RESUMO
Expression of the endoderm specific gene Endo16, was used to monitor endoderm specification in developmentally arrested and in dissociated embryos. beta-APN treatment halts gastrulation, however, in two species of sea urchins Endo16 mRNA is still expressed, suggesting that specification of the endodermal lineage has taken place in these developmentally arrested embryos. Endo16 mRNA is not expressed in embryos dissociated at the 4-8-cell stage unless they are reassociated shortly after the 16-cell stage. Interestingly, dissociation after the 16-cell stage also results in a lack of Endo16 expression. Lithium is unable to rescue Endo16 expression in these dissociated embryos. These results indicate that early signaling events mediated by cell-cell contact are required for the initial specification and maintenance of the endoderm in the developing embryo.
Assuntos
Embrião não Mamífero/fisiologia , Endoderma/fisiologia , Ouriços-do-Mar/embriologia , Sequência de Aminoácidos , Aminopropionitrilo/farmacologia , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Moléculas de Adesão Celular/genética , Comunicação Celular , Embrião não Mamífero/efeitos dos fármacos , Endoderma/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lítio/farmacologia , Dados de Sequência Molecular , Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacosRESUMO
Endo16 encodes a developmentally regulated protein restricted to cells participating in the formation of the archenteron during sea urchin gastrulation and to the stomach of the pluteus. The 4680-nt coding region of the Endo16 gene has been sequenced from overlapping cDNAs. Sequence analysis revealed that Endo16 is a large multidomain protein starting with a putative signal sequence at its amino terminus which is followed by a cysteine-rich region, two potential heparin-binding regions, an acidic domain of 5 clustered repeats, an RGD cell binding motif, and a group of 12 additional acidic repeats. Immunolocalization by confocal and electron microscopy demonstrate that the Endo16 protein is in the extracellular matrix and associated with the surface of endodermal cells in the mid and hindgut of the archenteron. The two distinct acidic repeat regions are similar to known calcium-binding sequences. A recombinant Endo16 protein containing both putative calcium-binding repeat regions has been shown to bind radioactive calcium. Tryptic digests of gastrula stage protein extracts in the presence and the absence of calcium have established that calcium stabilizes Endo16 protein against proteolytic degradation.
