RESUMEN
Adult skeletal muscles in vertebrates are composed of different types of myofibers endowed with distinct metabolic and contraction speed properties. Genesis of this fiber-type heterogeneity during development remains poorly known, at least in mammals. Six1 and Six4 homeoproteins of the Six/sine oculis family are expressed throughout muscle development in mice, and Six1 protein is enriched in the nuclei of adult fast-twitch myofibers. Furthermore, Six1/Six4 proteins are known to control the early activation of fast-type muscle genes in myocytes present in the mouse somitic myotome. Using double Six1:Six4 mutants (SixdKO) to dissect in vivo the genesis of muscle fiber-type heterogeneity, we analyzed here the phenotype of the dorsal/epaxial muscles remaining in SixdKO. We show by electron microscopy analysis that the absence of these homeoproteins precludes normal sarcomeric organization of the myofiber leading to a dystrophic aspect, and by immunohistochemistry experiments a deficiency in synaptogenesis. Affymetrix transcriptome analysis of the muscles remaining in E18.5 SixdKO identifies a major role for these homeoproteins in the control of genes that are specifically activated in the adult fast/glycolytic myofibers, particularly those controlling Ca(2+) homeostasis. Absence of Six1 and Six4 leads to the development of dorsal myofibers lacking expression of fast-type muscle genes, and mainly expressing a slow-type muscle program. The absence of restriction of the slow-type program during the fetal period in SixdKO back muscles is associated with a decreased HDAC4 protein level, and subcellular relocalization of the transcription repressor Sox6. Six genes thus behave as essential global regulators of muscle gene expression, as well as a central switch to drive the skeletal muscle fast phenotype during fetal development.
Asunto(s)
Proteínas de Drosophila/genética , Embrión de Mamíferos/metabolismo , Proteínas de Homeodominio/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas del Tejido Nervioso/genética , Factores de Transcripción/genética , Animales , Northern Blotting , Células Cultivadas , Proteínas de Drosophila/metabolismo , Embrión de Mamíferos/embriología , Embrión de Mamíferos/ultraestructura , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Desarrollo de Músculos/genética , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Rápida/ultraestructura , Fibras Musculares Esqueléticas/clasificación , Fibras Musculares Esqueléticas/citología , Fibras Musculares de Contracción Lenta/metabolismo , Fibras Musculares de Contracción Lenta/ultraestructura , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Tiempo , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
BACKGROUND/AIMS: To specify roles of HNF 4 alpha in mouse liver development, we have analyzed the ex vivo morphogenetic potential of HNF4 alpha-null embryonic hepatic cells. METHODS: Using mice with floxed or deficiency alleles of HNF4 alpha, hepatic cells lacking this transcription factor were explanted into primary culture and derived into cell lines. RESULTS: Contrary to behavior in vivo where HNF4 alpha-null liver cells fail to show normal polarity and epithelialization, e18.5 hepatic cells in primary culture from mutant embryos show restoration of apical expression of tight junction protein-1 and of transcripts for E-cadherin. Clones of control and HNF4 alpha-null cell lines were indistinguishable, even when differentiation of bile canalicular formation was induced. HNF4 alpha-null and control cell lines showed similar potential to colonize livers of the murine ALB-uPA/SCID model of liver regeneration, but null cells formed only bile ducts and not clusters of hepatocytes. Finally, analysis of mutant embryonic livers revealed a transcriptional signature consistent with a stress response, which could underlie the morphogenetic defects observed in vivo. CONCLUSIONS: We conclude that the lack of epithelialization characteristic of the HNF4 alpha-null embryonic liver is due, at least in part, to non-cell autonomous defects, and that null cells do not suffer intrinsic defects in polarization.