RESUMO
The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.
Assuntos
Coxins Endocárdicos , Endocárdio , Animais , Coxins Endocárdicos/metabolismo , Endocárdio/metabolismo , Valvas Cardíacas , Humanos , Mamíferos , Camundongos , Morfogênese , Miocárdio/metabolismoRESUMO
OBJECTIVES: Endocardial cushions are precursors of the valve septum complex that separates the four heart chambers. Several genes have been implicated in the development of endocardial cushions. Specifically, ERp44 has been found to play a role in the early secretory pathway, but its function in heart development has not been well studied. MATERIALS AND METHODS: In this study, we established conditional and tissue-specific knockout mouse models. The morphology, survival rate, the development of heart and endocardial cushion were under evaluation. The relationship between ERp44 and VEGFA was investigated by transcriptome, qPCR, WB, immunofluorescence and immunohistochemistry. RESULTS: ERp44 knockout (KO) mice were smaller in size, and most mice died during early postnatal life. KO hearts exhibited the typical phenotypes of congenital heart diseases, such as abnormal heart shapes and severe septal and valvular defects. Similar phenotypes were found in cTNT-Cre+/- ; ERp44fl / fl mice, which indicated that myocardial ERp44 principally controls endocardial cushion formation. Further studies demonstrated that the deletion of ERp44 significantly decreased the proliferation of cushion cells and impaired the endocardial-mesenchymal transition (EndMT), which was followed by endocardial cushion dysplasia. Finally, we found that ERp44 was directly bound to VEGFA and controlled its release, further regulating EndMT. CONCLUSION: We demonstrated that ERp44 plays a specific role in heart development. ERp44 contributes to the development of the endocardial cushion by affecting VEGFA-mediated EndMT.
Assuntos
Coxins Endocárdicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Miocárdio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Cardiopatias Congênitas/genética , Proteínas de Membrana/metabolismo , Mesoderma/metabolismo , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology. Among them, ERBB3 is a binding partner of ERBB2; the ERBB2/3 complex mediates downstream signaling for cell proliferation. ERBB3 has seven consensus binding sites to the p85 regulatory subunit of PI3K, which activates the downstream AKT pathway, leading to the proliferation of various cells. This study generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions. We determined that the proliferation of mesenchymal cells in the atrioventricular (AV) cushion in Erbb3 knock-in mutant embryos was temporarily reduced due to the decrease of AKT and ERK1/2 phosphorylation. Overall, our results suggest that AKT/ERK activation by the ERBB3-dependent PI3K signaling is crucial for AV cushion morphogenesis during embryonic heart development.
Assuntos
Comunicação Atrioventricular/genética , Coxins Endocárdicos/metabolismo , Receptor ErbB-3/metabolismo , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Comunicação Atrioventricular/metabolismo , Coxins Endocárdicos/embriologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/genética , Transdução de SinaisRESUMO
Smoothened is a key receptor of the hedgehog pathway, but the roles of Smoothened in cardiac development remain incompletely understood. In this study, we found that the conditional knockout of Smoothened from the mesoderm impaired the development of the venous pole of the heart and resulted in hypoplasia of the atrium/inflow tract (IFT) and a low heart rate. The blockage of Smoothened led to reduced expression of genes critical for sinoatrial node (SAN) development in the IFT. In a cardiac cell culture model, we identified a Gli2-Tbx5-Hcn4 pathway that controls SAN development. In the mutant embryos, the endocardial-to-mesenchymal transition (EndMT) in the atrioventricular cushion failed, and Bmp signalling was downregulated. The addition of Bmp2 rescued the EndMT in mutant explant cultures. Furthermore, we analysed Gli2+ scRNAseq and Tbx5-/- RNAseq data and explored the potential genes downstream of hedgehog signalling in posterior second heart field derivatives. In conclusion, our study reveals that Smoothened-mediated hedgehog signalling controls posterior cardiac progenitor commitment, which suggests that the mutation of Smoothened might be involved in the aetiology of congenital heart diseases related to the cardiac conduction system and heart valves.
