Phenotypic and Genome Editing Analysis of Stickleback Morphology

James Wilson, University of Miami, Marine Biology and Ecology, Coral Gables, United States; University of California, Berkeley, Molecular and Cell Biology, Berkeley, United States and Craig T Miller, University of California, Berkeley, Molecular and Cell Biology, United States
Understanding tissue and organ regeneration is an important goal due to its great potential for biomedical applications. Understanding the genetic basis of continuous tooth regeneration can help scientists understand both tooth and hair (homologous epithelial organs) regeneration. We sought to identify the genetic controls of continuous teeth regeneration using sticklebacks (Gasterosteus aculeatus) as a model. Ancestral marine sticklebacks have repeatedly colonized numerous freshwater environments, where many morphological adaptations evolve. Furthermore, genome editing methods can be used in both marine and freshwater populations to test the functions of specific candidate genes. In our first experiment, we compared tooth morphologies of sticklebacks between a wild ancestral marine population and a previously unstudied derived freshwater population to test the hypothesis that freshwater adaptation typically results in increased tooth number. We concluded that the freshwater population did possess increased tooth number in both the ventral tooth plate and one set of dorsal tooth plates. In our second experiment and to further develop genome editing methods to determine what genes govern these variations between tooth number and regeneration between populations, we directly compared Cas9 mRNA vs. protein efficacy in stickleback embryos. By injecting either Cas9 protein or mRNA along with guide RNAs to generate albino mutants, we directly compared lethality and effectiveness of Cas9 mRNA vs. protein. We determined that injecting Cas9 protein is more efficient at producing albino mutants. Establishing the most effective mode of Cas9 mutation generation will help further identify what mutations can lead to the up or down-regulation of tooth regeneration. By further understanding the genetic controls of tooth production, the scientific community can better tackle remediation of tooth, hair, and other epithelial organ loss via regeneration.