BACKGROUND
the concentration of dissolved oxygen  and temperature are the most important environmental variables that affect the overall biological processes of fishes.
temperature limits the rates of cellular biochemical reactions and dictates all aspects of fish life, including metabolism, development, growth, reproduction and behavior  <cit> .
adverse effects and even death can be caused when water temperature falls outside the species-specific thermal tolerance range  <cit> .
molecular oxygen is used by all eukaryotic cells as the terminal electron acceptor in aerobic energy production and the presence of adequate oxygen is essential to the survival of nearly all vertebrates  <cit> .
oxygen deficiency can impair cellular energy generation, induce the formation of reactive oxygen species  and lead to cell damage and apoptosis  <cit> .
in water environments, hypoxia  often occurs due to the inherent properties of water and the rapid fluctuations in the pattern of oxygen production and consumption  <cit> .
exposure of fish to hypoxia can suppress development, reduce growth, disturb endocrine function, impair reproductive performance, and cause mass mortality for wild populations  <cit> .
due to the importance of oxygen and temperature to their life, fishes have evolved versatile mechanisms to acclimate oxygen deficiency and temperature variations in their habitats  <cit> .
pre-acclimation of fishes to moderate hypoxia or thermal stress can activate the acclimation pathways and increase the tolerance to lethal hypoxia or thermal stress  <cit> , respectively.
the rationale of hypoxia acclimation in fish is to increase oxygen uptake and reduce oxygen demands.
upon hypoxia, fishes usually skim the surface water containing more oxygen, increase ventilation volume to absorb more oxygen, or reduce motility to spare oxygen consumption  <cit> .
except for behavioral actions, extensive physiological and biochemical modifications such as gill modifications to increase surface area  <cit> , increases in heart rate and hemoglobin content  <cit> , alterations in the structure or activity of specific ion channels  <cit> , and activation of anaerobic atp production via glycolysis  <cit> , are also involved in the process of hypoxia acclimation.
the acclimation of fish to thermal stresses was considered to be a process of “biochemical restructuration”  <cit> , including synthesizing temperature specific isoenzymes  <cit> , increasing the content of membrane lipid and the degree of fatty acid unsaturation  <cit> , recruiting different muscle fiber types  <cit> , generating molecular chaperones  <cit> , and changing mitochondrial densities and their properties  <cit> .
numerous studies have focused on the molecular mechanisms underlying the acclimation responses to environmental stressors including hypoxia and temperature fluctuations in the last decade.
hypoxia-regulated gene expression in the embryos and adult tissues of zebrafish   <cit> , goby   <cit> , medaka   <cit>  and xiphophorus maculatus has been characterized with microarrays  <cit> .
transcriptional responses to thermal stresses in species such as zebrafish  <cit> , common carp   <cit> , channel catfish   <cit> , annual killifish   <cit> , coral reef fish   <cit> , rainbow trout   <cit>  and antarctic plunderfish   <cit>  have been investigated using microarray as well.
very recently, rna-seq was applied to explore the transcriptional responses of fish to hypoxia and thermal stress at the whole genome level  <cit> .
these studies have demonstrated that hypoxia and thermal stresses can induce profound changes in gene expression profiles.
although the master factors responsible for mediating temperature stress-regulated gene expression remain unknown, hypoxia-inducible factor- <dig>  was revealed to be the key regulator for hypoxia-induced genes  <cit> .
effects of hypoxia and thermal stresses on fishes are often examined separately, but an increasing body of evidence indicates the interaction between temperature and oxygen on organismal performance  <cit> .
exposure of fish to both low and high temperature stresses can impair cardiovascular functions and decrease circulatory oxygen concentration, thus induce tissue hypoxia in spite of ample oxygen supply from the environment  <cit> .
therefore, oxygen was considered to be the limiting factor for thermal tolerance of fish due to restrictions in cardiovascular performance at extreme temperatures  <cit> .
thermal limitation in fish was interpreted as being caused first by limited oxygen supply capacity and second by transition to anaerobic metabolism  <cit> .
accordingly, acclimation of channel catfish to hypoxia can enhance the tolerance to acute heat stress through improving cardiovascular performance  <cit> .
furthermore, exposure of zoarcid fish   <cit>  and crucian carp   <cit>  to cold stress induced the expression and dna binding activity of hif- <dig>  suggesting the activation of hypoxia-induced pathways by cold stress.
however, it remains unclear whether fish can develop a cross-resistance to hypoxia and cold and how multiple-stress responses are co-regulated.
zebrafish is a good model to study the mechanisms of environmental acclimation and acclimation to both hypoxia and cold has been previously reported in this species  <cit> .
recently, rna-seq gradually substituted the microarray approach in exploring transcriptional responses of organisms to environmental stressors due to its high sensitivity and accuracy, digital expression and the ability to distinguish transcript isoforms  <cit> .
except for detecting transcript abundance, rna-seq identifies alternative splicing and alternative promoter usage events at the same time and provides a more holistic view of the transcriptome  <cit> .
although hypoxia-regulated gene expression has been extensively investigated in zebrafish, it remains to be characterized by rna-seq.
in this study, we investigated the effect of hypoxia acclimation on cold tolerance and vice versa in zebrafish larvae, characterized the transcriptional responses to hypoxia at the whole-genome level using rna-seq, and made a comparison of hypoxia- and cold-induced transcriptomes.
RESULTS
hypoxia acclimation increased the cold resistance of zebrafish larvae
to investigate the cross-resistance between hypoxia and cold stress, zebrafish larvae pre-acclimated to mild hypoxia or cold were exposed to lethal hypoxia and cold, respectively .
zebrafish larvae exposed to 5% o <dig> for 24 h demonstrated smaller intestine lumen, larger yolk sac and smaller body length when compared to the control larvae maintained in air , demonstrating an obvious effect of hypoxia exposure.
as shown in figure 1b and c, both hypoxia and cold acclimation enhanced the resistance of zebrafish larvae to the lethal level of the same stressor, indicating the activation of protective mechanisms upon mild hypoxia and cold.
moreover, hypoxia acclimation significantly increased the survival rate of zebrafish larvae after lethal cold exposure , suggesting hypoxia-inducible signaling pathways in zebrafish larvae are involved in the development of cold resistance.
however, pre-exposure to cold significantly decreased the survival rate of larvae upon lethal hypoxia exposure  and no mortality was observed when the pre-cold acclimated larvae were suddenly transferred to 28°c incubation in an additional experiment .
therefore, instead of enhancing resistance, cold-acclimation sensitized the larvae to hypoxia stress.figure  <dig> pre-acclimation to hypoxia increased the cold resistance of zebrafish larvae.
flowchart of stressor exposure.
