Fig 1.
Elevated numbers of TUNEL positive cells in Chmnull/WT.
(A, B) TUNEL assays were performed on flat mounted entire retinae from 6- month-old heterozygous null female mice (Chmnull/WT) and age-matched controls. The boxed region is magnified in the inset to reveal individual nuclei. Scale bars: 500μm (A, B) and 20μm (insets). C: TUNEL positive cells/mm2 of retina were quantitated. T test: *p< 0.05, n = 3.
Fig 2.
Elevated numbers of rods with swollen mitochondria in Chmnull/WT.
Eyes from 6-month-old heterozygous null female mice (Chmnull/WT) were processed for transmission electron microscopy. (A) Analysis along the full length of the retina (from peripheral through central to peripheral) revealed occasional cells with swollen mitochondria and degenerating outer segments (OS) in both Chmnull/WT and controls. Scale bars: 1μm. (B) Quantitative analysis revealed a greater number of such cells in Chmnull/WT. T test: **p<0.01, n = 8.
Fig 3.
Elevated numbers of Iba1 positive glia in Chmnull/WT.
(A) Flat mounted entire retinae from 6-month-old heterozygous null female mice were stained for the glial cell marker, Iba1. (B) High magnification of the boxed region in A indicates the stellate morphology of the glial cells. Scale bars: 500 μm (A) and 100μm (B). (C) Quantitation of glial cell numbers in heterozygous null female mice and age-matched controls reveals elevated numbers in Chmnull/WT. T-test: **p<0.01, n = 3.
Fig 4.
Chmnull/WT show normal ultrastructure of the inner segment, outer segment and connecting cilium.
Eyes from 6-month-old heterozygous null female mice (Chmnull/WT) were processed for transmission electron microscopy. High magnification examination of longitudinal sections through the eye cup revealed no abnormalities of the outer segment structure (OS) or connecting cilium (CC) or the Golgi apparatus (G) or mitochondria (mito) in the inner segment (IS) in the majority of cells. Scale bars: 500nm.
Fig 5.
Chmnull/WT have uniformly shortened outer segments.
Eyes from 2, 6 and 12 month-old heterozygous null female mice (Chmnull/WT) were processed for transmission electron microscopy. Low magnification examination of longitudinal sections throughout the length of the retina (from peripheral through central to peripheral), at 10 uniform intervals revealed uniform shortening of the OS (A-C). Higher magnification images revealed progressive increases in OS width (D-F). Scale bars: 20 μm (A,B), 1μm (D,E). T-test: ****p<0.001, n = 6 (C), *p<0.05, **p<0.01, †† p<0.01 n = 3 (F).
Fig 6.
Reduced rhodopsin in the outer segment is not accompanied by accumulation in the inner segment.
(A, B) Rhodopsin staining (green) of 12μm retinal sections of 6-month-old heterozygous null female mice (Chmnull/WT) revealed strongly positive outer segments (OS), a small number of rhodopsin positive punctae (phagosomes) in the retinal pigment epithelium (RPE) but no detectable staining in the inner segment (IS). This was despite evident photoreceptor loss, as indicated by reduced numbers of DAPI stained (blue) nuclei in the outer nuclear layer (ONL). (C-K) Eye cup-derived specimens from 6-month-old heterozygous null female mice (Chmnull/WT) and age-matched controls were stained for rhodopsin by cryo-immunoEM using antibodies to the C-terminus (1D4-5nm gold) and N-terminus (RetP1-15nm gold). Rhodopsin was concentrated on the outer segment (OS) discs and the limiting membrane of the connecting cilium (CC). Rhodopsin labelling (examples indicated by white arrowheads in F-I) was also present on the Golgi and on small vesicles within the inner segment. Scale bars: 20 μm (A,B) and 200nm (D-K). (L,M) Quantitation of the number of 1D4 rhodopsin gold particles/area of IS and OS revealed that rhodopsin concentration was reduced to a similar extent in IS and OS of heterozygous null female mice (Chmnull/WT). (N) Dividing the IS into equal basal (containing the Golgi), middle and apical domains revealed a shift in the distribution of gold particles in the IS to a greater proportion in the middle region (mainly vesicle-associated). T-test: *p<0.05, ****p<0.0001, n = 3.