The somatopleure is the amniotic primordium in amniote development, but its boundary to the embryonic body at early embryonic stages and the fate of cells constituting this structure are not well characterized. It also remains unclear how cells behave during the demarcation between intra- and extra-embryonic tissues. Here we identify cellular alignments, which indicate two streams towards the sites of dorsal amniotic closure and ventral thoracic wall formation. A subpopulation of mesodermal cells moving ventrally from the somatopleural region adjacent to the base of the head fold enter the body of the embryo and distribute to the thoracic wall, pharyngeal arches and heart. These cells are induced to differentiate into vascular endothelial cells and cardiomyocytes possibly by FGF and BMP signaling, respectively. These results indicate that the somatopleure acting as the amniotic primordium also serves as a source of embryonic cells, which may contribute to cardiovascular development.

Background

The somatopleure serves as the primordium of the amnion, an extraembryonic membrane surrounding the embryo. Recently, we have reported that amniogenic somatopleural cells (ASCs) not only form the amnion but also migrate into the embryo and differentiate into cardiomyocytes and vascular endothelial cells. However, detailed differentiation processes and final distributions of these intra-embryonic ASCs (hereafter referred to as iASCs) remain largely unknown.

Results

By quail-chick chimera analysis, we here show that iASCs differentiate into various cell types including cardiomyocytes, smooth muscle cells, cardiac interstitial cells, and vascular endothelial cells. In the pharyngeal region, they distribute selectively into the thyroid gland and differentiate into vascular endothelial cells to form intra-thyroid vasculature. Explant culture experiments indicated sequential requirement of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling for endothelial differentiation of iASCs. Single-cell transcriptome analysis further revealed heterogeneity and the presence of hemangioblast-like cell population within ASCs, with a switch from FGF to VEGF receptor gene expression.

Conclusion

The present study demonstrates novel roles of ASCss especially in heart and thyroid development. It will provide a novel clue for understanding the cardiovascular development of amniotes from embryological and evolutionary perspectives.

Hematopoietic progenitors are enriched in the endocardial cushion and contribute, in a Nkx2-5-dependent manner, to tissue macrophages required for the remodeling of cardiac valves and septa. However, little is known about the molecular mechanism of endocardial-hematopoietic transition. In the current study, we identified the regulatory network of endocardial hematopoiesis. Signal network analysis from scRNA-seq datasets revealed that genes in Notch and retinoic acid (RA) signaling are significantly downregulated in Nkx2-5-null endocardial cells. In vivo and ex vivo analyses validate that the Nkx2-5-Notch axis is essential for the generation of both hemogenic and cushion endocardial cells, and the suppression of RA signaling via Dhrs3 expression plays important roles in further differentiation into macrophages. Genetic ablation study revealed that these macrophages are essential in cardiac valve remodeling. In summary, the study demonstrates that the Nkx2-5/Notch/RA signaling plays a pivotal role in macrophage differentiation from hematopoietic progenitors.