Hemogenic Endothelium Specification Research Articles

Cpeb1b-mediated cytoplasmic polyadenylation of shha mRNA modulates zebrafish definitive hematopoiesis

During vertebrate embryogenesis, hematopoietic stem and progenitor cell (HSPC) production through endothelial-to-hematopoietic transition requires suitable developmental signals, but how these signals are accurately regulated remains incompletely understood. Cytoplasmic polyadenylation, which is one of the posttranscriptional regulations, plays a crucial role in RNA metabolism. Here, we report that Cpeb1b-mediated cytoplasmic polyadenylation is important for HSPC specification by translational control of Hedgehog (Hh) signaling during zebrafish early development. Cpeb1b is highly expressed in notochord and its deficiency results in defective HSPC production. Mechanistically, Cpeb1b regulates hemogenic endothelium specification by the Hedgehog-Vegf-Notch axis. We demonstrate that the cytoplasmic polyadenylation element motif-dependent interaction between Cpeb1b and shha messenger RNA (mRNA) in the liquid-like condensates, which are induced by Pabpc1b phase separation, is required for cytoplasmic polyadenylation of shha mRNA. Intriguingly, the cytoplasmic polyadenylation regulates translation but not stability of shha mRNA, which further enhances the Shha protein level and Hh signal transduction. Taken together, our findings uncover the role of Cpeb1b-mediated cytoplasmic polyadenylation in HSPC development and provide insights into how posttranscriptional regulation can direct developmental signals with high fidelity to translate them into cell fate transition.

Loss of KMT2C reprograms the epigenomic landscape in hPSCs resulting in NODAL overexpression and a failure of hemogenic endothelium specification

ABSTRACT Germline or somatic variation in the family of KMT2 lysine methyltransferases have been associated with a variety of congenital disorders and cancers. Notably, KMT2A-fusions are prevalent in 70% of infant leukaemias but fail to phenocopy short latency leukaemogenesis in mammalian models, suggesting additional factors are necessary for transformation. Given the lack of additional somatic mutation, the role of epigenetic regulation in cell specification, and our prior results of germline KMT2C variation in infant leukaemia patients, we hypothesized that germline dysfunction of KMT2C altered haematopoietic specification. In isogenic KMT2C KO hPSCs, we found genome-wide differences in histone modifications at active and poised enhancers, leading to gene expression profiles akin to mesendoderm rather than mesoderm highlighted by a significant increase in NODAL expression and WNT inhibition, ultimately resulting in a lack of in vitro hemogenic endothelium specification. These unbiased multi-omic results provide new evidence for germline mechanisms increasing risk of early leukaemogenesis.

2034 – RAB5C-MEDIATED ENDOCYTIC TRAFFICKING REGULATES HEMATOPOIETIC STEM AND PROGENITOR CELL DEVELOPMENT THROUGH NOTCH AND AKT SIGNALING

A better understanding of molecular mechanisms underlying hematopoietic stem and progenitor cell (HSPC) development may lead to improved protocols to generate functional and transplantable HSPCs ex vivo. Here we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium specification by endocytic trafficking of Notch ligand and receptor. We further show that the interaction between Rab5c and Appl1 in endosome is required for the survival of endothelial cells in the aorta-gonad-mesonephros region through AKT signaling. Interestingly, Rab5c over-activation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development, and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition.

Early Human Hemogenic Endothelium Generates Primitive and Definitive Hematopoiesis InVitro.

SummaryThe differentiation of human embryonic stem cells (hESCs) to hematopoietic lineages initiates with the specification of hemogenic endothelium, a transient specialized endothelial precursor of all blood cells. This in vitro system provides an invaluable model to dissect the emergence of hematopoiesis in humans. However, the study of hematopoiesis specification is hampered by a lack of consensus in the timing of hemogenic endothelium analysis and the full hematopoietic potential of this population. Here, our data reveal a sharp decline in the hemogenic potential of endothelium populations isolated over the course of hESC differentiation. Furthermore, by tracking the dynamic expression of CD31 and CD235a at the onset of hematopoiesis, we identified three populations of hematopoietic progenitors, representing primitive and definitive subsets that all emerge from the earliest specified hemogenic endothelium. Our data establish that hemogenic endothelium populations endowed with primitive and definitive hematopoietic potential are specified simultaneously from the mesoderm in differentiating hESCs.

GATA2 Is Dispensable for Specification of Hemogenic Endothelium but Promotes Endothelial-to-Hematopoietic Transition.

