Rod Photoreceptor Degeneration in mCFP-Transgenic Zebrafish

Purpose: In zebrafish transgenic for a membrane-targeted form of cyan fluorescent protein driven by the Xenopus rhodopsin promoter (XOPS-mCFP), rods begin degenerating at 3.5 days postfertilization (dpf) and are almost completely absent in mature zebrafish. The purpose of this study was to characterize this phenomenon and evaluate its effect, if any, on cones. Methods: The XOPS-mCFP line was bred to a zebrafish line with healthy rods expressing GFP (XOPS-GFP). Rod development, morphology and degeneration in the doubly transgenic fish were examined by fluorescence microscopy. Opsin localization was studied by microinjection of DNA encoding a vectorally-sorted rhodopsin-GFP fusion protein. Apoptosis was examined by TUNEL labeling. Expression of mCFP was blocked by injection of an antisense morpholino, and survival of rods at four dpf was assessed by fluorescence microscopy. The survival and organization of cone photoreceptors in this line was examined by immunolabeling of retinal sections from adult transgenic fish. Results: The onset of rod photoreceptor development in XOPS-mCFP transgenic larvae appeared similar to that of wild-type zebrafish. However, most of the rods had an abnormal morphology, and rod cell degeneration commenced around 3.5 dpf. Opsin mislocalization was also observed in rod photoreceptors from transgenic larvae at 3.5 dpf. By five dpf, no fluorescent rods were observed in the central retina of transgenic larvae, although some morphologically abnormal rods persisted near the proliferative zone at the retinal margin. TUNEL labeling revealed increased apoptosis in the photoreceptor cell layer of 3.5 dpf transgenic larvae. In histological sections from adult transgenic retinas, some immunolabeled rods were seen at the retinal margin, and a few, scattered rods were observed across the central retina. However, none of the remaining rods had intact outer segments. Injection of an antisense mCFP morpholino into transgenic embryos increased rod survival at four dpf, suggesting that expression of the mCFP transgene was the cause of rod cell death in this line. Immunolabeling of adult sections for cone photoreceptors revealed that survival and patterning of cones is unaffected in this line. <truncated: Gene Tools database limitation>