CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development

Abstract

ABSTRACTProper regulation of gene dosage is critical for the development of the early embryo and the extraembryonic tissues that support it. Specifically, loss ofCdx2 in vivoleads to stunted development of the allantois, an extraembryonic mesoderm-derived structure 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. We generate an allelic series forCDX2in human induced pluripotent stem cells (hiPSCs) consisting of WT, heterozygous, and homozygous nullCDX2genotypes, differentiate these cells in a 2D gastruloid model, and subject these cells to multiomic single nucleus RNA and ATAC sequencing. We identify several genes that CDX2 dose-dependently regulate cytoskeletal integrity and adhesiveness in the extraembryonic mesoderm population, including regulators of the VEGF, canonical WNT, and non-canonical WNT signaling pathways. 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 dosage is reduced. Finally, because the loss of CDX2 or TBXT phenocopy one anotherin 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 communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability, includingANK3andANGPT1. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and suggest these genes may underlie the defects in vascular development and allantoic elongation seen in the absence or reduction of CDX2in vivo.Summary StatementUsing 2D human gastruloids, CDX2 is shown to dose-dependently influence genes related to tissue permeability, cell-cell adhesions, and cytoskeletal architecture during extraembryonic mesoderm development.