RESUMO
The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart, we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially derived fibroblasts eventually largely replace the endocardially derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development.
Assuntos
Fibroblastos/citologia , Valvas Cardíacas/embriologia , Coração/embriologia , Pericárdio/citologia , Animais , Desenvolvimento Embrionário , Valvas Cardíacas/citologia , Ventrículos do Coração/citologia , Ventrículos do Coração/embriologia , Camundongos , OrganogêneseRESUMO
RATIONALE: The proepicardium is a transient structure comprising epicardial progenitor cells located at the posterior limit of the embryonic cardiac inflow. A network of signals regulates proepicardial cell fate and defines myocardial and nonmyocardial domains at the venous pole of the heart. During cardiac development, epicardial-derived cells also contribute to coronary vessel morphogenesis. OBJECTIVE: To study Notch function during proepicardium development and coronary vessel formation in the mouse. METHODS AND RESULTS: Using in situ hybridization, RT-PCR, and immunohistochemistry, we find that Notch pathway elements are differentially activated throughout the proepicardial-epicardial-coronary transition. Analysis of RBPJk-targeted embryos indicates that Notch ablation causes ectopic procardiogenic signaling in the proepicardium that in turn promotes myocardial differentiation in adjacent mesodermal progenitors, resulting in a premature muscularization of the sinus venosus horns. Epicardium-specific Notch1 ablation using a Wt1-Cre driver line disrupts coronary artery differentiation, reduces myocardium wall thickness and myocyte proliferation, and reduces Raldh2 expression. Ectopic Notch1 activation disrupts epicardium development and causes thinning of ventricular walls. CONCLUSIONS: Epicardial Notch modulates cell differentiation in the proepicardium and adjacent pericardial mesoderm. Notch1 is later required for arterial endothelium commitment and differentiation and for vessel wall maturation during coronary vessel development and myocardium growth.
Assuntos
Circulação Sanguínea/fisiologia , Vasos Coronários/embriologia , Morfogênese/fisiologia , Pericárdio/embriologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Vasos Coronários/citologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Modelos Animais , Mutação , Pericárdio/citologia , Receptor Notch1/genética , Receptor Notch1/fisiologia , Receptores Notch/genéticaRESUMO
BACKGROUND: The atrioventricular (AV) node is essential for the sequential excitation and optimized contraction of the adult multichambered heart; however, relatively little is known about its formation from the embryonic AV canal. A recent study demonstrated that signaling by Alk3, the type 1a receptor for bone morphogenetic proteins, in the myocardium of the AV canal was required for the development of both the AV valves and annulus fibrosus. To test the hypothesis that bone morphogenetic protein signaling also plays a role in AV node formation, we investigated conduction system function and AV node morphology in adult mice with conditional deletion of Alk3 in the AV canal. METHODS AND RESULTS: High-resolution optical mapping with correlative histological analysis of 28 mutant hearts revealed 4 basic phenotypic classes based on electrical activation patterns and volume-conducted ECGs. The frequency of AV node conduction and morphological abnormalities increased from no detectable anomalies (class I) to severe defects (class IV), which included the presence of bypass tracts, abnormal ventricular activation patterns, fibrosis of the AV node, and twin AV nodes. CONCLUSIONS: The present findings demonstrate that bone morphogenetic protein signaling is required in the myocardium of the AV canal for proper AV junction development, including the AV node.
Assuntos
Nó Atrioventricular/fisiopatologia , Mapeamento Potencial de Superfície Corporal , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Sistema de Condução Cardíaco/fisiopatologia , Animais , Nó Atrioventricular/crescimento & desenvolvimento , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/deficiência , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/fisiologia , Genótipo , Bloqueio Cardíaco , Camundongos , Camundongos Mutantes , Miocárdio/patologiaRESUMO
Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein's anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus.
