Pcdh19 Expression in Normal and Regenerating Adult Zebrafish Retinas

My Honors Research Project will test the function of the N-cadherin molecule on the zebrafish optic nerve regeneration. N-cadherin is a member of the cadherin superfamily. Cadherins are cell adhesion molecules important for animal development and maintenance of adult structures. Unlike mammals, fish and amphibians have the ability to regenerate their optic nerve after damages. Molecular mechanisms underlying the optic nerve regeneration are still under intense investigation. Studies in Dr. Liu’s laboratory showed that expression of several cadherins, including N-cadherin, was greatly increased during adult zebrafish optic nerve regeneration, suggesting that cadherins may be involved in the optic nerve regeneration process. This idea is supported by preliminary results recently obtained in Dr. Liu’s laboratory showing that blocking N-cadherin function using electroporation of a zebrafish N-cadherin specific morpholino antisense oligonucleotide (NcadMO) severely affected the optic nerve regeneration. Cadherins affect animal development by affecting cell-cell adhesion and expression and function of other molecules. As the first step in determining the molecular mechanism underlying N-cadherin function in the optic nerve regeneration, I will examine expression of two transcription factors, KLF6a and KLF7a known to be important regulators of the optic nerve regeneration, in adult zebrafish retina with N-cadherin function blocked by the NcadhMO. Adult zebrafish (Danio rerio) will be used in my proposed experiments. The fish will be anesthetized and the optic nerve of the left eye will be crushed using a fine-tipped forceps. In one group of animals, the NcadMO will be electroporated into the left eye one day after the optic nerve crush. In another group of fish, a control MO (standard control MO that does not bind to N-cadherin mRNA) will be electroporated to the left eye also one day after the optic nerve crush. The animals will be allowed to survive for 4 or 7 days. After those few days, the fish will be anesthetized, and the left eye will be dissected out quickly, and fixed in 4% paraformaldehyde. After washing with phosphate buffered saline, the tissues will be cryoprotected (20% sucrose), embedded, sectioned using a cryostat and processed for in situ hybridization to assess expression of KLF6a and KLF7a mRNA expression. My hypothesis is that if N-cadherin controls the optic nerve regeneration via regulating KLF6a and KLF7a expression, their mRNA expression levels will be decreased in the retinas treated with the NcadMO compared to the retinas treated with the control MO. By pursuing this project, I intend to gain a broader understanding of the organization of the vertebrate retina and the basis of the nervous tissue regeneration. Specifically, using a model species such as the zebrafish, I hope to learn how N-cadherin exerts its function on the optic nerve regeneration. Also, this research topic has much potential for the medical research field, since it may provide us with insight into reasons why fish can successfully regenerate their optic nerves while the mammals including humans cannot. Results from this study may help us develop therapeutic strategies (e.g. supply synthetic N-cadherin or its interacting molecules to patients with optic nerve injuries) to help eye and/or optic nerve regeneration in mammals. Results of this project will be presented as a poster at local scientific meetings (e.g. UA Annual Undergraduate Research Symposium) and/or in a peer-reviewed publication in a scientific journal in the field