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Anand Swaroop, Dustin Thad Whitaker, Soo-Young Kim, Passley Hargrove, Hannah Fann; Molecular determinants of photoreceptor pre-synapse morphogenesis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2598. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In neuronal circuits, precise morphology and connectivity are essential to proper functionality and transmission of signals. Retinal photoreceptors provide an excellent model to study circuit formation as the two classes provide similar information but have different structures; this enables us to identify a gene pool that controls presynapse morphogenesis.
We identified 720 genes by filtering developmental transcriptomes of wild type rods and Nrl-knockout cone-like cells with ChIP-seq data for NRL and CRX. We selected ~10% of the genes (78 genes) by G.O. analysis and manual curation. After in vivo electroporation of RNAi and reporter constructs on postnatal day (P)1, the retinas were collected, sectioned, and imaged at P21. Rescue experiments were performed by co-injecting altered cDNA, where shRNA binding site was changed, with the shRNA and measured as above. Analysis of wild type and knockout littermates was performed as above with only a reporter construct.
We analyzed two features of the rod synaptic terminal, the spherule, after RNAi knockdown. Of 78 assayed genes, knockdown of seven (9%) resulted in increased size of spherules, whereas 25 genes (32%) showed significantly different position of the spherule in the outer plexiform layer (OPL). Five genes controlled both spherule size and synapse terminal location, and the knockdown resulted in a more cone-like phenotype. Reintroduction of 6/7 cDNAs alongwith the shRNA recovered the original spherule morphology, validating that the targeted gene caused the original screen phenotype. We also obtained knockout mouse models for five genes. All three mutants that were analyzed for synaptic terminal morphology demonstrated results similar to our electroporation screen.
We have identified at least 27 genes that appear to control the morphology and/or connectivity of rod spherules during retinal development. We are currently constructing a protein interaction network to identify other molecules that might be involved in pre-synapse morphogenesis. Our studies provide the foundation for elucidating the architecture of synaptic circuits in the OPL of the mammalian retina and should help in stem cell-based photoreceptor replacement therapies for retinal degeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Distinct presynapse morphology of rod spherules (green) and cone pedicles (blue)
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