Dr. Eric Birgbauer
College of Arts and Sciences
Department of Chemistry, Physics, and Geology
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The visual system is developed throughout the embryonic stage and is dictated by axon guidance molecules that tell the growing fibers of nerve cells, axons, where to grow. The ends of axons, called growth cones, continuously sprout out or collapse based on the axon guidance molecules. Research has shown that lysophosphatidic acid (LPA) causes growth cone collapse, so it is hypothesized that it is an axon guidance molecule. LPA binds to specific receptors, known as Lpars of which there are at least five known. Previous research indicates that Lpar 1,2, and 3 are not required in mice for growth cone responses to LPA. Furthermore, Lpar5 is not expressed in retinal tissue, so our research has focused on Lpar4. Our research is trying to determine how axon guidance molecules help shape the visual system that we have today so we start at the embryonic stage where it begins. By working with an embryo that is somewhat similar to ours, we can further figure out how the system works and how our visual system builds itself. We are working to mutate the receptor that we believe to do a substantial amount of axon guidance by using CRISPR as a tool and then see how growth cones respond to LPA without Lpar4. Chicken eggs are used since we easily could take them out at embryonic ages in an artificial culture system in a cup. We have validated guide RNAs that can mutate chicken Lpar4 by using CRISPR. We will inject plasmids expressing these guide RNAs as well as Cas9 along with a tdTomato fluorescent tag into the developing embryonic chicken eye. After developing further, we dissect the chicken retina to see if retinal cells still respond to LPA with growth cone collapse in the absence of Lpar4. We have been optimizing injection and electroporation of plasmid DNA into early embryonic chicken retina as well as dissecting and removing embryonic retina tissue to determine if our sample has transfected properly. We have also been obtaining baseline data on growth cone collapse by different concentrations of LPA.
Gonzalez, Thomas A., "Mutating Lpar4 in the Visual System using CRISPR" (2020). S.U.R.E Posters. 9.
This project was supported by SC INBRE grant from the National Institute of General Medical Sciences (8 P20 GM103499) of the National Institutes of Health