Hematopoietic Stem Cells Formation Research Articles

Notch3 is required in the somitic microenvironment for the specification of hematopoietic stem cells

Hematopoietic stem cells (HSCs) are generated during embryonic development and possess the ability to reconstitute all adult blood lineages and self-renew for the life of an organism. Notch signaling is essential for HSC formation, as evidenced by the fact that many Notch pathway proteins are required for this process including the specific Notch1 receptor that functions cell-autonomously in mouse. Additionally, recent data from the zebrafish indicate that Notch signaling downstream of the non-canonical Wnt protein Wnt16 is also required in the somitic environment for HSC and sclerotome specification, suggesting that these two developmental events are related.

Declined Presentation: Inhibition of endothelial ERK signalling by SMAD1/5 is essential for haematopoietic stem cell emergence

The ontogeny of embryonic hematopoietic stem cells (HSCs) is principally documented to be governed by transcriptional factors and cytokines. However, the regulation of such dynamic process by other types of molecules remains poorly defined. Here, we report that disruption of Wip1, the wild type P53-induced phosphatase, results in markedly reduced number and defective function of HSCs in E12.5 fetal liver. Most strikingly, adult-reconstituting HSCs are nearly absent in E11.5-12.5 Wip1-deficient AGM region. Wip1 deletion leads to significant reduction in number of CD31+CD45+ cells (enrichment of HSCs and type 2 pre-HSCs) when that of CD31+CD41+CD45- cells (enrichment of type 1 pre-HSCs) remains unchanged, suggesting blocked maturation of HSCs in vivo. Incubation of type 1 (CD31+CD41+CD45-) and type 2 (CD31+CD45+) pre-HSCs with CCT, the specific inhibitor of Wip1, abrogates and severely impairs HSC formation, respectively. In contrast, the endothelial population (CD31+CD41-CD45-Ter119-) from E10.5 Wip1-deficient AGM region harbors comparable number of hemogenic endothelial cells, albeit with compromised B lymphoid potential in vitro. Quite interestingly, the HSCs with long-term and efficient engraftment potential can be detected in E11.5-12.5 Wip1-/- yolk sac. In Wip1 knockout embryos, expression of hematopoietic related transcriptional factors, such as Runx1 and GATA-2, is significantly reduced in AGM region but up-regulated in yolk sac. Taken together, our study reveals the Wip1 phosphatase is indispensable for specifically the Pre-HSC/HSC maturation in the AGM region.

Distinct regulation of the anterior and posterior myeloperoxidase expression by Etv2 and Gata1 during primitive Granulopoiesis in zebrafish

Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.

Translation of branched-chain aminotransferase-1 transcripts is impaired in cells haploinsufficient for ribosomal protein genes

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells.

Assessment of hematopoietic failure due to Rpl11 deficiency in a zebrafish model of Diamond-Blackfan anemia by deep sequencing

BackgroundDiamond–Blackfan anemia is a rare congenital red blood cell dysplasia that develops soon after birth. RPL11 mutations account for approximately 4.8% of human DBA cases with defective hematopoietic phenotypes. However, the mechanisms by which RPL11 regulates hematopoiesis in DBA remain elusive. In this study, we analyzed the transcriptome using deep sequencing data from an Rpl11-deficient zebrafish model to identify Rpl11-mediated hematopoietic failure and investigate the underlying mechanisms.ResultsWe characterized hematological defects in Rpl11-deficient zebrafish embryos by identifying affected hematological genes, hematopoiesis-associated pathways, and regulatory networks. We found that hemoglobin biosynthetic and hematological defects in Rpl11-deficient zebrafish were related to dysregulation of iron metabolism-related genes, including tfa, tfr1b, alas2 and slc25a37, which are involved in heme and hemoglobin biosynthesis. In addition, we found reduced expression of the hematopoietic stem cells (HSC) marker cmyb and HSC transcription factors tal1 and hoxb4a in Rpl11-deficient zebrafish embryos, indicating that the hematopoietic defects may be related to impaired HSC formation, differentiation, and proliferation. However, Rpl11 deficiency did not affect the development of other blood cell lineages such as granulocytes and myelocytes.ConclusionWe identified hematopoietic failure of Rpl11-deficient zebrafish embryos using transcriptome deep sequencing and elucidated potential underlying mechanisms. The present analyses demonstrate that Rpl11-deficient zebrafish may serve as a model of DBA and may provide insights into the pathogenesis of mutant RPL11-mediated human DBA disease.

