Carr BJ et al. (2024), Prominin-1 null Xenopus laevis develop subretin...

XB-ART-60968

J Cell Sci 2024 Oct 02; doi: 10.1242/jcs.262298.

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Prominin-1 null Xenopus laevis develop subretinal drusenoid-like deposits, cone-rod dystrophy, and RPE atrophy.

Carr BJ , Skitsko D , Kriese LM , Song J , Li Z , Ju MJ , Moritz OL .

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PROMININ-1 (PROM1) mutations are associated with inherited, non-syndromic vision loss. We used CRISPR/Cas9 to induce prom1-null mutations in Xenopus laevis and then tracked retinal disease progression from the ages of 6 weeks to 3 years old. Prom1-null associated retinal degeneration in frogs is age-dependent and involves RPE dysfunction preceding photoreceptor degeneration. Before photoreceptor degeneration occurs, aging prom1-null frogs develop increasing size and numbers of cellular debris deposits in the subretinal space and outer segment layer, which resemble subretinal drusenoid deposits (SDD) in their location, histology, and representation in color fundus photography and optical coherence tomography (OCT). Evidence for an RPE origin of these deposits includes infiltration of pigment granules into the deposits, thinning of RPE as measured by OCT, and RPE disorganization as measured by histology and OCT. The appearance and accumulation of SDD-like deposits and RPE thinning and disorganization in our animal model suggests an underlying disease mechanism for prom1-null mediated blindness of death and dysfunction of the RPE preceding photoreceptor degeneration, instead of direct effects upon photoreceptor outer segment morphogenesis, as was previously hypothesized.

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Species referenced: Xenopus laevis
Genes referenced: calb1 ctbp2 erg prom1 rho rpe65 vim
GO keywords: eye development [+]