zebrafish embryos were incubated in air at 28°c from fertilization to  <dig> hpf and then pre-acclimated to hypoxia or cold for 24 h. sample collection for rna-seq and lethal hypoxia and cold exposure were performed at  <dig> hpf.
survival rates of zebrafish larvae after lethal cold  and hypoxia .
data was shown as mean ± standard deviation .
different letters above the error bars indicate significant difference  between treatment groups.
photographs of zebrafish larvae after lethal cold  and hypoxia  challenge.
dead fish displayed an obvious body curvature.
hypoxia-regulated gene expression
in order to disclose the molecular basis underlying the protective effect of hypoxia acclimation on zebrafish larvae against lethal cold stress, gene expression in larvae acclimated to 5% o <dig> for 24 h was characterized with rna-seq.
the low quality reads were filtered and the remaining clean reads were then mapped to the zebrafish genomic sequence.
the statistics for read pre-processing and mapping were displayed in table  <dig>  the total number of raw read pairs ranged from  <dig>  to  <dig>  million and about 96% of the raw reads passed the quality threshold.
about 91% of the clean reads were mapped to the genomic sequence by tophat and the number of reads mapped to splice junctions was quite similar among different samples .
finally,  <dig>  -  <dig> % of the alignment was unique in the genome.
these results suggest the high quality of our sequencing datasets.table  <dig>
statistics for read filtering and mapping
sample name
control1
control2
control3
hypoxia
hypoxia
hypoxia
after read mapping, gene expression was calculated using cufflinks and the abundances of genes were expressed as fpkm   <cit> .
as shown in figure 2a and b, most of the genes were of low and medium abundance according to their fpkm values and no obvious difference was found in the abundance distribution curve between the control and hypoxia-acclimated samples, suggesting a smaller effect of hypoxia on gene expression when compared to cold stress  <cit> .
the correlation between the abundance of genes under control and hypoxia is displayed in figure 2c.
genes with a fold change ≥  <dig>  and a q-value ≤  <dig>  were considered to be differentially expressed.
the number of up- and down-regulated genes was  <dig> and  <dig>  respectively .figure  <dig> gene expression regulated by hypoxia.
distribution of fpkm values for genes expressed in the control  and hypoxia-acclimated  zebrafish larvae.
the red interpolation line denotes a bimodal distribution of the frequency of fpkm.
correlation of gene expression between the control and hypoxia-treated group.
the up- and down-regulated genes were shown in red and blue, respectively.
genes not regulated by hypoxia treatment were shown in green.
the most prominent hypoxia-induced gene was hbz , followed by ponzr <dig> , egln <dig> , hpx , ponzr <dig> , hbm , alas <dig> , hbbe <dig> , ankrd <dig> , p4ha1b  and two uncharacterized genes si:dkey-202 l <dig>  and loc <dig>  these genes were up-regulated more than 5-fold by hypoxia .
the gene most highly inhibited by hypoxia was he1a , the expression of which was reduced 51-fold by hypoxia.
the expression of genes including lifrb , clca <dig>  loc <dig> and si:ch73-362 m <dig>  was inhibited 3-fold upon hypoxia .
validation of rna-seq data with qpcr
the qpcr assays were performed to validate the rna-seq results.
to identify internal reference genes appropriate for hypoxia exposure, two commonly used reference genes  and seven genes  demonstrating considerable expression stability after hypoxia treatment were selected as candidates .
the relative expression of these genes was detected by qpcr and their expression stability upon hypoxia exposure was analyzed using genorm  <cit>  and normfinder  <cit> , respectively.
both genorm and normfinder revealed that smarce1and erp <dig> are the most stable genes .
therefore, the geometric average of their expression was used as normalization factor for the analysis of hypoxia-related qpcr data.
the expression of  <dig> genes  was selected to be detected by qpcr to validate the rna-seq data.
as shown in table  <dig> and figure  <dig>  results from qpcr agreed very well with those of rna-seq for both up- and down-regulated genes.
a spearman bivariate correlation analysis revealed that the data of rna-seq and qpcr was significantly correlated , indicating the reliability of rna-seq data.table  <dig>
validation of rna-seq data with qpcr
gene symbol
fold change
rna-seq
qpcr
ak3
cpox
hif1an
ncoa4
osgn1
hbm
hephl1
mb
steap3
p4ha1b
tfr1a
urod
fads2
mep1b
elov2
slc34a2a
slc13a2
functional classification of hypoxia-regulated genes
go enrichment analyses indicated that the most enriched biological processes for hypoxia-induced genes include oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development, cellular iron ion homeostasis, protoporphyrinogen ix biosynthetic process and peptidyl-pyrromethane cofactor linkage .
these enriched processes are tightly associated with oxygen uptake and delivery, which is consistent with the demand to enhance oxygen uptake and delivery efficiency upon hypoxia.
hypoxia-inhibited genes are mainly involved in inorganic anion transport, sodium ion transport, very long-chain fatty acid biosynthetic process, glycolysis and cytidine deamination .figure  <dig> go enrichment analysis of genes up-regulated by hypoxia.
the size of circles is proportional to the number of genes associated with the go term.
the arrows represent the relationship between parent–child terms.
the color scale indicates corrected p-value of enrichment analysis.
genes co-regulated by hypoxia and cold stress
the protection of hypoxia-acclimation against adverse effects elicited by lethal cold prompts us to characterize the transcriptional events co-regulated by hypoxia and cold.
a comparison with our previous cold-regulated gene expression dataset  <cit>  identified  <dig> genes that were induced by both hypoxia and cold .
the expression of six genes induced by both hypoxia and cold exposure were investigated using qpcr to validate the co-regulation.
as shown in figure  <dig>  the expression of hephl <dig> , mb , tfr1a , urod  and steap <dig>  was up-regulated upon both hypoxia and cold.
a search for the function annotation in the gene ontology database  indicates that these co-regulated genes are mainly involved in oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis , suggesting that the activation of these processes are associated with the hypoxia-induced protection against cold stress.table  <dig>
genes co-induced by hypoxia and cold
genes
fold change
biological process
hypoxia
cold
cyp46a1
hephl1
sqrdl
bbox1
cox7a2
cox6b1
mb
hbae3
tspo
tfr1a
tfr1b
hmbsa
urod
steap3
slc16a1
abcb10
acta1b
klf4b
ncoa4
eno1a
zgc:161979
zgc:113232
znf292b
alternative promoter usage of hmbsb upon hypoxia and cold
except for differential gene expression, alternative promoter usage of hmbsb  upon hypoxia was identified by cufflinks.
dataset comparison demonstrated that promoter transition of hmbsb was also induced by cold stress.
due to the significance of alternative promoter usage in transcriptome diversity, we analyzed this event in detail and confirmed it using 5′ race.
hmbsb is involved in the biosynthetic process of tetrapyrrole, a source material of heme biosynthesis.
alternative promoter usage of hmbsb results in alternative first exons that are spliced to the common second exon and the start codon is present within the alternative first exon .
the corresponding transcripts of alternative promoters  were designated as hmbsb-p <dig> and hmbsb-p <dig>  respectively.
the read coverage of the first exon for hmbsb-p <dig> was obviously higher in hypoxia and cold-treated samples than that of control .
the alternative promoter usage of hmbsb results in different n-terminus of encoded peptides .
hmbsb-p <dig> is the main transcript under control condition; however, the main transcript was changed to hmbsb-p <dig> when exposed to hypoxia and cold .figure  <dig> alternative promoter usage of hmbsb upon hypoxia and cold.
read coverage at hmbsb locus.
the upper panel shows the read coverage of representative sample from each treatment group and the bottom panel indicates the structure of hmbsb transcripts.
the green box indicates the first exon determined by the alternative promoter.
partial sequence alignment of hmbsb peptides.
relative abundance of hmbsb transcripts detected by rna-seq.
