RESUMO
The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm and is regulated by transcription factor Pax6 and secreted BMP4. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2, a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.
Assuntos
Matriz Extracelular , Regulação da Expressão Gênica no Desenvolvimento , Cristalino , Fator de Transcrição PAX6 , Animais , Matriz Extracelular/metabolismo , Camundongos , Cristalino/metabolismo , Cristalino/crescimento & desenvolvimento , Cristalino/citologia , Fator de Transcrição PAX6/metabolismo , Fator de Transcrição PAX6/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/genética , Embrião de Galinha , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Galinhas/genética , Olho/metabolismo , Olho/crescimento & desenvolvimento , Olho/embriologiaRESUMO
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.
Assuntos
Organoides , Células Fotorreceptoras Retinianas Cones , Transcriptoma/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias , Humanos , Organoides/química , Organoides/citologia , Organoides/metabolismo , Organoides/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/química , Retina/citologia , Retina/metabolismo , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/química , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologia , Análise de Célula ÚnicaRESUMO
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.
Assuntos
Diferenciação Celular , Glicólise , Mitofagia , Retina/embriologia , Células Ganglionares da Retina/fisiologia , Animais , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/metabolismoRESUMO
Cellular differentiation is marked by temporally and spatially regulated gene expression. The ocular lens is one of the most powerful mammalian model system since it is composed from only two cell subtypes, called lens epithelial and fiber cells. Lens epithelial cells differentiate into fiber cells through a series of spatially and temporally orchestrated processes, including massive production of crystallins, cellular elongation and the coordinated degradation of nuclei and other organelles. Studies of transcriptional and posttranscriptional gene regulatory mechanisms in lens provide a wide range of opportunities to understand global molecular mechanisms of gene expression as steady-state levels of crystallin mRNAs reach very high levels comparable to globin genes in erythrocytes. Importantly, dysregulation of crystallin gene expression results in lens structural abnormalities and cataracts. The mRNA life cycle is comprised of multiple stages, including transcription, splicing, nuclear export into cytoplasm, stabilization, localization, translation and ultimate decay. In recent years, development of modern mRNA detection methods with single molecule and single cell resolution enabled transformative studies to visualize the mRNA life cycle to generate novel insights into the sequential regulatory mechanisms of gene expression during embryogenesis. This review is focused on recent major advancements in studies of transcriptional bursting in differentiating lens fiber cells, analysis of nascent mRNA expression from bi-directional promoters, transient nuclear accumulation of specific mRNAs, condensation of chromatin prior lens fiber cell denucleation, and outlines future studies to probe the interactions of individual mRNAs with specific RNA-binding proteins (RBPs) in the cytoplasm and regulation of translation and mRNA decay.
Assuntos
Cristalinas/genética , Regulação da Expressão Gênica/fisiologia , Cristalino/metabolismo , Transcrição Gênica , Animais , Diferenciação Celular , Humanos , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Ativação TranscricionalRESUMO
Congenital aniridia is caused by heterozygous mutations in the PAX6 gene. In this disease, congenital iris and foveal hypoplasia is associated with juvenile onset cataract, glaucoma, and corneal keratopathy. In rodents, Pax6 mutations result in a congenital reduction in ocular size that is not typically described in human aniridia. Here, the ocular morphometry of aniridia patients is compared with the lens phenotype of Pax6+/tm1/Pgr mice to reveal whether there are species differences in Pax6 regulation of lens development and homeostasis. Ultrasound biometry (UBM) revealed that eleven percent of aniridia patients exhibited mild microphthalmia while the anterior chamber depth of aniridic eyes was significantly reduced from 6 months of age onward. Although aniridic lens thickness was normal from birth, it was significantly decreased in aniridic lenses older than 30. Notably, 86% of aniridic lenses exhibited cataractous changes in this cohort. In addition, a significant proportion of aniridia patients develop lens subluxation as they age associated with reduced lens diameter as measured by anterior segment optical coherence tomography (AS-OCT). Analysis of young adult Pax6+/tm1/Pgr mouse lenses by micro-computed tomography (microCT), bright field and dark field imaging revealed that they are reduced in size but did not exhibit overt cataracts at this age. Overall, this study reveals that congenital microphthalmia as assessed by axial length, or microphakia, as assessed by lens thickness, are not typical in human aniridia, although these are primary manifestations of Pax6 mutations in mice, suggesting that PAX6 regulates some aspects of lens development differently between these species.
