Thesis (Ph. D.)--Florida State University, 2003. / Advisor: Dr. Joseph Travis, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Sept. 29, 2003). Includes bibliographical references.

As a study organism, annual killifish (Austrofundulus limnaeus) provide a well suited study system for examining the effects of environmental temperature fluctuations at the cellular level. A. limnaeus persist in the harsh high desert climate of the Maracaibo Basin, Venezuela where they live in small, ephemeral freshwater pools. Temperatures in these waters can vary as much as 20 degrees C daily and reach maximums of over 40 degrees C due to the semi-arid climate. Previous cDNA microarray studies on killifish revealed the mRNA pattern for High Mobility Group Protein 1 (HMGB1) to be strongly affected by temperature perturbations. Specifically, peaks in hmgb1 transcript abundance were negatively correlated with temperature during temperature cycling, and experienced over a 10 fold difference in expression in response to the temperature cycle. Using the same temperature cycling experimental setup, this study's aim was three-fold: (1) to characterize the total amount of HMGB1 protein in adult male killifish livers, (2) to describe the subcellular localization of the HMGB1 protein in adult male killifish livers and (3) to sequence the 5' upstream region of the hmgb1 gene to identify possible stress responsive elements. We detected no significant difference in total HMGB1 protein levels as a consequence of temperature cycling. The data for subcellular localization of HGMB1 protein do not support a strong change in subcellular localization of the protein in response to temperature cycling; most of the HMGB1 protein is found in the cytoplasmic compartment in liver tissue. Although overall patterns of subcellular localization did not change significantly, we found a significant difference between nuclear HMGB1 protein levels in temperature cycled fish versus control (constant temperature) fish. This could suggest a muting of the natural translocation of HMGB1 into the nucleus observed in control fish at around 9:00 at night. Finally, the upstream region of the hgmb1 gene does reveal a number of putative stress responsive transcription factor binding sites.

The annual killifish, Austrofundulus limnaeus, may enter embryonic diapause at three distinct points of development, termed diapause I, II, and III. Previous studies suggest a role for steroid hormones in the regulation of diapause in annual killifish. This study concerns the hormonal and genomic components involved in the developmental decision to enter or escape diapause II from both a maternal and embryonic perspective. Steroid hormone levels were measured in tissues isolated from adult female fish that were producing either high or low proportions of escape embryos. Levels of steroid hormones were also measured in new fertilized embryos that were known to be on either an escape or diapausing developmental trajectory. In addition, cDNA microarray gene expression analysis was used to identify gene sequences that may be associated with the regulation of entry into diapause in this species. Decreases in maternal estrogen levels associated with aging are correlated with decreasing escape embryo production, but there is no direct association between measured steroid hormone levels and escape embryo production. However, maternal production of escape embryos is correlated with increased ratios of 17 ß-estradiol to testosterone in ovary tissue, and cDNA microarray gene expression analysis indicates differentially regulated sequences associated with escape embryo production in maternal tissues. Both of these independent measures suggest hormonal involvement in the regulation of diapause. Embryonic levels of steroid hormones in newly fertilized embryos are not correlated with entry or escape from diapause II, although incubation in exogenous cortisol and 17 ß-estradiol causes an increase in the proportion of escape embryos. Gene expression analysis again suggests hormonal involvement. Interestingly, genes involved in epigenetic control of gene expression though chromatin condensation are differentially regulated in both maternal tissues producing escape embryos, and in embryos on the different developmental trajectories. These data suggest that hormonal control of gene expression through alterations of chromatin condensation may regulate the decision to enter or escape diapause II.

Embryos from the annual killifish Austrofundulus limnaeus have a unique and unequalled ability among vertebrates to withstand extended periods of anoxia (maximum lethal time to 50% mortality of 65 days at 25°C). In addition, tolerance of anoxia is gained and subsequently lost during the normal development of this species. Thus, anoxia tolerant and anoxia sensitive individuals can be compared within the same species, making A. limnaeus an excellent model for studying the molecular changes associated with survival of oxygen deprivation in vertebrates. The aim of this project is to analyze the molecular changes associated with anoxia tolerance in the embryos of A. limnaeus. Understanding how the cells of these embryos become tolerant to anoxia will aid in identifying novel therapeutic targets to reduce cell death following periods of ischemia in heart, brain or other tissues. Three major analyses were used to investigate the molecular changes associated with anoxia tolerance in this species. The first was a cell cycle and cell cycle arrest analysis using flow cytometry along with an immunoblot analysis of both positive and negative regulators of cell cycle progression. The second was a cell death analysis utilizing caspase-3/7 activity as well as TUNEL staining. The third was an immunoblot analysis of three different post-translational modifers (ubiquitin, SUMO-1 and SUMO-2/3). The overall findings from this study indicate that the embryos of A. limnaeus do indeed experience some degree of cellular stress (i.e. increase in ubiquitinated proteins, increase in p53 expression, evidence of DNA damage from TUNEL staining and increases in caspase activity) in response to anoxic treatment, even in their most protective state of diapause II. However, despite these observations, the whole organisms are still able to recover from anoxia and do not succumb to death. The overall low levels of TUNEL-positive cells and caspase activity relative to the positive controls indicates that the damage accrued in response to anoxic treatment is minimal. It appears that embryos are able to either "sacrifice" a certain portion of cells or they are able to repair the damage required for resumed development following anoxia.