5′ ends of hmbsb cdnas.
the 5′race assay based on rna adaptor ligation was performed to analyze the 5′ ends of hmbsb cdnas.
electrophoresis identified two bands for the control and one band for both hypoxia- and cold-treated samples .
the resulted pcr fragments of different samples were cloned and sequenced.
a total of seven transcription start sites  were identified for the two alternative promoters.
tss <dig> to tss <dig> is determined by p <dig> and tss <dig> and tss <dig> belong to p <dig> .
consistent with figure 6d, all clones from the control sample represented transcripts driven by p <dig>  and the transcript of tss <dig> is  <dig> base pairs longer than that of tss <dig>  p <dig> was hypoxia- and cold-specific and most of the clones from the hypoxia-treated sample were transcripts driven by p <dig>  and the ratio of p1/p <dig> for clones derived from the cold-treated sample was 3/ <dig> .
these results indicate that the alternative promoter p <dig> of hmbsb gene is specifically activated by hypoxia and cold and the activating effect of hypoxia is stronger than that of cold.figure  <dig> transcription start sites of hmbsb under different conditions.
total rna samples from different treatment group were subjected to 5′ race, respectively.
the 5′ ends of hmbsb cdna were cloned and sequenced.
the obtained sequences were aligned and classified into promoter and transcription start site groups.
cold and hypoxia pre-acclimation alleviated lipid peroxidation damage
since most of the cold and hypoxia co-regulated genes are involved in improving cellular oxygen availability and oxidation-reduction process, we performed lipid peroxidation assays to test whether cold or hypoxia-acclimation can alleviate oxidation damage upon lethal cold exposure.
the results indicate that exposure to mild cold or low oxygen stress has no effect on the content of malondialdehyde , the product of lipid peroxidation .
after the exposure of larvae to lethal cold for 12 h, the mda concentration of cold- and hypoxia-acclimated samples was significantly lower than that of the control .
this is consistent with the protective effect of cold- and hypoxia-acclimation against lethal cold stress.figure  <dig> cold- and hypoxia-acclimation alleviated oxidation damage upon lethal cold exposure.
larvae at  <dig> hpf were subjected to cold- or hypoxia-acclimation for 24 h and then incubated at 10°c for 12 h. the larvae in the same plate were homogenized for lipid peroxidation assays with a kit based on the reaction of thiobarbituric acid  with malondialdehyde .
the mda content was normalized to protein concentration.
data was shown as mean ± standard deviation .
different letters above the error bars indicate significant differences  among treatment groups.
discussion
oxygen and temperature are the most important environmental factors for the life of fishes and both hypoxia and temperature stress can cause deleterious effects on the organismal performance.
due to the importance of these factors, fishes have developed acclimation mechanisms to survive the daily and seasonal fluctuations in oxygen concentration or temperature in their habitats during the process of evolution.
the interdependence between oxygen availability and thermal resistance has been well documented in fishes, i.e.
exposure of fish to both low and high temperature stresses can induce tissue hypoxia through impairing the cardiovascular function and oxygen will subsequently limit the thermal tolerance of fish  <cit> .
although the protection of hypoxia acclimation against the adverse effect of heat stress has been reported  <cit> , it is not clear whether there is a cross-resistance between hypoxia and cold stress.
in this study, pre-acclimation of zebrafish larvae to mild hypoxia significantly increased the survival rates after lethal cold exposure, indicating the roles of hypoxia-inducible pathways in the establishment of cold resistance in fish.
however, pre-acclimation to mild cold significantly reduced the survival rate of zebrafish larvae upon lethal hypoxia exposure.
this is consistent with a previous study in mammal that cold acclimation decreased the hypoxia resistance of rats  <cit> .
the increased incidence of fatty changes in the striated muscle and the marked depletion of liver glycogen of cold-acclimated rats upon low oxygen may be responsible for the hypoxia-susceptibility  <cit> .
to investigate the molecular mechanisms underlying the protection of hypoxia-acclimation against cold stress, gene expression in hypoxia-acclimated zebrafish larvae was characterized using rna-seq and compared with that of cold-acclimated samples.
although hypoxia-elicited gene expression in zebrafish has been explored by microarray in several previous studies  <cit> , rna-seq characterization may give new information due to its advantages over microarray.
a total of  <dig> genes were found to be regulated by hypoxia in 96-hpf zebrafish larvae, including  <dig> up-regulated and  <dig> down-regulated genes.
a large part of these genes were not found to be regulated by hypoxia in previous microarray studies.
the number of genes influenced by hypoxia in this study is markedly smaller than that of cold-regulated genes  <cit> , but is quite similar with the previous studies characterizing hypoxia-regulated gene expression using microarray  <cit> .
this is consistent with the partial protection of hypoxia-acclimation against cold stress.
it is possible that more complicated mechanisms are involved in the development of cold tolerance in fish.
most of the hypoxia-induced genes identified in this study are involved in biosynthesis of the source material of hemoglobin, hematopoiesis and oxidation-reduction processes.
hemoglobin genes including hbz, hbm, hbae <dig> , hbae <dig> , hbbe <dig>  and hbbe <dig> were among the most prominent gene families induced by hypoxia .
although hbz was the most highly up-regulated gene under hypoxia, hbae <dig>  hbbe <dig> and hbbe <dig>  were the most abundant hemoglobin genes in both control and hypoxia-treated samples.
however, hemoglobin genes were not found to be up-regulated in 24-hpf zebrafish embryos exposed to hypoxia  <cit> .
genes involved in erythrocyte differentiation such as alas <dig> , tfr1a , slc4a1a  and gata1a  were also highly induced by hypoxia.
alas <dig> encodes 5-aminolevulinate synthase, the rate-controlling enzyme of erythroid heme synthesis; mutations of this gene are the causative of the x-linked sideroblastic anemia in human  <cit> .
