Extremely low-frequency electromagnetic fields facilitate both osteoblast and osteoclast activity through Wnt/ß-catenin signaling in the zebrafish scale.

Electromagnetic fields (EMFs) have received widespread attention as effective, noninvasive, and safe therapies across a range of clinical applications for bone disorders. However, due to the various frequencies of devices, their effects on tissues/cells are vary, which has been a bottleneck in understanding the effects of EMFs on bone tissue. Here, we developed an in vivo model system using zebrafish scales to investigate the effects of extremely low-frequency EMFs (ELF-EMFs) on fracture healing. Exposure to 10 millitesla (mT) of ELF-EMFs at 60 Hz increased the number of both osteoblasts and osteoclasts in the fractured scale, whereas 3 or 30 mT did not. Gene expression analysis revealed that exposure to 10 mT ELF-EMFs upregulated wnt10b and Wnt target genes in the fractured scale. Moreover, ß-catenin expression was enhanced by ELF-EMFs predominantly at the fracture site of the zebrafish scale. Inhibition of Wnt/ß-catenin signaling by IWR-1-endo treatment reduced both osteoblasts and osteoclasts in the fractured scale exposed to ELF-EMFs. These results suggest that ELF-EMFs promote both osteoblast and osteoclast activity through activation of Wnt/ß-catenin signaling in fracture healing. Our data provide in vivo evidence that ELF-EMFs generated with a widely used commercial AC power supply have a facilitative effect on fracture healing.

The sinusoidal hematopoietic niche is formed by Jam1a via Notch signaling in the zebrafish kidney.

The zebrafish is a unique model to understand hematopoietic niches as hematopoietic stem/progenitor cells are maintained in the kidney. However, little is known about which cell types in the kidney play a role in hematopoietic niches. Here, we demonstrate that the sinusoidal endothelium is an essential and conserved niche component in the zebrafish kidney. Histological analysis revealed that runx1:mCherry + hematopoietic cells were predominantly detected in the dorsolateral region of the kidney where sinusoids are highly developed. Loss of Junctional adhesion molecule 1a (Jam1a), which is expressed in both sinusoidal endothelial cells and hematopoietic cells, resulted in a remarkable reduction in sinusoids and a defect in hematopoietic niches. We found that Jam1a regulates jagged-1a expression in vascular endothelial cells to form a sinusoidal structure in the kidney. Collectively, these data suggest that sinusoids are formed by Jam1a via endothelial Notch signaling to provide hematopoietic niches in the zebrafish kidney.

The utility of zebrafish cardiac arrhythmia model to predict the pathogenicity of KCNQ1 variants.

Genetic testing for inherited arrhythmias and discriminating pathogenic or benign variants from variants of unknown significance (VUS) is essential for gene-based medicine. KCNQ1 is a causative gene of type 1 long QT syndrome (LQTS), and approximately 30% of the variants found in type 1 LQTS are classified as VUS. We studied the role of zebrafish cardiac arrhythmia model in determining the clinical significance of KCNQ1 variants. We generated homozygous kcnq1 deletion zebrafish (kcnq1del/del) using the CRISPR/Cas9 and expressed human Kv7.1/MinK channels in kcnq1del/del embryos. We dissected the hearts from the thorax at 48 h post-fertilization and measured the transmembrane potential of the ventricle in the zebrafish heart. Action potential duration was calculated as the time interval between peak maximum upstroke velocity and 90% repolarization (APD90). The APD90 of kcnq1del/del embryos was 280 ± 47 ms, which was significantly shortened by injecting KCNQ1 wild-type (WT) cRNA and KCNE1 cRNA (168 ± 26 ms, P < 0.01 vs. kcnq1del/del). A study of two pathogenic variants (S277L and T587M) and one VUS (R451Q) associated with clinically definite LQTS showed that the APD90 of kcnq1del/del embryos with these mutant Kv7.1/MinK channels was significantly longer than that of Kv7.1 WT/MinK channels. Given the functional results of the zebrafish model, R451Q could be reevaluated physiologically from VUS to likely pathogenic. In conclusion, functional analysis using in vivo zebrafish cardiac arrhythmia model can be useful for determining the pathogenicity of loss-of-function variants in patients with LQTS.

Inhibition of Canonical Wnt Signaling Promotes Ex Vivo Maintenance and Proliferation of Hematopoietic Stem Cells in Zebrafish.

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. Coculture 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 with 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.

Hematopoietic Cell Isolation by Antibody-Free Flow Cytometry in the Zebrafish Embryo.

