Functional screening in zebrafish reveals Sslc39a10 as a potentially important zinc transporter in early hematopoiesis. A) Schematic diagram depicting the strategy for performing functional screening in zebrafish. B,C) Relative levels of the indicated mt2 and slc39a family genes were measured in cmyb⁺ cells treated at 60–72 hpf with 100 µm TPEN or/and 100 µm ZnSO₄ (n = 3 per group). D) Representative images of embryos at 3dpf following injection of a control morpholino (MO), slc39a6 MO, or slc39a10 MO. The arrows indicate the heart region. Note the visibly smaller head and presence of cardiac edema in the slc39a10 morphant embryo. E) Representative images of o‐dianisidine‐stained embryos at 3dpf following injection of the control MO, slc39a6 MO, or slc39a10 MO. The arrows indicate the heart region. F) Whole‐mount in situ hybridization (WISH) of cmyb in 2dpf embryos after injection of the control MO, slc39a6 MO, or slc39a10 MO. Note the extremely low expression of cmyb in the caudal hematopoietic tissue (CHT) region (arrows) in the slc39a10 morphant. G) DNA and corresponding amino acid sequences of the wild‐type (WT) slc39a6 allele and the mutant allele after a 4‐nucleotide deletion (‐4 bp) using CRISPR/Cas9‐based editing, introducing a premature stop codon. H) Representative images of a WT embryo and slc39a6 mutant sibling at 5dpf. The arrows indicate the heart region. I) DNA and corresponding amino acid sequences of the WT slc39a10 allele and mutant allele after a 4‐nucleotide deletion (‐4 bp) using CRISPR/Cas9‐based editing, introducing a premature stop codon. J) Representative images of WT embryos and slc39a10 mutant siblings at the indicated stages. The arrows indicate the heart region. The data in (B) and (C) are presented as the mean ± SD. p values were determined using one‐way ANOVA with Tukey's post hoc test (for multi‐group comparisons). *p < 0.05, ***p < 0.001, and ns, not significant.

Slc39a10 mutant zebrafish have defective hematopoiesis. A) WISH of slc39a10 in WT embryos at the indicated stages. The arrows indicate a concentrated expression of slc39a10 in the hematopoietic region. B) Mating strategy for producing offspring from slc39a10 heterozygous crosses. The offspring were born at the expected Mendelian ratio. Note that the homozygous slc39a10 mutant embryos have a small head, small eyes, and cardiac edema. C) Kaplan–Meier survival curve of WT and slc39a10 mutant sibling (n = 15 and 13 embryos, respectively). D) Summary of zinc concentration measured in 3dpf WT and slc39a10 mutant sibling embryos loaded with the fluorescent zinc probe FluoZin‐3, AM (n = 3 per group). E) WISH of cmyb in WT and slc39a10 mutant siblings at the indicated stages. The arrows indicate the CHT region. F,G) Representative images of WT and Tg(cmyb:eGFP) slc39a10 mutant sibling embryos either uninjected or injected with slc39a10 mRNA of zebrafish, mouse, and human gene orthologs (F), and quantification of cmyb⁺ cells in the CHT region at 3dpf (G). The dashed boxes indicated the region of HSPC counting. Scale bar, 50 µm. H) Schematic illustration of the construct of full‐length slc39a10 driven by the runx1 enhancer. I) Confocal imaging showing the expression of Slc39a10 protein indicated by the green fluorescence (GFP) in runx1⁺ HSPCs in the CHT region at 2 dpf. The white arrows indicated runx1⁺ HSPC in the CHT. Scale bar, 50 µm. J) qPCR showing the mRNA level of slc39a10 in EGFP⁻ and EGFP⁺ embryos at 4 dpf (n = 3 per group). K) WISH showing that the decreased expression of cmyb in slc39a10 mutant embryos was partially rescued by overexpression of slc39a10 in runx1⁺HSPCs. The red arrows indicate cmyb⁺ HSPCs in the CHT. Scale bar, 50 µm. L) Representative images of WT and slc39a10 mutant sibling embryos in Tg(globinLCR:GFP) background at the indicated stages. M,N) Example FACS analysis plots of GFP fluorescence measured in erythrocytes obtained from WT and slc39a10 mutant sibling embryos at the indicated stages and summary of the percentage of GFP⁺ erythrocytes (n = 4 per group). O) WISH of hbae3, l‐plastin, lyz, mpo, and rag1 mRNA in WT and slc39a10 mutant sibling embryos at the indicated stages. The red arrows indicate HSPCs in the CHT, and the black arrowheads indicate T cells in the thymus. Data in (D), (G), (J), (M), and (N) as mean ± SD. p values of survival in (C) were determined using the Log‐rank test, in (D), (G), (J), (M), and (N) using 2‐tailed unpaired Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001, and ns, not significant.

