Summary Notch signalling plays a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development1-3 and requires intimate contact between signal emitting and receiving cells, although little is known regarding when, where, and how these intercellular events occur. We previously reported that the somitic Notch ligands, Dlc and Dld, are essential for HSC specification4. It has remained unclear, however, how these somitic requirements are connected to the later emergence of HSCs from the dorsal aorta (DA). Here we show that Notch signalling establishes HSC fate as their shared vascular precursors migrate across the ventral face of the somite and that Junctional adhesion molecules (JAMs) mediate this required Notch signal transduction. HSC precursors express jam1a and migrate axially across the ventral somite, where Jam2a and Notch ligands Dlc and Dld are expressed. Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs. Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos. Together, these results indicate that Jam1a – Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the DA.

Differentiation of osteoclasts (OCs) from hematopoietic cells requires cellular interaction with osteoblasts (OBs). Due to the difficulty of live-imaging in the bone, however, the cellular and molecular mechanisms underlying intercellular communication involved in OC differentiation are still elusive. Here, we develop a fracture healing model using the scale of trap: GFP; osterix:mCherry transgenic zebrafish to visualize the interaction between OCs and OBs. Transplantation assays followed by flow cytometric analysis reveal that most trap:GFP high OCs in the fractured scale are detected in the osterix:mCherry + fraction because of uptake of OB-derived extracellular vesicles (EVs). In vivo live-imaging shows that immature OCs actively interact with osterix:mCherry + OBs and engulf EVs prior to convergence at the fracture site. In vitro cell culture assays show that OB-derived EVs promote OC differentiation via Rankl signaling. Collectively, these data suggest that EV-mediated intercellular communication with OBs plays an important role in the differentiation of OCs in bone tissue.

Hematopoietic stem cells (HScs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. it has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry. the double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a + runx1 + ) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. transcriptome analysis revealed that gata2a + runx1 + cells showed typical molecular signatures of HScs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. transplantation assays demonstrated that long-term repopulating HScs were highly enriched within the gata2a + runx1 + fraction. in contrast, colony-forming assays showed that gata2a − runx1 + cells abundantly contain erythroid-and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.Hematopoietic stem cells (HSCs) are self-renewing multipotent cells that can generate all types of blood cells over the lifetime of an individual and can be used therapeutically to treat hematopoietic diseases 1 . In the adult, most HSCs present in bone marrow are quiescent and divide rarely under homeostatic conditions. HSCs produce a heterogeneous pool of hematopoietic progenitor cells (HPCs), which have limited or no self-renewal ability, but rapidly proliferate and differentiate to satisfy the requirements for new mature blood cells 2,3 . Although the frequency of HSCs is extremely rare in bone marrow, HSC potential can be evaluated by transplantation assays, whereby the relative hematopoietic reconstitution activity of co-transplanted donor and competitor cells are compared in a recipient 4 . Purification of HSCs from murine and human bone marrow has been facilitated via transplantation assays using combinations of multiple cell-surface markers [5][6][7][8][9] . Studies in mice revealed that a single CD150 + CD34 − c-kit + Sca-1 + Lineage-marker − cell in the bone marrow showed long-term and multilineage hematopoietic reconstitution following transplantation 10,11 . Prospective isolation of highly purified HSCs thus elucidated many aspects of HSC biology, including self-renewal, differentiation, and HSC niches.The zebrafish is an excellent model for the study of HSCs due to its many unique advantages. Many valuable tools and experimental methods have been established for the study of hematopoietic cells in zebrafish (e.g. transgenic/mutant animals, transplantation assays, cell culture assays, etc.) 12,13 . Moreover, genome-editing technology based on...

The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow, however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Co-culture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared to candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (> 2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.