Distribution Patterns Of Neural Crest Cells And ISL-1Positive Cells During The Morphogenesis Of Arterial Pole Of Mouse Embryonic Heart

Background: Cardiac neural crest cells, together with ISL-1positive cardiacprogenitor cells from the second heart field(SHF) participate in the morphogenesis ofarch arteries and the outflow tract(OFT) of the vertebrate. Transcription factor ISL-1is considered to be the main marker protein of the SHF that promotes proliferation,survival and migration of the cardiac precursor cells in SHF. ISL-1positive cells inthe SHF are multi-potent cells, regulated by diverse transcription factors and signalingsystems in different spatiotemporal stages of embryonic development, and candifferentiate into cardiomyocytes, smooth muscle cells and other cell types. Afterformation of the linear heart tube from primary heart field, ISL-1positivemesenchymal cells in the SHF and the cardiac neural crest cells between otic placodeand the third somite migrate to arterial pole of the linear heart tube to take part in themorphogenesis of arch arteries and OFT. Deletion of cardiac crest may interfere withthe development of SHF and may result in malformation of arch arteries and OFT,including persistent truncus arteriosus, pulmonary stenosis and double outlet rightventricle, but little is known about the distribution relationship between ISL-1positivecells in SHF and cardiac neural crest cells during the morphogenesis of embryoniccardiac OFT. Transcriptional factor AP-2a gene is utilized as a marker forpremigratory and migratory neural crest cells, the date of spatiotemporal distributionrelationship between AP-2α expressing neural crest cells during migration and ISL-1positive precursor cells in SHF is also lacking. In present study, the spatiotemporaldistribution relationship between ISL-1positive cells and AP-2α positive cardiacneural crest cells during the development of arch arteries and the OFT is observedwith the method of immunohistochemical staining, the results provide reliableexperimental evidence for further investigating the mechanism underlying thecongenital heart disease due to the defect in the development of OFT.Objective: To explore the distribution patterns of neural crest cells and ISL-1positivecells during the development of arterial pole of mouse embryonic heart.Methods：Serial sections of mouse embryonic hearts from embryonic day(ED)8toED12were stained immunohistochemically with antibodies against α-smooth muscle actin(α-SMA), myosin heavy chain(MHC), AP-2α and ISL-1.Results: At ED8, AP-2α positive neural crest cells were mainly distributed in neuralfold ectoderm and otic placode ectoderm. ISL-1positive cells forming the secondheart field were found in the mesoderm on both sides of neural groove that extendedalong the splanchnic mesoderm of dosal percardial cavity wall to the heart tube, andcontinued with MHC and α-SMA positive cardiomyocytes. At ED9to ED10,migration of AP-2α positive crest cells was detected along the aortic arch arteriestowards the endocardial cushion at the arterial pole of the outflow tract of theembryonic heart to take part in the formation of aorto-pulmonary septum (AP septum).At ED11, AP-2α positive cells in the mesenchyme surrounding the foregut endodermwere found projecting into the aortic sac cavity to form AP-2α positive AP septum. AtED9, ISL-1positive cells in the mesenchyme on both sides of foregut and branchialarche core were found adding cardiomyocytes to OFT. At ED10to ED11, ISL-1positive cells in the mesenchyme ventral to the foregut were detected participating inthe formation of AP septum together with AP-2α positive cells, but less ISl-1positivecells were found in the mesenchyme around the arch arteries. ISL-1positive cells inthe splanchnic mesoderm of the dorsal wall of the cardiac cavity and coremesenchyme in the branchial arches continued with α-SMA and MHC positive OFTmyocardium. At ED12, expression of AP-2α and ISL-1in AP septum and endocardialcushion of the outflow tract disappeared and began to express α-SMA. ISL-1positivecells ventral to the foregut that were continuous with the wall of aorta and pulmonarytrunk also began to show α-SMA expression.Conclusion: AP-2α is the specific marker of cardiac neural crest cells, AP-2α positivecardiac neural crest cells and ISL-1positive cardiac progenitor cells cooperate toparticipate in the morphogenesis of arterial pole of mouse embryonic heart, both ofwhich are necessary for the development of the cardiac outflow tract, deletion ofAP-2α results in malformation of the outflow tract of mouse embryonic heart. At ED10to ED11, ISL-1positive cardiac progenitor cells and AP-2α positive cells areinvolved in the formation of AP septum and endocardial cushion. After ED11, AP-2αpositive cardiac neural crest cells can only be found around the sixth arch arteries,with the fusion of AP septum with outflow cushion(outflow tract crest), AP septumand the wall of the aorta and pulmonary trunk begin to lose expression of ISL-1andobtain the expression of α-SMA.