The Function Of Transcription Elongation In Hematopoietic Stem Cell Development

The specification of hematopoietic stem cells (HSCs) in vertebrate embryos is tightly regulated by RNA polymerase II (Pol II)-mediated transcription, which proceeds through multiple steps including initiation/activation, elongation and termination. Increasing evidences in recent years have demonstrated that transcriptionally engaged Pol II often pauses shortly after initiation by pausing factors DSIF and NELF, and its release requires positive elongation factors such as p-TEFb. Little is known about how pausing-to-elongation switch contribute to cell fate determination. In a zebrafish spt5 mutant, we detected a dramatic loss of runx1+ HSCs in the AGM region in 36-hour embryos. Spt5 is an essential subunit of DSIF, which pauses Pol II in early elongation stage but stimulates Pol II elongation upon phosphorylated by p-TEFb. The zebrafish mutant carries a single amino acid change in the Spt5 protein that disrupts the pausing function but leaves the stimulatory function intact, suggesting a requirement of Pol II pausing in HSC formation. Similarly, knocking down the other pausing factor NELF by morpholinos also greatly reduced the number of runx1+ HSCs in zebrafish embryos. This loss of HSCs is not caused by general developmental defects, as markers for vessels and aorta endothelium remain normal in mutants. We further demonstrated that reducing Pol II elongation by either p-TEFb inhibitors or morpholino knockdown of CDK9, the essential subunit of p-TEFb, could rescue the HSC defect in spt5 mutants. The rescue by cdk9 morpholino is dose-dependent. Low-dose morpholino that presumably caused partial knockdown of CDK9 was able to rescue spt5 mutants, whereas high-dose morpholino completely wiped out HSCs in both wild type and mutant embryos. In conclusion, our data suggested that HSC formation during embryonic development requires a balanced transcription that is regulated by both negative and positive elongation factors. Such mechanisms may also be employed in other tissues to regulate cell fate determination. Disclosures: No relevant conflicts of interest to declare.

