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.

Blog 3: Interneuromast Chain Ablation in Transgenic Zebrafish


      Over the course of the previous two months, Dr Steiner and I have continued to push forward in our laser ablation experiments. The transgenic Zebrafish that our studies revolve around have been breeding consistently every week, which has been excellent and enabled us to continue to conduct experimental trials. We have maintained the same controls pertaining to the DAPI laser, which are a laser power of 75% and wavelength of 405 nm (high energy), but have made some specific changes in the actual ablation. During our imaging on the Confocal microscope, Dr. Steiner began to notice some rather discrete cells that were very dim in terms of fluorescence but clearly visible. These cells were located very close to the site of ablation on the interneuromast chain, and have the potential to completely confound our ablations. We want to be absolutely definitive regarding the fact that we have successfully ablated the interneuromast chain, which entails that all of the cells at that site have been killed. In an effort to prevent these cells from surviving the ablation and confounding our results, Dr. Steiner and I have been zeroing in on them during our experimental trials.

We first began with maintaining the aforementioned laser controls and annihilating these background cells for 10-15 seconds, which was followed by careful interpretation of the subsequent images. We wanted to be sure that we have successfully ablated those cells, and that they would be out of the picture. These additional preliminary 10-15 second ablations enabled us to eliminate these target cells, but have had some blowback on our experimental trials. In the experimental trials where we hit the background cells for 10-15 seconds, we frequently observed that the interneuromast chain failed to close the gap in the 24 hour Time Lapse Microscopy footage. Dr. Steiner and I both feel that the failure of the interneuromast chains to close may have been due to excessive ablation emanating from the additional targeting of these background cells.

        These background cells have become a rather challenging dynamic, but we were still able to obtain an additional 2 data points over the course of the last two months. Both of these data points were fairly strong, and made good contributions to our data. Dr. Steiner and I plan to continue to conduct ablation experiments on a weekly basis, but will be setting our sights on using fish that are slightly older. We have been conducting experiments on fish that are 2 days post fertilization, and will be conducting experiments on fish that are 3 days post fertilization in the coming weeks. We are excited to see if there is any difference in our results of experimental trials centered around fish that are slightly older.

Blog #2: Path Finding Mechanisms in Transgenic Zebrafish

Throughout the course of the previous 2 months, Dr. Steiner and I have had the opportunity to conduct multiple ablation experiments involving the new Confocal microscope. The assembly of this new microscope that Dr. Steiner was able to get for our research was recently completed, and we were very eager to try it out for the purposes of our 24 hour time lapse microscopy films. All of our previous 24 hour Time lapse microscopy films were conducted on a different Confocal microscope that has been in the lab for quite some time. In the conduction of numerous experimental trials on this microscope, we have never ran into any problems  regarding the imaging procedure, DAPI laser ablations, and 24 hour time lapse that we couldn’t solve. We’ve certainly encountered some challenges, particularly in establishing optimal DAPI laser control for the ablations, but have been able to strategically work through them in experimentation. The adversity we faced early on in the semester has certainly spilled over into our attempts to get the new Confocal microscope up and running as a primary component of our experimental trials.

During our analysis of the 24 hour time lapse films conducted on the new Confocal microscope, we have been consistently confronted with a major problem. All of the 24 hour time lapse films taken on the new Confocal microscope showed that the gaps produced in the interneuromast chains from the laser ablations are not closing. The projections from both sides of the ablated interneuromast chain showed minimal degrees of activity early on in the majority of the films that we reviewed, and failed to find each other and close the gap every single time. This failure of the gap produced in the interneuromast chain to close at this level was something that we never observed when conducting trials on the other Confocal microscope. Dr. Steiner and I are determined to find out why this problem is arising in the 24 hour time lapse films conducted on the new Confocal microscope. Our plan of action is to perform laser ablations on 6 Zebrafish during one morning trial, and image 3 of the Zebrafish on the older Confocal microscope while simultaneously imaging the other 3 on the new Confocal microscope. Review of all 6 of these 24 hour time lapse films should cast light on whether this failed gap closure is emanating from a component of new Confocal microscope or if it has an ulterior origin.

Aside from this problem that we are currently staring into the eyes of, my abilities and competency in carrying out full scale ablations has continued to grow under the direction of Dr. Steiner. Throughout the month of November, Dr. Steiner presented me with multiple opportunities to independently conduct ablation experiments on the Confocal microscope which was very exciting. I had a chance to really test my understanding and ability to apply the things that Dr. Steiner taught me about the microscope during live trials. To me, opportunities of this nature are inherently the times in which real, tremendous growth can happen. In terms of our game plan for the remainder of this month, we plan to solve the problem pertaining to the 24 hour lapse films captured on the new Confocal microscope and to continue to make strides in gathering data points.

Interneuromast Chain Regeneration in Zebrafish

Throughout non-mammilian vertebrates, the regeneration and recovery of hair cells that have sensory implications following damage has been observed; whereas mammalian hair cells tremendously lack this unique capability. Zebrafish, a non-mammalian vertebrate, is inherently capable of regenerating sensory hair cells, which exist within the organism’s ear as well as in its sensory lateral line system. Comprehension of the molecular basis of this unique capability has potential to define therapy for hearing loss in human beings. The focal point of our work and experimentation is the interneuromast chain of the lateral line system of the Zebrafish.

The lateral line is a mechanosensory system comprised of small sensory patches called neuromasts that are linked together by an interneuromast chain. Each sensory neuromast possesses hair cells extending their stereocillia beyond the epidermis where they may be deflected by water from which they can transduce external stimuli. These neuromast cells are directly supported by cells located beneath them called inner support cells as well as additional cells called mantle cells forming a ring around them. The mantle cells are particularly significant in that they may be involved in neuromast regeneration, and can be defined as multipotent progenitor cells of the lateral line system.  Interestingly enough, mantle cells of the neuromasts at the tail-end of Zebrafish have been demonstrated to enter the cell cycle in their contribution to regeneration following amputation of the tail.

In the First component of our experimentation, we set out to characterize the regenerative capacity of the interneuromast chain through the implementation of Confocal Microscopy. In the transgenic line of Zebrafish that we are using in this experiment, the interneuromast chain is reported upon by Green fluorescent protein. Being that the interneuromast chain is labeled with Green fluorescent protein, we can visualize it with ease under the Confocal Microscope. In addition to its purposes in effectually visualizing the interneuromast chain, we have been able to utilize the Confocal Microscope to conduct our ablation experiments. We have implemented the DAPI Laser of the Microscope to completely ablate the interneuromast chain; and the DAPI laser is set at a high energy wavelength of 405 nm at a laser power of 75%. Additionally, we allow the laser to annihilate the chain for exactly 60 seconds during each experimental trial.

The controls for our DAPI laser were determined over the course of several different experimental trials in which we previously struggled to optimize our controls. Following our ablations of the interneuromast chain, we have further conducted 24 hour Time Lapse experiments in an effort to capture the events following the interneuromast chain ablation. We have successfully captured 3 separate movies that portray complete interneuromast chain regeneration, and were able to characterize the gaps we created in terms of microns.

Throughout the course of September and into October, we encountered some adversity with regards to the mating behaviors of our Zebrafish in which there was absolutely no breeding. However, our luck turned around this past week and we had successful breeding. We were able to conduct an ablation experiment, and produced what appears to be the most impressive gap in the interneuromast chain created following ablation thus far. We are optimistic and excited to see what our 24 hour time lapse experiment reveals later this week.