Transcription Factor Induction of Ectopic Vascular Blood Stem Cell Niches In Vivo
Authors: Hagedorn Elliott J. , Perlin Julie R. , Freeman Rebecca J. , Mao Clara , Fernández-Maestre Inés , Daily Madeleine L. , D'Amato Christopher R. , Wattrus Samuel J. , Han Tianxiao , Fairchild Michael J. , Riquelme Raquel , Li Brian , Redfield Shelby E. , Collins Samantha H. , Lichtig Asher , Yang Song , Zhou Yi , Kunar Balvir , Gomez-Salinero Jesus M , Dinh Theresa T. , Pan Junliang , Holler Karoline , Butcher Eugene , Van Oudenaarden Alexander , Rafii Shahin , Junker Jan Philipp , Zon Leonard I 
Published: February 2020

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.