A Network Of Epigenetic Regulators Guide Developmental Hematopoiesis In Vivo

Long-term hematopoietic stem cells (HSCs) are capable of self-renewal and differentiation into all mature hematopoietic lineages. This process is regulated by transcription factors interact with co-factors to orchestrate chromatin structure and facilitate gene expression. To generate a compendium of factors that establish the epigenetic code in HSCs, we have undertaken the first large-scale in vivo reverse genetic screen targeting chromatin factors. We have designed and injected antisense morpholinos to knockdown expression of 488 zebrafish orthologs of conserved human chromatin factors. The resultant morphants were analyzed by whole embryo in situ hybridization at 36 hours post fertilization for expression of two HSC marker genes, c-myb and runx1, which are expressed in the developing blood stem cells. Morphants were categorized into five groups based on HSC marker expression, ranging from no change to mild, intermediate, or strong reduction in expression or an increase in expression. 29 morpholinos caused a complete or near complete knockdown of HSC marker expression, while 4 were found to increase HSC marker expression. As ubiquitous knockdown of chromatin factors could interfere with vascular development and the establishment of proper arterial identity, a crucial upstream event for HSC formation, we subsequently analyzed morphants with the most robust HSC phenotypes using two vascular markers: kdr for overall vasculogenesis and ephrinb2a for arterial formation. We found that of the 29 morpholinos that caused reduced marker expression, only 9 showed reduced overall vascular or arterial marker staining, suggesting that the majority of morphants with HSC phenotypes are specific to HSC formation. For the 4 morphants with increased HSC marker expression, vasculature appeared normal. These factors likely function as potent negative regulators of HSC development. Several genes known to be essential for HSC self-renewal and maintenance were identified in the screen. For example, knockdown of Mll or Dot1, which are also present in leukemia fusion proteins, fail to specify HSCs, as indicated by a nearly complete reduction in expression of the HSC markers in embryos tested. Of the remaining hits, many represent factors with no previous function ascribed in hematopoiesis. By incorporating protein interaction data, we have defined a handful of complexes necessary for HSC specification, including the SWI/SNF, ISWI, SET1/MLL, CBP/P300/HBO1/NuA4, HDAC/NuRD, and Polycomb complexes. As chromatin factors associated with the same complex likely share target binding sites, we analyzed 34 published ChIP-seq datasets in K562 erythroleukemia cells of chromatin factors tested in the screen, including hits from our screen: SIN3A, CHD4, HDAC1, TAF1, and JARID1C associated with the HDAC/NuRD complex and RNF2, SUZ12, CBX2, and CBX8 from the Polycomb complexes. We ranked triplet combinations of these factors together with all other groups of three factors based on the percent overlap of target genes. The HDAC/NuRD and PRC1/2 complex combinations predicted from our screen fell within the top 20% of all possible combinations of 3 factors, suggesting that our screen has identified chromatin factors that function in distinct complexes to regulate hematopoietic development. Our work has been compiled into a web-based database that will be made publicly available upon publication. Within this database, users can search by gene names and aliases, chromatin domain names and human or zebrafish genes. All experimental data, including experimental design, materials, protocols, images, and all further analyses of the 33 most robust morphants is included. Our large-scale genetic analysis of chromatin factors involved in HSC development provides a comprehensive view of the programs involved in epigenetic regulation of the blood program, offering new avenues to pursue in the study of histone modifications in HSCs and for therapeutic alternatives for patients with blood disorders and leukemia. Disclosures:Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

AGM-Derived Endothelial Cells and Notch Ligands Provide Embryonic Hematopoietic Stem Cell-Supportive Niches In Vitro

Greater knowledge of embryonic niche signals regulating the establishment, maintenance, and expansion of hematopoietic stem cells (HSC) during development will be essential in deriving therapeutically useful HSC from pluripotent stem cells (PSC). To this end, we have used the murine embryo model to dissect components of embryonic hematopoietic microenvironments which are sufficient to support nascent HSC and their precursors in vitro. We demonstrate that Akt pathway-activated endothelial cells (ECs) derived from the AGM (aorta-gonad-mesonephros) region, a critical site of HSC emergence during development, can substantially increase short and long-term multilineage engraftment potential from isolated embryonic day 11 (E11) VE-Cadherin+/CD45+ AGM-derived hematopoietic cells by co-culture in vitro. Furthermore, preliminary experiments show that co-culture with AGM-ECs also promotes high level, multilineage engraftment capacity from VE-cadherin+/c-kit+ precursors isolated from younger embryos (E9-E10). These results suggest that endothelial cells from an embryonic HSC-producing niche provide signals sufficient to promote maturation of HSC from embryonic precursors and subsequently support early HSC expansion in vitro. Further dissection of required signals for embryonic HSC expansion identified a unique combination of Notch activation by immobilized Notch ligands, cytokines, and small molecule inhibition of the TGF-β pathway, which is sufficient to inhibit differentiation and enhance self-renewal of embryonic, definitive-stage hematopoietic precursors in vitro. Notably, these conditions significantly increased short and long-term, multilineage repopulating HSC from E11 VE-Cadherin+/CD45+, but not E9-10 VE-Cadherin+/c-kit+ AGM-derived hematopoietic cells, indicating AGM-ECs provide additional, yet to be identified, signals for HSC maturation from developmental precursors. These findings have important implications for dissecting critical niche signals for HSC formation and expansion that will be essential for addressing the elusive goal of deriving HSC from pluripotent precursors. Disclosures:Rafii:Angiocrine Bioscience: Founder Other.

