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Activation of the TGFβ pathway impairs endothelial to haematopoietic transition.

Vargel Ö, Zhang Y, Kosim K, Ganter K, Foehr S, Mardenborough Y, Shvartsman M, Enright AJ, Krijgsveld J, Lancrin C - Sci Rep (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Mouse Biology Unit, Via Ercole Ramarini 32, 00015 Monterotondo, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

TGFβ treatment during BL-CFC culture favours the vascular lineage over the haematopoietic one.(A) Experimental workflow of the quantitative secretome analysis by LC-MS/MS. (B) Flow cytometry analysis of VE-Cad and CD41 expression after 6 hours of culture following the addition of TGFβ2 compared to control condition. Upper panel shows one representative example of flow cytometry analysis while the bottom one shows a bar graph representing the average frequency of the EC (VE-Cad+CD41−) and HPC (VE-Cad−CD41+) populations from 3 independent experiments. The p-values were calculated with Student’s t-test (2 tails, type 3). EC population: **Control versus TGFβ2 p-value = 0.004 (n = 3); HPC population: *Control versus TGFβ2 p-value = 0.03 (n = 3). (C) Heatmap representing Log2 fold change (Log2 FC) of protein expression between TGFβ2 treated and non-treated samples for all the proteins detected in the 3 biological replicates. Each column represents one independent secretome experiment. (D) STRING representation of a network involving the proteins detected in C. Nine proteins out of 33 were not part of the network. Different line colours represent the types of evidence for the association between proteins.
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f2: TGFβ treatment during BL-CFC culture favours the vascular lineage over the haematopoietic one.(A) Experimental workflow of the quantitative secretome analysis by LC-MS/MS. (B) Flow cytometry analysis of VE-Cad and CD41 expression after 6 hours of culture following the addition of TGFβ2 compared to control condition. Upper panel shows one representative example of flow cytometry analysis while the bottom one shows a bar graph representing the average frequency of the EC (VE-Cad+CD41−) and HPC (VE-Cad−CD41+) populations from 3 independent experiments. The p-values were calculated with Student’s t-test (2 tails, type 3). EC population: **Control versus TGFβ2 p-value = 0.004 (n = 3); HPC population: *Control versus TGFβ2 p-value = 0.03 (n = 3). (C) Heatmap representing Log2 fold change (Log2 FC) of protein expression between TGFβ2 treated and non-treated samples for all the proteins detected in the 3 biological replicates. Each column represents one independent secretome experiment. (D) STRING representation of a network involving the proteins detected in C. Nine proteins out of 33 were not part of the network. Different line colours represent the types of evidence for the association between proteins.

Mentions: After 1 day of BL-CFC culture, cells were treated for 6 hours with TGFβ211 or with the vehicle as a negative control in presence of AHA, ‘intermediate’ or ‘heavy’ isotopes of lysine and arginine. Supernatants from the 2 conditions were harvested and pooled; AHA-containing proteins were isolated with click chemistry and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) (Fig. 2A). In the meantime the cells were harvested for flow cytometry analysis and examined for the expression of CD41 and VE-Cad markers. Surprisingly we noticed that the 6 hours TGFβ2 treatment increased the frequency of EC (VE-Cad+CD41−) by 4-fold compared to the non-treated condition whereas the frequency of HPC (VE-Cad−CD41+) was reduced by roughly 2-fold (Fig. 2B).


Activation of the TGFβ pathway impairs endothelial to haematopoietic transition.

Vargel Ö, Zhang Y, Kosim K, Ganter K, Foehr S, Mardenborough Y, Shvartsman M, Enright AJ, Krijgsveld J, Lancrin C - Sci Rep (2016)

TGFβ treatment during BL-CFC culture favours the vascular lineage over the haematopoietic one.(A) Experimental workflow of the quantitative secretome analysis by LC-MS/MS. (B) Flow cytometry analysis of VE-Cad and CD41 expression after 6 hours of culture following the addition of TGFβ2 compared to control condition. Upper panel shows one representative example of flow cytometry analysis while the bottom one shows a bar graph representing the average frequency of the EC (VE-Cad+CD41−) and HPC (VE-Cad−CD41+) populations from 3 independent experiments. The p-values were calculated with Student’s t-test (2 tails, type 3). EC population: **Control versus TGFβ2 p-value = 0.004 (n = 3); HPC population: *Control versus TGFβ2 p-value = 0.03 (n = 3). (C) Heatmap representing Log2 fold change (Log2 FC) of protein expression between TGFβ2 treated and non-treated samples for all the proteins detected in the 3 biological replicates. Each column represents one independent secretome experiment. (D) STRING representation of a network involving the proteins detected in C. Nine proteins out of 33 were not part of the network. Different line colours represent the types of evidence for the association between proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759586&req=5

f2: TGFβ treatment during BL-CFC culture favours the vascular lineage over the haematopoietic one.(A) Experimental workflow of the quantitative secretome analysis by LC-MS/MS. (B) Flow cytometry analysis of VE-Cad and CD41 expression after 6 hours of culture following the addition of TGFβ2 compared to control condition. Upper panel shows one representative example of flow cytometry analysis while the bottom one shows a bar graph representing the average frequency of the EC (VE-Cad+CD41−) and HPC (VE-Cad−CD41+) populations from 3 independent experiments. The p-values were calculated with Student’s t-test (2 tails, type 3). EC population: **Control versus TGFβ2 p-value = 0.004 (n = 3); HPC population: *Control versus TGFβ2 p-value = 0.03 (n = 3). (C) Heatmap representing Log2 fold change (Log2 FC) of protein expression between TGFβ2 treated and non-treated samples for all the proteins detected in the 3 biological replicates. Each column represents one independent secretome experiment. (D) STRING representation of a network involving the proteins detected in C. Nine proteins out of 33 were not part of the network. Different line colours represent the types of evidence for the association between proteins.
Mentions: After 1 day of BL-CFC culture, cells were treated for 6 hours with TGFβ211 or with the vehicle as a negative control in presence of AHA, ‘intermediate’ or ‘heavy’ isotopes of lysine and arginine. Supernatants from the 2 conditions were harvested and pooled; AHA-containing proteins were isolated with click chemistry and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) (Fig. 2A). In the meantime the cells were harvested for flow cytometry analysis and examined for the expression of CD41 and VE-Cad markers. Surprisingly we noticed that the 6 hours TGFβ2 treatment increased the frequency of EC (VE-Cad+CD41−) by 4-fold compared to the non-treated condition whereas the frequency of HPC (VE-Cad−CD41+) was reduced by roughly 2-fold (Fig. 2B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Mouse Biology Unit, Via Ercole Ramarini 32, 00015 Monterotondo, Italy.

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.

No MeSH data available.


Related in: MedlinePlus