Retrograde intraflagellar transport by cytoplasmic dynein-2 is required for outer segment extension in vertebrate photoreceptors but not arrestin translocation.
OBJECTIVE
Anterograde intraflagellar transport (IFT) is essential for photoreceptor outer segment formation and maintenance, as well as for opsin trafficking. However, the role of retrograde IFT in vertebrate photoreceptors remains unclear. The purpose of this study was to evaluate zebrafish photoreceptors lacking the retrograde IFT motor, cytoplasmic dynein-2.


METHODS
Morpholino oligonucleotides against the heavy chain (dync2-h1), light intermediate chain (dync2-li1), and intermediate chain (dync2-i1) subunits of cytoplasmic dynein-2 were injected into zebrafish embryos. Retinas and ciliated cells of these zebrafish morphants were analyzed by immunohistochemistry and transmission electron microscopy. Whole-field electroretinograms (ERGs) were performed on dynein morphants at 5 to 6 days after fertilization (dpf).


RESULTS
Zebrafish lacking cytoplasmic dynein-2 function exhibited small eyes, kidney cysts, and short photoreceptor outer segments, some of which were disorganized with accumulated vesicles. Morphant photoreceptor connecting cilia were swollen, but neither opsin nor arrestin was mislocalized, although IFT88 accumulated in the distal region of the connecting cilium. Nasal cilia were shortened and displayed cytoplasmic swelling along the axoneme. Loss of cytoplasmic dynein-2 function resulted in a significant reduction in the amplitude of ERG a-, b-, and d-waves but no change in threshold response.


CONCLUSIONS
Retrograde IFT is essential for outer segment extension and IFT protein recycling in vertebrate photoreceptors. The results show, for the first time, that the dync2-i1 subunit of cytoplasmic dynein-2 is necessary for retrograde IFT. In addition, arrestin translocation does not require retrograde IFT. Finally, the ERG results indicate that loss of cytoplasmic dynein-2 reduces the photoreceptor light response.
Krock
Bryan L
BL
Department of Biology, Texas A&M University, College Station, Texas 77843, USA.
Mills-Henry
Ishara
I
Perkins
Brian D
BD
D000818:Animals; D019393:Arrestin / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000196:embryology; D056445:Cytoplasmic Dyneins / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004596:Electroretinography; D004625:Embryo, Nonmammalian / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017403:In Situ Hybridization; D007668:Kidney / Q000196:embryology; D020419:Photoreceptor Cells, Vertebrate; D021381:Protein Transport; D055214:Retinal Photoreceptor Cell Outer Segment; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000502:physiology*
D019393:Arrestin; D029961:Zebrafish Proteins; D056445:Cytoplasmic Dyneins