| Background: It is known that in the early stage of embryonic heart development,the cardiac precursor cells from outside the heart are continuously migrating into the heart through the arterial pole and the venous pole of cardiac tube,differentiate into the working myocardium or the conduction system myocardium under the control of related transcription factors or signaling molecules and participate in the development of the four-chambers and conduction systems.Studies have demonstrated that the cardiac precursor cells of the second heart field(SHF)can migrate into the cardiac tube through the dorsal mesocardium to participate in the formation of the outflow tract,right ventricle,part of the atria and atrial septum,atrial dorsal mesenchyme,the aorta and pulmonary trunk.Insulin enhancer binding protein 1(Islet-1,Isl-1)is one of the markers of SHF precursor cells.The observation of temporal and spatial distribution characteristics of Isl-1 positive cells and the proliferation and migration mechanisms of the SHF precursor cells provide important morphological data for the mechanisms by which SHF precursor cells participate in early heart development.Our previous study shows that in the early stage of mouse and human embryonic heart development,Isl-1 may enable SHF mesenchymal cells to maintain the status of myocardial precursor cell and the ability of proliferation and migration to participate in the separation of aortic sac and the development of ascending aorta and pulmonary artery.Within the SHF mesenchyme,Isl-1 positive pulmonary endoderm is closely related to the surrounding pulmonary endoderm associated Isl-1 positive cell field(PE-Isl-1F),and under the induction of pulmonary endoderm,PE-Isl-1F is formed a characteristic cone-shaped distribution pattern.The Isl-1 positive cone-shaped structure is well defined,but the surrounding SHF mesenchyme is negative for Isl-1 expression,so other transcription factors may regulate the formation of characteristic cone-shaped structures.Whether the Isl-1 negative mesenchyme participates in the development of the aortic sac and outflow tract needs further study.Our previous study showed that the primordium of the sinoatrial node(SAN)of central conduction system was positively expressed by Isl-1,suggesting that the sinoatrial cardiomyocytes may be derived from the SHF myocardial precursor cells in the venous pole,and later under the regulation of the transcription factor Tbx3,differentiated into pacemaker cells to form the well-functioning sinoatrial node.However,the mesenchyme in the wall of SVC connected to SAN primordium maintains a negative expression of Isl-1,so the source of sinoatrial node primordial cardiac precursor cells needs further investigation.The expression of transcription factor Tbx3 was observed in the atrioventricular canal(AVC)in the early stage of cardiac tube development.It continuously expressed during the development of AVC and atrioventricular node and moved into SAN primordium through the venous valve to regulate the development of SAN primordium.However,the mesenchyme of the dorsal pericardial cavity and the mesenchyme surrounding the foregut are also Tbx3-positive,so whether the SHF mesenchymal precursor cells are the source of Tbx3-positive cells in AVC and whether they continuously migrate into AVC during the cardiac tube development need further investigation.This study systematically observed the temporal and spatial expression patterns of two members of the Iroquois homeobox gene family,Irx1 and Irx2,in the early development of mouse embryo and human embryonic heart.The first part focuses on the temporal and spatial distribution of Irx1 and Irx2-positive SHF precursor cells in the cardiac artery pole.By comparing with the distribution characteristics of Isl-1 positive SHF precursor cells,the different pathways of SHF precursor cells take part in the development of aortic sac and outflow tract are discussed.The second part focuses on the distribution of Irx1 and Irx2-positive SHF precursor cells during the development of the conduction system,in order to provide a morphological evidence for the origin of sinoatrial node and the development of atrioventricular node and ventricular conduction system.Objective: To observe the temporal and spatial expression patterns of Irx1 and Irx2 in the early development of mouse embryo and human embryonic heart,in order to investigate the development of Irx1 and Irx2-positive SHF precursor cells in the cardiac aortic sac and outflow tract,as well as the origin of sinoatrial node,atrioventricular node and ventricular conduction to provide a morphological evidence for morphological research.Methods: The collected early human embryo specimens after medical abortion and the mouse embryo specimens with embryonic age of ED8 to ED14 were serially sectioned for immunohistochemical staining and immunofluorescence staining.Isl-1 labeled second