Assuntos
Valvas Cardíacas/embriologia , Coração/embriologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA6 , Valvas Cardíacas/anormalidades , Integrases/genética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/fisiologia , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
During folding of the embryo, lateroanterior visceral mesoderm forms the embryonic tubular heart at the midline, just ventral to the foregut. In mice, this nascent tube contains the future left ventricle and atrioventricular canal. Mesenchymal cells subsequently recruited to the cardiac lineage at the intake and the outflow of the tube will form the atria and the right ventricle and outflow tract, respectively. Shortly after its emergence, the embryonic heart tube starts to loop, and the first signs of left ventricular chamber differentiation become visible on the outer curvature of the middle portion of the tube. Subsequently, the right ventricle differentiates cranially, and the atria caudally, while the inflow tract, atrioventricular canal, inner curvatures, and outflow tract form recognizable components flanking the chambers. The latter, nonchamber regions in turn provide signals for the formation of the cushion mesenchyme, are involved in remodeling of the heart, and form the nodes of the conduction system. This review discusses how the patterning of the heart tube relates to the localized differentiation of atrial and ventricular chambers, why some parts of the heart do not form chambers, and how this relates to the formation of the conduction system.
Assuntos
Expressão Gênica , Coração/embriologia , Camundongos/embriologia , Morfogênese/genética , Animais , Átrios do Coração/embriologia , Sistema de Condução Cardíaco/embriologia , Ventrículos do Coração/embriologia , Humanos , Células-Tronco Mesenquimais , Morfogênese/fisiologia , Miócitos CardíacosRESUMO
We identified a GATA6 gene enhancer that selectively marks the developing atrioventricular conduction system (AVCS) in transgenic mice. This enhancer reads anterior/posterior and medial/lateral positional information early in the cardiogenic programme and remains active in progressively more restricted subsets of heart cells leading up to AVCS formation. Additional experiments will be required to determine if the potential to be recruited into the AVCS is similarly restricted to a subset of myocardial cells early in the cardiogenic programme or if this enhancer can also be activated de novo in cells that initially reside outside this field. We are using several strategies to identify factors that regulate this and other AVCS enhancers and hence govern AVCS function. We are also using this enhancer to make transgenic mice that express Cre, or an inducible form of Cre, to track lineages and to delete floxed genes in the developing or mature AVCS. This Cre/lox approach provides a means to deconstruct complex congenital heart phenotypes that involve the conduction system and to test whether genes are required to form the AVCS or to maintain AVCS function. Lastly, we are exploring strategies to isolate and analyse AVCS cells from normal and affected hearts.
Assuntos
Regulação da Expressão Gênica , Sistema de Condução Cardíaco/fisiologia , Coração/embriologia , Transcrição Gênica , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Fator de Transcrição GATA6 , Coração/anatomia & histologia , Sistema de Condução Cardíaco/embriologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Morfogênese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The mouse is the animal of choice for the study of molecular mechanisms involved in the regulation of cardiovascular morphogenesis and function. Recently, a series of genetically engineered mouse models have been reported (e.g. cGATA6/lacZ, MinK/lacZ knock-in/knock-out, engrailed2/lacZ, Cardiac troponin I/lacZ) that provide new and exciting information on the development of the atrioventricular conduction system (AVCS). On the basis of these and ongoing studies, concepts for the formation of the AVCS are continuously being adjusted. A proper understanding of the normal developmental mechanisms underlying the cardiac remodelling leading to the formation of the AVCS is imperative for the interpretation of cardiac abnormalities, including conduction disturbances, as observed in some genetically perturbed (knockout) mice. In this paper information on murine AVCS development will be integrated with published and unpublished results from studies in other vertebrates, including human and rabbit. We will illustrate that although many pieces of the puzzle still remain to be gathered, the outline of a very complex and critical event in cardiac morphogenesis is slowly emerging. Specifically, we will re-evaluate the concept of the 'primary ring' in the context of the new insights in the development of the AV junction as provided by the respective mouse models described above.
Assuntos
Sistema de Condução Cardíaco/embriologia , Sistema de Condução Cardíaco/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Animais , Coração/anatomia & histologia , Coração/fisiologia , Sistema de Condução Cardíaco/fisiologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Morfogênese , Miocárdio/citologia , Miocárdio/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransgenesRESUMO
The cGATA-6 gene is flanked by an enhancer that selectively marks the atrioventricular conduction system (AVCS) in transgenic mice. This enhancer reads anterior/posterior and medial/lateral positional information very early in the cardiogenic program and remains active in progressively more restricted regions of primary myocardium leading up to the emergence of a histologically distinct AVCS. We undertook to parse this enhancer to resolve how the respective AVCS-specific transcription program is regulated at the molecular level. We determined that this AVCS enhancer includes a 102 bp module that is sufficient to restrict expression to primary nonchamber myocardium. This offers a novel tool to analyze the early molecular delineation of primary and chamber myocardium, which subsequently give rise to components of the central and peripheral conduction system, respectively. Furthermore, we show that this 102 bp module in turn contains a nested 47 bp core module that has the potential to direct expression specifically to the AVCS domain of primary myocardium, albeit with low efficiency. Accordingly, we show that a GATA site and a GC-rich site in the 102 bp region bolster the activity of the nested 47 bp AVCS core region even within the context of the parental 1,478 bp enhancer. These are the first functional elements to be reported for a cardiac conduction system-specific control region.
