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• 1. [Article] Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus

  Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene ...

  Citation × Citation Citation Abstract Copy Title: Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus Author: DeYoung, Melissa, Thayer, Michelle, Van der Oost, John, Stedman, Kenneth M. Year: 2011 Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1- based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55a core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55a core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea. Title: Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus Author: DeYoung, Melissa, Thayer, Michelle, Van der Oost, John, Stedman, Kenneth M. Year: 2011 Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1- based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55a core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55a core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea. Close

• 2. [Article] The Cul3 Ubiquitin Ligase: An Essential Regulator of Diverse Cellular Processes

  Cul3 forms E3 ubiquitin ligase complexes that regulate a variety of cellular processes. This dissertation describes Cul3‘s role in several of these pathways and provides new mechanistic details regarding ...

  Citation × Citation Citation Abstract Copy Title: The Cul3 Ubiquitin Ligase: An Essential Regulator of Diverse Cellular Processes Author: Davidge, Brittney Marie Year: 2017 Cul3 forms E3 ubiquitin ligase complexes that regulate a variety of cellular processes. This dissertation describes Cul3‘s role in several of these pathways and provides new mechanistic details regarding the role of Cul3 in eukaryotic cells. Cyclin E is an example of a protein that is regulated in a Cul3-dependent manner. Cyclin E is a cell cycle regulator that controls the beginning of DNA replication in mammalian cells. Increased levels of cyclin E are found in some cancers, in addition, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with tumorigenesis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligase complexes, Cul1 and Cul3. Cul1 mediated degradation of cyclin E is triggered by cyclin E phosphorylation, however the mechanism Cul3 uses to ubiquitinate cyclin E is poorly understood. In order to gain a better understanding of how Cul3 mediates cyclin E destruction we identified the degron on cyclin E that is important in Cul3 dependent degradation. In addition, we show this degron is lacking in LMW cyclin E (found in abundance in breast cancer), providing a novel mechanism for how these cyclin E modifications result in increased cyclin E levels by avoiding the Cul3 degradation pathway. Title: The Cul3 Ubiquitin Ligase: An Essential Regulator of Diverse Cellular Processes Author: Davidge, Brittney Marie Year: 2017 Cul3 forms E3 ubiquitin ligase complexes that regulate a variety of cellular processes. This dissertation describes Cul3‘s role in several of these pathways and provides new mechanistic details regarding the role of Cul3 in eukaryotic cells. Cyclin E is an example of a protein that is regulated in a Cul3-dependent manner. Cyclin E is a cell cycle regulator that controls the beginning of DNA replication in mammalian cells. Increased levels of cyclin E are found in some cancers, in addition, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with tumorigenesis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligase complexes, Cul1 and Cul3. Cul1 mediated degradation of cyclin E is triggered by cyclin E phosphorylation, however the mechanism Cul3 uses to ubiquitinate cyclin E is poorly understood. In order to gain a better understanding of how Cul3 mediates cyclin E destruction we identified the degron on cyclin E that is important in Cul3 dependent degradation. In addition, we show this degron is lacking in LMW cyclin E (found in abundance in breast cancer), providing a novel mechanism for how these cyclin E modifications result in increased cyclin E levels by avoiding the Cul3 degradation pathway. Close

• 3. [Article] Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i>

  The Annual Killifish, Austrofundulus limnaeus, survives the seasonal drying of their pond habitat in the form of embryos entering diapause midway through development. The diapause trajectory is one of ...

  Citation × Citation Citation Abstract Copy Title: Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i> Author: Romney, Amie Lynn Thomas Year: 2017 The Annual Killifish, Austrofundulus limnaeus, survives the seasonal drying of their pond habitat in the form of embryos entering diapause midway through development. The diapause trajectory is one of two developmental phenotypes. Alternatively, individuals can "escape" entry into diapause and develop continuously until hatching. The alternative phenotypes of A. limnaeus are a form of developmental plasticity that provides this species with a physiological adaption for surviving stressful environments. The developmental trajectory of an embryo is not distinguishable morphologically upon fertilization and phenotype is believed to be influenced by maternal provisioning within the egg based on observations of offspring phenotype production. However, incubation temperature may override any such maternal pattern suggesting an environmental influence on the regulation of developmental trajectory. We hypothesize that maternally packaged gene products coordinate the cellular events prior to the maternal-to-zygotic transition (MTZ) that determine developmental trajectory in embryos of A. limnaeus. In addition, we propose that environmentally responsive gene expression after the MTZ can sustain or override any such maternal provisioning. Using high-throughput RNA-sequencing, we have generated transcriptomic profiles of protein-coding messenger RNA and noncoding RNA during development in A. limnaeus. Embryos destined for either the diapause or escape phenotypes display unique expression profiles immediately upon fertilization that support hormone synthesis, well before the stage when phenotypes are morphologically distinct. At stages when the trajectories diverge from one another, differential expression of the vitamin D receptor signaling pathway suggests that vitamin D signaling may be a key regulator of developmental phenotype in this species. These data provide a critical link between maternal and environmental influences on the genetic regulation of phenotypic plasticity. These results will not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression and development. Uncovering genetic mechanisms in a system exhibiting alternative developmental trajectories will elucidate the role of maternal packaging in regulating developmental decisions, and in sustaining metabolic depression during diapause. Title: Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish <i>Austrofundulus limnaeus</i> Author: Romney, Amie Lynn Thomas Year: 2017 The Annual Killifish, Austrofundulus limnaeus, survives the seasonal drying of their pond habitat in the form of embryos entering diapause midway through development. The diapause trajectory is one of two developmental phenotypes. Alternatively, individuals can "escape" entry into diapause and develop continuously until hatching. The alternative phenotypes of A. limnaeus are a form of developmental plasticity that provides this species with a physiological adaption for surviving stressful environments. The developmental trajectory of an embryo is not distinguishable morphologically upon fertilization and phenotype is believed to be influenced by maternal provisioning within the egg based on observations of offspring phenotype production. However, incubation temperature may override any such maternal pattern suggesting an environmental influence on the regulation of developmental trajectory. We hypothesize that maternally packaged gene products coordinate the cellular events prior to the maternal-to-zygotic transition (MTZ) that determine developmental trajectory in embryos of A. limnaeus. In addition, we propose that environmentally responsive gene expression after the MTZ can sustain or override any such maternal provisioning. Using high-throughput RNA-sequencing, we have generated transcriptomic profiles of protein-coding messenger RNA and noncoding RNA during development in A. limnaeus. Embryos destined for either the diapause or escape phenotypes display unique expression profiles immediately upon fertilization that support hormone synthesis, well before the stage when phenotypes are morphologically distinct. At stages when the trajectories diverge from one another, differential expression of the vitamin D receptor signaling pathway suggests that vitamin D signaling may be a key regulator of developmental phenotype in this species. These data provide a critical link between maternal and environmental influences on the genetic regulation of phenotypic plasticity. These results will not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression and development. Uncovering genetic mechanisms in a system exhibiting alternative developmental trajectories will elucidate the role of maternal packaging in regulating developmental decisions, and in sustaining metabolic depression during diapause. Close

