Male fetal sex affects uteroplacental angiogenesis in growth restriction mouse model†.

Abnormally increased angiotensin II activity related to maternal angiotensinogen (AGT) genetic variants, or aberrant receptor activation, is associated with small-for-gestational-age babies and abnormal uterine spiral artery remodeling in humans. Our group studies a murine AGT gene titration transgenic (TG; 3-copies of the AGT gene) model, which has a 20% increase in AGT expression mimicking a common human AGT genetic variant (A[-6]G) associated with intrauterine growth restriction (IUGR) and spiral artery pathology. We hypothesized that aberrant maternal AGT expression impacts pregnancy-induced uterine spiral artery angiogenesis in this mouse model leading to IUGR. We controlled for fetal sex and fetal genotype (e.g., only 2-copy wild-type [WT] progeny from WT and TG dams were included). Uteroplacental samples from WT and TG dams from early (days 6.5 and 8.5), mid (d12.5), and late (d16.5) gestation were studied to assess uterine natural killer (uNK) cell phenotypes, decidual metrial triangle angiogenic factors, placental growth and capillary density, placental transcriptomics, and placental nutrient transport. Spiral artery architecture was evaluated at day 16.5 by contrast-perfused three-dimensional microcomputed tomography (3D microCT). Our results suggest that uteroplacental angiogenesis is significantly reduced in TG dams at day 16.5. Males from TG dams are associated with significantly reduced uteroplacental angiogenesis from early to late gestation compared with their female littermates and WT controls. Angiogenesis was not different between fetal sexes from WT dams. We conclude that male fetal sex compounds the pathologic impact of maternal genotype in this mouse model of growth restriction.

Temperature-dependent vitamin D signaling regulates developmental trajectory associated with diapause in an annual killifish.

The mechanisms that integrate environmental signals into developmental programs remain largely uncharacterized. Nuclear receptors (NRs) are ligand-regulated transcription factors that orchestrate the expression of complex phenotypes. The vitamin D receptor (VDR) is an NR activated by 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], a hormone derived from 7-dehydrocholesterol (7-DHC). VDR signaling is best known for regulating calcium homeostasis in mammals, but recent evidence suggests a diversity of uncharacterized roles. In response to incubation temperature, embryos of the annual killifish Austrofundulus limnaeus can develop along two alternative trajectories: active development and diapause. These trajectories diverge early in development, from a biochemical, morphological, and physiological perspective. We manipulated incubation temperature to induce the two trajectories and profiled changes in gene expression using RNA sequencing and weighted gene coexpression network analysis. We report that transcripts involved in 1,25(OH)2D3 synthesis and signaling are expressed in a trajectory-specific manner. Furthermore, exposure of embryos to vitamin D3 analogs and Δ4-dafachronic acid directs continuous development under diapause-inducing conditions. Conversely, blocking synthesis of 1,25(OH)2D3 induces diapause in A. limnaeus and a diapause-like state in zebrafish, suggesting vitamin D signaling is critical for normal vertebrate development. These data support vitamin D signaling as a molecular pathway that can regulate developmental trajectory and metabolic dormancy in a vertebrate. Interestingly, the VDR is homologous to the daf-12 and ecdysone NRs that regulate dormancy in Caenorhabditis elegans and Drosophila We suggest that 7-DHC-derived hormones and their associated NRs represent a conserved pathway for the integration of environmental information into developmental programs associated with life history transitions in animals.

Metabolomics analysis of annual killifish (Austrofundulus limnaeus) embryos during aerial dehydration stress.

The annual killifish, Austrofundulus limnaeus, survives in ephemeral ponds in the coastal deserts of Venezuela. Persistence through the dry season is dependent on drought-resistant eggs embedded in the pond sediments during the rainy season. The ability of these embryos to enter drastic metabolic dormancy (diapause) during normal development enables A. limnaeus to survive conditions lethal to most other aquatic vertebrates; critical to the survival of the species is the ability of embryos to survive months and perhaps years without access to liquid water. Little is known about the molecular mechanisms that aid in survival of the dry season. This study aims to gain insight into the mechanisms facilitating survival of dehydration stress due to aerial exposure by examining metabolite profiles of dormant and developing embryos. There is strong evidence for unique metabolic profiles based on developmental stage and length of aerial exposure. Actively developing embryos exhibit more robust changes; however, dormant embryos respond in an active manner and significantly alter their metabolic profile. A number of metabolites accumulate in aerial-exposed embryos that may play an important role in survival, including the identification of known antioxidants and neuroprotectants. In addition, a number of unique metabolites not yet discussed in the dehydration literature are identified, such as lanthionine and 2-hydroxyglutarate. Despite high oxygen availability, embryos accumulate the anaerobic end product lactate. This paper offers an overview of the metabolic changes occurring that may support embryonic survival during dehydration stress due to aerial incubation, which can be functionally tested using genetic and pharmacological approaches.

