During life, blood is regenerated by multipotent and self-renewable hematopoietic stem and progenitor cells (HSPCs) through hematopoiesis. In vertebrates, these cells originally emerge from the endothelium of the embryonic aorta trough the endothelial-to-hematopoietic transition (EHT). The zebrafish has become a key model to study this process, thanks to its optical transparency, its rapid development and conserved developmental programs with mammals.
We identified two undescribed genes, si:ch211-214p16.1 and si:ch211-214p16.2 (referred to as p16.1 and p16.2), which are specifically expressed by emerging HSPCs in the dorsal aorta of the zebrafish embryo as early as 30-36 hours post-fertilization (hpf). During development, both genes are then expressed in other hematopoietic organs of the zebrafish embryo, such as the caudal hematopoietic tissue (CHT) where HSPCs expand, and the thymus where HSPCs differentiate into T-cells. The expression of p16.1 is eventually detected in the kidney glomeruli that will develop into the whole kidney marrow (WKM), the fish equivalent of the mammal bone marrow, where HSPCs reside in adult animals.
Given this expression pattern, we hypothesized that p16.1 and p16.2 might play a key function in HSPC biology. By performing transient knock-down of both genes, I have determined that p16.1 and p16.2 were important for T-cell differentiation in the zebrafish embryo, as observed by a decrease of the T-cell marker rag1 at 5dpf in the thymus, which was linked to a decrease of lymphoid-committed progenitors in the CHT at 48hpf and a subsequent delay in thymus colonization. Surprisingly, double knock-out animals did not recapitulate this delayed thymopoiesis phenotype. By performing bulk RNA sequencing on T-cell progenitors from double knock-out animals, I found out that several genetic factors known to be involved in T-cell differentiation in mammals were upregulated in double knock-out thymocytes. I uncovered that one of these factors, ccr9b, played a key role in the compensatory mechanism to achieve thymopoiesis in double mutants.
I also characterized the p16.1:GFPLT reporter line, previously established in the laboratory, to show expression mainly in the lymphoid compartment. By combining this new line with the CD45:DsRed line, that marks T cells and myeloid cells, this allows to discriminate of all major hematopoietic cell types found in adult hematolymphoid organs by flow cytometry. I took advantage of this combination to study the hematopoietic recovery observed in adult runx1 mutants, and found that the recovery of each mutant does not follow a precise pattern, even if most adult mutants always lack neutrophils and T cells, in agreement with the known roles of runx1 during hematopoietic differentiation.
