clpxb mutant zebrafish embryos develop normal numbers of red cells, but are heme-deficient and have dysregulated red cell morphology. A, qPCR analysis of clpxb mRNA in WT and clpxb mutant embryos. clpxb mRNA is decreased by approximately 50% in mutant embryos (p-value < 0.0001). B, clpxb is required for erythroid hemoglobinization. clpxb mutant embryos are anemic at 48 hpf. C, clpxb is not required for erythroid specification. 72 hpf clpxb/clpxb; Tg(lcr:GFP) zebrafish embryos had similar percentages of GFP+ erythroid cells to control Tg(lcr:GFP) embryos as assessed by FACS. D, Compared with control erythroid cells, erythroid cells from clpxb mutant embryos were more variable in appearance, had smaller cytoplasmic areas and larger nuclei. E, clpxb erythroid cells had higher nuclear:cytoplasmic ratios (p-value < 0.0001). The nuclear:cytoplasmic ratios of clpxb erythroid cells exhibited higher variability (F test: p < 0.05). F, The increased nuclear:cytoplasmic ratio was caused by increased nuclear area in clpxb erythroid cells (p-value < 0.0001) and G, decreased cytoplasmic area (p-value<0.0001). ∗p < 0.05, Student's t test; error bars indicate SD.