Fig. 4

Fig. 4 ttn.1 heterozygosity induces a severe phenotype in homozygous srpk3-null mutant zebrafish larvae.

a–h, Lateral view of Alexa Fluor phalloidin filamentous actin (green) and α-actinin Z-band marker (red) staining in skeletal fast muscle fibers in WT (a,e), srpk3^+/−; ttn.1^+/− (b,f), srpk3^−/−; ttn.1^+/+ (c,g) and srpk3^−/−; ttn.1^+/− larvae (d,h) at 5 dpf. Compared to WT (a,e) or double heterozygotes (srpk3^+/−; ttn.1^+/−; b,f), homozygous srpk3-null alone only causes very mild muscle fiber defects (c,g), while ttn.1 heterozygosity in homozygous srpk3^−/− larvae severely affects muscle fiber integrity (d,h). i–t, Isolated myofiber immunostaining and electron microscopy (EM) in skeletal fast muscle fibers in WT (i,m,q), srpk3^+/+; ttn.1^+/− (j,n,r), srpk3^−/−; ttn.1^+/+ (k,o,s) and srpk3^−/−; ttn.1^+/− (l,p,t) larvae at 5 dpf. Isolated myofiber immunostaining showed that titin expression is largely reduced in the double mutant (srpk3^−/−; ttn.1^+/−; l,p) but not in the single heterozygous ttn.1 mutant (srpk3^+/+; ttn.1^+/−; j,n) or the srpk3-null (srpk3^−/−; ttn.1^+/+; k,o). EM showed that srpk3-null zebrafish (srpk3^−/−; ttn.1^+/+; s) had well-defined sarcomeres, with mildly disorganized myofibrils. The double-mutant fish (srpk3^−/−; ttn.1^+/−; t) displayed pronounced disruption of the sarcomere structure. White scale bars are 25 µm. Black scale bar is 500 nm. Representative images from >15 pooled fish per genotype.