CDX2 dose-dependently influences the GRN underlying vascularization in human extraembryonic mesoderm-like cells
Proper regulation of gene dosage is critical for the development of the early embryo and the extraembryonic tissues that support it. Specifically, loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure that is critical for nutrient delivery and waste removal in the early embryo. In this study, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By generating an allelic series for CDX2 in human induced pluripotent stem cells consisting of WT, heterozygous, and null CDX2 genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to multiomic single nucleus RNA and ATAC sequencing, we identify several genes regulating cytoskeletal integrity, adhesiveness, and polarity of the extraembryonic mesoderm and in a dose-dependent manner, including CDH1 and WNT5B. Despite these dose-dependent gene expression patterns, snATAC-seq reveals that heterozygous CDX2 expression is capable of inducing a WT-like chromatin accessibility profile, suggesting accessibility is not sufficient to drive gene expression when the CDX2 dose is reduced. Finally, because the loss of CDX2 or TBXT phenocopy one another in vivo, we compare differentially expressed genes in our CDX2 knock-out model to those from TBXT knock-out hiPSCs differentiated in an analogous experiment. This comparison identifies several genes critical for cytoskeletal integrity and vasculogenesis, including ANK3 and ANGPT1. Taken together, these results inform how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and suggest these genes may underlie defects in vascular development and allantoic elongation seen in the absence or reduction of CDX2 in vivo. Overall design: 2D gastruloids derived from WT, CDX2-Het, or CDX2-KO human iPSCs were isolated after 48hrs of BMP4 treatment for multiomic snRNA-seq and snATAC-seq. Nuclei from two bioreplicates of about 288 2D gastruloids per genotype per replicate were sequenced.