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

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. Hematopoietic cells (expressing fluorescent reporters under the control of cd41 and gata2b promoters) from 5dpf Tg(gata2b:Gal4;UAS:RFP; cd41:eGFP) larvae were collected after dissection (separating the anterior and posterior region along a transversal section at the posterior limit of the elongated yolk), dissociation and sorting using a BD FACS AriaIII cell sorter. Transcriptomic libraries were generated using the 10X Genomics Chromium Next GEM Single Cell 3ʹ Kit v3.1 kit, and sequenced on a Novaseq6000 platform to generate 150-bp paired-end reads, and a theoretical count of 40 000 reads per cell. Alignment and demultiplexing was performed using the CellRanger (version v6.1.2) and reads were aligned to the zebrafish genome annotated by the Lawson lab (version 4.3.2).