RESUMO
Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study and now provide a detailed analysis on a how high-glucose milieu downregulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other coregulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1), were enriched at the Tug1 promoter under high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high-glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1 and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Podócitos/metabolismo , RNA Longo não Codificante/biossíntese , Transcrição Gênica , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular Tumoral , Glucose/genética , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Camundongos , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Defective anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. Only a few molecular cues have been identified in early formation of the female urogenital system. Here we describe a novel long non-coding RNA molecule known as Leat1 (long non-coding RNA, EphrinB2 associated transcript 1). This lncRNA is syntenic with EfnB2 (which encodes EphrinB2) and expressed during embryonic development of the genital tubercle. While lncRNAs have varied functions, many are known to regulate their neighbouring genes. Eph/Ephrin bidirectional signaling molecules mediate many patterning pathways in early embryonic development, including cloacal septation and urethral development. Here we investigate the role of Leat1 and its possible regulation of EphrinB2 during development of the female reproductive tract. We show that a loss of Leat1 leads to reduced EfnB2 expression in the developing female genital tubercle, reduced anogenital distance and decreased fertility.
Assuntos
Efrina-B2/genética , Organogênese/genética , RNA Longo não Codificante/genética , Anormalidades Urogenitais/genética , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , CamundongosRESUMO
Ciliopathies form a group of inherited disorders sharing several clinical manifestations because of abnormal cilia formation or function, and few treatments have been successful against these disorders. Here, we report a mouse model with mutated Sclt1 gene, which encodes a centriole distal appendage protein important for ciliogenesis. Sodium channel and clathrin linker 1 (SCLT1) mutations were associated with the oral-facial-digital syndrome (OFD), an autosomal recessive ciliopathy. The Sclt1-/- mice exhibit typical ciliopathy phenotypes, including cystic kidney, cleft palate and polydactyly. Sclt1-loss decreases the number of cilia in kidney; increases proliferation and apoptosis of renal tubule epithelial cells; elevates protein kinase A, extracellular signal-regulated kinases, SMAD and signal transducer and activator of transcription 3 (STAT3) pathways; and enhances pro-inflammation and pro-fibrosis pathways with disease progression. Embryonic kidney cyst formation of Sclt1-/- mice was effectively reduced by an anti-STAT3 treatment using pyrimethamine. Overall, we reported a new mouse model for the OFD; and our data suggest that STAT3 inhibition may be a promising treatment for SCLT1-associated cystic kidney.
Assuntos
Fator de Transcrição STAT3/metabolismo , Canais de Sódio/metabolismo , Animais , Cílios/metabolismo , Ciliopatias/genética , Ciliopatias/metabolismo , Cistos/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Rim/metabolismo , Doenças Renais Císticas/etiologia , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Modelos Animais , Mutação , Fenótipo , Fator de Transcrição STAT3/genética , Transdução de Sinais , Canais de Sódio/genéticaRESUMO
While increased mitochondrial reactive oxygen species have been commonly implicated in a variety of disease states, their in vivo role in the pathogenesis of diabetic nephropathy remains controversial. Using a two-photon imaging approach with a genetically encoded redox biosensor, we monitored mitochondrial redox state in the kidneys of experimental models of diabetes in real-time in vivo. Diabetic (db/db) mice that express a redox-sensitive Green Fluorescent Protein biosensor (roGFP) specifically in the mitochondrial matrix (db/dbmt-roGFP) were generated, allowing dynamic monitoring of redox changes in the kidneys. These db/dbmt-roGFP mice exhibited a marked increase in mitochondrial reactive oxygen species in the kidneys. Yeast NADH-dehydrogenase, a mammalian Complex I homolog, was ectopically expressed in cultured podocytes, and this forced expression in roGFP-expressing podocytes prevented high glucose-induced increases in mitochondrial reactive oxygen species. Thus, in vivo monitoring of mitochondrial roGFP in diabetic mice confirms increased production of mitochondrial reactive oxygen species in the kidneys.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/patologia , Rim/patologia , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Técnicas Biossensoriais , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/etiologia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Oxirredução , PodócitosRESUMO
Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Mutação , Proteínas Nucleares/genética , Insuficiência Ovariana Primária/genética , Animais , Modelos Animais de Doenças , Feminino , Humanos , Infertilidade Feminina/genética , Camundongos , Linhagem , CoesinasRESUMO
The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.
