Fig 2: 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.