Fig. 3

rgs3 morphant phenotypes and functional rescue.

Schematic of zebrafish rgs3 mRNA/protein composite (A). Numbers refer to the amino acid number of the encoded Rgs3 protein, while the locations of morpholino binding sites employed in later experiments are indicated by red lines above the transcript. MO = rgs3 MO, MOb = rgs3 MOb and SA = rgs3 MOsa. The RGS domain of Rgs3, amino acids 43–158, is highlighted by the black box (A). Western analysis demonstrates that myc-tagged rgs3 and rgs3^N109A proteins are detectable from 5 hpf to 24 hpf (B). Antisense morpholino-mediated gene knockdown of rgs3 results in embryonic defects. Lateral views of 28 hpf wild-type (C,E) and rgs3 MO injected (D,F) embryos illustrate that rgs3 morphants have a reduced body length (D) and altered somite formation (F). rgs3 was co-injected with rgs3 MO to monitor rescue of gene knockdown. The molecular markers krox20, myoD and pax2 were used to evaluate rgs3 morphant rescue (G–Q). krox 20 labels rhombomeres 3 and 5, myoD labels the developing somites and adaxial cells while, pax2 labels the otic vesicle (o), midbrain-hindbrain boundary (MHB) and eye (E). Lateral (G and O–Q) and dorsal (H–N) views, anterior to the right, of 15 hpf (G–N) and 20 hpf (O–Q) wt embryos injected with Control MO (G,H,K,L,O), rgs3 MO (I,M,P) and rgs3 MO+rgs3 (J,N,Q). Boxed regions in G and K represent the areas magnified in H–J and L–N respectively. Asterisks indicate the spacing and width of three representative somites (L–N). krox20, myoD and pax2 expression patterns indicate that rgs3 is able to suppress the morpholino-induced defect (J,N,Q). For structural functional analyses, rgs3^N109A was evaluated for rescue of knockdown. Morphological analyses reveals that rgs3 is able to suppress the MO induced defect (R) while, rgs3^(N109A) is unable to suppress the MO induced defect (R).