Misguidance in Path finding Mechanisms in Twitch-Twice-Roundabout3 Mutant Zebrafish – Final Report

Our research focuses specifically on the regenerative capacity of the interneuromast chain following ablation experiments conducted on a Confocal Microscope through the usage of the DAPI laser. The Roundabout (ROBO) family is an important family of cell receptors that function in response to their Slit ligands; which have interestingly been implicated in axonal guidance in the past. Studies have zeroed in on and noted the Twitch Twice gene in the encoding of Robo3 within Zebrafish, and have related the Robo 3 receptor to the mechanism of guidance in interneuromast chain regeneration. The ends of interneuromast chains have specialized structures called growth cones that send out dynamical filamentous projections called filopodia that can respond to chemically based guidance stimuli. Following direct characterization of the interneuromast chain, Dr. Steiner and I will demonstrate that a mutation in the Twitch Twice gene encoding Robo3 results in misguidance during the rengenerative process ascertained by the interneuromast chain.

Dr. Steiner and I were able to complete our first successful ablation experiment this past week on our Confocal Microscope. The primary objective of our ablation experiments is to produce a definitive gap in the interneuromast chain of the Zebrafish through the usage of a DAPI laser of 75% power with a high energy wavelength of 405nm. We established that this specific laser power is sufficient to completely annihilate the interneuromast chain for exactly 60 seconds through multiple experimental trials. Previous laser powers of lesser magnitude at the same wavelength that had been implemented were insufficient in successfully ablating the interneuromast chain, and resulted in photobleaching. In an effort to characterize the regenerative capacity of the interneuromast chain, we incorporated a 24 hour Time Lapse Microscopy component into our experimental procedure. In our successful ablation experiment, the 24 Hour Time Lapse movie demonstrated clear and complete interneuromast regeneration within the first 8.5 hours. Additionally, we analyzed the interneuromast chain on a computerized 3-dimensional platform to define the gap that we produced in the ablation. We measured the gap to be exactly 36.8 microns, and determined the rate of interneuromast gap closure to be 4.33 microns/hour.

With our first successful ablation experiment and interneuromast gap characterization analysis behind us, we can maintain the experimental conditions and continue to conduct ablations on the Confocal Microscope on a weekly basis. However, our ability to conduct weekly ablation experiments is contingent upon successful and consistent mating regarding the Zebrafish. After conducting a minimum of an additional 10 experiments, we will conduct specific statistical measurements to analyze the significance of the data that we have generated. Our statistical analysis will focus on the size of the gap in microns produced by the DAPI laser in the actual ablation, as well as the rate of closure of the gap in terms of microns/hour. We plan to generate statistically significant data, and are extremely optimistic about the future of this experiment moving forward.

Dr. Steiner has been an outstanding instructor throughout the course of my time in his lab, and I am very grateful to have him as a research mentor. When it came down to teaching me how to operate all of the microscopes and carry out major parts of the experimental procedure, he was very patient and effective in teaching me. He allotted me the time to ask as many questions as necessary, and as many attempts as I needed in learning how to use the microscopes. Without all of the guidance and instruction that he has provided me, I would not be where I am today in this experiment and in the lab as a whole.

I remember the first time that I watched Dr. Steiner carry out an ablation experiment on the confocal microscope, and thinking, I can’t wait until the day comes that I can do this on my own. Fast forward 2.5 months later, and I am preparing to make my first independent attempt at conducting an entire experiment this coming Thursday. To me, the greatest thing that we can do with our time is breathe life into our thoughts and visions for ourselves when presented with the opportunity to do so. This program has provided with a chance to do exactly that, and I plan on chasing it until I’ve made it.

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