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Single-cell dissection of long non-coding RNAs pivotal for embryonic hematopoietic stem cell development

ABSTRACT: In mammalian embryos, the stepwise generation of hematopoietic stem cells (HSCs) from endothelial precursors and pre-hematopoietic stem cells (pre-HSCs) is stringently modulated by signaling pathways and transcription factors. Nevertheless, the understanding of regulatory strength and mechanism by non-coding RNA (ncRNA) remains entirely lacking. Taking advantage of our capacity to capture rare single pre-HSCs and HSCs in vivo and accordingly generate the single-cell transcriptome datasets, here we presented the first long non-coding RNA (lncRNA) landscape of HSC development at single-cell resolution. Combining bioinformatics analysis and functional screening, we identified 6 lncRNAs showing influence on hematopoietic progenitor generation in vitro. Moreover, endothelial disruption of H19 in vivo resulted in defective emergence of functional pre-HSCs and HSCs in aorta-gonad-mesonephros region, albeit with unperturbed development of erythro-myeloid progenitors in yolk sac. Single-cell RNA sequencing revealed that H19 deficient pre-HSCs (The DMR region of H19 was knocked out in the "KO" samples) possessed similar molecular pattern with endothelial cells, suggesting their severe blockade during hematopoietic transition. Methylation sequencing of pre-HSCs further demonstrated hyper methylation on promoter region of several master hematopoietic transcription factors, including Runx1 and Spi1, by H19 deficiency. Taken together, our study identified H19 as the first non-coding gene indispensable for endothelial-to-HSC transition, and the single-cell lncRNAomics map should be valuable for exploring epigenetic control of HSC generation and regeneration.

ORGANISM(S): Mus musculus

PROVIDER: GSE108653 | GEO | 2020/12/01

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.

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Single-cell dissection of long non-coding RNAs pivotal for embryonic hematopoietic stem cell development

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