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	Fig. 1. Representative histology of wildtype and F0 prom1-null retinas in animals aged 6 weeks to 1 year examined with Hoechst and WGA labeling (A-F) and 4-channel labelless autofluorescence combining 405 nm, 488 nm, 555 nm, and 647 nm excitation wavelengths (G-L). Between 6 weeks and 1 year of age, deposits of cellular debris in the outer segment layer accumulate and increase significantly in size (white arrows). These deposits are strongly labelled with Hoechst dye (white; A-F) and are not present in wildtype control animals. Hoechst dye also demonstrates the progressive disorganization of RPE nuclei (F, black arrows). When examined using autofluorescence (G-L), the deposits fluoresce strongly at different excitation spectra than the surrounding outer segment material, and there is an increase in autofluorescent puncta in the RPE layer of prom1-null frogs (H, J, L) compared to the wildtype controls (G, I, K). Autofluorescence images are color-coded to correspond to the detected emission spectra: cyan (405 nm), green (488 nm), orange (555 nm), and red (647 nm). Scale bars = 50 µm. Labels (A-F): Blue = wheat germ agglutinin (WGA); White = Hoechst nuclear dye. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium. Number of animals: (A, n = 10; B, n = 13; C, n = 5; D, n = 5; E, n = 3; F, n = 6; G, n = 4; H, n = 4; I, n = 3; J, n = 5; K, n = 3; L, n = 6).
	Fig. 2. Representative images of Oil Red O, BODPIPY, and E06 labeling in 1-3 year old wildtype (A,C,E) and prom1-null (B,D,F) retinas. Both Oil Red O (A,B) and BODIPY (C,D) label the oil droplets in the cone photoreceptors (white arrows), but neither labels the SDD-like deposits in the prom1-null retinas strongly (black arrows). Red autofluorescence is detected in the RPE of wildtype frogs and in the RPE and large deposits in prom1-null frogs (C,D; magenta). E06 labels the RPE moderately in wildtype frogs (E). In prom1-null frogs, the RPE is labeled more strongly, and it looks similar to red autofluorescence (D) in that it penetrates more deeply into the outer segment layer. The deposits are labeled heterogeneously with E06, and they are more immunopositive than the surrounding outer segments, where no deposits appear (asterisks, F). The RPE is disrupted in prom1-null retinas; pigment granules infiltrate into the SDD-like deposits (B,F) and migrate further into the outer segment layer than in wildtype animals (B,D,F). Scale bars = 50 µm. Number of animals: Wildtype, n = 5; prom1-null, n = 9. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium; ChrC, choriocapillaris; Chr, choroid.
	Fig. 3. Labeling of SDD like deposits with markers for (A) outer segments, (B) RPE, and (C) vimentin. SDD-like deposits do not show appreciable fluorescent signal for outer segment markers WGA, rhodopsin, or cone opsin (A, merge). Cone opsin was not detected in the deposits, but cone opsin positive-label surrounded the deposits in a honeycomb-like pattern (A, cone opsin, merge). Large deposits containing multiple nuclei are negative for RPE65 (B, dotted line), but single nuclei displaced from the RPE into the outer segment layer are surrounded by a ring of strongly RPE65-positive immunoreactivity (B; white arrow). Vimentin-positive immunoreactivity is increased in the RPE of prom1-null animals as well as the SDD-like deposits (black arrows), but stays relatively consistent in the cytoskeleton of the Müller glia in the inner plexiform layer (C). Scale bars = 25 µm. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segments; RPE, retinal pigment epithelium. Numbers of animals: Wildtype, n = 3-6; prom1-null, n = 3-5.
	Fig. 4. Representative images prom1-null F1 frogs compared to wildtype animals in color fundus photography and infrared OCT (A-C). F1 frogs have SDD-like deposits of cellular debris in the outer segment layer (B,C; white arrowheads), but one phenotype has a large number of these deposits and no window defects or penetration of the OCT beam through the dense RPE layer and into the choroid (B), while the other has fewer or no SDD-like deposits and large window defects that reveal the underlying choroid and choroidal vasculature (black arrowheads, C). Black arrows mark blood vessels on the vitreal side of the retina and the white arrow marks a large choroidal vessel that can be seen through the RPE in both the color fundus photo and the OCT scan. Fundus field of view = 1.8 mm. OCT Scale bars = 100 µm. Comparison of a wildtype animal and a 3 year old prom1-null adult frog with significant deposits using DOPU OCT in B-scan (D,E), en face (F,G), and 3D reconstructions (H,I).Deposits in prom1-null frogs have a significantly altered DOPU signal and are easily detectible as bright yellow or green spots in B-scan and en face orientations (E,G; white arrows). When projected in 3D, DOPU signal is not uniform within the deposits. Instead, green signal that indicates altered polarization caused by RPE pigment granules or other light-reflecting structures exists as a core within the deposit (I, black arrow). DOPU OCT detects loss of structural integrity of the ELM as evidenced by the projection of the large deposits through this layer, and loss of a strong band of red signal in the B-scan (E), en face (G), and 3D reconstructions (I). Evidence of RPE integrity breakdown is shown by breakdown of bright green bands in the B-Scan (E) and patchiness in the yellow en face image (G). The large black vessels in the prom1-null animal en face ELM, RPE, and choroid images (G) is not an anatomical defect, but is instead caused by shadowing from the vitreal vessels blocking the DOPU OCT signal. Scale bars = 200 µm. Abbreviations: NFL, nerve fibre layer; ELM, external limiting membrane; RPE, retinal pigment epithelium; CHR, choroid. Number of animals: wildtype = 6, prom1-null = 9.
