FIG 4.

GFI1 SUMOylation on K239 is required for zebrafish primitive erythropoiesis. (A) One-cell-stage zebrafish embryos were injected with gfi1aa splice-blocking morpholino, alone or in combination with RNA expressing wild-type GFI1, GFI1-K239R, or GFI1-E241Q, each with a C-terminal Flag epitope tag. At 48 h postfertilization (hpf), primitive erythropoiesis was revealed by o-dianisidine staining. Four representative embryos are shown out of 200 embryos scored for each knockdown/rescue combination. Arrows in uninjected controls show the zone of primitive erythropoiesis. Primitive erythropoiesis was quantified using a graded scoring system. Scores of 1 to 4 were assigned for each embryo to indicate none, modest, moderate, or complete hemoglobinization, respectively. Statistical significance was assessed using a Wilcoxon-Mann-Whitney test (***, P < 0.0005). (B) Morpholino-induced splicing blockade of gfi1aa in zebrafish. Total RNA was isolated from uninjected (U) zebrafish embryos or after injection of a scrambled control (Sc) or gfi1aa splice-blocking morpholino (SB) targeting the boundary between intron 1 and exon 2, as shown (thick line). Unspliced gfi1aa cDNA was amplified with primers a and b spanning the intron 1-exon 2 boundary to yield a 169-bp amplimer. Spliced gfi1aa mRNA was amplified using primers c and b to yield a 263-bp amplimer. (C) Expression of Flag-tagged GFI1, GFI1-K239R (K>R), and GFI1-E241Q (E>Q) derivatives in extracts prepared from the equivalent of eight zebrafish embryos at 24 h postinjection.