• 4. [Article] ImpL2 Represses Insulin Signaling in Response to Hypoxia

  xiii, 56 p. : ill. (some col.)

  Citation × Citation Citation Abstract Copy Title: ImpL2 Represses Insulin Signaling in Response to Hypoxia Author: Allee, John Paul, 1970- Year: 2011 xiii, 56 p. : ill. (some col.) Title: ImpL2 Represses Insulin Signaling in Response to Hypoxia Author: Allee, John Paul, 1970- Year: 2011 xiii, 56 p. : ill. (some col.) Close

• 5. [Article] The effect of plant growth regulators on the growth of Closterium moniliferum

  Physiologic responses to Gibberellic Acid (GA), I-Naphthalene Acetic Acid (NAA), Benzylaminopurine (BAP), and Abscisic Acid (ABA). suggest that Oosterium monilfferum has the ability to utilize these plant ...

  Citation × Citation Citation Abstract Copy Title: The effect of plant growth regulators on the growth of Closterium moniliferum Author: Christensen, Cynthia Lehua Warnock Year: 1990 Physiologic responses to Gibberellic Acid (GA), I-Naphthalene Acetic Acid (NAA), Benzylaminopurine (BAP), and Abscisic Acid (ABA). suggest that Oosterium monilfferum has the ability to utilize these plant growth factors. The growth promoters NAA and GA both increased growth when added to the media. The cell division regulator BAP (a synthetic cytokinin). also had a promotive effect on growth. Abscisic acid was found to be inhibitive to growth. Title: The effect of plant growth regulators on the growth of Closterium moniliferum Author: Christensen, Cynthia Lehua Warnock Year: 1990 Physiologic responses to Gibberellic Acid (GA), I-Naphthalene Acetic Acid (NAA), Benzylaminopurine (BAP), and Abscisic Acid (ABA). suggest that Oosterium monilfferum has the ability to utilize these plant growth factors. The growth promoters NAA and GA both increased growth when added to the media. The cell division regulator BAP (a synthetic cytokinin). also had a promotive effect on growth. Abscisic acid was found to be inhibitive to growth. Close

• 6. [Article] Robustness of a model microbial community emerges from population structure among single cells of a clonal population

  Microbial populations can withstand, overcome and persist in the face of environmental fluctuation. Previously, we demonstrated how conditional gene regulation in a fluctuating environment drives dilution ...

  Citation × Citation Citation Abstract Copy Title: Robustness of a model microbial community emerges from population structure among single cells of a clonal population Author: Thompson, Anne W., Turkarslan, Serdar, Arens, Christina E., de Lomana, Adrian Lopez Garcia, Raman, Arjun V., Stahl, David A., Baliga, Nitin S. Year: 2017 Microbial populations can withstand, overcome and persist in the face of environmental fluctuation. Previously, we demonstrated how conditional gene regulation in a fluctuating environment drives dilution of condition-specific transcripts, causing a population of Desulfovibrio vulgaris Hildenborough (DvH) to collapse after repeatedly transitioning from sulfate respiration to syntrophic conditions with the methanogen Methanococcus maripaludis. Failure of the DvH to successfully transition contributed to the collapse of this model community. We investigated the mechanistic basis for loss of robustness by examining whether conditional gene regulation altered heterogeneity in gene expression across individual DvH cells. We discovered that robustness of a microbial population across environmental transitions was attributable to the retention of cells in two states that exhibited different condition-specific gene expression patterns. In our experiments, a population with disrupted conditional regulation successfully alternated between cell states. Meanwhile, a population with intact conditional regulation successfully switched between cell states initially, but collapsed after repeated transitions, possibly due to the high energy requirements of regulation. These results demonstrate that the survival of this entire model microbial community is dependent on the regulatory system's influence on the distribution of distinct cell states among individual cells within a clonal population. Title: Robustness of a model microbial community emerges from population structure among single cells of a clonal population Author: Thompson, Anne W., Turkarslan, Serdar, Arens, Christina E., de Lomana, Adrian Lopez Garcia, Raman, Arjun V., Stahl, David A., Baliga, Nitin S. Year: 2017 Microbial populations can withstand, overcome and persist in the face of environmental fluctuation. Previously, we demonstrated how conditional gene regulation in a fluctuating environment drives dilution of condition-specific transcripts, causing a population of Desulfovibrio vulgaris Hildenborough (DvH) to collapse after repeatedly transitioning from sulfate respiration to syntrophic conditions with the methanogen Methanococcus maripaludis. Failure of the DvH to successfully transition contributed to the collapse of this model community. We investigated the mechanistic basis for loss of robustness by examining whether conditional gene regulation altered heterogeneity in gene expression across individual DvH cells. We discovered that robustness of a microbial population across environmental transitions was attributable to the retention of cells in two states that exhibited different condition-specific gene expression patterns. In our experiments, a population with disrupted conditional regulation successfully alternated between cell states. Meanwhile, a population with intact conditional regulation successfully switched between cell states initially, but collapsed after repeated transitions, possibly due to the high energy requirements of regulation. These results demonstrate that the survival of this entire model microbial community is dependent on the regulatory system's influence on the distribution of distinct cell states among individual cells within a clonal population. Close

• 7. [Article] Effects of dehydration on hemoglobin oxygen affinity and blood cell volume in two anurans

  Two aspects of possible adaptation In cardiovascular performance caused by Increased plasma electrolytes were examined. Cells In anisotonic plasma may either act as osmometers or volume regulate. Blood ...

