CRISPR/Cas9 mediated mutation of the mtnr1a melatonin receptor gene causes rod photoreceptor degeneration in developing Xenopus tropicalis

Allan F Wiechmann,Marko E Horb,Teryn A Martin

doi:10.1038/s41598-020-70735-2

Allan F Wiechmann, Marko E Horb + Show 1 more

Open Access

https://doi.org/10.1038/s41598-020-70735-2

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Abstract

Nighttime surges in melatonin levels activate melatonin receptors, which synchronize cellular activities with the natural light/dark cycle. Melatonin receptors are expressed in several cell types in the retina, including the photon-sensitive rods and cones. Previous studies suggest that long-term photoreceptor survival and retinal health is in part reliant on melatonin orchestration of circadian homeostatic activities. This scenario would accordingly envisage that disruption of melatonin receptor signaling is detrimental to photoreceptor health. Using in vivo CRISPR/Cas9 genomic editing, we discovered that a small deletion mutation of the Mel1a melatonin receptor (mtnr1a) gene causes a loss of rod photoreceptors in retinas of developing Xenopus tropicalis heterozygous, but not homozygous mutant tadpoles. Cones were relatively spared from degeneration, and the rod loss phenotype was not obvious after metamorphosis. Localization of Mel1a receptor protein appeared to be about the same in wild type and mutant retinas, suggesting that the mutant protein is expressed at some level in mutant retinal cells. The severe impact on early rod photoreceptor viability may signify a previously underestimated critical role in circadian influences on long-term retinal health and preservation of sight. These data offer evidence that disturbance of homeostatic, circadian signaling, conveyed through a mutated melatonin receptor, is incompatible with rod photoreceptor survival.

Highlights

Circadian rhythms synchronize to the natural solar cycle via photoreceptive cells in the retina of the eye that detect radiant light energy and convey that information to inner retinal cells, some of which project to the brain
We examined the impacts of disrupted melatonin receptor signaling on retinal cell function and viability, by mutating mtnr1a receptor gene expression of the Western clawed frog Xenopus (Silurana) tropicalis
To examine the role of melatonin receptor signaling on retinal circadian functions, we mutated the mtnr1a melatonin receptor gene of X. tropicalis using CRISPR/Cas[9] and generated heterozygous F1 progeny, and analyzed their retinal morphology

Summary

Introduction

Circadian (daily) rhythms synchronize to the natural solar cycle via photoreceptive cells in the retina of the eye that detect radiant light energy and convey that information to inner retinal cells, some of which project to the brain. The increased vulnerability of photoreceptors to light damage during subjective n ight[7] is consistent with reports that melatonin receptor activation exacerbates light-induced rod cell death[8,9]. Together, these studies support the overarching hypothesis that unabated activation of melatonin receptors is detrimental to long-term photoreceptor health. These studies support the overarching hypothesis that unabated activation of melatonin receptors is detrimental to long-term photoreceptor health Another key role of retinal melatonin is to synchronize the daily shedding of photoreceptor outer segment distal tips and their phagocytosis by the adjacent retinal pigment epithelial (RPE) cells[10]. The early studies by Bagnara and co-workers with X. laevis tadpoles confirmed melatonin as the skinblanching signal of the pineal gland in vivo[25,26,27]

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