Zebrafish Cbfb Is Required For The Mobilization, But Not The Emergence, Of Hematopoietic Stem Cells In Embryos

CBFβ and RUNX1 form a DNA-binding heterodimer that plays a crucial role during definitive hematopoiesis at the stage of hematopoietic stem cells (HSCs). Both of them are targets of recurrent chromosomal translocations in human leukemia. In mammals and zebrafish, RUNX1 is required for the emergence of definitive HSCs from the hemogenic endothelium. Mouse knockouts for either Runx1 or Cbfb show similar phenotypes with complete lack of definitive hematopoiesis. Therefore, the impairment of all definitive hematopoietic lineages in both Runx1-/- and Cbfb-/- embryos suggested that the CBF heterodimer is required for HSC formation. However the exact role of the CBF complex in the development of HSCs remains unclear. The cellular mechanisms and the genetic pathways driving the HSC generation are highly conserved across vertebrates. Thus, we used the zebrafish model to dissect the role of cbfb and the CBF complex in the emergence and the maintenance of HSCs. We generated two independent cbfb knockouts (cbfb-/-) by zinc-finger nuclease (ZFN) - mediated targeted mutagenesis. The analysis of cbfb-/- embryos revealed a previously unknown role of cbfb during definitive hematopoiesis. Similar to the published zebrafish runx1 mutant embryos (runx1W84X/W84X), cbfb-/- embryos underwent primitive hematopoiesis and developed erythromyeloid progenitors (EMPs), but they lacked definitive hematopoiesis as the expression of markers for differentiated blood lineages such as rag1, lplastin and αe1globin was completely abrogated. Moreover, circulating thrombocytes were almost undetectable in cbfb-/-/tg(cd41:GFP) embryos. Unlike the runx1 mutants in which HSCs are not formed, however, the emergence of runx1+/c-myb+ HSCs from the hemogenic endothelium along the ventral wall of the dorsal aorta was unaffected in the cbfb-/- mutants. Rather, the subsequent translocation of the HSCs from aorta-gonad-mesonephros (AGM) to the caudal hematopoietic tissue (CHT) was blocked, as evidenced by the accumulation of runx1+ HSCs in the AGM and the concomitant absence of such cells in the CHT. Live imaging analysis of cbfb-/-/tg(c-myb:eGFP) embryos confirmed that HSCs egressed from the dorsal aorta but did not enter circulation through the axial vein. Moreover, embryos treated with a specific inhibitor of RUNX1-CBFβ interaction, Ro5-3335, phenocopied the hematopoietic defects observed in the cbfb-/- mutants, confirming that the function of RUNX1 and CBFβ during HSC development could be uncoupled. The Notch-Runx1 pathway is critical for the initial specification of HSCs during definitive hematopoiesis. Therefore, in order to gain insight into the genetic mechanisms that regulate cbfb expression we investigated the Notch pathway. We found that transient Notch activation enhanced cbfb expression and expanded it ectopically. On the other hand, in the Notch signaling mutant mind bomb, cbfb expression in hematopoietic regions was abrogated. Thus, our results suggest that cbfb is also downstream of the Notch pathway during hematopoiesis. Overall our data indicate that CBFβ and functional CBFβ-RUNX1 heterodimers are not required for the emergence of HSCs, but are essential for the mobilization of HSCs during early definitive hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

MiR-142-3p regulates the formation and differentiation of hematopoietic stem cells in vertebrates

Previous studies on developmental hematopoiesis have mainly focused on signaling and transcription factors, while the appreciation of epigenetic regulation including that of microRNAs is recent. Here, we show that in zebrafish and mouse, miR-142-3p is specifically expressed in hematopoietic stem cells (HSCs). Knockdown of miR-142a-3p in zebrafish led to a reduced population of HSCs in the aorta-gonad-mesonephros (AGM) region as well as T-cell defects in the thymus. Mechanistically, miR-142a-3p regulates HSC formation and differentiation through the repression of interferon regulatory factor 7 (irf7)-mediated inflammation signaling. Finally, we show that miR-142-3p is also involved in the development of HSCs in mouse AGM, suggesting that it has a highly conserved role in vertebrates. Together, these findings unveil the pivotal roles that miR-142a-3p plays in the formation and differentiation of HSCs by repressing irf7 signaling.

