Single-cell and in situ spatial analyses reveal the diversity of newly born hematopoietic stem cells and of their niches [scRNA-Seq]

Hematopoietic stem cells (HSCs) and more committed progenitors (collectively referred to as HSPCs) emerge from vessels during development, via Endothelial-to-Hematopoietic Transition (EHT). Recently, using the zebrafish embryo, we showed that two EHT cell types emerge from the dorsal aorta, raising the question of their subsequent fate. To address this issue, we established a complex pipeline based on single-cell photoconversion and transgenic lines to characterize the abilities of EHT cell progenies to conquer hematopoietic organs and to obtain their transcriptomic profiles. We show that the two EHT cell types lead to partly differentially fated cells, with significant differences in thymus colonization and T-lymphoid lineage commitment. In addition, we investigated implantation of HSPCs in niches, with the support of HSPC signatures (gata2b and cd34/podocalyxin), retrieved from our single-cell datasets. This revealed, at unprecedented resolution, the homing of HSPCs in niches of entire early larvae, including the pronephros, the sub-aortic and caudal regions, as well as the area contacting the supra-intestinal artery. Our work provides new insights on fundamental aspects of HSPC fate acquisition, from their emergence to their homing in specific niches. Single-cell transcriptomics analysis of newly born hematopoietic stem and progenitor cells. This dataset comprises progenies of cells photoconverted at 2dpf and sorted by FACS at 5dpf, using the Tg(kdrl:nls-kikume) transgenic line, as well as sorted (FACS) hematopoietic cells from transgenic reporter lines, namely from the Tg(runx1+23:eGFP) and the outcross of the Tg(cd41:eGFP) and Tg(gata2b:RFP) lines. Larvae were collected after dissection (separating the rostral, anterior and posterior region along a transversal section at the posterior limit of the elongated yolk and at the posterior limit of the swim bladder), dissociation and sorting using a BD FACS AriaIII cell sorter. Progenies of photoconverted cells comprise progenies of single-EHT photoconversion (either EHT pol+ or EHT pol-) or whole hemogenic endothelium photoconversion. Cells were sorted in 384 wells plates (28 plates, in 2 independent sessions of 14 plates each) and the transcriptomic library was prepared according to the MARS-seq protocol 2.0 (Keren-Shaul et al. 2019). Libraries were then sequenced using the NextSeq 500/550 High Output Kit v2 (75 cycles) (Illumina). Raw reads were converted to fastq files using bcl2fastq package (2.20.0). Reads were demultiplexed and mapped to the Lawson Lab curated genome [(Lawson et al., 2020), v4.3.2] using the MARS-seq pipeline scripts (Keren-Shaul et al., 2019).