Fig. 3: Kat6b overexpression restores transplantable haematopoietic stem cells that are absent in Kat6a^–/– mice.

a Flow cytometry gating strategy for the assessment of haematopoietic stem cells (HSCs) in E14.5 foetal livers. HSCs were identified as the CD48^–CD150⁺ cell population within the lineage marker negative (LIN^–) cKIT and SCA1 positive cell population (LSK; gated on single, viable cells, lacking expression of lineage (LIN) markers B220, CD19, CD4, CD8, GR1 and TER119 and expressing SCA1 and cKIT). Representative plots for HSCs are shown for each genotype. b Total number of HSCs per foetal liver of N = 3 Kat6a^–/–Kat6b^+/+, N = 8 Kat6a^+/+Kat6b^+/+, N = 6 Kat6a^–/–Tg(Kat6b) and N = 4 Kat6a^+/+Tg(Kat6b) foetuses. c Log₂ fold-change in Kit mRNA levels in E9.5 embryos as assessed by RNA-sequencing across the comparisons Kat6a^–/–Kat6b^+/+ vs. Kat6a^+/+Kat6b^+/+, Kat6a^–/–Tg(Kat6b) vs. Kat6a^+/+Kat6b^+/+ and Kat6a^–/–Tg(Kat6b) vs. Kat6a^–/–Kat6b^+/+ embryos. N = 4 embryos per genotype. The FDRs are shown above the bars. Data were analysed as described in the ‘methods’ section under RNA sequencing and analysis. d Percentage survival of irradiated recipient mice after transplantation of 1 × 10⁶ foetal liver cells from N = 3 Kat6a^–/–Kat6b^+/+, N = 4 Kat6a^–/–Tg(Kat6b), N = 3 Kat6a^+/+TgKat6b) or N = 5 Kat6a^+/+Kat6b^+/+ E14.5 mouse foetuses; each foetal liver sample was transplanted into 3 lethally irradiated recipients. Data are displayed in a Kaplan-Meier plot and were analysed using a Mantel-Cox test. e Automated haematological analyser (ADVIA) assessment of red blood cells per μl peripheral blood in recipient mice of E14.5 foetal liver donor cells from N = 5 Kat6a^+/+Kat6b^+/+, N = 3 Kat6a^+/+Tg(Kat6b), N = 4 Kat6a^–/–Tg(Kat6b) or N = 3 Kat6a^–/–Kat6b^+/+ foetuses, at a time 3–4 weeks after transplantation when the recipient mice of Kat6a^–/–Kat6b^+/+ donor cells in each set reached the ethical end-point. Each foetal liver sample was transplanted into 3 recipient mice. Each circle represents the average of the recipient mice of an individual foetal liver donor. Data are displayed as mean ± s.e.m. and were analysed using a one-way ANOVA with Dunnett post-hoc correction. f Gating strategy for the assessment of peripheral blood of foetal liver transplant recipient mice. B cells were defined as B220⁺CD19⁺, T cells as CD4⁺ or CD8⁺, granulocytes as GR1^hi MAC1⁺ and monocytes as GR1^loMAC1⁺. Foetal liver donor-derived cells (CD45.1⁺) were distinguished from residual recipient cells (CD45.1/2⁺). g, h Contribution of foetal liver (donor)-derived cells to peripheral blood populations at 4 weeks (f) and 20 weeks (g) post transplantation of the recipients described in (e). i Gating strategy for the assessment of stem and progenitor cells in the bone marrow of foetal liver transplant recipient mice. Cell types as indicated on the plots were distinguished by CD48 vs. CD150, CD34 vs. CD16.32 or IL7Rα expression. (j–k) Contribution of foetal liver (donor)-derived cells to stem and progenitor populations in the bone marrow of foetal liver cell recipients at 20 weeks post-transplantation in CD48 vs. CD150 (i), CD34 vs CD16/32 (j) cell populations. l Gating strategy for the assessment of B cell progenitors in the bone marrow of foetal liver transplant recipient mice. Cells were gated on single, viable cells co-expressing B220 and CD19 and distinguished by cKIT, IgM and IgD expression to identify the cell populations indicated in the plots. m Contribution of foetal liver (donor)-derived cells to bone marrow B cell progenitors 20 weeks post-transplant. Circles represent individual foetuses (b) or the mean of three recipients of a single foetal liver donor (g, h, j, k, m). Data are presented as mean ± s.e.m. and were analysed using a one-way ANOVA (b) or two-way ANOVA with Tukey post hoc correction (g, h, j, k, m). HSCs, haematopoietic stem cells; LK; lineage marker negative cKIT⁺ cells; LSK, lineage marker negative SCA1⁺cKIT⁺ cells; HPC-1 (haematopoietic progenitor 1), HPC-2 (haematopoietic progenitor 2), MPP (multipotent progenitor), CLP (common lymphoid progenitor), CMP (common myeloid progenitor), GMP (granulocyte macrophage progenitors), MEP (megakaryocyte/erythroid progenitor). Granulo (granulocytes), Mono (monocytes); superscript: int, intermediate.