Activation of the TGFβ pathway impairs endothelial to haematopoietic transition

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.

PAR1 Scaffolds TGFβRII to Downregulate TGF-β Signaling and Activate ESC Differentiation to Endothelial Cells

“…Intriguingly, downregulation of PAR1 expression as well as direct agonist activation of PAR1 suppressed neovascularization through forcing the association of TGFβRII to TGFβRI, and thereby activating TGF-β signaling (Figure 4H). PAR1 in its inactive state prevented TGF-β signaling by binding TGFβRII, and thus blocked the TGFβRII interaction with TGFβRI required for activation of the TGF-β pathway (Vargel et al., 2016). However, in the absence of PAR1, TGFβRII was free to bind TGFβRI resulting in unfettered TGF-β signaling, which also blocked mESC differentiation to ECs.…”

Quantification of Mouse Hematopoietic Progenitors’ Formation Using Time-lapse Microscopy and Image Analysis

“…Here, by combining in vitro time-lapse imaging of an adherent BL-CFC culture with automatic image analysis we introduce a simple and efficient method to quantify those round cells during a culture period, which gives a direct measure of the number of cells undergoing EHT. This protocol enables us to easily test novel parameters affecting EHT rate such as over-expression of certain transcription factors (Bergiers et al ., 2018) or testing pathway inhibiting small molecules in the culture media (Vargel et al ., 2016). Below, we describe the details of the time-lapse microscopy of BL-CFC culture and image analysis to assess the number of cells underwent EHT.…”

Cells with hematopoietic potential reside within mouse proepicardium

“…These hematopoietic cells arise from a specific subpopulation of endothelial cells (EC) called the hemogenic endothelium (HE) (Dzierzak and Speck 2008; Bertrand et al 2010; Hirschi 2012), which is capable of endothelial-to-hematopoietic transition (EHT) (Kissa and Herbomel 2010; Vargel et al 2016). The presence of HE and the EHT occurrence during early period of development have been demonstrated in the ventral wall of the dorsal aorta at the level of the developing gonad/mesonephros, in the placenta, in the vitelline and umbilical arteries, as well as in the embryonic head and the somites (de Bruijn et al 2000; Gekas et al 2005; Li et al 2012; Antas et al 2013; Nakano et al 2013; Yzaguirre and Speck 2016).…”