RESUMO
We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract. Here, we have generated conditional p110α/PTEN double-knockout mice, and evaluated epithelial cell proliferation and lens homeostasis. The double deletion of p110α and PTEN rescued the defect in lens size seen after the single knockout of p110α, but accelerated the lens rupture phenotype seen in PTEN single-knockout mice. Levels of phosphorylated Akt in double-knockout lenses were significantly higher than in wild-type lenses, but not as elevated as those reported for PTEN single-knockout lenses. These results showed that the double deletion of the p110α catalytic subunit of PI3K and its opposing phosphatase, PTEN, exacerbated the rupture defect seen in the single PTEN knockout and alleviated the growth defect observed in the single p110α knockout. Thus, the integrity of the PI3K signaling pathway was absolutely essential for proper lens homeostasis, but not for lens growth.
Assuntos
Cristalino , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinase , Animais , Homeostase , Cristalino/crescimento & desenvolvimento , Cristalino/metabolismo , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4-/- mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-ß-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Fatores de Transcrição de Choque Térmico/deficiência , Cristalino/patologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Envelhecimento/genética , Animais , Animais Geneticamente Modificados , Catarata/genética , Catarata/patologia , Linhagem Celular , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Metilação de DNA , Modelos Animais de Doenças , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Fatores de Transcrição de Choque Térmico/genética , Histonas/genética , Histonas/metabolismo , Humanos , Cristalino/citologia , Cristalino/crescimento & desenvolvimento , Cristalino/efeitos da radiação , Camundongos , Regiões Promotoras Genéticas , Raios Ultravioleta/efeitos adversos , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
High myopia is a leading cause of blindness worldwide. Myopia progression may lead to pathological changes of lens and affect the outcome of lens surgery, but the underlying mechanism remains unclear. Here, we find an increased lens size in highly myopic eyes associated with up-regulation of ß/γ-crystallin expressions. Similar findings are replicated in two independent mouse models of high myopia. Mechanistic studies show that the transcription factor MAF plays an essential role in up-regulating ß/γ-crystallins in high myopia, by direct activation of the crystallin gene promoters and by activation of TGF-ß1-Smad signaling. Our results establish lens morphological and molecular changes as a characteristic feature of high myopia, and point to the dysregulation of the MAF-TGF-ß1-crystallin axis as an underlying mechanism, providing an insight for therapeutic interventions.
Assuntos
Cristalino/patologia , Fatores de Transcrição Maf/metabolismo , Miopia Degenerativa/patologia , Fator de Crescimento Transformador beta1/metabolismo , beta-Cristalinas/biossíntese , gama-Cristalinas/biossíntese , Animais , Feminino , Humanos , Cristalino/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Proteínas Smad/metabolismo , Regulação para Cima/genéticaRESUMO
The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos and larvae at single cell resolution through five days of development, identifying a number of novel putative regulators of lens development. Our data address open questions about the temperospatial expression of α-crystallins during lens development that will support future studies of their function and provide the first detailed view of ß- and γ-crystallin expression in and outside the lens. We describe divergent expression in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, membrane associated, RNA-binding, and transcription factor genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.
