Figure 4.

Adamtsl4^tvrm267 mice develop RPE lesions. (A) Bright field fundus image of a WT (B6) mouse at 3 months of age. (B) Detail of boxed area in (A) showing the normal appearance of the RPE. (C) Bright lesions, consistent with hypopigmentation, in a 3-month-old Adamtsl4^tvrm267 mutant. (D) Detail of boxed area in (C) showing an alteration in pigmentation among RPE cells associated with bright lesions. (E–H) Fluorescence micrographs of RPE flatmounts stained for F-actin (rhodamine phalloidin, red) and nuclei (DAPI, blue). (E) WT flatmount showing the normal polygonal architecture of the RPE as revealed by F-actin staining at cell boundaries. (F) Detail of panel (E) showing mono- and binucleated cells, and phalloidin staining within cell boundaries due to F-actin in apical processes. (G) Flatmount of the homozygous Adamtsl4^tvrm267 eye shown in panels (C) and (D) indicating a precise correspondence of the lesions. (H) Detail of panel (G) showing that in areas associated with lesions, many cells are enlarged and have diminished phalloidin staining (n = 3). (I) Morphometric analysis of Adamtsl4^tvrm267 mice at 3 months of age indicating an increase in area and Feret diameter of RPE cells within lesions. Cells within lesions (n = 55) were compared with cells in the area surrounding the lesions (n = 372). Asterisks: P < 0.0001; error bars indicate mean ± SD. (J and K) Fundus images (J, right eye; K, left eye) showing the variability in lesion severity within an individual homozygous Adamtsl4^tvrm267 mouse. (L) Incidence of RPE lesions with age. The splatter phenotype was graded in homozygous Adamtsl4^tvrm267 eyes by indirect ophthalmoscopic evaluation (severity of phenotype: N, none; L, low; M, medium; H, high; see grading scale in Supplementary Material, Fig. S1). The number of mice with a grade in both eyes of no (NN) or high (HH), or any other combination (mixed) was determined in ranges up to ∼10 months of age. The total number of mice examined within each age range is indicated. Mice were free of lesions up to 1 month of age. The fraction with a mixed or bilateral high splatter phenotype increased dramatically between 1 and 3 months and progressed slowly thereafter. (M) Whole flatmount from a homozygous Adamtsl4^tvrm267 eye showing a regional distribution of lesions in the mutant near the optic nerve head with a bias toward the inferior fundus. Quadrants are designated S, superior; I, inferior; T, temporal; N, nasal. B6 controls: (A, B, E and F); tvrm267 mutants: (C, D, G, H, J, K, M). Scale bars: (E and G) 50 µm; (F and H) 25 µm; (M) 500 µm.