Final Report Summary - FRINGEHE (Uncovering the Role of Notch Modifiers in the Generation of Hematopoietic Stem Cells in the Embryo)
Hematopoietic stem cells (HSCs) are first detected around day 10.5 of mouse embryonic gestation in the dorsal aorta of the Aorta-Gonad-Mesonephros (AGM) region. The specialization of an aortic hemogenic endothelium and the consequence formation of intra-aortic hematopoietic clusters precede the generation of HSCs. This endothelial-to-hematopoietic transition (EHT) is governed by a complex genetic network that is still poorly understood.
One of the remaining challenges in Embryonic Stem Cell and induced iPS-cell research is the generation and maintenance of functional HSCs that are crucial for cellular therapies involving hematopoietic transplantation. Consequently, it is fundamental to fully identify the regulators of definitive HSC emergence that takes place in the embryo. Only after understanding the molecular sequence of events that leads to the generation of a HSC during embryonic life, it will be possible to reproduce this process in vitro.
This project was elaborated and executed in order to generate novel and relevant data on the molecular mechanisms that control the generation of HSCs during embryonic gestation, in particular in disclosing the role of Notch signalling pathway on embryonic hematopoiesis. It is known that Notch signaling plays a dual function in embryonic dorsal aorta and both Notch ligands Dll4 and Jag1 are expressed in the aortic endothelium. While Notch1/Dll4 axis is essential for the specification of arterial identity of the dorsal aorta, Notch1/Jag1 axis is indispensable for the establishment of the hematopoietic determination. As both ligands are expressed in the aortic endothelium, it remains elusive how specific signals are transduced into different biological fates.
To better understand the Jag1-dependent gene expression pattern associated with the acquisition of the hematopoietic fate, we analysed the transcriptional program of specific AGM-derived subpopulations before the acquisition of the CD45 hematopoietic marker. Interestingly, incubation of endothelial cells on Jag1-, but not Dll4-expressing stroma, first imposed on these cells a significant decrease of their endothelial-specific signature, therefore resembling the a hematopoietic precursor population.
By analysing AGM cell populations in the Jag1-deficient embryos, we found an increased expression of Notch target genes, concomitant with an up-regulated endothelial transcriptional program, both in the endothelium and in hematopoietic-fated cells, whereas expression of Dll4 ligand remained similar to wild-type counterpart populations.
With the accomplishment of this project, it is uncovered a Notch ligand-specific transcriptional response by exposing purified subpopulations of the AGM region to Jag1 or Dll4 ligands. Applying both in vitro and in vivo complex methodologies, it is for the first time demonstrated that Jag1 counteracts Dll4-induced Notch activation governing the loss of endothelial identity during HSC specification in the dorsal aorta.
This is a pioneering project in the field of embryonic hematopoiesis addressing very pertinent and specific questions that have never been addressed so far. The repercussion of the results generated on elucidation of definitive hematopoiesis will undoubtedly advance the current available knowledge in this area of research. Finally, knowledge generated by the accomplishment of this project has great importance in the design of protocols for in vitro generation of HSCs, a relevant issue in regenerative medicine, in particular as a source of cells suitable for transplantation in patients without compatible blood donors.
One of the remaining challenges in Embryonic Stem Cell and induced iPS-cell research is the generation and maintenance of functional HSCs that are crucial for cellular therapies involving hematopoietic transplantation. Consequently, it is fundamental to fully identify the regulators of definitive HSC emergence that takes place in the embryo. Only after understanding the molecular sequence of events that leads to the generation of a HSC during embryonic life, it will be possible to reproduce this process in vitro.
This project was elaborated and executed in order to generate novel and relevant data on the molecular mechanisms that control the generation of HSCs during embryonic gestation, in particular in disclosing the role of Notch signalling pathway on embryonic hematopoiesis. It is known that Notch signaling plays a dual function in embryonic dorsal aorta and both Notch ligands Dll4 and Jag1 are expressed in the aortic endothelium. While Notch1/Dll4 axis is essential for the specification of arterial identity of the dorsal aorta, Notch1/Jag1 axis is indispensable for the establishment of the hematopoietic determination. As both ligands are expressed in the aortic endothelium, it remains elusive how specific signals are transduced into different biological fates.
To better understand the Jag1-dependent gene expression pattern associated with the acquisition of the hematopoietic fate, we analysed the transcriptional program of specific AGM-derived subpopulations before the acquisition of the CD45 hematopoietic marker. Interestingly, incubation of endothelial cells on Jag1-, but not Dll4-expressing stroma, first imposed on these cells a significant decrease of their endothelial-specific signature, therefore resembling the a hematopoietic precursor population.
By analysing AGM cell populations in the Jag1-deficient embryos, we found an increased expression of Notch target genes, concomitant with an up-regulated endothelial transcriptional program, both in the endothelium and in hematopoietic-fated cells, whereas expression of Dll4 ligand remained similar to wild-type counterpart populations.
With the accomplishment of this project, it is uncovered a Notch ligand-specific transcriptional response by exposing purified subpopulations of the AGM region to Jag1 or Dll4 ligands. Applying both in vitro and in vivo complex methodologies, it is for the first time demonstrated that Jag1 counteracts Dll4-induced Notch activation governing the loss of endothelial identity during HSC specification in the dorsal aorta.
This is a pioneering project in the field of embryonic hematopoiesis addressing very pertinent and specific questions that have never been addressed so far. The repercussion of the results generated on elucidation of definitive hematopoiesis will undoubtedly advance the current available knowledge in this area of research. Finally, knowledge generated by the accomplishment of this project has great importance in the design of protocols for in vitro generation of HSCs, a relevant issue in regenerative medicine, in particular as a source of cells suitable for transplantation in patients without compatible blood donors.