Testing the potential for vascularization between the chick chorioallantoic membrane and an in-vitro 3D mouse model of early post-implantation development

Early post-implantation development is difficult to study in vivo because key events occur deep within the uterus and can only be captured through fixed embryo snapshots. Stem cell-derived ETiX embryoids offer a useful alternative, as they self-organize into epiblast-, visceral endoderm-, and trophoblast-like tissues and reproduce several features of peri- and post-implantation development. In this project, I generated a triple-labeled ETiX system in which each lineage expresses a distinct nuclear fluorescent reporter (H2B-GFP, H2B-RFP, H2B-CFP). After establishing and validating the individual reporter cell lines, I showed that triple-labeled ETiX assemble correctly, maintain appropriate lineage identity, and display strong correlation between the expression of each reporter in the appropriate tissue and the corresponding molecular markers used to identify it. I then adapted the chick chorioallantoic membrane (CAM) assay to test whether ETiX can survive and interact with a living vascularized tissue. ETiX embedded in agarose remained viable for several days, preserved reporter expression, and were found in areas of close contact with CAM-derived tissue, although it remains an open question whether they induce vascular remodeling or interact with the developing vasculature. This work provides an initial framework for combining triple-labeled embryoids with the CAM assay. Future experiments will be essential to determine whether ETiX can actively induce vascular remodeling and to further assess their potential as a platform for studying early extraembryonic/vascular interactions in the early post-implantation embryo.