CDX2 regulates multiple trophoblast genes in bovine trophectoderm CT‐1 cells

Abstract

The bovine trophectoderm (TE) undergoes a dramatic morphogenetic transition prior to uterine endometrial attachment. Many studies have documented trophoblast-specific gene expression profiles at various pre-attachment stages, yet genetic interactions within the transitioning TE gene regulatory network are not well characterized. During bovine embryogenesis, transcription factors OCT4 and CDX2 are co-expressed during early trophoblast elongation. In this study, the bovine trophectoderm-derived CT-1 cell line was utilized as a genetic model to examine the roles of CDX2 and OCT4 within the bovine trophoblast gene regulatory network. An RT-PCR screen for TE-lineage transcription factors identified expression of CDX2, ERRB, ID2, SOX15, ELF5, HAND1, and ASCL2. CT-1 cells also express a nuclear-localized, 360 amino acid OCT4 ortholog of the pluripotency-specific human OCT4A. To delineate the roles of CDX2 and OCT4 within the CT-1 gene network, CDX2 and OCT4 levels were manipulated via overexpression and siRNA-mediated knockdown. An increase in CDX2 negatively regulated OCT4 expression, but increased expression of IFNT, HAND1, ASCL2, SOX15, and ELF5. A reduction of CDX2 levels exhibited a reciprocal effect, resulting in decreased expression of IFNT, HAND1, ASCL2, and SOX15. Both overexpression and knockdown of CDX2 increased ETS2 transcription. In contrast to CDX2, manipulation of OCT4 levels only revealed a positive autoregulatory mechanism and upregulation of ASCL2. Together, these results suggest that CDX2 is a core regulator of multiple trophoblast genes within CT-1 cells.

Single-cell gene expression of the bovine blastocyst

“…2009; Kuijk et al . 2012; Schiffmacher & Keefer 2013; Denicol et al . 2014) and was found to be highly expressed in all four TE populations in the present study.…”

“…The most commonly used marker for TE in bovine is CDX2 (Dobbs et al . 2013; Schiffmacher & Keefer 2013; Denicol et al . 2014) but the observation that outer cells of the blastocyst only gradually become committed to the TE lineage (Berg et al .…”

Transcriptional control of IFNT expression

“…Later, potential CDX2 binding sites were noted in a more proximal site at −248 to −47 bp of boIFNT-c1 (Sakurai, et al 2009) and between −414 to −235 bp in an ovine IFNT (Sakurai, et al 2010). CDX2 knockdown in bovine trophectoderm BT-1 (Sakurai, et al 2009) and CT-1 cells (Schiffmacher and Keefer 2013) (Fig 5B) has also implicated a role for CDX2 in controlling IFNT expression.…”

Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation

“…In addition, despite sharing the “pre-implanting” descriptor, cells derived from these stages can vary significantly, as developmental differences are considerable between D7 and D18: D7-D10 embryos are spherical blastocysts while in D12-D18, they are already highly differentiated [36], and can in fact be 5–15 days post-implantation in rodents, primates, or lagomorphs. Therefore, cell derivation at these stages does not lead to equivalent phenotypes, as confirmed by a comparison of trophoblast CT-1 cells (derived from D10-D11 embryos [37]) with ours.…”

“…Therefore, cell derivation at these stages does not lead to equivalent phenotypes, as confirmed by a comparison of trophoblast CT-1 cells (derived from D10-D11 embryos [37]) with ours. The former resembled those of an ovoid stage (D12-D13 [36]), while the latter resembled those of a filamentous stage (D18) (bi-nucleated cells; DLX3 , PAG1 , PRL expression). Similarly, some of the bXECs displayed a multi-nucleated phenotype (S2 Fig) that had not previously been observed in endoderm cell cultures (D10-12; cow or pig endoderm: CE-1, CE-2 [37]; PE-1, PE2 [38]).…”