Investigating the role of Apelin signaling during hematopoietic stem cell development in zebrafish

Hematopoietic stem and progenitor cells (HSPCs) are multipotential stem cells which can self-renew and generate all blood lineages throughout life. Only a very limited population of HSPCs emerges during embryonic development. Consequently, defects in the development of HSPCs lead to a variety of hematopoietic diseases in adulthood. Early in embryonic development HSPCs arise in the ventral floor of the dorsal aorta (VDA) from a distinct subpopulation of arterial endothelial cells (ECs), so called hemogenic endothelial cells (HECs). In a process called endothelial-to-hematopoietic transition (EHT) these arterial endothelial cells (ECs) lose their arterial identity and acquire a hematopoietic fate. The nascent HSPCs bud out of the vessel wall into the subaortic space to eventually enter circulation via the posterior cardinal vein (PCV) to colonize the transient vascular stem cell niche, the caudal hematopoietic tissue (CHT) where they proliferate and partially differentiate before they mobilize again. Via the bloodstream, they travel to the adult niches for hematopoietic and lymphopoietic organs, the kidney marrow and the thymus, respectively. These organs constitute all blood lineages during life. The G-protein-coupled receptor Apelin Receptor (Aplnr) has been shown to play an important role in vasculogenesis and angiogenesis. However, little is known about the role of Apelin signaling in hematopoiesis. Here, we show the zonation of the DA into apelin receptor (aplnr) expressing and aplnr non-expressing ECs by live confocal imaging of novel transgenic reporters. Of interest, aplnr non-expressing ECs are exclusively located in the VDA. Molecular analysis of HSPC marker genes confirmed that these aplnr-negative ECs give rise to HSPCs. Furthermore, zonation by aplnr expression in the DA functionally restricts the hemogenic endothelium. Loss of Apelin signaling leads to an extension of the hemogenic endothelium and subsequently to an increased formation of HSPCs within the DA. Conversely, forced activation of Apelin signaling in the vasculature leads to a decrease in HSPC numbers in the DA by maintaining EC identity. Furthermore, we could observe reduced expression of tbx20, a transcription factor, upon loss of Apelin signaling. Morpholino injection experiments showed that loss of tbx20 results in increased runx1 expression, phenocopying the loss of Apelin signaling. Our experiments therefore indicate Tbx20 as a potential downstream effector of Apelin signaling. In addition, we show that HSPCs exhibit a skew towards the erythroid lineage in Apelin deficient larvae. Our results discover Apelin signaling as a critical regulator of cell fates in the DA, acting as a molecular switch to balance vascular and hematopoietic identities. Besides the involvement of Apelin signaling during HEC specification and HSPC emergence, our results suggest that Apelin plays an additional role in HSPC/CHT niche interaction. Nascent HSPCs start to express apln after budding from the DA. We could show that the apln mRNA expressing HSPCs lodge in contact with aplnr expressing niche ECs, suggesting a possible interaction between these cell types. Our experiments showed that only a fraction of HSPCs is expressing apln. Further analysis of this population is still pending. Two hypotheses are proposed: (1) They represent a specialized subpopulation of HSPCs, and (2) HSPCs only express apln in distinct time frames during niche interaction, e.g. only during homing, during niche remodeling or during mobilization. Further analysis of this population is still pending. Genetic experiments could also show, that loss of Apelin signaling results in an altered morphology and increased volume of the CHT vascular stem cell niche. In summary, our study identifies Apelin signaling as a key upstream regulator of vascular and hematopoietic cell fate within the DA. By precisely modulating the spatial and temporal dynamics of Apelin signaling, this pathway influences the balance between vascular maintenance, angiogenesis, and HSPC formation. Furthermore, Apelin signaling seems to be involved in HSPC/niche interaction, but more research must be conducted to fully uncover the exact role. These insights enhance our understanding of the hierarchical acquisition of hematovascular programs and could inform approaches for the de novo generation of functional HSPCs for therapeutic use.