In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. The annual killifish Austrofundulus limnaeus is possibly the most well described extremophile vertebrate and its embryos have been shown to tolerate extremes in oxygen, salinity, and water availability. This phenotype is likely a result of the annual killifish life history, which includes periods of temporary habitat desiccation and oxygen deprivation, and requires the production of stress-tolerant embryos that depress metabolism in a state of suspended animation, known as diapause. Over the past several decades, the basic morphology and physiology of annual killifish development has become better characterized. However, there are still basic cellular processes that remain to be described in annual killifish such as A. limnaeus. Specifically, changes in DNA structure, expression, and copy number are known to have profound impacts on the phenotype and survival of an organism. Little is known as to how A. limnaeus maintains genome integrity during cell stress, nor how the A. limnaeus nuclear and mitochondrial genomes may have evolved under the unpredictable conditions in which A. limnaeus thrive. Early annual killifish embryonic development is also characterized by a complete dispersion and subsequent reaggregation of embryonic blastomeres prior to formation of the embryonic axis. This unusual period of early development may provide a functional adaptation that allows annual killifish embryos to survive these extreme conditions.
The overall goals of this project were to (1) characterize the ability of A. limnaeus to tolerate and repair DNA damage through enzymatic and developmental mechanisms, (2) to determine possible consequences of mitochondrial DNA sequence and copy number on the metabolism of A. limnaeus, and (3) to establish a draft genome of A. limnaeus for future comparative genome studies. The results of this project show that embryos of A. limnaeus have an impressive ability to survive and reverse high doses of DNA damage induced by ultraviolet-C (UV-C) radiation, especially when allowed to recover under photoreactivating light. Surprisingly, embryos that survived irradiation during blastomere dispersion phases were able to develop normally. Characterization of gene expression during embryonic development for genes important for axis formation and cellular differentiation suggests that A. limnaeus embryos may delay axis formation until several days after epiboly is complete, thus allowing time for cells that become damaged to be replaced by surrounding pluripotent cells. This outcome would represent first case of a developmental buffering stage in a vertebrate. A. limnaeus embryos are also unique in their mitochondrial response to anoxia. Whereas in other species the amount of mitochondrial DNA (mtDNA) copy number fluctuates following extremes in oxygen availability, A. limnaeus embryonic mtDNA remains stable. Additionally, characterization of the fully sequenced A. limnaeus mitochondrial genome reveals possible evolutionary adaptations that may have facilitated dormancy and anoxia tolerance when compared to other species within the Order Cyprinodontiformes. The final chapter of this project characterizes the draft genome of A. limnaeus and I provide evidence suggesting that epigenetic DNA methylation that may be involved in regulating diapause.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-3525 |
| Date | 18 September 2015 |
| Creators | Wagner, Josiah Tad |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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