Loss of slc39a10 reduces the number of HSPCs in zebrafish via increased cell death. A) Developmental timeline showing the sites of hematopoiesis in zebrafish. The primitive wave begins at 12 hpf in two locations, the anterior lateral mesoderm (ALM) and the intermediate cellular mass (ICM), generating the majority of monocytes and erythrocytes before 24 hpf. In the definitive wave, which begins at 36 hpf, HSPCs arise from the AGM region and then migrate to the CHT region, where they undergo transitional proliferation and differentiation. The red lines indicate the shifting sites of hematopoiesis, and the green dots indicate HSPCs. B) Representative brightfield images of the AGM region, showing WISH of runx1 and cmyb in 36 hpf embryos injected with the control MO or the slc39a10 MO. C,D) Example fluorescence images of 28, 52, and 72 hpf Tg(cmyb:eGFP) embryos injected with the control MO or slc39a10 MO (C), and quantification of cmyb⁺ cells in the CHT measured at 36dpf and 48 hpf (D; n = 26 for control and n = 29 for slc39a10 morphants at 36 hpf, n = 35 for control and n = 40 for slc39a10 morphants at 48 hpf). E,F) Representative images (E) and quantification (F) of γ‐H2aX immunostaining in the CHT region of 48 hpf Tg(cmyb:eGFP) embryos injected with the control MO or slc39a10 MO (n = 10 each). G,H) Representative images (G) and quantification (H) of TUNEL immunostaining in the CHT region of 48 hpf Tg(cmyb:eGFP) embryos injected with the control MO or slc39a10 MO (n = 18 for control and n = 21 for slc39a10 morphants). I,J) Representative images (I) and quantification (J) of pH3 immunostaining in the CHT region of 48 hpf Tg(cmyb:eGFP) embryos injected with the control MO or slc39a10 MO (n = 13 for control and n = 21 for slc39a10 morphants). Data in (D), (F), (H), and (J) are presented as mean ± SD. p values in (D), (F), (H), and (J) using 2‐tailed unpaired Student's t‐test. ***p < 0.001 and ns, not significant.

Mice lacking Slc39a10 in hematopoietic cells have defective hematopoiesis and die within 1 day of birth. A) Alignment of the amino acid sequences of zebrafish (Danio rerio) slc39a10, mouse (Mus musculus) Slc39a10, and human (Homo sapiens) SLC39A10 proteins, as well as the scoring for protein homology obtained using ClustalW. B) Slc39a10 mRNA levels were measured in FL LT‐HSCs obtained from control and Slc39a10 cKO mice (n = 5 mice per group). C) Representative images of newborn (P1) control and cKO mouse pups. D) Kaplan–Meier survival curve of control and cKO mice (n = 8 and 10 for control and cKO, respectively). E) Example images of peripheral blood taken from control and cKO mice. F–H) Summary of red blood cell (RBC; F), white blood cell (WBC; G), and platelet (PLT; H) counts in the peripheral blood of control and cKO mice (n = 3 mice per group). I,J) Wright–Giemsa‐stained peripheral blood smears (I) and bone marrow smears (J) taken from control and cKO mice. Scale bars: 100 µm. K) Gross appearance of the whole body (top) and fetal liver (bottom) of control, heterozygous (het), and homozygous cKO embryos at E14.5. L) Absolute numbers of liver cells in control and cKO embryos measured at E14.5 (n = 10 and 7 for control and cKO, respectively). M) Fetal liver cells were obtained from E14.5 embryos, and long‐term repopulating HSCs (LT‐HSCs) were gated using flow cytometry. N,O) Summary of the percentage and absolute number of LT‐HSCs in the liver of control and cKO embryos (n = 10 and 7 for control and cKO, respectively). P) Schematic depiction of hematopoiesis showing the proliferation and differentiation of HSCs in the fetal mouse liver. Q,R) Gating strategy (Q) and absolute numbers (R) of the indicated erythrocyte developmental stages in the liver of control and cKO embryos were measured at E14.5 (n = 4 and 3 for control and cKO, respectively). S–U) Gating strategy (S), frequency (T), and absolute numbers (U) of myeloid cells (defined as Mac1⁺Gr1⁺), B cells (defined as CD19⁺), and T cells (defined as CD3⁺) in the liver of control and cKO embryos measured at E14.5 (n = 4 and 3 for control and cKO, respectively). Data in this figure are represented as mean ± SD. p values of survival in (D) were determined using the Log‐rank test, in (B), (F), (G), (H), (L), (N), (O), (R), (T), and (U) using 2‐tailed unpaired Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001, and ns, not significant.

