Function of apico-basal polarity establishment and intercellular junction dynamics in the emergence and fate of hematopoietic stem cells in the zebrafish embryo

Overview & Synopsis

Long-term hematopoietic stem cells (LT-HSC) are cells with long-term regenerative potential that give rise to cells of all hematopoietic lineages. In vertebrates, they are generated during a short window of the embryonic development via a developmental process called the Endothelial-to-Hematopoietic Transition (EHT). During this transition, placed under the control of the transcription factor Runx1, a subpopulation of aortic cells — the hemogenic endothelium — extrudes from the floor of the aorta. My lab previously reported, using the zebrafish embryo model, that this extrusion involves the invagination of the cell’s luminal membrane, the contraction of an apical acto-myosin belt, and is characterized by the anisotropic organization of junction components. After emergence, newly generated HSC precursors (pre-HSC) migrate to seed embryonic hematopoietic organs (namely the Caudal Hematopoietic Tissue (CHT), thymus and pronephros in the zebrafish). Unpublished data from my lab showed that, surprisingly and contrary to what is observed in many cell extrusion processes, EHT-undergoing cells maintain/reinforce an apico-basal polarity throughout their emergence. Moreover, we reported the existence of a hitherto undescribed second type of EHT. This second type of EHT is characterised by an absence of luminal invagination, an absence of apico-basal polarity, and an apparent crawling of neighboring endothelial cells on its luminal membrane. In this context, the two goals of my PhD were: first to characterize some critical steps of the dynamic interplay between emerging Hematopoietic Stem Cells precursors and endothelial neighbours before and during the EHT, and second to investigate whether the two types of EHT give rise to progenies with (dis)similar migration, proliferation, differentiation and stemness potential. In the first line of results, I investigated the role of potential regulators of EHT, linking contractile actomyosin to apico-basal polarity, i.e. Rho GEF proteins recruited at apical junctions via a PDZ domain. I used interfering approaches coupled to phenotypic analysis using high-resolution live imaging as well as quantitative analysis of junction components renewal by FRAP. We uncovered the sensitivity of ArhGEF11/PDZ-RhoGEF mRNA expression and alternative splicing to Runx1 interference. Interfering with ArhGEF11 alternative splicing alters the dynamics of junction components around hemogenic cells, thus hindering the intercalation of endothelial cells around hemogenic cells, a necessary step for extrusion completion. This modification of the interplay between endothelial and hemogenic cells leads to an accumulation of hemogenic cells in the aorta. For the second line of results, I developed a single cell tracing protocol that allowed me to photoconvert single cells during their emergence and follow their progeny three days after. I coupled this approach to in vivo tracing using confocal microscopy and single cell sorting followed by transcriptomic analysis (MARS-seq). This strategy allowed me to study in depth the respective fates of both EHT cell types, spatially, temporally and transcriptionally. I show that both types of EHT give rise to multipotent hematopoietic cells and are able to generate cells of all hematopoietic lineages (erythroid/megakaryocyte, myeloid and lymphoid). However, they display a discrepancy in their ability to niche in the thymus and bias in their ability to generate different cell types of the lymphoid lineage. Moreover, contrary to previous investigations suggesting that hematopoietic cells directly enter the circulation after their emergence, we observed that some cells stay in the sub-artic region for long periods of time (up to 3days). Transcriptomic analysis of those cells shows the presence of all hematopoietic lineages as well as multiple HSC subpopulations. This suggests that the sub-aortic space might be an overlooked niche that could contribute to the specification of HSC.