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

摘要

class="head no_bottom_margin" id="sec1title">IntroductionHematopoietic stem cells (HSCs) are specified during embryonic development from a subset of arterial endothelial cells located in the floor of the dorsal aorta (DA). HSCs emerge by a process termed the endothelial-to-hematopoietic transition (EHT) (, , ). In zebrafish, the hematopoietic stem and progenitor cells (HSPCs) generated by EHT migrate to the caudal hematopoietic tissue (CHT), where they proliferate and undergo differentiation into erythroid and myeloid lineages (, ). Some will exit the CHT and migrate to the thymus to give rise to T cells, and others move to the kidney, the adult site of hematopoiesis in the zebrafish, equivalent to the bone marrow in mammals ().The transcription factor Runx1 is required for EHT in mice and zebrafish (, ). Its expression in the floor of the DA is initiated by 23 hpf in zebrafish () and marks a cell population committed to the hemogenic fate, the hemogenic endothelium (HE). Several signaling pathways including Hedgehog, VEGF, Notch and BMP are required sequentially to regulate programming of the arterial endothelium and HSPC emergence (, , , ). The Notch receptor Notch1 is the main driver of HSPC emergence from HE, likely downstream of its ligand Jagged1 (, , href="#bib28" rid="bib28" class=" bibr popnode">Jang et al., 2015) and is thought to drive runx1 expression via Gata2 (href="#bib56" rid="bib56" class=" bibr popnode">Robert-Moreno et al., 2005). Jagged1 is dispensable for arterial programming but required in the endothelium for the specification of HSPCs (href="#bib17" rid="bib17" class=" bibr popnode">Espin-Palazon et al., 2014, href="#bib18" rid="bib18" class=" bibr popnode">Gama-Norton et al., 2015, href="#bib57" rid="bib57" class=" bibr popnode">Robert-Moreno et al., 2008).In humans, defective transforming growth factor β (TGFβ) signaling is associated with proliferative disorders of HSPCs such as acute myeloid leukemia and T cell acute lymphoblastic leukemia (href="#bib32" rid="bib32" class=" bibr popnode">Kim and Letterio, 2003). More recently, it has been shown that paracrine TGFβ signaling in the bone marrow niche maintains quiescence of the resident HSC pool (href="#bib68" rid="bib68" class=" bibr popnode">Zhao et al., 2014) and may also direct differentiation of lineage-biased HSC subtypes (href="#bib7" rid="bib7" class=" bibr popnode">Challen et al., 2010), positioning TGFβ as a critical regulator of proliferation and differentiation of adult HSCs. Whether TGFβ plays a role in the formation of HSCs is however not known. Mutants for the ligand TGFβ1 or its receptor TGFβR2, including endothelial-specific conditional knockout mice, die between E9.5 and E10.5 due to defective recruitment of mural cells to the yolk sac vasculature and the subsequent loss of vessel integrity (href="#bib69" rid="bib69" class=" bibr popnode">Carvalho et al., 2004, href="#bib15" rid="bib15" class=" bibr popnode">Dickson et al., 1995, href="#bib52" rid="bib52" class=" bibr popnode">Oshima et al., 1996). This is before the emergence of HSPCs in the embryo proper (href="#bib13" rid="bib13" class=" bibr popnode">de Bruijn et al., 2002), effectively precluding the analysis of the role of TGFβ signaling in HSPC specification in mice. Zebrafish, however, develop externally and do not depend on extraembryonic tissues for survival. In addition, recruitment of mural cells to the endothelium does not happen until 72 hpf (href="#bib60" rid="bib60" class=" bibr popnode">Santoro et al., 2009), 2 days after the HSPCs are specified in the DA. Thus, we can address the role of TGFβ in HSPC emergence in zebrafish without the inherent limitations of the mouse models.The TGFβ superfamily comprises BMPs, Activins, Nodals, and TGFβs. There are three TGFβ ligands in the mouse: TGFβ1, TGFβ2, and TGFβ3 (href="#bib23" rid="bib23" class=" bibr popnode">Goumans and Mummery, 2000), and they all signal through a single type II serine-threonine kinase receptor (TGFβR2) that recruits the type I receptors Activin-like kinase 1 (Alk1) or Alk5 (href="#bib61" rid="bib61" class=" bibr popnode">Shi and Massague, 2003). Alk1 expression is essentially restricted to endothelial cells (ECs) (href="#bib51" rid="bib51" class=" bibr popnode">Oh et al., 2000), whereas Alk5 is more broadly expressed (href="#bib24" rid="bib24" class=" bibr popnode">Goumans et al., 2002) but also present in ECs. Activated Alk1 phosphorylates Smad1, Smad5, and Smad9, whereas activated Alk5 phosphorylates Smad2 and Smad3 (href="#bib61" rid="bib61" class=" bibr popnode">Shi and Massague, 2003). Activated Smads migrate to the nucleus together with the co-Smad Smad4 and regulate transcription together with co-activators or co-repressors (href="#bib61" rid="bib61" class=" bibr popnode">Shi and Massague, 2003). In addition, TGFβ can signal through the non-canonical Erk, JNK, and p38 MAPK kinase pathways to instigate transcriptional responses (href="#bib14" rid="bib14" class=" bibr popnode">Derynck and Zhang, 2003). Thus, to circumvent the complexity of the intracellular signaling elicited by TGFβ, we focused our attention on TgfβR2, the type II receptor for TGFβ. Abrogation of TGFβR2 activity revealed that TGFβ signaling plays a key role in the formation of HSPCs. TGFβ is required for the correct programming of the HE downstream of Vegf and independently of arterial programming. We demonstrate that Jag1a is a target of TGFβ signaling, and jag1a overexpression in endothelium rescues the loss of HSPCs in tgfbR2-depleted embryos. Finally, we identified two independent sources of ligand: TGFβ1a and TGFβ1b in the endothelium and TGFβ3 in the nearby notochord. Both inputs contribute to the regulation of jag1a in endothelium through the TgfβR2 receptor and thus enable Notch signaling to program the HE prior to specification of HSPCs.