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The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

The hematopoietic niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses, we define a conserved gene expression signature and cis-regulatory landscape unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code involving members of the Ets, Sox and Nuclear Hormone Receptor families that is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

U.S. National Science Foundation [EAR-1331408]; Catalina-Jemez Critical Zone Observatory; Research Experience for Undergraduates (REU) program at Biosphere [2]; [EAR-1263251]

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

Epoxyeicosatrienoic acids (EETs) are endogenous lipid signaling molecules with cardioprotective and vasodilatory actions. We recently showed that exogenous addition of 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via a G-protein coupled receptor(s), and significant research supports the existence of a specific high-affinity receptor. Identification of a hematopoietic specific EET receptor would enable genetic interrogation of the EET signaling pathway and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify the EET receptor based on the expression of GPCRs in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are commonly expressed in three EET-responsive human cell lines, but not expressed in an EET-unresponsive line. Of these candidates, only GPR132 showed EET-responsivenessin vitrousing a luminescence-based assay for β-arrestin recruitment. Knockdown of zebrafishgpr132bprevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to the putative high-affinity EET receptor, GPR132 is reported to have affinity for additional fatty acidsin vitro,and we found that these same fatty acids enhance hematopoietic stem cell specification in the zebrafish. We conducted structure-activity relationship analyses using bothin vitroandin vivoassays on diverse medium chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, while unoxygenated or saturated fatty acids had lower activity. Absence of the carboxylic acid moiety prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of polyunsaturated, oxygenated fatty acids to enhance both embryonic and adult hematopoiesis.

The hematopoietic stem and progenitor cell (HSPC) niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells (ECs) that directly interact with HSPCs and promote stem cell function. Utilizing spatial transcriptomics, in combination with tissue-specific RNA-seq, we identified 29 genes selectively enriched in ECs of the zebrafish fetal hematopoietic niche. Using upstream regulatory sequences for two of these genes, mrc1a and selectin E (sele), we generated GFP reporter lines that allowed us to selectively isolate niche ECs for ATAC-seq. This analysis identified 6,848 regions of chromatin that were accessible in niche ECs but not ECs from other tissues. Several of these regions were associated with the 29 genes. To evaluate whether these regions might be enhancers we coupled them to GFP and injected them into embryos. 12/15 sequences drove GFP expression in niche ECs. Upon closer examination of the mrc1a and sele genes, we identified enhancer sequences as short as 125 bp and 158 bp, respectively, which drove niche EC-specific expression. A genome-wide motif enrichment analysis of the 6,848 uniquely open chromatin regions revealed that Ets, SoxF and Nuclear Hormone Receptor (RXRA/NR2F2, specifically) sites were most enriched. In contrast, 4,522 pan-EC elements were enriched for Ets sites but not SoxF or NHR motifs. Using mutant variants of the 125 bp and 158 bp enhancer sequences, we demonstrated that Ets, SoxF and RXRA/NR2F2 sites were independently required for specific transgene expression. Gel shift experiments demonstrated that NR2F2 could bind the 125 bp and 158 bp zebrafish enhancers and this binding was disrupted upon mutation of the NR2F2 binding sites. Knockdown of the endogenous zebrafish nr2f2 gene resulted in a loss of expression of the 125 bp mrc1a enhancer-GFP construct and a significantly reduced number of HSPCs in the fetal niche. We next injected pools of human transcription factors, including at least one member from each of the three families, under the control of a ubiquitous promoter. Strikingly, we found that a combination of ETV2 or ETS1 with SOX7 and NR2F2 generated ectopic patches of mrc1a+ niche ECs that recruited runx1+ HSPCs outside of the endogenous niche. Using high-resolution live cell imaging we could observe HSPCs initially arriving at the ectopic sites, lodging for several hours and then eventually dividing and migrating away from the site through circulation. HSPCs localized to the ectopic regions were found in both intravascular and extravascular spaces, and were often enwrapped by ECs and in contact with cxcl12a+ stromal cells, similar to what is observed in the endogenous niche. Ectopic regions of niche EC gene expression were similarly observed when alternative regulatory elements were used for transcription factor overexpression, including a pan-EC enhancer (nrp1b), a muscle promoter (mylz2) and a heat shock promoter (hsp70). These results suggest the three-factor combinations are sufficient to reprogram niche EC identify in vivo. Lastly, we evaluated by RNA-seq the expression of our niche EC signature in the zebrafish kidney marrow (the site of adult hematopoiesis) and in ECs from multiple organs of the mouse, including the heart, kidney, liver, lung and bone marrow, at multiple stages of development (E11-13, E14-15, E16-17, P2-P4 and adult). Strikingly, 23/29 genes were highly expressed in ECs of the zebrafish kidney and 21/29 genes were enriched in the ECs of a mammalian hematopoietic organ - the fetal liver and/or adult bone marrow - relative to their expression in ECs from non-hematopoietic organs at the same stage. Notably, for a subset of the genes the expression patterns mirrored the temporal dynamics of HSPC ontogeny in the mouse, showing robust expression in fetal liver ECs and then later in adult bone marrow ECs with a concomitant reduction in liver ECs. An analysis of transcription factor expression within these EC populations revealed that Ets1, the SoxF factor Sox18, and Nr2f2 were the most highly expressed members of the Ets, Sox and NHR families. Collectively our work has uncovered a conserved gene expression signature and transcriptional regulatory program unique to the vascular niche of hematopoietic organs. These findings have important implications for designing a synthetic vascular niche for blood stem cells or for modulating the niche in a therapeutic context. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.