Transcriptional Factor GATA4 Expression During Primary Heart Tube Formation And Venous Pole Development Of The Mouse Embryonic Heart

Only the left ventricle originated from the primary heart tube. The outflow tract and right ventricle of the embryonic heart developed from the addition of cardiomyocytes from the mesenchyme rostral to the outflow tract and dorsal to the primary heart tube which was called the second heart field . Recently, it was reported that the mesenchyme between the foregut and venous pole of the heart continuously differentiated into cardiomyocytes and incorporated into the venous pole of the primary heart tube to form the atrium,sinus venous and sinoatrial node. Up to date, the mechanism underlying the development of the venous pole was unclear. GATA4 regulated the differentiation of mesenchymal cells into cardiomyocytes and normal morphogenesis of the developing heart but the relationship of GATA4 expression with the formation of primary heart tube and venous pole development of the mouse embryonic heart was not reported. In our present study, GATA4 expression pattern during the normal formation of primary heart tube and venous pole development might illuminate the mechanism of cardiamorphia appeared in GATA4 knockout embryos.In adult, pulmonary vein orifice located on the left atrium while during the development of embryonic heart, the relationship of venous sinus,pulmonary vein and atrium was disputed. It was reported that pulmonary vein developed from venous sinus. The others argued that pulmonary vein directly entered left atrium through dorsal mesocardium.In chapterⅠ, serial sections of embryonic day(ED)7.5 ~12 mouse hearts were stained with antibodies against transcriptional factor GATA4,α-smooth muscle actin(α-SMA),α-sarcomeric actin(α-SCA) and myosin heavy chain(MHC) to investigate the relationship of GATA4 expression with the formation of primary heart tube and venous pole development of the mouse embryonic heart. In chapterⅡ, mouse embryonic hearts from embryonic day 9 (ED9) to embryonic day 17 (ED17) were incubated with antibodies againstα-SCA andα-SMA to explore the distribution pattern of theα-sarcomeric actin (α-SCA) andα-smooth muscle actin (α-SMA) positive cells in the pulmonary vein of the mouse embryonic heart and to inquire the origin and myocardium formation of the pulmonary vein. ChapterⅠGATA4 expression during primary heart tube formation and venous pole development of the mouse embryonic heartSerial sections of embryonic day(ED)7.5 ~12 mouse hearts were stained with antibodies against transcriptional factor GATA4,α-smooth muscle actin(α-SMA),α-sarcomeric actin(α-SCA) and myosin heavy chain(MHC). We found that strong expression of GATA4 in the cardiogenic plate and fusing heart tube at ED7.5 was detected preceding that of MHC andα-SCA which were weakly expressed in a few cells, whereasα-SMA expression was still negative. At ED8.5, outflow tract and venous pole originated from second heart field were recognized clearly, strong expression ofα-SMA was seen extending to the distal ends of outflow tract and venous pole at the reflection with splanchnic epithelium on the dorsal wall of the pericardial cavity whereas GATA4 and MHC expression was mainly concentrated on the left and right ventricles of"S"-shaped heart. Gradual extension of GATA4 expression towards the distal ends of the outflow tract and venous pole was seen at late stage, and was always in advance of MHC andα-SCA expression. Morphogenesis and myocardialization of sinus venosus took place after ED9.5 during which sinus venosus was incorporating into the right atrium. The GATA4 andα-SMA positive anlage of sinoatrial node was found at ED11 in the wall of the right superior cardinal vein close to the right atrium due to proliferation of mesenchymal cells, and at ED12, began to show strong expression of MHC. Differentiation of mesenchymal cells into cardiomyocytes also could be detected in the wall of right inferior cardinal vein. We suggest that GATA4 is an early marker indicating differentiation of cardioblast towards cardiomyocytes in the primary heart field.α-SMA is a marker indicating early differentiation of cardiomyocyte from second heart field and its expression during heart development is probably not regulated by GATA4. GATA4 may not influence the differentiation of mesenchymal cells derived from second heart field into cardiomyocytes and migration to the two poles of heart tube, but it might mainly regulate proliferation and further differentiation of committed cardiomyocytes and normal looping morphogenesis of the developing heart. Moreover, mesenchymal wall of right cardinal vein close to the right atrium should be included in the second heart field. ChapterⅡThe origin and myocardium formation of the mouse pulmonary veinPregnant mice were killed after ethyl ether anaesthesia to obtain embryos from embryonic day 9 (ED9) to embryonic day 17 (ED17). Serial paraffin sections of mouse embryonic hearts were incubated with antibodies againstα-SCA andα-SMA by Immunohistochemistry PAP staining. We found that endothelial pulmonary vein appeared without expression ofα-SCA andα-SMA and the pulmonary vein orifice located on the left of the primary atrial septum at ED11. At ED12,α-SCA andα-SMA positive cells were observed around the endothelial pulmonary vein. After ED12, with the pulmonary vein lengthened,α-SMA positive cells appeared in the adjacent mesenchymal cells andα-SCA,α-SMA positive cells around pulmonary vein increased gradually, especially between ED12 and ED13 and the expression peak at ED14 and ED15. At ED16 and ED17, the pulmonary vein was more mature and the expression ofα-SMA decreased obviously butα-SCA maintained. We suggest that Pulmonary vein is not a part of venous sinus but is linked with left atrium directly; the myocardium aroud pulmonary vein derives from the adjacent mesenchymal cells.