Assuntos
Células Epiteliais/metabolismo , Cristalino/metabolismo , Transcriptoma/genética , alfa-Cristalinas/genética , gama-Cristalinas/genética , Animais , Células Epiteliais/citologia , Cristalino/crescimento & desenvolvimento , Peixe-Zebra , alfa-Cristalinas/metabolismo , gama-Cristalinas/metabolismoRESUMO
The homeodomain transcription factors (TFs) Pax6 (OMIM: 607108) and Prox1 (OMIM: 601546) critically regulate gene expression in lens development. While PAX6 mutations in humans can cause cataract, aniridia, microphthalmia, and anophthalmia, among other defects, Prox1 deletion in mice causes severe lens abnormalities, in addition to other organ defects. Furthermore, the optimal dosage/spatiotemporal expression of these key TFs is essential for development. In lens development, Pax6 expression is elevated in cells of the anterior epithelium compared to fiber cells, while Prox1 exhibits the opposite pattern. Whether post-transcriptional regulatory mechanisms control these precise TF expression patterns is unknown. Here, we report the unprecedented finding that the cataract-linked RNA-binding protein (RBP), Celf1 (OMIM: 601074), post-transcriptionally regulates Pax6 and Prox1 protein expression in lens development. Immunostaining shows that Celf1 lens-specific conditional knockout (Celf1cKO) mice exhibit abnormal elevation of Pax6 protein in fiber cells and abnormal Prox1 protein levels in epithelial cells-directly opposite to their normal expression patterns in development. Furthermore, RT-qPCR shows no change in Pax6 and Prox1 transcript levels in Celf1cKO lenses, suggesting that Celf1 regulates these TFs on the translational level. Indeed, RNA-immunoprecipitation assays using Celf1 antibody indicate that Celf1 protein binds to Pax6 and Prox1 transcripts. Furthermore, reporter assays in Celf1 knockdown and Celf1-overexpression cells demonstrate that Celf1 negatively controls Pax6 and Prox1 translation via their 3' UTRs. These data define a new mechanism of RBP-based post-transcriptional regulation that enables precise control over spatiotemporal expression of Pax6 and Prox1 in lens development, thereby uncovering a new etiological mechanism for Celf1 deficiency-based cataract.
Assuntos
Proteínas CELF1/genética , Catarata/genética , Proteínas de Homeodomínio/genética , Cristalino/metabolismo , Fator de Transcrição PAX6/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteínas CELF1/antagonistas & inibidores , Proteínas CELF1/deficiência , Catarata/patologia , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Cristalino/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genéticaRESUMO
The signal regulated transcription factors (SRTFs) control the ultimate transcriptional output of signaling pathways. Here, we examined a family of FGF-induced SRTFs - Etv1, Etv 4, and Etv 5 - in murine lens development. Contrary to FGF receptor mutants that displayed loss of ERK signaling and defective cell differentiation, Etv deficiency augmented ERK phosphorylation without disrupting the normal lens fiber gene expression. Instead, the transitional zone for lens differentiation was shifted anteriorly as a result of reduced Jag1-Notch signaling. We also showed that Etv proteins suppresses mTOR activity by promoting Tsc2 expression, which is necessary for the nuclei clearance in mature lens. These results revealed the functional divergence between Etv and FGF in lens development, demonstrating that these SRTFs can operate outside the confine of their upstream signaling.
Many cells contain proteins known as signal-induced transcription factors, which are poised to receive messages from the environment and then react by activating genes required for the cell to respond appropriately. It is commonly thought that these transcription factors faithfully follow the instructions they receive from the external signal: for instance, if the message was to encourage the cell to grow, the transcription factors would switch on growth-related genes. As the eyes of mice and other mammals develop, a signal known as FGF is required for certain cells to specialize into lens fiber cells: these long, thin, transparent cells form the bulk of the lens, the structure that allows focused vision. Previous studies suggest that FGF activates three transcription factors known as Etv1, Etv4 and Etv5, but their precise roles in the development of the lens has remained unclear. Here, Garg, Hannan, Wang et al. confirm that FGF signaling does indeed activate all three proteins. However, mutant mice that lacked Etv1, Etv4 and Etv5 still created lens fiber cells, suggesting that the transcription factors are largely unnecessary for lens fiber cells formation. Instead, the Etv proteins participated in a cascade of molecular events involving a protein called Notch; as a result, if the transcription factors were absent, the lens fiber cells formed prematurely. In addition, deactivating Etv1, Etv4 and Etv5 also promoted the activity of a protein which interfered with the removal of internal cell compartments, a process required for lens fiber cells to mature properly. These findings reveal that the roles of Etv1, Etv4 and Etv5 deviate from and even oppose FGF signaling in the lenses of mice. Transcription factors control the ultimate fate of a cell, and there is therefore increased interest in targeting them for therapy. The work by Garg, Hannan, Wang et al. reveals an unexpected complexity in how these proteins respond to upstream signals, highlighting the importance of further dissecting these relationships.