Figure 1.
runx1 mutants show robust engraftment after HCT. (A) Experimental schema for hematopoietic cell transplantation (HCT): donor marrow cells from ubi:GFP transgenic adult zebrafish were harvested and transplanted into runx1-mutant embryos at 2 dpf. Survival was assessed for 2 months posttransplant until the time of engraftment analysis. Fluorescent imaging of transplanted fish was also performed to qualitatively assess engraftment. (B) Kaplan-Meier curves showing survival of transplanted runx1 mutants (Txp runx1 mut) in comparison with sham-injected or uninjected mutants as well as transplanted heterozygotes (Txp hets) and uninjected heterozygotes. Survival analysis was performed until 2 months posttransplant. Statistical analysis performed with log-rank (Mantel-Cox) test. (C) Images of transplanted runx1-mutant larvae 5 days posttransplant (7 dpf); the inset highlights the seeding of the developing thymus (arrowhead) and kidney (arrow) with donor-derived GFP+ cells. (D) Bright field (left) and fluorescent (center and right) images of adult zebrafish showing levels of GFP positivity in ubi:GFP donors (top), a transplanted runx1 mutant (middle), and a negative untransplanted wild-type control (Neg Ctrl, bottom). Center images are at 0.63× magnification and lower camera exposure; right images are at 2× magnification and longer exposure times. Note that donor GFP+ cells are detectable in transplanted runx1 mutant adults via fluorescent microscopy with lower GFP intensity compared with ubi:GFP donors because only the blood cells are fluorescent. (E) Donor engraftment rate at 8 weeks posttransplant in the surviving runx1 mutants (Muts txp) compared with heterozygotes (Hets txp). Engraftment was defined as myeloid chimerism ≥5%. Mutants display a higher engraftment rate of 59% compared with 8% in heterozygotes. The result is significant (Fisher’s exact test P < .01). (F) Donor myeloid chimerism at 8 weeks posttransplant for all surviving transplanted fish. Each dot represents the chimerism level of individual transplanted fish. The yellow line indicates 5% chimerism, the cutoff defined as engraftment. Transplanted homozygous runx1 mutants have significantly higher chimerism values than heterozygotes, with a mean of 42% (± 44%) compared with 3% (± 9%), respectively. Statistical analysis was performed with Mann-Whitney U test. (G) Donor myeloid chimerism at 8 weeks posttransplant for all fish that reached the cutoff for engraftment. Each dot represents the chimerism level of individual transplanted fish. The yellow line indicates 5% chimerism, the cutoff defined as engraftment. Engrafted homozygous runx1 mutants have significantly higher chimerism values than engrafted heterozygotes, with a mean of 70% (± 36%) compared with 27% (± 17%), respectively. Error bars are standard deviation. Statistical analysis performed with Mann-Whitney U test. *P = .01-.05; **P = .001-.01; **** P < .0001.

runx1 mutants show robust engraftment after HCT. (A) Experimental schema for hematopoietic cell transplantation (HCT): donor marrow cells from ubi:GFP transgenic adult zebrafish were harvested and transplanted into runx1-mutant embryos at 2 dpf. Survival was assessed for 2 months posttransplant until the time of engraftment analysis. Fluorescent imaging of transplanted fish was also performed to qualitatively assess engraftment. (B) Kaplan-Meier curves showing survival of transplanted runx1 mutants (Txp runx1 mut) in comparison with sham-injected or uninjected mutants as well as transplanted heterozygotes (Txp hets) and uninjected heterozygotes. Survival analysis was performed until 2 months posttransplant. Statistical analysis performed with log-rank (Mantel-Cox) test. (C) Images of transplanted runx1-mutant larvae 5 days posttransplant (7 dpf); the inset highlights the seeding of the developing thymus (arrowhead) and kidney (arrow) with donor-derived GFP+ cells. (D) Bright field (left) and fluorescent (center and right) images of adult zebrafish showing levels of GFP positivity in ubi:GFP donors (top), a transplanted runx1 mutant (middle), and a negative untransplanted wild-type control (Neg Ctrl, bottom). Center images are at 0.63× magnification and lower camera exposure; right images are at 2× magnification and longer exposure times. Note that donor GFP+ cells are detectable in transplanted runx1 mutant adults via fluorescent microscopy with lower GFP intensity compared with ubi:GFP donors because only the blood cells are fluorescent. (E) Donor engraftment rate at 8 weeks posttransplant in the surviving runx1 mutants (Muts txp) compared with heterozygotes (Hets txp). Engraftment was defined as myeloid chimerism ≥5%. Mutants display a higher engraftment rate of 59% compared with 8% in heterozygotes. The result is significant (Fisher’s exact test P < .01). (F) Donor myeloid chimerism at 8 weeks posttransplant for all surviving transplanted fish. Each dot represents the chimerism level of individual transplanted fish. The yellow line indicates 5% chimerism, the cutoff defined as engraftment. Transplanted homozygous runx1 mutants have significantly higher chimerism values than heterozygotes, with a mean of 42% (± 44%) compared with 3% (± 9%), respectively. Statistical analysis was performed with Mann-Whitney U test. (G) Donor myeloid chimerism at 8 weeks posttransplant for all fish that reached the cutoff for engraftment. Each dot represents the chimerism level of individual transplanted fish. The yellow line indicates 5% chimerism, the cutoff defined as engraftment. Engrafted homozygous runx1 mutants have significantly higher chimerism values than engrafted heterozygotes, with a mean of 70% (± 36%) compared with 27% (± 17%), respectively. Error bars are standard deviation. Statistical analysis performed with Mann-Whitney U test. *P = .01-.05; **P = .001-.01; **** P < .0001.

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