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...

The Toll/NF-B pathway is a common, evolutionarily conserved innate immune pathway that modulates the responses of animal cells to microbe-associated molecular patterns (MAMPs). Because MAMPs have been implicated as critical elements in the signaling of symbiont-induced development, an expressed sequence tag library from the juvenile light organ of Euprymna scolopes was used to identify members of the Toll/NF-B pathway. Full-length transcripts were identified by using 5 and 3 RACE PCR. Seven transcripts critical for MAMP-induced triggering of the Toll/NF-B phosphorylation cascade have been identified, including receptors, signal transducers, and a transcription factor. Further investigations should elucidate the role of the Toll/NF-B pathway in the initiation of the beneficial symbiosis between E. scolopes and Vibrio fischeri.

SummaryIn horizontally transmitted mutualisms between marine animals and their bacterial partners, the host environment promotes the initial colonization by specific symbionts that it harvests from the surrounding bacterioplankton. Subsequently, the host must develop long-term tolerance to immunogenic bacterial molecules, such as peptidoglycan and lipopolysaccaride derivatives. We describe the characterization of the activity of a host peptidoglycan recognition protein (EsPGRP2) during establishment of the symbiosis between the squid Euprymna scolopes and its luminous bacterial symbiont Vibrio fischeri. Using confocal immunocytochemistry, we localized EsPGRP2 to all epithelial surfaces of the animal, and determined that it is exported in association with mucus shedding. Most notably, EsPGRP2 was released by the crypt epithelia into the extracellular spaces housing the symbionts. This translocation occurred only after the symbionts had triggered host morphogenesis, a process that is induced by exposure to the peptidoglycan monomer tracheal cytotoxin (TCT), a bacterial 'toxin' that is constitutively exported by V. fischeri. Enzymatic analyses demonstrated that, like many described PGRPs, EsPGRP2 has a TCT-degrading amidase activity. The timing of EsPGRP2 export into the crypts provides evidence that the host does not export this protein until after TCT induces morphogenesis, and thereafter EsPGRP2 is constantly present in the crypts ameliorating the effects of V. fischeri TCT.