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Cristalino/crescimento & desenvolvimento , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/fisiologia , Animais , Cristalinas/metabolismo , Células Epiteliais/fisiologia , Proteína Jagged-1/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Proto-Oncogênicas c-maf/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Mammals possess four Sall transcription factors that play various roles in organogenesis. Previously, we found that Sall1 is expressed in microglia in the central nervous system, and it plays pivotal roles in microglia maturation. In the eye, Sall1 was also expressed in the developing lens, and we examined its role in lens development. A knock-in mouse harboring the EGFP gene in the Sall1 locus (Sall1-gfp) was used to analyze the Sall1 expression pattern. In Sall1-gfp/wild, EGFP was expressed throughout the presumptive lens at E11.5, and subsequently the expression in the lens epithelium became weaker. After birth, signals were observed in the equator region. The effects of Sall1 knockout on lens development were examined in Sall1-gfp/gfp. Lens sections revealed small vacuole-like holes and gaps in the center of the lens fibers at E14.5. Subsequently, the vacuoles appeared in most regions of the fiber cells. Electron microscopic analysis indicated that the vacuoles were between the fiber cells, leading to huge gaps. In addition, contact between the lens epithelium and apical end of the fiber cell was disrupted, and there were gaps between the adjoining lens epithelial cells. However, gap junction structure was observed by electron microscopic analysis, and immunostaining of Zo1 showed rather appropriate expression pattern. Immunohistochemistry indicated that the major lens transcription factors Prox1 and Pax6 were expressed in relatively normal patterns. However, although the expression of Prox1 and Pax6 decreased in nuclei in the control lens, it remained in Sall1-gfp/gfp. In addition, lower expression level of c-Maf protein was observed. Therefore, Sall1 is strongly expressed in the lens from the early developmental stage and plays an essential role in the maintenance of fiber cell and lens epithelium adhesion.
Assuntos
Cristalino/citologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Cristalino/patologia , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Vacúolos/patologiaRESUMO
OBJECTIVE: It has been well established that sumoylation acts as an important regulatory mechanism that controls many different cellular processes. We and others have shown that sumoylation plays an indispensable role during mouse eye development. Whether sumoylation is implicated in ocular pathogenesis remains to be further studied. In the present study, we have examined the expression patterns of the de-sumoylation enzymes (SENPs) in the in vitro cataract models induced by glucose oxidase and UVA irradiation. METHODS: Four-week-old C57BL/6J mice were used in our experiments. Lenses were carefully dissected out from mouse eyes and cultured in M199 medium for 12 hours. Transparent lenses (without surgical damage) were selected for experimentation. The lenses were exposed to UVA for 60 min or treated with 20 mU/mL glucose oxidase (GO) to induce cataract formation. The mRNA levels were analyzed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: GO treatment and UVA irradiation can induce cataract formation in lens cultured in vitro. GO treatment significantly down-regulated the mRNA levels for SENPs from 50% to 85%; on the other hand, expression of seven SENP proteins under GO treatment appeared in 3 situations: upregulation for SENP1, 2 and 6; downregulation for SENP 5 and 8; and unchanged for SENP3 and 7. UVA irradiation upregulates the mRNAs for all seven SENPs; In contrast to the mRNA levels for 7 SENPs, the expression levels for 6 SENPs (SENP1-3, 5-6 and 8) appeared down-regulated from 10% to 50%, and only SENP7 was slightly upregulated. CONCLUSION: Our results for the first time established the differentiation expression patterns of 7 de-sumoylation enzymes (SENPs) under treatment by GO or UVA, which provide preliminary data to link sumoylation to stress-induced cataractogenesis.
