Brainbow Imaging of Neuromast Regeneration on the Lateral Line

The project Dr. Steiner and I are working on utilizes mutant and transgenic zebrafish in order to better understand the regeneration of sensory organs in vertebrates. Curiously, sensory hair cells—the cells responsible for hearing and motion detection in the lateral line system— are known to not regenerate in mammals; damage to cochlear hair cells can lead to permanent hearing loss or deafness, as these cells do not get repaired. It is our hope that in researching the molecular cues for regeneration of the zebrafish neuromast, the sensory organ of the lateral-line system, we will add to the growing body of knowledge on hair cell regeneration and potentially contribute to noninvasive treatments for individuals with damaged hearing.

In the research lab this summer, I have utilized genetic principles to create multi-transgenic zebrafish through breeding. Three transgenic lines of interest are maintained through multiple generations of zebrafish, and carriers of these transgenes are confirmed with fluorescence microscopy and genotyping PCR with subsequent gel electrophoresis. These transgenes allow for chemical initiation of a mosaic pattern of fluorescence throughout the cells of embryonic zebrafish. With confocal imaging, I will be able to examine the colors the cells are expressing, tracing them back to the initial mantle cell exhibiting the same color. This aims to provide some of the first evidence of mantle cells differentiating into hair cells during regeneration, long suspected by other researchers.

Because the fluorescence is initiated within the cells’ genes, the color is maintained throughout multiple cell divisions. This also has the novel effect of cells fluorescing different colors depending on the extent of recombination during chemical treatment, and the number of copies of the gene present. This transgenic toolbox is known as Brainbow, as it was initially used to label neurons with different colors in order to trace their individual neurites; our procedure is sometimes referred to as ‘Zebrabow’. The final effect of the procedure, when viewing the neuromast through a confocal microscope, is neuromast and muscle cells fluorescing multiple different colors. Dr. Steiner and I have been trying to perfect our Zebrabow procedure in order to create the ideal treatment for our intended result, which is a neuromast containing clearly colored and differentiated mantle (supporting progenitor cells) and hair cells. Our zebrafish also have a transgene expressing green fluorescent protein in macrophages, in order to better understand the role of macrophages in neuromast regeneration; this is observed simultaneously as Zebrabow mosaics.

Hurdles this summer have included propagating the transgenes in order to create embryos with the appropriate zebrabow and GFP genes; zebrafish can be fickle about their breeding, and it can be difficult to find a carrier of the desired genes. At one point I had to carry out enzymatic digests of tail fin clippings to extract genomic DNA and analyzed these with PCR in order to confirm a carrier, producing negative results the first PCR cycle (the entire process having taken a week). I also have no prior experience with confocal or fluorescent microscopes, but Dr. Steiner has been a great source of knowledge on microscopy. I’m now using the confocal microscope independently, which is what I’m proudest of thus far. I’ve only been working on the project for a month and a half, and I’ve experienced a lot already; I’m eager to continue this project into the next semester and learn much more about confocal microscopy while also producing valuable results.

 

 

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