RESUMO
Sonic Hedgehog (Shh) is a critical signaling factor for a variety of developmental pathways during embryogenesis, including the specification of left-right asymmetry in the heart. Mice that lack Hedgehog signaling show a delay in the induction of cardiomyogenesis, as indicated by a delayed expression of Nkx2-5. To further examine a role for Shh in cardiomyogenesis, clonal populations of P19 cells that stably express Shh, termed P19(Shh) cells, were isolated. In monolayer P19(Shh) cultures the Shh pathway was functional as shown by the up-regulation of Ptc1 and Gli1 expression, but no cardiac muscle markers were activated. However, Shh expression induced cardiomyogenesis following cellular aggregation, resulting in the expression of factors expressed in cardiac muscle including GATA-4, MEF2C, and Nkx2-5. Furthermore, aggregated P19 cell lines expressing Gli2 or Meox1 also up-regulated the expression of cardiac muscle factors, leading to cardiomyogenesis. Meox1 up-regulated the expression of Gli1 and Gli2 and, thus, can modify the Shh signaling pathway. Finally, Shh, Gli2, and Meox1 all up-regulated BMP-4 expression, implying that activation of the Hedgehog pathway can regulate bone morphogenetic protein signals. Taken together, we propose a model in which Shh, functioning via Gli1/2, can specify mesodermal cells into the cardiac muscle lineage.
Assuntos
Transativadores/fisiologia , Actinas/metabolismo , Animais , Northern Blotting , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Primers do DNA/química , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Fator de Transcrição GATA4 , Proteínas Hedgehog , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like , Fatores de Transcrição MEF2 , Mesoderma/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Regulação Miogênica/metabolismo , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Gli2 and Meox1 are transcription factors that are expressed in the developing somite and play roles in the commitment of cells to the skeletal muscle lineage. To further define their roles in regulating myogenesis, the function of wild type and dominant-negative forms of Gli2 and Meox1 were examined in the context of differentiating P19 stem cells. We found that Gli2 overexpression up-regulated transcript levels of Meox1 and, conversely, Meox1 overexpression resulted in the upregulation of Gli2 transcripts. Furthermore, dominant-negative forms of either Meox1 or Gli2 disrupted the ability of P19 cells to commit to the muscle lineage and to properly express either Gli2 or Meox1, respectively. Finally, Pax3 transcripts were induced by Gli2 overexpression and lost in the presence of either mutants Meox1 or Gli2. Taken together, these results support the existence of a regulatory loop between Gli2, Meox1, and Pax3 that is essential for specification of mesodermal cells into the muscle lineage.