Assuntos
Nó Atrioventricular/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Elementos Facilitadores Genéticos , Genes Reguladores , Miocárdio/metabolismo , Fatores de Transcrição/fisiologia , Animais , Nó Atrioventricular/embriologia , Sequência de Bases , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fator de Transcrição GATA6 , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Miocárdio/citologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
AIMS: During development, the heart tube grows by differentiation of Isl1(+)/Nkx2-5(+) progenitors to the arterial and venous pole and dorsal mesocardium. However, after the establishment of the heart tube, Tbx18(+) progenitors were proposed to form the Tbx18(+)/Nkx2-5(-) sinus venosus and proepicardium. To elucidate the relationship between these contributions, we investigated the origin of the Tbx18(+) sinus venosus progenitor population in the cardiogenic mesoderm and its spatial and temporal relation to the second heart field during murine heart development. METHODS AND RESULTS: Explant culture revealed that the Tbx18(+) cell population has the potential to form Nkx2-5(-) sinus venosus myocardium. Three-dimensional reconstruction of expression patterns showed that during heart tube elongation, the Tbx18(+) progenitors remained spatially and temporally separate from the Isl1(+) second heart field, only overlapping with the Isl1(+) domain at the right lateral side of the inflow tract, where the sinus node developed. Consistently, genetic lineage analysis revealed that the Tbx18(+) descendants formed the sinus venosus myocardium, but did not contribute to the pulmonary vein myocardium that developed in the Isl1(+) second heart field. By means of DiI labelling and expression analysis, the origin of the sinus venosus progenitor population was traced to the lateral rim of splanchnic mesoderm that down-regulated Nkx2-5 expression approximately 2 days before its differentiation into sinus venosus myocardium. CONCLUSION: Our data indicate that the cardiogenic mesoderm contains an additional progenitor subpopulation that contributes to the sinus venosus myocardium. After patterning of the cardiogenic mesoderm, this progenitor population remains spatially separated and genetically distinctive from the second heart field subpopulation.
Assuntos
Coração/embriologia , Mesoderma/metabolismo , Miócitos Cardíacos/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Idade Gestacional , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM , Óperon Lac/genética , Mesoderma/citologia , Camundongos , Camundongos Transgênicos , Morfogênese , Proteínas/genética , Veias Pulmonares/embriologia , Veias Pulmonares/metabolismo , RNA não Traduzido , Proteínas Recombinantes de Fusão/metabolismo , Nó Sinoatrial/embriologia , Nó Sinoatrial/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Técnicas de Cultura de Tecidos , Fatores de Transcrição/metabolismoRESUMO
Periostin is a fasciclin-containing adhesive glycoprotein that facilitates the migration and differentiation of cells that have undergone epithelial-mesenchymal transformation during embryogenesis and in pathological conditions. Despite the importance of post-transformational differentiation as a general developmental mechanism, little is known how periostin's embryonic expression is regulated. To help resolve this deficiency, a 3.9-kb periostin proximal promoter was isolated and shown to drive tissue-specific expression in the neural crest-derived Schwann cell lineage and in a subpopulation of periostin-expressing cells in the cardiac outflow tract endocardial cushions. In order to identify the enhancer and associated DNA binding factor(s) responsible, in vitro promoter dissection was undertaken in a Schwannoma line. Ultimately a 304-bp(peri) enhancer was identified and shown to be capable of recapitulating 3.9 kb(peri-lacZ)in vivo spatiotemporal patterns. Further mutational and EMSA analysis helped identify a minimal 37-bp region that is bound by the YY1 transcription factor. The 37-bp enhancer was subsequently shown to be essential for in vivo 3.9 kb(peri-lacZ) promoter activity. Taken together, these studies identify an evolutionary-conserved YY1-binding 37-bp region within a 304-bp periostin core enhancer that is capable of regulating simultaneous novel tissue-specific periostin expression in the cardiac outflow-tract cushion mesenchyme and Schwann cell lineages.
