RPE degeneration precedes photoreceptor degeneration in rpgra−/− zebrafish. (A,B) Immunostaining of the retinas (A) and flat mounted eyecups (B) from 2 mpf zebrafish using an antibody against Rpgra (green). Scale bar = 30 µm. (C) Immunofluorescence analysis of flat-mounted eyecups from 1.5, 2, and 4 mpf zebrafish using the anti-ZO-1 antibody (green). We defined cells exhibiting loss of polygonal cellular architecture and junctional integrity, or those with irregular nuclear morphology, as abnormal RPE cells. The proportion of RPE cells with abnormal morphology was quantified by manual counting. White arrows indicate the aberrant RPE cells. Scale bar = 30 µm. (D) The number of abnormal RPE cells were quantized for each of the images (n = 8). (E) Hematoxylin and eosin analysis of cryosections in WT and rpgra−/− zebrafish at 2, 4, and 8 mpf. White lines indicate the length of photoreceptor outer segments. Scale bar = 30 µm. INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment epithelium. (F) The quantitative results of the thickness of OS layer and ONL layer. Three parallel samples were tested for each group (n = 3 biological replicates). Data were indicated as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
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