MICAL-2 Regulates SRF/MRTF-A-Dependent Gene Transcription during Zebrafish Development

(A,B) Representative control embryo derived from transgenic myl7:egfp reporter fish at 24 hpf, showing the size and position of a normal looping heart tube. By 48 hpf the control heart is fully looped. The myl7:egfp transgene directs eGFP expression to myocardial cells and facilitates detection of alterations in heart morphology.

(C,D) mical2b regulates cardiogenesis during zebrafish development. The mical2b splice-blocking morphant embryo at 24 hpf shows a smaller heart tube that fails to loop normally and thus is positionally displaced and leads to a significant pericardial edema (PE). By 48 hpf the morphant heart tube is linear and dysmorphic. Similar results were obtained with the translation blocking morpholino. For A-D n > 100.

(E) Higher magnification view of the normal control heart at 52 hfp highlights the evenly spaced cardiomyocytes (white arrows) focused on the atrial chamber.

(F-G) In contrast, cells in the smaller morphant atrium are irregularly spaced (white arrows). The representative embryonic hearts shown in F and G are for embryos injected with 2 or 4 ng of the splice-blocking morpholino, respectively (n > 50).

(H) mical2b knockdown impairs SRF/MRTF-A-dependent, but not SRF/MRTF-A-independent gene transcription. Levels are compared to control wild-type (WT) embryos for the mical2b morphants (2B), and in embryos rescued by coinjected RNA encoding the full-length wild-type murine MICAL-2 or the MICAL-2GV mutant protein. Experimental gene levels were normalized by comparison to housekeeping gene EF1α. ANOVA (^∗∗∗p < 0.0005) with Dunnett posttest. ^∗∗∗p < 0.0005, ^∗∗p < 0.005, ^∗∗p < 0.05, n ≥ 6. Mean ± SEM.

See also Figure S5 and Table S2.