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

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. 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. Cyclic oscillations of intracellular activity occur in many cells of the body, and are especially robust in retinal cells since they need to anticipate the 12 magnitudes in ambient light intensity changes that can occur over a 24-h period 1. The key circadian signaling molecule of darkness, melatonin, is produced by retinal photoreceptors at nighttime to drive circadian events in the eye and is secreted into the cerebrospinal fluid and general circulation from the pineal gland 2. Melatonin binds to integral membrane G protein-coupled receptors (GPCRs) expressed in target cells throughout the body to harmonize cellular activities with the predictable 24-h day/night cycle 3. Melatonin receptor subtypes are designated as Mel1a, Mel1b, and Mel1c in amphibians, fish and birds 4 , and as MT1, MT2, and GPR50 (which does not bind melatonin) in mammals, respectively 3. One primary function of melatonin in the retina is to enhance visual sensitivity to light at nighttime 5. If the solar light cycle is disengaged from circadian cellular readiness, the metabolic imbalance imposed on darkadapted photoreceptors by radiant light energy causes cell damage 6. The increased vulnerability of photoreceptors to light damage during subjective night 7 is consistent with reports that melatonin receptor activation exacerbates light-induced rod cell death...

CRISPR/Cas9-Mediated Models of Retinitis Pigmentosa Reveal Differential Proliferative Response of Müller Cells between Xenopus laevis and Xenopus tropicalis

“…Four of them led to frameshifts (probably resulting in truncated proteins or nonsense-mediated decay), and four others generated amino acid deletions ( Figure 1 B). This percentage is similar to previous CRISPR/Cas9 reports in X. tropicalis [ 12 , 16 , 17 ] and reveals an efficient rho gene editing in this species using this technology.…”