heart zone(SHF)precursor cells,Tbx3 labeled embryonic heart central conduction system,transcription factor Nkx2.5 and anti-myocardial myosin heavy chain(MHC)markers of developing atrial and ventricular myocytes The systematic observation of the temporal and spatial expression patterns of Irx1 and Irx2 in early cardiac development.Results: As early as the mouse embryonic age of day8(ED8),the endoderm,the mesoderm of the dorsal wall of the pericardium and the dorsal mesocardium showed strong positive expression of Irx1 and Isl-1.From ED8.5 to ED10.5,Irx1 and Isl-1 were co-expressed in the ventral wall of the endoderm and the solid cell mass,the wall of the distal end of the outflow tract(OFT)and the mesoderm of the dorsal wall of the pericardium.Irx1 positive cells,which were also observed in a small amount of mesenchyme under the aortic sac endothelium and the endothelium of the aortic sac and OFT,continued with Irx1 positive cells in the dorsal mesenchyme of the pericardial cavity.At ED11 to ED12,the endoderm cells were double positive for Irx1 and Isl-1 expression.The surrounding mesenchyme of the endoderm formed a characteristic cone structure with Irx1 and Isl-1 double positive.The tip protruded into the aortic sac cavity and continued with the myocardium and cushion of distal end of OFT to participate in the separation of OFT.At ED13 to ED14,the outflow tract is divided into ascending aorta and pulmonary trunk.The esophagus is completely separated from the trachea,and only a small amount of Irx1 positive cells are retained around the trachea.At human embryonic stage C11 to C12(C11~C12),the positive expression of Irx1 and Irx2 was found in the dorsal wall and ventral wall of endoderm.The positive expression of Isl-1 and Nkx2.5 was only found in the ventral wall of endoderm.At C13~C14,pulmonary endoderm and solid cell cords were positively expressed by Irx1,Irx2 and Isl-1,and the characteristic Isl-1 positive cone structure was recognized.The tip protruded into the aortic sac cavity and then continued with the distal end of OFT to participates in the development of the separation of OFT.The Irx1 positive cells in the dorsal mesenchyme of the pericardial cavity also flowed into the OFT and diffusely distributed in the cardiac jelly.At C15,the tip of the Isl-1 positive cone structure under the ventral wall of trachea protruded into the aortic sac to form the septum of aorta and pulmonary artery,separating the aortic sac into the ascending aorta and pulmonary trunk.Isl-1 and Irx1 double positive cells can be seen in the tracheal wall and extend to the ventral side mesenchyme.A large number of double positive cells also can be seen in the arterial wall of the pulmonary trunk and the ascending aorta and the septum of aorta and pulmonary artery.The co-expression patterns of Isl-1 and Irx2 are consistent with the expression patterns of Isl-1 and Irx1.MHC-negative myocardium of the OFT showed strong expression of Isl-1,Irx1 and Irx2,and MHC-positive OFT myocardium showed only weaker expression of Isl-1 and Irx1.The endocardial cushion and endothelium were positive for Irx1 and Irx2,but the expression of Isl-1 was negative.At C16,Irx1 is still widely expressed in the esophagus,trachea and surrounding mesenchyme,and extended to the septum of aorta and pulmonary artery,wrapping the ascending aorta and pulmonary trunk.Nkx2.5-positive OFT myocardium was negative for Isl-1 and Irx1.Irx1 was positively expressed in the endocardial cushion of the OFT but Nkx2.5 and Isl-1 were negative.At the mouse embryonic stage ED8.5 to ED9.5,the wall of the atrioventricular canal(AVC)of primitive cardiac tube was positive for Irx1 and Tbx3,and a small number of double positive cells were observed.The endothelium of AVC was also positive for Irx1 expression.At ED10 to ED10.5,the inner side wall of the right superior vena cava(SVC)was locally thickened and strongly expressed by Isl-1,and it was consistent with the Isl-1 strong positive myocardial dorsal wall of the atrium.The primordium of the sinoatrial node(SAN)gradually developed.A large number of strongly positive Irx1 cells extend from the inner side wall of the right SVC to the primordium of SAN.The myocardium of the wall of AVC showed positive expression of Irx1 and Tbx3,and some double positive cells were observed.Irx1 and Tbx3 double positive cells extend from the posterior wall of AVC to the ventricular septum and reach to the ventral side wall of the ventricle.At ED11 to ED12,the primordium of SAN was strongly positive for Isl-1 and Irx1,weakly positive for Tbx3 and negative for NKX2.5.The positive expression of Irx1 in the wall of the right SVC was also significantly enhanced,continued with the positive expression of Irx1 in SAN.The expression of Tbx3 in the anterior wall of AVC was stronger than that in Irx1,but the expression of Irx1 in the lateral wall of AVC was enhanced.The expression of Irx1 was also