  Citation × Citation Citation Abstract Copy Title: Effects of dehydration on hemoglobin oxygen affinity and blood cell volume in two anurans Author: Zygmunt, Andrew Christopher Year: 1984 Two aspects of possible adaptation In cardiovascular performance caused by Increased plasma electrolytes were examined. Cells In anisotonic plasma may either act as osmometers or volume regulate. Blood flow rate Is dependent upon cell viscosity, which in turn is a consequence of cell volume and membrane deform-ability. Cell volume changes which Increase membrane deform-ability will thus potentially extend the limits of dehydration tolerance. It was found in R. catesbeiana and B. marinus that red blood cell Is maintain constant volume during dehydration. Cells in vitro Initially lose water, but then sodium, potassium and water move Into the cell. Cell viscosity within the physiologic range of hematocrits was higher In salt loaded non-regulating cell Is of B. marinus than In regulating isotonic cells. Title: Effects of dehydration on hemoglobin oxygen affinity and blood cell volume in two anurans Author: Zygmunt, Andrew Christopher Year: 1984 Two aspects of possible adaptation In cardiovascular performance caused by Increased plasma electrolytes were examined. Cells In anisotonic plasma may either act as osmometers or volume regulate. Blood flow rate Is dependent upon cell viscosity, which in turn is a consequence of cell volume and membrane deform-ability. Cell volume changes which Increase membrane deform-ability will thus potentially extend the limits of dehydration tolerance. It was found in R. catesbeiana and B. marinus that red blood cell Is maintain constant volume during dehydration. Cells in vitro Initially lose water, but then sodium, potassium and water move Into the cell. Cell viscosity within the physiologic range of hematocrits was higher In salt loaded non-regulating cell Is of B. marinus than In regulating isotonic cells. Close

• 8. [Article] Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i>

  Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although ...

  Citation × Citation Citation Abstract Copy Title: Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i> Author: Kacharia, Fenil Rashmin Year: 2016 Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria. Title: Investigating the Origin and Functions of a Novel Small RNA in <i>Escherichia coli</i> Author: Kacharia, Fenil Rashmin Year: 2016 Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria. Close

• 9. [Article] Behavioral Temperature Regulation in the Turtle Pseudemys Scripta Elegans

  A group of red-eared turtles (Pseudemys scripta elegans) was acclimated to 18-20˚C and artificial photo-period for 21 days. The animals were then individually placed in a thermal gradient and their selected ...

  Citation × Citation Citation Abstract Copy Title: Behavioral Temperature Regulation in the Turtle Pseudemys Scripta Elegans Author: Johnston, Michael H. Year: 1977 A group of red-eared turtles (Pseudemys scripta elegans) was acclimated to 18-20˚C and artificial photo-period for 21 days. The animals were then individually placed in a thermal gradient and their selected temperatures were continuously recorded for 24 hours. A second group of turtles was acclimatized to 7.5-11.0˚C and natural photoperiods for 28 days, and subsequently tested in the same manner as the first group. Lastly, the turtles in the first group, after being acclimated to 3.0˚C and the previous artificial photoperiod for 14 days, were retested in the gradient. Title: Behavioral Temperature Regulation in the Turtle Pseudemys Scripta Elegans Author: Johnston, Michael H. Year: 1977 A group of red-eared turtles (Pseudemys scripta elegans) was acclimated to 18-20˚C and artificial photo-period for 21 days. The animals were then individually placed in a thermal gradient and their selected temperatures were continuously recorded for 24 hours. A second group of turtles was acclimatized to 7.5-11.0˚C and natural photoperiods for 28 days, and subsequently tested in the same manner as the first group. Lastly, the turtles in the first group, after being acclimated to 3.0˚C and the previous artificial photoperiod for 14 days, were retested in the gradient. Close

• 10. [Article] Neurobiology of Seasonal Life-history Transitions

  Many animals exhibit seasonal changes in life-history stages, and these seasonal transitions are often accompanied by dramatic switches in behavior. While the neuroendocrine mechanisms that regulate such ...

