RESUMO
Fibroblast growth factor 2 (FGF2) plays an important role in cortical development. However, the genes downstream of FGF2 that mediate its effect are largely unknown. We have performed a microarray screening of genes regulated by FGF2 using primary cortical neuron culture derived from embryonic day 14.5 (E14.5) mouse forebrains. In this study, we have analysed a previously uncharacterised gene encoding a 180-amino acid protein, hereby named 'coiled-coil protein 1 (ccp1)', that showed a modest up-regulation upon FGF2 stimulation. Northern blots and RT-PCR showed specific expression of ccp1 in multiple tissues including adult and embryonic brains. In situ hybridizations revealed that ccp1 was expressed in the cortical plate between Reelin and Tbr1-positive layers in the dorsal cortex at E15.5. Furthermore, the expression pattern of ccp1 at E13.5-E14.5 reflected some of the aspects of tangential migration of cortical progenitors during the early phase. We observed that the expressed ccp1 protein was localised to endo/lysosomal compartment in the cell body as well as to vesicles present in the processes of primary cortical neurons and oligodendrocyte cell line.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Sequência Conservada , Éxons , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Hibridização In Situ , Íntrons , Zíper de Leucina , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Especificidade de Órgãos , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Proteína Reelina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
Phototransduction in Drosophila is a phosphoinositide-mediated signalling pathway. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in this process, and its levels are tightly regulated. A photoreceptor-specific form of the enzyme CDP-diacylglycerol synthase (CDS), which catalyzes the formation of CDP-diacylglycerol from phosphatidic acid, is a key regulator of the amount of PIP2 available for signalling. cds mutants develop light-induced retinal degeneration. We report here the isolation and characterization of two murine genes encoding this enzyme, Cds1 and Cds2. The genes encode proteins that are 73% identical and 92% similar but exhibit very different expression patterns. Cds1 shows a very restricted expression pattern but is expressed in the inner segments of the photoreceptors whilst Cds2 shows a ubiquitous pattern of expression. Using fluorescent in situ hybridization we have mapped Cds1 and Cds2 to chromosomes 5E3 and 2G1 respectively. These are regions of synteny with the corresponding human gene localization (4q21 and 20p13). Transient transfection experiments with epitope tagged proteins have also demonstrated that both are associated with the endoplasmic reticulum.
Assuntos
Diacilglicerol Colinofosfotransferase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Mapeamento Cromossômico , Cromossomos de Mamíferos/genética , Cricetinae , Cricetulus , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Diacilglicerol Colinofosfotransferase/metabolismo , Retículo Endoplasmático/metabolismo , Éxons , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Genes/genética , Hibridização in Situ Fluorescente , Íntrons , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Mice with the K644E kinase domain mutation in fibroblast growth factor receptor 3 (Fgfr3) (EIIa;Fgfr3(+/K644E)) exhibited a marked enlargement of the brain. The brain size was increased as early as E11.5, not secondary to the possible effect of Fgfr3 activity in the skeleton. Furthermore, the mutant brains showed a dramatic increase in cortical thickness, a phenotype opposite to that in FGF2 knockout mice. Despite this increased thickness, cortical layer formation was largely unaffected and no cortical folding was observed during embryonic days 11.5-18.5 (E11.5-E18.5). Measurement of cortical thickness revealed an increase of 38.1% in the EIIa;Fgfr3(+/K644E) mice at E14.5 and the advanced appearance of the cortical plate was frequently observed at this stage. Unbiased stereological analysis revealed that the volume of the ventricular zone (VZ) was increased by more than two fold in the EIIa;Fgfr3(+/K644E) mutants at E14.5. A relatively mild increase in progenitor cell proliferation and a profound decrease in developmental apoptosis during E11.5-E14.5 most likely accounts for the dramatic increase in total telecephalic cell number. Taken together, our data suggest a novel function of Fgfr3 in controlling the development of the cortex, by regulating proliferation and apoptosis of cortical progenitors.