Figure 2. Prdm16 is required for survival, cell cycle regulation, and maintenance in fetal and adult HSCs.

a) The cellularity of the liver and spleen and b) the frequencies of mature hematopoietic cells in the liver were normal in newborn Prdm16^LacZ/LacZ mice (3 independent experiments with a total of 5–12 mice/genotype; the exact number of mice is indicated in the panel legend). c) The frequencies of most colony-forming progenitors were normal in the liver of Prdm16^LacZ/LacZ mice, but CFU-GEMM and CFU-GM were significantly depleted. d) HSCs were depleted in the fetal (E14.5) and newborn (P0) liver of Prdm16^LacZ/LacZ mice (4 independent experiments with a total of 3–12 mice/genotype; the exact number of mice is indicated under each bar). e) c-kit and Sca-1 staining is shown in the right column for CD150⁺CD48⁻CD41⁻ cells gated in the left column for representative mice of each genotype. f) Irradiated CD45.1⁺ recipient mice were competitively reconstituted with 3×10⁵ CD45.2⁺ neonatal liver cells from Prdm16^+/+ (black lines), Prdm16^LacZ/+ (green lines) or Prdm16^LacZ/LacZ mice (red lines) along with 3×10⁵ CD45.1⁺ recipient bone marrow cells. Each line represents average donor cell reconstitution levels (mean±SD; 3 independent experiments with 2–5 recipients/treatment/experiment; the total number of mice transplanted with cells of each genotype is inducted in panel legends for f–h). g) Donor cell reconstitution in an independent experiment in which recipients were transplanted with 3×10⁵ CD45.2⁺ neonatal liver cells from a Prdm16^+/+ donor (black lines), or 3×10⁵ cells from a Prdm16^LacZ/LacZ donor (red lines), or 6×10⁶ cells from Prdm16^LacZ/LacZ donor (blue lines) along with 3×10⁵ young adult CD45.1⁺ bone marrow cells (1 experiment with at least 5 recipients/treatment). h) Donor cell reconstitution in 2 experiments in which recipients were transplanted with 20 CD45.2⁺ HSCs from Prdm16^+/+ (black lines), Prdm16^LacZ/+ (green lines), or Prdm16^LacZ/LacZ donors (red lines) along with 3×10⁵ CD45.1⁺ bone marrow cells (2 independent experiments). i) Bone marrow HSC frequency in adult Prdm16^+/+ and Prdm16^LacZ/+ mice (2 experiments with 5 or 6 mice/genotype). j) The frequency of annexin V+/DAPI+ unfractionated newborn liver cells or c-kit⁺Sca-1⁺ cells (5 independent experiments with 6 or 9 mice/genotype). k) Cell cycle distribution of cells from newborn liver (3 independent experiments with 3–9 mice/genotype). In panels i–k, the exact number of mice of each genotype is indicated under each bar in each panel (*, P<0.05; **, P<0.01; ***, P<0.001, error bars always represent SD).