transferrin receptor trf <dig> plays a crucial role in cellular iron uptake and is previously reported to be induced by hypoxia in human cell lines  <cit> .
anion exchanger slc4a <dig> functions as a transporter that mediates anion exchange across the cell membrane and as a structural protein, which is required for normal flexibility and stability of the erythrocyte membrane via the interactions of its cytoplasmic domain with cytoskeletal proteins  <cit> .
hematopoietic transcription factor gata <dig> is indispensible for the maturation of erythrocytes  <cit> .
the up-regulation of these genes underlies the increased resistance of zebrafish larvae to lethal hypoxia during the acclimation process.
hif- <dig> is the critical regulator for hypoxia response and plays key roles in cellular hypoxia acclimation  <cit> .
the activity of hif- <dig> is tightly regulated by the egl- <dig> family hypoxia-inducible factors  and the hypoxia-inducible factor  <dig> alpha subunit inhibitor .
egln genes encode proline hydroxylases  which mediate proteosomal degradation of hif1α under normoxic oxygen conditions  <cit> .
egln genes were reported to be regulated by hif- <dig> and suggested to be a negative feedback regulatory mechanism for limiting accumulation of hif1α in hypoxia  <cit> .
like egln members, hif1an  hydroxylates a conserved asparaginyl residue within hif1α when oxygen is available, and thus prevents the recruitment of co-activators and suppresses the activity  <cit> .
because the activities of both egln members and hif1an are oxygen-dependent, hif1α is stabilized under hypoxia and dimerizes with hif1β to form active hif- <dig>  zebrafish has four hif1a genes, including hif1aa, hif1ab, hif1al and hif1al <dig>  but hif1al <dig> was the only one up-regulated by hypoxia in this study .
however, hif1aa, hif1ab and hif1al were slightly but significantly induced by cold in our previous study .
it is interesting that all hif1α-suppressing genes, including egln genes and hif1an were induced by hypoxia  but none of them was up-regulated by cold stress.
it is possible that the stress response of hypoxia under 18°c is weaker than that of 5% oxygen and therefore the negative regulatory mechanism is not activated.
the protective effects of hypoxia acclimation against cold stress in zebrafish larvae prompts us to explore the mechanisms activated under both hypoxia and cold.
genes co-induced by hypoxia and cold are mainly involved in oxidation-reduction processes, hemoglobin biosynthetic process and oxygen transport.
cold-induced oxidative stress was widely found in insects  <cit> , fishes  <cit>  and mammals like rat  <cit> , suggesting that establishment of defense systems against oxidative damage would be a common task for cold acclimation in animals.
among the genes co-induced by hypoxia and cold, sqrdl  functions to catalyze the conversion of sulfide to persulfides, thereby decreasing toxic concentrations of sulfide accumulated upon hypoxia/ischemia  <cit> .
hephl <dig>  is an analog of ceruloplasmin that functions as a multicopper ferroxidase to convert fe2+ to less toxic fe3+ and aids in counteracting the deleterious effects of iron/ros-mediated oxidative damage  <cit> .
cyp46a <dig>  is the cholesterol 24-hydroxylase which catalyzes the oxidation of cholesterol into 24s-hydroxycholesterol to facilitate the efflux of cholesterol across the blood–brain barrier  <cit> .
since the content of cholesterol is negatively related to the fluidity of cell membrane  <cit> , elimination of cholesterol from cells may be necessary for the increment of membrane fluidity at low temperature.
myoglobin is important for intracellular oxygen transportation upon oxygen scarcity and was reported to be up-regulated by hypoxia in various carp tissues  <cit> .
up-regulation of mb by cold stress further suggests that improving oxygen supply is an important aspect of cold acclimation.
furthermore, genes involved in oxygen transport, iron homeostasis and hemoglobin biosynthetic process such as hbae <dig>  tfr1a, tfr1b, urod  and steap <dig>  were up-regulated upon both hypoxia and cold.
nearly all these co-regulated genes are involved in improving cellular oxygen availability and oxidation-reduction process.
furthermore, the results of lipid peroxidation assays demonstrated that both cold and hypoxia pre-acclimation alleviate oxidation damage caused by lethal cold stress, suggesting the association of these genes with cold acclimation.
however, the increase in the abundance of transcripts under cold stress is not necessarily resulted from activated transcription since the turnover of rna can be affected by temperature.
the post-transcriptional regulation mechanisms reducing mrna decay may contribute to the up-regulation of certain genes as well.
in addition to co-induced genes, we identified and confirmed the alternative promoter usage of hmbsb under hypoxia and cold.
alternative promoter usage is a versatile mechanism to create diversity and flexibility in the regulation of gene expression besides alternative splicing.
messenger rna molecules derived from alternative promoters may differ in the level of transcription initiation, stability and translation efficiency.
alternative promoters can have different tissue specificity, react differently to environmental signals and lead to the generation of protein isoforms differing at the amino terminus  <cit> .
subsequently, a different amino terminus can lead to alterations in protein levels, functions, or subcellular distribution  <cit> .
zebrafish hmbsb gene is involved in the tetrapyrrole biosynthetic process and is specifically expressed in blood  <cit> .
alternative promoter usage of zebrafish hmbsb gene leads to peptides differing in the initial ten amino acids.
promoter transition was also found for human hmbs  gene: the housekeeping promoter is active in all cells and the alternative promoter is present only in erythroid cells  <cit> .
although the alternative promoter of human hmbs gene is associated with tissue specificity, the biological significance and mechanisms underlying the hypoxia/cold-inducibility of the alternative promoter of zebrafish hmbsb gene remains to be characterized.
CONCLUSIONS
hypoxia acclimation increased the survival rate of zebrafish larvae after lethal cold exposure, indicating that hypoxia-inducible signaling pathways play important roles in the establishment of fish cold resistance.
hypoxia acclimation of zebrafish larvae is intimately associated with biological processes including oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development and cellular iron ion homeostasis.
genes co-induced by hypoxia and cold are mainly involved in oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis.
an alternative promoter of hmbsb is specifically activated by hypoxia and cold.
the transcriptional events co-regulated by hypoxia and cold represent the molecular basis of hypoxia-induced protection against cold stress.

