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Leah Middleton, Ralph F Nelson; Transgenic trβ2 expression does not induce full red cone morphology despite inducing red cone physiology. Invest. Ophthalmol. Vis. Sci. 2018;59(9):584.
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Thyroxin β2 nuclear receptor (trβ2) is necessary for expression of red opsin in long-wavelength cones (Ng et al., 2001). The four cone types in zebrafish (red, green, blue, and UV) are distinguished both by opsin expression and by shape. We examine whether trβ2 also determines the characteristic "long-single" red cone shape using transgenics which express trβ2 in all cone types.
Two, gain-of-function, zebrafish transgenics express trβ2 either in all cone progenitors (crx:mYFP-2A- trβ2), or in mature cones of all spectral types (gnat2:mYFP-2A- trβ2). Transgenic zebrafish larvae were raised at 28°C in 300µM PTU to prevent pigment development. At 6dpf, larvae were mounted in 1.5% agarose in an 8-chamber slide. Right eyes were imaged in z-stacks on a Zeiss 880 confocal at either 25x or 40x magnification at 1024 x 1024 pixel resolution. Cone cell body width, length of axonal projection, and cone density were measured in ImageJ. Differences were evaluated with one-way ANOVA and Tukey’s posthoc test.
Cones with trβ2 transgenes were greater in cell body width and shorter axon length than wildtype red cones. crx:mYFP-2a-trβ2 cones are narrower than wildtype (WT) UV cones but similar in width to WT blue cones; gnat2:mYFP-2a-trβ2 cones are narrower than both UV and blue cones. Axons of cones in both transgenics are shorter than those of WT red cones, similar in length to WT UV and blue cones. The densities of gnat2 and crx transgenic cones, promoters we anticipated having expression in all cones, are significantly less than a calculated estimate of total WT cone density. Labeled cones of transgenic animals are unevenly distributed through the cone layer.
Despite a shift towards both red opsin expression and red cone physiology in cones with crx-driven trβ2 expression, the cones are morphologically distinct from WT red cones. trβ2, when expressed in all mature cones with gnat or crx promoters, is therefore not sufficient to induce full red cone, or "long single," morphology, but must play a role in morphology because trβ2 cones lack resemblance to any type of WT cone. The reduction in cone density detected in the trβ2 transgenics needs further investigation. Nonetheless it is clear that cones with transgenic expression of trβ2 show significantly altered morphology, and that trβ2 plays a role not only in red-opsin expression, but in the development of cone shape.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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