Figure 5.

Mitochondrial AIF deficiency-induced murine embryonic lethality linked to OXPHOS deficiency and energy loss. (A) To generate AIF KO mice, we crossed AIF^+/- females with C57BL/6 WT males. After genetic identification, the result of the crossing (progeny and E8.5 to E13.5 embryos) was reported in a table. (B) Light microscope images of E7.5 to E9.5 AIF^+/Y and AIF^-/Y embryos illustrating the progressive growth delay of the AIF KO embryos. Bar: 500 μm. (C) Hematoxylin/eosin staining performed on the sagittal sections of E8.5 and E9.5 AIF^+/Y and AIF^-/Y embryos, underlining the morphology and the abnormal nervous development of the AIF KO embryos. Bar: 500 μm. (D) Electron microscopy picturing mitochondria of E9.5 AIF^+/Y and AIF^-/Y embryos. Representative microphotographs are shown. Arrowheads in the picture mark the mitochondria. White squares show characteristic mitochondria. Bar: 0.5 μm. (E) Immunoblot of AIF and key proteins of the ETC complexes I to V performed in whole protein extracts from E9.5 AIF^+/Y and AIF^-/Y embryos. Equal loading was confirmed by β-Actin probing. This experiment was repeated 4 times with similar results. (F) Representative results of the histochemical assessment of mitochondrial OXPHOS activity in E9.5 AIF^+/Y and AIF^-/Y embryos using cytochrome c oxidase/succinate dehydrogenase (COX/SDH) double labeling. Bar: 50 μm. Whereas OXPHOS activity is normal in AIF^+/Y embryos, as demonstrated by the brown color of the section, AIF^-/Y showed defective OXPHOS activity (absence of brown staining). This experiment was repeated 3 times with similar results. (G) Total ATP levels recorded, as described in the Methods section, in E9.5 AIF^+/Y and AIF^-/Y embryos (n = 6). Results are expressed as RLU (relative light units). (H) Lactate release measured, as described in the Methods section, in E9.5 AIF^+/Y and AIF^-/Y embryos (n = 6). Statistical significance in (G) and (H) was calculated by the student t test. Bars represent mean ± SEM.