The embryonic epicardium, originating from the proepicardial organ (PEO), provides a source of multipotent progenitors for cardiac lineages, including pericytes, fibroblasts, and vascular smooth muscle cells. Maximizing the regenerative capacity of the adult epicardium depends on recapitulating embryonic cell fates. The potential of the epicardium to contribute coronary endothelium is unclear, due to conflicting Cre-based lineage trace data. Controversy also surrounds when epicardial cell fate becomes restricted. Here, we systematically investigate expression of five widely used epicardial markers, Wt1, Tcf21, Tbx18, Sema3d, and Scx, over the course of development. We show overlap of markers in all PEO and epicardial cells until E13.5, and find no evidence for discrete proepicardial sub-compartments that might contribute coronary endothelium via the epicardial layer. Our findings clarify a number of prevailing discrepancies and support the notion that epicardium-derived cell fate, to form fibroblasts or mural cells, is specified after epithelial-mesenchymal transition, not pre-determined within the PEO.
RESULTSWt1, Sema3d, Tbx18, Scx, and Tcf21 Overlap in the PEO, but Their Expression Domains Are Not Confined to This Tissue First, we used multiplexed single-molecule RNA in situ hybridization (RNAscope) on E9.5 sagittal mouse sections to simultaneously detect expression of the PEO markers:
Macrophages are components of the innate immune system with key roles in tissue inflammation and repair. It is now evident that macrophages also support organogenesis, but few studies have characterized their identity, ontogeny and function during heart development. Here, we show that the distribution and prevalence of resident macrophages in the subepicardial compartment of the developing heart coincides with the emergence of new lymphatics, and that macrophages interact closely with the nascent lymphatic capillaries. Consequently, global macrophage deficiency led to extensive vessel disruption, with mutant hearts exhibiting shortened and mis-patterned lymphatics. The origin of cardiac macrophages was linked to the yolk sac and foetal liver. Moreover, the Cx3cr1+ myeloid lineage was found to play essential functions in the remodelling of the lymphatic endothelium. Mechanistically, macrophage hyaluronan was required for lymphatic sprouting by mediating direct macrophage-lymphatic endothelial cell interactions. Together, these findings reveal insight into the role of macrophages as indispensable mediators of lymphatic growth during the development of the mammalian cardiac vasculature.
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