  Citation × Citation Citation Abstract Copy Title: Neurobiology of Seasonal Life-history Transitions Author: Lucas, Ashley Rae Year: 2015 Many animals exhibit seasonal changes in life-history stages, and these seasonal transitions are often accompanied by dramatic switches in behavior. While the neuroendocrine mechanisms that regulate such behavioral transitions are poorly understood, arginine vasotocin (AVT) and neuropeptide Y (NPY) are excellent candidates because they regulate reproductive and feeding behavior, respectively. In this study, I asked if seasonal changes in AVT and/or NPY are concomitant with spring migration away from the breeding grounds, as male and female red-sided garter snakes (Thamnophis sirtalis parietalis) are transitioning from reproductive to non-reproductive behavior during this time. To address this question, I collected male and female snakes in different migratory stages during the spring and fall. Brains were processed for AVT and NPY immunohistochemistry and the total number of immunoreactive (-ir) cells quantified for each individual. As predicted, males had significantly more AVT-ir cells in the preoptic area and bed nucleus of the stria terminalis, brain regions important for courtship behavior, during the spring mating season compared to the fall. Females had significantly more AVT-ir cells in the preoptic area during the spring compared to the fall and, surprisingly, did not exhibit seasonal changes in NPY. In contrast, males had significantly more NPY-ir cells in the cortex, a region important for spatial memory, and in the posterior hypothalamus during the fall compared to the spring, which likely reflects increased feeding behavior during the summer foraging period. Neither AVT- nor NPY-ir cell number varied significantly with migratory status, indicating that seasonal changes in these neuropeptides are not directly related to migration. I then asked if the observed seasonal changes in AVT and NPY in males and females are related to the transition from reproductive to non-reproductive states. Compared to courting males, non-courting males had significantly more AVT-ir cells in the supraoptic nucleus and more NPY-ir cells in the cortex. AVT- and NPY-ir cells did not differ between unmated and mated females. Collectively, my results suggest that AVT and NPY play a role in regulating seasonal transitions in male reproductive behavior, rather than regulating migration per se. Further, these data indicate that both AVT and NPY are regulating reproductive behavior differently in males versus females. These data provide the framework for future studies examining the mechanisms regulating transitions between reproductive, migratory and foraging behaviors. Title: Neurobiology of Seasonal Life-history Transitions Author: Lucas, Ashley Rae Year: 2015 Many animals exhibit seasonal changes in life-history stages, and these seasonal transitions are often accompanied by dramatic switches in behavior. While the neuroendocrine mechanisms that regulate such behavioral transitions are poorly understood, arginine vasotocin (AVT) and neuropeptide Y (NPY) are excellent candidates because they regulate reproductive and feeding behavior, respectively. In this study, I asked if seasonal changes in AVT and/or NPY are concomitant with spring migration away from the breeding grounds, as male and female red-sided garter snakes (Thamnophis sirtalis parietalis) are transitioning from reproductive to non-reproductive behavior during this time. To address this question, I collected male and female snakes in different migratory stages during the spring and fall. Brains were processed for AVT and NPY immunohistochemistry and the total number of immunoreactive (-ir) cells quantified for each individual. As predicted, males had significantly more AVT-ir cells in the preoptic area and bed nucleus of the stria terminalis, brain regions important for courtship behavior, during the spring mating season compared to the fall. Females had significantly more AVT-ir cells in the preoptic area during the spring compared to the fall and, surprisingly, did not exhibit seasonal changes in NPY. In contrast, males had significantly more NPY-ir cells in the cortex, a region important for spatial memory, and in the posterior hypothalamus during the fall compared to the spring, which likely reflects increased feeding behavior during the summer foraging period. Neither AVT- nor NPY-ir cell number varied significantly with migratory status, indicating that seasonal changes in these neuropeptides are not directly related to migration. I then asked if the observed seasonal changes in AVT and NPY in males and females are related to the transition from reproductive to non-reproductive states. Compared to courting males, non-courting males had significantly more AVT-ir cells in the supraoptic nucleus and more NPY-ir cells in the cortex. AVT- and NPY-ir cells did not differ between unmated and mated females. Collectively, my results suggest that AVT and NPY play a role in regulating seasonal transitions in male reproductive behavior, rather than regulating migration per se. Further, these data indicate that both AVT and NPY are regulating reproductive behavior differently in males versus females. These data provide the framework for future studies examining the mechanisms regulating transitions between reproductive, migratory and foraging behaviors. Close

• 11. [Article] Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation

  Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations ...

  Citation × Citation Citation Abstract Copy Title: Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation Author: Cole, Jessica, Ferguson, Amanda, Segarra, Verónica A., Walsh, Susan Year: 2017 Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST)-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions. Title: Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation Author: Cole, Jessica, Ferguson, Amanda, Segarra, Verónica A., Walsh, Susan Year: 2017 Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST)-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions. Close

• 12. [Article] Mechanisms of Animal Diapause: Recent Developments from Nematodes, Crustaceans, Insects, and Fish

  Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, ...

  Citation × Citation Citation Abstract Copy Title: Mechanisms of Animal Diapause: Recent Developments from Nematodes, Crustaceans, Insects, and Fish Author: Hand, Steven C., Denlinger, David L., Podrabsky, Jason E., Roy, Richard Year: 2016 Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in post-dauer animals have been documented in Caenorhabditis elegans It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages. Title: Mechanisms of Animal Diapause: Recent Developments from Nematodes, Crustaceans, Insects, and Fish Author: Hand, Steven C., Denlinger, David L., Podrabsky, Jason E., Roy, Richard Year: 2016 Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in post-dauer animals have been documented in Caenorhabditis elegans It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages. Close

• 13. [Article] Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i>

  Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus ...

  Citation × Citation Citation Abstract Copy Title: Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i> Author: Romney, Amie L., Podrabsky, Jason E. Year: 2017 Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus is unique among vertebrates because the embryos have distinct cell movements that separate epiboly from axis formation during early development, can enter into a state of metabolic dormancy known as diapause and can survive extreme environmental conditions. The ability to enter into diapause can be maternally programmed, with young females producing embryos that do not enter into diapause. Alternately, embryos can be programmed to “escape” from diapause and develop directly by both maternal factors and embryonic incubation conditions. Thus, maternally packaged gene products are hypothesized to regulate developmental trajectory and perhaps the other unique developmental characters in this species. Results: Using high-throughput RNA sequencing, we generated transcriptomic profles of mRNAs, long non-coding RNAs and small non-coding RNAs (sncRNAs) in 1–2 cell stage embryos of A. limnaeus. Transcriptomic analyses suggest maternal programming of embryos through alternatively spliced mRNAs and antisense sncRNAs. Comparison of these results to those of comparable studies on zebrafsh and other fshes reveals a surprisingly high abundance of transcripts involved in the cellular response to stress and a relatively lower expression of genes required for rapid transition through the cell cycle. Conclusions: Maternal programming of developmental trajectory is unlikely accomplished by diferential expression of diapause-specifc genes. Rather, evidence suggests a role for trajectory-specifc splice variants of genes expressed in both phenotypes. In addition, based on comparative studies with zebrafsh, the A. limnaeus 1–2 cell stage transcriptome is unique in ways that are consistent with their unique life history. These results not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression during development Title: Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i> Author: Romney, Amie L., Podrabsky, Jason E. Year: 2017 Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus is unique among vertebrates because the embryos have distinct cell movements that separate epiboly from axis formation during early development, can enter into a state of metabolic dormancy known as diapause and can survive extreme environmental conditions. The ability to enter into diapause can be maternally programmed, with young females producing embryos that do not enter into diapause. Alternately, embryos can be programmed to “escape” from diapause and develop directly by both maternal factors and embryonic incubation conditions. Thus, maternally packaged gene products are hypothesized to regulate developmental trajectory and perhaps the other unique developmental characters in this species. Results: Using high-throughput RNA sequencing, we generated transcriptomic profles of mRNAs, long non-coding RNAs and small non-coding RNAs (sncRNAs) in 1–2 cell stage embryos of A. limnaeus. Transcriptomic analyses suggest maternal programming of embryos through alternatively spliced mRNAs and antisense sncRNAs. Comparison of these results to those of comparable studies on zebrafsh and other fshes reveals a surprisingly high abundance of transcripts involved in the cellular response to stress and a relatively lower expression of genes required for rapid transition through the cell cycle. Conclusions: Maternal programming of developmental trajectory is unlikely accomplished by diferential expression of diapause-specifc genes. Rather, evidence suggests a role for trajectory-specifc splice variants of genes expressed in both phenotypes. In addition, based on comparative studies with zebrafsh, the A. limnaeus 1–2 cell stage transcriptome is unique in ways that are consistent with their unique life history. These results not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression during development Close

• 14. [Article] Insulin-like growth factor signaling regulates developmental trajectory associated with diapause in embryos of the annual killifish Austrofundulus limnaeus

  Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the ...