Assuntos
Catarata/genética , Olho/metabolismo , Sumoilação/genética , Animais , Catarata/induzido quimicamente , Catarata/patologia , Cisteína Endopeptidases/genética , Endopeptidases/genética , Olho/crescimento & desenvolvimento , Olho/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Glucose Oxidase/toxicidade , Humanos , Cristalino/efeitos dos fármacos , Cristalino/crescimento & desenvolvimento , Cristalino/metabolismo , Cristalino/efeitos da radiação , Camundongos , RNA Mensageiro/genética , Raios Ultravioleta/efeitos adversosRESUMO
The Integrator complex subunit 1 (INTS1) is a component of the integrator complex that comprises 14 subunits and associates with RPB1 to catalyze endonucleolytic cleavage of nascent snRNAs and assist RNA polymerase II in promoter-proximal pause-release on protein-coding genes. We present five patients, including two sib pairs, with biallelic sequence variants in INTS1. The patients manifested absent or severely limited speech, an abnormal gait, hypotonia and cataracts. Exome sequencing revealed biallelic variants in INTS1 in all patients. One sib pair demonstrated a missense variant, p.(Arg77Cys), and a frameshift variant, p.(Arg1800Profs*20), another sib pair had a homozygous missense variant, p.(Pro1874Leu), and the fifth patient had a frameshift variant, p.(Leu1764Cysfs*16) and a missense variant, p.(Leu2164Pro). We also report additional clinical data on three previously described individuals with a homozygous, loss of function variant, p.(Ser1784*) in INTS1 that shared cognitive delays, cataracts and dysmorphic features with these patients. Several of the variants affected the protein C-terminus and preliminary modeling showed that the p.(Pro1874Leu) and p.(Leu2164Pro) variants may interfere with INTS1 helix folding. In view of the cataracts observed, we performed in-situ hybridization and demonstrated expression of ints1 in the zebrafish eye. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 to make larvae with biallelic insertion/deletion (indel) variants in ints1. The mutant larvae developed typically through gastrulation, but sections of the eye showed abnormal lens development. The distinctive phenotype associated with biallelic variants in INTS1 points to dysfunction of the integrator complex as a mechanism for intellectual disability, eye defects and craniofacial anomalies.
Assuntos
Catarata/genética , Anormalidades Craniofaciais/genética , Deficiências do Desenvolvimento/genética , Proteína Wnt1/genética , Adolescente , Adulto , Animais , Catarata/fisiopatologia , Criança , Pré-Escolar , Anormalidades Craniofaciais/fisiopatologia , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Mutação da Fase de Leitura/genética , Gastrulação/genética , Humanos , Lactente , Cristalino/crescimento & desenvolvimento , Cristalino/patologia , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Dobramento de Proteína , Sequenciamento do Exoma , Proteína Wnt1/química , Adulto Jovem , Peixe-Zebra/genéticaRESUMO
Epithelial cell polarity, adhesion, proliferation, differentiation and survival are essential for morphogenesis of various organs and tissues including the ocular lens. The molecular mechanisms regulating the lens epithelial phenotype however, are not well understood. Here we investigated the role of scaffolding protein ankyrin-G (AnkG) in mouse lens development by conditional suppression of AnkG expression using the Cre-LoxP recombination approach. AnkG, which serves to link integral membrane proteins to the spectrin/actin cytoskeleton, was found to distribute predominantly to the lateral membranes of lens epithelium with several isoforms of the protein being detected in the mouse lens. Conditional deficiency of AnkG impaired mouse lens morphogenesis starting from embryonic stage E15.5, with neonatal (P1) AnkG cKO lenses exhibiting overt abnormalities in shape, size, epithelial cell height, sheet length and lateral membrane assembly together with defective fiber cell orientation relative to lenses from littermate AnkG floxed or Cre expressing mice. Severe disruptions in E-cadherin/ß-catenin-based adherens junctions, and the membrane organization of spectrin-actin cytoskeleton, ZO-1, connexin-50 and Na+-K+-ATPase were noted in AnkG deficient lenses, along with detection in lens epithelium of α-smooth muscle actin, a marker of epithelial to mesenchymal transition. Moreover, lens epithelial cell proliferation and survival were severely compromised while differentiation appears to be normal in AnkG deficient mouse lenses. Collectively, these results indicate that AnkG regulates establishment of the epithelial phenotype via lateral membrane assembly, stabilization of E-cadherin-based cell-cell junctions, polarity and membrane organization of transport and adhesion proteins and the spectrin-actin skeleton, and provide evidence for an obligatory role for AnkG in lens morphogenesis and growth.