SummaryThe transcriptional factor GATA2 is required for blood and hematopoietic stem cell formation during the hemogenic endothelium (HE) stage of development in the embryo. However, it is unclear if GATA2 controls HE lineage specification or if it solely regulates endothelial-to-hematopoietic transition (EHT). To address this problem, we innovated a unique system, which involved generating GATA2 knockout human embryonic stem cell (hESC) lines with conditional GATA2 expression (iG2−/− hESCs). We demonstrated that GATA2 activity is not required for VE-cadherin+CD43−CD73+ non-HE or VE-cadherin+CD43−CD73– HE generation and subsequent HE diversification into DLL4+ arterial and DLL4– non-arterial lineages. However, GATA2 is primarily needed for HE to undergo EHT. Forced expression of GATA2 in non-HE failed to induce blood formation. The lack of GATA2 requirement for generation of HE and non-HE indicates the critical role of GATA2-independent pathways in specification of these two distinct endothelial lineages.

Cells with hematopoietic potential reside within mouse proepicardium

During embryonic development, hematopoietic cells are present in areas of blood-vessel differentiation. These hematopoietic cells emerge from a specific subpopulation of endothelial cells called the hemogenic endothelium. We have previously found that mouse proepicardium contained its own population of endothelial cells forming a network of vascular tubules. We hypothesize that this EC population contains cells of hematopoietic potential. Therefore, we investigated an in vitro hematopoietic potential of proepicardial cell populations. The CD31+/CD45−/CD71− cell population cultured for 10 days in MethocultTM gave numerous colonies of CFU-GEMM, CFU-GM, and CFU-E type. These colonies consisted of various cell types. Flk-1+/CD31−/CD45−/CD71−, and CD45+ and/or CD71+ cell populations produced CFU-GEMM and CFU-GM, or CFU-GM and CFU-E colonies, respectively. Immunohistochemical evaluations of smears prepared from colonies revealed the presence of cells of different hematopoietic lineages. These cells were characterized by labeling with various combinations of antibodies directed against CD31, CD41, CD71, c-kit, Mpl, Fli1, Gata-2, and Zeb1 markers. Furthermore, we found that proepicardium-specific marker WT1 co-localized with Runx1 and Zeb1 and that single endothelial cells bearing CD31 molecule expressed Runx1 in the proepicardial area of embryonic tissue sections. We have shown that cells of endothelial and/or hematopoietic phenotypes isolated from mouse proepicardium possess hematopoietic potential in vitro and in situ. These results are supported by RT-PCR analyses of proepicardial extract, which revealed the expression of mRNA for crucial regulatory factors for hemogenic endothelium specification, i.e., Runx1, Notch1, Gata2, and Sox17. Our data are in line with previous observation on hemangioblast derivation from the quail PE.

Human Definitive Hematopoietic Specification from Pluripotent Stem Cells Is Regulated By Mesodermal Expression of CDX4

