srf mRNA expression in developing zebrafish. Day 3 (A,C,E,G) and day 7 (B,D,F,H) developing zebrafish embryos/larvae were prepared for whole mount in situ hybridization as described in Materials and Methods with antisense riboprobes to srf1 (A,B), srf2 (C,D), sm22α (E,F) and myh11 (G,H). Sections of day 7 larvae validated probe penetrance and sense control probes all showed no staining (data not shown).

Adult tissue expression of srf mRNA. Indicated tissues were harvested from 3 month-old zebrafish and cDNA templates prepared with primers specific to each target mRNA as described in Materials and Methods. Abbreviations: Br, brain; He, heart; SB, swim bladder; Sk, skeletal muscle; Li, liver. ZF4 is an embryonic zebrafish fibroblast cell line. hprt is a control mRNA to validate presence of relatively equal amounts of total RNA. These results are representative of two other independent studies.

Adult tissue expression of Srf1 protein. Adult (3 month) tissues were processed for immunohistochemistry as described in Materials and Methods. Arrows point to Srf1 nuclear staining (in red) in cardiomyocytes of the heart (A), skeletal muscle of posterior trunk (B), visceral SMC of gut (C), vascular SMC of dorsal aorta (D) and cortical neurons of brain (E). The asterisk in (A) indicates nucleated red blood cells within the ventricular chamber of the heart. Note the peri-aortic melanin (dark staining rim in panel D) that has recently been described and validated (Miano et al., 2006). Staining specificity was demonstrated with a competing peptide to human SRF (data not shown). Magnifications are 400x.

Modulating endogenous Srf genes. Expression of srf1 and srf2 genes was studied in the ZF4 embryonic zebrafish fibroblast cell line following transduction with the indicated adenoviral transgenes. Total RNA was isolated for RT-PCR three days post-viral transduction. Note attenuated expression of both srf transcripts with shRNA to mammalian Srf and increases with a human MYOCD transgene. Increases in endogenous myh11 (a known mammalian SRF target gene restricted only to SMC lineages; Miano et al., 1994) are also evident with Ad-MYOCD. As in Fig. 3, the hprt housekeeping gene serves as a loading control. Results were repeated in an independent study.