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R. A. Gorsuch, D. R. Hyde; Investigating the Role of MMP2 in Retinal Regeneration. Invest. Ophthalmol. Vis. Sci. 2010;51(13):730.
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In response to light-induced photoreceptor apoptosis, the zebrafish retina undergoes a robust regenerative response that results in complete recovery of lost or damaged neurons. This response initiates with a subset of Müller glia dividing asymmetrically to produce a transiently amplifying neuronal progenitor cell (NPC) population. NPCs then migrate from the inner nuclear layer to the outer nuclear layer, where they differentiate into new rod and cone photoreceptors. Currently, little is known about the basic cell biology of migrating NPCs or of the factors necessary for proper migration. Studies from mouse models of retinal regeneration suggest that matrix metalloproteinase (MMP) family members are required for proper migration of NPCs. Additionally, microarray experiments performed by our lab revealed that several mmp family members, including mmp2, significantly increase in expression during the regeneration time-course. This study aims to identify components necessary for proper NPC migration in the regenerating zebrafish retina.
Real-time quantitative PCR was used to confirm the increased expression of mmp2 during the constant intense light treatment. To determine the spatial and temporal expression of MMP2 protein during regeneration, immunohistochemistry (IHC) was performed on light-damaged retinas using anti-MMP2 antibodies. MMP2 expression levels were also analyzed via immunoblots of light-damaged retinal lysates. Morpholino-mediated knockdown of MMP2 was used to determine the role of MMP2 in retinal regeneration.
MMP2 expression was observed primarily around the soma of dividing Müller glia by 31 hr of light. By 68 hr of light, stronger MMP2 expression was apparent and surrounded clusters of NPCs that were beginning to migrate to the ONL. Western blot analysis confirmed that MMP2 expression levels increased steadily during the regeneration time-course, reaching a maximum at 68 hr of constant light treatment. Immunolocalization and immunoblots of the MMP2 protein confirmed that electroporation of the anti-mmp2 morpholino efficiently knocked down MMP protein expression during the constant intense light treatment.
MMP2 expression is strongly upregulated both at the transcript and protein level during the regeneration time-points when many NPCs appear to begin migrating to the ONL. Furthermore, MMP2 seems to localize very strongly to columns of migrating NPCs. These data provide the first evidence that the MMP family of proteases may be necessary components for proper cell migration in the regenerating zebrafish retina.
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