Assuntos
Aniridia/patologia , Catarata/patologia , Cristalino/patologia , Microftalmia/patologia , Adolescente , Adulto , Idoso , Animais , Aniridia/genética , Câmara Anterior/patologia , Comprimento Axial do Olho/patologia , Catarata/genética , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos Mutantes , Microftalmia/genética , Microscopia Acústica , Pessoa de Meia-Idade , Fator de Transcrição PAX6/genética , Fenótipo , Microscopia com Lâmpada de Fenda , Tomografia de Coerência Óptica , Adulto JovemRESUMO
BACKGROUND: Homeodomain transcription factor Otx2 is essential for embryonic development of multiple head tissues, including retinal pigment epithelium (RPE) and neuroretina. Temporospatial regulation of Otx2 expression is critical for its functions. Molecular dissection of the cis-acting enhancers will help elucidate how Otx2 expression is regulated. RESULTS: We comprehensively characterized distal enhancer hs1150 that was previously identified in a high throughput study. We established multiple transgenic mouse lines in which human hs1150, corresponding mouse hs1150, and two highly conserved sub-fragments in the mouse hs1150 were individually fused to a minimal hsp68 promoter to drive reporter expression. We found that hs1150 enhancer directed reporter expression in the RPE, neuroretina, and brain in a developmentally regulated manner. Human hs1150-directed reporter expression largely recapitulated Otx2 expression in the RPE, in the early neuroretina, and to a lesser degree in the early brain. Mouse hs1150, although shorter than human hs1150, exhibited similar enhancer activity, indicating functional conservation of hs1150 enhancer across species. Both of the highly conserved subfragments in mouse hs1150 enhancer directed reporter expression in the early neuroretina, indicating that the hs1150 enhancer has two functional components. CONCLUSIONS: Our findings provide insight into the molecular mechanisms underlying the regulation of Otx2 retinal expression.
Assuntos
Fatores de Transcrição Otx/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Proteínas do Olho/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
Ocular lens development represents an advantageous system in which to study regulatory mechanisms governing cell fate decisions, extracellular signaling, cell and tissue organization, and the underlying gene regulatory networks. Spatiotemporally regulated domains of BMP, FGF, and other signaling molecules in late gastrula-early neurula stage embryos generate the border region between the neural plate and non-neural ectoderm from which multiple cell types, including lens progenitor cells, emerge and undergo initial tissue formation. Extracellular signaling and DNA-binding transcription factors govern lens and optic cup morphogenesis. Pax6, c-Maf, Hsf4, Prox1, Sox1, and a few additional factors regulate the expression of the lens structural proteins, the crystallins. Extensive crosstalk between a diverse array of signaling pathways controls the complexity and order of lens morphogenetic processes and lens transparency.
Assuntos
Redes Reguladoras de Genes , Cristalino/crescimento & desenvolvimento , Mamíferos/genética , Animais , Sistemas CRISPR-CasRESUMO
Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction (i.e. the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell differentiation. In the ocular lens, the positions of lens fiber cells correlate precisely with their differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the ß-actin and lens differentiation-specific α-, ß-, and γ-crystallin genes by RNA fluorescent in situ hybridization (FISH) in the lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular differentiation dramatically alters the burst fraction in synchronized waves across the lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration.