DNA methylation of Runx1 regulatory regions correlates with transition from primitive to definitive hematopoietic potential in vitro and in vivo

AbstractThe transcription factor Runx1 (AML1) is a central regulator of hematopoiesis and is required for the formation of definitive hematopoietic stem cells (HSCs). Runx1 is alternatively expressed from two promoters: the proximal (P2) prevails during primitive hematopoiesis, while the distal (P1) dominates in definitive HSCs. Although some transcription factor binding sites and cis-regulatory elements have been identified, a mechanistic explanation for the alternative promoter usage remains elusive. We investigated DNA methylation of known Runx1 cis-elements at stages of hematopoietic development in vivo and during differentiation of murine embryonic stem cells (ESCs) in vitro. In vivo, we find loss of methylation correlated with the primitive to definitive transition at the P1 promoter. In vitro, hypomethylation, acquisition of active chromatin modifications, and increased transcriptional activity at P1 are promoted by direct interaction with HOXB4, a transcription factor that confers definitive repopulation status on primitive hematopoietic progenitors. These data demonstrate a novel role for DNA methylation in the alternative promoter usage at the Runx1 locus and identify HOXB4 as a direct activator of the P1 promoter. This epigenetic signature should serve as a novel biomarker of HSC potential in vivo, and during ESC differentiation in vitro.

Hemogenic endothelium specification and hematopoietic stem cell maintenance employ distinct Scl isoforms

Recent studies have shown that nascent hematopoietic stem cells (HSCs) derive directly from the ventral aortic endothelium (VAE) via endothelial to hematopoietic transition (EHT). However, whether EHT initiates from a random or predetermined subpopulation of VAE, as well as the molecular mechanism underlying this process, remain unclear. We previously reported that different zebrafish stem cell leukemia (scl) isoforms are differentially required for HSC formation in the ventral wall of the dorsal aorta. However, the exact stage at which these isoforms impact HSC development was not defined. Here, using in vivo time-lapse imaging of scl isoform-specific reporter transgenic zebrafish lines, we show that prior to EHT scl-β is selectively expressed in hemogenic endothelial cells, a unique subset of VAE cells possessing hemogenic potential, whereas scl-α is expressed later in nascent HSCs as they egress from VAE cells. In accordance with their expression, loss-of-function studies coupled with in vivo imaging analysis reveal that scl-β acts earlier to specify hemogenic endothelium, which is later transformed by runx1 into HSCs. Our results also reveal a previously unexpected role of scl-α in maintaining newly born HSCs in the aorta-gonads-mesonephros. Thus, our data suggest that a defined hemogenic endothelial population preset by scl-β supports the deterministic emergence of HSCs, and unravel the cellular mechanisms by which scl isoforms regulate HSC development.

Distinct temporal requirements for Runx1 in hematopoietic progenitors and stem cells

The transcription factor Runx1 is essential for the formation of yolk sac-derived erythroid/myeloid progenitors (EMPs) and hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, long-term repopulating HSCs (LT-HSCs) persist when Runx1 is conditionally deleted in fetal liver cells, demonstrating that the requirement for Runx1 changes over time. To define more precisely when Runx1 transitions from an essential factor to a homeostatic regulator of EMPs and HSCs, and whether that transition requires fetal liver colonization, we performed conditional, timed deletions of Runx1 between E7.5 and E13.5. We determined that Runx1 loss reduces the formation or function of EMPs up through E10.5. The Runx1 requirement in HSCs ends later, as deletion up to E11.5 eliminates HSCs. At E11.5, there is an abrupt transition to Runx1 independence in at least a subset of HSCs that does not require fetal liver colonization. The transition to Runx1 independence in EMPs is not mediated by other core binding factors (Runx2 and/or Runx3); however, deleting the common non-DNA-binding β subunit (CBFβ) severely compromises LT-HSC function. Hence, the requirements for Runx1 in EMP and HSC formation are temporally distinct, and LT-HSC function is highly reliant on continued core binding factor activity.