 the concentration of dissolved oxygen and temperature are the most important environmental variables that affect the overall biological processes of fishes. temperature limits the rates of cellular biochemical reactions and dictates all aspects of fish life, including metabolism, development, growth, reproduction and behavior. adverse effects and even death can be caused when water temperature falls outside the species-specific thermal tolerance range. molecular oxygen is used by all eukaryotic cells as the terminal electron acceptor in aerobic energy production and the presence of adequate oxygen is essential to the survival of nearly all vertebrates. oxygen deficiency can impair cellular energy generation, induce the formation of reactive oxygen species and lead to cell damage and apoptosis. in water environments, hypoxia often occurs due to the inherent properties of water and the rapid fluctuations in the pattern of oxygen production and consumption. exposure of fish to hypoxia can suppress development, reduce growth, disturb endocrine function, impair reproductive performance, and cause mass mortality for wild populations. due to the importance of oxygen and temperature to their life, fishes have evolved versatile mechanisms to acclimate oxygen deficiency and temperature variations in their habitats. pre-acclimation of fishes to moderate hypoxia or thermal stress can activate the acclimation pathways and increase the tolerance to lethal hypoxia or thermal stress, respectively. the rationale of hypoxia acclimation in fish is to increase oxygen uptake and reduce oxygen demands. upon hypoxia, fishes usually skim the surface water containing more oxygen, increase ventilation volume to absorb more oxygen, or reduce motility to spare oxygen consumption. except for behavioral actions, extensive physiological and biochemical modifications such as gill modifications to increase surface area, increases in heart rate and hemoglobin content, alterations in the structure or activity of specific ion channels, and activation of anaerobic atp production via glycolysis, are also involved in the process of hypoxia acclimation. the acclimation of fish to thermal stresses was considered to be a process of “biochemical restructuration”, including synthesizing temperature specific isoenzymes, increasing the content of membrane lipid and the degree of fatty acid unsaturation, recruiting different muscle fiber types, generating molecular chaperones, and changing mitochondrial densities and their properties. numerous studies have focused on the molecular mechanisms underlying the acclimation responses to environmental stressors including hypoxia and temperature fluctuations in the last decade. hypoxia-regulated gene expression in the embryos and adult tissues of zebrafish, goby, medaka and xiphophorus maculatus has been characterized with microarrays. transcriptional responses to thermal stresses in species such as zebrafish, common carp, channel catfish, annual killifish, coral reef fish, rainbow trout and antarctic plunderfish have been investigated using microarray as well. very recently, rna-seq was applied to explore the transcriptional responses of fish to hypoxia and thermal stress at the whole genome level. these studies have demonstrated that hypoxia and thermal stresses can induce profound changes in gene expression profiles. although the master factors responsible for mediating temperature stress-regulated gene expression remain unknown, hypoxia-inducible factor- was revealed to be the key regulator for hypoxia-induced genes. effects of hypoxia and thermal stresses on fishes are often examined separately, but an increasing body of evidence indicates the interaction between temperature and oxygen on organismal performance. exposure of fish to both low and high temperature stresses can impair cardiovascular functions and decrease circulatory oxygen concentration, thus induce tissue hypoxia in spite of ample oxygen supply from the environment. therefore, oxygen was considered to be the limiting factor for thermal tolerance of fish due to restrictions in cardiovascular performance at extreme temperatures. thermal limitation in fish was interpreted as being caused first by limited oxygen supply capacity and second by transition to anaerobic metabolism. accordingly, acclimation of channel catfish to hypoxia can enhance the tolerance to acute heat stress through improving cardiovascular performance. furthermore, exposure of zoarcid fish and crucian carp to cold stress induced the expression and dna binding activity of hif- suggesting the activation of hypoxia-induced pathways by cold stress. however, it remains unclear whether fish can develop a cross-resistance to hypoxia and cold and how multiple-stress responses are co-regulated. zebrafish is a good model to study the mechanisms of environmental acclimation and acclimation to both hypoxia and cold has been previously reported in this species. recently, rna-seq gradually substituted the microarray approach in exploring transcriptional responses of organisms to environmental stressors due to its high sensitivity and accuracy, digital expression and the ability to distinguish transcript isoforms. except for detecting transcript abundance, rna-seq identifies alternative splicing and alternative promoter usage events at the same time and provides a more holistic view of the transcriptome. although hypoxia-regulated gene expression has been extensively investigated in zebrafish, it remains to be characterized by rna-seq. in this study, we investigated the effect of hypoxia acclimation on cold tolerance and vice versa in zebrafish larvae, characterized the transcriptional responses to hypoxia at the whole-genome level using rna-seq, and made a comparison of hypoxia- and cold-induced transcriptomes.  hypoxia acclimation increased the cold resistance of zebrafish larvae to investigate the cross-resistance between hypoxia and cold stress, zebrafish larvae pre-acclimated to mild hypoxia or cold were exposed to lethal hypoxia and cold, respectively. zebrafish larvae exposed to 5% o for 24 h demonstrated smaller intestine lumen, larger yolk sac and smaller body length when compared to the control larvae maintained in air, demonstrating an obvious effect of hypoxia exposure. as shown in figure 1b and c, both hypoxia and cold acclimation enhanced the resistance of zebrafish larvae to the lethal level of the same stressor, indicating the activation of protective mechanisms upon mild hypoxia and cold. moreover, hypoxia acclimation significantly increased the survival rate of zebrafish larvae after lethal cold exposure, suggesting hypoxia-inducible signaling pathways in zebrafish larvae are involved in the development of cold resistance. however, pre-exposure to cold significantly decreased the survival rate of larvae upon lethal hypoxia exposure and no mortality was observed when the pre-cold acclimated larvae were suddenly transferred to 28°c incubation in an additional experiment. therefore, instead of enhancing resistance, cold-acclimation sensitized the larvae to hypoxia stress.figure pre-acclimation to hypoxia increased the cold resistance of zebrafish larvae. flowchart of stressor exposure. zebrafish embryos were incubated in air at 28°c from fertilization to hpf and then pre-acclimated to hypoxia or cold for 24 h. sample collection for rna-seq and lethal hypoxia and cold exposure were performed at hpf. survival rates of zebrafish larvae after lethal cold and hypoxia. data was shown as mean ± standard deviation. different letters above the error bars indicate significant difference between treatment groups. photographs of zebrafish larvae after lethal cold and hypoxia challenge. dead fish displayed an obvious body curvature. hypoxia-regulated gene expression in order to disclose the molecular basis underlying the protective effect of hypoxia acclimation on zebrafish larvae against lethal cold stress, gene expression in larvae acclimated to 5% o for 24 h was characterized with rna-seq. the low quality reads were filtered and the remaining clean reads were then mapped to the zebrafish genomic sequence. the statistics for read pre-processing and mapping were displayed in table  the total number of raw read pairs