The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine, as it will provide an unlimited source of these cells for transplantation, and a unique platform for the study of both normal and disease hematopoietic processes. To reproducibly achieve this goal in all hPSC lines, we must first fully understand hematopoietic ontogeny.Understanding hematopoietic development is complicated by the existence of at least two distinct programs during development that are difficult to distinguish: a transient "primitive" extra-embryonic hematopoietic program that does not give rise to HSCs, and a "definitive" program that gives rise to HSCs and all hematopoietic lineages. We have recently developed a novel method to obtain and identify, from hPSCs, mesoderm harboring exclusively primitive or exclusively definitive hematopoietic potential, by the differential expression of CD235a within KDR+ mesoderm. With this, we were able to determine that stage-specific canonical Wnt signaling specified the definitive hematopoietic program, while simultaneously repressing the primitive hematopoietic program. Further, we have now found that definitive hematopoietic specification is also dependent on bFGF signaling during this same window of time. We then asked if this tractable system could help us understand the genetic regulation of definitive hematopoietic specification from hPSCs. We isolated Wnt-dependent KDR+CD235a- definitive hematopoietic mesoderm and Wnt-independent KDR+CD235a+ primitive hematopoietic mesoderm and performed whole-transcriptome gene expression analysis, which revealed strong CDX and HOX gene enrichment exclusively within KDR+CD235a- definitive hematopoietic mesoderm. Monitoring CDX expression over time in the differentiation cultures revealed that both CDX1 and CDX2 were expressed in a bFGF-independent manner, prior to Wnt-dependent definitive hematopoietic KDR+CD235a- mesoderm specification. In contrast, CDX4 was expressed exclusively within definitive hematopoietic KDR+CD235a- mesoderm in a Wnt- and bFGF-dependent manner. This expression pattern suggested that CDX4 expression is specific to definitive, but not primitive, hematopoietic specification.To determine whether CDX4 expression plays a role in definitive hematopoietic specification, we generated an inducible CDX4 expression hPSC line using the "safe-harbor" AAVS1 locus. We manipulated exogenous CDX4 expression during the same stage of the differentiation culture that Wnt signaling is critical for definitive hematopoietic specification. Interestingly, in the absence of Wnt stimulation, exogenous CDX4 expression caused a >90% repression in primitive hematopoietic potential. Critically, CDX4 expression during this same time conferred 10-fold greater definitive hematopoietic potential within CD34+CD73-CD184- hemogenic endothelium, giving rise to definitive erythroid-myeloid-lymphoid multilineage progenitors. This is consistent with CDX4 being the transcriptional effector of Wnt signaling during early hematopoietic specification within mesoderm.We next generated a CDX4 knockout hPSC line by CRISPR/Cas9, and a CDX4 knockdown hPSC line via shRNA expression from the AAVS1 locus. As expected, the absence of CDX4 expression did not reduce primitive hematopoietic potential in comparison to control lines. However, when Wnt signaling was stimulated to specify definitive hematopoiesis, hPSCs lacking CDX4 expression exhibited a 10-fold decrease in definitive CD34+CD73-CD184- hemogenic endothelium specification, indicating its expression is critical for definitive hematopoietic specification. Taken together, these findings indicate that CDX4 is the earliest identified transcription factor that is a critical regulator of human definitive hematopoietic specification, and provide a mechanistic basis for Wnt-mediated definitive hematopoietic specification from hPSCs. By understanding the genetic regulation of early definitive hematopoietic specification from hPSCs, we can now identify the additional signal pathways required for efficient HSC specification from hPSCs. DisclosuresNo relevant conflicts of interest to declare.

Accumulation of the Vitamin D Precursor Cholecalciferol Antagonizes Hedgehog Signaling to Impair Hemogenic Endothelium Formation

SummaryHematopoietic stem and progenitor cells (HSPCs) are born from hemogenic endothelium in the dorsal aorta. Specification of this hematopoietic niche is regulated by a signaling axis using Hedgehog (Hh) and Notch, which culminates in expression of Runx1 in the ventral wall of the artery. Here, we demonstrate that the vitamin D precursor cholecalciferol (D3) modulates HSPC production by impairing hemogenic vascular niche formation. Accumulation of D3 through exogenous treatment or inhibition of Cyp2r1, the enzyme required for D3 25-hydroxylation, results in Hh pathway antagonism marked by loss of Gli-reporter activation, defects in vascular niche identity, and reduced HSPCs. Mechanistic studies indicated the effect was specific to D3, and not active 1,25-dihydroxy vitamin D3, acting on the extracellular sterol-binding domain of Smoothened. These findings highlight a direct impact of inefficient vitamin D synthesis on cell fate commitment and maturation in Hh-regulated tissues, which may have implications beyond hemogenic endothelium specification.

Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium

AbstractHematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial–cadherin+ hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis.

Estrogens Act As a Dorsal-Ventral Limiting Factor To Pattern The Hematopoietic Stem Cell Niche

