N-myc and c-myc double-deficient lens demonstrated that these Myc genes cooperate to drive lens growth prior to lens vesicle stage. Together, these findings provide evidence for exclusive and cooperative functions of Myc transcription factors in mouse lens development and identify novel mechanisms by which N-myc regulates cell differentiation during eye morphogenesis.

Programmed mitophagy is essential for the glycolytic switch during cell differentiation.

Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis-associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX-deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX-dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts.

Chromatin remodeling enzyme Snf2h regulates embryonic lens differentiation and denucleation.

Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIß, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation.

Intercellular Adhesion-Dependent Cell Survival and ROCK-Regulated Actomyosin-Driven Forces Mediate Self-Formation of a Retinal Organoid.

In this study we dissected retinal organoid morphogenesis in human embryonic stem cell (hESC)-derived cultures and established a convenient method for isolating large quantities of retinal organoids for modeling human retinal development and disease. Epithelialized cysts were generated via floating culture of clumps of Matrigel/hESCs. Upon spontaneous attachment and spreading of the cysts, patterned retinal monolayers with tight junctions formed. Dispase-mediated detachment of the monolayers and subsequent floating culture led to self-formation of retinal organoids comprising patterned neuroretina, ciliary margin, and retinal pigment epithelium. Intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces are required for the self-organization. Our data supports a hypothesis that newly specified neuroretina progenitors form characteristic structures in equilibrium through minimization of cell surface tension. In long-term culture, the retinal organoids autonomously generated stratified retinal tissues, including photoreceptors with ultrastructure of outer segments. Our system requires minimal manual manipulation, has been validated in two lines of human pluripotent stem cells, and provides insight into optic cup invagination in vivo.

Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling.

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.

Unfolded-protein response-associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract.

Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei.

Constitutive overexpression of Norrin activates Wnt/ß-catenin and endothelin-2 signaling to protect photoreceptors from light damage.

Norrin is a retinal signaling molecule which is expressed in Müller glia and binds to Frizzled-4 to activate canonical Wnt/ß-catenin signaling. Norrin is part of an essential signaling system that controls the formation of retinal capillaries during development. To evaluate neuroprotective properties of Norrin independently from its function during retinal angiogenesis, we generated transgenic mice (Rpe65-Norrin) that constitutively express Norrin in the retinal pigmented epithelium. Substantial amounts of Norrin were secreted into the outer retina, which triggered retinal Wnt/ß-catenin signaling in conjunction with an increase in the expression of endothelin-2 (EDN2), endothelin receptor B (EDNRB), and glial fibrillary acidic protein (GFAP). Photoreceptors of Norrin-overexpressing mice were significantly less vulnerable to light-induced damage compared to their wild-type littermates. Following light damage, we observed less apoptotic death of photoreceptors and a better retinal function than in controls. The protective effects were abolished if either Wnt/ß-catenin or EDN2 signaling was blocked by intravitreal injection of Dickkopf-1 or BQ788, respectively. Light-damaged retinae from transgenic mice contained higher amounts of brain-derived neurotrophic factor (BDNF) and pAkt than those of wild-type littermates. We conclude that constitutive overexpression of Norrin protects photoreceptors from light damage, an effect that is mediated by Wnt/ß-catenin and EDN2 signaling and involves neurotrophic activities of BDNF. The findings suggest that Norrin and its associated signaling pathways have strong potentials to attenuate photoreceptor death following injury.

Spatial expression patterns of autophagy genes in the eye lens and induction of autophagy in lens cells.