GABA metabolism is crucial for long-term survival of anoxia in annual killifish embryos.

In most vertebrates, a lack of oxygen quickly leads to irreparable damages to vital organs, such as the brain and heart. However, there are some vertebrates that have evolved mechanisms to survive periods of no oxygen (anoxia). The annual killifish (Austrofundulus limnaeus) survives in ephemeral ponds in the coastal deserts of Venezuela and their embryos have the remarkable ability to tolerate anoxia for months. When exposed to anoxia, embryos of A. limnaeus respond by producing significant amounts of γ-aminobutyric acid (GABA). This study aims to understand the role of GABA in supporting the metabolic response to anoxia. To explore this, we investigated four developmentally distinct stages of A. limnaeus embryos that vary in their anoxia tolerance. We measured GABA and lactate concentrations across development in response to anoxia and aerobic recovery. We then inhibited enzymes responsible for the production and degradation of GABA and observed GABA and lactate concentrations, as well as embryo mortality. Here, we show for the first time that GABA metabolism affects anoxia tolerance in A. limnaeus embryos. Inhibition of enzymes responsible for GABA production (glutamate decarboxylase) and degradation (GABA-transaminase and succinic acid semialdehyde dehydrogenase) led to increased mortality, supporting a role for GABA as an intermediate product and not a metabolic end-product. We propose multiple roles for GABA during anoxia and aerobic recovery in A. limnaeus embryos, serving as a neurotransmitter, an energy source, and an anti-oxidant.

No water, no problem: stage-specific metabolic responses to dehydration stress in annual killifish embryos.

Annual killifish survive in temporary ponds by producing drought-tolerant embryos that can enter metabolic dormancy (diapause). Survival of dehydration stress is achieved through severe reduction of evaporative water loss. We assessed dehydration stress tolerance in diapausing and developing Austrofundulus limnaeus embryos. We measured oxygen consumption rates under aquatic and aerial conditions to test the hypothesis that there is a trade-off between water retention and oxygen permeability. Diapausing embryos survive dehydrating conditions for over 1.5 years, and post-diapause stages can survive for over 100 days. Diapausing embryos respond to dehydration stress by increasing oxygen consumption rates while post-diapause embryos exhibit the same or reduced rates compared with aquatic embryos. Thus, water retention does not always limit oxygen diffusion. Aerial incubation coupled with hypoxia causes some embryos to arrest development. The observed stage-specific responses are consistent with an intrinsic bet-hedging strategy in embryos that would increase developmental variation in a potentially adaptive manner.

Antioxidant capacity and anoxia tolerance in Austrofundulus limnaeus embryos.

Embryos of Austrofundulus limnaeus can tolerate extreme environmental stresses by entering into a state of metabolic and developmental arrest known as diapause. Oxidative stress is ubiquitous in aerobic organisms and the unique biology and ecology of A. limnaeus likely results in frequent and repeated exposures to oxidative stress during development. The antioxidant capacity of A. limnaeus was explored during development by measuring antioxidant capacity due to small molecules and several enzymatic antioxidant systems. Diapause II embryos can survive for several days in 1% hydrogen peroxide without indications of negative effects. Surprisingly, both small and large molecule antioxidant systems have the highest capacity during early development, which may be due to maternal provisioning. Antioxidant capacity is largely invested in small molecules during early development and in enzymatic systems during late development. The switch in antioxidant mechanisms and decline in small molecule antioxidants during development correlates with the loss of extreme anoxia tolerance.

The genome of Austrofundulus limnaeus offers insights into extreme vertebrate stress tolerance and embryonic development.

BACKGROUND: The annual killifish Austrofundulus limnaeus inhabits ephemeral ponds in northern Venezuela, South America, and is an emerging extremophile model for vertebrate diapause, stress tolerance, and evolution. Embryos of A. limnaeus regularly experience extended periods of desiccation and anoxia as a part of their natural history and have unique metabolic and developmental adaptations. Currently, there are limited genomic resources available for gene expression and evolutionary studies that can take advantage of A. limnaeus as a unique model system. RESULTS: We describe the first draft genome sequence of A. limnaeus. The genome was assembled de novo using a merged assembly strategy and was annotated using the NCBI Eukaryotic Annotation Pipeline. We show that the assembled genome has a high degree of completeness in genic regions that is on par with several other teleost genomes. Using RNA-seq and phylogenetic-based approaches, we identify several candidate genes that may be important for embryonic stress tolerance and post-diapause development in A. limnaeus. Several of these genes include heat shock proteins that have unique expression patterns in A. limnaeus embryos and at least one of these may be under positive selection. CONCLUSION: The A. limnaeus genome is the first South American annual killifish genome made publicly available. This genome will be a valuable resource for comparative genomics to determine the genetic and evolutionary mechanisms that support the unique biology of annual killifishes. In a broader context, this genome will be a valuable tool for exploring genome-environment interactions and their impacts on vertebrate physiology and evolution.

