Zebrafish aggf1 and cloche act independently during differentiation of hemangioblasts. (A-H) Heterozygous cloche^m39 mutant zebrafish were mated with each other. The offspring embryos were injected with 100 pg of aggf1 mRNA and used for WISH at 10 somites with antisense probes for etsrp (A-D) and scl (E-H). (A-B) Uninjected control embryos (23.8%, 29/122) showed no detectable etsrp expression, whereas 76.2% (93/122) embryos showed nearly normal expression of etsrp at 10 somites. (C-D) No detectable etsrp expression was displayed in 24.2% (24/99) of the embryos, whereas 75.8% (75/99) embryos showed nearly normal expression of etsrp at 10 somites. (E-F) Uninjected control embryos (76.5%, 117/153) showed normal expression of scl, whereas 23.5% (36/153) of embryos did not display expression of scl at 10 somites. (G-H) No detectable scl expression was displayed in 25.8% (30/116) of the embryos, whereas 74.2% (86/116) of the embryos showed nearly normal expression of scl at 10 somites. The ratios are consistent with the autosomal recessive inheritance pattern (3:1), suggesting that injection of aggf1 mRNA did not alter the 3:1 ratio and did not rescue the expression of hemangioblast markers in cloche^m39 mutant embryos. (I-Q) Overexpression of lycat mRNA did not rescue abnormal hemangioblast phenotypes in aggf1 morphants. Zebrafish embryos (1- to 2-cell stage) were injected with Std-MO (2 ng; I,L,O), aggf1 MO1 (2 ng; J,M,P), or aggf1 MO1 (2 ng) together with lycat mRNA (100 pg) (K,N,Q) and used for WISH at 6 somites with antisense probes for fli1 (I-K), scl (L-N), and lmo2 (O-Q). Injection of lycat mRNA failed to rescue the reduced expression of fli1, scl, and lmo2 in PLPM by aggf1 MO1. Embryos are shown in a dorsal view with the anterior at the top. (R-W) Overexpression of lycat-egfp mRNA did not rescue abnormal hemangioblast phenotypes in aggf1 morphants, that is, reduced expression of fli1 (R-T) or scl (U-W).