Association Of Neural Crest Cells And ISL-1 Positive Cells With Outflow Tract Development Of Mouse Embryonic Heart

Background: The primary heart tube is developed from cardiogenic mesoderm of embryonic disc of three germ layers, which is only the primordium of the left ventricle, while the right ventricle, outflow tract (OFT) and part of atria are formed by addition of cardiomyocytes from mesenchyme of the second heart field (SHF) dorsal to the primary heart tube to the arterial pole of the primary heart tube. Transcriptional factor ISL-1 is considered to be the marker protein of the cardiac precursor cells of SHF and the important node protein to regulate the development of SHF, including the proliferation and the differentiation of the cardiac precursor cells. Deletion of ISL-1 gene results in the absence of right ventricle and OFT of embryonic heart tube as well as the death of embryos between embryonic day (ED) 9 and ED10. Under the regulation of diverse signal system and transcriptional factors, the neural crest cells between the otic placode and the third somite migrate along the 3, 4 and 6 branchial arches into the OFT of the heart tube and interact with the cardiac precursor cells of SHF to participate in the normal development and septation of OFT. The failure of myocardial cell addition from SHF to OFT after neural crest ablation generates the same developmental malformation of OFT as those produced in the defect of SHF development. Little is known about the spatial-temporal distribution of SHF and the differentiation potency of ISL-1 positive cells during development of normal mouse embryos, the data of spatial-temporal distribution relationship between the ISL-1 positive cells and the neural crest cells during development of heart tube is also lacking. In present study, the spatial-temporal distribution pattern of migrating neural crest cells expressing cellular retinoic acid binding protein 1 (CRABP1) and the ISL-1 positive cells during different developmental periods is observed to provide reliable morphologic experimental evidence for further investigating the interaction between two kinds of cells during the development of OFT and the mechanism by which the congenital heart disease is resulted due to the defect in the development of OFT.Objective: To explore the relationship of migrating neural crest cells expressing CRABP1 and ISL-1 positive cardiac progenitor cells with the development of OFT of mouse embryonic heart.Methods: Serial sections of mouse embryonic hearts from ED 7.5 to ED 13 were stained immunohistochemically or immunofluorescently with antibodies againstα-smooth muscle actin (α-SMA), myosin heavy chain (MHC), ISL-1, CRABP1 and phosphorylated histone H3 (PHH3).Results: At ED7.5, CRABP1 and ISL-1 positive cells were found in the mesoderm, while the expression of heart specific proteins,α-SMA and MHC could not be detected. At ED8.5, ISL-1 positive cardiac progenitor cells were detected in the mesenchyme of dorsal mesocardium, in the splanchnic mesoderm dorsal to pericardial cavity, mesenchyme ventral and lateral to the primary pharynx. From ED9 to ED10, ISL-1 positive cells were sequentially detected in the craniofacial mesenchyme and core mesenchyme in the branchial arches. ISL-1 positive mesenchyme constituted SHF for the development of OFT of heart tube to add cardiomyocytes to OFT. From ED11 to ED13, ISL-1 positive cells ventral to pharynx formed a cone-shaped structure from which ISL-1 positive cells extended towards the developing intrapericardial aorta, pulmonary trunk and aorto-pulmonary septum (AP septum). CRABP1 positive neural crest cells were interspersed among the ISL-1 positive cells at early stages, a few CRABP1 and ISL-1 double-positive cells were found at the distal pole of OFT. As embryos developed into ED10, CRABP1 positive cells were uniquely distributed surrounding the ISL-1 positive core mesenchyme in branchial arches. Thereafter, the gradual disappearance of CRABP1 expression in the wall of arch arteries and other regions was followed by expression ofα-SMA.Conclusion: The SHF is a dynamic structure, during different spatial-temporal periods, craniofacial mesenchyme, splanchnic mesoderm dorsal to pericardial cavity, the core mesenchyme in the branchial arch and the mesenchyme around the pharynx sequentially develop into the ISL-1 positive SHF, and add myocardial cells to the distal pole of OFT. After ED11, ISL-1 positive cells are involved in the development of the smooth muscle cells in the wall of proximal ascending aorta and pulmonary trunk as well as the formation of AP septum. Before ED11, neural crest cells cooperate with ISL-1 positive cells gradually to form a unique distribution pattern and guarantee normal development of the heart. From ED11 onward, neural crest cells have migrated into the presumptive position and differentiate into different cell types.