ranged from to million and about 96% of the raw reads passed the quality threshold. about 91% of the clean reads were mapped to the genomic sequence by tophat and the number of reads mapped to splice junctions was quite similar among different samples. finally, - % of the alignment was unique in the genome. these results suggest the high quality of our sequencing datasets.table statistics for read filtering and mapping sample name control1 control2 control3 hypoxia hypoxia hypoxia after read mapping, gene expression was calculated using cufflinks and the abundances of genes were expressed as fpkm. as shown in figure 2a and b, most of the genes were of low and medium abundance according to their fpkm values and no obvious difference was found in the abundance distribution curve between the control and hypoxia-acclimated samples, suggesting a smaller effect of hypoxia on gene expression when compared to cold stress. the correlation between the abundance of genes under control and hypoxia is displayed in figure 2c. genes with a fold change ≥  and a q-value ≤  were considered to be differentially expressed. the number of up- and down-regulated genes was and respectively.figure gene expression regulated by hypoxia. distribution of fpkm values for genes expressed in the control and hypoxia-acclimated zebrafish larvae. the red interpolation line denotes a bimodal distribution of the frequency of fpkm. correlation of gene expression between the control and hypoxia-treated group. the up- and down-regulated genes were shown in red and blue, respectively. genes not regulated by hypoxia treatment were shown in green. the most prominent hypoxia-induced gene was hbz, followed by ponzr, egln, hpx, ponzr, hbm, alas, hbbe, ankrd, p4ha1b and two uncharacterized genes si:dkey-202 l and loc these genes were up-regulated more than 5-fold by hypoxia. the gene most highly inhibited by hypoxia was he1a, the expression of which was reduced 51-fold by hypoxia. the expression of genes including lifrb, clca loc and si:ch73-362 m was inhibited 3-fold upon hypoxia. validation of rna-seq data with qpcr the qpcr assays were performed to validate the rna-seq results. to identify internal reference genes appropriate for hypoxia exposure, two commonly used reference genes and seven genes demonstrating considerable expression stability after hypoxia treatment were selected as candidates. the relative expression of these genes was detected by qpcr and their expression stability upon hypoxia exposure was analyzed using genorm and normfinder, respectively. both genorm and normfinder revealed that smarce1and erp are the most stable genes. therefore, the geometric average of their expression was used as normalization factor for the analysis of hypoxia-related qpcr data. the expression of genes was selected to be detected by qpcr to validate the rna-seq data. as shown in table  and figure  results from qpcr agreed very well with those of rna-seq for both up- and down-regulated genes. a spearman bivariate correlation analysis revealed that the data of rna-seq and qpcr was significantly correlated, indicating the reliability of rna-seq data.table validation of rna-seq data with qpcr gene symbol fold change rna-seq qpcr ak3 cpox hif1an ncoa4 osgn1 hbm hephl1 mb steap3 p4ha1b tfr1a urod fads2 mep1b elov2 slc34a2a slc13a2 functional classification of hypoxia-regulated genes go enrichment analyses indicated that the most enriched biological processes for hypoxia-induced genes include oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development, cellular iron ion homeostasis, protoporphyrinogen ix biosynthetic process and peptidyl-pyrromethane cofactor linkage. these enriched processes are tightly associated with oxygen uptake and delivery, which is consistent with the demand to enhance oxygen uptake and delivery efficiency upon hypoxia. hypoxia-inhibited genes are mainly involved in inorganic anion transport, sodium ion transport, very long-chain fatty acid biosynthetic process, glycolysis and cytidine deamination.figure go enrichment analysis of genes up-regulated by hypoxia. the size of circles is proportional to the number of genes associated with the go term. the arrows represent the relationship between parent–child terms. the color scale indicates corrected p-value of enrichment analysis. genes co-regulated by hypoxia and cold stress the protection of hypoxia-acclimation against adverse effects elicited by lethal cold prompts us to characterize the transcriptional events co-regulated by hypoxia and cold. a comparison with our previous cold-regulated gene expression dataset identified genes that were induced by both hypoxia and cold. the expression of six genes induced by both hypoxia and cold exposure were investigated using qpcr to validate the co-regulation. as shown in figure  the expression of hephl, mb, tfr1a, urod and steap was up-regulated upon both hypoxia and cold. a search for the function annotation in the gene ontology database indicates that these co-regulated genes are mainly involved in oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis, suggesting that the activation of these processes are associated with the hypoxia-induced protection against cold stress.table genes co-induced by hypoxia and cold genes fold change biological process hypoxia cold cyp46a1 hephl1 sqrdl bbox1 cox7a2 cox6b1 mb hbae3 tspo tfr1a tfr1b hmbsa urod steap3 slc16a1 abcb10 acta1b klf4b ncoa4 eno1a zgc:161979 zgc:113232 znf292b alternative promoter usage of hmbsb upon hypoxia and cold except for differential gene expression, alternative promoter usage of hmbsb upon hypoxia was identified by cufflinks. dataset comparison demonstrated that promoter transition of hmbsb was also induced by cold stress. due to the significance of alternative promoter usage in transcriptome diversity, we analyzed this event in detail and confirmed it using 5′ race. hmbsb is involved in the biosynthetic process of tetrapyrrole, a source material of heme biosynthesis. alternative promoter usage of hmbsb results in alternative first exons that are spliced to the common second exon and the start codon is present within the alternative first exon. the corresponding transcripts of alternative promoters were designated as hmbsb-p and hmbsb-p respectively. the read coverage of the first exon for hmbsb-p was obviously higher in hypoxia and cold-treated samples than that of control. the alternative promoter usage of hmbsb results in different n-terminus of encoded peptides. hmbsb-p is the main transcript under control condition; however, the main transcript was changed to hmbsb-p when exposed to hypoxia and cold.figure alternative promoter usage of hmbsb upon hypoxia and cold. read coverage at hmbsb locus. the upper panel shows the read coverage of representative sample from each treatment group and the bottom panel indicates the structure of hmbsb transcripts. the green box indicates the first exon determined by the alternative promoter. partial sequence alignment of hmbsb peptides. relative abundance of hmbsb transcripts detected by rna-seq. 5′ ends of hmbsb cdnas. the 5′race assay based on rna adaptor ligation was performed to analyze the 5′ ends of hmbsb cdnas. electrophoresis identified two bands for the control and one band for both hypoxia- and cold-treated samples. the resulted pcr fragments of different samples were cloned and sequenced. a total of seven transcription start sites were identified for the two alternative promoters. tss to tss is determined by p and tss and tss belong to p. consistent with figure 6d, all clones from the control sample represented transcripts driven by p and the transcript of tss is base pairs longer than that of tss p was hypoxia- and cold-specific and most of the clones from the hypoxia-treated sample were transcripts driven by p and the ratio of p1/p for clones derived from the cold-treated sample was 3/. these results indicate that the alternative promoter p of hmbsb gene is specifically activated by hypoxia and cold and the activating effect of hypoxia is stronger than that of cold.figure transcription start sites of hmbsb under different conditions. total rna samples from different treatment group were subjected to 5′ race, respectively. the 5′ ends of hmbsb cdna