Fig. 3

Reduction of rpS7 to haploinsufficient levels in zebrafish embryos induces autophagy in RBCs.

(A) Western blot analysis of rpS7 protein levels in zebrafish carrying viral inserts in the rpS7 gene at 1 and 2 dpf. The amount of rpS7 is calculated by densitometer analysis measuring the rpS7/actin ratio (N = 3). (B) Electron micrographs of 1 dpf embryos. Representative morphology of RBCs is shown in wild type (upper left panel) and rpS7-deficient embryos (upper right and lower panels). Arrowheads indicate double-membrane autophagosomes, arrows indicate autolysosomes. N = nucleus. Size bars upper and bottom left panels = 1 μM, upper right = 500 nM, lower right = 200 nM. (C) Quantification of double-membrane structures in the RBCs of micrographs in (B). In each case 28 cells were examined. (D) O-dianisidine staining of embryos at 2 dpf. Embryos (N>100) were scored in (E) and genotyped to confirm mutation status. (F) Representative mitochondrial morphology (m) displayed in wild type (upper left panel) and rpS7-deficient embryos at 2dpf (upper right). Typical Golgi complex (G) in wild type embryos shown (lower left panel) compared to Golgi complex in rpS7-deficient embryos (lower right). Arrows denote double membranes sequestering cytosolic material, an indication for autophagosome formation. *Denotes engulfed cytoplasmic material. Size bars = 200 nM.