Assuntos
Moléculas de Adesão Celular/genética , Endocárdio/embriologia , Endocárdio/metabolismo , Elementos Facilitadores Genéticos , Células de Schwann/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Sequência Conservada , Sondas de DNA/genética , Endocárdio/citologia , Coração Fetal/citologia , Coração Fetal/embriologia , Coração Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Óperon Lac , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Células de Schwann/citologia , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Fator de Transcrição YY1/metabolismoRESUMO
Since rates of cardiomyocyte generation in the embryo are much higher than within the adult, we explored whether the embryonic heart would serve as useful experimental system for examining the myocardial potential of adult stem cells. Previously, we reported that the long-term culturing of adult mouse bone marrow produced a cell population that was both highly enriched for macrophages and cardiac competent. In this study, the myocardial potential of this cell population was analyzed in greater detail using the embryonic chick heart as recipient tissue. Experiments involving the co-incubation of labeled bone marrow cells with embryonic heart tissue showed that bone marrow (BM) cells incorporated into the myocardium and immunostained for myocyte proteins. Reverse transcription-polymerase chain reaction analysis demonstrated that the heart tissue induced bone marrow cells to express the differentiated cardiomyocyte marker alpha-cardiac myosin heavy chain. The cardiomyocyte conversion of the bone marrow cells was verified by harvesting donor cells from mice that were genetically labeled with a myocardial-specific beta-galactosidase reporter. Embryonic hearts exposed to the transgenic bone marrow in culture exhibited significant numbers of beta-galactosidase-positive cells, indicating the presence of bone marrow-derived cells that had converted to a myocardial phenotype. Furthermore, when transgenic mouse BM cells were injected into living chick embryos, donor cells incorporated into the developing heart and exhibited a myocardial phenotype. Immunofluorescence analysis demonstrated that donor BM cells exhibiting myocyte markers contained only nuclei from mouse cells, indicating that differentiation and not cell fusion was the predominant mechanism for the acquisition of a myocyte phenotype. These data confirm that adult mouse bone marrow contain cells with the ability to form cardiomyocytes. In addition, the predominance of the macrophage phenotype within the donor bone marrow cell population suggests that transdifferentiation of immune response cells may play a role in cellular regeneration in the adult.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Proliferação de Células , Coração/embriologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Animais , Transplante de Medula Óssea , Embrião de Galinha , Meios de Cultivo Condicionados , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Macrófagos/citologia , Camundongos , Camundongos Transgênicos , Transplante HeterólogoRESUMO
Apoptosis occurs at high frequency in the myocardium of the developing avian cardiac outflow tract (OFT). Up- or down-regulating apoptosis results in defects resembling human conotruncal heart anomalies. This finding suggested that regulated levels of apoptosis are critical for normal morphogenesis of the four-chambered heart. Recent evidence supports an important role for hypoxia of the OFT myocardium in regulating cell death and vasculogenesis. The purpose of this study was to determine whether apoptosis in the outflow tract myocardium occurs in the mouse heart during developmental stages comparable to the avian heart and to determine whether differential hypoxia is also present at this site in the murine heart. Apoptosis was detected using a fluorescent vital dye, Lysotracker Red (LTR), in the OFT myocardium of the mouse starting at embryonic day (E) 12.5, peaking at E13.5-14.5, and declining thereafter to low or background levels by E18.5. In addition, high levels of apoptosis were detected in other cardiac regions, including the apices of the ventricles and along the interventricular sulcus. Apoptosis in the myocardium was detected by double-labeling with LTR and cardiomyocyte markers. Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) and immunostaining for cleaved Caspase-3 were used to confirm the LTR results. At the peak of OFT apoptosis in the mouse, the OFT myocardium was relatively hypoxic, as indicated by specific and intense EF5 staining and HIF1alpha nuclear localization, and was surrounded by the developing vasculature as in the chicken embryo. These findings suggest that cardiomyocyte apoptosis is an evolutionarily conserved mechanism for normal morphogenesis of the outflow tract myocardium in avian and mammalian species.