PURPOSE: Mutation of the autophagy gene FYVE (named after the four cysteine-rich proteins: Fab 1 [yeast orthologue of PIKfyve], YOTB, Vac 1 [vesicle transport protein], and EEA1) and coiled coil containing 1 (fyco1) causes human cataract suggesting a role for autophagy in lens function. Here, we analyzed the range and spatial expression patterns of lens autophagy genes and we evaluated whether autophagy could be induced in lens cells exposed to stress. METHODS: Autophagy gene expression levels and their spatial distribution patterns were evaluated between microdissected human lens epithelium and fibers at the mRNA and protein levels by microarray data analysis, real-time PCR and western blot analysis. Selected autophagy protein spatial expression patterns were also examined in newborn mouse lenses by immunohistochemistry. The autophagosomal content of cultured human lens epithelial cells was determined by counting the number of microtubule-associated protein 1 light chain 3B (LC3B)-positive puncta in cells cultured in the presence or absence of serum. RESULTS: A total of 42 autophagy genes were detected as being expressed by human lens epithelium and fibers. The autophagosomal markers LC3B and FYCO1 were detected throughout the newborn mouse lens. Consistently, the autophagy active form of LC3B (LC3B II) was detected in microdissected human lens fibers. An increased number of LC3B-positive puncta was detected in cultured lens cells upon serum starvation suggesting induction of autophagy in lens cells under stress conditions. CONCLUSIONS: The data provide evidence that autophagy is an important component for the function of lens epithelial and fiber cells. The data are consistent with the notion that disruption of lens autophagy through mutation or inactivation of specific autophagy proteins could lead to loss of lens resistance to stress and/or loss of lens differentiation resulting in cataract formation.

The orchestration of mammalian tissue morphogenesis through a series of coherent feed-forward loops.

Tissue morphogenesis requires intricate temporal and spatial control of gene expression that is executed through specific gene regulatory networks (GRNs). GRNs are comprised from individual subcircuits of different levels of complexity. An important question is to elucidate the mutual relationship between those genes encoding DNA-binding factors that trigger the subcircuit with those that play major "later" roles during terminal differentiation via expression of specific genes that constitute the phenotype of individual tissues. The ocular lens is a classical model system to study tissue morphogenesis. Pax6 is essential for both lens placode formation and subsequent stages of lens morphogenesis, whereas c-Maf controls terminal differentiation of lens fibers, including regulation of crystallins, key lens structural proteins required for its transparency and refraction. Here, we show that Pax6 directly regulates c-Maf expression during lens development. A 1.3-kb c-Maf promoter with a 1.6-kb upstream enhancer (CR1) recapitulated the endogenous c-Maf expression pattern in lens and retinal pigmented epithelium. ChIP assays revealed binding of Pax6 and c-Maf to multiple regions of the c-Maf locus in lens chromatin. To predict functional Pax6-binding sites, nine novel variants of Pax6 DNA-binding motifs were identified and characterized. Two of these motifs predicted a pair of Pax6-binding sites in the CR1. Mutagenesis of these Pax6-binding sites inactivated transgenic expression in the lens but not in retinal pigmented epithelium. These data establish a novel regulatory role for Pax6 during lens development, link together the Pax6/c-Maf/crystallin regulatory network, and suggest a novel type of GRN subcircuit that controls a major part of embryonic lens development.

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

BACKGROUND: Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2ß) 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. RESULTS: To 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 IIß, 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 IIß, for lens fiber cell denucleation and indirectly for retinal development. CONCLUSIONS: These studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase IIß 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.

The transcription factor Pax6 regulates survival of dopaminergic olfactory bulb neurons via crystallin αA.

Most neurons in the adult mammalian brain survive for the entire life of an individual. However, it is not known which transcriptional pathways regulate this survival in a healthy brain. Here, we identify a pathway regulating neuronal survival in a highly subtype-specific manner. We show that the transcription factor Pax6 expressed in dopaminergic neurons of the olfactory bulb regulates the survival of these neurons by directly controlling the expression of crystallin αA (CryαA), which blocks apoptosis by inhibition of procaspase-3 activation. Re-expression of CryαA fully rescues survival of Pax6-deficient dopaminergic interneurons in vivo and knockdown of CryαA by shRNA in wild-type mice reduces the number of dopaminergic OB interneurons. Strikingly, Pax6 utilizes different DNA-binding domains for its well-known role in fate specification and this role of regulating the survival of specific neuronal subtypes in the mature, healthy brain.

Lens fiber cell differentiation and denucleation are disrupted through expression of the N-terminal nuclear receptor box of NCOA6 and 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.