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In vivo haematopoietic cell production by bone marrow-derived hemogenic endothelium

ABSTRACT: By tracing the VE-cadherin expression in the newborn bone marrow hematopoietic LSK (lineage minus/Sca-positive/Kit-positive) cells, we demonstrated that the late foetal/newborn BM hemogenic endothelial cells produce a small cohort of hematopoietic stem and progenitor cells (HSPCs) capable of circulating and colonizing the secondary haematopoietic organs. Phenotypic and functional analyses disclosed that BM endothelium-derived HSPCs are mainly Multipotent Progenitors (MPPs) and a few Hematopoietic Stem Cells. We used microarrays to detail the global programme of gene expression underlying the endothelial origin of LSK cells in the newborn bone marrow.

ORGANISM(S): Mus musculus

PROVIDER: GSE122857 | GEO | 2018/11/24

REPOSITORIES: GEO

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Project description:ETS Variant 6 (ETV6) encodes an essential transcriptional repressor abundantly expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with Thrombocytopenia 5 (T5), a poorly-understood genetic condition predisposing to thrombocytopenia and hematologic malignancies. To elucidate how germline ETV6 variants impact the HSPC compartment and contribute to disease, we generated a knock-in mouse harboring an Etv6R355X loss-of-function variant, which represents the mouse equivalent to the T5-associated variant ETV6R359X. All HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice under homeostatic conditions; however, these animals exhibit subtle shifts in the proportions and/or numbers of specific progenitor subtypes. To examine whether the Etv6R355X/+ mutation impacts HSPC function, we carried out serial competitive transplantation and observed that Etv6R355X/+ lineagesca1+cKit+ (LSK) cells exhibit significantly impaired reconstitution, with near complete failure to repopulate irradiated-recipients by the tertiary transplant. Mechanistic studies incorporating CUT&RUN, ATAC-Seq and Hi-C identify ETV6 binding at inflammatory gene loci, including those within the TNF pathway in Etv6+/+ HSPCs, the mouse BM-progenitor derived HPC5 cell line, and G-CSF-mobilized human CD34+ cells. Further, single-cell RNA sequencing of mouse LSK cells isolated six-weeks post-competitive transplantation reveals upregulation of inflammatory gene pathways. Corroborating these findings, we observe significantly increased production of TNF by Etv6R355X/+ versus Etv6+/+ HSPCs post-transplantation. From these studies, we conclude that ETV6 represses inflammatory response genes within HSPCs under conditions of hematopoietic stress, and that this mechanism may be critical to sustain HSPC function. Functionally, Etv6R355X/+ bone marrow (BM) cells exhibited reduced colony formation in later rounds of serial replating, while Etv6R355X/+ LSKs exhibited significantly impaired engraftment in serial competitive transplantation. RNA-seq of Etv6R355X/+ LSKs and/or sort-purified HSPCs revealed upregulation of genes associated with an active stem cell phenotype and pro-inflammatory signaling, including increased expression of the cytokine Tnf. CUT&RUN of Etv6+/+ LSKs and HPC5 hematopoietic progenitors showed that ETV6 binds to the Tnf locus at regions of open chromatin, while CUT&RUN of human CD34+ cells identified ETV6 targets enriched in TNF signaling. In line with these findings, Etv6R355X/+ HSPCs exhibited increased cell-cycling and TNF expression post-transplantation. Finally, genetic deletion of Tnf restored the serial replating capacity of Etv6R355X/+ BM cells.

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In vivo haematopoietic cell production by bone marrow-derived hemogenic endothelium

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