Tcea3 regulates the vascular differentiation potential of mouse embryonic stem cells

Abstract

Tcea3 is present in high concentrations in mouse embryonic stem cells (mESCs) and functions to activate Lefly1, a negative regulator of Nodal signaling. The Nodal pathway has numerous biological activities, including mesoderm induction and patterning in early embryogenesis. Here, we demonstrate that the suppression of Tcea3 in mESCs shifts the cells from pluripotency into enhanced mesoderm development. Vascular endothelial growth factor A (VEGFA) and VEGFC, major transcription factors that regulate vasculogenesis, are activated in Tcea3 knocked down (Tcea3 KD) mESCs. Moreover, differentiating Tcea3 KD mESCs have perturbed gene expression profiles with suppressed ectoderm and activated mesoderm lineage markers. Most early differentiating Tcea3 KD cells expressed Brachyury-T, a mesoderm marker, whereas control cells did not express the gene. Finally, development of chimeric embryos that included Tcea3 KD mESCs was perturbed.

Figure 1

Gene expression profiles of Tcea3 KD mESCs. (A) Scatter plots of cDNA microarray analysis of Tcea3 KD mESCs (R ² = correlation coefficients) showing that 117 genes are activated and 94 genes are suppressed in Tcea3 KD mESCs. (B) Panther classification of 211 genes showed that major regulators of vasculogenesis are activated in Tcea3 KD mESCs. (C) Scatter plots of cDNA microarray analysis of differentiating Tcea3 KD mESCs (R ² = correlation coefficients) showing that 105 genes are activated and 170 genes are suppressed in Tcea3 KD mESCs. Differentiation of mESCs was induced by culturing in medium lacking LIF for 3 days. (D) Panther classification of 275 genes showed that 18 of 21 ectoderm lineage marker genes were suppressed, whereas 7 of 9 mesoderm lineage marker genes were activated.

Figure 2

Tcea3 KD mESCs have an enhanced vascular differentiation potential. (A) Tcea3 KD and WT mESCs were induced to differentiate by culturing in medium lacking LIF for 3 days, and differentiating cells were immunostained with the mesoderm marker Brachyury-T. (B) Tcea3 KD and WT mESCs were induced to differentiate by culturing in medium lacking LIF, in the presence or absence of VEGF and bFGF, for 3 days. Differentiating cell morphology was examined by light microscopy. (C) Tube formation of endothelial-like cells differentiated from J1 and Tcea3 KD mESCs. After endothelial differentiation of mESCs for 7 days, the cells were plated on Matrigel to evaluate the vessel formation potential of the differentiated endothelial-like cells. More vessel-like structures were observed in endothelial-like cells differentiated from Tcea3 KD cells compared to those from WT ESCs.

Figure 3

Embryos that developed from Tcea3 KD mESCs exhibit abnormal phenotypes including highly vascularized skin. (A) Comparison of chimeric embryos generated by injecting GFP-expressing WT or Tcea3 KD mESCs. Left panels, morphological features of representative chimeric embryos; middle panels, GFP signals from the corresponding embryos; right panels, H&E-stained embryo midsagittal sections. All embryos generated from WT cells appeared normal. By contrast, highly vascularized tissues were detected from some embryos generated from Tcea3 KD as indicated by the asterisks. (B) The efficiency of chimeric embryo formation from blastocysts containing WT or Tcea3 KD mESCs. Whereas all embryos generated by WT cells look normal, some chimeric embryos generated by Tcea3 KD cells appeared developmentally abnormal.