  Citation × Citation Citation Abstract Copy Title: Insulin-like growth factor signaling regulates developmental trajectory associated with diapause in embryos of the annual killifish Austrofundulus limnaeus Author: Woll, S. Cody, Podrabsky, Jason E. Year: 2017 Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the embryonic blastomeres, and a short postembryonic life span. Insulin-like growth factor (IGF) signaling is known to play a role in the regulation of metabolic dormancy in a number of animals but has not been explored in annual killifishes. The abundance of IGF proteins during development and the developmental effects of blocking IGF signaling by pharmacological inhibition of the insulin-like growth factor I receptor (IGF1R) were explored in embryos of the annual killifish Austrofundulus limnaeus. Blocking of IGF signaling in embryos that would normally escape entrance into diapause resulted in a phenotype that was remarkably similar to that of embryos entering diapause. IGF-I protein abundance spikes during early development in embryos that will not enter diapause. In contrast, IGF-I levels remain low during early development in embryos that will enter diapause II. IGF-II protein is packaged at higher levels in escape-bound embryos compared with diapause-bound embryos. However, IGF-II levels quickly decrease and remain low during early development and only increase substantially during late development in both developmental trajectories. Developmental patterns of IGF-I and IGF-II protein abundance under conditions that would either induce or bypass entrance into diapause are consistent with a role for IGF signaling in the regulation of developmental trajectory and entrance into diapause in this species. We propose that IGF signaling may be a unifying regulatory pathway that explains the larger suite of characters that are associated with the complex life history of annual killifishes. Title: Insulin-like growth factor signaling regulates developmental trajectory associated with diapause in embryos of the annual killifish Austrofundulus limnaeus Author: Woll, S. Cody, Podrabsky, Jason E. Year: 2017 Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the embryonic blastomeres, and a short postembryonic life span. Insulin-like growth factor (IGF) signaling is known to play a role in the regulation of metabolic dormancy in a number of animals but has not been explored in annual killifishes. The abundance of IGF proteins during development and the developmental effects of blocking IGF signaling by pharmacological inhibition of the insulin-like growth factor I receptor (IGF1R) were explored in embryos of the annual killifish Austrofundulus limnaeus. Blocking of IGF signaling in embryos that would normally escape entrance into diapause resulted in a phenotype that was remarkably similar to that of embryos entering diapause. IGF-I protein abundance spikes during early development in embryos that will not enter diapause. In contrast, IGF-I levels remain low during early development in embryos that will enter diapause II. IGF-II protein is packaged at higher levels in escape-bound embryos compared with diapause-bound embryos. However, IGF-II levels quickly decrease and remain low during early development and only increase substantially during late development in both developmental trajectories. Developmental patterns of IGF-I and IGF-II protein abundance under conditions that would either induce or bypass entrance into diapause are consistent with a role for IGF signaling in the regulation of developmental trajectory and entrance into diapause in this species. We propose that IGF signaling may be a unifying regulatory pathway that explains the larger suite of characters that are associated with the complex life history of annual killifishes. Close

• 15. [Article] Epigenetic Regulation of Alternative Developmental Trajectories

  The annual killifish, Austrofundulus limnaeus, is capable of entering into a state of metabolic and developmental dormancy during embryogenesis termed diapause. However, individuals may also follow an ...

  Citation × Citation Citation Abstract Copy Title: Epigenetic Regulation of Alternative Developmental Trajectories Author: Romney, Arnie Lynn T., Podrabsky, Jason E. Year: 2017 The annual killifish, Austrofundulus limnaeus, is capable of entering into a state of metabolic and developmental dormancy during embryogenesis termed diapause. However, individuals may also follow an alternative phenotypic trajectory and instead "escaped" entry into diapause and develop continuously until hatching. Early development along the two trajectories is morphologically indistinguishable, and gene regulatory mechanisms are currently unknown. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately escape embryos and older females produce diapausing embryos. Additionally, the incubation temperature of embryos can override this maternal pattern and alter trajectory. Maternal provisioning is known in many vertebrates to direct early development prior to the initiation of embryonic gene transcription. Using RNA-seq, we have generated transcriptomic profiles of messenger RNAs (mRNAs) during embryogenesis of A. limnaeus. Embryos that are destined for either the diapause or escape phenotypes have unique profiles of mRNA isoforms at fertilization; well before they are morphologically distinct. We hypothesize that maternally packaged gene products coordinate the cellular events that determine developmental trajectory in A. limnaeus. In addition, we propose the expression of environmentally responsive microRNAs during development that can override maternal provisioning. RNA-seq data during early development, after the maternal-to-zygotic transition, suggest distinct profiles of microRNA expression that are phenotype specific. Our findings suggest maternal programming of diapause through the packaging of specific mRNAs as well as temperature induced microRNAs that target maternal transcripts allowing an escape from the diapause trajectory. This presents a novel example of maternal-embryonic-conflict in gene regulation that provides the embryo with alternatives in developmental trajectories in the face of fluctuating environmental patterns. These results will not only impact our understanding of genetic mechanisms that regulate entrance into diapause, but will also provide insight into epigenetic control of development. Title: Epigenetic Regulation of Alternative Developmental Trajectories Author: Romney, Arnie Lynn T., Podrabsky, Jason E. Year: 2017 The annual killifish, Austrofundulus limnaeus, is capable of entering into a state of metabolic and developmental dormancy during embryogenesis termed diapause. However, individuals may also follow an alternative phenotypic trajectory and instead "escaped" entry into diapause and develop continuously until hatching. Early development along the two trajectories is morphologically indistinguishable, and gene regulatory mechanisms are currently unknown. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately escape embryos and older females produce diapausing embryos. Additionally, the incubation temperature of embryos can override this maternal pattern and alter trajectory. Maternal provisioning is known in many vertebrates to direct early development prior to the initiation of embryonic gene transcription. Using RNA-seq, we have generated transcriptomic profiles of messenger RNAs (mRNAs) during embryogenesis of A. limnaeus. Embryos that are destined for either the diapause or escape phenotypes have unique profiles of mRNA isoforms at fertilization; well before they are morphologically distinct. We hypothesize that maternally packaged gene products coordinate the cellular events that determine developmental trajectory in A. limnaeus. In addition, we propose the expression of environmentally responsive microRNAs during development that can override maternal provisioning. RNA-seq data during early development, after the maternal-to-zygotic transition, suggest distinct profiles of microRNA expression that are phenotype specific. Our findings suggest maternal programming of diapause through the packaging of specific mRNAs as well as temperature induced microRNAs that target maternal transcripts allowing an escape from the diapause trajectory. This presents a novel example of maternal-embryonic-conflict in gene regulation that provides the embryo with alternatives in developmental trajectories in the face of fluctuating environmental patterns. These results will not only impact our understanding of genetic mechanisms that regulate entrance into diapause, but will also provide insight into epigenetic control of development. Close

