Molecular Reproduction and Development, October 2013, Vol.80(10), pp.826-839
Byline: Andrew T. Schiffmacher, Carol L. Keefer SUMMARY 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. Mol. Reprod. Dev. 80: 826-839, 2013. [c] 2013 Wiley Periodicals, Inc. Supporting information: Additional Supporting Information may be found in the online version of this article Additional supporting information may be found in the online version of this article at the publisher's web-site. CAPTION(S): Figure S1. Expression profile of selected genes in the CT-1 cell line that are also expressed during bovine or mouse trophoblast development. Three replicates of cDNAs from CT-1 cells grown on a collagen substrate or substrate-free CellBIND[R] were tested. cDNAs derived from CT-1 cells cultured on STO mouse embryonic fibroblasts or STO cells alone were analyzed as controls for STO cDNA contamination. cDNAs from mid-gestation cotyledon and pooled IVP blastocysts served as positive controls where appropriate. Figure S2. siRNA mediated downregulation of OCT4 reduces endogenous OCT4 levels, but does not significantly regulate gene expression in a subset of bovine trophoblast-specific genes. Data are represented as the siRNA treatment means and standard errors of normalized Ct values relative to the ACTG1-normalized, non-transfected-control treatment Ct mean (set at 1) for each gene. Means are generated from 3 independent experimental cDNA replicates. Asterisks (*) located above values denote a significant difference between that treatment value and all other values of the corresponding gene (P 0.05). Black bars indicate nontransfected control levels, dark gray bars indicate mis-match siRNA-transfected control levels, and light gray bars indicate OCT4 siRNA-transfected levels. Figure S3. mRNA levels of transcription factors CDX2 (A) and OCT4 (B) following siRNA knockdown in CT-1 cells. Two siRNA concentrations (50 vs. 100nM) were tested at two Lipitoid/DNA ([+ or -]) charge ratios (3/1 vs. 4/1). For each treatment, a non-specific control siRNA was also transfected to serve as a negative control. Each transfection treatment is expressed as the mean and standard error (n=3) of the siRNA target relative expression level normalized to treatment control levels. An asterisk (*) denotes a significant difference between treatment effect and untransfected control (P 0.05).
Transcription (Genetics) -- Analysis ; Genetic Research -- Analysis ; Genes -- Analysis;