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Research Presenter ~ Bridgett K. Kelly
Research Presenter ~ Bridgett K. Kelly
Department of Biology, Miami University
About Me: My name is Bridgett Kelly, I am from Canfield, Ohio and I am a Junior this year at Miami University. I am a double-major in Biology and Psychology and I have two co-majors in Neuroscience and Pre-Medical Studies. After graduating next spring, I plan to attend medical school to become a physician.
Mentors: Erika Grajales-Esquivel, Dr. Katia Del Rio-Tsonis
Background
Background
Today, 1 million Americans 40 years and older are blind, with another 39 million living with vision impairment. The main causes of age-related vision impairment are eye diseases such as macular degeneration, glaucoma, and cataracts (1). One of the main tissues damaged by these conditions is the retina, the part of the eye that turns visual stimuli into neural impulses to be sent to the brain and allow vision. Unfortunately, humans have limited ability to regenerate tissues and organs that have been damaged. Other vertebrates, like newts, have the miraculous ability to regenerate several parts of their body throughout their lifespan, including limbs, tail, brain, spinal cord, heart, retina, and lens (2-6). Studying the mechanisms by which newt retina regeneration occurs will provide us with knowledge that can lead to having the ability to induce regeneration of the retina in other animals, particularly humans.
In the Spanish newt, the mechanism of retina regeneration begins with the cells of the retinal pigmented epithelium (RPE) located in the most posterior part of the eye. This layer of cells dedifferentiates to become neural progenitor cells that proliferate and eventually reprogram into the several cell types present in the retina: ganglion, amacrine, horizontal, bipolar, muller glia, and photoreceptors, as well as a new RPE cells monolayer.
Research Questions and Rationale
Research Questions and Rationale
This project investigates the regeneration process of the retina in the Spanish newt. In this organism, the mechanism of retina regeneration begins with the cells of the retinal pigment epithelium (RPE) located in the most posterior part of the eye. This layer of cells dedifferentiates into neural progenitor cells that proliferate and eventually reprogram into the several cell types present in the retina: ganglion, amacrine, horizontal, bipolar, Muller glia and photoreceptors.
This research aims to answer: How long does it take for the retina to fully regenerate? At what points in the regeneration process are cells proliferating and reprograming? Once the retina has been regenerated, what cell types are present?
Methods
Methods
Figure 1: Optimized surgery method to remove the retina.
Figure 2: Experimental procedure to characterize regenerated retina.
Results
Results
Phase 1: Visualization of retina regeneration process on the eight-month-old newt via histology and EdU staining.
Phase 1: Visualization of retina regeneration process on the eight-month-old newt via histology and EdU staining.
Figure 3: Histological sections of Newt eyes at consecutive weeks after the optimized retinectomy procedure. At 2 weeks post retinectomy, some depigmentation of the RPE occurs, but is not fully observable until a week later. After 8 weeks post retinectomy, the regenerated retina has been fully formed.
Figure 4: Confocal images of EdU staining the S-phase of the cell cycle. Weeks 2 and 3 show RPE cells that have incorporated EdU into newly synthesized DNA. At week 8, only the ciliary margin shows EdU incorporation, the regenerated retina contains newly differentiated neural cells that are not proliferating. DAPI was used as a counterstain to visualize nuclei (blue).
Phase 2: Characterization of the regenerated retina in three-and-a-half-month-old newts via immunofluorescence staining.
Phase 2: Characterization of the regenerated retina in three-and-a-half-month-old newts via immunofluorescence staining.
Figure 5: Confocal images of immunofluorescence staining of neural epithelium (NE) collected 20 days post retinectomy. DAPI (blue) stains the nuclei, RPE65 (red) on the top panel marks the presence of Retinal Pigment Epithelium, and PAX6 (red) on the bottom panel marks the presence of neural progenitor cells. At 20 dPR, the RPE is almost fully reformed and there is a distinct layer of neural progenitor cells.
Figure 6: Confocal images of immunofluorescence staining of neural retina (NR) collected 30 days post retinectomy. DAPI (blue) stains the nuclei, PAX6 (red) on the top panel marks the presence of ganglion, amacrine, and horizontal cells, AP-2 (magenta) in the bottom panel specifically marks the presence of amacrine cells. At 30 dPR, the neural retina contains ganglion, horizontal, and amacrine cells.
Figure 7: Confocal images of immunofluorescence staining of neural retina collected 40 days post retinectomy. DAPI (blue) stains the nuclei, PAX6 (red) in the top panel mark ganglion, amacrine, and horizontal cells, AP-2 (magenta) in the middle panel specifically marks amacrine cells, and Vimentin (green) in the bottom panel marks Muller glial cells. At 40 dPR, the neural retina has almost fully formed and is laminated.
Future Directions
Future Directions
To have a more comprehensive picture of this regenerative process, different immunofluorescent markers need to be used to observe the presence of other cell types, including photoreceptors and bipolar cells. Another way to characterize the retina is by observing the mRNA present in the cells via HCR-FISH. In addition, single-cell RNA sequencing needs to be done to understand the molecular changes associated with physical changes that are happening in the retina. With the results of future experiments, a comprehensive atlas of the retina regeneration process of the Spanish newt can be created and used as a reference for all.
Career Readiness Skills
Career Readiness Skills
Conducting this research has strengthened my ability to gather and analyze new information, problem-solve, and anticipate the needs of my team as I have learned and utilized several techniques to investigate this biological process. I have also learned to work in a fast-paced lab setting and effectively communicate with my team members and mentors in order to stay on track to meet our goals. Presenting my research to people with diverse educational backgrounds has taught me to organize and communicate my findings in a way that anyone can understand.
Acknowledgments
Acknowledgments
Center for Visual Sciences at Miami University (CVSMU)
Dr. Katia Del-Rio Tsonis: Principal Investigator
Erika Grajales-Esquivel: Research Associate
KDRT Lab Members
Animal Care Facility
References
References
Centers for Disease Control and Prevention. (n.d.). Fast facts of common eye disorders. U.S. Department of Health & Human Services. https://www.cdc.gov/visionhealth/basics/ced/fastfacts.htm
Garza-Garcia, A., Driscoll, P. C., & Brockes, J. P. (2010). Evidence for the Local Evolution of Mechanisms Underlying Limb Regeneration in Salamanders. Integrative and Comparative Biology, Vol. 50, pp. 528–535.
Tsonis, P. A., & Del Rio-Tsonis, K. (2004). Lens and retina regeneration: transdifferentiation, stem cells and clinical applications. Experimental Eye Research, 78(2), 161–172.
Kirkham, M., & Joven, A. (2015). Studying newt brain regeneration following subtype specific neuronal ablation. Methods in Molecular Biology , 1290, 91–99.
Zhang F., Clarke J. D., Santos-Ruiz L., Ferretti P.. 2002, Differential regulation of fibroblast growth factor receptors in the regenerating amphibian spinal cord in vivo, Neuroscience, 114, 837–48
Matsunami, M., Suzuki, M., Haramoto, Y., Fukui, A., Inoue, T., Yamaguchi, K., … Hayashi, T. (2019). A comprehensive reference transcriptome resource for the Iberian ribbed newt Pleurodeles waltl, an emerging model for developmental and regeneration biology. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 26(3), 217–229.
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