• 16. [Article] The metabolic cost of behavioral thermoregulation of body temperature in the northern alligator lizard Gerrhonotus coeruleus, and how it affects the classical concept of eurythermality

  The effect of total metabolic cost expenditures on the precision of behavioral thermoregulation was investigated for the purportedly eurythermic Northern Alligator lizard (Gerrhonotus coeruleus). An operant ...

  Citation × Citation Citation Abstract Copy Title: The metabolic cost of behavioral thermoregulation of body temperature in the northern alligator lizard Gerrhonotus coeruleus, and how it affects the classical concept of eurythermality Author: Campbell, James Dudley Year: 1981 The effect of total metabolic cost expenditures on the precision of behavioral thermoregulation was investigated for the purportedly eurythermic Northern Alligator lizard (Gerrhonotus coeruleus). An operant apparatus was designed to test metabolic output at different heat reinforcement magnitudes. The mean TB reflected in each trial was positively correlated to the length of reinforcement. The shuttle rate during each trial was inversely correlated to the length of reinforcement. The standard deviation and total metabolic costs did not vary significantly between trials undertaken at the same ambient temperature. Eurythermality in G. coeruleus is caused by fluctuations in preferred body temperature and not by fluctuations around this temperature. The metabolic cost of behavioral thermoregulation did not change with corresponding changes in reinforcement magnitude. This indicates that eurythermality is the recorded effect of lizards behaviorally regulating to different TB in a stenothermic manner, rather than of random TB fluctuations in a wide range of normal activity (the classical view of eurythermality). Title: The metabolic cost of behavioral thermoregulation of body temperature in the northern alligator lizard Gerrhonotus coeruleus, and how it affects the classical concept of eurythermality Author: Campbell, James Dudley Year: 1981 The effect of total metabolic cost expenditures on the precision of behavioral thermoregulation was investigated for the purportedly eurythermic Northern Alligator lizard (Gerrhonotus coeruleus). An operant apparatus was designed to test metabolic output at different heat reinforcement magnitudes. The mean TB reflected in each trial was positively correlated to the length of reinforcement. The shuttle rate during each trial was inversely correlated to the length of reinforcement. The standard deviation and total metabolic costs did not vary significantly between trials undertaken at the same ambient temperature. Eurythermality in G. coeruleus is caused by fluctuations in preferred body temperature and not by fluctuations around this temperature. The metabolic cost of behavioral thermoregulation did not change with corresponding changes in reinforcement magnitude. This indicates that eurythermality is the recorded effect of lizards behaviorally regulating to different TB in a stenothermic manner, rather than of random TB fluctuations in a wide range of normal activity (the classical view of eurythermality). Close

• 17. [Article] Biological Rhythms: Melatonin Shapes the Space-Time Continuum of Social Communication

  A new study shows that nocturnal courtship vocalization is regulated by a circadian rhythm and potentiated by melatonin at multiple timescales. These findings are in contrast to those in diurnal vocalizers ...

  Citation × Citation Citation Abstract Copy Title: Biological Rhythms: Melatonin Shapes the Space-Time Continuum of Social Communication Author: Wilczynski, Walter, Lutterschmidt, Deborah I. Year: 2016 A new study shows that nocturnal courtship vocalization is regulated by a circadian rhythm and potentiated by melatonin at multiple timescales. These findings are in contrast to those in diurnal vocalizers and provide a striking example of melatonin's niche-specific functions. Title: Biological Rhythms: Melatonin Shapes the Space-Time Continuum of Social Communication Author: Wilczynski, Walter, Lutterschmidt, Deborah I. Year: 2016 A new study shows that nocturnal courtship vocalization is regulated by a circadian rhythm and potentiated by melatonin at multiple timescales. These findings are in contrast to those in diurnal vocalizers and provide a striking example of melatonin's niche-specific functions. Close

• 18. [Article] Investigating the Role of Small Noncoding RNAs in Vertebrate Anoxia Tolerance

  Very few vertebrates survive extended periods of time without oxygen. Entry into metabolic depression is central to surviving anoxia, which is supported by overall suppression of protein synthesis, yet ...

