Coupling Of Heart Outflow Tract Morphogenesis With Development Of Pulmonary Endoderm In Embryo

Cardiagenic medoderm located on the rostral and both bilateral of embryonic disc of three germ layers, which is called primary heart field (PHF) is fused to form the primary heart tube, which is only the primordium of the left ventricle, while the outflow tract, right ventricle and part of atrium of the embryonic heart are formed by addition of cardiomyocytes, which are originated from the mesenchyme of the second heart field (SHF) dorsal to the primary heart tube to the arterial pole of the primary heart tube. Little is known about the morphological relationship of the PHF with the SHF during early development of embryonic mouse heart. The differentiation of the cardiac precursor cells into the cardiomyocytes and the embryonic heart morphogenesis are regulated by different signal system and transcription factors. Transcription factor ISL-1is considered to be the better marker protein of cardiac precursor cells of SHF. Transcription factor GATA4and Nkx2.5are considered to be the better marker protein of cardiac precursor cells and cardiomyocytes. Transcription factors GATA4, Nkx2.5and ISL-1are considered to regulate the hyperplasia and the differentiation of the cardiac precursor cells into the cardiomyocytes in the PHF and SHF respectively. GATA4, Nkx2.5and ISL-1knockout or mutation result in the congenital heart malformations or embryo death. The endoderm induction is required for the the differentiation of the cardiac precursor cells into the cardiomyocytes both in the PHF and SHF, and for normal cardiac morphogenesis. Beating heart tube couldn't form if the endoderm were removed on the early stage of cardiac development. The researches about the foregut endoderm induction on the heart tube mainly focus on diverse signal molecules and transcription factors. Some signal molecule or transcription factors genes of forgut endoderm conditional knockout result in the embryonic mouse heart malformations, including the development of the outflow tract, right ventricle and aortic arch. So, it is considered that foregut endoderm could induce the differentiation of the cardiomyocytes and the heart morphogenesis via diverse signal molecules or transcription factors affected on the cardiac precursor cells. 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 embryo, amphibians, et al, via the deletion of endoderm genes or the culture in vitro. But the foregut endoderm morphogenesis and the morphological relationship of the foregut endoderm with the PHF and the SHF during the induction on the differentiation of the cardiomyocytes and the heart morphogenesis of wide-type embryonic mouse heart were not reported. In present study, we explored the morphological relationship of the pulmonary endoderm with PHF, SHF and the heart outflow tract during early development of embryonic mouse heart, which provide morphologic experimental evidence for the elucidation of the foregut endoderm induction on the normal heart development, and provide morphologic and theoretical basis for further investigating the congenital malformation, such as persistent truncus arteriosus resulted by abnormal septation of the outflow tract.The morphological research of GATA4during the development of the embryonic heart were focused on the observation of the phenotype by gene knockout, transgene and cell or tissue culture in vitro, restricted at the level of mRNA. Little is known about the spatial-temporal expression pattern of GATA4in the heart-forming field at the level of the protein, and about the relationship of the GATA4positive cells with the arterial pole early development, and via Western blotting technique. Few is reported about the comparison of the expression patterns of GATA4and Nkx2.5. Endoderm induction is required for the differentiation of the cardiac precursor cells into the cardiomyocytes in the primary heart field and second heart field and for the heart morphogenesis. GATA4is essentisl for the foregut endoderm cells proliferation. GATA4knockout on the foregut endoderm results in cardiamorphia. The research of GATA4effection on the foregut endoderm during the development of the embryonic mouse heart was restricted in the observation of the phenotype by gene knockout, cell or tissue culture in vitro or on the level of mRNA. The expression pattern of GATA4on the foregut endoderm in the wide-type mouse has not been reported. In present study, we explored the morphological relationship of the expression patterns of transcription factor GATA4in the heart-forming field and the foregut endoderm with the development and septation of the arterial pole of embryonic mouse heart via the technique of the immunohistochemistry and Western blotting to provide reliable morphologic evidence for further investigating the role of GATA4during the arterial pole early development and the theory that the foregut endoderm induction is required for the heart development.In part I, serial sections of mouse