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Nicholas Silva, Laura Kakuk-Atkins, Milad Ashrafzadeh, Peter Hitchcock; Mmp-9 regulates proliferation and photoreceptor regeneration in the zebrafish. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5569.
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© ARVO (1962-2015); The Authors (2016-present)
Müller glia serve as resident stem cells in the retina of the zebrafish. Photoreceptor death activates a complex network of signaling molecules expressed by dying neurons, surviving neurons, and Müller glia. Gene array data show that matrix metalloproteinase 9 (mmp-9) is strongly upregulated after a photolytic lesion (Calinescu et al., 2009). mmp-9 has many functions following nervous tissue injury, however, the expression and function of Mmp-9 during photoreceptor regeneration are unknown.
qPCR was used to quantify the expression of mmp-9 and genes encoding components of the inflammatory response, nfk-a, tnf-b, il-8, nfkb1 and nfkb2, at 8, 16, 24, 48, 72, 120, and 168 hours post lesion (hpl). Mitotically active cells were labeled with BrdU between 24 and 48hpl. In situ hybridization for mmp-9 was performed on Tg[gfap:EGFP]mi2002 transgenic zebrafish followed by immunohistochemistry for BrdU and GFP. Zymogen assays were performed with protein lysates to evaluate Mmp-9 catalytic activity. To study Mmp9 function, CRISPR/Cas9 gene editing was used to create loss-of-function mutants. Images were collected using confocal microscopy and BrdU- and PCNA-labeled cells were counted.
Photolytic lesions strongly induce the expression of mmp-9, tnf-a, tnf-b, il-8, nfkb1 and nfkb2. The expression of these genes peaks at 24hpl, which corresponds to when Müller glia re-enter the cell cycle. In situ hybridizations show that throughout this time course, mmp-9 is expressed by Müller glia and Müller glia-derived photoreceptor progenitors. Zymogen assays show that in wildtype fish between Mmp-9 is catalytically active. In contrast, there is no catalytic activity associated with mmp-9 mutants. Further, compared to wildtype fish, mmp-9 mutants have significantly more BrdU-labeled cells in the inner nuclear layer following photoreceptor death. This hyperproliferation within the inner nuclear layer at 72hpl is evident at 168 hpl by an increase in the number of BrdU-labeled cells within the outer nuclear layer.
Our results show that photoreceptor death induces the expression of genes encoding inflammatory molecules, and the expression of mmp-9 is restricted to Müller glia and photoreceptor progenitors. Photolytic lesions induce hyperprolifieration in mmp-9 mutants, demonstrating a critical role for mmp-9 in regulating injury-induced proliferation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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