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A critical requirement for IκBα in controlling dormancy in Hematopoietic stem cells via retinoic acid during embryonic development [Cut&Tag]

ABSTRACT: Hematopoietic Stem Cells (HSCs) originate from the E11.5 aorta-gonads-and mesonephros (AGM) region during development before they migrate to the foetal liver for proliferation and maturation, and finally seed the bone marrow around birth, their final site of residence. In the AGM, HSCs reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). Molecular pathways that determine HSC fate instead of HPCs are still unknown, although inflammatory signalling has been implicated in the development of all blood cells, including NF-κB. Here, we describe a dormant phenotype of LT-HSCs in the IκBα KO. Although IκBα is critical for retaining inactive NF-κB complexes in the cytoplasm, it can regulate stem cell related genes by interacting with the PRC2 complex in the nucleus. Accordingly, we find decreased PRC2 dependent H3K27me3 accumulation at the promoters of PI3K and retinoic acid signalling molecules by cut and tag assay in AGM derived CD31+ cells, which includes HE/IAHC derived from IκBα KO embryos. Furthermore, this regulation of the retinoic acid signalling by IκBα is further confirmed by cut and tag assay for IκBα itself in CD31+ cells of the AGM and more specifically also in sorted LT-HSCs from the E14.5 foetal liver. Over-activation of the retinoic acid/PI3K levels in LT-HSCs of the IκBα KO is evident in their dormant molecular profile. Functionally, IκBα KO LT-HSCs are less proliferative and respond with delayed activation upon transplantation. Overall, we identify nuclear IκBα as an essential player specifically for HSC specification/proliferation from the onset of HSPC emergence in the AGM.

ORGANISM(S): Mus musculus

PROVIDER: GSE188524 | GEO | 2024/04/15

REPOSITORIES: GEO

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Project description:Haematopoietic stem cells (HSCs) are derived early from embryonic precursor cells, such as haemogenic endothelial cells and pre-HSCs. However, the identity of precursor cells remains elusive due to their rareness, transience, and inability to be isolated efficiently. Here we employed potent surface markers to capture the nascent pre-HSCs at 30% purity, as rigorously validated by single-cell-initiated serial transplantation assay. Then we applied single-cell RNA-Seq technique to analyse five populations closely related to HSC formation: endothelial cells, CD45- and CD45+ pre-HSCs in E11 aorta-gonad-mesonephros (AGM) region, and mature HSCs in E12 and E14 foetal liver. In comparison, the pre-HSCs showed unique features in transcriptional machinery, apoptosis, metabolism state, signalling pathway, transcription factor network, and lncRNA expression pattern. Among signalling pathways enriched in pre-HSCs, the mTOR activation was uncovered indispensable for the emergence of HSCs but not haematopoietic progenitors from endothelial cells in vivo. By comparing with proximal populations without HSC potential, the core molecular signature of pre-HSCs was identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating the step-wise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical application. RNA-Seq of 181 single-cell samples from 8 FACS sorted cell types: 1. endothelial cells (samples E11.0_EC_xxxx. CD31+ VE-cadherin+CD41-CD43-CD45-Ter119-); 2. T1 pre-HSCs (samples E11.0_T1_xxxx. CD31+CD45-CD41low c-Kit+CD201high); 3. T1 CD201- cells (samples E11.0_T1CD201neg_xxxx, CD31+CD45-CD41low c-Kit+CD201low/-) ; 4. T2 pre-HSCs (samples E11.0_T2_35xx. CD31+CD45+c-Kit+CD201high), 5. T2 CD41low (samples E11.0_T2_21xx, E11.0_T2_24xx and E11.0_T2_27xx. CD31+CD45+CD41low); 6. E12 HSCs (samples E12.5_FL_xxxx. Lin-Sca-1+Mac-1lowCD201+); 7. E14 HSCs (samples E14.5_FL_xxxx. CD45+CD150+CD48-CD201+); 8. Adult HSCs (samples Adult_HSC_xxxx. CD45+CD150+CD48-CD201+). ECs, T1 pre-HSCs, T1 CD201- cells, T2 pre-HSCs, T2 CD41low cells were sorted from E11 AGM region. Mature HSCs were sorted from E12 or E14 fetal liver and adult bonemarrow.

Project description:During embryonic development, the first hematopoietic stem cells (HSCs) arise from a transient population of endothelial cells lining the ventral surface of the dorsal aorta, via a process of endothelial to hematopoietic transition (EHT) at embryonic day (E) 10.5. This region contains resident PDGFRA+ stromal cells (PSCs), but their identity and role in HSC generation in the AGM are not well understood. Using a library of compound transgenic mice, we identified a population of PDGFRA+/Nestin-GFP (N-GFP)-/PDGFRB-/CD31- cells with PSC activity in the E10.5 and E11.5 mouse AGM. Freshly isolated PSCs were adept at forming blood vessels with CD31+ luminal endothelium enveloped by PDGFRB+ pericytes, when transplanted subcutaneously into mice. Conditional ablation of PDGFRA+ or Nestin+ cells led to either complete or partial loss of PSCs respectively, with severe loss of endothelial and pericyte-like cells, and concomitant loss of blood formation in the AGM. Lineage tracing studies using tamoxifen induction in PDGFRACreERT2/R26eYFP embryos showed that stromal, sub-endothelial, endothelial and long-term repopulating hematopoietic stem cells (LT-HSCs) cells in the E11.5 AGM were progeny of PDGFRA cells. Using transgenic reporter mice, we showed that MesP1 (mesoderm) derived PDGFRA+ cells dominated the sub-endothelial and deeper ventral stroma in the AGM at E10.5 and E11.5 but were replaced by Wnt1 (neural crest) derived cells at E13.5. Re-aggregation of E11.5 Mesp1 derived PSC cells with E13.5 aortic or adult cardiac non-hemogenic endothelial cells resulted in the generation of endothelial cell derived LT-HSCs. RNA-sequencing analysis of non-hemogenic E13.5 endothelial cells showed up-regulation of EHT genes, WNT, BMP and Notch cell signalling pathways when re-aggregated with E11.5 Mesp1 derived PSCs. LT-HSC generation from these re-aggregates was suppressed by dose-dependent inhibition of PDGF-AA/PDGFRA signalling. Taken together, we report that PSC populations in the AGM are temporally dynamic, and that MesP1-derived PSCs regulate hemogenic potential of the endothelium and that this cooperativity is dependent on PDGF-AA/PDGFRA signalling. This submission represents the RNAseq component of the study.

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A critical requirement for IκBα in controlling dormancy in Hematopoietic stem cells via retinoic acid during embryonic development [Cut&Tag]

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