Assuntos
Anquirinas/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/metabolismo , Morfogênese/genética , Animais , Animais Recém-Nascidos , Anquirinas/deficiência , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/genética , Polaridade Celular/genética , Transição Epitelial-Mesenquimal/genética , Epitélio/embriologia , Epitélio/metabolismo , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , FenótipoRESUMO
Spred, like Sprouty (Spry) and also Sef proteins, have been identified as important regulators of receptor tyrosine kinase (RTK)-mediated MAPK/ERK-signaling in various developmental systems, controlling cellular processes such as proliferation, migration and differentiation. Spreds are widely expressed during early embryogenesis, and in the eye lens, become more localised in the lens epithelium with later development, overlapping with other antagonists including Spry. Given the synexpression of Spreds and Spry in lens, in order to gain a better understanding of their specific roles in regulating growth factor mediated-signaling and cell behavior, we established and characterised lines of transgenic mice overexpressing Spred1 or Spred2, specifically in the lens. This overexpression of Spreds resulted in a small lens phenotype during ocular morphogenesis, retarding its growth by compromising epithelial cell proliferation and fiber differentiation. These in situ findings were shown to be dependent on the ability of Spreds to suppress MAPK-signaling, in particular FGF-induced ERK1/2-signaling in lens cells. This was validated in vitro using lens epithelial explants, that highlighted the overlapping role of Spreds with Spry2, but not Spry1. This study provides insights into the putative function of Spreds and Spry in situ, some overlapping and some distinct, and their importance in regulating lens cell proliferation and fiber differentiation contributing to lens and eye growth.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Epiteliais/citologia , Cristalino/crescimento & desenvolvimento , Proteínas Repressoras/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Adenoviridae/genética , Animais , Western Blotting , Fatores de Crescimento de Fibroblastos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , TransfecçãoRESUMO
Dysfunction of HSF4 is associated with congenital cataracts. HSF4 transcription activity is turned on and regulated by phosphorylation during early postnatal lens development. Our previous data suggested that mutation HSF4b/S299A can upregulate HSF4 transcription activity in vitro, but the biological significance of posttranslational modification on HSF4/S299 during lens development remains unclear. Here, we found that the mutation HSF4/S299A can upregulate the expression of HSP25 and alpha B-crystallin at both protein and mRNA levels in mouse the lens epithelial cell line, but HSF4/S299D does not. Using the rabbit polyclonal antibody against phospho-S299 of HSF4, we found that EGF and ectopic expression of MEK1 can increase the phosphorylation of HSF4/S299 and induce HSF4 sumoylation, and these effects are inhibited by U0126. ERK1/2 can phosphorylate the S299 in HSF4/wt but not in HSF4/S299A in the in vitro kinase assay. Functionally, ectopic MEK1 can inhibit HSF4-controled alpha B-crystallin expression but has less effect on HSF4/S299A. EGF can upregulate phospho-HSF4/S299 and downregulate alpha B-crystallin expression in P3 mouse lens, and this downregulation is suppressed by U0126. During mouse lens development, phosphorylation of HSF4/S299 is downregulated in P3 lens and upregulated in P7 and P14 lens. However, in 2 months old lens, both phosphorylation of HSF4/S299 and total HSF4 protein are decreased. Interestingly, ERK1/2 activity is lower in P3 lens than in P7 and P14 lens, which is in line with the phosphorylation of HSF4/S299. Taken together, our data demonstrate that HSF4/299 is a phosphorylation target of MEK1-ERK1/2, and phosphorylation of S299 is responsible for tuning down HSF4 transcription activity during postnatal lens development.