were cloned and sequenced. the obtained sequences were aligned and classified into promoter and transcription start site groups. cold and hypoxia pre-acclimation alleviated lipid peroxidation damage since most of the cold and hypoxia co-regulated genes are involved in improving cellular oxygen availability and oxidation-reduction process, we performed lipid peroxidation assays to test whether cold or hypoxia-acclimation can alleviate oxidation damage upon lethal cold exposure. the results indicate that exposure to mild cold or low oxygen stress has no effect on the content of malondialdehyde, the product of lipid peroxidation. after the exposure of larvae to lethal cold for 12 h, the mda concentration of cold- and hypoxia-acclimated samples was significantly lower than that of the control. this is consistent with the protective effect of cold- and hypoxia-acclimation against lethal cold stress.figure cold- and hypoxia-acclimation alleviated oxidation damage upon lethal cold exposure. larvae at hpf were subjected to cold- or hypoxia-acclimation for 24 h and then incubated at 10°c for 12 h. the larvae in the same plate were homogenized for lipid peroxidation assays with a kit based on the reaction of thiobarbituric acid with malondialdehyde. the mda content was normalized to protein concentration. data was shown as mean ± standard deviation. different letters above the error bars indicate significant differences among treatment groups. discussion oxygen and temperature are the most important environmental factors for the life of fishes and both hypoxia and temperature stress can cause deleterious effects on the organismal performance. due to the importance of these factors, fishes have developed acclimation mechanisms to survive the daily and seasonal fluctuations in oxygen concentration or temperature in their habitats during the process of evolution. the interdependence between oxygen availability and thermal resistance has been well documented in fishes, i.e. exposure of fish to both low and high temperature stresses can induce tissue hypoxia through impairing the cardiovascular function and oxygen will subsequently limit the thermal tolerance of fish. although the protection of hypoxia acclimation against the adverse effect of heat stress has been reported, it is not clear whether there is a cross-resistance between hypoxia and cold stress. in this study, pre-acclimation of zebrafish larvae to mild hypoxia significantly increased the survival rates after lethal cold exposure, indicating the roles of hypoxia-inducible pathways in the establishment of cold resistance in fish. however, pre-acclimation to mild cold significantly reduced the survival rate of zebrafish larvae upon lethal hypoxia exposure. this is consistent with a previous study in mammal that cold acclimation decreased the hypoxia resistance of rats. the increased incidence of fatty changes in the striated muscle and the marked depletion of liver glycogen of cold-acclimated rats upon low oxygen may be responsible for the hypoxia-susceptibility. to investigate the molecular mechanisms underlying the protection of hypoxia-acclimation against cold stress, gene expression in hypoxia-acclimated zebrafish larvae was characterized using rna-seq and compared with that of cold-acclimated samples. although hypoxia-elicited gene expression in zebrafish has been explored by microarray in several previous studies, rna-seq characterization may give new information due to its advantages over microarray. a total of genes were found to be regulated by hypoxia in 96-hpf zebrafish larvae, including up-regulated and down-regulated genes. a large part of these genes were not found to be regulated by hypoxia in previous microarray studies. the number of genes influenced by hypoxia in this study is markedly smaller than that of cold-regulated genes, but is quite similar with the previous studies characterizing hypoxia-regulated gene expression using microarray. this is consistent with the partial protection of hypoxia-acclimation against cold stress. it is possible that more complicated mechanisms are involved in the development of cold tolerance in fish. most of the hypoxia-induced genes identified in this study are involved in biosynthesis of the source material of hemoglobin, hematopoiesis and oxidation-reduction processes. hemoglobin genes including hbz, hbm, hbae, hbae, hbbe and hbbe were among the most prominent gene families induced by hypoxia. although hbz was the most highly up-regulated gene under hypoxia, hbae hbbe and hbbe were the most abundant hemoglobin genes in both control and hypoxia-treated samples. however, hemoglobin genes were not found to be up-regulated in 24-hpf zebrafish embryos exposed to hypoxia. genes involved in erythrocyte differentiation such as alas, tfr1a, slc4a1a and gata1a were also highly induced by hypoxia. alas encodes 5-aminolevulinate synthase, the rate-controlling enzyme of erythroid heme synthesis; mutations of this gene are the causative of the x-linked sideroblastic anemia in human. transferrin receptor trf plays a crucial role in cellular iron uptake and is previously reported to be induced by hypoxia in human cell lines. anion exchanger slc4a functions as a transporter that mediates anion exchange across the cell membrane and as a structural protein, which is required for normal flexibility and stability of the erythrocyte membrane via the interactions of its cytoplasmic domain with cytoskeletal proteins. hematopoietic transcription factor gata is indispensible for the maturation of erythrocytes. the up-regulation of these genes underlies the increased resistance of zebrafish larvae to lethal hypoxia during the acclimation process. hif- is the critical regulator for hypoxia response and plays key roles in cellular hypoxia acclimation. the activity of hif- is tightly regulated by the egl- family hypoxia-inducible factors and the hypoxia-inducible factor alpha subunit inhibitor. egln genes encode proline hydroxylases which mediate proteosomal degradation of hif1α under normoxic oxygen conditions. egln genes were reported to be regulated by hif- and suggested to be a negative feedback regulatory mechanism for limiting accumulation of hif1α in hypoxia. like egln members, hif1an hydroxylates a conserved asparaginyl residue within hif1α when oxygen is available, and thus prevents the recruitment of co-activators and suppresses the activity. because the activities of both egln members and hif1an are oxygen-dependent, hif1α is stabilized under hypoxia and dimerizes with hif1β to form active hif- zebrafish has four hif1a genes, including hif1aa, hif1ab, hif1al and hif1al but hif1al was the only one up-regulated by hypoxia in this study. however, hif1aa, hif1ab and hif1al were slightly but significantly induced by cold in our previous study. it is interesting that all hif1α-suppressing genes, including egln genes and hif1an were induced by hypoxia but none of them was up-regulated by cold stress. it is possible that the stress response of hypoxia under 18°c is weaker than that of 5% oxygen and therefore the negative regulatory mechanism is not activated. the protective effects of hypoxia acclimation against cold stress in zebrafish larvae prompts us to explore the mechanisms activated under both hypoxia and cold. genes co-induced by hypoxia and cold are mainly involved in oxidation-reduction processes, hemoglobin biosynthetic process and oxygen transport. cold-induced oxidative stress was widely found in insects, fishes and mammals like rat, suggesting that establishment of defense systems against oxidative damage would be a common task for cold acclimation in animals. among the genes co-induced by hypoxia and cold, sqrdl functions to catalyze the conversion of sulfide to persulfides, thereby decreasing toxic concentrations of sulfide accumulated upon hypoxia/ischemia. hephl is an analog of ceruloplasmin that functions as a multicopper ferroxidase to convert fe2+ to less toxic fe3+ and aids in counteracting the deleterious effects of iron/ros-mediated oxidative damage. cyp46a is the cholesterol 24-hydroxylase which catalyzes the oxidation of cholesterol into 24s-hydroxycholesterol to facilitate the efflux of cholesterol across the blood–brain barrier. since