Assuntos
Apoptose , Coração Fetal/citologia , Animais , Caspase 3 , Caspases/metabolismo , Embrião de Galinha , Feminino , Coração Fetal/metabolismo , Idade Gestacional , Coração/embriologia , Humanos , Hipóxia/metabolismo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Especificidade da Espécie , Coloração e RotulagemRESUMO
GATA factors regulate critical events in hematopoietic lineages (GATA-1/2/3), the heart and gut (GATA-4/5/6) and various other tissues. Transgenic approaches have revealed that GATA genes are regulated in a modular fashion by sets of enhancers that govern distinct temporal and/or spatial facets of the overall expression patterns. Efforts are underway to resolve how these GATA gene enhancers are themselves regulated in order to elucidate the genetic and molecular hierarchies that govern GATA expression in particular developmental contexts. These enhancers also afford a raft of tools that can be used to selectively perturb and probe various developmental events in transgenic animals.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/genética , Vertebrados/crescimento & desenvolvimento , Animais , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Fatores de Ligação de DNA Eritroide Específicos , Peixes/crescimento & desenvolvimento , Fator de Transcrição GATA2 , Fator de Transcrição GATA3 , Fator de Transcrição GATA4 , Fator de Transcrição GATA5 , Fator de Transcrição GATA6 , Coração/crescimento & desenvolvimento , Intestinos/crescimento & desenvolvimento , Transativadores/genética , Fatores de Transcrição/metabolismo , Vertebrados/metabolismoRESUMO
Most internal organs are situated in a coelomic cavity and are covered by a mesothelium. During heart development, epicardial cells (a mesothelium) move to and over the heart, undergo epithelial-mesenchymal transition (EMT), and subsequently differentiate into endothelial and vascular smooth muscle cells. This is thought to be a unique process in blood vessel formation. Still, structural and developmental similarities between the heart and gut led us to test the hypothesis that a conserved or related mechanism may regulate blood vessel development to the gut, which, similar to the heart, is housed in a coelomic cavity. By using a combination of molecular genetics, vital dye fate mapping, organ culture and immunohistochemistry, we demonstrate that the serosal mesothelium is the major source of vasculogenic cells in developing mouse gut. Our studies show that the gut is initially devoid of a mesothelium but that serosal mesothelial cells expressing the Wilm's tumor protein (Wt1) move to and over the gut. Subsequently, a subset of these cells undergoes EMT and migrates throughout the gut. Using Wt1-Cre genetic lineage marking of serosal cells and their progeny, we demonstrate that these cells differentiate to smooth muscle of all major blood vessels in the mesenteries and gut. Our data reveal a conserved mechanism in blood vessel formation to coelomic organs, and have major implications for our understanding of vertebrate organogenesis and vascular deficiencies of the gut.
Assuntos
Indução Embrionária , Epitélio/fisiologia , Intestinos/irrigação sanguínea , Miócitos de Músculo Liso/citologia , Membrana Serosa/citologia , Animais , Biomarcadores/análise , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/crescimento & desenvolvimento , Desenvolvimento Embrionário , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Intestinos/embriologia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Organogênese , Proteínas WT1/análiseRESUMO
Mouse embryos lacking the retinoic acid receptor RXRalpha properly undergo the early steps of heart development, but then fail to initiate a proliferative expansion of cardiomyocytes that normally results in the formation of the compact zone of the ventricular chamber wall. RXRalpha(-/-) embryos have a hypoplastic ventricular chamber and die in midgestation from cardiac insufficiency. In this study, we have investigated the underlying mechanistic basis of this phenotype. We find that interference with retinoic acid receptor function in the epicardium of transgenic embryos recapitulates the hypoplastic phenotype of RXRalpha deficient embryos. We further show that wild type primary epicardial cells, and an established epicardial cell line (EMC cells), secrete trophic protein factors into conditioned media that stimulate thymidine incorporation in primary fetal cardiomyocytes, and thymidine incorporation, cell cycle progression, and induction of cyclin D1 and E activity in NIH3T3 cells. In contrast, primary epicardial cells derived from RXRalpha(-/-) embryos and an EMC subline constitutively expressing a dominant negative receptor construct both fail to secrete activity into conditioned media. The production of trophic factors is induced by retinoic acid treatment and is inhibited by a retinoid receptor antagonist. Fetal atrial and ventricular myocytes both respond to epicardial-derived trophic signaling, although postnatal cardiomyocytes are nonresponsive. We therefore propose that the fetal epicardium, in response to retinoic acid and in a manner requiring the activity of RXRalpha, secretes trophic factors which drive fetal cardiomyocyte proliferation and promote ventricular chamber morphogenesis.