17beta-estradiol has a biphasic effect on the formation of hematopoietic stem cells

While the mechanisms regulating hematopoietic stem cells (HSCs) are increasingly understood, less is known about the impact of environmental factors on this process. Estrogenic compounds were isolated as modifiers of runx1 expression in a zebrafish chemical screen. Exposure to 17beta-estradiol (E2) significantly decreased runx1+ HSCs in the AGM compared to controls by in situ hybridization and qPCR (p<0.01) which was confirmed by CD41 FACS (p<0.0001). E2 treatment from 12-24 hours post-fertilization disrupted vessel formation and angiogenic sprouting, as indicated by flk1 (kdrl) and altered the assignment of ephrinb2+ arterial identity.

Activated N-Ras signaling regulates arterial-venous specification in zebrafish

BackgroundThe aberrant activation of Ras signaling is associated with human diseases including hematological malignancies and vascular disorders. So far the pathological roles of activated Ras signaling in hematopoiesis and vasculogenesis are largely unknown.MethodsA conditional Cre/loxP transgenic strategy was used to mediate the specific expression of a constitutively active form of human N-Ras in zebrafish endothelial and hematopoietic cells driven by the zebrafish lmo2 promoter. The expression of hematopoietic and endothelial marker genes was analyzed both via whole mount in situ hybridization (WISH) assay and real-time quantitative PCR (qPCR). The embryonic vascular morphogenesis was characterized both by living imaging and immunofluorescence on the sections with a confocal microscopy, and the number of endothelial cells in the embryos was quantified by flow cytometry. The functional analyses of the blood circulation were carried out by fluorescence microangiography assay and morpholino injection.ResultsIn the activated N-Ras transgenic embryos, the primitive hematopoiesis appeared normal, however, the definitive hematopoiesis of these embryos was completely absent. Further analysis of endothelial cell markers confirmed that transcription of arterial marker ephrinB2 was significantly decreased and expression of venous marker flt4 excessively increased, indicating the activated N-Ras signaling promotes the venous development at the expense of arteriogenesis during zebrafish embryogenesis. The activated N-Ras-expressing embryos showed atrophic axial arteries and expansive axial veins, leading to no definitive hematopoietic stem cell formation, the blood circulation failure and subsequently embryonic lethality.ConclusionsOur studies revealed for the first time that activated N-Ras signaling during the endothelial differentiation in vertebrates can disrupt the balance of arterial-venous specification, thus providing new insights into the pathogenesis of the congenital human vascular disease and tumorigenic angiogenesis.

Glucose metabolism impacts the spatiotemporal onset and magnitude of HSC induction in vivo

AbstractMany pathways regulating blood formation have been elucidated, yet how each coordinates with embryonic biophysiology to modulate the spatiotemporal production of hematopoietic stem cells (HSCs) is currently unresolved. Here, we report that glucose metabolism impacts the onset and magnitude of HSC induction in vivo. In zebrafish, transient elevations in physiological glucose levels elicited dose-dependent effects on HSC development, including enhanced runx1 expression and hematopoietic cluster formation in the aorta-gonad-mesonephros region; embryonic-to-adult transplantation studies confirmed glucose increased functional HSCs. Glucose uptake was required to mediate the enhancement in HSC development; likewise, metabolic inhibitors diminished nascent HSC production and reversed glucose-mediated effects on HSCs. Increased glucose metabolism preferentially impacted hematopoietic and vascular targets, as determined by gene expression analysis, through mitochondrial-derived reactive oxygen species (ROS)–mediated stimulation of hypoxia-inducible factor 1α (hif1α). Epistasis assays demonstrated that hif1α regulates HSC formation in vivo and mediates the dose-dependent effects of glucose metabolism on the timing and magnitude of HSC production. We propose that this fundamental metabolic-sensing mechanism enables the embryo to respond to changes in environmental energy input and adjust hematopoietic output to maintain embryonic growth and ensure viability.