Assuntos
Coxins Endocárdicos/embriologia , Coxins Endocárdicos/metabolismo , Proteínas Hedgehog/metabolismo , Organogênese , Transdução de Sinais , Nó Sinoatrial/embriologia , Nó Sinoatrial/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Biologia Computacional/métodos , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptor Smoothened/genética , Receptor Smoothened/metabolismoRESUMO
Cushion tissues, the primordia of valves and septa of the adult heart, are formed in the atrioventricular (AV) and outflow tract (OFT) regions of the embryonic heart. The cushion tissues are generated by the endothelial-mesenchymal transition (EMT), involving many soluble factors, extracellular matrix, and transcription factors. Moreover, neural crest-derived mesenchymal cells also migrate into the OFT cushion. The transcription factor Msx1 is known to be expressed in the endothelial and mesenchymal cells during cushion tissue formation. However, its exact role in EMT during cushion tissue formation is still unknown. In this study, we investigated the expression patterns of Msx1 mRNA and protein during chick heart development. Msx1 mRNA was localized in endothelial cells of the AV region at Stage 14, and its protein was first detected at Stage 15. Thereafter, Msx1 mRNA and protein were observed in the endothelial and mesenchymal cells of the OFT and AV regions. in vitro assays showed that ectopic Msx1 expression in endothelial cells induced p27, a cell-cycle inhibitor, expression and inhibited fibroblast growth factor 4 (FGF4)-induced cell proliferation. Although the FGF signal reduced the EMT-inducing activities of transforming growth factor ß (TGFß), ectopic Msx1 expression in endothelial cells enhanced TGFß signaling-induced αSMA, an EMT marker, expression. These results suggest that Msx1 may support the transformation of endothelial cells due to a TGFß signal in EMT during cushion tissue formation.
Assuntos
Proliferação de Células/fisiologia , Coxins Endocárdicos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Fator de Transcrição MSX1/metabolismo , Miocárdio/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Embrião de Galinha , Coxins Endocárdicos/metabolismo , Fator de Transcrição MSX1/genética , Antígeno Nuclear de Célula em Proliferação/genética , Transdução de Sinais/fisiologiaRESUMO
NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of Nox2 disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of Nox2-/- neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, Nox2-/- embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, Nox2 deficiency disrupted EndMT of atrioventricular cushion explants ex vivo. Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of Nox2 was associated with reduced expression of Gata4, Tgfß2, Bmp2, Bmp4, and Snail1, which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Cardiopatias Congênitas/patologia , Coração/embriologia , NADPH Oxidase 2/metabolismo , Animais , Apoptose , Proliferação de Células , Coxins Endocárdicos/embriologia , Coxins Endocárdicos/metabolismo , Coxins Endocárdicos/patologia , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Camundongos , NADPH Oxidase 2/deficiência , NADPH Oxidase 2/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Heart formation involves a complex series of tissue rearrangements, during which regions of the developing organ expand, bend, converge, and protrude in order to create the specific shapes of important cardiac components. Much of this morphogenesis takes place while cardiac function is underway, with blood flowing through the rapidly contracting chambers. Fluid forces are therefore likely to influence the regulation of cardiac morphogenesis, but it is not yet clear how these biomechanical cues direct specific cellular behaviors. In recent years, the optical accessibility and genetic amenability of zebrafish embryos have facilitated unique opportunities to integrate the analysis of flow parameters with the molecular and cellular dynamics underlying cardiogenesis. Consequently, we are making progress toward a comprehensive view of the biomechanical regulation of cardiac chamber emergence, atrioventricular canal differentiation, and ventricular trabeculation. In this review, we highlight a series of studies in zebrafish that have provided new insight into how cardiac function can shape cardiac morphology, with a particular focus on how hemodynamics can impact cardiac cell behavior. Over the long-term, this knowledge will undoubtedly guide our consideration of the potential causes of congenital heart disease.
