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.


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