Expression of IFT Proteins in Vertebrate Rod Photoreceptors

Abstract

Abstract: : Purpose: Intraflagellar transport of a 17S particle containing at least 15 distinct polypeptides (IFTs) has been shown to be important for the assembly and maintenance of cilia and flagella. Mice with mutations or deletions in the gene for IFT 88 (Tg737/polaris) show photoreceptor degeneration, suggesting a role for IFT in maintaining vertebrate rod photoreceptors, which contain a modified cilium linking the inner and outer segments. Using Xenopus laevis and Danio rerio (zebrafish) as model systems, we have tested this hypothesis by analyzing the expression of the endogenous proteins, and by the rod-specific overexpression of IFT proteins in transgenic animals. Methods: To examine the expression and localization of endogenous IFT proteins, Western blotting and indirect immunofluorescence were carried out on rod photoreceptor outer segments isolated from adult Xenopus, using rabbit antisera directed against IFT 20, 52, 57 and 88. For rod-specific overexpression of GFP fusions of these four IFT proteins, transgenic Xenopus and zebrafish embryos were made using species-specific rod opsin promoters. Transgenesis was confirmed by genomic PCR. Fluorescent specimens were observed by Hoffman modulation contrast microscopy, wide-field epifluorescence miscroscopy, confocal microscopy and multiphoton microscopy. Some transgenic embryos were fixed and embedded for frozen sectioning or electron microscopy. Results: Immunofluorescence showed that endogenous IFT 20, 52, 57 and 88 were localized in discrete particles on the connecting cilium and axoneme of Xenopus laevis rod photoreceptors. When overexpressed in transgenic Xenopus or zebrafish, IFT 52-GFP and IFT 57-GFP were concentrated at the connecting cilium and were present in the axoneme of rod outer segments. IFT 20-GFP formed large aggregates in the inner and outer segments with no apparent localization to the connecting cilium. IFT 88-GFP expression was almost never detected in whole embryos of either species, although transgenesis was confirmed by PCR of genomic DNA. Frozen sections of Xenopus embryos showed rare cells that expressed low levels of IFT 88-GFP concentrated at the connecting cilium. Preliminary light and electron microscopic examination of these retinas showed pyknotic nuclei and malformed outer segments not observed in the retinas of embryos expressing GFP alone. Conclusions: Our data demonstrate targeting of IFT proteins to cilia in rod cells and suggest that overexpression of IFT 88-GFP can lead to retinal degeneration.

Keywords: photoreceptors • cytoskeleton • microscopy: confocal/tunneling