As the summer winds down, I have been reflecting on the project Dr. Steiner and I began working on in June. Dr. Steiner and I have been utilizing multi-transgenic zebrafish to deeply explore the regeneratory process of zebrafish sensory hair cells. The current body of zebrafish research suggests supporting mantle cells divide to produce hair cells during regeneration of neuromast sensory organs along the lateral line, although evidence of such is lacking. Together, we created a plan to manipulate three transgenic lines to image the regeneration of these neuromast cells, with the end-goal of better understanding this regeneratory process and the factors that control it.
With these research goals in mind, I’ve been learning about the complexities of conducting biological research with living specimens. Many times during the semester, my colleagues and I have sacrificed time to come in on weekends to feed the fish; there have been several weeks the fish didn’t cooperate with breeding, and we had to quickly work on a new plan for the week. Truthfully, though, working with live zebrafish brings an element of physicality and life to my research. It’s a pleasure starting my day seeing our entire population of zebrafish greet me with frantic swimming.
Also challenging, but extremely rewarding, is developing the Zebrabow process I’ve been using to visualize cells of zebrafish. At its best, this method makes the cells of zebrafish fluoresce a beautiful mosaic of colors, allowing a researcher to better understand the cell divisions that potentially lead to new hair cells. No single procedure will work for every lab; Dr. Steiner has greatly helped me in creating a Zebrabow process that works for our fish in particular. At the start of the summer, we had very little mosaic fluorescence amongst the cells. This provided me several opportunities to review the process we had used, and modify it for better performance in the future. Such modifications make this research project feel like it is truly our own; over the next few semesters, I hope to refine this procedure to obtain consistent and repeatable results.
This summer project has not only taught me about valuable lab techniques, such as confocal & fluorescent microscopy and taking care of living specimens; working with Dr. Steiner this summer made me increasingly comfortable with the research process and environment, which I hope to be a part of for years to come. Being able to pave my own way through a new set of procedures is fantastic experience for my future endeavors. Most of all, I’m excited to continue my research with Dr. Steiner over the upcoming year, building upon everything I’ve learned over the past months.
Included in this blog post is an image of one of the Zebrabow treatments I’ve done, in order for readers to better understand the process. This image was taken as numerous ‘slices’ of images from the confocal microscope, then compressed into a single image. On the bottom right is the neuromast, the organ containing hair cells that Dr. Steiner & I study. One can see numerous flourescent cells, as well as some macrophages (depicted by amorphous green-flourescent projections).
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.