	Fig. 5. TEM micrographs of complex, lipofuscin-like deposits in the RPE of prom1-null mutants with a moderate disease phenotype (age 8 months). These deposits are localized in (A,B) or near (C) the RPE and contain electron dense material, membranes (mem), multivesicular bodies (mvb), lysosomes (lys), and numerous vacuoles (v). The yellow inset shows a higher magnification of a putative lysosome fusing to a disorganized multivesicular body in a large deposit located within the RPE. Scale bar = 0.5 µm, inset scale bar = 1 µm. Abbreviations: ROS, rod outer segment; RPE, retinal pigment epithelium.
	Fig. 6. Retinal function as measured by gross scotopic ERG response amplitude and the difference between wildtype and prom1-null animal response amplitude across three different ages: 6 weeks (green circles), 1 year (blue triangles), and 2 years (pink squares). A) Waterfall plots of scotopic ERGs for 6 weeks, 1 year, and 2-year-old frogs. Wildtype traces are black while the mutant traces are colored. B) Scotopic A-wave response amplitude from wildtype and prom1-null animals. C) Scotopic B-Wave response from wildtype and prom1-null animals. D) The difference in A-wave response amplitude between wildtype and prom1-null animals. E) The difference in scotopic Bwave response amplitude between wildtype and prom1-null animals. Number of animals and respective symbols are denoted under the waterfall plots. Difference data were calculated and graphed as the mean difference of wildtype minus prom1-null animals ± SEM. Data were analyzed using Two-Way ANOVA to segregate effects of age and light intensity. For differences data (D,E), statistical significance is denoted above the group that is different, with colours of asterisks indicating the statistical significance for the comparison group. Asterisks: * p < 0.05. Data from 6 week old animals were published previously (Carr et al., 2021).
	Fig. 7. Retinal function as measured by gross photopic ERG response and the difference between wildtype and prom1-null animal response amplitude across three different ages: 6 weeks (green circles), 1 year (blue triangles), and 2 years (pink squares). A) Waterfall plots of photopic ERG and flicker responses for 6 week, 1 year, and 2 year old frogs. Wildtype traces are black while the mutant traces are colored. B)Gross photopic A-wave response amplitude of wildtype and prom1-null animals. C) Gross photopic B-wave response amplitude of wildtype and prom1-null animals. D) Gross photopic flicker response amplitude of wildtype and prom1-null animals. E) The difference in photopic A-wave response between wildtype and prom1-null animals. F) The difference in photopic B-wave response amplitude between wildtype and prom1- null animals. G) The difference in photopic flicker response amplitude between wildtype and prom1-null animal. Number of animals and respective symbols are denoted under the waterfall plots. Difference data were calculated and graphed as the mean difference of wildtype minus prom1-null animals ± SEM. For gross photopic amplitude response (B,C,D), statistical differences on the graphs denote differences between wildtype and prom1-null animals for the same age group; color indicates the age. For differences data (E,F,G), statistical significance is denoted above the group that is different, with colours of asterisks indicating the statistical significance for the comparison group. Asterisks: * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001. Data from 6 week old animals were published previously (Carr et al., 2021).
	Fig. 8. Wildtype and prom1-null retinal immunoreactivity to CTBP2 (A), calbindin (B), rhodopsin, and cone opsin (C). A) There is significant loss of CTBP2 immunoreactivity in the central retinas and RPE of prom1-null frogs aged 6 months. B) In animals aged ≥ 2 years, there is significant loss of calbindin immunoreactivity that starts off as just loss in the cone outer segments with calbindin labelling in healthy retina on the left, but loss of calbindin in mutant retina with SDD-like deposit () on the right (ii; mosaic F0 animal). This loss of calbindin expands to immunoreactivity in a subset of bipolar cells concurrent with inner plexiform and nerve fibre layer thinning (iv, F1 animal). There is also increased vimentin labeling in cellular debris deposits (iv, grey; ). C) Severely degenerated retinas lose calbindin immunoreactivity completely and have significant thinning of the inner plexiform layer. Some cone opsin immunoreactivity remains within the outer segment layer, but it is greatly reduced. Rhodopsin and wheat germ agglutinin immunoreactivity are preserved. i,ii are taken with the Leica DM600B epifluorescence microscope. Scale bars = 50 µm. Abbreviations: NFL, nerve fibre layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium. Labels: (A): green = wheat germ agglutinin, blue = Hoechst, white = CTBP2. (B): green = calbindin (cone inner segments and bipolar cells), blue = Hoechst, magenta = wheat germ agglutinin, white = vimentin. Number of Animals: Wildtype, n = 4; prom1- null, n = 5.
