Fig 2.

Obscurin A RhoGEF morphant phenotype. (A) Morphant embryos were assessed at 72 hpf and scored for head size (small head), eye morphology (small eye, poorly developed optic cup), ear morphology (one or no otoliths), cardiac structure and function (ventricular hypoplasia or dilatation, pericardial edema) and skeletal muscle appearance (reduced tail length and width). (B-D) Gross morphology of control (B), obscurin A RhoGEF morphant (C) (0.1 mM of each morpholino), and rescued obscurin A RhoGEF morphant embryos (D) (0.1 mM of each morpholino + 2250 pg RhoGEF/Ank expression construct). The obscurin A RhoGEF morphant embryos demonstrate a high incidence of pericardial edema (>), moderate (^) to severe (*) eye hypoplasia, and smaller heads than controls. These defects were rescued or markedly diminished by co-injection of an obscurin A expression construct that included the RhoGEF and ankyrin binding domains (RhoGEF/Ank). (E) RT-PCR of mRNA extracted from 4, 24 and 72 hpf embryos injected with the expression construct demonstrating correct amplification of the expression tag (<). (F) Western analysis (Ab: anti-EGFP) of 72 hpf whole embryo lysates demonstrating expression of a fusion protein (>) of the expected size only in injected embryos (R) and not in control (C). (G) Percentage of obscurin A RhoGEF morphant embryos demonstrating one or more of the phenotypic abnormalities upon co-injection with the indicated rescue construct (*: p < 0.05 compared to empty vector control). Note the high efficiency of rescue with the RhoGEF/Ank and the absence of rescue with the non-sense and RhoGEF-m/Ank constructs. (H) Diagram of the amino acid sequence change introduced into the RhoGEF-m/Ank construct that replaces a conserved KY (Lysine-Tyrosine) motif with TM (Threonine-Methionine). Underlined nucleotides were changed using site-directed mutagenesis.