Hypoxia is important in both biomedical and environmental contexts and necessitates rapid adaptive changes in metabolic organization. Mammals, as air breathers, have a limited capacity to withstand sustained exposure to hypoxia. By contrast, some aquatic animals, such as certain fishes, are routinely exposed and resistant to severe environmental hypoxia. Understanding the changes in gene expression in fishes exposed to hypoxic stress could reveal novel mechanisms of tolerance that may shed new light on hypoxia and ischemia in higher vertebrates. Using cDNA microarrays, we have studied gene expression in a hypoxia-tolerant burrow-dwelling goby fish, Gillichthys mirabilis. We show that a coherent picture of a complex transcriptional response can be generated for a nonmodel organism for which sequence data were unavailable. We demonstrate that: (i) although certain shifts in gene expression mirror changes in mammals, novel genes are differentially expressed in fish; and (ii) tissue-specific patterns of expression reflect the different metabolic roles of tissues during hypoxia. L imitation in availability of oxygen (hypoxia) is a stress important in both biomedical and environmental contexts (1). In humans and other air-breathing vertebrates, hypoxia leads to rapid adaptive changes in metabolic organization, for instance in activation of anaerobic ATP-generating pathways like glycolysis. In mammals, a central role for a specific transcription factor, the hypoxia-inducible factor (HIF)-1␣ has been demonstrated. HIF-1␣ mediates the expression of a series of genes involved in both cellular and systemic responses to hypoxia, leading to enhanced anaerobic metabolism and induced erythropoiesis and angiogenesis (2).The patterns of differential gene expression associated with hypoxic stress in aquatic animals, for instance fishes, remain largely unknown. Understanding the tissue-specific and temporal changes in gene expression in fishes exposed to hypoxia could reveal new mechanisms of hypoxia tolerance and shed light on the evolution of this adaptive response in vertebrates. The long-jaw mudsucker Gillichthys mirabilis is a hypoxia-tolerant species that inhabits estuaries along the coastline of central and southern California, where it lives in burrows that characteristically have low levels of dissolved oxygen (3). Here, we exploit DNA microarray technology to investigate the response of this euryoxic fish to prolonged (up to 6 d) hypoxia. The application of DNA microarrays allows the expression of hundreds to many thousands of genes to be monitored simultaneously, providing a broad and integrated picture of the way an organism responds to a changing environment (4). To date, however, microarray analyses have been applied almost exclusively to model species for which gene sequence data are abundant. We show the utility of microarray approaches for the study of gene expression in a species for which, at the onset of our investigation, sequence data were unavailable. Our study thus provides a road map for exploiting DNA mic...

How do organisms respond adaptively to environmental stress? Although some gene-specific responses have been explored, others remain to be identified, and there is a very poor understanding of the system-wide integration of response, particularly in complex, multitissue animals. Here, we adopt a transcript screening approach to explore the mechanisms underpinning a major, wholebody phenotypic transition in a vertebrate animal that naturally experiences extreme environmental stress. Carp were exposed to increasing levels of cold, and responses across seven tissues were assessed by using a microarray composed of 13,440 cDNA probes. A large set of unique cDNAs (Ϸ3,400) were affected by cold. These cDNAs included an expression signature common to all tissues of 252 up-regulated genes involved in RNA processing, translation initiation, mitochondrial metabolism, proteasomal function, and modification of higher-order structures of lipid membranes and chromosomes. Also identified were large numbers of transcripts with highly tissue-specific patterns of regulation. By unbiased profiling of gene ontologies, we have identified the distinctive functional features of each tissue's response and integrate them into a comprehensive view of the whole-body transition from one strongly adaptive phenotype to another. This approach revealed an expression signature suggestive of atrophy in cooled skeletal muscle. This environmental genomics approach by using a well studied but nongenomic species has identified a range of candidate genes endowing thermotolerance and reveals a previously unrecognized scale and complexity of responses that impacts at the level of cellular and tissue function.fish ͉ microarray ͉ stress

The physiological strategies that enable organisms to thrive in habitats where environmental factors vary dramatically on a daily basis are poorly understood. One of the most variable and unpredictable habitats on earth is the marine rocky intertidal zone located at the boundary between the terrestrial and marine environments. Mussels dominate rocky intertidal habitats throughout the world and, being sessile, endure wide variations in temperature, salinity, oxygen, and food availability due to diurnal, tidal, and climatic cycles. Analysis of gene-expression changes in the California ribbed mussel (Mytilus californianus) at different phases in the tidal cycle reveals that intertidal mussels exist in at least four distinct physiological states, corresponding to a metabolism and respiration phase, a cell-division phase, and two stress-response signatures linked to moderate and severe heat-stress events. The metabolism and cell-division phases appear to be functionally linked and are anticorrelated in time. The magnitudes and timings of these states varied by vertical position on the shore and appear to be driven by microhabitat conditions. The results provide new insights into the strategies that allow life to flourish in fluctuating environments and demonstrate the importance of time course data collected from field animals in situ in understanding organism-environment interactions.