observed in the endocardial cushion of AVC.In the posterior wall of AVC,Tbx3 positive expression was limited,but Irx1 strong positive cells extended to the septum of the ventricle.At ED13 to ED14,the size of SAN increased,and it was positive for Isl-1 and Tbx3,but Irx1 was negative.The distribution of Irx1 and Tbx3 positive cells was observed in the left and right venous valves,and the atrioventricular node(AVN)with positive of Irx1 and Tbx3.At hunan embryonic stage C11~C12,the primitive atrioventricular wall showed strong Irx1 positive expression and the dorsal wall of the primitive atrium showed strong Irx2 positive expression.At C13~C14,the primordium of SAN was formed showed Isl-1 positive and Nkx2.5 negative expression.Irx1 showed strong positive expression in SAN primordium and venous valve.Part of the primordial cells in SAN primordium were positive for Irx2.The positive expression of Irx1 in the wall of AVC was enhanced,and the endocardial cushion and endothelium of AVC were also strongly positive for Irx1 and Irx2.Irx1-positive cells extended from the mesenchyme of the ventral wall of the trachea to the dorsal wall of the atrium,connected with Irx1 positive cells in the atrioventricular wall and endocardial cushion through the left and right venous valves and dorsal mesenchymal protrusion(DMP)of the atrium.At C15 to C16,the SAN primordium contained a large number of Irx1 positive cells and some of them simultaneously expressed Isl-1.The myocardium and mesenchyme of the atrioventricular wall were positive for Irx1,but the positive expression of Nkx2.5 was restricted to the myocardium.Irx1-positive cells extended from the atrial wall to the DMP and connected to the endocardial cushion of AVC.Irx2 was weakly expressed in the atrial wall,venous valves and atrial septum,but negatively expressed in the atrioventricular wall,endocardial cushion and DMP.Conclusion: 1.Irx1 and Irx2 are widely expressed in different germ layers in early embryonic development,and they overlap with the expression of Isl-1,which supports Irx1 and Irx2 may participate in the development of a variety of organs including the heart.2.The temporal and spatial expression pattern of Irx1 in SHF is very similar to that of Isl-1.The characteristic Isl-1 positive cone-shaped structure is simultaneously expressed by Irx1 and Isl-1,but the expression range of Irx1 is more extensive than Isl-1,so it is speculated that Irx1 is a widely expressed transcription factor in SHF precursor cells.Under certain regulatory factors,part of the mesenchyme proliferates and simultaneously expresses Irx1 and Isl-1,forming a characteristic cone-shaped structure,and then plays an important role in the process of precursor cells migrating into the two poles of cardiac tube.3.Irx1 and Irx2 positive SHF precursor cells are closely related to the separation of aortic sac and OFT.During the continuous addition of SHF precursor cells to the distal pole of the OFT,Irx1 and Irx2 may act synergistically with Isl-1 to determine the different differentiation directions of SHF precursor cells,in the early stages of aortic sac and OFT separation.Involved in the development of the ascending aorta and pulmonary trunk wall,on the other hand,through the main pulmonary artery septum extending into the OFT endocardial cushion,participating in the separation of the OFT.4.The Irx1 and Irx2-positive endoderm cells are closely adjacent to the surrounding positive mesenchymal cells and the endoderm expression intensity is higher than that of the surrounding mesenchyme,suggesting that Irx1 and Irx2 are also the important regulators of proliferation and differentiation of Irx1 and Isl-1 double positive SHF precursor cell induced by foregut endoderm.5.Irx1-positive SHF myocardial precursor cells may be the source of SAN.Irx1-positive precursor cells added through both the SVC wall and the dorsal mesocardium to SAN regulate the morphogenesis of SAN with Isl-1 positive precursor cells.Irx1 participates in the process of Tbx3 promoting the differentiation of precursor cells into pacing myocardium for a limited time.6.The development of AVC myocardium is heterogeneous.The expression of Tbx3 in the anterior wall of AVC was stronger than that in Irx1.In the posterior wall of AVC,the Irx1-positive mesenchyme continued with the Irx1 strong positive cells in the left SVC wall.In addition,Irx1 and Tbx3 positive cells in the atrioventricular wall are involved in the development of ventricular conduction.7.The development of morphology and function of AVN are later than SAN.When the Tbx3 positive expression in SAN primordium was enhanced,and the positive expression of Irx1 and Irx2 disappeared.Only the developing atrioventricular node and the top of the atrioventricular septum remained Irx1 positive,suggesting that AVN still retained the characteristic of the precursor cells. |
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