Final Report- Interneuromast Chain Regeneration in ET20 Transgenic Zebrafish

The Zebrafish is a non-mammalian vertebrate that ascertains an inherent regenerative capacity pertaining to the sensory hair cells that are involved in the transduction of extrinsic stimuli. Anatomically speaking, the Zebrafish has a mechanosensory lateral line system that is comprised of neuromasts containing sensory hair cells, and these neuromasts are linked together by the interneuromast chain. Interestingly enough, the interneuromast chain has stem cell like capabilities in being able to give rise to sensory hair cell containing organs such the neuromasts that function as integral components of the lateral line system. Unlike the Zebrafish, human beings completely lack the ability to independently regenerate sensory hair cells. Once the hair cells of human beings have been destroyed, they are damaged for life and rendered incapable of transduction relative to auditory stimuli. Interestingly enough, the environment in which the hair cells of the Zebrafish exist within, namely the mechanosensory lateral line system, is biologically analogous to that of the cochlea of the inner ear of human beings. Thus, we can easily extrapolate significant findings relative to sensory hair cells in Zebrafish and apply them to human beings in an effort to unravel key biological underpinnings of hair cell destruction.

Dr. Steiner and I will be specifically studying the interneuromast chain of transgenic Zebrafish within the dimensions of Confocal Microscopy. The transgene that our Zebrafish possess has Tol 2 sequences flanking the enhancer element of the transgene that can be recognized by a Tol 2 Transposable element. Upon recognition by the Tol 2 Transposable element, our transgene with be randomly integrated into genome of the Zebrafish with the hope that it lands near the germ line. With succcesful integration of this transgene into the genome comes an interneuromast chain as well as neuromasts that are labeled with green fluorescent protein providing for astounding visualization under the Confocal microscope. The green fluorescent label enables us to visualize exactly what is going on throughout the entire experimental trial.  In part 1 of our experimentation, we plan to decisively establish a baseline regarding the regenerative capacity of the interneuromast chain through laser ablation trials. The Confocal microscope is equipped with a DAPI laser that can produce a very high energy wavelength of 405 nm at a laser power of 75%, which can annihilate the interneuromast chain with ease. The idea behind the complete destruction of the interneuromast chain is that a definitive gap in the chain will be produced, further providing an opportunity for the interneuromast chain to showcase its regenerative abilities. During each experimental trial, Dr. Steiner and I annihilate the interneuormast chains of Zebrafish utilizing the aforementioned conditions for exactly 45 seconds. Prior to the ablation, we capture a pre-ablation image demonstrating what the chain looked like before the ablation. Immediately after the ablation, we capture a post-ablation image demonstrating the clear gap in the interneuromast chain and death of the cells at the hands of our DAPI Laser.

Directly after the laser ablations have been completed, Dr. Steiner and I move to setting a 24 Hour Time Lapse Microscopy experiment into play that is intended to capture the interneuromast chain’s regenerative process as a function of time. Throughout the course of the academic year, Dr. Steiner and I have been successful in accruing primary data points pertaining interneuromast chain regrowth as evidenced by our 24 Hour-Time Lapse movies. These films have been an incredible tool in not only capturing the entire regenerative process as a whole, but have enabled us to zero in on some rather minute details regarding finite projections extending outward from both sides of the interneuromast chain. We hypothesize that these projections are reaching outward into space in search of chemical cues that function as directional guides for gap closure. We have captured multiple images from some of our 24 hour Time Lapse movies demonstrating projections extending forward from a side of a previously ablated interneuormast chain. Moreover, we’ve noticed a very intriguing structural motif in these projections that we refer to as the “fork” given its fork-like shape. Our analyses have yielded some very exciting results, and I look forward to striving to further understand the biological significance of these projections.

Dr. Steiner and I currently have accrued 7 data points over the course of the previous few months, and have our sights set on accruing a minimum of 15 data points in an effort to establish a strong baseline for interneuromast chain regeneration. Following the establishment of this baseline, we plan to introduce an additional transgenic line of Zebrafish that have a mutation in the Twitch Twice gene encoding the Robo3 receptor. Dr. Steiner and I hypothesize that the Robo3 receptor has a major role in interneuromast chain gap closure, and a mutation in the Twitch Twice gene would render the Robo3 receptor protein non-functional. We plan to conduct the exact same laser ablation experiments, but in the face of a non-functional Robo3 receptor protein. Our current hypothesis is that interneuromast chain gap closure will fail in the presence of a non-functional Robo3 receptor protein emanating from a mutation in the Twitch Twice gene. Dr. Steiner and I are excited to continue to accrue primary data in the completion of phase 1 of the experiment and move into this second phase in our quest to understand interneuromast chain regeneration on a molecular genetic level.

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