Fig 2: Morphological organization of 21EM15 organoids partially recapitulates eye lens patterning. (A) Immunofluorescence (IF) microscopy reveals the localization of Laminin on the outer region of the organoid. (B) Multiplex microscopic images indicate the localization of the expressed proteins, PAX6, PROX1 and JAG1. The red and blue squares correspond to the typical areas (respectively, the outer and inner regions) used to display the magnification insets presented below the grey level image of PROX1 and to quantify the PROX1 nuclear labeling shown in (C). The white dashed line symbolizes the central axis of the organoid. (C) Violin plots combined with jittered scattered plots showing that PROX1 is enriched in the nuclei of cells located in the inner region. This graph is representative of three independent experiments; n = 30 organoids. p < 2.2 × 10−16; Wilcoxon rank sum test with continuity correction. (D) IF images showing the localization of αB-Crystallin. Insets show the enlargement of the outer region, the central axis and the core of the organoid. Data are representative of at least three independent experiments; n = 30 organoids.

Fig 3: Model for 21EM15 lens organoid organization. This model shows that, unlike the lens, 21EM15 organoids lack the typical lens capsule and anterior epithelium. Instead, there is a zone that we term “capsuloid”, comprising the outer layers of proliferative cells embedded in a Laminin layer. Further inwards and from the outside in, 21EM15 organoids recapitulate several aspects of the organization of the lens, with a region corresponding to the germinal zone, where Ki67 and Jag1 are expressed, followed by a transition zone that expresses Pax6 and where PROX1 progressively becomes nuclear. Along the central axis of the organoid, PROX1 is predominantly nuclear, and cells progressively degrade their organelles, such as nuclei (loss of Lamin B and Fibrillarin) and mitochondria (loss of TOMM20 signal), and begin to express Hsf4 and Cryab. These events correspond closely to those described in the normal lens cortex.

Fig 4: Intravitreal MPTP administration drives retinal ganglion cell loss. A Retinal ganglion cells were fluorescently labelled in whole-mounted retina with anti-RBPMS (magenta). B There were no significant differences between the naïve and vehicle control (green) groups. RBPMS density significantly decreased relative to the vehicle control at 7, 14 and 21 days post-injection for 5 mg/mL (orange) and 50 mg/mL (red) MPTP. C The thickness of all retinal layers (GCL, IPL, INL, OPL, and ONL) was measured from cross-sections. For all retinal layers, there were no significant differences between the vehicle control (green) and 5 mg/mL MPTP (orange) or 5 mg/mL MPTP (red) at 7 days post MPTP injection. D All amacrine cells were fluorescently labelled in retinal cross-sections with anti-Prox1 antibodies. Images captured with a 5 × objective were analyzed for Prox1 signal intensity, images captured with a 20 × objective were analyzed for Prox1+ cell soma counts. This data was plotted. There were no significant differences in signal intensity between vehicle control (green) and 5 mg/mL MPTP (orange) or 50 mg/mL MPTP (red). The number of Prox1+ cells, the total number of INL cells (relative to the measured INL area), and the relative density of Prox1+ cells (Prox1+ cells/total cell number) did not significantly change over the different conditions. There were no significant differences in between vehicle control (green) and 5 mg/mL MPTP (orange) or 50 mg/mL MPTP (red). Scale bar = 20 µm in A and D (20 ×), 200 µm in D (5 ×)

Fig 5: Immunofluorescence staining of primary mouse RGCs. (a) Brn3a (green), (b) RBPMS (green), and (c) Prox1 (green) staining, each shown alongside DAPI (blue) nuclear staining and merged images. Scale bar = 50 μm.