FIG. 3.

Forced expression of asb11 mRNA interferes with muscle differentiation in vivo. (A–D) Zebrafish embryos were injected at the 2-cell stage in 1 of the 2 blastomeres (marked with asterisk) with encoding full-length MT-d-asb11 mRNA (300 pg). The numbers indicate the percentages of injected embryos displaying the particular phenotype (total n=82). Panel A shows an uninjected wild-type embryo. (E, F) Immunolabeling of MT-Asb11 overlay with myoD in situ hybridization. (G–O) Embryos were injected at 1-cell stage with Asb11 mRNA and fixed at 16 hours postfertilization (hpf ). Whole mount in situ hybridization was performed using (G–I) myoD, (J–L) mck, and (M–O) myoG riboprobe. Expression was compared between uninjected (G, J, M) and injected embryos (H, I, K, L, N, O). (P–S) Abnormal axial muscle formation at 36 hpf in response to d-asb11 mRNA injections. Fast and slow muscle fibers were stained by immunolabeling as described earlier [33]. (T) Confirmation of d-asb11-forced expression effects on mck at the protein levels. Zygotes were injected MT-asb11 mRNA, MT only, or MT-asb11ΔSOCS-injected embryos, serving as controls. Total cell lysates were isolated at 16 hpf. The protein was analyzed using an anti-Mck antibody. Anti-MT antibody was used to verify induced expression of asb11 variants, and as a loading control an anti-Hsp70 antibody was used. A representative image from 3 independent experiments is shown. Color images available online at www.liebertpub.com/scd