Assuntos
Fatores de Transcrição de Choque Térmico/genética , Cristalino/metabolismo , Sistema de Sinalização das MAP Quinases , Substituição de Aminoácidos , Animais , Células Cultivadas , Regulação para Baixo , Técnicas de Inativação de Genes , Proteínas de Choque Térmico HSP27/genética , Fatores de Transcrição de Choque Térmico/química , Fatores de Transcrição de Choque Térmico/deficiência , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Cristalino/crescimento & desenvolvimento , Camundongos , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Fosforilação , Mutação Puntual , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/metabolismo , Transcrição Gênica , Regulação para Cima , Cadeia B de alfa-Cristalina/genéticaRESUMO
We previously investigated the transcriptome and proteome profiles of the murine ocular lens at six developmental time points including two embryonic (E15 and E18) and four postnatal time points (P0, P3, P6, and P9). Here, we extend our analyses to identify novel transcripts and peptides in developing mouse lens. We identified a total of 9,707 novel transcripts and 325 novel fusion genes in developing mouse lens. Additionally, we identified 13,281 novel alternative splicing (AS) events in mouse lens including 6,990 exon skipping (ES), 2,447 alternative 3' splice site (A3SS), 1,900 alternative 5' splice site (A5SS), 1,771 mutually exclusive exons (MXE), and 173 intron retention (IR). Finally, we integrated our OMIC (Transcriptome and Proteome) datasets identifying 20 novel peptides in mouse lens. All 20 peptides were validated through matching MS/MS spectra of synthetic peptides. To the best of our knowledge, this is the first report integrating OMIC datasets to identify novel peptides in developing murine lens.
Assuntos
Processamento Alternativo/genética , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Organogênese/genética , Peptídeos/genética , Proteoma/genética , Transcriptoma/genética , Algoritmos , Animais , Cromatografia Líquida , Bases de Dados Genéticas , Éxons/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Íntrons/genética , Camundongos , Gravidez , Sítios de Splice de RNA/genética , Análise de Sequência de RNA , Espectrometria de Massas em TandemRESUMO
Cataract refers to opacities of the lens that impede the passage of light. Mutations in heat shock transcription factor 4 (HSF4) have been associated with cataract; however, the mechanisms regarding how mutations in HSF4 cause cataract are still obscure. In this study, we generated an hsf4 knockout zebrafish model using TALEN technology. The mutant zebrafish developed an early-onset cataract with multiple developmental defects in lens. The epithelial cells of the lens were overproliferated, resulting in the overabundance of lens fiber cells in hsf4null zebrafish lens. Consequently, the arrangement of the lens fiber cells became more disordered and irregular with age. More importantly, the terminal differentiation of the lens fiber cell was interrupted as the organelles cannot be cleaved in due time. In the cultured human lens epithelial cells, HSF4 could stabilize and retain p53 in the nucleus to activate its target genes such as fas cell surface death receptor (Fas) and Bcl-2-associated X apoptosis regulator (Bax). In the hsf4null fish, both p53 and activated-caspase3 were significantly decreased. Combined with the finding that the denucleation defect could be partially rescued through microinjection of p53, fas and bax mRNA into the mutant embryos, we directly proved that HSF4 promotes lens fiber cell differentiation by activating p53 and its downstream regulators. The data we presented suggest that apoptosis-related genes are involved in the lens fiber cell differentiation. Our finding that HSF4 functions in the upstream to activate these genes highlighted the new regulatory modes of HSF4 in the terminal differentiation of lens fiber cell.
