Fig 2: Progressive RGC loss after IA injury. In these whole-mount retinas stained with the RGC marker ?-synuclein (SNCG) 2 or 4 weeks after sham (a) or IA injury (b-c), there is evident RGC loss. Insets in a and c show the retinal flat mounts from which images in main panels originated. Note the progressive decrease in cell density from a to c. d. A bar graph with densities of surviving SNCG (+) RGCs 2 and 4 weeks post-injury, as compared to sham. Data were analyzed with one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05. Scale bars: a-c, 50 µm; inserts in a and c, 100 µm

Fig 3: Identification of hESC-derived RGC markers. (a) Expression of representative RGC markers, ATHO7, BRN3B, TUJ1, ?-synuclein (SNCG), and ISLET1 are shown. Real-time PCR analysis was performed on total mRNA extracted from hESC-derived RGCs on D30. BRN3B is expressed at high levels (>1,000×) at D30, as compared with that analysed on D0. Each mRNA expression level was first normalised to HPRT1 expression and then compared to mRNA expression on D0. (b) Immunohistochemical detection of BRN3B, ATOH7, TUJ1, SNCG, and ISLET1 and double-staining of ATOH7 and TUJ1 on D30. Whole-mount staining was performed for TUJ1; serial section staining was performed for the other targets. BRN3B and SNCG staining is mostly visible at the peripheral margins of the attached OV, while ATOH7 and ISLET1 staining is also visible at the centre of the OV. ATOH7 stains the nucleus of the RGCs. Axons stained by TUJ1 emerges from ATOH7-positive cells. Scale bars, 100 µm. Scale bar for double-staining, 15 µm. Error bars, standard deviation.

Fig 4: Quantitative assessment of DLK-JNK activation after IA injury based on counts of p-JUN (+) RGCs. a-c. In whole-mount retinas dually stained for the RGC marker ?-synuclein (SNCG) (red) and the DLK-JNK marker p-JUN (green), there is induction of p-JUN in RGCs at day 1 (b) and attenuation of labeling by day 14 (c). No p-JUN (+) RGCS are seen in the sham scenario (a). Left-sided images have been acquired with green filter combination for red SNCG immunofluorescence, images at the center have been acquired with blue filter combination for green p-JUN immunofluorescence, and panels on the right are merged images in which double-labeled profiles appear orange. Note the extensive colocalization at day 1, based on the large number of orange profiles (b, right panel). Most p-JUN (+) nuclei at day 14 seem to belong to smaller or atrophic RGCs (c). d-e. Bar graphs of densities of p-JUN (+) RGCs in the retinas of sham and injured animals at 1, 3, 7, and 14 days post-injury (d), and of the ratios of densities of p-JUN (+) RGCs over densities of total RGCs from two representative time points, i.e. 1 and 14 days (e). Significant differences are indicated with asterisks. The comparison in (e) was done to ensure that reduction in numbers of double labeled profiles in (d) was not simply an artifact of the progressive death of RGCs. Data were analyzed with one-way ANOVA followed by Tukey’s post hoc test for (d) and with student’s t-test for (e). * p < 0.05. Scale bars: 25 µm