SUMMARY The cellular response to stress relies on the rapid induction of genes encoding proteins involved in preventing and repairing macromolecular damage incurred as a consequence of environmental insult. To increase our understanding of the scope of this response, a cDNA microarray, consisting of 9207 cDNA clones, was used to monitor gene expression changes in the gill and white muscle tissues of a eurythermic fish, Gillichthys mirabilis(Gobiidae) exposed to ecologically relevant heat stress. In each tissue, the induction or repression of over 200 genes was observed. These genes are associated with numerous biological processes, including the maintenance of protein homeostasis, cell cycle control, cytoskeletal reorganization,metabolic regulation and signal transduction, among many others. In both tissues, the molecular chaperones, certain transcription factors and a set of additional genes with various functions were induced in a similar manner;however, the majority of genes displayed tissue-specific responses. In gill,thermal stress induced the expression of the major structural components of the cytoskeleton, whereas these same genes did not respond to heat in muscle. In muscle, many genes involved in promoting cell growth and proliferation were repressed, perhaps to conserve energy for repair and replacement of damaged macromolecules, but a similar repression was not observed in the gill. Many of the observed changes in gene expression were similar to those described in model species whereas many others were unexpected. Measurements of the concentrations of the protein products of selected genes revealed that in each case an induction in mRNA synthesis correlated with an increase in protein production, though the timing and magnitude of the increase in protein was not consistently predicted by mRNA concentration, an important consideration in assessing the condition of the stressed cell using transcriptomic analysis.

Poikilothermic animals respond to chronic cold by increasing phosphoglyceride unsaturation to restore the fluidity of cold-rigidified membranes. Despite the importance of this compensatory response, the enzymes involved have not been clearly identified, and the mechanisms that control their activity are unknown. In carp liver, cold induces an 8- to 10-fold increase in specific activity of the microsomal stearoyl coenzyme A desaturase. Cold-induced up-regulation of gene transcription resulted in a 10-fold increase in desaturase transcript amounts after 48 to 60 hours. However, this increase was preceded by the activation of latent desaturase, probably by a posttranslational mechanism. These two mechanisms may act sequentially to match desaturase expression to the demands imposed by a progressive decrease in temperature.

Myoglobin (Myg) is an oxygen-binding hemoprotein that is widely thought to be expressed exclusively in oxidative skeletal and cardiac myocytes, where it plays a key role in coping with chronic hypoxia. We now show in a hypoxia-tolerant fish model, that Myg is also expressed in a range of other tissues, including liver, gill, and brain. Moreover, expression of Myg transcript was substantially enhanced during chronic hypoxia, the fold-change induction being far greater in liver than muscle. By using 2D gel electrophoresis, we have confirmed that liver expresses a protein corresponding to the Myg-1 transcript and that it is significantly up-regulated during hypoxia. We have also discovered a second, unique Myg isoform, distinct from neuroglobin, which is expressed exclusively in the neural tissue but whose transcript expression was unaffected by environmental hypoxia. Both observations of nonmuscle expression and a brain-specific isoform are unprecedented, indicating that Myg may play a much wider role than previously understood and that Myg might function in the protection of tissues from deep hypoxia and ischemia as well as in reoxygenation and reperfusion injury.

Residents in the marine intertidal, the zone where terrestrial and marine habitats converge, inhabit an environment that is subject to both the 24-h day and night daily rhythm of the terrestrial earth and also the 12.4-h ebb and flow of the tidal cycle. Here, we investigate the relative contribution of the daily and tidal cycle on the physiology of intertidal mussels, Mytilus californianus, by monitoring rhythms of gene expression in both simulated and natural tidal environments. We report that >40% of the transcriptome exhibits rhythmic gene expression, and that depending on the specific tidal conditions, between 80% and 90% of the rhythmic transcripts follow a circadian expression pattern with a period of 24 to 26 h. Consistent with the dominant effect of the circadian cycle we show that the expression of clock genes oscillates with a 24-h period. Our data indicate that the circadian 24-h cycle is the dominant driver of rhythmic gene expression in this intertidal inhabitant despite the profound environmental and physiological changes associated with aerial exposure during tidal emergence.

We conducted a large-scale gene expression screen using the 3,200 cDNA probe microarray developed specifically for Ursus americanus to detect expression differences in liver and skeletal muscle that occur during winter hibernation compared with animals sampled during summer. The expression of 12 genes, including RNA binding protein motif 3 (Rbm3), that are mostly involved in protein biosynthesis, was induced during hibernation in both liver and muscle. The Gene Ontology and Gene Set Enrichment analysis consistently showed a highly significant enrichment of the protein biosynthesis category by overexpressed genes in both liver and skeletal muscle during hibernation. Coordinated induction in transcriptional level of genes involved in protein biosynthesis is a distinctive feature of the transcriptome in hibernating black bears. This finding implies induction of translation and suggests an adaptive mechanism that contributes to a unique ability to reduce muscle atrophy over prolonged periods of immobility during hibernation. Comparing expression profiles in bears to small mammalian hibernators shows a general trend during hibernation of transcriptional changes that include induction of genes involved in lipid metabolism and carbohydrate synthesis as well as depression of genes involved in the urea cycle and detoxification function in liver. gene expression; hibernation; translation; RNA binding protein motif 3 MAMMALIAN HIBERNATION IS a physiological and behavioral adaptation involving a coordinated suppression of heat production and body temperature wherein whole body metabolism and energy demand are significantly reduced over several days to several months. During hibernation, heart and respiration rates, blood flow, and oxygen consumption decrease dramatically to Ͻ10% of normal or basal rates (2, 4). These physiological changes do not represent a loss of homeostasis but instead are precisely controlled and spontaneously reversible, and they allow individual animals to survive highly seasonal or unpredictable environments where food availability becomes lacking (7, 12). The molecular and genetic basis of hibernation in small mammals has only recently begun to be described, and little is known about its evolutionary history. Hibernating species have been found in diverse families among seven orders of mammals (26), however, and the interspersed phylogenetic distribution of hibernating and nonhibernating species has led to the hypothesis that rather than requiring the creation of novel gene products, hibernation results from the differential expression of genes that exist widely among mammals (35).Microarray technology provides powerful means for the unbiased detection of differences in expression of thousands of genes in a single hybridization experiment (14). In contrast to single-gene expression analysis, the genome-wide approach also allows for identification of coordinated transcriptional changes in functional groups of regulatory genes within metabolic and signaling pathways. Recent studies of differential gen...