Assuntos
Líquidos Corporais/fisiologia , Coração/embriologia , Coração/fisiologia , Morfogênese , Peixe-Zebra/embriologia , Animais , Fenômenos Biomecânicos , Diferenciação Celular/genética , Coxins Endocárdicos/citologia , Coxins Endocárdicos/embriologia , Coxins Endocárdicos/metabolismo , Endocárdio/citologia , Endocárdio/embriologia , Endocárdio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Coração/anatomia & histologia , Peixe-Zebra/genéticaRESUMO
The bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, belongs to the most prevalent congenital heart diseases in the world, occurring in 0.5-2% of the general population. We aimed to understand how changes in early cellular contributions result in BAV formation and impact cardiovascular outflow tract development. Detailed 3D reconstructions, immunohistochemistry and morphometrics determined that, during valvulogenesis, the non-coronary leaflet separates from the parietal outflow tract cushion instead of originating from an intercalated cushion. Nos3-/- mice develop a BAV without a raphe as a result of incomplete separation of the parietal outflow tract cushion into the right and non-coronary leaflet. Genetic lineage tracing of endothelial, second heart field and neural crest cells revealed altered deposition of neural crest cells and second heart field cells within the parietal outflow tract cushion of Nos3-/- embryos. The abnormal cell lineage distributions also affected the positioning of the aortic and pulmonary valves at the orifice level. The results demonstrate that the development of the right and non-coronary leaflets are closely related. A small deviation in the distribution of neural crest and second heart field populations affects normal valve formation and results in the predominant right-non-type BAV in Nos3-/- mice.
Assuntos
Valva Aórtica/anormalidades , Linhagem da Célula , Doenças das Valvas Cardíacas/embriologia , Mutação/genética , Crista Neural/patologia , Óxido Nítrico Sintase Tipo III/genética , Animais , Aorta/metabolismo , Valva Aórtica/embriologia , Doença da Válvula Aórtica Bicúspide , Embrião de Mamíferos/metabolismo , Coxins Endocárdicos/metabolismo , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Crista Neural/metabolismo , Óxido Nítrico Sintase Tipo III/deficiênciaRESUMO
Abnormal endocardial cushion formation is a major cause of congenital heart valve disease, which is a common birth defect with significant morbidity and mortality. Although ß-catenin and BMP2 are two well-known regulators of endocardial cushion formation, their interaction in this process is largely unknown. Here, we report that deletion of ß-catenin in myocardium results in formation of hypoplastic endocardial cushions accompanying a decrease of mesenchymal cell proliferation. Loss of ß-catenin reduced Bmp2 expression in myocardium and SMAD signaling in cushion mesenchyme. Exogenous BMP2 recombinant proteins fully rescued the proliferation defect of mesenchymal cells in cultured heart explants from myocardial ß-catenin knockout embryos. Using a canonical WNT signaling reporter mouse line, we showed that cushion myocardium exhibited high WNT/ß-catenin activities during endocardial cushion growth. Selective disruption of the signaling function of ß-catenin resulted in a cushion growth defect similar to that caused by the complete loss of ß-catenin. Together, these observations demonstrate that myocardial ß-catenin signaling function promotes mesenchymal cell proliferation and endocardial cushion expansion through inducing BMP signaling.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Coxins Endocárdicos/metabolismo , Miocárdio/metabolismo , Organogênese , Transdução de Sinais , beta Catenina/metabolismo , Animais , Proliferação de Células , Coxins Endocárdicos/embriologia , Endocárdio/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Modelos Biológicos , Comunicação Parácrina , Ratos , Via de Sinalização WntRESUMO
