Fig 1: Emc3 transcription pattern in C57BL/6 mouse retinas at different stages of development. For E11.5 (A, A’), E15.5 (B, B’), P0.5 (C, C’), and P7 (D, D’) C57BL/6 mouse retina, RNAscope indicates that Emc3 was widely expressed in all layers of the retina. Red dots represent Emc3 mRNA signal. Left column A–D: 400 × magnification; right column A’–D’: outlined region in left column at 1000 × magnification. Emc3, endoplasmic reticulum membrane protein complex subunit 3; NBL, neuroblastic layer; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Fig 2: Retina-specific Emc3 knockout caused disruption of the retinal structure and function. (A) Strategy for CKO mouse generation. CKO mice were obtained by cross breeding Emc3flox/flox mice and Six3-cre mice. (B, upper) Western blot detected the efficiency of the EMC3 knockout in P7 mouse retinas. Grayscale analysis (B, lower) showed that EMC3 was decreased by 56%. The α-tubulin was the normalized standard. ***P < 0.001, unpaired, 2-tailed Student's t-test. (C) ERG recordings of 5W mice at a stimulus intensity of 2 cd/m2. CKO mice had neither a scotopic nor photopic wave, which means that the CKO mice completely lost visual function (n = 5). (D) Color fundus photographs and OCT of 5W mice. CKO mouse fundus images had changes in fundus pigments. CKO mice OCT images revealed severe thinning, and the fundus blood vessels were almost completely absent. (E) H&E staining showed morphological changes in 5W mice. CKO mouse retinas were extremely thin, and the RCL, ONL, and INL had almost disappeared. RCL, rod and cone layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 40 µm.
Fig 3: Loss of EMC3 affected the neurogenesis and positional changes of photoreceptor cells and horizontal cells. (A) Recoverin-marked (green) photoreceptors in P0.5 mice. Photoreceptors appeared in the retinal apical region in control mice. DAPI (blue) was used as a nuclear marker. The histogram on the right represents the number of cells per field. RPE, retinal pigment epithelium; NBL, neuroblastic layer; Scale bars: 50 µm. (B) Immunofluorescence indicates that horizontal cells (Prox1+, green) were dislocated in E15.5 and P0.5 CKO mice. Prox1+ cells are arranged close to the dotted line in the control mice. (C) Ki67-marked (green) cells (RPCs) in E15.5, E17.5, and P0.5 mice. Ki67+ cells disappeared in the apical region of the P0.5 CKO mouse retina. (D) Immunofluorescence indicates that cells in mitosis (pH3+, green) were dislocated in E15.5 and P0.5 CKO mice. (E) Heatmap of the differential proteins involved in the regulation of neural differentiation in E17.5 mouse retina.
Fig 4: Deletion of EMC3 activates the ERS response and apoptosis. (A) E15.5, E17.5, and P0.5 retinal cryostat sections were immunostained with an antibody for pIRE1α (red). White arrows indicate the cells with activated IRE1α. (B) Western blot and quantitation were performed to detect ATF6 (50 kDa). (C) TUNEL assay (green) indicated apoptosis, which appeared in CKO mouse retinas beginning at E15.5. White circles show that apoptotic cells appeared in the center of the rosettes. Scale bars: 50 µm.
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