Figure 2

Down-regulation of raw causes cardial apoptosis. Epifluorescence micrographs showed localized cell death in the raw mutant. (A-C) Wild-type (WT) embryos, (D-F) raw¹ mutant embryos. (A) Acridine orange (AO) positive cells were detected mainly in cephalic ganglia and head regions at stage 14. (D) raw¹ mutant embryos of the same age displayed a similar AO staining pattern. Normal PCD detected using (B) AO or (C) TUNEL staining in wild-type embryos at stage 16. Excessive cell death (brackets) was observed in dorsal mesoderm of raw¹ mutant embryos using (E) AO or (F) TUNEL staining. (G) Real time PCR quantification of raw mRNA level relatively to rps17 mRNA. (H) Knocking down the expression of raw in the ectoderm caused excessive cell death (brackets) in the dorsal mesoderm. (I-L) Double-labeling of dying cells using immunostaining of (J) him-GFP expressing cells (green) and (K) TUNEL (red) in raw¹ mutant was assessed by confocol microscopy. (I) High magnification view of TUNEL and anti-GFP immunostained embryos from marked area (white box) in (L) merged image. Dying cells (yellow nuclei surrounded with green cytoplasma); Healthy him-GFP-positive cells (green); Unidentified dying cells (red, white arrowheads). WT, wild-type; st, stage.