Hand2-mediated Epithelial Maintenance And Integrity In Cardiac Outflow Tract Morphogenesis

During embryogenesis,epithelial organization is the prerequisite for organogenesis where epithelial-to-mesenchymal transition(EMT)and reversed MET occur alternately to accomplish organ morphogenesis and remodeling.While EMT in organogenesis has been extensively investigated,the understanding of MET process is largely lacking.Recent studies provided hints about MET in early heart development,which has not been systemically explored.During heart tube fusion,cardiac progenitor cells,giving rise to the cardiac crescent,form an epithelial sheet in the ventral midline.The epithelial transition of mesenchymal-progenitor cells from the anterior second heart field also happens with atypical apical-basal polarity formation in the anterior dorsal pericardial wall(a DPW).By progressive addition to the arterial pole of heart tube,these a SHF progenitors contribute to the developing outflow tract and right ventricle.Maximal elongation of OFT is crucial for its remodeling during cardiac septation.Direct or indirect perturbation cause a range of heart diseases.Polarity establishment of a SHF progenitor cells has been shown playing an important role for cell deployment to OFT.Hand2,as the important cardiac transcription factor in heart development.But its role in second heart field and OFT morphology remains unclear.Here,we revealed a gradient of Hand2 protein in the cardiac progenitors in the dorsal pericardial wall(DPW)and adjacent transition zone(TZ)in the most distal outflow tract(OFT).Deletion of Hand2 in a SHF using Mef2c-AHF-Cre caused cell arrest and accumulation in the TZ leading to defective morphogenesis.We elaborately characterized the epithelial features of the a DPW and TZ in early mouse embryos through in-depth investigation of the alterations in Hand2 mutant mice.We found that as cells moving to OFT,cell adhesion is remolded and strengthened,including extended cell adherens junction and new incorporated cell-ECM adhesion to form maturation epithelium in the TZ,representing the stronger cell-cell and cell-ECM cohesiveness required for structure integrity of OFT.Inside the cells,the powerful actin cytoskeleton architecture containing co-localized NMMII,as the foundation of cell adhesion complex,is constructed in the TZ as well compared to a DPW.When Hand2 is deleted,while cell apical-basal polarity was unaffected,the key epithelial elements of adherens junction and cell-matrix adhesion were disrupted in the TZ of Hand2 mutant mice,indicating poorly formed epithelium.Our study improved an unconventional MET process in SHF development.The robustly proliferative AHF progenitors with mesenchymal property enter the a DPW,acquiring atypical epithelial transition with punctate and apical location of cell adhesion molecules(N-cad and E-cad)that then is remolded and strengthened to form mature epithelium when starting to undergoing myocardial differentiation in the TZ,the nescent epithelium is continuously generated in the TZ,which may push the caudal extension of the coherent epithelial sheet of primary OFT bulb.Furthermore,RNA-seq analysis revealed altered regulation of the contractile fiber and actin cytoskeleton in mutant mice.Furthermore,we have identified Stars transcriptionally controlled by Hand2.STARS facilitates actin polymerization that is essential for cell adhesion.Thus,we have uncovered a new mechanism of Hand2-mediated epithelium maintenance and integrity which is crucial for OFT morphogenesis in early heart development.Meanwhile,this study provides insights to understanding cardiac progenitor contribution to OFT development.