  Citation × Citation Citation Abstract Copy Title: Investigating the Role of Small Noncoding RNAs in Vertebrate Anoxia Tolerance Author: Riggs, Claire Louise Year: 2017 Very few vertebrates survive extended periods of time without oxygen. Entry into metabolic depression is central to surviving anoxia, which is supported by overall suppression of protein synthesis, yet requires increased expression of specific proteins. Studying the rapid and complex regulation of gene expression associated with survival of anoxia may uncover new mechanisms of cellular biology and transform our understanding of cells, as well as inform prevention and treatment of heart attack and stroke in humans. Small non-coding RNAs (sncRNAs) have emerged as regulators of gene expression that can be rapidly employed, can target individual genes or suites of genes, and are highly conserved across species. There are diverse types of sncRNAs, some coopted from degradation of longer RNAs in the cell. The sncRNA revolution has yielded a large body of literature revealing the roles of sncRNAs in a myriad of biological processes, from development to regulation of the cell cycle and apoptosis, to responding to stress, including freezing, dehydration, ischemia, and anoxia. Given the regulatory complexity required to survive anoxia, examining sncRNAs in the context of extreme anoxia tolerance has the potential to expand our understanding of the role that sncRNAs may play in basic cell biology, as well as in response to stresses such as anoxia. A comparative model including anoxia-tolerant and anoxia-sensitive phenotypes allows us to better identify sncRNAs that likely play a critical role in anoxia tolerance. Embryos of A. limnaeus are the most anoxia tolerant vertebrate known and are comprised of a range of anoxia-tolerance phenotypes. These characteristics create a unique opportunity for comparative study of the role of sncRNAs in anoxia tolerance in phenotypes with a common genomic background. The overall goals of this project were to: (1) describe the sncRNA transcriptome and changes in its expression in response to anoxia in the embryos of A. limnaeus and in other anoxia-tolerant vertebrates, and (2) to identify specific sncRNAs of interest based on these sequencing projects and to follow-up on their biogenesis, localization, and function in A. limnaeus embryos and a continuous cell line derived from A. limnaeus embryos. Chapter 2 focuses on the identity and expression of sncRNAs in embryos of A. limnaeus in 4 embryonic stages that differ in their anoxia tolerance and physiology. Chapter 3 explores sncRNA expression in brain tissue (the most oxygen-sensitive organ) in other anoxia-tolerant vertebrates: the crucian carp, western painted turtle, leopard frog, and epaulette shark. This allows us to assess the similarities and differences in sncRNA biology in species that evolved anoxia independently, and put the findings from A. limnaeus in an evolutionary context. Chapter 4 describes the establishment of the AL4 anoxia-tolerant cell line derived from A. limnaeus embryos, which allows for more detailed study of particular sncRNAs of interest in Chapter 5. Using whole embryos of A. limnaeus and the AL4 cell line, Chapter 5 describes the expression, localization, and possible biogenesis and mechanism of action of mitochondria-derived sncRNAs, known as mitosRNAs. Chapter 6 summarizes the findings and discusses potential future directions. The work in this dissertation represents the first global survey of sncRNA expression in anoxia tolerant vertebrates. While many interesting patterns of expression were identified, the most interesting discovery is the expression of sncRNAs that are generated in the mitochondria, but have the potential to function in other compartments of the cell. This discovery could transform the way we view the role of the mitochondria in regulating gene expression in eukaryotic cells. Title: Investigating the Role of Small Noncoding RNAs in Vertebrate Anoxia Tolerance Author: Riggs, Claire Louise Year: 2017 Very few vertebrates survive extended periods of time without oxygen. Entry into metabolic depression is central to surviving anoxia, which is supported by overall suppression of protein synthesis, yet requires increased expression of specific proteins. Studying the rapid and complex regulation of gene expression associated with survival of anoxia may uncover new mechanisms of cellular biology and transform our understanding of cells, as well as inform prevention and treatment of heart attack and stroke in humans. Small non-coding RNAs (sncRNAs) have emerged as regulators of gene expression that can be rapidly employed, can target individual genes or suites of genes, and are highly conserved across species. There are diverse types of sncRNAs, some coopted from degradation of longer RNAs in the cell. The sncRNA revolution has yielded a large body of literature revealing the roles of sncRNAs in a myriad of biological processes, from development to regulation of the cell cycle and apoptosis, to responding to stress, including freezing, dehydration, ischemia, and anoxia. Given the regulatory complexity required to survive anoxia, examining sncRNAs in the context of extreme anoxia tolerance has the potential to expand our understanding of the role that sncRNAs may play in basic cell biology, as well as in response to stresses such as anoxia. A comparative model including anoxia-tolerant and anoxia-sensitive phenotypes allows us to better identify sncRNAs that likely play a critical role in anoxia tolerance. Embryos of A. limnaeus are the most anoxia tolerant vertebrate known and are comprised of a range of anoxia-tolerance phenotypes. These characteristics create a unique opportunity for comparative study of the role of sncRNAs in anoxia tolerance in phenotypes with a common genomic background. The overall goals of this project were to: (1) describe the sncRNA transcriptome and changes in its expression in response to anoxia in the embryos of A. limnaeus and in other anoxia-tolerant vertebrates, and (2) to identify specific sncRNAs of interest based on these sequencing projects and to follow-up on their biogenesis, localization, and function in A. limnaeus embryos and a continuous cell line derived from A. limnaeus embryos. Chapter 2 focuses on the identity and expression of sncRNAs in embryos of A. limnaeus in 4 embryonic stages that differ in their anoxia tolerance and physiology. Chapter 3 explores sncRNA expression in brain tissue (the most oxygen-sensitive organ) in other anoxia-tolerant vertebrates: the crucian carp, western painted turtle, leopard frog, and epaulette shark. This allows us to assess the similarities and differences in sncRNA biology in species that evolved anoxia independently, and put the findings from A. limnaeus in an evolutionary context. Chapter 4 describes the establishment of the AL4 anoxia-tolerant cell line derived from A. limnaeus embryos, which allows for more detailed study of particular sncRNAs of interest in Chapter 5. Using whole embryos of A. limnaeus and the AL4 cell line, Chapter 5 describes the expression, localization, and possible biogenesis and mechanism of action of mitochondria-derived sncRNAs, known as mitosRNAs. Chapter 6 summarizes the findings and discusses potential future directions. The work in this dissertation represents the first global survey of sncRNA expression in anoxia tolerant vertebrates. While many interesting patterns of expression were identified, the most interesting discovery is the expression of sncRNAs that are generated in the mitochondria, but have the potential to function in other compartments of the cell. This discovery could transform the way we view the role of the mitochondria in regulating gene expression in eukaryotic cells. Close

• 19. [Article] Gene Expression Patterns of Bone Morphogenetic Proteins (BMPs) During Early Embryonic Development in The Annual Killifish Austrofundulus Limnaeus

  The developmental pattern of the annual killifish Austrofundulus limnaeus is unusual compared to other fish. First, the cell movements associated with gastrulation are separated from formation of the embryonic ...