Phospholipase C gamma-1 is required for granulocyte maturation in zebrafish

The regulation of hematopoiesis is generally evolutionarily conserved from zebrafish to mammals, including hematopoietic stem cell formation and blood cell lineage differentiation. In zebrafish, primitive granulocytes originate at two distinct regions, the anterior lateral plate mesoderm (A-LPM) and the intermediate cell mass (ICM). Few studies in the zebrafish have examined genes specifically required for the granulocytic lineage. In this study, we identified the responsible gene for a zebrafish mutant that has relatively normal hematopoiesis, except decreased expression of the granulocyte-specific gene mpx. Positional cloning revealed that phospholipase C gamma-1 (plcg1) was mutated. Deficiency of plcg1 function specifically affected development of granulocytes, especially the maturation process. These results suggested that plcg1 functioned specifically in zebrafish ICM granulopoiesis for the first time. Our studies suggest that specific pathways regulate the differentiation of the hematopoietic lineages.

Concise Review: Blood Relatives: Formation and regulation of hematopoietic stem cells by the basic helix-loop-helix transcription factors stem cell leukemia and lymphoblastic leukemia-derived sequence 1

The basic helix-loop-helix (bHLH) proteins are a large family of transcription factors that regulate the formation and fate of tissue stem cells. In hematopoiesis, the two major bHLH factors are stem cell leukemia (SCL) and lymphoblastic leukemia-derived sequence 1 (LYL1), both identified more than 20 years ago in chromosomal translocations occurring in T-cell acute lymphoblastic leukemia. SCL was termed the master regulator of hematopoiesis following the observation that SCL knockout mice die from complete lack of blood formation. However, once established, SCL is no longer required for maintenance of hematopoiesis. Pull-down experiments together with add-back experiments in SCL-null embryonic stem cells and generation of mice carrying a germline DNA binding mutation of SCL demonstrates that most of SCL function is mediated through the formation of a large DNA binding multiprotein complex with both repressor and activator potential. Recent genome-wide binding studies in a hematopoietic stem progenitor cell line suggest that SCL and LYL1 preferentially bind target DNA sequences as components of a heptad of transcription factors. LYL1, a paralog of SCL has been the forgotten sibling until recent mouse studies demonstrated that LYL1 replaced the function of SCL in adult hematopoiesis. Why LYL1 can replace the function of SCL for the maintenance but not formation of hematopoiesis remains a fundamental question. This review will compare and contrast the roles of these two transcription factors in hematopoiesis focusing on recent functional and genome-wide binding studies.

In VivoChemical Screening for Modulators of Hematopoiesis and Hematological Diseases

In vivo chemical screening is a broadly applicable approach not only for dissecting genetic pathways governing hematopoiesis and hematological diseases, but also for finding critical components in those pathways that may be pharmacologically modulated. Both high-throughput chemical screening and facile detection of blood-cell-related phenotypes are feasible in embryonic/larval zebrafish. Two recent studies utilizing phenotypic chemical screens in zebrafish have identified several compounds that promote hematopoietic stem cell formation and reverse the hematopoietic phenotypes of a leukemia oncogene, respectively. These studies illustrate efficient drug discovery processes in zebrafish and reveal novel biological roles of prostaglandin E2 in hematopoietic and leukemia stem cells. Furthermore, the compounds discovered in zebrafish screens have become promising therapeutic candidates against leukemia and included in a clinical trial for enhancing hematopoietic stem cells during hematopoietic cell transplantation.

Erythroid/Myeloid Progenitors and Hematopoietic Stem Cells Originate from Distinct Populations of Endothelial Cells

Hematopoietic stem cells (HSCs) and an earlier wave of definitive erythroid/myeloid progenitors (EMPs) differentiate from hemogenic endothelial cells in the conceptus. EMPs can be generated in vitro from embryonic or induced pluripotent stem cells, but efforts to produce HSCs have largely failed. The formation of both EMPs and HSCs requires the transcription factor Runx1 and its non-DNA binding partner core binding factor β (CBFβ). Here we show that the requirements for CBFβ in EMP and HSC formation in the conceptus are temporally and spatially distinct. Panendothelial expression of CBFβ in Tek-expressing cells was sufficient for EMP formation, but was not adequate for HSC formation. Expression of CBFβ in Ly6a-expressing cells, on the other hand, was sufficient for HSC, but not EMP, formation. The data indicate that EMPs and HSCs differentiate from distinct populations of hemogenic endothelial cells, with Ly6a expression specifically marking the HSC-generating hemogenic endothelium.