Deletions of chromosome 1p36 are associated with a high incidence of congenital heart defects (CHDs). The arginine-glutamic acid dipeptide repeats gene (RERE) is located in a critical region for CHD on chromosome 1p36 and encodes a cardiac-expressed nuclear receptor co-regulator. Mutations affecting RERE cause atrial and ventricular septal defects (VSDs) in humans, and RERE-deficient mice also develop VSDs. During cardiac development, mesenchymal cells destined to form part of the atrioventricular (AV) septum are generated when endocardial cells in the AV canal undergo epithelial-to-mesenchymal transition (EMT) and migrate into the space between the endocardium and the myocardium. These newly generated mesenchymal cells then proliferate to fill the developing AV endocardial cushions. Here, we demonstrate that RERE-deficient mouse embryos have reduced numbers of mesenchymal cells in their AV endocardial cushions owing to decreased levels of EMT and mesenchymal cell proliferation. In the endocardium, RERE colocalizes with GATA4, a transcription factor required for normal levels of EMT and mesenchymal cell proliferation. Using a combination of in vivo and in vitro studies, we show that Rere and Gata4 interact genetically in the development of CHDs, RERE positively regulates transcription from the Gata4 promoter and GATA4 levels are reduced in the AV canals of RERE-deficient embryos. Tissue-specific ablation of Rere in the endocardium leads to hypocellularity of the AV endocardial cushions, defective EMT and VSDs, but does not result in decreased GATA4 expression. We conclude that RERE functions in the AV canal to positively regulate the expression of GATA4, and that deficiency of RERE leads to the development of VSDs through its effects on EMT and mesenchymal cell proliferation. However, the cell-autonomous role of RERE in promoting EMT in the endocardium must be mediated by its effects on the expression of proteins other than GATA4.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Transporte/metabolismo , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica no Desenvolvimento , Comunicação Interventricular/embriologia , Comunicação Interventricular/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas Repressoras/deficiência , Alelos , Animais , Proliferação de Células , Embrião de Mamíferos/metabolismo , Coxins Endocárdicos/embriologia , Coxins Endocárdicos/metabolismo , Coxins Endocárdicos/patologia , Endocárdio/embriologia , Endocárdio/metabolismo , Endocárdio/patologia , Transição Epitelial-Mesenquimal/genética , Fator de Transcrição GATA4/metabolismo , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Proteínas Repressoras/genéticaRESUMO
Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKOEndo) by crossing conditionally-targeted Bmp2flox/flox mice with a Cre-driver line, Nfatc1Cre, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKOEndo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5-11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKOEndo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKOEndo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKOEndo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKOEndo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKOEndo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Linhagem da Célula , Coxins Endocárdicos/citologia , Coxins Endocárdicos/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Proteína Morfogenética Óssea 2/genética , Moléculas de Adesão Celular/metabolismo , Morte Celular , Proliferação de Células , Colágeno/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Coxins Endocárdicos/metabolismo , Deleção de Genes , Comunicação Interventricular/metabolismo , Comunicação Interventricular/patologia , Imageamento Tridimensional , Imuno-Histoquímica , Mesoderma/citologia , Camundongos Knockout , Valva Mitral/patologia , Fatores de Transcrição NFATC/metabolismo , Proteoglicanas/metabolismo , Fatores de Transcrição/metabolismo , Transformação GenéticaRESUMO
Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin-associated matrix protein-1 (hLAMP-1) is a matrix protein expressed within Hensen's node at Hamburger-Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5-6 and enhanced within the left pre-cardiac field at HH stage 7. At HH stages 15-16, hLAMP-1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP-1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8-14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8-14 with antibodies specific to hLAMP-1. Our results showed prominent expression of hLAMP-1-positive particles in the extracellular matrix associated with the pre-cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8-9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP-1-stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP-1 may be involved in the formation of the endocardial tube.
