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Transforming Growth Factor β Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells

View Article: PubMed Central - PubMed

ABSTRACT

Hematopoietic stem cells (HSCs) are self-renewing multipotent stem cells that generate mature blood lineages throughout life. They, together with hematopoietic progenitor cells (collectively known as HSPCs), emerge from hemogenic endothelium in the floor of the embryonic dorsal aorta by an endothelial-to-hematopoietic transition (EHT). Here we demonstrate that transforming growth factor β (TGFβ) is required for HSPC specification and that it regulates the expression of the Notch ligand Jagged1a in endothelial cells prior to EHT, in a striking parallel with the epithelial-to-mesenchymal transition (EMT). The requirement for TGFβ is two fold and sequential: autocrine via Tgfβ1a and Tgfβ1b produced in the endothelial cells themselves, followed by a paracrine input of Tgfβ3 from the notochord, suggesting that the former programs the hemogenic endothelium and the latter drives EHT. Our findings have important implications for the generation of HSPCs from pluripotent cells in vitro.

No MeSH data available.


Jag1a Is Required Downstream of TGFβ Signaling for HSC Specification(A) Expression of arterial markers in jag1aMO is unaffected when compared to wild-type embryos.(B) Expression of runx1, cmyb, and gfi1aa in wild-type and jag1a morphants (MO) at 28 hpf. All the markers analyzed are reduced or absent in jag1a morphants. Red arrowheads indicate the remaining gene expression in the floor of the DA of jag1a morphants.(C) HSPCs (yellow arrowheads) are severely reduced in the CHT of itga2b:GFP;Kdrl:HsRas-mCherry transgenic embryos at 48 hpf injected with the jag1aMO. Itga2b:GFP+ cells, magenta; Kdrl-HsRas:mCherry+ cells, green.(D) Overexpression of jag1a with a Kdrl:jag1-V5 construct partially rescues the loss of runx1 and cmyb in the floor of the DA. 15 pg of the construct was used for this experiment. The numbers of embryos are shown in each panel as the number of embryos with phenotype/total number analyzed.(E) Quantitation of the rescue effect observed in (D).See also Figure S6.
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fig6: Jag1a Is Required Downstream of TGFβ Signaling for HSC Specification(A) Expression of arterial markers in jag1aMO is unaffected when compared to wild-type embryos.(B) Expression of runx1, cmyb, and gfi1aa in wild-type and jag1a morphants (MO) at 28 hpf. All the markers analyzed are reduced or absent in jag1a morphants. Red arrowheads indicate the remaining gene expression in the floor of the DA of jag1a morphants.(C) HSPCs (yellow arrowheads) are severely reduced in the CHT of itga2b:GFP;Kdrl:HsRas-mCherry transgenic embryos at 48 hpf injected with the jag1aMO. Itga2b:GFP+ cells, magenta; Kdrl-HsRas:mCherry+ cells, green.(D) Overexpression of jag1a with a Kdrl:jag1-V5 construct partially rescues the loss of runx1 and cmyb in the floor of the DA. 15 pg of the construct was used for this experiment. The numbers of embryos are shown in each panel as the number of embryos with phenotype/total number analyzed.(E) Quantitation of the rescue effect observed in (D).See also Figure S6.

Mentions: To determine if Jag1a is required for arterial programming or HSPC specification in zebrafish, we knocked down jag1a with a specific morpholino (Yamamoto et al., 2010) and found no obvious defects in arterial programming compared with wild-type embryos (Figure 6A). However, expression of the HSPC markers runx1, cmyb, and gfi1aa was severely downregulated in jag1a morphants at 28 hpf (Figure 6B). Furthermore, itga2b:GFP+ HSPCs were nearly absent in the CHT of jag1a morphants by 48 hpf (Figure 6C) suggesting that the HE was mis-programmed and failed to give rise to HSPCs in the absence of Jag1a. A recent study showed that jag1a is regulated by TNFα through the TnfR2 receptor in ECs (Espin-Palazon et al., 2014). Expression of tnfR2 in ECs was unaffected in tgfbR2 morphants (Figure S6), suggesting that TGFβ does not regulate jag1a indirectly via regulation of tnfr2. To determine if Jag1a is the main target of TGFβ signaling in HSPC specification, we restored Jag1a expression specifically in the endothelium of tgfbr2MO1 morphants using a Kdrl:jag1a construct. Wild-type embryos overexpressing jag1a in ECs showed little effect on expression of runx1 or cmyb at 28 hpf (Figures 6D and 6E). However, forced expression of jag1a in the endothelium of tgfbR2 morphants rescued the loss of runx1 and cmyb expression (Figures 6D and 6E), confirming that the hematopoietic defects in tgfbR2 morphants are mainly due to loss of jag1a. We conclude that autocrine TGFβ1 and paracrine TGFβ3 signal to the endothelium through TgfβR2, inducing jag1a expression, which in turn induces HE programming and HSPC emergence.