Assuntos
Processamento Alternativo/genética , Orelha Interna/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Receptores Notch/genética , Animais , Padronização Corporal/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cóclea/crescimento & desenvolvimento , Cóclea/patologia , Orelha Interna/metabolismo , Orelha Interna/patologia , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Serrate-Jagged , Transdução de Sinais/genética , Vestíbulo do Labirinto/crescimento & desenvolvimento , Vestíbulo do Labirinto/patologiaRESUMO
Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300(-/-) ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens.
Assuntos
Proteína de Ligação a CREB/fisiologia , Proteína p300 Associada a E1A/fisiologia , Histonas/metabolismo , Cristalino/embriologia , Acetilação , Animais , Apoptose , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Indução Embrionária , Expressão Gênica , Cristalino/anatomia & histologia , Cristalino/enzimologia , Camundongos , Mutação , Processamento de Proteína Pós-Traducional , Fase SRESUMO
Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development.
Assuntos
Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Cerebelo/anormalidades , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Cor de Cabelo/genética , Histona Desacetilases/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Monofenol Mono-Oxigenase/genética , Mutagênese Insercional , Proteínas Proto-Oncogênicas/deficiência , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Proteínas Repressoras/deficiência , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Transposases/genéticaRESUMO
Fertilization is the process that leads to the formation of a diploid zygote from two haploid gametes. This is achieved through a complex series of cell-to-cell interactions between a sperm and an egg. The final event of fertilization is the fusion of the gametes' membranes, which allows the delivery of the sperm genetic material into the egg cytoplasm. In vivo studies in the laboratory mouse have led to the discovery of membrane proteins that are essential for the fusion process in both the sperm and egg. Specifically, the sperm protein Izumo1 was shown to be necessary for normal fertility. Izumo1-deficient spermatozoa fail to fuse with the egg plasma membrane. Izumo1 is a member of the Immunoglobulin Superfamily of proteins, which are known to be involved in cell adhesion. Here, we describe BART97b, a new mouse line with a recessive mutation that displays a fertilization block associated with a failure of sperm fusion. BART97b mutants carry a deletion that inactivates Spaca6, a previously uncharacterized gene expressed in testis. Similar to Izumo1, Spaca6 encodes an immunoglobulin-like protein. We propose that the Spaca6 gene product may, together with Izumo1, mediate sperm fusion by binding an as yet unidentified egg membrane receptor.
Assuntos
Elementos de DNA Transponíveis/genética , Fertilização/genética , Imunoglobulinas/genética , Proteínas de Membrana/genética , Camundongos Mutantes/genética , Camundongos Transgênicos/genética , Interações Espermatozoide-Óvulo/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Feminino , Fertilização/fisiologia , Deleção de Genes , Inativação Gênica , Masculino , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Plasma Seminal/genética , Análise de Sequência de DNA , Interações Espermatozoide-Óvulo/fisiologiaRESUMO
The novel long non-coding RNA (lncRNA) Leat1 is extraordinarily conserved in both its location (syntenic with EfnB2, an essential gene in anogenital patterning) and sequence. Here we show that Leat1 is upregulated following the testosterone surge from the developing testis and directly interacts with EfnB2, positively regulating its expression. Leat1 expression is suppressed by estrogen, which in turn suppresses the expression of EfnB2. Moreover, the loss of Leat1 leads to reduced EfnB2, resulting in a severe hypospadias phenotype. The human LEAT1 gene is also co-expressed with EFNB2 in the developing human penis suggesting a conserved function for this gene in urethral closure. Together our data identify Leat1 as a novel molecular regulator of urethral closure and implicate it as a target of endocrine disruption in the etiology of hypospadias.
