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.

Misguidance in Path Finding Mechanisms in Twitch-Twice-Roundabout3 Mutant Zebrafish

Over the course of the previous few weeks, Dr. Steiner and I have continued to conduct Laser ablation experiments on the Confocal Microscope. Our primary objective has been to completely ablate the interneuromast chain in an effort to characterize the regenerative process through a 24 hour Time-Lapse Microscopy movie. We have been strategically ablating significant components of the interneuromast chain including cell bodies between neuromasts L3 and L4. In order to appropriately gauge laser power, we developed 3 primary controls based on previous experimental findings that all ascertained a high energy wavelength of 405 nm. In our last experimental trial, we annihilated the interneuromast chain with a laser power of 75% for exactly 60 seconds; and were successful in producing a small-scale gap in the chain. The gap we created confirmed that the control we implemented regarding Laser Power was optimal, and our Time-Lapse movie revealed very interesting results emanating from the ablation itself.

Following a careful reviewing of our 24 hour Time-Lapse Microscopy movie, we were able to clearly identify the gap in the interneuromast chain that we produced and observe its complete regeneration. This particular Time Lapse movie was important not only because it is definitive evidence of the interneuromast chain’s regenerative capacity, but also due to the fact that we observed cell body migration. The cell body migration that we witnessed on the film was slightly odd, and we will remain cognitive of this migration as we progress in experimentation. An understanding of the cell body migration and its function in the regeneration that we observed is something we will be striving for. While this experiment in itself was moderately successful, we will be focusing on creating larger-scale gaps in our continued ablation experiments. Upon successfully ablating the interneuormast to a greater extent, we will maintain the conditions in completing an additional 10-12 experiments to generate statistically significant data.

We have encountered some challenges throughout the course of our experimentation pertaining specifically to the interneuromast chain ablations. We have had some difficultly with actually ablating the interneuromast chain completely, and have noticed that some of our laser power controls only resulted in photobleaching of the chain. Incomplete ablations resulting in mere photobleaching are insufficient, and cannot be used to paint a picture of regeneration. Increasing the laser power to 75% for an additional 60 seconds appears to be a step in the right direction, and is a control that will be built upon. An additional confocal microscope with a spinning disk system is in the process of being assembled, which will function as a definitive solution in later weeks. Additionally, the mating behaviors and patterns of the Zebrafish, our model organism, have presented some adversity as well. In attempting to keep their breeding consistent, they receive multiple feedings on a daily basis.

The past few weeks have been particularly exciting for me, as my degree of competency on the Confocal Microscope is continuing to grow under Dr. Steiner’s mentorship and instruction. I am becoming progressively more comfortable in operating the Microscope during the experimental trials, and will soon be able to carry out experiments independently. The ability to conduct experiments on the Confocal Microscope confidently and soundly is a skill that I consider to be invaluable due to the sheer power of the microscope itself. The immense potential that it brings to the table relative to all walks of scientific exploration is truly incomprehensible. I believe that enhancing my capability and comfortability with the Confocal microscope is something that I can greatly draw upon on in my future in research. I plan to learn absolutely as much as I can about the nature of the microscope, and will take my skills as far they can possibly go. With regards to the future of this experiment, we will continue to work diligently in conducting experimental trials until we have arrived at characterization of interneuromast chain regeneration.

Relevant Questions:

What biological mechanisms are driving the cell body migration that we observed?

How can we continue to adjust the laser power to avoid photobleaching and achieve complete ablation?

Misguidance in Path Finding Mechanisms in Twitch-Twice-Roundabout3 Mutant Zebrafish

Over the course of the previous few weeks, Dr. Steiner and I have continued to conduct Laser ablation experiments on the Confocal Microscope. Our primary objective has been to completely ablate the interneuromast chain in an effort to characterize the regenerative process through a 24 hour Time-Lapse Microscopy movie. We have been strategically ablating significant components of the interneuromast chain including cell bodies between neuromasts L3 and L4. In order to appropriately gage laser power, we developed 3 primary controls based on previous experimental findings, that all ascertained a high energy wavelength of 405 nm. In our last experimental trial, we annihilated the interneuromast chain with a laser power of 75% for exactly 60 seconds, and were successful in producing a small-scale gap in the chain. The gap we created confirmed that the control we implemented regarding Laser Power was optimal, and our Time-Lapse movie revealed some very interesting results emanating from the ablation itself.

Following careful reviewing of our 24 hour Time-Lapse Microscopy movie, we were able to clearly identify the gap in the interneuromast chain that we produced and observe its complete regeneration. This particular Time Lapse movie was important not only because it is definitive evidence of the interneuromast chain’s regenerative capacity, but also due to the fact that we observed cell body migration. The cell body migration that we witnessed on the film was slightly odd, and we will remain cognitive of this migration as we progress in experimentation. An understanding of the cell body migration and its function in regeneration that we observed is something we will be striving for. While this experiment in itself was moderately successful, we will be focusing on creating larger-scale gaps in our continued ablation experiments. Upon successfully ablating the interneuormast to a greater extent, we will maintain the conditions in completing an additional10-12 experiments to generate statistically significant data.

We have encountered some challenges throughout the course of our experimentation pertaining specifically to the interneuromast chain ablations. We have had some difficultly with actually ablating the interneuromast chain completely, and have noticed that some of our laser power controls only resulted in photobleaching of the chain. Incomplete ablations resulting in mere photobleaching are insufficient, and cannot be used to paint a picture of regeneration. Increasing the laser power to 75% for an additional 30 seconds appears to be a step in the right direction, and is a control that will be built upon. An additional confocal microscope with a spinning disk system is in the process of being assembled, which will function as a definitive solution in later weeks. Additionally, the mating behaviors and patterns of the Zebrafish, our model organism, has presented some adversity as well. In attempting to keep their breeding consistent, they have received multiple feedings on a daily basis.

