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Jingling Li, Josh Hobgood, David R Hyde; ADAM17a and ADAM17b perform different functions to initiate zebrafish retinal regeneration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):415.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated that repressing Notch signaling and expressing TNFα are necessary and sufficient to induce Müller glial proliferation in the intact zebrafish retina (Conner et al., 2014). Activation of both TNFα and Notch require processing cleavage by the A Disintegrin And Metalloproteases (ADAM) protein family. We tested the hypothesis that two ADAM17 paralogs, ADAM17a and ADAM17b, function during regeneration by processing either TNFα or Notch to initiate Müller glia dedifferentiation and proliferation.
Morpholino-mediated knockdown of either ADAM17a or ADAM17b expression were done using albino Tg[gfap:EGFP] zebrafish, followed by constant light damage treatment as previously described (Vihtelic and Hyde, 2000). Standard control morpholino (S.C. MO) was used as the control. TNFα protein was intravitreally injected to rescue the morphant phenotype. Intraperitoneal injection of the γ-secretase inhibitor RO4929097 into morphant zebrafish was performed to block Notch processing. Eyes for immunostaining were formaldehyde fixed and cryosectioned. The proliferating Müller glia were labeled by PCNA and examined using confocal microscopy. PCNA-positive cells or cell clusters in the inner nuclear layer (INL) were then counted and analyzed using Student’s t-test for significant differences.
Both adam17a and adam17b morphant retinas had significantly fewer (p<0.01) number of proliferating Müller glial cells relative to the standard control morphant (S.C. MO, 103.9±8.0; adam17a MO, 15±5.1; adam17b MO, 16.3±4.1). Intravitreal injection of TNFα protein rescued Müller glial proliferation in adam17b morphant eyes (106.3±8.8), but not in adam17a morphant eyes (6.5±1.6). The number of PCNA-positive cell clusters also significantly increased (p<0.01) in adam17b morphant retinas after TNFα injection. Inhibiting Notch signaling induced Müller glial proliferation in both adam17a (63.9±6.5) and adam17b (80.3±4.9) morphant retinas, but without continued progenitor cell proliferation.
Our results suggest that both ADAM17a and ADAM17b are required for initiation of zebrafish retinal regeneration and serve different functions. ADAM17b is necessary for TNFα processing and activation. Neither ADAM17a nor ADAM17b cleave the Notch receptor in Müller glia quiescence. The function of ADAM17a remains unclear.
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