Figure 6.
Figure 6. Primitive erythropoiesis is delayed in Gfi1aa/Gfi1b-depleted embryos. Images of embryos stained by RNA whole-mount in situ hybridization are shown in panels A-D and J-K. Embryos in panel E were stained with o-dianisidine. Live embryos are shown in panels F and I. Views of embryos are lateral in panels A-D,F,I,J. Ventral and dorsal views of embryos are shown in panels E and K, respectively. Cohorts of embryos derived from the crosses indicated were stained in panels A-D. Examples of embryos with high and low gene expression are depicted. Bar charts summarize the genotyping data in panels A-C. In panel D, 6/96 22- to 23-hpf and 9/89 23- to 24-hpf embryos had very low slc4a1a expression. Genotyping 11 of these showed that all of them were qmc551/qmc554 double-homozygous mutants. Embryos with slightly stronger staining turned out to be gfi1aaqmc551hom;gfi1bqmc554het carriers. A more comprehensive overview of the WISH data on the 23- to 24-hpf embryos is presented in supplemental Figure 5. In panel E, 98 2-day-old-embryos from an incross of gfi1aaqmc551het;gfi1bqmc554het parents were stained with o-dianisidine (o-dia.) for the presence of hemoglobin. Four embryos displayed reduced staining. Genotyping showed that they were double homozygous mutants. In panel F, 3-day-old embryos with normal (red arrow) and hypochromic (red arrowhead) prRBCs are shown. The bar charts summarize the genotyping results. (G) RBCs were isolated from 2- and 3-day-old wt and double-mutant embryos. Cells were fixed with methanol and stained with Giemsa. (H) The size of the RBC nuclei (n>15 cells per embryo) was determined with Image J for wt and gfi1aaqmc551;gfi1bqmc554 double-mutant embryos at 2 and 3 dpf (n ≥ 10 embryos for every type of embryos and every stage). Average values per embryo are plotted. Mean values and standard deviations are indicated. A 1-way analysis of variance (ANOVA) test revealed that the differences between the mean values were statistically significant for the comparisons indicated (ANOVA test performed on GraphPad Prism: *P < .05; ***P < .001; ****P < .0001). (I) Fluorescent images of the trunk and tail of representative embryos with hypochromic (top) and normal (bottom) blood as shown in panel F. The genotypes of the representative embryos are shown. In panel J, cmyb WISH experiments on GFP-low (ie, gfi1aaqmc551het, embryos from a gfi1aaqmc551/gfi1bqmc554 double heterozygous incross) revealed no differences in staining between embryos. Genotyping revealed that 4 of the 24 embryos were gfi1bqmc554hom. In panel K, 20 5-dpf embryos derived from a gfi1bqmc554het incross were stained for the expression of the T-cell gene rag1. All embryos showed the same level of rag1 expression, including the 5 gfi1bqmc554hom embryos.

Primitive erythropoiesis is delayed in Gfi1aa/Gfi1b-depleted embryos. Images of embryos stained by RNA whole-mount in situ hybridization are shown in panels A-D and J-K. Embryos in panel E were stained with o-dianisidine. Live embryos are shown in panels F and I. Views of embryos are lateral in panels A-D,F,I,J. Ventral and dorsal views of embryos are shown in panels E and K, respectively. Cohorts of embryos derived from the crosses indicated were stained in panels A-D. Examples of embryos with high and low gene expression are depicted. Bar charts summarize the genotyping data in panels A-C. In panel D, 6/96 22- to 23-hpf and 9/89 23- to 24-hpf embryos had very low slc4a1a expression. Genotyping 11 of these showed that all of them were qmc551/qmc554 double-homozygous mutants. Embryos with slightly stronger staining turned out to be gfi1aaqmc551hom;gfi1bqmc554het carriers. A more comprehensive overview of the WISH data on the 23- to 24-hpf embryos is presented in supplemental Figure 5. In panel E, 98 2-day-old-embryos from an incross of gfi1aaqmc551het;gfi1bqmc554het parents were stained with o-dianisidine (o-dia.) for the presence of hemoglobin. Four embryos displayed reduced staining. Genotyping showed that they were double homozygous mutants. In panel F, 3-day-old embryos with normal (red arrow) and hypochromic (red arrowhead) prRBCs are shown. The bar charts summarize the genotyping results. (G) RBCs were isolated from 2- and 3-day-old wt and double-mutant embryos. Cells were fixed with methanol and stained with Giemsa. (H) The size of the RBC nuclei (n>15 cells per embryo) was determined with Image J for wt and gfi1aaqmc551;gfi1bqmc554 double-mutant embryos at 2 and 3 dpf (n ≥ 10 embryos for every type of embryos and every stage). Average values per embryo are plotted. Mean values and standard deviations are indicated. A 1-way analysis of variance (ANOVA) test revealed that the differences between the mean values were statistically significant for the comparisons indicated (ANOVA test performed on GraphPad Prism: *P < .05; ***P < .001; ****P < .0001). (I) Fluorescent images of the trunk and tail of representative embryos with hypochromic (top) and normal (bottom) blood as shown in panel F. The genotypes of the representative embryos are shown. In panel J, cmyb WISH experiments on GFP-low (ie, gfi1aaqmc551het, embryos from a gfi1aaqmc551/gfi1bqmc554 double heterozygous incross) revealed no differences in staining between embryos. Genotyping revealed that 4 of the 24 embryos were gfi1bqmc554hom. In panel K, 20 5-dpf embryos derived from a gfi1bqmc554het incross were stained for the expression of the T-cell gene rag1. All embryos showed the same level of rag1 expression, including the 5 gfi1bqmc554hom embryos.

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