Slc39a10‐deficient HSCs have increased ROS levels, increased DNA damage, a higher prevalence of apoptosis, and impaired repopulation capacity. A,B) Representative FACS profiles (A) and quantification (B) of MitoSOX mean fluorescence intensity (MFI) measured in HSCs (Lin⁻Sca1⁺Mac1⁺) obtained from control and cKO embryos at E14.5 (n = 3 per group). C,D) Representative FACS profiles (C) and quantification (D) of intracellular p‐Kap1 staining in HSCs (Lin⁻Sca1⁺Mac1⁺) obtained from control and cKO embryos at E14.5 (n = 4 and 3 for control and cKO, respectively). E,F) Representative FACS profiles (E) and quantification (F) of annexin V staining in LT‐HSCs (Lin⁻Sca1⁺CD48⁻CD150⁺Mac1⁺) obtained from control and cKO embryos at E14.5 (n = 4 per group). G,H) Representative FACS profiles of intracellular Ki67 staining in HSCs (Lin⁻Sca1⁺Mac1⁺) obtained from control and cKO embryos at E14.5 (G), and summary of the percentage of cells in the indicated cell cycles (H; n = 3 per group). I,J) Representative images of in vitro colony assays performed using fetal liver cells obtained from control and cKO embryos at E14.5 (I), and summary of the number of primitive erythroid progenitor cells (BFU‐E), granulocyte‐macrophage progenitor cells (CFU‐GM), and multipotent granulocytes, erythroid, macrophage, and megakaryocyte progenitor cells (CFU‐GEMM) measured on day 12 (n = 3 per group). K) Schematic diagram depicting the transplantation strategy in which lethally irradiated CD45.1⁺/CD45.2⁺ recipient mice received donor‐derived (CD45.2⁺) fetal liver cells. L) Kaplan–Meier survival curve of CD45.1⁺/45.2⁺ mice that were lethally irradiated and then transplanted with vehicle or fetal liver cells obtained from either CD45.2⁺ control or cKO embryos (n = 6 recipients per group). M) Schematic diagram depicting the strategy for competitive transplantation in which lethally irradiated CD45.1⁺/CD45.2⁺ recipient mice received the indicated donor‐derived fetal liver (CD45.2⁺) cells and competitor (CD45.1⁺) cells at a 1:1 ratio. N) Time course showing the percentage of donor‐derived CD45.2⁺ cells in the peripheral blood of recipient mice measured at the indicated time points following transplantation (n = 5 recipients per group). O) Summary of the percentage of donor‐derived myeloid cells, B cells, and T cells in the peripheral blood of recipient mice 16 weeks after co‐transplantation with control or cKO cells (n = 5 recipients per group). P) Summary of the percentage of donor‐derived cells (CD45.2⁺) in the bone marrow of recipient mouse mice 16 weeks after transplantation (n = 5 recipients per group). Q) Summary of the percentage of donor‐derived LT‐HSCs, short‐term HSCs (ST‐HSCs), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), GMP (granulocyte/monocyte progenitor cells), MEP (megakaryocyte/erythrocyte progenitor cells), and CLP (common lymphoid progenitor cells) 16 weeks after co‐transplantation with control or cKO donor cells (n = 5 recipients per group). R) Summary of the percentage of donor‐derived myeloid cells, B cells, and T cells in the bone marrow of recipient mice 16 weeks after co‐transplantation with control or cKO cells (n = 5 recipients per group). Data in this figure are represented as mean ± SD. p values of survival in (L) were determined using the Log‐rank test, in (B), (D), (F), (H), (J), (N), (O), (P), (Q), and (R) using 2‐tailed unpaired Student's t‐test. *p < 0.05, ***p < 0.001, and ns, not significant.

