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Research Presenter ~ Bridgett Kelly
Research Presenter ~ Bridgett Kelly
Katia Del Rio-Tsonis Regeneration Lab, Department of Biology, Miami University
About Me: My name is Bridgett Kelly, I am from Canfield, Ohio and I am a Senior 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 this spring, I will apply to medical school to pursue a career as a physician.
Mentors: Erika Grajales-Esquivel and 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 Question and Rationale
Research Question 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?
This project also aims to explore a new protocol for characterization of the Spanish newt retina: HCR-FISH. In the last two years, I and other members of the KDRT lab have worked to optimize this protocol including the tissue retrieval, probe design, and solution administration to the sectioned tissues.
Methods
Methods
Figure 1. Procedures for retina characterization. EdU is administered to observe the proliferation of cells at different timepoints, as it stains cells in the beginning of the cell cycle. Hematoxylin and Eosin staining allows us to look at tissue morphology, allowing visualization of the regenerated structure. Immunofluorescence staining uses antibodies to localize proteins within the tissue, thus displays gene expression. HCR-FISH is a highly specific techniques that uses probes to identify mRNA presence in tissues, giving a clear look at gene expression in the intact newt eye.
HCR-FISH Probe Design
HCR-FISH Probe Design
As a part of optimizing the HCR-FISH protocol, our lab has developed a software to design complementary probes specifically for our experimentation. This process involves using the fully sequenced pleurodeles waltl genome on the National Institute of Health website to retrieve the desired sequence of the mRNA we were investigating. We then used multiple data bases to verify the conservation and coverage of the probe, including NCBI BLASTX, Multiple Genome Viewer, and checked the phylogenetic orthologs using SHOOT.bio. After confirming the identity of the probe, we used a software developed by members of the KDRT lab to design and order the probes.
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 2: 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 3: 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 4: 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 5: 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 6: 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.
Phase 3: Characterization of retina using HCR-FISH.
Phase 3: Characterization of retina using HCR-FISH.
Figure 7. Confocal images of HCR-FISH on intact eight-month-old newt eye. DAPI (blue)stains the nuclei and RPE65 (pink) marks the preseansce of RPE cell layer. This signal is seen in the most posterior region of the retina.
Figure 8. Confocal images of HCR-FISH of an intact eight-month-old newt eye. DAPI stains the nuclei and PAX6 (red) marks the presence of ganglion, amacrine, and horizontal cells in the neural retina.
Future Directions
Future Directions
Look at the entire regenerative process using HCR to further characterize regeneration.
Test different antibodies with immunofluorescence staining and more probes using HCR-FISH.
Start single cell RNA sequencing at different regeneration time points.
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.
Compliance
Compliance
All procedures done on experimental subjects in compliance with Institutional Animal Care and Use Committee (IACUC) and Miami University protocols.
Acknowledgements
Acknowledgements
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
Funding from Undergraduate Research Award
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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