RESUMO
The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo.
Assuntos
Anormalidades do Sistema Digestório/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Rim/anormalidades , Proteínas de Membrana/deficiência , Proteínas Proto-Oncogênicas c-ret/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Análise de Variância , Animais , Western Blotting , Primers do DNA/genética , Anormalidades do Sistema Digestório/metabolismo , Sistema Nervoso Entérico/patologia , Técnicas de Introdução de Genes , Genótipo , Técnicas Histológicas , Hiperplasia/etiologia , Hiperplasia/patologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Mutantes , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-ret/genética , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Transdução de Sinais/genéticaRESUMO
Disrupted-In-Schizophrenia 1 (DISC1), a susceptibility gene for major psychiatric disorders, regulates neuronal migration and differentiation during mammalian brain development. Although roles for DISC1 in postnatal neurogenesis in the dentate gyrus (DG) have recently emerged, it is not known how DISC1 and its interacting proteins govern the migration, positioning, and differentiation of dentate granule cells (DGCs). Here, we report that DISC1 interacts with the actin-binding protein girdin to regulate axonal development. DGCs in girdin-deficient neonatal mice exhibit deficits in axonal sprouting in the cornu ammonis 3 region of the hippocampus. Girdin deficiency, RNA interference-mediated knockdown, and inhibition of the DISC1/girdin interaction lead to overextended migration and mispositioning of the DGCs resulting in profound cytoarchitectural disorganization of the DG. These findings identify girdin as an intrinsic factor in postnatal development of the DG and provide insights into the critical role of the DISC1/girdin interaction in postnatal neurogenesis in the DG.
Assuntos
Giro Denteado/embriologia , Giro Denteado/crescimento & desenvolvimento , Proteínas dos Microfilamentos/metabolismo , Neurogênese/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Bromodesoxiuridina/metabolismo , Diferenciação Celular/genética , Movimento Celular/genética , Células Cultivadas , Chlorocebus aethiops , Giro Denteado/citologia , Estimulação Elétrica/métodos , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Cones de Crescimento/fisiologia , Humanos , Imunoprecipitação/métodos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Interferência de RNA/fisiologia , Ratos , Transfecção/métodos , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/genéticaRESUMO
Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.
Assuntos
Mutação , Doenças Renais Policísticas/genética , Fatores de Transcrição/genética , Dedos de Zinco , Animais , Sequência de Bases , Proliferação de Células , Primers do DNA , Corantes Fluorescentes , Técnicas de Silenciamento de Genes , Hibridização In Situ , Oryzias , Pâncreas/metabolismo , Doenças Renais Policísticas/patologia , Frações Subcelulares/metabolismoRESUMO
Glis3 is a member of the Gli-similar subfamily. GLIS3 mutations in humans lead to neonatal diabetes, hypothyroidism, and cystic kidney disease. We generated Glis3-deficient mice by gene-targeting. The Glis3(-/-) mice had significant increases in the basal blood sugar level during the first few days after birth. The high levels of blood sugar are attributed to a decrease in the Insulin mRNA level in the pancreas that is caused by impaired islet development and the subsequent impairment of Insulin-producing cell formation. The pancreatic phenotypes indicate that the Glis3-deficient mice are a model for GLIS3 mutation and diabetes mellitus in humans.
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Transativadores/deficiência , Animais , Animais Recém-Nascidos , Sequência de Bases , Glicemia/metabolismo , Carboxipeptidases A/metabolismo , Primers do DNA/genética , Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Humanos , Recém-Nascido , Insulina/genética , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Knockout , Mutação , Pâncreas/metabolismo , Pâncreas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Transativadores/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
We provide an overview of glomerulogenesis in medaka from the embryo to the adult by means of in situ hybridization with the wt1 gene as a marker as well as histology and three-dimensional images. The pronephric glomus starts to develop in the intermediate mesoderm during early somitogenesis, is completed before hatching, and persists throughout the lifetime of the fish. Within 5 days after hatching, mesonephric glomerulus formation begins in the caudomedial end of the pronephric sinus and duct area. The number of glomeruli reaches approximately 200-300 in each kidney within 2 months after hatching. wt1 expression during nephron maturation served as a marker for the formation of the mesenchymal condensate and the nephrogenic body. Existence of mesenchymal condensates and persistence of wt1 expression in the adult kidney suggest that the mesonephros retains precursor cells that may be capable of contributing to neoglomerulogenesis during adulthood.
