Endothelio-mesenchymal interaction controls runx1 expression and modulates the notch pathway to initiate aortic hematopoiesis.
ABSTRACT: Hematopoietic stem cells (HSCs) are produced by a small cohort of hemogenic endothelial cells (ECs) during development through the formation of intra-aortic hematopoietic cell (HC) clusters. The Runx1 transcription factor plays a key role in the EC-to-HC and -HSC transition. We show that Runx1 expression in hemogenic ECs and the subsequent initiation of HC formation are tightly controlled by the subaortic mesenchyme, although the mesenchyme is not a source of HCs. Runx1 and Notch signaling are involved in this process, with Notch signaling decreasing with time in HCs. Inhibiting Notch signaling readily increases HC production in mouse and chicken embryos. In the mouse, however, this increase is transient. Collectively, we show complementary roles of hemogenic ECs and mesenchymal compartments in triggering aortic hematopoiesis. The subaortic mesenchyme induces Runx1 expression in hemogenic-primed ECs and collaborates with Notch dynamics to control aortic hematopoiesis.
Project description:Hematopoietic stem/progenitor cells (HSPC) in zebrafish emerge from the aortic hemogenic endothelium (HE) and migrate towards the caudal hematopoietic tissue (CHT), where they expand and differentiate during definitive hematopoiesis. Phospholipase C gamma 1 (Plc?1) has been implicated for hematopoiesis in vivo and in vitro and is also required to drive arterial and HSPC formation. Genetic mutation in plcg1-/- (y10 allele) completely disrupts the aortic blood flow, specification of arterial fate, and HSPC formation in zebrafish embryos. We previously demonstrated that ginger treatment promoted definitive hematopoiesis via Bmp signaling. In this paper, we focus on HSPC development in plcg1-/- mutants and show that ginger/10-gingerol (10-G) can rescue the expression of arterial and HSPC markers in the HE and CHT in plcg1-/- mutant embryos. We demonstrate that ginger can induce scl/runx1 expression, and that rescued HE fate is dependent on Bmp and Notch. Bmp and Notch are known to regulate nitric oxide (NO) production and NO can induce hematopoietic stem cell fate. We show that ginger produces a robust up-regulation of NO. Taken together, we suggest in this paper that Bmp, Notch and NO are potential players that mediate the effect of ginger/10-G for rescuing the genetic defects in blood vessel specification and HSPC formation in plcg1-/- mutants. Understanding the molecular mechanisms of HSPC development in vivo is critical for understanding HSPC expansion, which will have a positive impact in regenerative medicine.
Project description:The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.
Project description:The human placenta is a source of hematopoietic stem and progenitor cells (HSPCs). The RUNX1 transcription factor is required for the formation of functional HSPCs. The impact of preeclampsia (PE) and preterm labor (PTL, spontaneous preterm labor [sPTL] and inflammatory preterm labor [iPTL]) on HSPC localization and RUNX1 expression in the human placenta is unknown.We compared the frequency and density of HSPC in control samples from sPTL (n = 6) versus PE (n = 6) and iPTL (n = 6). We examined RUNX1 protein and RNA expression in placentas from normal pregnancies (5-22 weeks, n = 8 total) and in placentas from the aforementioned pregnancy complications (n = 5/group).Hematopoietic stem and progenitor cells were rare cell types, associated predominantly with the vasculature of placental villi. The HSPC density was greater in the chorionic plate (CP) compared to the villi (P < .001) and greater in PE and iPTL samples as compared to controls within the CP (not significant) and overall (P < .05). During the fetal period, RUNX1 was expressed in the mesenchyme of the CP and villi. Inflammatory PTL samples were more likely to exhibit intraluminal RUNX1(+) cell populations (P < .001) and RUNX1(+) cell clusters attached to arterial endothelial cells.Placental HSPCs likely arise from hematopoietic niches comprised RUNX1(+) mesenchyme and vascular endothelium. Pregnancy complications that result in preterm birth differentially affect placental HSPC localization and RUNX1 expression. Our results support previous findings that inflammation positively regulates hematopoiesis. We present new evidence that hemogenic endothelium may be active at later stages of human fetal development in the context of inflammation.
Project description:During ontogeny, hematopoietic stem and progenitor cells arise from hemogenic endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in hemogenic endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects hemogenic endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFβ allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in hemogenic endothelium maturation and the establishment of the hematopoietic system.
Project description:Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC-derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina-induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor ? chain-null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood-derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence.
