Analysing the contribution of Wt1-expressing cells in the development of the vascular and visceral smooth muscle in mouse embryos and gastruloids

Abstract

During embryonic development, the epicardium, the mesothelium of the heart, contributes vascular smooth muscle cells (VSMCs) to the coronary vessels; however, the developmental origin of VSMCs in the embryo is not clearly defined. A previous study had reported that cells expressing the Wilms’ tumour protein (Wt1), a key marker of mesothelia, give rise to VSMCs in the intestine, mesentery, and coronary vessels, using genetic lineage tracing experiments (Wilm et al., 2005). Because Wt1 is expressed in the mesothelium of the body cavities, we concluded that Wt1-expressing mesothelial cells contributed to the VSMC compartment in the respective organs. Further studies analysing Wt1 lineage contribution led to the finding that Wt1 is switched on and remains expressed in the visceral mesothelium from around E9.5 onwards, while earlier transient expression of Wt1 in the nascent mesoderm specifies the future vascular and visceral smooth muscle of the intestine and mesentery (Wilm et al., 2021, Alghamdi et al., 2020). These results have revealed that the VSMC lineage is more complicated. In order to determine the contribution of Wt1-expressing cells to the VSMC lineage, this study used temporally controlled lineage-tracing experiments based on a Tamoxifen-inducible reporter system for different embryonic stages between 18.5 and 8.5 of embryonic development. Our lineage tracing of Wt1-expressing cells tagged at E8.5 and followed to different stages of embryonic development revealed their VSMC fate, with only a few visceral mesothelial cells found to be GFP-positive. Data of this thesis suggests that Wt1 expression before or during the initial phase of mesothelium formation in the intermediate and lateral plate mesoderm marks the cells that will become VSMCs. Furthermore, analysis of Wt1GFP/+ embryos at E8.5 was found to have some cells in the mesoderm and the primitive streak. This suggests a more intricate role for Wt1 in the lineage tracing of VSMCs that expressed Wt1. Further analysis of the Wt1-expressing mesoderm population was performed by using gastruloids generated from mouse embryonic stem cells (ESCs) as a valuable in vitro tool to study early embryonic development. We labelled gastruloid with different intermediate, lateral plate, and paraxial mesodermal markers to identify possible co-expression in the cells that expressed Wt1. Interestingly, gastruloids cultured for 72h and 96h showed co-expression of Wt1 and the mesodermal markers in the same region, while after 96h of culture, some of these cells appeared to have migrated to the anterior part of the gastruloid while other cells remained in the posterior part identified by expression of the early mesoderm marker Brachyury. This separation seemed to be completed in 120h gastruloids, which are thought to be equivalent to embryos at between E7.5 and E8.5, as the Wt1 expression only presented in the anterior side of the gastruloid. These findings led us to conclude that the origin of the Wt1-expressing cells that give rise to VSMSC in the intestine and the mesentery appears to be a posterior mesodermal-derived progenitor cell population. These progenitor cells then follow a specific journey during the next stages of embryonic development.