Assuntos
Embrião de Galinha/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Coração/embriologia , Miocárdio/metabolismo , Animais , Embrião de Galinha/embriologia , Criopreservação/veterinária , Coxins Endocárdicos/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica/veterinária , Mesoderma/embriologia , Mesoderma/metabolismoRESUMO
The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Coxins Endocárdicos/embriologia , Coxins Endocárdicos/metabolismo , Redes Reguladoras de Genes , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Animais , Sequência de Bases , Movimento Celular/genética , Cromatina/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Development of the valves and septa of the heart depends on the formation and remodeling of the endocardial cushions in the atrioventricular canal and outflow tract. These cushions are populated by mesenchyme produced from the endocardium by epithelial-mesenchymal transition (EMT). The endocardial cushions are remodeled into the valves at post-EMT stages via differentiation of the mesenchyme and changes in the extracellular matrix (ECM). Transforming growth factor ß (TGFß) signaling has been implicated in both the induction of EMT in the endocardial cushions and the remodeling of the valves at post-EMT stages. We previously identified the RNA binding protein muscleblind-like 1 (MBNL1) as a negative regulator of TGFß signaling and EMT in chicken endocardial cushions ex vivo. Here, we investigate the role of MBNL1 in endocardial cushion development and valvulogenesis in Mbnl1(∆E3/∆E3) mice, which are null for MBNL1 protein. METHODS: Collagen gel invasion assays, histology, immunohistochemistry, real-time RT-PCR, optical coherence tomography, and echocardiography were used to evaluate EMT and TGFß signaling in the endocardial cushions, and morphogenesis, ECM composition, and function of the heart valves. RESULTS: As in chicken, the loss of MBNL1 promotes precocious TGFß signaling and EMT in the endocardial cushions. Surprisingly, this does not lead to the production of excess mesenchyme, but later valve morphogenesis is aberrant. Adult Mbnl1(∆E3/∆E3) mice exhibit valve dysmorphia with elevated TGFß signaling, changes in ECM composition, and increased pigmentation. This is accompanied by a high incidence of regurgitation across both inflow and outflow valves. Mbnl1(∆E3/∆E3) mice also have a high incidence of ostium secundum septal defects accompanied by atrial communication, but do not develop overt cardiomyopathy. CONCLUSIONS: Together, these data indicate that MBNL1 plays a conserved role in negatively regulating TGFß signaling, and is required for normal valve morphogenesis and homeostasis in vivo.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Coxins Endocárdicos/embriologia , Valvas Cardíacas/embriologia , Organogênese , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Animais , Proteínas de Ligação a DNA/genética , Coxins Endocárdicos/metabolismo , Transição Epitelial-Mesenquimal , Coração/embriologia , Valvas Cardíacas/citologia , Valvas Cardíacas/metabolismo , Camundongos , Proteínas de Ligação a RNA/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The Pitx2 gene is involved in the establishment of vertebrate left-right axis with an important role in subsequent heart organogenesis. Mutations in the Pitx2 gene have been associated with Axenfeld-Rieger syndrome, which is characterized by ocular, craniofacial, and umbilical anomalies, as well as cardiac defects. In addition, recent data have unravelled a molecular link between PITX2 loss of function and atrial fibrillation (AF), supporting an important role of Pitx2 not only in development but also in heart homeostasis. Three PITX2 isoforms have been described in mice: PITX2A, PITX2B, and PITX2C. During heart organogenesis, PITX2C seems to play a determinant role in left-right signalling from early somitogenesis onwards. However the participation of the PITX2A and/or PITX2B isoforms during cardiogenesis is controversial. Here we report for the first time that the Pitx2a and Pitx2b isoforms are jointly expressed with the Pitx2c isoform during heart development. Interestingly, in terms of relative quantification of mRNA, the Pitx2b and Pitx2c isoforms display similar expression profiles during cardiogenesis, decreasing with further development but maintaining their expression until adult stages. Moreover, a detailed analysis of PITX2B protein during cardiac development shows that PITX2B is dynamically expressed in the developing ventricular septum and asymmetrically expressed in the tricuspid valve primordia, suggesting a putative role of the PITX2B isoform during ventricular septation as well as in the maturation of the right portion of the atrioventricular canal.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Miocárdio/metabolismo , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Coxins Endocárdicos/embriologia , Coxins Endocárdicos/crescimento & desenvolvimento , Coxins Endocárdicos/metabolismo , Feminino , Coração/embriologia , Coração/crescimento & desenvolvimento , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Camundongos Endogâmicos BALB C , Organogênese/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Proteína Homeobox PITX2RESUMO
