RESUMO
BACKGROUND: Many human gene mutations have been linked to congenital heart disease (CHD), yet CHD remains a major health issue worldwide due in part to an incomplete understanding of the molecular basis for cardiac malformation. RESULTS: Here we identify the orthologous mouse Pou6f1 and zebrafish pouC as POU homeodomain transcription factors enriched in the developing heart. We find that pouC is a multi-functional transcriptional regulator containing separable activation, repression, protein-protein interaction, and DNA binding domains. Using zebrafish heart development as a model system, we demonstrate that pouC knockdown impairs cardiac morphogenesis and affects cardiovascular function. We also find that levels of pouC expression must be fine-tuned to enable proper heart formation. At the cellular level, we demonstrate that pouC knockdown disrupts atrioventricular canal (AVC) cardiomyocyte maintenance, although chamber myocyte specification remains intact. Mechanistically, we show that pouC binds a bmp4 intronic regulatory element to mediate transcriptional activation. CONCLUSIONS: Taken together, our study establishes pouC as a novel transcriptional input into the regulatory hierarchy that drives AVC morphogenesis in zebrafish. We anticipate that these findings will inform future efforts to explore functional conservation in mammals and potential association with atrioventricular septal defects in humans. Developmental Dynamics 248:173-188, 2019. © 2018 Wiley Periodicals, Inc.
Assuntos
Proteína Morfogenética Óssea 4/genética , Regulação da Expressão Gênica no Desenvolvimento , Septos Cardíacos/crescimento & desenvolvimento , Fatores do Domínio POU/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Proteína Morfogenética Óssea 4/metabolismo , Coração/embriologia , Coração/crescimento & desenvolvimento , Defeitos dos Septos Cardíacos , Septos Cardíacos/embriologia , Camundongos , Fatores do Domínio POU/metabolismo , Ligação Proteica , Fatores de Transcrição , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5(+) progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.
Assuntos
Diferenciação Celular/fisiologia , Indução Embrionária/fisiologia , Fibroblastos/citologia , Coração/embriologia , Miócitos Cardíacos/fisiologia , Fatores de Transcrição/metabolismo , Potenciais de Ação/fisiologia , Análise de Variância , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Primers do DNA/genética , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Imuno-Histoquímica , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genéticaRESUMO
The cardiac conduction system coordinates electrical activation through a series of interconnected structures, including the atrioventricular node (AVN), the central connection point that delays impulse propagation to optimize cardiac performance. Although recent studies have uncovered important molecular details of AVN formation, relatively little is known about the transcriptional mechanisms that regulate AV delay, the primary function of the mature AVN. We identify here MyoR as a novel transcription factor expressed in Cx30.2(+) cells of the AVN. We show that MyoR specifically inhibits a Cx30.2 enhancer required for AVN-specific gene expression. Furthermore, we demonstrate that MyoR interacts directly with Gata4 to mediate transcriptional repression. Our studies reveal that MyoR contains two nonequivalent repression domains. While the MyoR C-terminal repression domain inhibits transcription in a context-dependent manner, the N-terminal repression domain can function in a heterologous context to convert the Hand2 activator into a repressor. In addition, we show that genetic deletion of MyoR in mice increases Cx30.2 expression by 50% and prolongs AV delay by 13%. Taken together, we conclude that MyoR modulates a Gata4-dependent regulatory circuit that establishes proper AV delay, and these findings may have wider implications for the variability of cardiac rhythm observed in the general population.
Assuntos
Nó Atrioventricular/metabolismo , Fator de Transcrição GATA4/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Animais , Nó Atrioventricular/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Células COS , Chlorocebus aethiops , Conexinas/genética , Conexinas/metabolismo , Embrião de Mamíferos , Feminino , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica , Genes Reporter , Frequência Cardíaca/fisiologia , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/genética , Transcrição Gênica , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors. METHODOLOGY/PRINCIPAL FINDINGS: The localization pattern of Spectraplakin in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin's gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test. CONCLUSIONS/SIGNIFICANCE: In summary, we found a unique localization of Spectraplakin in photoreceptors, and identified the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions through genetic mutational analysis. Our data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis.