Endogenous Opsin 3 (OPN3) Protein Expression in the Adult Brain Using a Novel OPN3-mCherry Knock-In Mouse Model

Abstract

The opsins have been studied extensively for their functions in visual phototransduction; however, the mechanisms underlying extraocular opsin signaling remain poorly understood. The first mammalian extraocular opsin to be discovered, opsin 3 (OPN3), was found in the brain more than two decades ago, yet its function remains unknown. A significant hindrance to studying OPN3 has been a lack of specific antibodies against mammalian OPN3, resulting in an incomplete understanding of its expression in the brain. Although Opn3 promoter-driven reporter mice have been generated to examine general OPN3 localization, they lack the regulated expression of the endogenous protein and the ability to study its subcellular localization. To circumvent these issues, we have created a knock-in OPN3-mCherry mouse model in which the fusion protein OPN3-mCherry is expressed Significance Statement Before the current report, there had been a significant lack of encephalic opsin 3 (OPN3) protein characterization, likely driven by the absence of murine OPN3 antibodies. This study describes a novel OPN3-mCherry knock-in mouse that we used to analyze endogenous OPN3 protein expression across the brain. We have uncovered new aspects of OPN3: localization to previously undocumented brain subregions, expression in GABAergic neurons and non-neuronal cells, and punctate subcellular localization in the soma. Our OPN3 expression map is an invaluable step toward discovering its elusive encephalic functions. The OPN3-mCherry mouse will facilitate investigation of OPN3 not only in the brain, but also across the entire organism, a useful feature as OPN3 is emerging as a possible mediator of phototransduction outside the brain.

Distinct Opsin 3 (Opn3) Expression in the Developing Nervous System during Mammalian Embryogenesis

“…Notably, our study identified 25 new Opn3 -eGFP + structures in the CNS and PNS, including sensory ganglions, the olfactory epithelium, vestibular nuclei and parafascicular nucleus, that have not been previously reported to express Opn3 ( Table 1 ). The analyzed Opn3 -eGFP knock-in mouse line possesses two intact Opn3 wild-type alleles that are consistent with an observed normal phenotype, which is in contrast to the recently developed Opn3::mCherry fusion protein mouse line ( Olinski et al, 2020b ). Being under the influence of the native Opn3 promoter, both detected eGFP and Opn3::mCherry expression patterns appear to mirror the cellular expression profile of endogenous Opn3 from earlier reports.…”

“…Studies of adult Opn3::mCherry reporter mice have reported that Opn3 is expressed also in non-neuronal astrocytes ( Olinski et al, 2020b ). In mouse, the differentiation of astrocytes begins ∼E17–E18 ( Reemst et al, 2016 ; Matsue et al, 2018 ).…”

“…Whereas most visual and non-visual opsins appear to be expressed in more or less restricted patterns, Opn3 has been shown to be expressed in a broad range of multiple adult tissues ( Blackshaw and Snyder, 1999 ; Nissilä et al, 2012 ; Olinski et al, 2020b ). Opn3 mRNA was first discovered in the cerebral cortex and cerebellum of the adult mouse brain ( Blackshaw and Snyder, 1999 ), results that have been subsequently verified and expanded, most recently in studies using an Opn3 ::mCherry mouse model ( Olinski et al, 2020b ). Despite more detailed knowledge about the spatial expression of Opn3 in the adult mouse brain, there is a significant lack of information concerning when and where in the nervous system Opn3 is expressed during embryogenesis.…”

“…For example, Opn4 has been detected in melanocytes ( Provencio et al, 1998 ), the iris ( Xue et al, 2011 ; Sghari et al, 2020 ), cornea and associated trigeminal ganglia ( Delwig et al, 2018 ), blood vessels and heart ( Regard et al, 2008 ; Sikka et al, 2014 ), and Opn5 being found in the eye, brain, testes, ear, and skin ( Kojima et al, 2011 ; Buhr et al, 2015 , 2019 ; Haltaufderhyde et al, 2015 ). The initial identification and expression of Opn3 in the adult mouse brain, including regions like the cerebral cortex and cerebellum ( Blackshaw and Snyder, 1999 ), have been verified and extended ( Nissilä et al, 2012 ; Olinski et al, 2020b ). Recent studies in the adult mammalian brain, especially that of rodents and some primates, indicate that Opn3 exhibits a far wider expression profile ( Nissilä et al, 2012 ; Olinski et al, 2020b ), which suggests the potential for many unknown roles of light signaling in the brain and other organs.…”

“…The initial identification and expression of Opn3 in the adult mouse brain, including regions like the cerebral cortex and cerebellum ( Blackshaw and Snyder, 1999 ), have been verified and extended ( Nissilä et al, 2012 ; Olinski et al, 2020b ). Recent studies in the adult mammalian brain, especially that of rodents and some primates, indicate that Opn3 exhibits a far wider expression profile ( Nissilä et al, 2012 ; Olinski et al, 2020b ), which suggests the potential for many unknown roles of light signaling in the brain and other organs. Notably in adult vertebrates, including higher mammals, Opn3 is expressed in adipocytes and several tissues, such as, heart, intestine, kidneys, liver, ovaries, pancreas, placenta, pulmonary arteries, retina and associated eye structures, skeletal muscle, skin, testes, uterus, trachea, and lungs ( Blackshaw and Snyder, 1999 ; Halford et al, 2001 ; Tarttelin et al, 2003a ; Haltaufderhyde et al, 2015 ; Ortiz et al, 2018 ; Nayak et al, 2020 ; Olinski et al, 2020a ; Yim et al, 2020 ; Wu et al, 2021 ).…”