Project description:Intraembryonic hematopoiesis occurs at two different sites, the floor of the aorta and subaortic patches (SAPs) of the para-aortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) region. Notch1 and RBP-jkappa are critical for the specification of hematopoietic stem cells (HSCs) in Notch signal-receiving cells. However, the mechanism by which Notch signaling is triggered from the Notch signal-sending cells to support embryonic hematopoiesis remains to be determined. We previously reported that Mind bomb-1 (Mib1) regulates Notch ligands in the Notch signal-sending cells (B. K. Koo, M. J. Yoon, K. J. Yoon, S. K. Im, Y. Y. Kim, C. H. Kim, P. G. Suh, Y. N. Jan, and Y. Y. Kong, PLoS ONE 2:e1221, 2007). Here, we show that intraembryonic hematopoietic progenitors were absent in the P-Sp of Mib1(-/-) embryos, whereas they were partly preserved in the Tie2-cre; Mib1(f)(/f) P-Sps, suggesting that Mib1 plays a role in the endothelium and the SAPs. Interestingly, dll1 and dll4/Jag1 are expressed in the SAPs and the endothelium of the AGM, respectively, where mib1 is detected. Indeed, Notch signaling was activated in the nascent HSCs at both sites. In the P-Sp explant culture, the overexpression of Dll1 in OP9 stromal cells rescued the failed production of hematopoietic progenitors in the Mib1(-/-) P-Sp, while its activity was abolished by Mib1 knockdown. These results suggest that Mib1 is important for intraembryonic hematopoiesis not only in the aortic endothelium but also in the SAPs.
Project description:During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial-to-hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch-mediated EHT In vivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT Global or endothelial-specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1-pAKT as a novel molecular pathway that, in conjunction with the shh-vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs.
Project description:Runt-related transcription factor 1 (RUNX1) is required for definitive hematopoiesis; however, the functions of most human RUNX1 isoforms are unclear. In particular, the effects of RUNX1-205 (a novel splice variant that lacks exon 6 in comparison with RUNX1b) on human hematopoiesis are not clear. In this study, a human embryonic stem cell (hESC) line with inducible RUNX1-205 overexpression was established. Analyses of these cells revealed that induction of RUNX1-205 overexpression at early stage did not influence the induction of mesoderm but blocked the emergence of CD34+ cells, and the production of hematopoietic stem/progenitor cells was significantly reduced. In addition, the expression of hematopoiesis-related factors was downregulated. However, these effects were abolished when RUNX1-205 overexpression was induced after Day 6 in co-cultures of hESCs and AGM-S3 cells, indicating that the inhibitory effect occurred prior to generation of hemogenic endothelial cells, while the promotive effect could be observed during the late stage of hematopoiesis. This is very similar to that of RUNX1b. Interestingly, the mRNA expression profile of RUNX1-205 during hematopoiesis was distinct from that of RUNX1b, and the protein stability of RUNX1-205 was much higher than that of RUNX1b. Thus, the function of RUNX1-205 in normal and diseased models should be further explored.
Project description:Tracing the emergence of the first hematopoietic stem cells (HSCs) in human embryos, particularly the scarce and transient precursors thereof, is so far challenging, largely due to the technical limitations and the material rarity. Here, using single-cell RNA sequencing, we constructed the first genome-scale gene expression landscape covering the entire course of endothelial-to-HSC transition during human embryogenesis. The transcriptomically defined HSC-primed hemogenic endothelial cells (HECs) were captured at Carnegie stage (CS) 12-14 in an unbiased way, showing an unambiguous feature of arterial endothelial cells (ECs) with the up-regulation of RUNX1, MYB and ANGPT1. Importantly, subcategorizing CD34+CD45- ECs into a CD44+ population strikingly enriched HECs by over 10-fold. We further mapped the developmental path from arterial ECs via HSC-primed HECs to hematopoietic stem progenitor cells, and revealed a distinct expression pattern of genes that were transiently over-represented upon the hemogenic fate choice of arterial ECs, including EMCN, PROCR and RUNX1T1. We also uncovered another temporally and molecularly distinct intra-embryonic HEC population, which was detected mainly at earlier CS 10 and lacked the arterial feature. Finally, we revealed the cellular components of the putative aortic niche and potential cellular interactions acting on the HSC-primed HECs. The cellular and molecular programs that underlie the generation of the first HSCs from HECs in human embryos, together with the ability to distinguish the HSC-primed HECs from others, will shed light on the strategies for the production of clinically useful HSCs from pluripotent stem cells.
Project description:Haematopoietic stem cells (HSCs) are produced during embryogenesis from the floor of the dorsal aorta. The localization of HSCs is dependent on the presence of instructive signals on the ventral side of the vessel. The nature of the extrinsic molecular signals that control the aortic haematopoietic niche is currently poorly understood. Here we demonstrate a novel requirement for FGF signalling in the specification of aortic haemogenic endothelium. Our results demonstrate that FGF signalling normally acts to repress BMP activity in the subaortic mesenchyme through transcriptional inhibition of bmp4, as well as through activation of two BMP antagonists, noggin2 and gremlin1a. Taken together, these findings demonstrate a key role for FGF signalling in establishment of the developmental HSC niche via its regulation of BMP activity in the subaortic mesenchyme. These results should help inform strategies to recapitulate the development of HSCs in vitro from pluripotent precursors.