The zebrafish is a useful model to identify genes functioning in hematopoiesis, owing to high conservation of hematopoiesis. Flow cytometry is widely used to isolate and quantitatively characterize human and mouse hematopoietic cells, often using fluorescently labeled antibodies. However, such analysis is limited in zebrafish due to lack of antibodies that recognize zebrafish hematopoietic cells. We here describe methods for isolation of hematopoietic cells by antibody-free flow cytometry in the zebrafish embryo. Hematopoietic stem cells (HSCs) are specified from a specific subset of vascular endothelial cells, the hemogenic endothelial cell (HEC), by a high level of Notch signaling. In combination with a Notch reporter line (Tp1:GFP) and a vascular specific line (fli1a:dsRed), HECs can be isolated as Tp1:GFPhigh fli1a:dsRed+ cells at 20-22 hours post-fertilization (hpf). Zebrafish erythrocytes can be purified using 1,5-bis{[2-(dimethylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9,10-dione (DRAQ5), a DNA-staining fluorescent dye, and gata1:dsRed (erythroid marker line). DRAQ5high dsRed+ cells are morphologically erythrocyte-like, hemoglobin-stained positive, and express erythropoiesis-related genes. Zebrafish neutrophils can be also isolated using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5. PHA-Elow DRAQ5low cells have myeloperoxidase activity, are Sudan Black B-positive, and express neutrophil-related genes. These methods will help to perform genetical and functional assays for various types of hematopoietic cells in zebrafish embryos.

Melatonin suppresses both osteoblast and osteoclast differentiation through repression of epidermal Erk signaling in the zebrafish scale.

Melatonin has been implicated in the regulation of bone metabolism; however, the molecular mechanisms underlying its involvement in fracture healing are still obscure. We previously developed an in vivo fracture healing model using the scale of a double-transgenic zebrafish, trap:GFP; osterix:mCherry, which labels osteoclasts and osteoblasts with GFP and mCherry, respectively. Here we show using this model that melatonin inhibits both osteoblast and osteoclast differentiation under fracture stress through the repression of Erk signaling in epidermal cells of the scale. Melatonin treatment resulted in reduced numbers of both osteoblasts and osteoclasts in the fractured scale. Immunochemistry analysis revealed that Erk signals in epidermal cells, which express melatonin receptors, were greatly enhanced in response to fracture stress, but this enhancement was blocked by melatonin treatment. Moreover, inhibition of Erk signaling phenocopied the effects of melatonin treatment in the fractured scale. Collectively, these data suggest that the activation of epidermal Erk signaling is required for both osteoblast and osteoclast differentiation in the early stage of fracture healing, and melatonin suppresses epidermal Erk signaling, leading to impaired fracture healing.

Uptake of osteoblast-derived extracellular vesicles promotes the differentiation of osteoclasts in the zebrafish scale.

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:GFPhigh 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.

Dual role of Jam3b in early hematopoietic and vascular development.

In order to efficiently derive hematopoietic stem cells (HSCs) from pluripotent precursors, it is crucial to understand how mesodermal cells acquire hematopoietic and endothelial identities: two divergent, but closely related, cell fates. Although Npas4 has been recently identified as a conserved master regulator of hemato-vascular development, the molecular mechanisms underlying cell fate divergence between hematopoietic and vascular endothelial cells are still unclear. Here, we show in zebrafish that mesodermal cell differentiation into hematopoietic and vascular endothelial cells is regulated by Junctional adhesion molecule 3b (Jam3b) via two independent signaling pathways. Mutation of jam3b led to a reduction in npas4l expression in the posterior lateral plate mesoderm and defects in both hematopoietic and vascular development. Mechanistically, we show that Jam3b promotes endothelial specification by regulating npas4l expression through repression of the Rap1a-Erk signaling cascade. Jam3b subsequently promotes hematopoietic development, including HSCs, by regulating lrrc15 expression in endothelial precursors through the activation of an integrin-dependent signaling cascade. Our data provide insight into the divergent mechanisms for instructing hematopoietic or vascular fates from mesodermal cells.

Enrichment of hematopoietic stem/progenitor cells in the zebrafish kidney.

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.

Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling.

Notch signalling plays a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development and requires intimate contact between signal-emitting and signal-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 specification. It has remained unclear, however, how these somitic requirements are connected to the later emergence of HSCs from the dorsal aorta. Here we show in zebrafish 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 (also known as f11r) and migrate axially across the ventral somite, where Jam2a and the 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 dorsal aorta.