Hit pause: Developmental arrest in annual killifishes and their close relatives.

Killifishes survive and persist in extreme environments by exploiting both aquatic and terrestrial habitats for egg deposition, and by adjusting the length of development to match availability of water to support larval growth and maturation. Annual killifishes persist in ephemeral bodies of water through the production of drought-tolerant embryos. Survival of the environmental stresses associated with their highly variable and seasonal habitat is supported by their ability to enter into at least two states of metabolic and developmental dormancy, diapause or quiescence. There are three stages of diapause in annual killifishes, one occurring prior to gastrulation, one about midway through development, and one in late pre-hatching embryos. Quiescence may occur at any developmental stage. In addition, delayed hatching is known to occur in close relatives of the annual killifishes, and may be superficially confused with pre-hatching diapause. These types of developmental delay are induced by different cues and serve different purposes in the life history of the species. Thus, it is likely that the molecular mechanisms that induce dormancy and support survival are unique in each case. It is imperative that we properly define these forms of developmental dormancy in our studies in order to put our results into the proper ecological and evolutionary context. Here the unique characteristics of these distinct categories of developmental delay are reviewed. Developmental Dynamics 246:858-866, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Embryonic development of the annual killifish Austrofundulus limnaeus: An emerging model for ecological and evolutionary developmental biology research and instruction.

BACKGROUND: Austrofundulus limnaeus is an annual killifish from the Maracaibo basin of Venezuela. Annual killifishes are unique among vertebrates in their ability to enter into a state of dormancy at up to three distinct developmental stages termed diapause I, II, and III. These embryos are tolerant of a wide variety of environmental stresses and develop relatively slowly compared with nonannual fishes. RESULTS: These traits make them an excellent model for research on interactions between the genome and the environment during development, and an excellent choice for developmental biology laboratories. Furthermore, A. limnaeus is relatively easy to maintain in a laboratory setting and has a high fecundity, making it an excellent candidate as an emerging model for studies of development, and for defining the limits of developmental buffering in vertebrates. CONCLUSIONS: This study reports for the first time on the detailed development of A. limnaeus and provides a photographic and illustrated atlas of embryos on the two developmental trajectories possible in this species. Developmental Dynamics 246:779-801, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

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 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.

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 embryonic blastomeres, and a short post-embryonic 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.

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, 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.

Physiological strategies during animal diapause: lessons from brine shrimp and annual killifish.

Diapause is a programmed state of developmental arrest that typically occurs as part of the natural developmental progression of organisms that inhabit seasonal environments. The brine shrimp Artemia franciscana and annual killifish Austrofundulus limnaeus share strikingly similar life histories that include embryonic diapause as a means to synchronize the growth and reproduction phases of their life history to favorable environmental conditions. In both species, respiration rate is severely depressed during diapause and thus alterations in mitochondrial physiology are a key component of the suite of characters associated with cessation of development. Here, we use these two species to illustrate the basic principles of metabolic depression at the physiological and biochemical levels. It is clear that these two species use divergent molecular mechanisms to achieve the same physiological and ecological outcomes. This pattern of convergent physiological strategies supports the importance of biochemical and physiological adaptations to cope with extreme environmental stress and suggests that inferring mechanism from transcriptomics or proteomics or metabolomics alone, without rigorous follow-up at the biochemical and physiological levels, could lead to erroneous conclusions.

Hormonal components of altered developmental pathways in the annual killifish, Austrofundulus limnaeus.

The annual killifish, Austrofundulus limnaeus, typically enters embryonic diapause at two distinct points of development, termed diapause II and III. This study explores the role of maternal and embryonic steroid hormones, including 17-ß-estradiol (E2), androstenedione (A4) and testosterone (T), in regulating the developmental decision to enter or escape diapause II. Steroid hormone levels were measured in tissues isolated from adult female killifish during the normal lifespan of this species and in individuals of the same age that were producing either high or low proportions of escape embryos. Levels of steroid hormones were also measured during early development and in fertilized eggs that were predicted to be on either an escape or diapausing developmental trajectory. Decreases in maternal E2 levels associated with age are correlated with decreasing escape embryo production. Maternal production of escape embryos is correlated with increased ratios of E2 to T in adult ovary tissue. Interestingly, neither hormone is significantly different in fish producing embryos on different developmental pathways when examined independently. Levels of steroid hormones in fertilized eggs are not correlated with entry or escape from diapause II, though levels of A4 tend to be higher in escape embryos. Escape embryos exhibit faster hormone metabolism and earlier hormone synthesis than embryos that will enter diapause II. Incubation of embryos in exogenous E2 is associated with a 7-fold increase in escape embryo production, and significantly elevated A4 levels. These data suggest that steroid hormones may be critical factors involved in determining developmental pathways in embryos of A. limnaeus.

