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Fig. 6
mtClpX Is Important for Vertebrate Heme Biosynthesis and Erythropoiesis
(A) Relative mRNA abundance for human CLPX, CLPP, ALAS2, and SLC25A38 (indicated as S25A38) throughout erythropoiesis, as indicated in a microarray dataset described in Novershtern et al. (2011). Erythroid development stages were defined by cell-type-specific markers as follows: 1, CD34+ CD71+ GlyA 2, CD34 CD71+ GlyA 3, CD34 CD71+ GlyA+; 4, CD34 CD71low GlyA+; 5, CD34 CD71 GlyA+.
(B) o-dianisidine staining (brown) for hemoglobinized red cells in zebrafish embyos. Embyros were grown from zygotes injected at the one- to two-cell stage with clpxa-targeting morpholinos (MOa and MOb) or uninjected zygotes (control).
(C) Erythrocyte development at 72 hpf was quantified by flow cytometry, using dissociated cells from Tg(globin-LCR:eGFP) zebrafish. p d 0.01 for erythrocyte reduction by clpxa knockdown with either morpholino.
(D) Rescue of clpxa MOb-induced anemia by ALA supplementation. Tg(globin-LCR:eGFP) zebrafish embryos were supplemented with 2 mM ALA from 24 to 72 hpf, upon which GFP+ erythrocytes were quantified by flow cytometry. p = 0.025 for rescue of anemia in clpxa knockdown embryos by ALA supplementation.
(E) Heterozygous sauternes (ALAS2 mutant) or frascati (SLC25A37 mutant) zebrafish were crossed, and progeny were grown for 72 hpf, with or without ALA supplementation as in (D). Anemia was assayed by o-dianisidine staining. p = 0.04 for rescue of anemia in sauternes+/ progeny by ALA. n = 52 for sauternes ALA; n = 43 for sauternes +ALA; n = 98 for frascati ALA; n = 122 for frascati +ALA.
Error bars represent mean ± SD. See also Figure S5.
Reprinted from Cell, 161, Kardon, J.R., Yien, Y.Y., Huston, N.C., Branco, D.S., Hildick-Smith, G.J., Rhee, K.Y., Paw, B.H., Baker, T.A., Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis, 858-867, Copyright (2015) with permission from Elsevier. Full text @ Cell