the content of cholesterol is negatively related to the fluidity of cell membrane, elimination of cholesterol from cells may be necessary for the increment of membrane fluidity at low temperature. myoglobin is important for intracellular oxygen transportation upon oxygen scarcity and was reported to be up-regulated by hypoxia in various carp tissues. up-regulation of mb by cold stress further suggests that improving oxygen supply is an important aspect of cold acclimation. furthermore, genes involved in oxygen transport, iron homeostasis and hemoglobin biosynthetic process such as hbae tfr1a, tfr1b, urod and steap were up-regulated upon both hypoxia and cold. nearly all these co-regulated genes are involved in improving cellular oxygen availability and oxidation-reduction process. furthermore, the results of lipid peroxidation assays demonstrated that both cold and hypoxia pre-acclimation alleviate oxidation damage caused by lethal cold stress, suggesting the association of these genes with cold acclimation. however, the increase in the abundance of transcripts under cold stress is not necessarily resulted from activated transcription since the turnover of rna can be affected by temperature. the post-transcriptional regulation mechanisms reducing mrna decay may contribute to the up-regulation of certain genes as well. in addition to co-induced genes, we identified and confirmed the alternative promoter usage of hmbsb under hypoxia and cold. alternative promoter usage is a versatile mechanism to create diversity and flexibility in the regulation of gene expression besides alternative splicing. messenger rna molecules derived from alternative promoters may differ in the level of transcription initiation, stability and translation efficiency. alternative promoters can have different tissue specificity, react differently to environmental signals and lead to the generation of protein isoforms differing at the amino terminus. subsequently, a different amino terminus can lead to alterations in protein levels, functions, or subcellular distribution. zebrafish hmbsb gene is involved in the tetrapyrrole biosynthetic process and is specifically expressed in blood. alternative promoter usage of zebrafish hmbsb gene leads to peptides differing in the initial ten amino acids. promoter transition was also found for human hmbs gene: the housekeeping promoter is active in all cells and the alternative promoter is present only in erythroid cells. although the alternative promoter of human hmbs gene is associated with tissue specificity, the biological significance and mechanisms underlying the hypoxia/cold-inducibility of the alternative promoter of zebrafish hmbsb gene remains to be characterized.  hypoxia acclimation increased the survival rate of zebrafish larvae after lethal cold exposure, indicating that hypoxia-inducible signaling pathways play important roles in the establishment of fish cold resistance. hypoxia acclimation of zebrafish larvae is intimately associated with biological processes including oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development and cellular iron ion homeostasis. genes co-induced by hypoxia and cold are mainly involved in oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis. an alternative promoter of hmbsb is specifically activated by hypoxia and cold. the transcriptional events co-regulated by hypoxia and cold represent the molecular basis of hypoxia-induced protection against cold stress. 
hypoxia-inhibited genes are enriched in inorganic anion transport, sodium ion transport, very long-chain fatty acid biosynthetic process and cytidine deamination. gene ontology enrichment analyses revealed that genes up-regulated by hypoxia are primarily involved in oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development and cellular iron ion homeostasis. a comparison with the dataset of cold-regulated gene expression identified genes co-induced by hypoxia and cold and these genes are mainly associated with oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis. rna-seq and bioinformatics analyses revealed the biological processes associated with hypoxia acclimation. the alleviation of lipid peroxidation damage by both cold- and hypoxia-acclimation upon lethal cold stress suggests the association of these genes with cold resistance. hypoxia and temperature stress are two major adverse environmental conditions often encountered by fishes. the interaction between hypoxia and temperature stresses has been well documented and oxygen is considered to be the limiting factor for the thermal tolerance of fish. although both high and low temperature stresses can impair the cardiovascular function and the cross-resistance between hypoxia and heat stress has been found, it is not clear whether hypoxia acclimation can protect fish from cold injury. pre-acclimation of 96-hpf zebrafish larvae to mild hypoxia significantly improved their resistance to lethal hypoxia and increased the survival rate of zebrafish larvae after lethal cold exposure. furthermore, the alternative promoter of hmbsb gene specifically activated by hypoxia and cold was identified and confirmed. zebrafishhypoxiacold stressrna-seqgene expressionissue-copyright-statement© the author 2015 acclimation responses to mild hypoxia and cold stress were found in zebrafish larvae and pre-acclimation to hypoxia significantly improved the tolerance of larvae to lethal cold stress. transcriptional events co-induced by hypoxia and cold may represent the molecular basis underlying the protection of hypoxia-acclimation against cold injury. however, pre-acclimation of 96-hpf larvae to cold decreased their tolerance to lethal hypoxia although their ability to endure lethal cold increased. rna-seq analysis identified up-regulated and down-regulated genes upon mild hypoxia exposure.
 hypoxia and temperature stress are two major adverse environmental conditions often encountered by fishes. the interaction between hypoxia and temperature stresses has been well documented and oxygen is considered to be the limiting factor for the thermal tolerance of fish. although both high and low temperature stresses can impair the cardiovascular function and the cross-resistance between hypoxia and heat stress has been found, it is not clear whether hypoxia acclimation can protect fish from cold injury.  pre-acclimation of 96-hpf zebrafish larvae to mild hypoxia significantly improved their resistance to lethal hypoxia and increased the survival rate of zebrafish larvae after lethal cold exposure. however, pre-acclimation of 96-hpf larvae to cold decreased their tolerance to lethal hypoxia although their ability to endure lethal cold increased. rna-seq analysis identified up-regulated and down-regulated genes upon mild hypoxia exposure. gene ontology enrichment analyses revealed that genes up-regulated by hypoxia are primarily involved in oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development and cellular iron ion homeostasis. hypoxia-inhibited genes are enriched in inorganic anion transport, sodium ion transport, very long-chain fatty acid biosynthetic process and cytidine deamination. a comparison with the dataset of cold-regulated gene expression identified genes co-induced by hypoxia and cold and these genes are mainly associated with oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis. the alleviation of lipid peroxidation damage by both cold- and hypoxia-acclimation upon lethal cold stress suggests the association of these genes with cold resistance. furthermore, the alternative promoter of hmbsb gene specifically activated by hypoxia and cold was identified and confirmed.  acclimation responses to mild hypoxia and cold stress were found in zebrafish larvae and pre-acclimation to hypoxia significantly improved the tolerance of larvae to lethal cold stress. rna-seq and bioinformatics analyses revealed the biological processes associated with hypoxia acclimation. transcriptional events co-induced by hypoxia and cold may represent the molecular basis underlying the protection of hypoxia-acclimation against cold injury. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords zebrafishhypoxiacold stressrna-seqgene expressionissue-copyright-statement© the author 2015 