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• 1. [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

• 2. [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

• 3. [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

• 4. [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

• 5. [Article] Mitochondria-derived Small Non-coding RNAs in Extreme Anoxia Tolerance

  Mitochondria are critical sensors of oxygen and play a major role in the regulation of the cellular response to oxygen deprivation. Understanding mitochondrial function during transitions into and out ...

  Citation × Citation Citation Abstract Copy Title: Mitochondria-derived Small Non-coding RNAs in Extreme Anoxia Tolerance Author: Riggs, Claire L., Podrabsky, Jason E. Year: 2017 Mitochondria are critical sensors of oxygen and play a major role in the regulation of the cellular response to oxygen deprivation. Understanding mitochondrial function during transitions into and out of anoxia in species that can tolerate anoxia may lend new insights into the role that mitochondria play in cellular homeostasis in response to fluctuating levels of oxygen. The objective of this study was to examine the potential role of small RNAs in long-term vertebrate anoxia tolerance. Expression patterns of small RNAs were determined using RNAseq in the most anoxia tolerant vertebrate, embryos of the annual killifishAustrofundulus limnaeus. Anoxia tolerance varies during development in this species in a way that allows for the comparison of developmental stages with extreme anoxia tolerance to stages with a more typical vertebrate anoxia tolerance. Embryos of 4 different developmental stages were exposed to anoxia, followed by aerobic recovery, and small RNA expression was determined with Illumina RNAseq under aerobic conditions, anoxia, and during aerobic recovery. A large proportion of the differentially expressed small RNAs were found to be encoded in the mitochondrial genome. About 12% of the unique small RNAs identified were encoded within the mitochondrial genome, and around 20% of the highly differentially expressed RNAs in response to anoxia were mitochondrial in origin. Of the differentially expressed sequences mapping to the mitochondrial genome, small RNAs derived from mitochondrial tRNAs were particularly enriched, with sequences up-regulated during anoxia and aerobic recovery from anoxia. These small RNAs of interest localize to anoxia sensitive tissues, such as the brain and heart, providing further evidence for a potential role in supporting anoxia tolerance. These same small RNAs have been detected in cell cultures derived from A. limnaeus embryos, by RNAseq and in situ hybridization. Cell culture expression patterns corroborated whole-embryo sequence expression patterns. This work highlights a potential new class of small RNAs derived from the mitochondrial genome that may act to mediate and coordinate the physiological response to anoxia in this species. Further understanding the biology of these mitochondrially-derived small RNAs and their role in anoxia tolerance may provide novel insight into combating cellular damage induced by heart attack and stroke, two lethal consequences of heart disease in humans. Title: Mitochondria-derived Small Non-coding RNAs in Extreme Anoxia Tolerance Author: Riggs, Claire L., Podrabsky, Jason E. Year: 2017 Mitochondria are critical sensors of oxygen and play a major role in the regulation of the cellular response to oxygen deprivation. Understanding mitochondrial function during transitions into and out of anoxia in species that can tolerate anoxia may lend new insights into the role that mitochondria play in cellular homeostasis in response to fluctuating levels of oxygen. The objective of this study was to examine the potential role of small RNAs in long-term vertebrate anoxia tolerance. Expression patterns of small RNAs were determined using RNAseq in the most anoxia tolerant vertebrate, embryos of the annual killifishAustrofundulus limnaeus. Anoxia tolerance varies during development in this species in a way that allows for the comparison of developmental stages with extreme anoxia tolerance to stages with a more typical vertebrate anoxia tolerance. Embryos of 4 different developmental stages were exposed to anoxia, followed by aerobic recovery, and small RNA expression was determined with Illumina RNAseq under aerobic conditions, anoxia, and during aerobic recovery. A large proportion of the differentially expressed small RNAs were found to be encoded in the mitochondrial genome. About 12% of the unique small RNAs identified were encoded within the mitochondrial genome, and around 20% of the highly differentially expressed RNAs in response to anoxia were mitochondrial in origin. Of the differentially expressed sequences mapping to the mitochondrial genome, small RNAs derived from mitochondrial tRNAs were particularly enriched, with sequences up-regulated during anoxia and aerobic recovery from anoxia. These small RNAs of interest localize to anoxia sensitive tissues, such as the brain and heart, providing further evidence for a potential role in supporting anoxia tolerance. These same small RNAs have been detected in cell cultures derived from A. limnaeus embryos, by RNAseq and in situ hybridization. Cell culture expression patterns corroborated whole-embryo sequence expression patterns. This work highlights a potential new class of small RNAs derived from the mitochondrial genome that may act to mediate and coordinate the physiological response to anoxia in this species. Further understanding the biology of these mitochondrially-derived small RNAs and their role in anoxia tolerance may provide novel insight into combating cellular damage induced by heart attack and stroke, two lethal consequences of heart disease in humans. Close