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.

Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Includes bibliographical references. Issued in print and online. Available via ProQuest Digital Dissertations.

Karyotypes of nine species of Venezuelan annual killifishes were compared. Karyotypic differences were found between species and between some genera. A chromosomal sexual dimorphism was found in <u>Pterolebias hoignei</u> (2N = 46). Males of this species possess a single large "Y" chromosome. No chromosomal divergence was detected among conspecific populations from different localities. <u>Pterolebias hoignei</u> and <u>Pterolebias zonatus</u> were found to differ both in diploid number (46 and 42 respectively) and in metacentric chromosome number (6 and 12 respectively). All <u>Rachovia</u>species were found to have a diploid number of 44. <u>Rachovia maculipinnis</u>and <u>R. brevis</u> were found to differ from one another in metacentric chromosome number (20 and 12 respectively). <u>Rachovia hummelincki</u> and <u>R. pyropunctata</u> both possess metacentric chromosome counts of 10. No karyotypic differences were found between <u>Austrofundulus transilis</u> and <u>Austrofundulus limnaeus</u> (2N = 44, metacentrics = 12). Several populations of the latter although divergent in male color patterns, did not differ karyotypically. All species examined, except <u>Rivulus stellifer</u>, departed from the presumptive ancestral teleost diploid number of 48. The degree of chromosomal variation appears not to be as high among the New World as among the Old World annuals. This difference among lineages with similar life histories and reproductive strategies suggests that stochastic events have not played a major role in mediating chromosomal divergence in annual fishes. / Master of Science

Little is known about the physiology or biochemistry of hypoxia (reduced levels of oxygen) tolerance during development in vertebrate embryos. In most species, relatively brief bouts of severe hypoxia are lethal or teratogenic. An exception to such hypoxia intolerance is the annual killifish Austrofundulus limnaeus, in which populations persist in hypoxic environments. This species inhabits seasonal ponds in Venezuela, surviving through the dry season in the form of diapausing embryos. Embedded in the pond sediment, embryos of A. limnaeus are routinely exposed to hypoxia and anoxia (lack of oxygen) as part of their normal development. Here, we exposed embryos to various levels of PO2 (21.2, 15.6, 10.8, 8.4, 6.1, 3.6, and 2.2 kPa) at two different temperatures (25°C and 30°C) to study the effects on developmental rate and heart rate. We also measured enzyme activity and quantified DNA content of individual embryos to compare differences among the varying levels of hypoxia and temperature. Hypoxia caused a significant decline in developmental rate and caused a stage-specific decline in heart rate. Higher temperature caused an increase in the developmental rate for those embryos incubated at PO2 of 6.1 kPa and greater. Temperature had a negative effect by hindering development below a PO2 of 6.1 kPa. Total embryonic DNA content was reduced at low partial pressures (15.6, 10.8, 8.4, 6.1, 3.6, and 2.2 kPa) of oxygen. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase were all down-regulated indicating a complete lack of enzymatic metabolic compensation to combat reduced oxygen levels.

Development in the annual killifish Austrofundulus limnaeus can follow two distinct developmental trajectories. Typical development includes the entrance of embryos into a state of metabolic and developmental arrest termed diapause. Alternately, embryos can escape diapause and develop directly without pause. These two trajectories are characterized by differences in the rate and timing of developmental, morphological, and physiological traits. Insulin and Insulin-like growth factor (IGF) signaling (IIS) is known to regulate entrance into diapause in a variety of invertebrates. In this thesis I explore the possible role of IGFs in the regulation of development and diapause in embryos of A. limnaeus. Here I report stage-specific expression of IGF-I and II proteins and their associated mRNA transcripts. Patterns of IGF-I protein expression are consistent with IGF signaling playing a major role in supporting the escape trajectory. In addition, treatment of embryos with a potent inhibitor of the IGF-I receptor (IGF1R) mimics the diapause developmental pattern even under conditions that should favor direct development. Evaluation of mRNA gene expression patterns in the two developmental trajectories suggests a role for IGF-I signaling through the RAS-MAPK-ERK pathway, which may be promoting the escape phenotype. Additionally, IGF-I activity may be enhanced in escape trajectory embryos though upregulation of IGF binding protein 2 (IGFBP-2) mRNA. These data suggest a major role for IGF signaling in the promotion of the escape trajectory, and thus we predict that specific mechanisms are in place in diapause-bound embryos that block IGF signaling and thus promote entrance into diapause. The data presented here suggest that blocking IGF signaling is critical for induction of diapause, but also suggests that other signaling pathways are likely also at play. Other pathways such at the TGF-beta signaling molecules and SMAD pathway, may also be involved in the direct regulation of the diapause phenotype, as has been shown for other animal models of developmental arrest.

Thesis (Ph. D.)--University of Kentucky, 2004. / Title from document title page (viewed Jan. 7, 2005). Document formatted into pages; contains ix, 141p. : ill. Includes abstract and vita. Includes bibliographical references (p. 128-139).