RESUMO
lncRNA taurine-upregulated gene 1 (Tug1) is a promising therapeutic target in the progression of diabetic nephropathy (DN), but the molecular basis of its protection remains poorly understood. Here, we generate a triple-mutant diabetic mouse model coupled with metabolomic profiling data to interrogate whether Tug1 interaction with peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) is required for mitochondrial remodeling and progression of DN in vivo. We find that, compared with diabetic conditional deletion of Pgc1α in podocytes alone (db/db; Pgc1αPod-f/f), diabetic Pgc1α knockout combined with podocyte-specific Tug1 overexpression (db/db; TugPodTg; Pgc1αPod-f/f) reverses the protective phenotype of Tug1 overexpression, suggesting that PGC1α is required for the renoprotective effect of Tug1. Using unbiased metabolomic profiling, we find that altered urea cycle metabolites and mitochondrial arginase 2 play an important role in Tug1/PGC1α-induced mitochondrial remodeling. Our work identifies a functional role of the Tug1/PGC1α axis on mitochondrial metabolic homeostasis and urea cycle metabolites in experimental models of diabetes.
Assuntos
Rim/metabolismo , Metaboloma , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Substâncias Protetoras/metabolismo , RNA Longo não Codificante/metabolismo , Ureia/metabolismo , Animais , Arginase/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Progressão da Doença , Deleção de Genes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/deficiência , Podócitos/metabolismo , RNA Longo não Codificante/genéticaRESUMO
Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.
Assuntos
Corpo Ciliar/embriologia , Corpo Ciliar/crescimento & desenvolvimento , Proteínas do Olho/biossíntese , Dosagem de Genes , Proteínas de Homeodomínio/biossíntese , Iris/embriologia , Iris/crescimento & desenvolvimento , Fatores de Transcrição Box Pareados/biossíntese , Proteínas Repressoras/biossíntese , Processamento Alternativo , Animais , Diferenciação Celular , Corpo Ciliar/citologia , Corpo Ciliar/metabolismo , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Iris/citologia , Iris/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: Mammalian 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. RESULTS: Ras 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. CONCLUSIONS: Collectively, 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.
Assuntos
Córnea/metabolismo , Cristalino/metabolismo , Proteínas ras/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Development of the secondary palate in mammals is a complex process that can be easily perturbed, leading to the common and distressing birth defect cleft palate. Animal models are particularly useful tools for dissecting underlying genetic components of cleft palate. We describe a new cleft palate model resulting from a transgene insertion mutation. Transgene insertional mutagenesis disrupts the genomic organization and expression of the Ap2ß1 gene located on chromosome 11. This gene encodes the ß2-adaptin subunit of the heterotetrameric adaptor protein 2 complex involved in clathrin-dependent endocytosis. Homozygous cleft palate mutant mice express no Ap2ß1 messenger RNA or ß2-adaptin protein and die during the perinatal period. Heterozygous mice are phenotypically normal despite expressing diminished ß2-adaptin messenger RNA and protein compared with wildtype. Remarkably, the paralogous ß1-adaptin subunit of the adaptor protein 1 complex partially substitutes for the missing ß2-adaptin in embryonic fibroblasts from homozygous mutant mice, resulting in assembly of reduced levels of an adaptor protein 2 complex bearing ß1-adaptin. This variant adaptor protein 2 complex is, therefore, apparently capable of maintaining viability of the homozygous mutant embryos until birth but insufficient to support palatogenesis. Nonsyndromic cleft palate in an animal model is associated with disruption of the Ap2ß1 gene.
Assuntos
Fissura Palatina/genética , Fator de Transcrição AP-2/deficiência , Fator de Transcrição AP-2/genética , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutagênese Insercional , SíndromeRESUMO
The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and alphaA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.