Transforming Growth Factor β Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells
Jag1a Is Required Downstream of TGFβ Signaling for HSC Specification(A) Expression of arterial markers in jag1aMO is unaffected when compared to wild-type embryos.(B) Expression of runx1, cmyb, and gfi1aa in wild-type and jag1a morphants (MO) at 28 hpf. All the markers analyzed are reduced or absent in jag1a morphants. Red arrowheads indicate the remaining gene expression in the floor of the DA of jag1a morphants.(C) HSPCs (yellow arrowheads) are severely reduced in the CHT of itga2b:GFP;Kdrl:HsRas-mCherry transgenic embryos at 48 hpf injected with the jag1aMO. Itga2b:GFP+ cells, magenta; Kdrl-HsRas:mCherry+ cells, green.(D) Overexpression of jag1a with a Kdrl:jag1-V5 construct partially rescues the loss of runx1 and cmyb in the floor of the DA. 15 pg of the construct was used for this experiment. The numbers of embryos are shown in each panel as the number of embryos with phenotype/total number analyzed.(E) Quantitation of the rescue effect observed in (D).See also Figure S6.
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fig6: Jag1a Is Required Downstream of TGFβ Signaling for HSC Specification(A) Expression of arterial markers in jag1aMO is unaffected when compared to wild-type embryos.(B) Expression of runx1, cmyb, and gfi1aa in wild-type and jag1a morphants (MO) at 28 hpf. All the markers analyzed are reduced or absent in jag1a morphants. Red arrowheads indicate the remaining gene expression in the floor of the DA of jag1a morphants.(C) HSPCs (yellow arrowheads) are severely reduced in the CHT of itga2b:GFP;Kdrl:HsRas-mCherry transgenic embryos at 48 hpf injected with the jag1aMO. Itga2b:GFP+ cells, magenta; Kdrl-HsRas:mCherry+ cells, green.(D) Overexpression of jag1a with a Kdrl:jag1-V5 construct partially rescues the loss of runx1 and cmyb in the floor of the DA. 15 pg of the construct was used for this experiment. The numbers of embryos are shown in each panel as the number of embryos with phenotype/total number analyzed.(E) Quantitation of the rescue effect observed in (D).See also Figure S6.
Mentions: To determine if Jag1a is required for arterial programming or HSPC specification in zebrafish, we knocked down jag1a with a specific morpholino (Yamamoto et al., 2010) and found no obvious defects in arterial programming compared with wild-type embryos (Figure 6A). However, expression of the HSPC markers runx1, cmyb, and gfi1aa was severely downregulated in jag1a morphants at 28 hpf (Figure 6B). Furthermore, itga2b:GFP+ HSPCs were nearly absent in the CHT of jag1a morphants by 48 hpf (Figure 6C) suggesting that the HE was mis-programmed and failed to give rise to HSPCs in the absence of Jag1a. A recent study showed that jag1a is regulated by TNFα through the TnfR2 receptor in ECs (Espin-Palazon et al., 2014). Expression of tnfR2 in ECs was unaffected in tgfbR2 morphants (Figure S6), suggesting that TGFβ does not regulate jag1a indirectly via regulation of tnfr2. To determine if Jag1a is the main target of TGFβ signaling in HSPC specification, we restored Jag1a expression specifically in the endothelium of tgfbr2MO1 morphants using a Kdrl:jag1a construct. Wild-type embryos overexpressing jag1a in ECs showed little effect on expression of runx1 or cmyb at 28 hpf (Figures 6D and 6E). However, forced expression of jag1a in the endothelium of tgfbR2 morphants rescued the loss of runx1 and cmyb expression (Figures 6D and 6E), confirming that the hematopoietic defects in tgfbR2 morphants are mainly due to loss of jag1a. We conclude that autocrine TGFβ1 and paracrine TGFβ3 signal to the endothelium through TgfβR2, inducing jag1a expression, which in turn induces HE programming and HSPC emergence.

View Article: PubMed Central - PubMed

ABSTRACT

Hematopoietic stem cells (HSCs) are self-renewing multipotent stem cells that generate mature blood lineages throughout life. They, together with hematopoietic progenitor cells (collectively known as HSPCs), emerge from hemogenic endothelium in the floor of the embryonic dorsal aorta by an endothelial-to-hematopoietic transition (EHT). Here we demonstrate that transforming growth factor β (TGFβ) is required for HSPC specification and that it regulates the expression of the Notch ligand Jagged1a in endothelial cells prior to EHT, in a striking parallel with the epithelial-to-mesenchymal transition (EMT). The requirement for TGFβ is two fold and sequential: autocrine via Tgfβ1a and Tgfβ1b produced in the endothelial cells themselves, followed by a paracrine input of Tgfβ3 from the notochord, suggesting that the former programs the hemogenic endothelium and the latter drives EHT. Our findings have important implications for the generation of HSPCs from pluripotent cells in vitro.

No MeSH data available.