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Abbie M. Jensen, John Willoughby; Generation Of A Genetically Encoded Measure Of Outer Segment Renewal In Zebrafish Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4404.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptors are morphologically specialized cells with distinct functional compartments. The rod outer segment compartment is a modified cilium that contains phototransduction machinery and intramembraneous discs packed with photon-capturing Rhodopsin. Photoreceptors have the remarkable ability to shed and renew the outer segment; the tips of outer segments are shed and are phagocytosed by neighboring RPE cells. Little is known about the cellular and molecular mechanisms that control outer segment shedding and renewal, such as what determines how much outer segment is made each day and what is the composition of the machinery that adds the new material. We are interested in indentifying the genes and cellular mechanisms that control outer segment homeostasis through the processes of shedding and renewal.
We created a new genetic tool to help identify the genes and cellular mechanisms involved in shedding and renewal. We generated a stable transgenic zebrafish line that expresses a transmembrane-bound mCherry (TM-mCherry) construct under the control of the Hsp70 promoter.
Within hours after heat shock, we observed high levels of TM-mCherry in the plasma membrane of the cell body, inner segment and base of the outer segment of all photoreceptors. At later time points, we observed a stripe of red fluorescence in rod outer segments, diffuse fluorescence in cone outer segments, and very low fluorescence in the plasma membrane of photoreceptor cell bodies and inner segments. With time, the stripe of red fluorescence was displaced apically/distally. We measured rod outer segment growth rates in young larvae and adult fish. In young larvae (3-8 days post fertilization), rod outer segments grow quickly (~3.6 microns/day) and shedding begins after 8 days post fertilization. In adults, rod outer segments grow about 1.5 microns/day and the outer segment is renewed in about 15 days.
With this new tool we can address our long-term goal to identify genes and molecular pathways involved outer segment homeostasis by quantitatively measuring rates of outer segment growth. Ultimately, we will develop computational models of outer segment size as a function of rates of growth and shedding.
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