Assuntos
Diferenciação Celular , Núcleo Celular/fisiologia , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/citologia , Organogênese , Transcrição Gênica , Animais , Animais Recém-Nascidos , Montagem e Desmontagem da Cromatina , Embrião de Mamíferos/fisiologia , Feminino , Cristalino/fisiologia , CamundongosRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos/metabolismo , Cristalino/citologia , Cristalino/embriologia , Animais , Autofagia , Compartimento Celular , Ciclo Celular , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Choque Térmico , Camundongos Knockout , Mitofagia , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX6/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Retina/citologia , Retina/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula , Imunoprecipitação da Cromatina , Feminino , Imunofluorescência , Camundongos , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Neurogênese/fisiologia , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Gravidez , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Expressão Gênica/fisiologia , Cristalino/metabolismo , Proteoma/fisiologia , Transcriptoma/fisiologia , Animais , Animais Recém-Nascidos , Cromatografia Líquida , Cristalinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Cristalino/citologia , Camundongos , Proteômica , RNA Mensageiro/genética , Espectrometria de Massas em Tandem , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The tissue-specific transcriptional programs during normal development require tight control by distal cis-regulatory elements, such as enhancers, with specific DNA sequences recognized by transcription factors, coactivators, and chromatin remodeling enzymes. Gata3 is a sequence-specific DNA-binding transcription factor that regulates formation of multiple tissues and organs, including inner ear, lens, mammary gland, T-cells, urogenital system, and thyroid gland. In the eye, Gata3 has a highly restricted expression domain in the posterior part of the lens vesicle; however, the underlying regulatory mechanisms are unknown. RESULTS: Here we describe the identification of a novel bipartite Gata3 lens-specific enhancer located â¼18 kb upstream from its transcriptional start site. We also found that a 5-kb Gata3 promoter possesses low activity in the lens. The bipartite enhancer contains arrays of AP-1, Ets-, and Smad1/5-binding sites as well as binding sites for lens-associated DNA-binding factors. Transient transfection studies of the promoter with the bipartite enhancer showed enhanced activation by BMP4 and FGF2. CONCLUSIONS: These studies identify a novel distal enhancer of Gata3 with high activity in lens and indicate that BMP and FGF signaling can up-regulate expression of Gata3 in differentiating lens fiber cells through the identified Gata3 enhancer and promoter elements. Developmental Dynamics 247:1186-1198, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
Assuntos
Elementos Facilitadores Genéticos , Fator de Transcrição GATA3/genética , Cristalino/embriologia , Animais , Sítios de Ligação , Proteína Morfogenética Óssea 4/fisiologia , Proteínas de Ligação a DNA , Fator 2 de Crescimento de Fibroblastos/fisiologia , Fator de Transcrição GATA3/química , Fator de Transcrição GATA3/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
Neuroretina and retinal pigment epithelium (RPE) are differentiated from the progenitors in optic vesicles, but it is unclear when and how the two lineages are segregated. Manipulation of chick embryos reveals that the early anteroventral optic vesicle is crucial for neuroretinal development, but the molecular mechanism is unclear. Homeodomain transcription factor Six3 is required for neuroretinal specification and is dispensable for RPE formation, but the cell fates of Six3-deficient progenitors and the origins of remnant RPE are unknown. Here, we performed lineage tracing of Six3-Cre positive cells in wild-type and Six3-deficient mouse embryos. Six3-Cre positive progenies were found in a population of progenitors in the anteroventral optic pits/vesicles starting at E8.5, and were found in neuroretina, optic stalk, ventral forebrain, but not RPE, at E10.5. Six3-deletion in the small population of progenitors at E8.5 was sufficient to cause rostral expansion of Wnt8b and drastic reduction of Fgf8/MAPK signaling, ablating neuroretinal specification without affecting RPE. Lineage tracing revealed Six3-deficient progenitors at E8.5 were eventually lost and the remnant RPE was derived from Six3-Cre negative cells. Thus, Six3 in a small population of progenitors expressing Six3-Cre at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice.
Assuntos
Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Cristalino/embriologia , Proteínas do Tecido Nervoso/genética , Hipófise/embriologia , Prosencéfalo/embriologia , Epitélio Pigmentado da Retina/embriologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Sobrevivência Celular/genética , Embrião de Galinha , Fator 8 de Crescimento de Fibroblasto/metabolismo , Camundongos , Camundongos Knockout , Comunicação Parácrina/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteína Homeobox SIX3RESUMO
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.
