Pulmonary Endoderm, Second Heart Field And The Morphogenesis Of Distal Outflow Tract In Mouse Embryonic Heart

The establishment of efficient systemic and pulmonary circulation relies on the normal morphogenesis and septation of the heart chambers. It has been demonstrated that the cardiac progenitor cells derived from the second heart field (SHF) migrate through dorsal mesocardium to form outflow tract (OFT), right ventricle, part of the atria, and the atrial septum, including the dorsal mesenchymal protrusion, and to populate the aorta and pulmonary trunk. The data from the transgenic mice and gene knockout mouse embryos have revealed that conditional deletion of transcription factors or signal proteins from the foregut endoderm result in the malformation of OFT septation, suggesting that foregut endoderm plays roles in regulation of normal development of OFT and SHF. Major insights on the relationship of foregut endoderm development with the heart development have been gained from the studies of a number of vertebrate and invertebrate models, including mouse, chick, amphibians, et al, via the culture in vitro or the deletion of endoderm genes in vivo. However, in wide-type embryonic mouse, the morphological relationship in the development of foregut endoderm and embryonic heart and the mechanism underlying the interaction between them are seldom described. Sonic hedgehog (SHH) signaling pathway coordinates multiple aspects of cardiovascular growth and development. As a secreted ligand, SHH can only function properly by binding to its receptors, including patchedl (Ptcl), patched2(Ptc2) and smoothened (Smo). Foregut endoderm has been regarded as one of the sources of SHH ligand and conditional abrogation of SHH signaling in foregut endoderm during the cardiac progenitor cell specification leads to the OFT malformation, including persistent truncus arterious, transposition of great arteries, etc. and the atrial and atrioventricular septal defect as well. However, the morphological relationship of expression of SHH signaling pathway members in developing foregut endoderm with development of surrounding SHF and OFT is seldom described. In order to get insight into the mechanism underlying the interaction between foregut endoderm and surrounding Islet-1(Isl-1) positive second heart field mesenchyme, we investigated the morphological relationship of developing trachea groove with SHF during normal OFT development of the mouse embryonic heart, and the expression patterns of Ptc1, Ptc2and Smo, critical receptors in SHH signaling pathway. The results demonstrated that the development of a subset of Isl-1positive field ventral to the foregut is closely coupled with pulmonary endoderm or tracheal groove, and the Isl-1positive cells surrounding the trachea groove is distributed in a special pattern. Because several lines of evidence have demonstrated that the Isl-1positive mesenchyme beneath the foregut floor takes part in the morphogenesis of the heart and therefore is considered to be a subset of the second heart field, we dubbed the subset of Isl-1positive field ventral to the foregut as pulmonary endoderm-associated Isl-1field (PE-Isl-1F). During PE-Isl-1F development, the SHH receptors Ptcl and Ptc2are exclusively expressed in the developing pulmonary endoderm. Close proximity of PE-Isl-1F to the developing trachea groove might be required for and facilitate the specification of the progenitor cells in PE-Isl-1F into cardiac lineage. In addition to taking part in the formation of the lateral walls of the intrapericardial aorta and pulmonary trunk, Isl-1positive cells of PE-Isl-1F surrounding the trachea groove protrude into the aortic sac, together with the distal end of the outflow trunk cushions, to form the transient aortic-pulmonary septum (AP septum). Our findings provide morphological evidence for interaction of endoderm with SHF and lend support to the hypothesis that the pulmonary endoderm patterns the morphogenesis of cardiac structural components required for establishing efficient systemic and pulmonary circulation.Part I Coupling of development of pulmonary endoderm with pulmonary endoderm-associated second heart field in mouse embryoBackground and objective:Second heat field (SHF), characterized by islet-1(Isl-1) expression, contribute the cardiac progenitor cells to outflow tract (OFT) and right ventricle for the arterial pole forming after the initial heart tube formation. Conditional deletion of transcription factors or signal proteins from the foregut endoderm result in the malformation of OFT, suggesting that foregut endoderm plays roles in regulation of normal development of OFT and SHF. In order to get insight into the mechanism underlying those OFT malformations, we investigated the morphological relationship of developing trachea groove from foregut endoderm with surrounding Isl-1positive mesenchyme of SHF during OFT development in the normal mouse embryonic heart.Methods:By immunohistochemistry and double immunofluorescence methods, serial sections of mouse embryos from ED9to ED13were stained with a series of marker antibodies for specifically highlighting SHF (Isl-1), endoderm(Foxa2), basement membrane (Laminin), epithelial polarity (E-cadherin), myocardium (MHC) and smooth muscle (α-SMA) respectively.Results:At ED9, locally thickened Isl-1positive endoderm in the ventral foregut wall predicted the initiation of pulmonary endoderm