Assuntos
Catarata/genética , Diferenciação Celular/genética , Fatores de Transcrição de Choque Térmico/genética , Proteína Supressora de Tumor p53/genética , Animais , Caspase 3/genética , Catarata/patologia , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/patologia , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cristalino/crescimento & desenvolvimento , Cristalino/metabolismo , Cristalino/patologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteína X Associada a bcl-2/genética , Receptor fas/genéticaRESUMO
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
Resumo Objetivo: Determinar a frequência da microftalmia associada à catarata congênita e sua frequência etiológica. Comparar o resultado visual após a cirurgia da catarata congênita em olhos microftálmicos, com o resultado visual obtido em olhos não microftálmicos. Método: Estudo retrospectivo de 76 pacientes portadores de microftalmia e catarata congênita, selecionados após análise de 1050 prontuários dos pacientes atendidos no ambulatório de catarata congênita da UNIFESP. A microftalmia foi determinada pela ecobiometria ultrassonica. Exames oculares e complementares foram feitos para esclarecer a causa etiológica. O resultado visual pós- operatório do Grupo I (com microftalmia) foi confrontado com o resultado visual obtido no Grupo II (sem microftalmia). Resultados: O diâmetro ântero-posterior dos olhos microftálmicos variou de 13 à 21 mm. A frequência etiológica da catarata congênita associada aos olhos microftálmicos foi assim distribuída: doenças infecciosas (55,3%); seguidos de idiopáticas (26,3%), colobomas (7,9%), hereditárias (6,6%), persistência do vítreo primário hiperplásico (2,6%) e associada à síndrome de Lenz (1,3%) .A frequência da microftalmia foi de 7,23 %. 68,3% de olhos afácicos microftálmicos atingiram visão melhor e ou igual à 20/200. Conclusão: A frequência da microftalmia associada à catarata congênita foi de 7,23%. A maior frequência etiológica ocorreu nas doenças infecciosas (55,3%), Embora os olhos microftálmicos tenham tendência para piores resultados visuais quando comparados aos não microftálmicos, nesta pesquisa os olhos microftálmicos afácicos que atingiram visão melhor ou igual a 20/200 foram de 68,3%.
Abstract Objective: To determine the frequency of microphthalmia associated with congenital cataract and its etiological frequency. Compare the result of visual acuity in aphakic microphthalmus eyes, with the visual acuity result obtained in non microphthalmus eyes. Methods: Retrospective study of 76 patients with microphthalmia and congenital cataract, selected after analysis of 1050 medical records of patients seen in congenital cataract clinic of UNIFESP. All patients underwent complete ophthalmologic examination and microphthalmia determined by ultrasound biometry. Investigations were made to clarify the etiological cause. The postoperative visual outcome of Group I (with microphthalmia) was faced with the visual results obtained in Group II (control group without microphthalmia). Results: The anteroposterior diameter of microphthalmus eyes ranged from 13 to 21 mm. The etiological frequency of microphthalmia and congenital cataract was distributed as follows: infectious diseases (55.3%), idiopathic (26.3%), colobomas (7.9%), hereditary (6.6%), persistent hyperplastic vitreous (2.6%) and linked to the Lenz's syndrome (1.3%). The visual acuity in aphakic eyes that reached better view and or equal to 20/200 was 68.3%. Conclusion: The frequency of microphthalmia associated with congenital cataract was 7.23%. The etiological occurred more frequently in infectious disease (55.3%). The aphakics eyes with microphthalmia tend to have worse visual acuity results than the eyes without microphthalmia. If we consider the visual results same and above 20/200 as successful in this search, aphakic eyes with microphthalmia that hit these indices are 68.3%.
Assuntos
Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Criança , Catarata/congênito , Extração de Catarata/métodos , Acuidade Visual , Microftalmia/etiologia , Microftalmia/epidemiologia , Afacia Pós-Catarata , Microftalmia/cirurgia , Estudos Retrospectivos , Seguimentos , Biometria , Resultado do Tratamento , Comprimento Axial do Olho , Cristalino/crescimento & desenvolvimentoRESUMO
The eye lens is a transparent and avascular organ in the front of the eye that is responsible for focusing light onto the retina in order to transmit a clear image. A monolayer of epithelial cells covers the anterior hemisphere of the lens, and the bulk of the lens is made up of elongated and differentiated fiber cells. Lens fiber cells are very long and thin cells that are supported by sophisticated cytoskeletal networks, including actin filaments at cell junctions and the spectrin-actin network of the membrane skeleton. In this review, we highlight the proteins that regulate diverse actin filament networks in the lens and discuss how these actin cytoskeletal structures assemble and function in epithelial and fiber cells. We then discuss methods that have been used to study actin in the lens and unanswered questions that can be addressed with novel techniques.