Assuntos
Afacia/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Dominantes , Cristalino/embriologia , Cristalino/metabolismo , Animais , Cristalinas/genética , Toxina Diftérica/genética , Embrião de Mamíferos , Endotélio Corneano/anormalidades , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Imuno-Histoquímica , Hibridização In Situ , Cristalino/patologia , Cristalino/fisiologia , Camundongos , Camundongos Transgênicos , TransgenesRESUMO
It is widely accepted that vitreous humor-derived FGFs are required for the differentiation of anterior lens epithelial cells into crystallin-rich fibers. We show that BMP2, 4, and 7 can induce the expression of markers of fiber differentiation in primary lens cell cultures to an extent equivalent to FGF or medium conditioned by intact vitreous bodies (VBCM). Abolishing BMP2/4/7 signaling with noggin inhibited VBCM from upregulating fiber marker expression. Remarkably, noggin and anti-BMP antibodies also prevented purified FGF (but not unrelated stimuli) from upregulating the same fiber-specific proteins. This effect is attributable to inhibition of BMPs produced by the lens cells themselves. Although BMP signaling is required for FGF to enhance fiber differentiation, the converse is not true. Expression of noggin in the lenses of transgenic mice resulted in a postnatal block of epithelial-to-secondary fiber differentiation, with extension of the epithelial monolayer to the posterior pole of the organ. These results reveal the central importance of BMP in secondary fiber formation and show that although FGF may be necessary for this process, it is not sufficient. Differentiation of fiber cells, and thus proper vision, is dependent on cross-talk between the FGF and BMP signaling pathways.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular , Fatores de Crescimento de Fibroblastos/metabolismo , Cristalino/embriologia , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas de Transporte/genética , Células Cultivadas , Embrião de Galinha , Meios de Cultivo Condicionados , Células Epiteliais/metabolismo , Proteínas do Olho/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Filamentos Intermediários/metabolismo , Cristalino/citologia , Cristalino/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Corpo Vítreo/metabolismo , delta-Cristalinas/metabolismoRESUMO
PURPOSE: The retinoblastoma (Rb) gene family member p130 binds preferentially to the E2F5 transcription factor and forms a repressive E2F5/p130 complex that inhibits cell cycle progression and tumor growth. It is unclear whether E2F5, either alone or in combination with p130, can interfere with the transcriptional activity of other E2F family members, such as E2F1 and E2F3a, in vivo. In this study, we used transgenic mice to test whether overexpression of E2F5 with/without p130 would be sufficient to inhibit E2F1 or E2F3a induced cell cycle reentry. METHODS: Transgenic mice were generated by microinjection of constructs containing different E2F cDNAs (E2F1, E2F3a, and E2F5) or the p130 cDNA linked to the mouse alphaA-crystallin promoter. The E2F5 single and E2F5/p130 double transgenic mice were cross-mated with E2F1 or E2F3a transgenic mice. The resulting double or triple transgenic mouse embryos were characterized by histology, in situ hybridization, immunohistochemistry, and BrdU incorporation assays. RESULTS: Overexpression of E2F5 alone was not sufficient to inhibit E2F1 or E2F3a induced cell cycle reentry in lens fiber cells. Transgenic mice coexpressing E2F5 and p130 in lens fiber cells did not show lens defects. However, coexpression of E2F5/p130 with E2F1 or E2F3a in lens fiber cells decreased the number of BrdU positive fiber cells induced by the E2F1 or E2F3a alone. Therefore, overexpression of E2F5/p130, but not E2F5 alone, can inhibit activator E2F-mediated cell proliferation in vivo, confirming that p130 plays a critical role in the repressive activity of E2F5/p130 complex. CONCLUSIONS: Overexpression of E2F5/p130 in post-mitotic lens fiber cells does not affect their normal differentiation program, but can inhibit inappropriate cell cycle reentry induced by the activator E2Fs. Since E2F5 alone cannot interfere with these E2F activities, we conclude that p130 is a key player in the inhibitory process.
Assuntos
Ciclo Celular/fisiologia , Fator de Transcrição E2F5/metabolismo , Cristalino/citologia , Proteína p130 Retinoblastoma-Like/metabolismo , Animais , Diferenciação Celular/fisiologia , Fatores de Transcrição E2F/metabolismo , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F3/metabolismo , Humanos , Camundongos , Camundongos TransgênicosRESUMO
PURPOSE: The purpose of this study was to reassess the role of the lens as an "embryonic organizer" of ocular tissues. METHODS: We ablated the lens in mice by lens-specific expression of an attenuated version of diphtheria toxin A subunit(Tox176) driven by a modified crystallin promoter. Alterations in the differentiation programs of ocular tissues were examined by hematoxylin and eosin staining, in situ hybridization, and immunohistochemistry. RESULTS: Transgenic mice in the family OVE1757 exhibited severe microphakia. Apoptotic lens fibers were seen by embryonic day 15 (E15) and the lenses were completely ablated by post natal day 8. Multiple defects were seen in the anterior chamber. Corneal endothelial cells did not differentiate properly. The mesenchymal cells that would normally give rise to the endothelial layer were found to express N-cadherin, but they failed to form tight junctions and undergo a mesenchymal-to-epithelial transition. Although early specification of the presumptive ciliary body and iris was detected, subsequent differentiation of the iris was blocked. No dramatic changes were seen in the development of the retina. CONCLUSIONS: These results support the hypothesis that an intact lens is essential for proper differentiation of both the corneal endothelium and the iris and that the lens "organizes" the development of tissues in the anterior chamber.
