Fig. 4.

Overexpression of Prdm1a using a global heat-shock Gal4/UAS system shows that proline/serine-rich and zinc finger domains are required for pectoral fin function. (A) Schematic of 4XnrUAS-modified prdm1a-2a-EGFP constructs that were injected into Tg(hsp70l:gal4FF);prdm1a^+/− intercrosses. Results for the ability to rescue the pectoral fin are shown. (B) Experimental design for heat-shock Gal4/UAS rescue experiments. Following injection with the UAS construct, embryos at 6 hpf (shield stage) were heat shocked, leading to activation of Gal4, expression of the 4XnrUAS-modified prdm1a-2a-EGFP construct, and cleavage of the 2a viral peptide from EGFP. Embryos were screened for mosaic EGFP expression at 24 hpf. (C,D) Representative images of 24 hpf embryos injected with the 4XnrUAS-modified prdm1a-2a-EGFP construct at the single-cell stage. (C) No heat shock (control). (D) Mosaic EGFP expression in embryos that were injected and heat shocked. The dotted box marks the pectoral fin. Scale bars: 200 µm. (E-L) Representative images of Alcian Blue-stained pectoral fins at 4 dpf are shown. (E) Uninjected WT (n=36). (F) Uninjected prdm1a^−/− mutants (n=11). (G-L) Mutants were injected with constructs containing (G) full-length Prdm1a (n=9), (H) Prdm1aΔSET (n=7), (I) Prdm1aΔP/S (n=13), (J) Prdm1aΔZnF (n=7), (K) Prdm1aΔP/S&ZnF (n=13) and (L) an EGFP negative control (n=16). Scale bars: 50 µm. The representative uninjected control images in E and F are also used in Fig. S4, which shows the effect of overexpression of modified Prdm1a in the WT background as part of the same experiment. (M-P) Measurements were taken for the length of the (M) cleithrum, (N) endoskeletal disk and (O) fin fold and (P) the area of the scapulocoracoid and postcoracoid. Each dot represents one independent biological replicate. Measurements for each individual were averaged and compared using a one-way ANOVA, followed by a Tukey's post-hoc test relative to uninjected, heat-shocked prdm1a^−/− mutants. prdm1a^−/− mutants injected with full-length Prdm1a exhibited a rescue in the endoskeletal disk (P=0.0654). Prdm1aΔSET also partially rescued the area of the scapulocoracoid/postcoracoid (P=0.0446). However, Prdm1aΔP/S, Prdm1aΔZnF and Prdm1aΔP/S&ZnF failed to rescue prdm1a^−/− mutants. Error bars represent the mean±s.d. Δ, deleted; Ant, anterior; cl, cleithrum; D, distal; dpf, days post fertilization; e, eye; ed, endoskeletal disk; ff, fin fold; hpf, hours post fertilization; MT, prdm1a^−/− mutant; P, proximal; pc, postcoracoid; pf, pectoral fin; Post, posterior; sc, scapulocoracoid; WT, wildtype; y, yolk.