The past few weeks have been particularly exciting for me, as my degree of competency on the Confocal Microscope is continuing to grow under Dr. Steiner’s mentorship and instruction. I am becoming progressively more comfortable in operating the Microscope during the experimental trials, and will soon be able to carry out experiments independently. The ability to conduct experiments on the Confocal Microscope confidently and soundly is a skill that I consider to be invaluable due to the sheer power of the microscope itself. The immense potential that it brings to the table relative to all walks of scientific exploration is truly incomprehensible. I believe that enhancing my capability and comfortability with the Confocal microscope is something that I can greatly draw upon on in my future in research. I plan to learn absolutely as much as I can about the nature of the microscope, and will take my skills as far they can possibly go. With regards to the future of this experiment, we will continue to work diligently in conducting experimental trials until we have arrived at characterization of interneuromast regeneration.

Misguidance in Path finding Mechanisms in Twitch-Twice-Roundabout3 Mutant Zebrafish

Hearing loss emanating from damage to the hair cells of the inner ear has significantly impacted the quality of life of over 48 million Americans. Ranging from newborns all the way to the geriatric population, hearing loss is an indiscriminate condition currently affecting every demographic. A common antidote to debilitating conditions that have biological roots, science, may hold a latent answer. Unfortunately, regeneration of the sensory hair cells of the inner ear in mammals does not occur as the damage is permanent. Thus, in our scientific exploration of the biological function and nature of hair cells, we will turn to a model organism that ascertains a definitive regenerative capacity. 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 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. With respect to the hair cells of human beings, the lateral line hair cells are functionally as well as morphologically similar. Mutations impacting the function and behavior of lateral line hair cells also result in hearing ailments and hearing loss in human beings. With such distinct biological similarity to humans and glaring potential for very sound experimental design, Zebrafish are our model organism of choice.

We intend to focus specifically on the regenerative capacity of the interneuromast chain following the conduction of ablation experiments through Confocal Microscopy. The Roundabout (Robo) family is a major family of cellular receptors that act in conjunction with their Slit ligands, and have been previously implicated in axonal guidance. Previous studies have cited the Twitch Twice gene in encoding Robo3 within Zebrafish, and have been able to correlate this Robo3 receptor to the mechanism of “guidance” in regeneration. The ends of developing interneuromast chains contain a specialized structure called growth cones. Growth cones send out dynamical filamentous projections called filopodia that respond to adhesion and guidance chemically-based sensory stimuli in the external environment. Following characterizing the regenerative process of the Interneuromast chain, we intend to demonstrate that a mutation in the Twitch Twice gene encoding Robo3 results in misguidance during the pathfinding mechanism in regrowth.

Our experiment is centered around a specific transgenic line of Zebrafish that have a genetic construct defined by a Myo6b promoter driving the expression of beta-actin-GFP (green fluorescent protein). Acting as a reporter component, GFP enables us to visualize the entire interneuromast chain of the Zebrafish within the dimensions of Confocal and Flourescent Microscopy. In completing our first major experimental step, we have been consistently screening Zebrafish that were 2 days post-fertilization under our Flourescent microscope. Zebrafish that were positive for our Myo6b genetic construct displayed a complete interneuromast chain that was fluorescing green as a result of our GFP reporter component. We have been isolating these positive Zebrafish on a weekly basis, and have moved to the ablation component of our experiment. Following our weekly screening of Zebrafish that are 2 days post Fertilization, we have been mounting three Zebrafish onto our Confocal Microscope.

Upon mounting this Zebrafish onto to a cover slide via Tricaine solution that anesthetizes them and low melting point agarose, we were able to begin our ablation experiments. Each week, we begin with capturing pre-ablation images of the interneuromast chain between neuromasts L3 and L4 of the Zebrafish. The pre ablation image is very precise in capturing the specific region of the interneuormast chain including the cell bodies that we plan to ablate. After capturing the pre-ablation image, we immediately move to ablating the interneuromast chain through a DAPI Laser, a component of the Confocal microscope. We have been annihilating the interneuromast chain at a power of 405 for exactly 30 seconds, and capture post-ablation images immediately after. This previous week, we moved to conducting a 24 hour time lapse microscopy experiment that was intended to capture the complete regenerative process of the interneuromast chain following ablation. However, after review of the 24 hour time lapse experiment, we noticed that we had only bleached the interneuormast chain, and had not completely ablated it. All of our other settings held up soundly, thus this coming week we plan to carry out the same experimental procedure with enhanced DAPI laser power.

This coming week marks a very exciting time for us, as we strongly believe that with our previous learnings from our attempted 24 hour time lapse experiment, we will be successful this time around. With enhanced DAPI Laser power, we hope to successfully complete the 24 hour time lapse experiment that captures the complete regenerative process of the interneuromast chain. If the experiment is successful, we will maintain the experimental conditions and carry out the same procedure an additional 9 times to validate our findings. Statistical measures will be conducted as well to further strengthen the results of the first major phase of our work. Following the completion of the Part 1, we will move to working with the Twitch Twice/Robo3 mutant Zebrafish. We plan to demonstrate through our 24 Time Lapse Microscopy following ablation that the mutation in Robo3 definitvely results in sensory misguidance during the regenerative process of the interneuromast chain. When we successfully complete this final part of the experiment, we will be able to validate our hypothesis that mutation in ROBO3 affects the sensory based guidance of the growth cone of the interneuromast chain during regeneration.