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Inge Van Hove, Els Janssens, Djoere Gaublomme, Kim Lemmens, Lieve Moons, Research Group Neural Circuit Development and Regeneration; Functional interactions between Mmp-2 and Mmp-14a during axonal innervation in the developing and regenerating zebrafish optic tectum. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3056.
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Matrix metalloproteinases (MMPs) cleave structural elements of the extracellular matrix and many molecules involved in signal transduction. Although an involvement of MMPs in the proper development and regeneration of the optic circuit has been sporadically reported, their role in pathfinding of retinal ganglion cell (RGCs) axons and in tectal (re)innervation are still largely unknown. Therefore, elucidating the contribution of Mmps in formation of retinotectal projections in the developing and regenerating visual system of zebrafish might unravel novel molecules, able to support mammalian CNS (re)innervation.
In situ hybridization (ISH), immunohistochemistry (IHC) and/or Western blotting were used to investigate Mmp-2 and Mmp-14 expression in the retina and optic tectum (OT) during tectal (re)innervation in zebrafish embryos and in regenerating adult zebrafish after optic nerve crush (ONC). Antisense morpholinos for both MMPs were applied to unravel their functions and Western blotting was used to investigate possible interactions between both Mmps in retinotectal development.
In developing zebrafish, ISH and IHC revealed expression of Mmp-14 in RGC axons and in the neuropil of the OT. Mmp-2 mRNA and protein were also found in the OT, more specifically in interneurons of the stratum periventriculare (SPV) and in the stratum fibrosum et grisum superficiale (SFGS) of the tectal neuropil. Knockdown of Mmp-14a with specific ATG or splice morpholinos (MOs) resulted in a reduced RGC axon innervation of the OT. Mmp-2 and Mmp-14a double knockdown, performed with suboptimal doses of ATG MOs, showed a significantly decreased tectal area, as compared to embryos after single Mmp-14a knockdown. Importantly, active Mmp-2 levels were reduced in Mmp14a knockdown embryos, indicating that, also in zebrafish, Mmp14a contributes to Mmp2 activation. After ONC in adult zebrafish, preliminary data revealed Mmp-14 downregulation during the axon outgrowth phase. However, when tectal reinnervation occurs, Mmp-14 levels were found to be upregulated in RGC axons. Currently, we are further investigating whether both Mmps are involved in tectal reinnervation after ONC.
Overall, these findings suggest a functional link or co-involvement between Mmp-2 and Mmp-14 in RGC axonal (re)innervation of the OT in the developing and regenerating zebrafish brain.
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