Figure 9.
Nfe2−/− Mks in the ABM generate proPlts but exhibit a failure of membrane budding.(A) Representative examples of optical sections from chimeric BM (femoral) harvested from recipients of Nfe2+/+ (i, n = 3 mice) and Nfe2−/− (ii, n = 3 mice) donor E13.5 FLs. GFP− donor-derived Mks are readily distinguished from recipient GFP+ cells (including Mks) according to GFP expression (ii, inset). Scale bars, 100 µm. (B) Total Plts (i) and RBC (ii) counts in recipients of Nfe2+/+ (n = 10 mice) and Nfe2−/− (n = 6 mice) E13.5 FL 4–5 wk after transplantation. For i, P = 0.007, two-tailed unpaired t test. Detection (iii–iv) and quantification (v–vi) of donor-derived (GFP−) Plts and RBCs. For v, P < 0.0001, two-tailed unpaired t test. (C) Frequency of observed donor (GFP−) Nfe2+/+ and Nfe2−/− Mk morphologies within the BM (i), spleen (ii), and lungs (iii) of recipient mice (n = 3 mice per donor genotype); ****, P < 0.0001 and *, P = 0.04, one-way ANOVA with Sidak’s correction (i); ****, P < 0.0001 and *, P = 0.18, one-way ANOVA with Sidak’s correction (ii); *, P = 0.04, one-way ANOVA with Sidak’s correction (iii). (D–F) Representative examples of donor-derived (GFP−) Nfe2+/+ and Nfe2−/− Mks budding in the BM (D) and undergoing proPlt formation in the spleen (E) and lungs (F). Scale bars, 20 µm. Yellow arrow indicates membrane bud, blue outline highlights proPlt-forming Mks; n = 3 replicates per donor genotype. (G) Quantification of proPlt fragments within the vasculature of lungs of Nfe2+/+ and Nfe2−/− fetuses. n = 4 independent experiments. P = 0.53, two-tailed unpaired t test. (H) Representative examples of free proPlts in the peripheral circulation of E13.5 Nfe2+/+ (i) and Nfe2−/− (ii) mice. Representative examples of free proPlt fragments in the peripheral circulation of E13.5 Nfe2+/+ (iii) and Nfe2−/− (iv) mice that are consistent in shape and size with proPlt fragments. Data are representative of eight independent experiments. Representative examples of free proPlts that were present in the BM of GFP+ adult mice that had been reconstituted with GFP−Nfe2+/+ cells (v) and GFP−Nfe2−/− cells (vi). n = 4 independent experiments. Scale bars, 10 µm.

Nfe2 −/− Mks in the ABM generate proPlts but exhibit a failure of membrane budding. (A) Representative examples of optical sections from chimeric BM (femoral) harvested from recipients of Nfe2+/+ (i, n = 3 mice) and Nfe2−/− (ii, n = 3 mice) donor E13.5 FLs. GFP− donor-derived Mks are readily distinguished from recipient GFP+ cells (including Mks) according to GFP expression (ii, inset). Scale bars, 100 µm. (B) Total Plts (i) and RBC (ii) counts in recipients of Nfe2+/+ (n = 10 mice) and Nfe2−/− (n = 6 mice) E13.5 FL 4–5 wk after transplantation. For i, P = 0.007, two-tailed unpaired t test. Detection (iii–iv) and quantification (v–vi) of donor-derived (GFP−) Plts and RBCs. For v, P < 0.0001, two-tailed unpaired t test. (C) Frequency of observed donor (GFP−) Nfe2+/+ and Nfe2−/− Mk morphologies within the BM (i), spleen (ii), and lungs (iii) of recipient mice (n = 3 mice per donor genotype); ****, P < 0.0001 and *, P = 0.04, one-way ANOVA with Sidak’s correction (i); ****, P < 0.0001 and *, P = 0.18, one-way ANOVA with Sidak’s correction (ii); *, P = 0.04, one-way ANOVA with Sidak’s correction (iii). (D–F) Representative examples of donor-derived (GFP−) Nfe2+/+ and Nfe2−/− Mks budding in the BM (D) and undergoing proPlt formation in the spleen (E) and lungs (F). Scale bars, 20 µm. Yellow arrow indicates membrane bud, blue outline highlights proPlt-forming Mks; n = 3 replicates per donor genotype. (G) Quantification of proPlt fragments within the vasculature of lungs of Nfe2+/+ and Nfe2−/− fetuses. n = 4 independent experiments. P = 0.53, two-tailed unpaired t test. (H) Representative examples of free proPlts in the peripheral circulation of E13.5 Nfe2+/+ (i) and Nfe2−/− (ii) mice. Representative examples of free proPlt fragments in the peripheral circulation of E13.5 Nfe2+/+ (iii) and Nfe2−/− (iv) mice that are consistent in shape and size with proPlt fragments. Data are representative of eight independent experiments. Representative examples of free proPlts that were present in the BM of GFP+ adult mice that had been reconstituted with GFP−Nfe2+/+ cells (v) and GFP−Nfe2−/− cells (vi). n = 4 independent experiments. Scale bars, 10 µm.

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