While the genetic control of hematopoietic stem cell (HSC) function is increasingly understood, less is known about factors that pattern the embryonic hematopoietic niche and specify the location of HSC emergence. 17beta-estradiol (E2) and related estrogenic compounds were identified in a zebrafish chemical genetic screen as modifiers of the number of runx1+ HSCs in the Aorta-Gonad-Mesonephros region. Exposure to exogenous E2 during hematopoietic specification significantly decreased production of runx1+ HSCs by in situ hybridization (ISH) and qPCR (p<0.01); this finding was confirmed by CD41 FACS (p<0.001). Morpholino knockdown of the estrogen receptors indicated E2-mediated loss of HSCs was due to Esr2a signaling. Zebrafish that express GFP under the control of the estrogen response element (ERE:GFP) showed endogenous estrogen activity and a 1.5 fold enhancement of GFP expression by FACS upon E2 treatment (p<0.01) confirming that zebrafish embryos contain endogenous estrogen and the transcriptional machinery to respond to exogenous E2; estradiol immunoassay revealed that endogenous estrogen is preferentially located in the yolk of developing embryos. E2 treatment from 12-24 hours post fertilization (hpf), the time in which hemogenic endothelium is specified, disrupted vessel formation as shown by flk1 (kdrl) and altered the assignment of hemogenic arterial identity as assessed by the arterial markers ephrinB2a and tbx20. As arterial specification is mediated by a cascade of sonic hedgehog, VEGF, and Notch, we examined if these pathways were altered. Consistent with a defect in the specification of hemogenic endothelium, E2 decreased expression of VEGFAa and arterial Notch pathway components deltaC and notch3 by ISH and qPCR (p<0.05). Induction of VEGFAa or Notch activity via heat-shock inducible lines rescued E2-mediated hematopoietic defects, suggesting that alterations in these networks underlie observed hematopoietic phenotypes. Further, repression of endogenous estrogen by the aromatase inhibitor anastrozole or the estrogen receptor antagonist fulvestrant increased levels of runx1, the arterial markers ephrinB2a and tbx20, and VEGFAa expression. Expression of venous flt4 was correspondingly decreased, demonstrating that endogenous estrogen signaling regulates arterial/venous identity. These results suggest that maternally deposited estrogens act as a novel morphogen organizer in patterning the HSC niche during early development. It further indicates that endogenous estrogen signaling acts to limit the ventral boundary of hemogenic endothelium by antagonizing somitic VEGFAa, thereby implicating estrogen as a critical regulator of the spatially restricted induction of HSCs. To determine if E2 could impact HSCs independently of its role in specification of hemogenic endothelium, we exposed embryos to E2 from 27-34 hpf, after arterial establishment and initiation of blood flow. Here, E2 enhanced HSCs by ISH and qPCR (runx1; p<0.05). qPCR levels of the cell cycle markers cyclinD1 (p<0.01) and c-myc (p<0.001) and AGM phospho-histone H3 immunoreactivity (p<0.01) increased, suggesting E2 enhances cell cycling. To determine if the ability of E2 to enhance proliferation was restricted to HSCs or was apparent on any blood progenitor population, we assessed the impact of E2 on primitive hematopoiesis. E2 treatment from 12-36 hpf enhanced expression of gata1 by ISH, FACS, and qPCR (p<0.05) while repressing the expression of globin (p<0.05), suggesting that while E2 enhances the self-renewal of progenitors, it impairs their differentiation into mature lineages. To analyze conservation of effect, we examined hematopoietic development in murine embryos with 5-alpha reductase deficiency. These embryos fail to metabolize testosterone into dihydroxytestosterone and as a result have more testosterone available for conversion by aromatase into estradiol, leading to increased estrogen levels. Homozygote pups show fewer phenotypic HSCs by FACS; heterozygotes display increased erythrocyte progenitors but no increase in mature erythrocytes, suggesting that the impact of estrogen on HSC induction and erythropoiesis is conserved in a mammalian system and is sensitive to dose. Together, these data indicate a novel role for estrogen in the regionalization of the hematopoietic niche and identify estrogen as an enhancer of HSC proliferation and maturation. Disclosures: No relevant conflicts of interest to declare.

Scl represses cardiogenesis via distant enhancers during hemogenic endothelium specification

Scl represses cardiogenesis via distant enhancers during hemogenic endothelium specification

Abstract 356: Scl Represses Cardiogenesis via Distant Enhancers during Hemogenic Endothelium Specification

Understanding the mechanisms directing mesoderm specification holds a great potential to advance the development of cell-based therapies for cardiovascular and blood disorders. The bHLH transcription factor Scl is known as the master regulator of the hematopoietic fate. We recently discovered that, in addition to its critical function in promoting the establishment of hemogenic endothelium during hematopoietic stem/progenitor cell (HS/PC) development, Scl is also required to repress cardiomyogenesis in endothelium in hematopoietic tissues and endocardium in the heart. However, the mechanisms for the cardiac repression have remained unknown. Using ChIP-sequencing and microarray analysis of Flk+ mesoderm differentiated from mouse ES cells, we show that Scl both directly activates a broad gene regulatory network required for hemogenic endothelium and HS/PC development (e.g. Runx1, cMyb, Lyl1, Mef2C, Sox7 etc.), and directly represses transcriptional regulators required for cardiogenesis (e.g. Gata4, Gata6, Myocd, etc.) and mesoderm development (Eomes, Mixl1, Etv2, etc.). Repression of cardiac and mesodermal programs occurs during a short developmental window through Scl binding to distant enhancers, while binding to hematopoietic regulators extends throughout HS/PC and red blood cell development and encompasses both distant and proximal binding sites. We also discovered that, surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification and Scl binding to majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors that facilitate HS/PC emergence from hemogenic endothelium. These results denote Scl as a true master regulator of hematopoietic vs. cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates.

