Figure 9: Role of Rab32/38 in Ciona pigment cell biogenesis.

Histograms showing the percentage of larvae with two pigment cells (red), one pigment cell (violet) and no pigment cells (orange) after electroporation of dominant-negative forms of Ci-Rab32/38 under control of PCP enhancer (ptyr>Rab32/38^G19V or ptyr>Rab32/38^G19V+T36N) (a) or short hairpin inhibiting construct against Ci-Rab32/38 mRNA (U6>shRab32/38-C-D and U6>shRab32/38-E-F) (b) compared with control larvae electroporated with ptyr>LacZ.Merged bright field/fluorescent images of transgenic larvae. (c,d) Double fluorescent WMISH was performed to test the ability of U6>shRab32/38 used to knock down the endogenous Ci-Rab32/38. The expression of Ci-Rab32/38 (red) is compared with Ci-tyrp1/2a (green) and with anti-GFP antibody to follow expression of the electroporated constructs. Embryos were electroporated with ptyrp1/2a>GFP plus ptyrp1/2a>LacZ (c) and ptyrp1/2a>GFP plus U6>shRab32/38-C-D (d). U6>shRab32/38-C–D is able to knock down the endogenous Ci-Rab32/38 transcripts (n=33/59, d) and to induce defects in pigmented sensory organ melanization (a,b), while Ci-tyrp1/2a expression resulted not affected (c) as compared with the control embryos (n=73/80, c). Anterior to the left; nuclear staining by DAPI (blue). Scale bar, 50 μm; scale bar, 20 μm for zoomed embryos. Within each histogram is the combined number of larvae counted during at least three trials; n>150 embryos scored for transgene expression; error bar, s.e.m.; oc, ocellus, ot, otolith.