• 6. [Article] Small Noncoding RNA Expression During Extreme Anoxia Tolerance of Annual Killifish (Austrofundulus limnaeus) Embryos

  Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially ...

  Citation × Citation Citation Abstract Copy Title: Small Noncoding RNA Expression During Extreme Anoxia Tolerance of Annual Killifish (Austrofundulus limnaeus) Embryos Author: Riggs, Claire L., Podrabsky, Jason E. Year: 2017 Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially expressed in diverse taxa in response to environmental stress, including anoxia. In most vertebrates, a brief period of oxygen deprivation results in severe tissue damage or death. Studies on sncRNA and anoxia have focused on these anoxia-sensitive species. Studying sncRNAs in anoxia-tolerant organisms may provide insight into adaptive mechanisms supporting anoxia tolerance. Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrates known, surviving over 100 days at their peak tolerance at 25°C. Their anoxia tolerance and physiology vary over development, such that both anoxia-tolerant and anoxia-sensitive phenotypes comprise the species. This allows for a robust comparison to identify sncRNAs essential to anoxia-tolerance. For this study, RNA sequencing was used to identify and quantify expression of sncRNAs in four embryonic stages of A. limnaeus in response to an exposure to anoxia and subsequent aerobic recovery. Unique stage-specific patterns of expression were identified that correlate with anoxia tolerance. In addition, embryos of A. limnaeus appear to constitutively express stress-responsive miRNAs. Most differentially expressed sncRNAs were expressed at higher levels during recovery. Many novel groups of sncRNAs with expression profiles suggesting a key role in anoxia tolerance were identified, including sncRNAs derived from mitochondrial tRNAs. This global analysis has revealed groups of candidate sncRNAs that we hypothesize support anoxia tolerance. Title: Small Noncoding RNA Expression During Extreme Anoxia Tolerance of Annual Killifish (Austrofundulus limnaeus) Embryos Author: Riggs, Claire L., Podrabsky, Jason E. Year: 2017 Small noncoding RNAs (sncRNA) have recently emerged as specific and rapid regulators of gene expression, involved in a myriad of cellular and organismal processes. MicroRNAs, a class of sncRNAs, are differentially expressed in diverse taxa in response to environmental stress, including anoxia. In most vertebrates, a brief period of oxygen deprivation results in severe tissue damage or death. Studies on sncRNA and anoxia have focused on these anoxia-sensitive species. Studying sncRNAs in anoxia-tolerant organisms may provide insight into adaptive mechanisms supporting anoxia tolerance. Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrates known, surviving over 100 days at their peak tolerance at 25°C. Their anoxia tolerance and physiology vary over development, such that both anoxia-tolerant and anoxia-sensitive phenotypes comprise the species. This allows for a robust comparison to identify sncRNAs essential to anoxia-tolerance. For this study, RNA sequencing was used to identify and quantify expression of sncRNAs in four embryonic stages of A. limnaeus in response to an exposure to anoxia and subsequent aerobic recovery. Unique stage-specific patterns of expression were identified that correlate with anoxia tolerance. In addition, embryos of A. limnaeus appear to constitutively express stress-responsive miRNAs. Most differentially expressed sncRNAs were expressed at higher levels during recovery. Many novel groups of sncRNAs with expression profiles suggesting a key role in anoxia tolerance were identified, including sncRNAs derived from mitochondrial tRNAs. This global analysis has revealed groups of candidate sncRNAs that we hypothesize support anoxia tolerance. Close