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1.
PLoS Genet ; 16(5): e1008782, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32421721

RESUMO

The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the jnk genes in cardiac morphogenesis remain unknown as mouse mutants exhibit functional redundancy, with early embryonic lethality of compound mutants. In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ventrículos do Coração/citologia , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Processamento Alternativo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Contagem de Células , Células Cultivadas , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/embriologia , Ventrículos do Coração/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
2.
Development ; 145(13)2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29853617

RESUMO

Signaling interactions between the myocardium and endocardium pattern embryonic cardiac regions, instructing their development to fulfill specific functions in the mature heart. We show that ectopic Bmp2 expression in the mouse chamber myocardium changes the transcriptional signature of adjacent chamber endocardial cells into valve tissue, and enables them to undergo epithelial-mesenchyme transition. This induction is independent of valve myocardium specification and requires high levels of Notch1 activity. Biochemical experiments suggest that Bmp2-mediated Notch1 induction is achieved through transcriptional activation of the Notch ligand Jag1, and physical interaction of Smad1/5 with the intracellular domain of the Notch1 receptor. Thus, widespread myocardial Bmp2 and endocardial Notch signaling drive presumptive ventricular endocardium to differentiate into valve endocardium. Understanding the molecular basis of valve development is instrumental to designing therapeutic strategies for congenital heart valve defects.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Embrião de Mamíferos/embriologia , Endocárdio/embriologia , Valvas Cardíacas/embriologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Embrião de Mamíferos/citologia , Endocárdio/citologia , Valvas Cardíacas/citologia , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Miocárdio/metabolismo , Receptores Notch/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo
3.
Circ Res ; 118(10): 1480-97, 2016 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-27056911

RESUMO

RATIONALE: The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial-mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. OBJECTIVE: The aim of this study is to determine the functional specificity of Notch in valve development. METHODS AND RESULTS: Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial-mesenchymal transition. Mice lacking endocardial Jag1, Notch1, or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post-epithelial-mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non-cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1-mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf, was markedly reduced in Jag1-mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1-mutant outflow tract explant cultures rescued the hyperproliferative phenotype. CONCLUSIONS: During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial-mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function.


Assuntos
Valva Mitral/metabolismo , Morfogênese , Receptor Notch1/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Transição Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Valva Mitral/anormalidades , Valva Mitral/embriologia , Receptor Notch1/metabolismo , Regulação para Cima
4.
Genesis ; 53(5): 337-45, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25950518

RESUMO

Heart valve development begins with the endothelial-to-mesenchymal transition (EMT) of endocardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate-mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock-in tamoxifen-inducible Cre line was recently generated (Msx1CreERT2) and characterized during embryonic development and after birth, and was shown to recapitulate the endogenous Msx1 expression pattern. Here, we further analyze this knock-in allele and track the Msx1-expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling.


Assuntos
Alelos , Técnicas de Introdução de Genes , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Integrases/genética , Fator de Transcrição MSX1/genética , Receptores de Estrogênio/genética , Animais , Endocárdio/enzimologia , Endocárdio/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Integrases/metabolismo , Fator de Transcrição MSX1/metabolismo , Camundongos , Organogênese/genética , Receptores de Estrogênio/metabolismo
5.
Cell Death Dis ; 12(8): 729, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294700

RESUMO

Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which ectopic Bmp2 expression is controlled by the Tie2 promoter. Tie2CRE/+;Bmp2tg/tg mice develop aortic valve dysfunction postnatally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2CRE/+;Bmp2tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and genetic bone disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2CRE/+;Bmp2tg/tg transgenic animals is accompanied by increased bone marrow hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type bone marrow hematopoietic stem cells into lethally irradiated Tie2CRE/+;Bmp2tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic bone marrow into wild-type recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic bone marrow colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell origin. Thus, Tie2CRE/+;Bmp2tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Linhagem da Célula , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Receptor TIE-2/metabolismo , Animais , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Transplante de Medula Óssea , Proteína Morfogenética Óssea 2/sangue , Calcinose/diagnóstico por imagem , Calcinose/patologia , Calcinose/fisiopatologia , Condrogênese , Células Endoteliais/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Estimativa de Kaplan-Meier , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Musculares/patologia , Ossificação Heterotópica/sangue , Ossificação Heterotópica/diagnóstico por imagem , Osteogênese , Tomografia Computadorizada por Raios X
6.
Cell Death Dis ; 9(3): 399, 2018 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-29540665

RESUMO

During mammalian heart development, restricted myocardial Bmp2 expression is a key patterning signal for atrioventricular canal specification and the epithelial-mesenchyme transition that gives rise to the valves. Using a mouse transgenic line conditionally expressing Bmp2, we show that widespread Bmp2 expression in the myocardium leads to valve and chamber dysmorphogenesis and embryonic death by E15.5. Transgenic embryos show thickened valves, ventricular septal defect, enlarged trabeculae and dilated ventricles, with an endocardium able to undergo EMT both in vivo and in vitro. Gene profiling and marker analysis indicate that cellular proliferation is increased in transgenic embryos, whereas chamber maturation and patterning are impaired. Similarly, forced Bmp2 expression stimulates proliferation and blocks cardiomyocyte differentiation of embryoid bodies. These data show that widespread myocardial Bmp2 expression directs ectopic valve primordium formation and maintains ventricular myocardium and cardiac progenitors in a primitive, proliferative state, identifying the potential of Bmp2 in the expansion of immature cardiomyocytes.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proliferação de Células , Transição Epitelial-Mesenquimal , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Animais , Proteína Morfogenética Óssea 2/genética , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Transdução de Sinais
7.
PLoS One ; 7(5): e37685, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22629443

RESUMO

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.


Assuntos
Sobrevivência Celular/fisiologia , Face/embriologia , Crista Neural/citologia , Quinases Associadas a rho/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Apoptose/fisiologia , Região Branquial/anormalidades , Anormalidades Congênitas/genética , Anormalidades Congênitas/metabolismo , Anormalidades Craniofaciais , Citoesqueleto/genética , Citoesqueleto/metabolismo , Face/anormalidades , Camundongos , Camundongos Transgênicos , Crista Neural/metabolismo , Quinases Associadas a rho/genética
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