Zinc supplementation significantly reduces the impaired properties of HSPCs in slc39a10 mutant zebrafish and HSCs in Slc39a10 cKO mice. A,B) Representative images of the CHT region (arrows) in WT and slc39a10 mutant sibling Tg(cmyb:eGFP) embryos in control conditions or exposed to 300 µm ZnSO₄ (A), and quantification of cmyb⁺ cells in the CHT region (B; n = 12 and 10 for wildtype sibling unexposed or exposed to 300 µm ZnSO₄, n = 4 and 6 for slc39a10 mutant unexposed or exposed to 300 µm ZnSO₄). C,D) Representative images of the CHT region (arrows) in WT and slc39a10 mutant sibling Tg(cmyb:eGFP) embryos treated with 100 µm TPEN and/or 100 µm TPEN+ZnSO₄ (C), and quantification of cmyb⁺ cells in the CHT region (D; n = 6, 12, and 8 for wildtype sibling unexposed or exposed to 100 µm TPEN and/or 100 µm TPEN+ZnSO₄). E,F) Quantification of Mt1 (E) and Mt2 (F) mRNA measured in LT‐HSCs obtained from control and cKO mouse embryos at E14.5 (n = 5 per group). G) Summary of zinc concentration measured in LT‐HSCs obtained from control and cKO mouse embryos at E14.5 (n = 3 per group). H) In vitro colony assays were performed using fetal liver cells obtained from control and cKO embryos at E14.5 and from cKO fetal liver cells treated with ZnSO₄ at the indicated concentrations (n = 3 per group). I) Schematic diagram depicting the strategy for in vitro cell culture of cKit⁺ cells obtained from WT fetal livers treated with or without TPEN. J–M) Summary of ROS levels (J), p‐Kap1 levels (K), the percentage of annexin V⁺ cells (L), and cell cycle distribution measured using Ki67 staining (M) in cKit⁺ cells obtained from WT mouse embryos at E16.5 and treated with 1.5 µm TPEN and/or zinc for 12 h (n = 3 per group). N) In vitro colony assays were performed using cKit⁺ cells obtained from WT embryos at E16.5 and treated with TPEN at the indicated concentration (n = 3 per group). Data in this figure are represented as mean ± SD. The data in (B), (E), (F), and (G) were analyzed using a 2‐tailed, unpaired Student's t‐test, and the data in (D), (H), (J), (K), (L), (M), and (N) were analyzed using a one‐way ANOVA with Tukey's post hoc test (for multi‐group comparisons). *p < 0.05; **p < 0.01; ***p < 0.001, and ns, not significant.

Inhibiting necroptosis partially restores the colony‐forming capacity of Slc39a10‐deficient HSCs. A) Schematic diagram depicting the strategy of sorting fetal LT‐HSCs from control and cKO embryos at E14.5 and performing RNA‐seq analysis. B) Heat map showing genes that are differentially expressed between fetal LT‐HSCs obtained from control and cKO mice; downregulation and upregulation (defined as a log2 fold change >1 or <−1 and p < 0.05) are shown in green and red, respectively. C) Gene Ontology (GO) analysis of the RNA‐seq data showing the top 15 enriched pathways in fetal LT‐HSCs between control and cKO mice (defined as a log2 fold change >1.3 or ←1.3 and p < 0.05). The pathways involved in metal ion homeostasis, erythrocyte development, and negative regulation of G1/S transition of the mitotic cell cycle are written in red. D) Volcano plot of the RNA‐seq data, showing the differentially expressed genes in fetal LT‐HSCs between control and cKO mice. The green and red dots indicate significantly downregulated and upregulated genes (defined as a log2 fold change >2 or <−2 and p < 0.05) respectively, while gray dots indicate genes that were not significantly upregulated or downregulated; the p21 gene is indicated. E,F) Summary of normalized p53 mRNA (E) and p53 protein (F) levels measured in LT‐HSCs obtained from control and cKO embryos at E14.5 (n = 5 and 3 for the mRNA and protein groups, respectively). G) In vitro colony assays were performed using fetal livers obtained from control, cKO, and Slc39a10^fl/fl; p53^fl/fl; Vav‐Cre⁺ double‐knockout embryos at E14.5; colony numbers were measured on day 12 (n = 4 per group). H‐I, Summary of normalized p21 mRNA (H) and p21 protein (I) levels measured in LT‐HSCs obtained from control and cKO embryos at E14.5 (n = 5 and 3 for the mRNA and protein groups, respectively). J,K) In vitro colony assays were performed using fetal livers obtained from control, cKO, cKO/p21^−/‐ (J), and cKO/p16^−/‐ (K) embryos at E14.5; colony numbers were measured on day 12 (n = 4 per group). L) Summary of CFUs formed by untreated control and cKO FL‐HSCs and cKO FL‐HSCs treated with 50 µm Z‐VAD‐FMK, 100 µm 3‐MA, 10 µm necrostatin‐1 (Nec‐1), 2.5 µm GSK’872, or 30 µm ferrostatin‐1 (Fer‐1) (n = 4 per group). M) Summary of CFUs formed by LT‐HSCs obtained from control, cKO, cKO/Ripk3^−/−, cKO/Mlkl^−/−, and cKO/Ripk3^−/−/Mlkl^−/− embryos at E14.5 (n = 3 per group). Data in this figure are represented as mean ± SD. The data in (E–I) were analyzed using a 2‐tailed, unpaired Student's t‐test, and the data in (J–M) were analyzed using a one‐way ANOVA with Tukey's post hoc test (for multi‐group comparisons). *p < 0.05; **p < 0.01; ***p < 0.001, and ns, not significant.