RESUMO
Novel regenerative therapies may stem from deeper understanding of the mechanisms governing cardiovascular lineage diversification. Using enhancer mapping and live imaging in avian embryos, and genetic lineage tracing in mice, we investigated the spatio-temporal dynamics of cardiovascular progenitor populations. We show that expression of the cardiac transcription factor Nkx2.5 marks a mesodermal population outside of the cardiac crescent in the extraembryonic and lateral plate mesoderm, with characteristics of hemogenic angioblasts. Extra-cardiac Nkx2.5 lineage progenitors migrate into the embryo and contribute to clusters of CD41+/CD45+ and RUNX1+ cells in the endocardium, the aorta-gonad-mesonephros region of the dorsal aorta and liver. We also demonstrated that ectopic expression of Nkx2.5 in chick embryos activates the hemoangiogenic gene expression program. Taken together, we identified a hemogenic angioblast cell lineage characterized by transient Nkx2.5 expression that contributes to hemogenic endothelium and endocardium, suggesting a novel role for Nkx2.5 in hemoangiogenic lineage specification and diversification.
Assuntos
Aorta/embriologia , Endocárdio/embriologia , Hemangioblastos/fisiologia , Proteína Homeobox Nkx-2.5/metabolismo , Animais , Embrião de Galinha , Camundongos , Análise Espaço-TemporalRESUMO
Neural crest development involves epithelial-mesenchymal transition (EMT), during which epithelial cells are converted into individual migratory cells. Notably, the same signaling pathways regulate EMT function during both development and tumor metastasis. p53 plays multiple roles in the prevention of tumor development; however, its precise roles during embryogenesis are less clear. We have investigated the role of p53 in early cranial neural crest (CNC) development in chick and mouse embryos. In the mouse, p53 knockout embryos displayed broad craniofacial defects in skeletal, neuronal and muscle tissues. In the chick, p53 is expressed in CNC progenitors and its expression decreases with their delamination from the neural tube. Stabilization of p53 protein using a pharmacological inhibitor of its negative regulator, MDM2, resulted in reduced SNAIL2 (SLUG) and ETS1 expression, fewer migrating CNC cells and in craniofacial defects. By contrast, electroporation of a dominant-negative p53 construct increased PAX7(+) SOX9(+) CNC progenitors and EMT/delamination of CNC from the neural tube, although the migration of these cells to the periphery was impaired. Investigating the underlying molecular mechanisms revealed that p53 coordinates CNC cell growth and EMT/delamination processes by affecting cell cycle gene expression and proliferation at discrete developmental stages; disruption of these processes can lead to craniofacial defects.
Assuntos
Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Laminina/metabolismo , Crista Neural/embriologia , Crânio/embriologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Células Cultivadas , Embrião de Galinha , Anormalidades Craniofaciais/complicações , Anormalidades Craniofaciais/embriologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Embrião de Mamíferos , Transição Epitelial-Mesenquimal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Anormalidades Musculoesqueléticas/complicações , Anormalidades Musculoesqueléticas/embriologia , Anormalidades Musculoesqueléticas/genética , Anormalidades Musculoesqueléticas/patologia , Crista Neural/citologia , Crista Neural/metabolismo , Crânio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The cells of the second heart field (SHF) contribute to the outflow tract and right ventricle, as well as to parts of the left ventricle and atria. Isl1, a member of the LIM-homeodomain transcription factor family, is expressed early in this cardiac progenitor population and functions near the top of a transcriptional pathway essential for heart development. Isl1 is required for the survival and migration of SHF-derived cells into the early developing heart at the inflow and outflow poles. Despite this important role for Isl1 in early heart formation, the transcriptional regulation of Isl1 has remained largely undefined. Therefore, to identify transcription factors that regulate Isl1 expression in vivo, we screened the conserved noncoding sequences from the mouse Isl1 locus for enhancer activity in transgenic mouse embryos. Here, we report the identification of an enhancer from the mouse Isl1 gene that is sufficient to direct expression to the SHF and its derivatives. The Isl1 SHF enhancer contains three consensus Forkhead transcription factor binding sites that are efficiently and specifically bound by Forkhead transcription factors. Importantly, the activity of the enhancer is dependent on these three Forkhead binding sites in transgenic mouse embryos. Thus, these studies demonstrate that Isl1 is a direct transcriptional target of Forkhead transcription factors in the SHF and establish a transcriptional pathway upstream of Isl1 in the SHF.