Is This Science? Students' Experiences of Failure Make a Research-Based Course Feel Authentic.

Course-based undergraduate research experiences (CUREs) and inquiry-based curricula both expose students to the scientific process. CUREs additionally engage students in novel and scientifically relevant research, with the intention of providing an "authentic" research experience. However, we have little understanding of which course design elements impact students' beliefs that they are experiencing "authentic" research. We designed a study to explore introductory biology students' perceptions of research authenticity in CURE and inquiry classes. Using the Laboratory Course Assessment Survey, we found that students in CURE sections perceived higher levels of authentic research elements than students in inquiry-based sections. To identify specific factors that impact perceptions of research authenticity, we administered weekly reflection questions to CURE students. Coding of reflection responses revealed that experiences of failure, iteration, using scientific practices, and the relevant discoveries in their projects enhanced students' perceived authenticity of their research experiences. Although failure and iteration can occur in both CUREs and inquiry-based curricula, our findings indicate these experiences-in conjunction with the Relevant Discovery element of a CURE-may be particularly powerful in enhancing student perceptions of research authenticity in a CURE.

Vertebrate cell death in energy-limited conditions and how to avoid it: what we might learn from mammalian hibernators and other stress-tolerant vertebrates.

Dormancy in vertebrates may expose cells to acidosis, hypoxia/anoxia, oxidative damage, and extremes in temperature. All of these insults are known to be pro-apoptotic in typical vertebrate cells, especially mammals. Since dormancy is presumably the result of a need for energy conservation, the inherent energetic demand of replenishing cells that underwent apoptosis seems at odds with this strategy. This review will discuss processes to mitigate apoptosis and how these processes might be regulated in stress-tolerant vertebrates such as mammalian hibernators. As data directly addressing such issues are scarce and often conflicting, an apparently complex regulation of apoptosis seems to be at work. For example, apoptosis is mitigated during dormancy, key signaling events including the activation of caspase-3 may still occur. However, both passive, temperature-induced depression of apoptotic signaling as well as active suppression of apoptosis appear to work in synergy in these systems. In many instances cell death is prevented by simply avoiding the cellular triggers (e.g. leakage of proteins from the mitochondria or increases in intracellular calcium) that initiate apoptotic signaling. In this review we discuss what is known about programmed cell death in these under-studied models and highlight features of their physiology that likely support survival in the face of conditions that would induce cell death in typical vertebrate cells.

Alternative developmental pathways associated with diapause regulated by temperature and maternal influences in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus enter a state of developmental arrest termed diapause as part of their normal developmental program. Diapause can occur at two distinct developmental stages in this species, termed diapause II and III. When incubated at 25°C, most embryos enter diapause II, whereas a small percentage of 'escape' embryos develop continuously past diapause II and enter diapause III. Control of entry into diapause II can be altered by maternal influences and the incubation environment experienced by the embryos. Young females produce a higher proportion of escape embryos than do older females. In addition, increasing the incubation temperature from 25 to 30°C induces all embryos to escape from diapause. Surprisingly, escape embryos follow a different morphological and physiological developmental trajectory than do embryos that enter diapause II. Development of anterior structures is advanced compared with that of posterior structures in escape embryos when compared with embryos that will enter diapause II. The difference in timing of development for these two trajectories is consistent with changes observed between two species but is very atypical of variation observed within a species. Importantly, the two developmental pathways diverge early in development, during the segmentation period, when, according to evolutionary theory, constraint on developmental pathways should be relatively high. The possession of alternative developmental pathways in a vertebrate embryo is a novel finding, the ecological and evolutionary importance of which is still unknown, but potentially significant in terms of life-history evolution.

Fish as model systems for the study of vertebrate apoptosis.

Apoptosis is a process of pivotal importance for multi-cellular organisms and due to its implication in the development of cancer and degenerative disease it is intensively studied in humans and mammalian model systems. Invertebrate models of apoptosis have been well-studied, especially in C. elegans and D. melanogaster, but as these are evolutionarily distant from mammals the relevance of findings for human research is sometimes limited. Presently, a non-mammalian vertebrate model for studying apoptosis is missing. However, in the past few years an increasing number of studies on cell death in fish have been published and thus new model systems may emerge. This review aims at highlighting the most important of these findings, showing similarities and dissimilarities between fish and mammals, and will suggest topics for future research. In addition, the outstanding usefulness of fish as research models will be pointed out, hoping to spark future research on this exciting, often underrated group of vertebrates.