Tissue organization alters gene expression in equine induced trophectoderm cells

Rapid morphological and gene expression changes occur during the early formation of a mammalian blastocyst. Critical to successful retention of the blastocyst and pregnancy is a functional trophectoderm (TE) that supplies the developing embryo with paracrine factors and hormones. The contribution of TE conformational changes to gene expression was examined in equine induced trophoblast (iTr) cells. Equine iTr cells were cultured as monolayers or in suspension to form spheres. The spheres are hollow and structurally reminiscent of native equine blastocysts. Total RNA was isolated from iTr monolayers and spheres and analyzed by RNA sequencing. An average of 32.2 and 31 million aligned reads were analyzed for the spheres and monolayers, respectively. Forty-four genes were unique to monolayers and 45 genes were expressed only in spheres. Conformation did not affect expression of CDX2, POU5F1, TEAD4, ETS2, ELF3, GATA2 or TFAP2A, the core gene network of native TE. Bioinformatic analysis was used to identify classes of genes differentially expressed in response to changes in tissue shape. In both iTr spheres and monolayers, the majority of the differentially expressed genes were associated with binding activity in cellular, developmental and metabolic processes. Inherent to protein:protein interactions, several receptor-ligand families were identified in iTr cells with enrichment of genes coding for PI3-kinase and MAPK signaling intermediates. Our results provide evidence for ligand initiated kinase signaling pathways that underlie early trophectoderm structural changes.