Temporal changes in Hox gene expression accompany endothelial cell differentiation of embryonic stem cells

Abstract

In pluripotent embryonic stem cells (ESCs), expression of the Hox master regulatory transcription factors that play essential roles in organogenesis, angiogenesis, and maintenance of differentiated tissues, is globally suppressed. We investigated whether differentiation of endothelial cells (ECs) from mouse ESCs was accompanied by activation of distinct Hox gene expression profiles. Differentiation was observed within 3 days, as indicated by the appearance of cells expressing specific endothelial marker genes (Flk-1+ /VE-Cadherin+ ). Expression of HoxA3 and HoxD3, which drive adult endothelial cell invasion and angiogenesis, peaked at day 3 and declined thereafter, whereas expression of HoxA5 and HoxD10, which maintain a mature quiescent EC phenotype, was low at day 3, but increased over time. The temporal and reciprocal changes in HoxD3 and HoxA5 expression were accompanied by corresponding changes in expression of established downstream target genes including integrin β3 and Thrombospondin-2. Our results indicate that differentiation and maturation of ECs derived from cultured ESCs mimic changes in Hox gene expression that accompany maturation of immature angiogenic endothelium into differentiated quiescent endothelium in vivo.

Figure 1

Differentiation of mouse embryonic stem cells (ESCs). Photomicrographs showing adherent cells emerging from embryoid bodies (EBs) cultured in differentiation media after one day (A) and 18 days (B). (C) Histogram showing the relative levels of Oct-4 mRNA expression in EBs cultured in differentiation media at various time points. Results are normalized to the GusB (internal standard) and are expressed relative to day zero. Error bars indicate standard deviation. “**” denotes a statistical difference (p < 0.005) between each individual time point and day zero (n = 3).

Figure 2

Differentiating ESCs express mesenchymal, epithelial and endothelial markers during differentiation. Histogram showing mRNA expression levels of the mesenchymal lineage markers BMPR (A) and K14 (B), during differentiation of ESCs, as measured by real time PCR. Corresponding mRNA levels of VE-Cadherin (C) and Flk-1 (D) in EBs on days 1–18 during culturing in differentiation media. Results are normalized to the GusB (internal standard) and are expressed relative to day zero. Error bars indicate standard deviation. “*” and “**” denote statistical differences (p < 0.05) and (p < 0.005) between each individual time point and day zero, respectively (n = 3).

Figure 3

Onset of endothelial lineage marker expression during differentiation of ESCs. Cell subsets expressing endothelial surface protein markers VE-Cadherin (A) and Flk-1 (B) during EC differentiation of ESCs, as measured by flow cytometry. Results are expressed relative to day zero. “*” and “**” denote statistical differences (p < 0.05) and (p < 0.005) between each individual time point and day zero, respectively (n = 3). Error bars indicate standard deviation. (C and D) Flow cytometry sorting of cells double-positive for endothelial cell markers (Flk-1⁺/VE-Cadherin⁺) before (C) and after differentiation for three days (D).

Figure 4

Early expression of the pro-angiogenic HoxA3 during EC differentiation. Histograms show mRNA levels of HoxA3 (A) and putative target genes uPAR (B) and CCL-2 (C) in sorted cells that were double-positive for endothelial markers (Flk-1⁺/VE-Cadherin⁺), as measured by real-time PCR. Results are normalized to the GusB (internal standard) and are expressed relative to day zero. Error bars indicate standard deviation. “*” denotes a statistical difference (p < 0.05) between each individual time point and day 0.

Figure 5

Early expression pro-angiogenic HoxD3 and integrin β3 in double-positive cells (Flk-1⁺/VE-Cadherin⁺) during EC differentiation. Histograms show mRNA levels of HoxD3 (A) and its target gene integrin β3 (B) in sorted double positive (Flk-1⁺/VE-Cadherin⁺) cells measured by real-time PCR. (C) Relative HoxD3 and Integrin β3 mRNA levels at day 3 following treatment with control (scrambled) or siRNA against HoxD3 and shows a marked reduction in HoxD3 mRNA and a corresponding loss of integrin β3 mRNA levels following knockdown of HoxD3. Results are normalized to the GusB (internal standard) and are expressed relative to day zero or control. Error bars indicate standard deviation. “*” and “**” denote a statistical difference (p < 0.05) and (p < 0.005) between each individual time point and day zero or control, respectively.

Figure 6

Delayed expression of Hox genes linked to maturation of EC phenotype during EC differentiation. Histograms show relative mRNA expression of Hox D10 (A) and Hox A5 (C), and respective target genes MMP14 (B) and Thrombospondin-2 (D), in sorted cells double-positive for endothelial markers (Flk-1⁺/VE-Cadherin⁺), as measured by real-time PCR at various time point during EC differentiation. (E) Relative mRNA levels for HoxA5 and Thsp-2 in differentiating ECs treated with scrambled (control) or siRNA against HoxA5, at day 17 and corresponding reduction in HoxA5 and Thrombospondin-2 mRNA levels. Results are normalized to the GusB (internal standard) and are expressed relative to day zero or control. Error bars indicate standard deviation. “*” and “**” denote statistical differences (p < 0.05) and (p < 0.005) between each individual time point and day 0 or control, respectively.

Figure 7

Temporal changes in Hox gene expression during endothelial differentiation of mouse ESCs. Schematic showing the temporal changes in Hox mRNA during EC differentiation and the corresponding angiogenic stage linked to expression of each Hox gene. Hox A3 and Hox D3, which promote migration and invasion of angiogenic sprouts, peak at early time points after induction of EC differentiation of ESCs. Expression of HoxA5 and HoxD10, which maintain a quiescent, mature EC phenotype, increases over time in culture.