embryos from embryonic day(ED)7.5to embryonic day(ED)13were stained immunohistochemically or immunofluorescently with antibodies against Islet-1(ISL-1), myosin heavy chain (MHC), a-smooth muscle actin (a-SMA), Nkx2.5and proliferating cell nuclear antigen (PCNA) to investigate the relationship of expression patterns of Nkx2.5and ISL-1in the PHF, the SHF and foregut endoderm with the embryonic mouse heart outflow tract morphogenesis. In part II, serial sections of mouse embryos from embryonic day(ED)7.5to embryonic day(ED)13were stained immunohistochemically or immunofluorescently with antibodies against GATA4, Nkx2.5, Islet-1(ISL-1), a-smooth muscle actin (a-SMA) and myosin heavy chain (MHC), and The expression of GATA4and PHH3on the embryonic mouse heart from ED11to ED14were tested via Western blotting to investigate the relationship of the spatial-temporal distribution patterns of GATA4, Nkx2.5and ISL-1positive cells on the primary heart field, the second heart field and the foregut endoderm with the arterial pole development of the embryonic mouse heart.Part I Coupling of heart outflow tract morphogenesis with development of pulmonary endoderm in mouse embryoSerial sections of mouse embryos from embryonic day(ED)7.5to embryonic day(ED)13were stained immunohistochemically or immunofluorescently with antibodies against Islet-1(ISL-1), myosin heavy chain (MHC), a-smooth muscle actin (a-SMA), Nkx2.5and proliferating cell nuclear antigen (PCNA). We found that, at ED7.5, ISL-1positive cells in the cardiogenic plate were continuous with that in the developing SHF. From ED10to ED13, ISL-1positive pulmonary endoderm cells showed strong proliferation, irregular arrangement, loss the polarity with apparent intercellular space and changed to mesenchyme-like cells without obvious border with surrounding ISL-1positive mesenchyme. Local proliferation of endoderm cells adjacent closely to the dorsal wall of aortic sac resulted in the formation of solid endoderm cord that extended towards aortic sac. Ventral to the foregut, ISL-1positive mesenchyme cells surrounding the pulmonary endoderm and endoderm cord formed a distinctive cone-shaped structure. We suggest that PHF and SHF is a continuum and ISL-1is an early marker for cardiac precursor cells in both structures. Pulmonary endoderm cells and the formation of the endoderm cord might play roles in inducing proliferation of ISL-1positive cells or might transform to mesenchymal cells via epithelium-mesenchyme transition to maintain certain cell density in cone-shaped structure to guarantee the normal septation of the outflow tract.Part II Association of transcription factor GATA4expression patterns with arterial pole development of embryonic mouse heartSerial sections of mouse embryos from embryonic day(ED)7.5to embryonic day(ED)13were stained immunohistochemically or immunofluorescently with antibodies against GATA4, Nkx2.5, Islet-1(ISL-1),α-smooth muscle actin (a-SMA) and myosin heavy chain (MHC). The expression of GATA4and PHH3were tested via Western blotting at the embryonic mouse heart from ED11to ED14. We found that:At ED7.5, positive cells for both transcription factors GATA4and Nkx2.5were found in the cardiogenic plate simultaneously, earlier than the expression of MHC, and partly overlapping with the expression of ISL-1. from ED8.5to ED10, further expression of GATA4and Nkx2.5were detected in the splanchnic mesoderm of the dorsal wall of the pericardia! cavity that constitutes the Second Heart Field(SHF). From ED11to ED12, GATA4positive cells was found in the outflow tract endocardial cushion. From ED11to ED13, GATA4and Nkx2.5expression were found descending from the reflection of dorsal pericardial wall with the distal pole of outflow tract to the base of the ascending aorta and pulmonary trunk. GATA4expression was also detected in the foregut endoderm and endoderm cord of ED11to ED13embryos. From ED11to ED13, ventral to the foregut, ISL-1positive cells of cone-shaped mesenchyme structure surrounded the GATA4and ISL-1positive pulmonary endoderm. At ED11, GATA4positive cells were found in the mesenchyme surrrounding the pulmonary endoderm. GATA4and PHH3expression in the embryonic mouse hearts reached the highest level between ED11and ED13. We suggest that:From ED7.5to ED13, transcription factor GATA4shows the same expression pattern as that of Nkx2.5during the early development of the embryonic mouse heart, transcription factor GATA4might play a role in the differentiation of the cardiac precursor cells in the primary heart field and the second heart field, GATA4might be involved in the development of the endocardial cushion, higher expression of GATA4during crucial stages of heart morphogenesis suggests that GATA4might play an important role in the proliferation of myocardial cells. The GATA4expression in the foregut endoderm might be involved in the development of the SHF ventral to the foregut. The spatial and temporal distribution of GATA4is unique during the early development of the embryonic mouse heart.