Assuntos
Diferenciação Celular , Cristalino/citologia , Cristalino/crescimento & desenvolvimento , Proteína Proto-Oncogênica N-Myc/metabolismo , Animais , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Transcriptoma/genéticaRESUMO
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.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Expressão Gênica/fisiologia , Cristalino/metabolismo , Análise Espaço-Temporal , Fatores de Transcrição/genética , Transcriptoma , Animais , Animais Recém-Nascidos , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cristalino/citologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Fator de Crescimento Transformador beta/genética , Via de Sinalização Wnt/genéticaRESUMO
The formation and life-long growth of the ocular lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To achieve their mature structure and transparent function, newly formed lens fiber cells undergo a series of cellular remodeling events including the complete elimination of cellular organelles to form the lens organelle-free zone (OFZ). To date, the mechanisms and requirements for organelle elimination by lens fiber cells remain to be fully elucidated. In previous studies, we detected the presence of mitochondria contained within autophagolysosomes throughout human and chick lenses suggesting that proteins targeting mitochondria for degradation by mitophagy could be required for the elimination of mitochondria during OFZ formation. Consistently, high-throughput RNA sequencing of microdissected embryonic chick lenses revealed that expression of a protein that targets mitochondria for elimination during erythrocyte formation, called BCL2 interacting protein 3-like (BNIP3L/NIX), peaks in the region of lens where organelle elimination occurs. To examine the potential role for BNIP3L in the elimination of mitochondria during lens fiber cell remodeling, we analyzed the expression pattern of BNIP3L in newborn mouse lenses, the effect of its deletion on organelle elimination and its co-localization with lens organelles. We demonstrate that the expression pattern of BNIP3L in the mouse lens is consistent with it playing an important role in the elimination of mitochondria during lens fiber cell organelle elimination. Importantly, we demonstrate that deletion of BNIP3L results in retention of mitochondria during lens fiber cell remodeling, and, surprisingly, that deletion of BNIP3L also results in the retention of endoplasmic reticulum and Golgi apparatus but not nuclei. Finally, we show that BNIP3L localizes to the endoplasmic reticulum and Golgi apparatus of wild-type newborn mouse lenses and is contained within mitochondria, endoplasmic reticulum and Golgi apparatus isolated from adult mouse liver. These data identify BNIP3L as a novel requirement for the elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell remodeling and they suggest a novel function for BNIP3L in the regulation of endoplasmic reticulum and Golgi apparatus populations in the lens and non-lens tissues.
Assuntos
Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Cristalino/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Animais , Western Blotting , Perfilação da Expressão Gênica , Cristalino/embriologia , Fígado/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Cristalinas/biossíntese , Fator 2 de Crescimento de Fibroblastos/metabolismo , Cristalino/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-maf/biossíntese , Animais , Embrião de Galinha , Cristalinas/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Cristalino/citologia , Células MCF-7 , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-maf/genética , Elementos de Resposta/fisiologia , Regulação para Cima/fisiologiaRESUMO
The ocular lens is a model system for understanding important aspects of embryonic development, such as cell specification and the spatiotemporally controlled formation of a three-dimensional structure. The lens, which is characterized by transparency, refraction and elasticity, is composed of a bulk mass of fiber cells attached to a sheet of lens epithelium. Although lens induction has been studied for over 100 years, recent findings have revealed a myriad of extracellular signaling pathways and gene regulatory networks, integrated and executed by the transcription factor Pax6, that are required for lens formation in vertebrates. This Review summarizes recent progress in the field, emphasizing the interplay between the diverse regulatory mechanisms employed to form lens progenitor and precursor cells and highlighting novel opportunities to fill gaps in our understanding of lens tissue morphogenesis.
Assuntos
Proteínas do Olho/metabolismo , Redes Reguladoras de Genes/fisiologia , Proteínas de Homeodomínio/metabolismo , Cristalino/embriologia , Modelos Biológicos , Organogênese/fisiologia , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Vertebrados/embriologia , Animais , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Humanos , Placa Neural/embriologia , Fator de Transcrição PAX6 , Especificidade da EspécieRESUMO
The DNA-binding transcription factor PAX6 was cloned 25 years ago by multiple teams pursuing identification of human and mouse eye disease causing genes, cloning vertebrate homologues of pattern-forming regulatory genes identified in Drosophila, or abundant eye-specific transcripts. Since its discovery in 1991, genetic, cellular, molecular and evolutionary studies on Pax6 mushroomed in the mid 1990s leading to the transformative thinking regarding the genetic program orchestrating both early and late stages of eye morphogenesis as well as the origin and evolution of diverse visual systems. Since Pax6 is also expressed outside of the eye, namely in the central nervous system and pancreas, a number of important insights into the development and function of these organs have been amassed. In most recent years, genome-wide technologies utilizing massively parallel DNA sequencing have begun to provide unbiased insights into the regulatory hierarchies of specification, determination and differentiation of ocular cells and neurogenesis in general. This review is focused on major advancements in studies on mammalian eye development driven by studies of Pax6 genes in model organisms and future challenges to harness the technology-driven opportunities to reconstruct, step-by-step, the transition from naïve ectoderm, neuroepithelium and periocular mesenchyme/neural crest cells into the three-dimensional architecture of the eye.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fator de Transcrição PAX6/genética , Animais , Aniversários e Eventos Especiais , Olho/crescimento & desenvolvimento , História do Século XX , História do Século XXI , Humanos , Fator de Transcrição PAX6/históriaRESUMO
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.