Investigating the role of the proteoglycan IMPG2 in retinal development and function, by generation of knockout zebrafish models.

The interphotoreceptor matrix (IPM) is the extracellular compartment surrounding retinal photoreceptor outer segments and participating in their differentiation, biological activity and survival. Recent studies have reported that in humans, mutations in the interphotoreceptor matrix proteoglycan 2 (IMPG2) gene are associated with autosomal recessive Retinitis pigmentosa (arRP) and autosomal dominant Vitelliform macular dystrophy (VMD). Both of these inherited retinal dystrophies are characterized by progressive degeneration of photoreceptors and vision loss. To investigate IMPG2 function we chose zebrafish as a model organism, as its retinal structure and organization are very similar to the human macula. We analysed the evolutionary conservation of IMPG2, finding that it appears only in vertebrates. In many teleosts including zebrafish there are two paralogues, Impg2a and Impg2b. Their expression as well as their role and possible differences are not yet known. Since IMPG2 protein structure has largely been unstudied, we performed homology modelling of IMPG2 conserved domains both in human and in zebrafish, showing structure similarity of conserved domains in the two species. We analysed Impg2a and Impg2b mRNA and protein expression in zebrafish during early embryonic development and in adult fish. RT-qPCR experiments revealed that Impg2a and Impg2b are transcribed from 3 days post fertilization (dpf), accompanying photoreceptor maturation, while in adults both Impg2 paralogues have an eye-specific expression. Western blots confirm a similar expression pattern for the proteins. Furthermore, immunohistochemistry (IHC) experiments performed on retina sections showed that the expression of Impg2 is detected starting from 7 dpf, when it localizes in the photoreceptor cell bodies. Over time its localization shifts, translocating to photoreceptor outer segments by 6 months post fertilization (mpf). Microinjection of antisense morpholino oligonucleotides (MOs) specific for each of the two paralogues provided preliminary evidence that in zebrafish Impg2 is involved in eye development and retinal pigmented epithelium (RPE) pigmentation. At 5 and 7 dpf we observed abnormalities in the photoreceptor layer of embryos microinjected with the combination of the two MOs by IHC, histological analyses and transmission electron microscopy (TEM). Moreover, morphant embryos showed an increase in cell proliferation in the ciliary marginal zone at 3 dpf. Finally, we generated impg2a-/- and impg2b-/- zebrafish single mutant lines by using CRISPR/Cas9 technology. Preliminary experiments on both single mutant lines showed an alteration of the photoreceptor layer but normal electroretinogram (ERG) response at 5 dpf and 1 mpf. We plan to functionally characterize the mutant phenotypes and perform testing of therapeutic compounds on this new zebrafish model to discover a possible treatment for IMPG2-related retinopathies.