	Fig. S1. Schematic of photoreceptor/RPE morphology and prom1 protein localization and expression in wildtype and prom1-null frogs. A) Wildtype outer segment morphology is highly organized in both rods and cones. Rods (magenta) comprise discrete discs of membrane whereas cones (green) comprise layered lamellae. B) In wildtype frogs, prom1 protein (cyan) is localized to the base of the rod outer segment (magenta) and one side of the cone outer segment (green). C) Outer segment morphology in prom1- null frogs is significantly dysmorphic; ROS are shortened and bulbous, COS are fragmented. There are also deposits of heterogeneous cellular material between the OS and RPE (asterisk). D-F) Prom1 protein immunoreactivity in wildtype animals aged 14- and 42-days post fertilization. Relative prom1 immunoreactivity decreases in the central retina as animals age and outer segments reach adult size. Scale bar = 50 µm. Bar graph demonstrates mean ± SEM. Number of animals: 14 dpf, n = 5; 42 dpf, n = 4. Micrograph labels: Magenta, wheat germ agglutinin; Green, cone opsin; blue, Hoechst 33342 nuclear stain; cyan/grey, prom1. Abbreviations: COS, cone outer segment; dfp, days post fertilization; ROS, rod outer segment; RPE, retinal pigment epithelium.
	Fig. S1. Schematic of photoreceptor/RPE morphology and prom1 protein localization and expression in wildtype and prom1-null frogs. A) Wildtype outer segment morphology is highly organized in both rods and cones. Rods (magenta) comprise discrete discs of membrane whereas cones (green) comprise layered lamellae. B) In wildtype frogs, prom1 protein (cyan) is localized to the base of the rod outer segment (magenta) and one side of the cone outer segment (green). C) Outer segment morphology in prom1- null frogs is significantly dysmorphic; ROS are shortened and bulbous, COS are fragmented. There are also deposits of heterogeneous cellular material between the OS and RPE (asterisk). D-F) Prom1 protein immunoreactivity in wildtype animals aged 14- and 42-days post fertilization. Relative prom1 immunoreactivity decreases in the central retina as animals age and outer segments reach adult size. Scale bar = 50 µm. Bar graph demonstrates mean ± SEM. Number of animals: 14 dpf, n = 5; 42 dpf, n = 4. Micrograph labels: Magenta, wheat germ agglutinin; Green, cone opsin; blue, Hoechst 33342 nuclear stain; cyan/grey, prom1. Abbreviations: COS, cone outer segment; dfp, days post fertilization; ROS, rod outer segment; RPE, retinal pigment epithelium.
	Fig. S2. A) OCT interpretation of wildtype Xenopus laevis retina. B) Retinal layer thickness as measured by OCT in 2-year-old wildtype and F0 prom1-null animals. The RPE represented the greatest contributor to overall thinning of the retinal in prom1-null frogs. Graphs represent the means ± SEM. Number of animals: Wildtype, n = 9; prom1- null, n = 7.
	Fig. S3. Comparison of the size and number of shed outer segment packets in wildtype versus prom1-null mutant tadpoles aged 14 dpf. Scale bars = 50 µm. Data are represented as mean ± SEM. Number of animals: wildtype, n = 5; prom1-null n = 6. Statistics: Student’s t-test, unpaired, two-tailed; * p < 0.05.
	Fig. S4. A comparison of morphology changes in photoreceptor synapses between a wildtype frog, a prom1-null frog, and a rabbit model of retinitis pigmentosa (P347L). The prom1-null frog and P347L rabbit have a severe phenotype and retinal synaptic remodelling. Remodelling features are very similar between the two animals and the two models of retinal degeneration, indicating similarities in the downstream processes of severe retinal degeneration. Scale bar = 0.5 µm.
	Fig. S5. Fluorescein angiography in 3-year-old wildtype and prom1-null mutant frogs. There was little observed difference in the vitreal blood vessel structure and there were no leaky vessels or significant bleed through to the choroidal vessels near the large lesions. Scale bars = 100 µm. Number of animals: Wildtype, n = 9, prom1-null, n = 8.
	Fig. S6. Lack of prom1 protein immunoreactivity in fully differentiated frog RPE and human pan retinal/RPE RNA seq data from the NEI eyeIntegration Database1–15 v2.12 (https:// eyeintegration.nei.nih.gov/). A) Representative micrograph of prom1 immunoreactivity in an adult frog retina and RPE. There is no detectable prom1 immunoreactivity in the RPE (white dotted lines). Red blood cell nuclei are denoted with an asterisk. B) GAMMA adjusted micrograph to increase the overall intensity of the prom1 protein signal against background to demonstrate lack of immunoreactivity in the RPE. Number of animals: n = 63. C) A heat map of RNA seq expression data for cone opsins, PROM1, PROM2, RHO, and RPE65 in the retina/ RPE. Samples are denoted by number at the bottom of the heat map and identified by the legend on the right. Abbreviations: RPE, retinal pigment epithelium; RBC, red blood cells.