| Objective:Second heart field cells and neural crest cells have revealed important roles for different cell lineages in the development of the cardiac arterial role.Neural crest cells have been reported to play roles in the morphogenesis of the pharyngeal arch arteries wall,however,how does the pharyngeal arch arteries separate from the aortic sac has not been elucidated.Methods:We studied the pharyngeal arch arteries development of normal mice between 9 and 12.5 days postconception(dpc),applying morphological observation and immunohistochemistry and 3D reconstructions,using antibodies against Isl-1 for the SHF cells,Nkx2.5 for the SHF cardiac precursor and myocardial cells,transcription factor Tbx3 to regulate SHF proliferation,transcription factor Ap2? for migrating neural crest cells,anti-Foxa2 antibody as a marker for endodermal cells,and anti ?-smooth muscle actin antibody ?-SMA for smooth muscle and marking the onset of cardiac differentiation,CRABP1 as a marker of neural crest cells.Results:We found that Isl-1 positive pharyngeal mesenchyme protrusions grow out from the dorsal wall of the aortic sac in succession and fuses with lateral wall of the aortic sac to separate the pharyngeal arch arteries from the aortic sac to form the independent arteries.The separation of the proximal regions of the left and right PAAs at the level of the aortic sac is performed by Isl-1 positive pharyngeal mesenchyme protrusion.No obvious Isl-1 positive pharyngeal mesenchyme protrusion was detected in the proximal regions of the first and second PAAs due to fast development.As development progresses,Isl-1 positive pharyngeal mesenchyme protrusion extends ventrally into the aortic sac cavity and fuses with the wall of the aortic sac to form a septum that divides the third PAAs proximal region at the level of aortic sac into left and right portions,and the wall of the nearby aortic sac wall also becomes part of the third PAA walls.The separation of the fourth and sixth PAAs proximal region at the level of aortic sac is also performed in this manner.The aortic sac wall is divided,becomes part of the wall of the PAAs,and moves caudally.After separation of the sixth PAAs proximal region at the level of aortic sac,the aortic sac translocation to the pericardial cavity continues to be divided.A wedge-shaped intrapericardial protrusion grows from the dorsal wall of the aortic sac to form the AP septum which divides the aortic sac into the ascending aorta and pulmonary trunk.At this time,the separation of the aortic sac is completed.The Isl-1 positive pharyngeal mesenchyme protrusion which is also present between the third PAAs and the fourth PAAs separates the root of the sixth PAAs prior to the formation of AP septum.The Isl-1 negative intrapericardial protrusion grows from the dorsal mesenchymal wall of the aortic sac and gradually acquires expression of a-SMA,and this intrapericardial protrusion will form the AP septum.The AP septum is fused with the tip of the Isl-1 positive pharyngeal mesenchyme protrusion.The protrusion of the pharyngeal mesenchyme was expressed as Isl-1+ Nkx2.5+,while the intrapericardial protrusion was negative for Isl-1 and Nkx2.5.At 11 days post-conception(dpc)in a mouse embryo,the Isl-1 positive mesenchyme protrusion appears as a heart-shaped structure where the Isl-1 positive pharyngeal ventral mesenchyme can be divided into different cell subpopulations,the Isl-1+Tbx3+ positive cell subpopulation near the dorsal region,and the Isl-1+ Nkx2.5+ positive cell subpopulation near the ventral region.Conclusion:Our results suggest that the aortic sac is a dynamic structure that is continuously divided during the migration from the pharyngeal mesenchyme to the pericardial cavity.The separation of the aortic sac is not completed until the AP septum divides the aortic sac into the ascending aorta and pulmonary trunk.Moreover,the midsagittal septum and the AP septum are distinct structures. |
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