  Citation × Citation Citation Abstract Copy Title: Gene Expression Patterns of Bone Morphogenetic Proteins (BMPs) During Early Embryonic Development in The Annual Killifish Austrofundulus Limnaeus Author: Chan, Eva Y Year: 2016 The developmental pattern of the annual killifish Austrofundulus limnaeus is unusual compared to other fish. First, the cell movements associated with gastrulation are separated from formation of the embryonic axis. In addition, embryonic diapause can occur. Diapause II and III are often observed when embryos are incubated at 25oC. Maternal influences and incubation environment are the factors that determine entrance into embryonic diapause, but the molecular mechanisms that regulate diapause are still unknown. Interestingly, embryonic diapause and the unusual cell movements observed during early development are always found together in species of annual killifish that exhibit diapause. Bone morphogenetic proteins (BMPS) are crucial in the development of various embryonic tissues. A recent study found the gene expressions pattern of BMP antagonists, noggin, chordin and follistatin, in early - embryonic development in A. limnaeus are different from zebrafish. This finding suggests BMPs may play roles in the regulation of diapause. In the present paper, the expression of BMP-2, BMP-4 and the BMP Receptor-2 were examined during early embryonic development in A. limnaeus using reverse transcription polymerase chain reaction (RT-PCR). Title: Gene Expression Patterns of Bone Morphogenetic Proteins (BMPs) During Early Embryonic Development in The Annual Killifish Austrofundulus Limnaeus Author: Chan, Eva Y Year: 2016 The developmental pattern of the annual killifish Austrofundulus limnaeus is unusual compared to other fish. First, the cell movements associated with gastrulation are separated from formation of the embryonic axis. In addition, embryonic diapause can occur. Diapause II and III are often observed when embryos are incubated at 25oC. Maternal influences and incubation environment are the factors that determine entrance into embryonic diapause, but the molecular mechanisms that regulate diapause are still unknown. Interestingly, embryonic diapause and the unusual cell movements observed during early development are always found together in species of annual killifish that exhibit diapause. Bone morphogenetic proteins (BMPS) are crucial in the development of various embryonic tissues. A recent study found the gene expressions pattern of BMP antagonists, noggin, chordin and follistatin, in early - embryonic development in A. limnaeus are different from zebrafish. This finding suggests BMPs may play roles in the regulation of diapause. In the present paper, the expression of BMP-2, BMP-4 and the BMP Receptor-2 were examined during early embryonic development in A. limnaeus using reverse transcription polymerase chain reaction (RT-PCR). Close

• 20. [Article] Effect of ethanol on thermoregulation in the goldfish, Carassius auratus

  In an attempt to elucidate the mechanism by which ethanol affects vertebrate thermoregulation, the effect of ethanol on temperature selection was studied in the goldfish, Carassius auratus. Ethanol was ...

  Citation × Citation Citation Abstract Copy Title: Effect of ethanol on thermoregulation in the goldfish, Carassius auratus Author: O'Connor, Candace Sharon Year: 1986 In an attempt to elucidate the mechanism by which ethanol affects vertebrate thermoregulation, the effect of ethanol on temperature selection was studied in the goldfish, Carassius auratus. Ethanol was administered to 10 to 15 g fish by mixing it in the water of a temperature gradient. The dose response curve was very steep between 0.5% (v/v) ethanol (no response) and 0.7% (significant lowering of selected temperature in treated fish). Fish were exposed to concentrations of ethanol as high as 1.7%, at which concentration most experimental fish lost their ability to swim upright in the water. At concentrations higher than 0.7%, the magnitude of the effect did not increase with increasing concentration of ethanol; treated animals continued to select temperatures about 2 C below temperatures selected by controls. Experiments alternating exposure to 1.0% ethanol and water showed that the rate of onset and disappearance of the ethanol effect was rapid (within 10 min). Other experiments exposing fish to 1.0% ethanol for up to 3 hr showed that the effect remained stable for this period of time. The thermoregulatory responses of fish are behavioral, and therefore relatively easy to observe and quantify. Ethanol produces a prompt, stable and reproducible depression of selected temperature in the goldfish. Because the temperature at which fish regulate is controlled by a central nervous system set point and not altered by effects on peripheral effector systems, it appears that ethanol may cause hypothermia in goldfish by directly acting to lower the set point. Title: Effect of ethanol on thermoregulation in the goldfish, Carassius auratus Author: O'Connor, Candace Sharon Year: 1986 In an attempt to elucidate the mechanism by which ethanol affects vertebrate thermoregulation, the effect of ethanol on temperature selection was studied in the goldfish, Carassius auratus. Ethanol was administered to 10 to 15 g fish by mixing it in the water of a temperature gradient. The dose response curve was very steep between 0.5% (v/v) ethanol (no response) and 0.7% (significant lowering of selected temperature in treated fish). Fish were exposed to concentrations of ethanol as high as 1.7%, at which concentration most experimental fish lost their ability to swim upright in the water. At concentrations higher than 0.7%, the magnitude of the effect did not increase with increasing concentration of ethanol; treated animals continued to select temperatures about 2 C below temperatures selected by controls. Experiments alternating exposure to 1.0% ethanol and water showed that the rate of onset and disappearance of the ethanol effect was rapid (within 10 min). Other experiments exposing fish to 1.0% ethanol for up to 3 hr showed that the effect remained stable for this period of time. The thermoregulatory responses of fish are behavioral, and therefore relatively easy to observe and quantify. Ethanol produces a prompt, stable and reproducible depression of selected temperature in the goldfish. Because the temperature at which fish regulate is controlled by a central nervous system set point and not altered by effects on peripheral effector systems, it appears that ethanol may cause hypothermia in goldfish by directly acting to lower the set point. Close