Fig. S5

mtClpX in Vertebrate Heme Biosynthesis and Erythropoiesis, Related to Figure 6

(A) Relative mRNA abundance for mouse clpx and tfrc (transferrin receptor; included as a positive control for genes strongly induced during erythropoiesis) throughout erythropoiesis. mRNA was collected from MEL cells at indicated times after induction of erythropoiesis by the addition of 1.5% DMSO to growth medium. mRNA was quantified by qPCR and normalized to a control mRNA (hprt). p < 0.05 for upregulation of clpx at days 2, 4, and 5, and for tfrc at days 2–5.

(B) In situ hybridization to determine expression pattern of ClpX isoforms in D. rerio embryos. gata-1 mRNA was used to delineate the ventral blood island (intermediate cell mass, ICM, arrows) in wt embryos. Dino mutants, which are ventralized due to a defect in chordin, exhibit expansion of the erythropoietic marker gata-1. D. rerio clpxa and clpxb exhibit nearly ubiquitous expression, with higher expression observed in neural tissues 24 hr post-fertilization.

(C) D. rerio clpxa morpholino-injected embryos have reduced clpxa mRNA levels. clpxa mRNA levels in uninjected and clpxa morpholino-injected D. rerio embryos at 72 hpf are displayed (as detected by qRT-PCR, normalized to hprt control). p < 0.001.

(D) clpxa mRNA levels in uninjected, nontargeting morpholino-injected, and clpxa morpholino-injected D. rerio embryos at 24 hpf are displayed. p < 0.01, ANOVA.

(E) Erythrocyte development at 72 hr post-fertiliztion (hpf) was quantified by flow cytometry, using dissociated cells from Tg(globin-LCR:eGFP) zebrafish, in which all erythrocytes are GFP-labeled. p < 0.01, ANOVA.

(F) FACS analysis of uninjected, nontargeting morpholino-injected and clpx MOb-injected Tg(gata1:GFP) zebrafish embryos at 24hpf. clpx morphants display a reduction in GFP+ erythroid progenitor cells. p < 0.0001, ANOVA. Error bars represent mean ± SD.