Fig. 3

Functional and structural rescue of fla mutant embryos by the transgenic reintroduction of Smyd1b. (A, B) Immunostaining of fla siblings (A) and mutants (B) with α-actinin-specific antibodies (red) and DAPI (blue). Normal sarcomeric striation was visible in siblings, whereas complete lack of sarcomeric organization was found in homozygous fla mutant embryos. (C, D) fla siblings show strong birefringence signals at 72 hpf (C) whereas fla mutants lack proper sarcomeric organization and thereby birefringence signal (D). (E, F) Representative overview of spontaneous movement assays (at 24 hpf) with false-colored and superimposed pictures of fla offspring derived from intercrossing heterozygous fla fish. Genotyping of these embryos revealed that all non-moving (yellow*) embryos are homozygous fla mutants (E). By contrast, all homozygous fla mutant embryos carrying the transgene (Tg(unc45b^min:smyd1b-gfp)) are able to move (F) (red to green shift). Red pictures 0 s, green pictures 10 s. (G) Transgenic reintroduction of Smyd1b (Tg(unc45b^min:smyd1b-gfp)) into homozygous fla mutant embryos leads to normal birefringence signal. (H) Statistical analysis of the spontaneous movement assay. 80 ± 3.6% of genotyped fla siblings (fla^+/+ and fla^+/-) showed normal motility after 24 hpf (n = 111; three independent experiments). By contrast, homozygous fla mutant embryos are completely paralyzed (n = 35; three independent experiments). In comparison, 82.7 ± 5.6% of fla sibs (fla^+/+ and fla^+/-) (n = 98; three independent experiments) and 80.7 ± 4.6% of homozygous fla mutants carrying the transgene that expresses smyd1b showed normal spontaneous movements (n = 32; three independent experiments). Error bars indicate sd. (I) Quantification of the touch-evoked flight response at 72 hpf. Homozygous fla mutants are completely paralyzed and do not flight upon tactile stimulation (n = 47; three independent experiments). 86.6% of fla siblings (fla^+/+ and fla^+/-) carrying the transgene (n = 61; three independent experiments) show regular motility at 72 hpf. Similarly, 88.2% of homozygous fla mutant embryos carrying the transgene (n = 26; three independent experiments) are able to respond regularly upon tactile stimulation. (J) α-actinin-specific immunostainings of skeletal muscles reveal normal sarcomeres in fla mutants carrying Tg(unc45b^min:smyd1b-gfp), indicating a complete structural rescue by the transgenic reintroduction of Smyd1b in homozygous fla mutants.