ETV2 expression marks blood and endothelium precursors, including hemogenic endothelium, at the onset of blood development

ETV2 has been identified as an important player in embryonic hematopoiesis. However, the cell populations in which this transcription factor is expressed and operates during blood specification remain to be fully characterized. Here we address these issues using ES cells and a transgenic mouse line expressing green fluorescent protein (GFP) under the control of ETV2 regulatory elements, allowing us to observe the tight association between ETV2 expression and the initiation of hematopoiesis. Both in differentiating ES cells and gastrulating embryos ETV2::GFP is mostly found co-expressed with endothelial markers and defines a subset of cells with greatly enriched primitive erythroid potential. Upon culture ETV2::GFP cells rapidly up-regulate CD41, down-regulate endothelium cell surface markers and generate definitive hematopoietic progenitors. Altogether these characteristics represent the hallmark of hemogenic endothelium cells, a specialized endothelium originating from the hemangioblast and giving rise to hematopoietic cells. Importantly, ETV2 deficiency results in a complete absence of hemogenic endothelium in differentiating ES cells and gastrulating embryos. Altogether our data reveal that ETV2 marks hemogenic endothelium in gastrulating embryos and is absolutely required for the formation of this precursor at the onset of hematopoiesis. These results enhance our understanding of embryonic hematopoiesis and the factors driving hemogenic endothelium specification.

Genetic Interaction Between Angiopoietin-Like Proteins 1 and 2 and Notch Signaling During HSC Development

AbstractAbstract 573Members of the angiopoietin-like protein (angptl) family have recently been shown to stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of angptls for HSCs during development and their mechanism of action have yet to be determined. To first recapitulate the effects of exogenous angptl on HSC expansion, we made a stable transgenic zebrafish line, Tg(hsp70:angptl2), that overexpresses angptl2 upon heatshock induction. Heatshocked Tg embryos showed a significant increase in cmyb- and runx1-positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf), suggesting that angptl2 can sufficiently expand definitive HSCs during development. We then assessed the effects of angptl1 and/or angptl2 morpholino (MO) knockdown. Single MO knockdown resulted in a decrease in cmyb- and runx1-positive HSCs in the AGM whereas embryos injected with combined angptl1 and 2 MOs (double morphants) exhibited more severe phenotype in which HSCs were completely absent. Angptl regulation of HSC development may occur by stimulating the production or specification of a patent hemogenic endothelium, as the double morphants displayed a disruption of vascular specification at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4 in the dorsal aorta. These double morphants also exhibited disrupted intersegmental blood vessel sprouting. Because proper patterning of the developing blood vessels is a prerequisite for subsequent HSC formation, these results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium specification. To dissect the mechanism of angptl signaling, we asked whether there is a genetic interaction between notch and angptl signaling since the observed phenotype in the angptl double morphants resembled that in the notch mutant mindbomb, mib. We first monitored notch signaling using a transgenic notch reporter zebrafish line that expresses EGFP when the active component of notch signaling, notch intracellular domain (NICD), is expressed. MO knockdown of angptls 1 and 2 resulted in an absence of notch signaling, particularly in the vasculature. Interestingly, overexpressing angptl2 by crossing Tg(hsp70:angptl2) into the mib mutant showed a significant rescue of the HSC phenotype, implying that angptl2 acts downstream of mib signaling. When MOs against angptls 1 and 2 were then injected into the NICD overexpressing Tg embryos, we found that NICD restored HSC formation in the angptl double morphants. Together, these data corroborate the hypothesis that angptls can regulate notch signaling by acting downstream of notch ligand-receptor interaction and upstream of NICD during definitive hematopoiesis. To further probe at the downstream signaling of angptls since their receptor is currently unknown, we found that both angptls can stimulate activation of akt in cultured endothelial cells. To investigate whether akt is involved in angptl signaling during development, we injected the constitutively active myr-akt into angptl double morphants and observed rescue of both HSC and vascular defects. This strongly indicates that akt is the key signaling component downstream of angptl signaling during these biological processes. Injection of myr-akt can also rescue HSC defects in mib, suggesting that notch signaling also requires akt activation for definitive HSC formation. Taken together, our data demonstrate that the angptls are required for HSC development through notch and akt signaling, by coordinating the production of a functional hemogenic endothelium. Disclosures:Zon:FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.