Abstract
The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFβ signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFβ activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFβ pathway. Quantitative proteomics analysis showed that TGFβ treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFβ blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFβ signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFβ signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFβ pathway does not enhance EHT as we hypothesised but instead impairs it.
Highlights
I receptors become active and phosphorylate and activate a group of receptor regulated SMAD (R-SMAD) proteins (SMAD2 and 3 for TGFBR1)
After isolating mesodermal Flk1+ containing Blast-Colony-Forming Cells (BL-CFC), common precursor of blood and endothelial cells[19,20], from day 3–3.25 embryoid body (EB) differentiation and putting them in culture in presence of VEGF and IL6, we can follow the formation of blood cells from endothelial cells (EC)
To find out if any of these populations could respond to TGFβ signalling, we performed quantitative RT-PCR (q-RT-PCR) to detect 15 genes coding for proteins involved in the TGFβ signalling pathway: 4 receptors, 3 ligands and 8 SMADs (Fig. 1 and Supplementary Table S1)
Summary
I receptors become active and phosphorylate and activate a group of receptor regulated SMAD (R-SMAD) proteins (SMAD2 and 3 for TGFBR1). The phosphorylated R-SMADs later form heterodimers with the common mediator SMAD proteins (SMAD4) and localize into the nucleus where they activate transcription of target genes. Another type of SMAD proteins, inhibitory SMAD (SMAD6 and 7), can block the phosphorylation of R-SMADs, which cuts off the down-stream relay of the signal[13]. Haematopoietic progenitor cells (HPC) are formed through the process of EHT in the ESC differentiation model[16,17] We have used this system to test whether or not activation of TGFβ signalling enhances EHT like it does for EMT and EndMT. Unlike the promoting effect of TGFβ in EMT and EndMT, we found that the TGFβ signalling inhibits EHT
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