Endothelial to mesenchymal transition (EMT) that occurs during cardiac outflow tract (OFT) development is critical for formation of the semilunar valves. Fibulin-1 (Fbln1) is an extracellular matrix protein that is present at several sites of EMT, including the OFT (i.e., E9.5-10.5). The aim of this study was to determine the role of Fbln1 in EMT during the earliest events of OFT development. Examination of proximal OFT cushions in Fbln1 null embryos detected hypercellularity at both E9.5 (93% increase; p = 0.002) and E10.5 (43% increase; p = 0.01) as compared to wild type, suggesting that Fbln1 normally suppresses OFT endocardial cushion EMT. This was supported by studies of proximal OFT cushion explants, which showed that explants from Fbln1 null embryos displayed a 58% increase in cells migrating from the explants as compared to wild type (p = 0.005). We next evaluated the effects of Fbln1 deficiency on the expression of factors that regulate proximal OFT EMT. At E9.5, Fbln1 null proximal OFT endocardium and EMT-derived mesenchyme showed increased TGFß2 (58% increase; p = 0.01) and increased Snail1-positive nuclei (27% increase; p = 0.0003). Histological examination of OFT cushions in Fbln1 null embryos (E9.5) also detected cells present in the cushion that were determined to be erythrocytes based on round morphology, autofluorescence, and positive staining for hemoglobin. Erythrocytes were also detected in Fbln1 null OFT cushions at E10.5. Together, the findings indicate that Fbln1 normally suppresses proximal OFT EMT preventing proximal cushion hypercellularity and blood cell accumulation.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Coxins Endocárdicos/metabolismo , Endocárdio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Miocárdio/metabolismo , Animais , Apoptose , Proteínas de Ligação ao Cálcio/genética , Proliferação de Células , Coxins Endocárdicos/citologia , Endocárdio/citologia , Proteínas da Matriz Extracelular/genética , Camundongos , Camundongos Knockout , Miocárdio/citologiaRESUMO
BACKGROUND: The basic helix-loop-helix transcription factor Twist1 has well-documented roles in progenitor populations of the developing embryo, including endocardial cushions (ECC) and limb buds, and also in cancer. Whether Twist1 regulates the same transcriptional targets in different tissue types is largely unknown. RESULTS: The tissue-specificity of Twist1 genomic occupancy was examined in mouse ECCs, limb buds, and peripheral nerve sheath tumor (PNST) cells using chromatin immunoprecipitation followed by sequencing (Chip-seq) analysis. Consistent with known Twist1 functions during development and in cancer cells, Twist1-DNA binding regions associated with genes related to cell migration and adhesion were detected in all three tissues. However, the vast majority of Twist1 binding regions were specific to individual tissue types. Thus, while Twist1 has similar functions in ECCs, limb buds, and PNST cells, the specific genomic sequences occupied by Twist1 were different depending on cellular context. Subgroups of shared genes, also predominantly related to cell adhesion and migration, were identified in pairwise comparisons of ECC, limb buds and PNST cells. Twist1 genomic occupancy was detected for six binding regions in all tissue types, and Twist1-binding sequences associated with Chst11, Litaf, Ror2, and Spata5 also bound the potential Twist1 cofactor RREB1. Pathway analysis of the genes associated with Twist1 binding suggests that Twist1 may regulate genes associated with the Wnt signaling pathway in ECCs and limb buds. CONCLUSIONS: Together, these data indicate that Twist1 interacts with genes that regulate adhesion and migration in different tissues, potentially through distinct sets of target genes. In addition, there is a small subset of genes occupied by Twist1 in all three tissues that may represent a core group of Twist1 target genes in multiple cell types.
Assuntos
Coxins Endocárdicos/metabolismo , Genoma , Botões de Extremidades/metabolismo , Neoplasias de Bainha Neural/genética , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genética , Animais , Sítios de Ligação , Células Cultivadas , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário/genética , Coxins Endocárdicos/citologia , Expressão Gênica , Botões de Extremidades/citologia , Camundongos , Neoplasias de Bainha Neural/patologia , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Análise de Sequência de DNA , Proteína 1 Relacionada a Twist/metabolismoRESUMO
AIMS: Valvular heart disease is responsible for considerable morbidity and mortality. Cardiac valves develop as the heart contracts, and they function throughout the lifetime of the organism to prevent retrograde blood flow. Their precise morphogenesis is crucial for cardiac function. Zebrafish is an ideal model to investigate cardiac valve development as it allows these studies to be carried out in vivo through non-invasive imaging. Accumulating evidence suggests a role for contractility and intracardiac flow dynamics in cardiac valve development. However, these two factors have proved difficult to uncouple, especially since altering myocardial function affects the intracardiac flow pattern. METHODS AND RESULTS: Here, we describe novel zebrafish models of developmental valve defects. We identified two mutant alleles of myosin heavy chain 6 that can be raised to adulthood despite having only one functional chamber-the ventricle. The adult mutant ventricle undergoes remodelling, and the atrioventricular (AV) valves fail to form four cuspids. In parallel, we characterized a novel mutant allele of southpaw, a nodal-related gene involved in the establishment of left-right asymmetry, which exhibits randomized heart and endoderm positioning. We first observed that in southpaw mutants the relative position of the two cardiac chambers is altered, affecting the geometry of the heart, while myocardial function appears unaffected. Mutant hearts that loop properly or exhibit situs inversus develop normally, whereas midline, unlooped hearts exhibit defects in their transvalvular flow pattern during AV valve development as well as defects in valve morphogenesis. CONCLUSION: Our data indicate that intracardiac flow dynamics regulate valve morphogenesis independently of myocardial contractility.
