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Topic

Quantification of Melanopsin Gene Expression During Sablefish (Anoplopoma fimbria) Development and Spatial Mapping of Opn4m Proteins on a Newly Established Brain Atlas

Department of Biology

Date & location

• Tuesday, December 17, 2024
• 10:00 A.M.
• Clearihue Building, Room B007

Examining Committee

Supervisory Committee

• Dr. John Taylor, Department of Biology, University of Victoria (Supervisor)
• Dr. Kerry Delaney, Department of Biology, UVic (Member)
• Dr. Raad Nashmi, Department of Biology, UVic (Member)
• Dr. Caren Helbing, Department of Biochemistry and Microbiology, UVic (Outside Member)

External Examiner

• Dr. Sarah McFarlane, Department of Cell Biology and Anatomy, University of Calgary

Chair of Oral Examination

• Dr. Richard Marcy, School of Public Administration, UVic

Abstract

In this research, I investigated opsins (light-sensitive proteins with conserved sequences expressed in all vertebrates, ranging from hagfish to humans) in sablefish (Anoplopoma fimbria). Sablefish is a slow-growing teleost species that experiences a unique developmental environment, traveling to the surface from a kilometer deep in the ocean across embryonic and larval stages. This transition through drastically different light and temperature conditions, along with transparency during these stages, makes sablefish a suitable animal model to investigate opsins. Teleost fish, in particular, have a high number of opsins due to the third genome duplication event and the retention of opsins after duplication. I uncovered 38 opsins in sablefish despite the fact that they spend most of their life in the ocean’s aphotic zone. I focused on melanopsin/opn4, an evolutionarily conserved subfamily of non-visual opsins, with a well-established role in the maintenance of circadian rhythm in mammals and a possible role in regulating melatonin pathways in zebrafish. Despite these well-researched roles, melanopsin shows expression in a diversity of cell and tissue types, suggesting the possibility of other functional roles. These proteins are activated by both light and temperature, and can regulate internal clock genes in response to both these external factors. Their conservation could indicate an important role in development, and their sensory potential may allow these proteins to serve a dynamic function in regulatory control of developmental processes. I examined these genes across four distinct life stages using qPCR and identified gene expression in whole larva at 30 days post-fertilization (dpf), in the larval brain at 82 dpf, and in the brain, eye, and heart of juveniles, as well as in the brain and heart of adults. Using fluorescent immunolabeling on sablefish optic tectum sections, I localized opn4m proteins for the first time in the endfeet of radial glial cells and likely in neuroepithelial cells within the juvenile optic tectum. The immunolabeling data on juvenile sablefish eye also revealed the expression of these proteins in all layers of the retina, including outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), and ganglion cell layer (GCL). Using qPCR, I have also quantified the expression of these genes throughout sablefish development from unfertilized eggs through all embryo stages and to the oldest larval stage. Furthermore, I examined the localization of three out of five melanopsin proteins in brain and eye of the four larval stages (18,29,36, and 47 dpf) to identify the cells that express these proteins. In this study, we report changes in expression of five melanopsin genes over embryonic and larval development, from unfertilized egg to 47 dpf, as well as in response to environmental changes in light and temperature. Using fluorescent immunolabeling and immunogold labeling, we also report for the first time, the expression of opn4m proteins in proliferative neural cells, radial glial cells, and potentially astroglia in the brain over larval development. Unsurprisingly, we also found these proteins expressed in amacrine, bipolar, horizontal, and photoreceptor cells in the retina of 47 dpf sablefish, a similar pattern of expression to that observed in larval and adult zebrafish and juvenile sablefish.

While melanopsin is best known for expression in intrinsically photosensitive retinal ganglion cells (ipRGCs) and for its role in circadian rhythm entrainment, an often-neglected observation is that when first described, melanopsin was detected in X. laevis retinal photoreceptors. It has since been found in photoreceptors of reptiles and ray-finned fish. In zebrafish (Danio rerio) an immunoprobe that binds to three of the five Opn4 paralogs showed a ring-like pattern of expression in cone cell inner-segments (Davies et al., 2011). No explanation for this was offered. I revisited this unusual pattern of expression using transmission electron microscopy and we looked for evidence of melanopsin expression in photoreceptors of a distant relative, the sablefish (Anoplopoma fimbria). Immunogold labeling was observed in outer segments and in ellipsoid megamitochondria of cone cells in both species.

Additionally I created the brain atlas for early sablefish brain development to guide my immunolabeling findings in larval stages. Using hemotaxylin and eosin staining (H&E) and ultramicrotomy, I have provided a developmental series of sablefish eye and brain sections, and created the partial brain atlas. The eye and brain atlas reported here includes three- and four-micron sections (942 in total) from 18, 29, 36, and 47 days dpf sablefish. Sections start at the olfactory bulb and run to the posterior medulla. Brain atlases with similar resolution are available for only a few other fishes, including zebrafish (Danio rerio) and medaka (Oryzias latipes). This resource is developed to provide a histological perspective on opsin gene expression data, but also makes an important contribution to the understanding of neurodevelopment in ray-finned fish. Sablefish take 47 days to reach the same stage of eye and brain development as 5 dpf zebrafish. Apart from this difference in rate, development is similar to both zebrafish and medaka, despite large differences in life history, morphology and behavior.