Assuntos
Elementos Facilitadores Genéticos/genética , Coração Fetal/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Mesoderma/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Movimento Celular/fisiologia , Coração Fetal/crescimento & desenvolvimento , Genes Reporter , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Óperon Lac , Mesoderma/citologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Fatores de TranscriçãoRESUMO
Adult skeletal muscle possesses a remarkable regenerative capacity, due to the presence of satellite cells, adult muscle stem cells. We used fate-mapping techniques in avian and mouse models to show that trunk (Pax3(+)) and cranial (MesP1(+)) skeletal muscle and satellite cells derive from separate genetic lineages. Similar lineage heterogeneity is seen within the head musculature and satellite cells, due to their shared, heterogenic embryonic origins. Lineage tracing experiments with Isl1Cre mice demonstrated the robust contribution of Isl1(+) cells to distinct jaw muscle-derived satellite cells. Transplantation of myofiber-associated, Isl1-derived satellite cells into damaged limb muscle contributed to muscle regeneration. In vitro experiments demonstrated the cardiogenic nature of cranial- but not trunk-derived satellite cells. Finally, overexpression of Isl1 in the branchiomeric muscles of chick embryos inhibited skeletal muscle differentiation in vitro and in vivo, suggesting that this gene plays a role in the specification of cardiovascular and skeletal muscle stem cell progenitors.
Assuntos
Linhagem da Célula , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Extremidades/patologia , Regulação da Expressão Gênica no Desenvolvimento , Heterogeneidade Genética , Cabeça , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM , Mesoderma/citologia , Mesoderma/transplante , Camundongos , Desenvolvimento Muscular , Músculos/patologia , Músculos/fisiologia , Miocárdio/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/metabolismo , Codorniz/embriologia , Regeneração , Fatores de TranscriçãoAssuntos
Proteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Animais , Ligação Competitiva/fisiologia , Evolução Molecular , Receptores Frizzled/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Ligação Proteica/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/fisiologia , Proteínas/químicaRESUMO
During embryogenesis, paraxial mesoderm cells contribute skeletal muscle progenitors, whereas cardiac progenitors originate in the lateral splanchnic mesoderm (SpM). Here we focus on a subset of the SpM that contributes to the anterior or secondary heart field (AHF/SHF), and lies adjacent to the cranial paraxial mesoderm (CPM), the precursors for the head musculature. Molecular analyses in chick embryos delineated the boundaries between the CPM, undifferentiated SpM progenitors of the AHF/SHF, and differentiating cardiac cells. We then revealed the regionalization of branchial arch mesoderm: CPM cells contribute to the proximal region of the myogenic core, which gives rise to the mandibular adductor muscle. SpM cells contribute to the myogenic cells in the distal region of the branchial arch that later form the intermandibular muscle. Gene expression analyses of these branchiomeric muscles in chick uncovered a distinct molecular signature for both CPM- and SpM-derived muscles. Islet1 (Isl1) is expressed in the SpM/AHF and branchial arch in both chick and mouse embryos. Lineage studies using Isl1-Cre mice revealed the significant contribution of Isl1(+) cells to ventral/distal branchiomeric (stylohyoid, mylohyoid and digastric) and laryngeal muscles. By contrast, the Isl1 lineage contributes to mastication muscles (masseter, pterygoid and temporalis) to a lesser extent, with virtually no contribution to intrinsic and extrinsic tongue muscles or extraocular muscles. In addition, in vivo activation of the Wnt/beta-catenin pathway in chick embryos resulted in marked inhibition of Isl1, whereas inhibition of this pathway increased Isl1 expression. Our findings demonstrate, for the first time, the contribution of Isl1(+) SpM cells to a subset of branchiomeric skeletal muscles.