Assuntos
Citoesqueleto de Actina/fisiologia , Cristalino/embriologia , Animais , Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Humanos , Cristalino/citologia , Cristalino/crescimento & desenvolvimento , Proteínas dos Microfilamentos/metabolismoRESUMO
Understanding how tissues and organs acquire and maintain an appropriate size and shape remains one of the most challenging areas in developmental biology. The eye lens represents an excellent system to provide insights into regulatory mechanisms because in addition to its relative simplicity in cellular composition (being made up of only two forms of cells, epithelial and fiber cells), these cells must become organized to generate the precise spheroidal arrangement that delivers normal lens function. Epithelial and fiber cells also represent spatially distinct proliferation and differentiation compartments, respectively, and an ongoing balance between these domains must be tightly regulated so that the lens achieves and maintains appropriate dimensions during growth and ageing. Recent research indicates that reciprocal inductive interactions mediated by Wnt-Frizzled and Notch-Jagged signaling pathways are important for maintaining and organizing these compartments. The Hippo-Yap pathway has also been implicated in maintaining the epithelial progenitor compartment and regulating growth processes. Thus, whilst some molecules and mechanisms have been identified, further work in this important area is needed to provide a clearer understanding of how lens size and shape is regulated.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Cristalino/crescimento & desenvolvimento , Morfogênese/fisiologia , Animais , Células Epiteliais/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Redes Reguladoras de Genes , Humanos , Cristalino/metabolismo , Transdução de Sinais/fisiologiaRESUMO
PURPOSE: A poor early life nutrition environment is well established to result in a range of cardiometabolic disorders in offspring in later life. These effects can be exacerbated via exposure to an obesogenic dietary environment. To date, the effect of maternal diet and/or a post-natal obesogenic nutritional environment on key characteristics related to lens growth and oxidative stress has not been undertaken. The present study, therefore, examined the characteristics and oxidative status of the lens. MATERIALS AND METHODS: Using a model of moderate maternal under-nutrition, rat dams were fed either a control diet (100% ad libitum, CON) or undernourished throughout pregnancy (50% of ad libitum intake, UN) and offspring fed either a control (5% fat, C) or high fat (30% fat, HF) diet post-weaning, resulting in four nutritional groups; CON-C, CON-HF, UN-C, and UN-HF. Offspring lenses were extracted at 160 days of age, weighed, imaged under dark and bright field microscopy, and then dissected into cortical and core fractions for biochemical analyses of oxidative stress markers. RESULTS: Our findings reveal that lenses from all groups were transparent. However, gender specific changes were evident at the biochemical level with increased oxidative stress detected in the cortex and core of female but not male UN-C lenses, and in the cortex of male but not female CON-HF lenses. The greatest increase in oxidative stress was detected in the UN-HF group in the cortex and core regions of the lens and for both genders. CONCLUSIONS: These findings show that oxidative stress is exacerbated in the lens as a result of a combination of altered pre-natal and post-natal diet. This demonstrates a novel interaction between the two developmental windows and warrants further investigations toward devising appropriate nutritional strategies for minimizing oxidative stress in the lens.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Doenças do Cristalino/etiologia , Cristalino/crescimento & desenvolvimento , Desnutrição/complicações , Fenômenos Fisiológicos da Nutrição Materna , Estresse Oxidativo/fisiologia , Adiposidade , Animais , Ácido Ascórbico/metabolismo , Biomarcadores/metabolismo , Glicemia/metabolismo , Peso Corporal , Feminino , Glutationa/metabolismo , Insulina/sangue , Doenças do Cristalino/metabolismo , Cristalino/metabolismo , Leptina/sangue , Masculino , Obesidade/etiologia , Obesidade/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Vitamina E/metabolismoRESUMO
Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.