differentiation. As soon as the formation of the lanryngo-tracheal groove is initiated at ED9.5, Isl-1positive cells begin to appear in the matrix surrounding the endoderm of the lanryngo-tracheal groove, which we dubbed pulmonary endoderm associated second heart field (PE-Isl-1F). With the elongation of the lanryngo-tracheal groove in the direction of the dorsal wall of the aortic sac from ED10to ED12, the number of Isl-1positive cells in PE-Isl-1F is increased and the distribution of the Isl-1positive cells is closely associated with the pulmonary endoderm in a distinct cone-shaped pattern with its broad base in close apposition to the floor of the foregut and ventral end protruding into the cavity of the aortic sac. During the development of lanryngo-tracheal groove, a solid endoderm cord could always be observed at the ventral end of the lanryngo-tracheal groove from ED10.5to ED12. Pulmonary endoderm of lanryngo-tracheal groove and its solid cord was located in the center of the Isl-1positive PE-Isl-1F. In lanryngo-tracheal groove and PE-Isl-1F development, Laminin positive basement membrane underlying the endoderm appeared as an interrupted or blurred fashion in some regions, accompanied by loss of Foxa2expression and apico-basal polarity in some pulmonary endoderm cells, especially at ED11. At ED13, separation of foregut at the level of outflow tract led to the formation of the trachea, endoderm cord could no longer be found, and expression intensity of Isl-1in PE-Isl-1F was reduced.Conclusion:The differentiation and development of pulmonary endoderm are closely associated with Isl-1positive mesenchyme aggregation of PE-Isl-1F, and the pulmonary endoderm patterns the PE-Isl-1F formation, between which EMT plays a role. Part Ⅱ Exclusive expression of SHH receptors in pulmonary endoderm and pulmonary endoderm-associated second heart fieldBackground and objective:Foregut endoderm has been regarded as one of the sources of SHH ligand and gene ablation or tissue specificity knockout of SHH or Smo in endoderm resulted in various developmental defects of OFT in mouse embryos, but the causes and mechanism of those OFT malformations have not been thoroughly elucidated. It has been demonstrated that the cardiac progenitor cells derived from SHF migrate and form OFT and right ventricle after the primitive cardiac tube formation. In order to demonstrate the morphological relationships of SHH, foregut endoderm and SHF in embryonic heart development, expression patterns of SHH receptors, Ptcl, Ptc2and Smo, were examined in the developing pulmonary endoderm and PE-Isl-1F.Methods:Serial sections of mouse embryos from ED9to ED13were stained by double immunofluorescence of Ptcl/Isl-1, Ptc2/Isl-1and Smo/Isl-1.Results:Ptcl and Ptc2began to express exclusively in pulmonary endoderm at ED10. With the ventral growth of pulmonary endoderm to form laryngo-tracheal groove, strong Ptcl and Ptc2expression were mainly confined to the pulmonary endoderm until ED12. Expression of Smo was broad in embryos from ED9to ED13, and mesenchymal cells of PE-Isl-1F surrounding the pulmonary endoderm co-expressed Isl-1and Smo.Conclusion:SHH signal pathway is involved in the pulmonary endoderm development and the formation of laryngo-tracheal groove and its solid cord by an autocrine or paracrine manner, and also associated with the formation of PE-Isl-1F surrounding the developing pulmonary endoderm. Part Ⅲ Pulmonary endoderm-associated second heart field and the morphogenesis of the distal outflow tract in mouse embryonic heartBackground and objective:The morphogenesis and septation of the distal outflow tract remains a controversial topic so far. Second heart field (SHF) is reported to contribute the myocardium and the smooth muscle required for OFT development. In this part, the morphological relationship between pulmonary endoderm-associated second heart field (PE-Isl-1F) and the morphogenesis of the intrapericardial trunks during the normal mouse embryos heart development was detected.Methods:By immunohistochemistry and double immunofluorescence methods, serial sections of mouse embryos from ED9to ED13were stained with a series of marker antibodies for specifically highlighting SHF (Isl-1), myocardium (MHC) and smooth muscle (α-SMA) respectively.Results:From ED10onward, development of PE-Isl-1F around the laryngo-tracheal groove promoted the continuous extension of its ventral end into the aortic sac to form obvious protrusion. By11.5, though the aortic sac and OFT were still not separated, the extension of PE-Isl-1F to the cranial and caudal myocardial wall of the outflow tract could be detected as Isl-1positive boluses at the level of4th and6th branchial arch arteries, respectively, which are the future lateral walls of intrapericardial aorta and pulmonary trunk. At ED12, expression of Isl-1in protrusion from PE-Isl-1F gradually disappeared and acquired strong a-SMA expression. Then the a-SMA positive protrusion fused with the a-SMA positive distal end of the outflow trunk cushions to form the transient aortic-pulmonary septum (AP septum) and divide aortic sac into the intrapericardial aorta and pulmonary trunks. At ED13, intrapericardial aorta and pulmonary trunk were characterized by disappearance of AP septum and appearance of a-SMA positive smooth muscle layers in their walls.Conclusion:PE-Isl-1F is involved in patterning the morphogenesis and septation of the intrapericardial arterial trunks.