Assuntos
Câmara Anterior/anormalidades , Afacia/congênito , Afacia/complicações , Cristalino/anormalidades , Animais , Animais Recém-Nascidos/anormalidades , Animais Recém-Nascidos/genética , Diferenciação Celular , Toxina Diftérica , Endotélio Corneano/anormalidades , Feminino , Maturidade dos Órgãos Fetais/genética , Iris/anormalidades , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Transgênicos , Microftalmia/etiologia , Organogênese/genética , Gravidez , Regiões Promotoras Genéticas , Retina/embriologiaRESUMO
PURPOSE: Insulin and insulin-like growth factors (IGFs) are putative regulators of cell proliferation and differentiation during lens development. Transgenic mice that overexpress IGF-1 in the lens have been previously described. To further understand the ocular functions of this growth factor family, the in vivo effects of insulin expression on lens development were investigated using transgenic mice. METHODS: Expression of insulin receptor (IR) and IGF-1 receptor (IGF-1R) in mouse lens was examined by reverse-transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. Transgenic mice that overexpress insulin in the lens were generated using two different promoters: a fiber-cell specific alphaA-crystallin (alphaA) promoter and a modified alphaA-promoter linked to the chicken delta1-crystallin enhancer (called the deltaenalphaA promoter). The deltaenalphaA promoter is active in both lens epithelial and fiber cells. The lens phenotypes were analyzed by histology and immunohistochemistry. Protein expression was examined by western blotting. RESULTS: Normal mouse lenses express both the insulin receptor (IR) and the IGF-1 receptor (IGF-1R), and their expression is highest at the lens periphery where the germinative and transitional zones are located. In transgenic mice, insulin expression in the lens induced cataract formation. The severity of the cataracts reflected the level of transgene expression, independent of the type of promoter used. In severely affected families, the spherical shape of the lens was altered and the lenses were smaller than normal. Histological analysis showed no evidence of premature differentiation of the anterior epithelial cells. In contrast to the IGF-1 mice, insulin transgenic mice exhibited an anterior shift in the location of the germinative and transitional zones, leading to a reduction of the lens epithelial compartment. Additional alterations included expansion of the lens transitional zone, variable nuclear positioning in the lens bow region, and inhibition of fiber cell denucleation and terminal differentiation. CONCLUSIONS: Elevated intraocular insulin does not enhance proliferation nor induce differentiation of mouse lens epithelial cells. Since an increase in IGF-1 causes a posterior shift of the lens geminative and transitional zones, while an increase in insulin causes an anterior shift of these zones, our results suggest that these two growth factors may work together to control the location of this structural domain during normal lens development. Our data also suggest that increased insulin-signaling activity in the lens can antagonize the endogenous signals that are responsible for fiber cell maturation and terminal differentiation.
Assuntos
Insulina/metabolismo , Cristalino/embriologia , Cristalino/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Catarata/etiologia , Catarata/metabolismo , Catarata/patologia , Diferenciação Celular , Proliferação de Células , Senescência Celular , Cristalinas/metabolismo , Embrião de Mamíferos/metabolismo , Hibridização In Situ , Insulina/genética , Cristalino/patologia , Camundongos , Camundongos Transgênicos , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição TecidualRESUMO
Neural tube closure is a critical feature of central nervous system morphogenesis during embryonic development. Failure of this process leads to neural tube defects, one of the most common forms of human congenital defects. Although molecular and genetic studies in model organisms have provided insights into the genes and proteins that are required for normal neural tube development, complications associated with live imaging of neural tube closure in mammals limit efficient morphological analyses. Here, we report the use of optical coherence tomography (OCT) for dynamic imaging and quantitative assessment of cranial neural tube closure in live mouse embryos in culture. Through time-lapse imaging, we captured two neural tube closure mechanisms in different cranial regions, zipper-like closure of the hindbrain region and button-like closure of the midbrain region. We also used OCT imaging for phenotypic characterization of a neural tube defect in a mouse mutant. These results suggest that the described approach is a useful tool for live dynamic analysis of normal neural tube closure and neural tube defects in the mouse model.