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foxn1/Casper Mutant Engrafts HSCs without Irradiation (A) Schematic representation of the workflow of HSC transplantation. CD41-GFPlo cells were sorted from the KM of Tg (CD41:GFP), and 1 × 104 CD41-GFPlo cells were injected into each recipient (Casper, 25-Gy-irradiated Casper, foxn1/Casper, and 15-Gy-irradiated foxn1/Casper). (B) The survival rate of each recipient at 60 days post transplantation (dpt). The irradiated foxn1/Casper showed a low survival rate after transplantation (data presented as days-percentage survival, t test; ∗p < 0.05, ∗∗p < 0.01; n (CD41-GFPlo > Casper) = 10, n (CD41-GFPlo > irradiated Casper) = 19, n (CD41-GFPlo > foxn1/Casper) = 23, and n (CD41-GFPlo > irradiated foxn1/Casper) = 18). (C) Imaging of representative recipients at 60 dpt. CD41-GFP+ cells could be visualized under a GFP field in the KM of nonirradiated foxn1/Casper mutant. The red dashed lines indicate the KM regions. n = 5; scale bar, 1 mm (kidney). (D) Confocal imaging of engrafted CD41-GFP+ cells at 60 dpt in tail region. White arrows indicate the CD41-GFPhi and CD41-GFPlo cells (top), and two magnified pictures show the CD41-GFPhi and CD41-GFPlo cells, respectively (bottom). Scale bar, 100 μm (top), 10 μm (bottom). (E) Representative flow-cytometric analysis of recipient KM at 60 dpt. The dots in the rectangles indicate engrafted CD41-GFP+ cells (mean ± SD, n [CD41-GFPlo > Casper] = 10, n [CD41-GFPlo > irradiated Casper] = 8, n [CD41-GFPlo > foxn1/Casper] = 8, and n [CD41-GFPlo > irradiated foxn1/Casper] = 4). (F) The statistical analysis of (E) (mean ± SD, one-way ANOVA, Tukey's; N.S., nonsignificant; ∗p < 0.05, ∗∗p < 0.01). See also Figure S2.

Allogeneic MDS-like Cell Transplantation with Tg (cmybhyper:GFP) (A) The pathologic external features of zebrafish Tg (cmybhyper:GFP). Some of the transgenic zebrafish display MDS-like phenotypes (exophthalmos, cachexia, and abdominal mass). The red arrows denote diseased areas. Scale bar, 1 mm. (B) Flow-cytometric analysis of 24-month-old WT (no transgenic background) and Tg (cmybhyper:GFP) KM. The representative percentages of myelocytes in WT, siblings, and MDS-like zebrafish are indicated. Total cell numbers in whole KM are indicated (mean ± SD; n [WT] = 3, n [siblings] = 7, n [MDS-like zebrafish] = 6). (C) Statistical analysis of the percentage of different blood lineages in (B) (mean ± SD, one-way ANOVA, Tukey's; ∗p < 0.05, ∗∗p < 0.01, n [WT] = 3, n [siblings] = 7, n [MDS-like zebrafish] = 6). (D) Direct observation of engrafted MDS-like cells. At 30 dpt, MDS-like cmybhyper-GFP+ cells could be visualized in foxn1/Casper recipient KM, not in the Casper. The red dashed lines indicate the KM regions. The red arrow indicates the engrafted MDS-like cmybhyper-GFP+ cells in the head. n (Casper) = 0/10, n (foxn1/Casper) = 9/12. Scale bar, 1 mm. (E) Statistical analysis of the percentage of different blood lineages in MDS-like and sibling cmybhyper-GFP+ cell-engrafted KMs. The cmybhyper-GFP+ cells in recipient KM were gated for analysis (mean ± SD, t test; N.S., non-significant; ∗p < 0.05; n > 3 in each group). (F) Wright-Giemsa staining of blood cells in recipient KM after MDS-like and sibling cmybhyper-GFP+ cell transplantation. The red arrows indicate the MDS-like myelocytes in recipients. Scale bar, 5 μm.