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Jingling Li, David R Hyde; ADAM17b performs functions through TNFα signaling to initiate zebrafish retinal regeneration. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
Tumor necrosis factor α (TNFα) is a known positive regulator in zebrafish retinal regeneration. However, the activation and upstream regulator of TNFα signaling remains unclear. We tested the hypothesis that the A Disintegrin And Metalloproteases 17b (ADAM17b) functions during regeneration by processing TNFα to initiate Müller glia proliferation.
Morpholino-mediated knockdown of ADAM17b expression was performed on one-year-old adult albino Tg[gfap:EGFP] zebrafish, followed by constant light treatment to damage the photoreceptors. Standard control morpholino (S.C. MO), which lacks any known complementary sequence in zebrafish genome, was used as the technical control. Eyes from each group (N=15) were cryosectioned (14µm), immunostained with a monoclonal antibody to proliferating cell nuclear antigen (PCNA), and imaged by confocal microscopy (z-stack image, 10µm). Intravitreal injections of zebrafish recombinant TNFα protein (1 mg/ml) were done to rescue the morphant phenotypes. Both Tg[ath5:GFP] and Tg[olig2:GFP] adult fish (N=9) were used to examine the Müller glia-derived neural progenitors. The numbers of PCNA or GFP-positive cells were quantified and statistically analyzed using either the Student’s t-test or one-way ANOVA and a Tukey’s post hoc test.
The adam17b morphant retinas had significantly fewer (p<0.0001) numbers of proliferating Müller glial cells relative to the S.C. MO retinas (adam17b MO, 16.3±4.1; S.C. MO, 103.9±8.0). Interestingly, intravitreal injection of TNFα protein rescued Müller glial proliferation in adam17b morphant eyes (106.3±8.8). However, these rescued proliferating cells failed to commit to a neural cell fate. Injection of TNFα into adam17b morphants significantly increased the number of Müller glia-derived neural progenitors (ath5:GFP, 9.4±2.5; olig2:GFP, 29.6±2.8) relative to the adam17b morphant retina (ath5:GFP, 2.3±2.4; p<0.011; olig2:GFP, 3.9±1.6; p<0.001), but remained significantly lower relative to the S.C. morphant retinas (ath5:GFP, 42.3±5.7; olig2:GFP, 44.1±2.9; p<0.002).
Our results suggest that ADAM17b is required for initiation of zebrafish retinal regeneration. Consistent to our hypothesis, ADAM17b is necessary for TNFα processing and activation during Müller glia proliferation. However, ADAM17b may have other substrates and also play an important role in progenitor cell fate commitment.
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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