Assuntos
Circulação Coronária , Comunicação Atrioventricular/embriologia , Coxins Endocárdicos/embriologia , Valvas Cardíacas/anormalidades , Hemodinâmica , Mecanotransdução Celular , Animais , Animais Geneticamente Modificados , Função Atrial , Comunicação Atrioventricular/genética , Comunicação Atrioventricular/metabolismo , Comunicação Atrioventricular/fisiopatologia , Coxins Endocárdicos/metabolismo , Coxins Endocárdicos/fisiopatologia , Genótipo , Valvas Cardíacas/metabolismo , Valvas Cardíacas/fisiopatologia , Morfogênese , Mutação , Contração Miocárdica , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The initial step of atrioventricular (AV) valve development involves the deposition of extracellular matrix (ECM) components of the endocardial cushion and the endocardial-mesenchymal transition. While the appropriately regulated expression of the major ECM components, Versican and Hyaluronan, that form the endocardial cushion is important for heart valve development, the underlying mechanism that regulates ECM gene expression remains unclear. We found that zebrafish crip2 expression is restricted to a subset of cells in the AV canal (AVC) endocardium at 55 hours post-fertilization (hpf). Knockdown of crip2 induced a heart-looping defect in zebrafish embryos, although the development of cardiac chambers appeared to be normal. In the AVC of Crip2-deficient embryos, the expression of both versican a and hyaluronan synthase 2 (has2) was highly upregulated, but the expression of bone morphogenetic protein 4 (bmp4) and T-box 2b (tbx2b) in the myocardium and of notch1b in the endocardium in the AVC did not change. Taken together, these results indicate that crip2 plays an important role in AV valve development by downregulating the expression of ECM components in the endocardial cushion.
Assuntos
Coxins Endocárdicos/metabolismo , Endocárdio/embriologia , Proteínas da Matriz Extracelular/genética , Valvas Cardíacas/embriologia , Proteínas com Domínio LIM/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Regulação para Baixo , Coxins Endocárdicos/embriologia , Endocárdio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
BACKGROUND: Valvuloseptal defects are the most common congenital heart defects. Notch signaling-induced endothelial-to-mesenchymal transition (EMT) in the atrioventricular canal (AVC) cushions at murine embryonic day (E)9.5 is a required step during early valve development. Insights to the transcriptional network that is activated in endocardial cells (EC) during EMT and how these pathways direct valve maturation are lacking. RESULTS: We show that at E11.5, AVC-EC retain the ability to undergo Notch-dependent EMT when explanted on collagen. EC-Notch inhibition at E10.5 blocks expression of known mesenchymal genes in E11.5 AVC-EC. To understand the genetic network and AVC development downstream of Notch signaling beyond E9.5, we constructed Tag-Seq libraries corresponding to different cell types of the E11.5 AVC and atrium in wild-type mice and in EC-Notch inhibited mice. We identified 1,400 potential Notch targets in the AVC-EC, of which 124 are transcription factors (TF). From the 124 TFs, we constructed a transcriptional hierarchy and identify 10 upstream TFs within the network. CONCLUSIONS: We validated 4 of the upstream TFs as Notch targets that are enriched in AVC-EC. Functionally, we show these 4 TFs regulate EMT in AVC explant assays. These novel signaling pathways downstream of Notch are potentially relevant to valve development.