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Jared David Chrispell, D Joshua Cameron, Shoji Osawa, Ellen R Weiss; Contributions of Grk1b and Grk7a to the Cone Photoresponse in Zebrafish Larvae. Invest. Ophthalmol. Vis. Sci. 2016;57(12):575.
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© ARVO (1962-2015); The Authors (2016-present)
In the vertebrate retina, termination of the photoresponse is essential for sustained function of photoreceptor cells when exposed to repeated visual stimuli. Abrogation of visual signaling requires phosphorylation of photoactivated visual pigments in rods and cones by the G protein-coupled receptor kinases (GRKs), GRK1 and GRK7. We previously demonstrated that GRK1 and GRK7 are co-expressed in cones in humans and other vertebrates including zebrafish. Electroretinogram (ERG) analysis detects cone photoresponses as early as 4 days post fertilization (dpf), whereas responses in rods appear between 15 and 21 dpf. Therefore, the zebrafish retina at 4-7 dpf serves functionally as an all-cone retina. To determine the contribution of these GRKs to visual signaling in cones, we have generated zebrafish lines in which Grk1b and Grk7a have been selectively deleted.
Transcription Activator-Like Effector Nuclease (TALEN) genome editing was used to knock out Grk1b or Grk7a. Western blotting and immunohistochemistry were used to confirm deletion of the proteins. These larvae were evaluated by ERG for recovery in response to light using a dual flash paradigm with increasing interstimulus intervals. In addition, visual acuity was measured via the optokinetic response (OKR).
Immunoblots of 5 dpf grk7a-/- larvae demonstrate complete elimination of Grk7. Trace levels of Grk1 observed in grk1b-/- larvae are localized only in nascent rods and probably represent Grk1a expression. Deletion of grk1b or grk7a results in a significant decrease in recovery to successive stimuli in each knockout line compared to wild type larvae. This decrease in the rate of recovery was similar in grk1b-/- and grk7a-/- zebrafish larvae. The grk1b-/- larvae also exhibited a significant reduction in visual acuity.
Elimination of either Grk1b or Grk7a in zebrafish larvae results in significantly lower rates of recovery in response to successive flashes of light. This decrease was comparable for grk1b-/- and grk7a-/- larvae and suggests for the first time that Grk1b, as well as Grk7a, plays a significant role in zebrafish cone visual signal termination. Interestingly, grk1b-/- larvae also exhibited a decrease in visual acuity. Future studies will evaluate grk7a/grk1b double knockout fish as well as measurements of spatial contrast sensitivity to determine the distinct or overlapping roles of Grk1b and Grk7a in cone vision.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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