Fig. 2

hrg1 DKO zebrafish survives to adulthood without overt hematological phenotypes.

(A) Yeast growth assay showing that zebrafish Hrg1a^iq261 and Hrg1b^iq361 alleles are incapable of mediating heme transport in yeast in contrast to WT forms of Hrg1a and Hrg1b. The hem1Δ strain transformed with empty vector pYes-DEST52, hrg1a, hrg1b, predicted hrg1a^iq261 and hrg1b^iq361 were cultivated overnight and spotted in serial dilutions on SC plates supplemented with 250μM ALA and indicated concentration of heme. (B) Immunoblot of Hrg1 in membrane fractionation lysates from WT Tü, hrg1a^iq261/iq261, hrg1b^iq361/iq361 and double mutant DKO. Each crude membrane fraction lysate was from pooled ~30 embryos. Each lane represents 100 μg of protein from membrane fractionation lysates. Ponceau is used as a loading control for membrane fraction. (C) O-dianisidine staining of RBCs in 3dpf embryos from Tü, hrg1a^iq261/iq261, hrg1b^iq361/iq361 and DKO (n = 50 for each genotype). Scale bar: 200μm. (D) Quantitative percentages of GFP⁺ cells in Tü, hrg1a^iq261/iq261, hrg1b^iq361/iq361 and DKO mutants in GlobinLCR-GFP transgenic background. Error bars indicate SEM of 3 independent experiments (two-way ANOVA, p >0.05). (E) Lateral view of ße1 and gata1 expression by WISH at 1dpf embryos from Tü, hrg1a^iq261/iq261, hrg1b^iq361/iq361 and DKO. Anterior is to the left. N = 50 for each genotype. Scale bar: 200μm. (F) May-Grünwald-Giemsa staining of isolated peripheral RBCs at 3dpf embryos from Tü, hrg1a^iq261/iq261, hrg1b^iq361/iq361 and hrg1a^iq261/iq261; hrg1b^iq361/iq36 (N = 30). Scale bar: 20μm. (G) May-Grünwald-Giemsa staining of isolated peripheral RBCs from adult Tü, hrg1a^iq261/iq261, hrg1b^iq361/iq361 and DKO. Scale bar: 20μm.