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Hypoxia-modulated gene expression profiling in sea urchin (Strongylocentrotus nudus) immune cells
Ecotoxicology and Environmental Safety 109 (2014) 63–69
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Hypoxia-modulated gene expression profiling in sea urchin (Strongylocentrotus nudus) immune cells Sung-Suk Suh a, Jinik Hwang a, Mirye Park a, So Yun Park a, Tae Kwon Ryu b, Sukchan Lee c, Taek-Kyun Lee a,n a
South Sea Environment Research Department, Korea Institute of Ocean Science and Technology, Geoje 656-830, Republic of Korea Accident Prevention and Assessment Division, Yuseong-gu, Daejeon, 305-343, Republic of Korea c Department of Genetic Engineering, SungKyunKwan University, Suwon 440-746, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 January 2014 Received in revised form 7 August 2014 Accepted 11 August 2014 Available online 24 August 2014
Hypoxia is an issue that affects ocean coastal waters worldwide. It has severe consequences for marine organisms, including death and rapid adaptive changes in metabolic organization. Although some aquatic animals are routinely exposed and resistant to severe environmental hypoxia, others such as sea urchins (Strongylocentrotus nudus) have a limited capacity to withstand this stress. In this study, hypoxia induced a significant increase in the number of red spherule cells among coelomocytes, which function as immune cells. This suggests that sea urchin immune cells could be used as a biological indicator of hypoxic stress. In the current study, we used cDNA microarrays to investigate the differential expression patterns of hypoxia-regulated genes to better understand the molecular mechanisms underlying the response of immune cells to hypoxia. Surprisingly, the predominant major effect of hypoxia was the widespread suppression of gene expression. In particular, the expression of RNA helicase and GATA-4/5/6 was decreased significantly in response to hypoxia, even in field conditions, suggesting that they could be utilized as sensitive bioindicators of hypoxic stress in the sea urchin. & 2014 Elsevier Inc. All rights reserved.
Keywords: Hypoxia Coelomocytes Strongylocentrotus nudus RNA helicase GATA-4/5/6
1. Introduction Rapid changes in dissolved oxygen (DO) levels in coastal waters have occurred over recent decades. The combined effects of the continued spread of coastal eutrophication and global warming have led to the widespread occurrence of hypoxia, a condition in which DO is below the level necessary to sustain organismal life, defined as o2 mg/l (Vaquer-Sunyer and Duarte, 2008). The area of oceanic hypoxic zones has increased continuously since 1960, with a current total area of 245,000 square kilometers worldwide (Diaz and Rosenberg, 2008). This has caused severe changes in marine ecosystems in the adjacent waters, which has resulted in the mass death of organisms including fishes, echinoderms, crustaceans, mollusks, and cnidarians (Vaquer-Sunyer and Duarte, 2008). Recently, molecular studies were performed to investigate the effects of hypoxia on aquatic organisms, including invertebrates and fish. Data revealed that the hypoxic response upregulated genes related to glycolysis, iron metabolism, amino acid metabolism and growth suppression, whereas genes related to
n
Corresponding author. Fax: þ 82 55 639 8539. E-mail address: [email protected] (T.-K. Lee).
http://dx.doi.org/10.1016/j.ecoenv.2014.08.011 0147-6513/& 2014 Elsevier Inc. All rights reserved.
translation were downregulated in aquatic organisms (Gracey et al., 2001; Padilla and Roth, 2001; Holm et al., 2008). In addition, a decrease in the expression of the GTP-binding protein Rab, which is relevant to cell growth and proliferation, and increases in HSP70 and glutathione reductase (GR) levels were observed in the embryos of zebrafish and sea stars exposed to hypoxia (Padilla and Roth, 2001; Holm et al., 2008). The consumption of green sea urchins was found to be decreased dramatically in oxygen-depleted environments, suggesting altered expression patterns of genes associated with translation (Morales et al., 2006; Siikavuopio et al., 2007). Nevertheless, limited studies have assessed the molecular aspects of the responses of marine organisms, including sea urchins, to hypoxia. Because they are suitable biological organisms for investigating the quality of seawater, sea urchins are becoming widely used to assess seawater pollution and the effects of marine environmental changes on aquatic organisms. For example, genome studies in purple sea urchins were performed recently (Hood and Fernandez, 2008; Hibino et al., 2006). Strongylocentrotus nudus belongs to the family Strongylocentrotidae and mostly exists on the coasts of South Korea. S. nudus is advantageous as a model organism for studies assessing the initial effects of external stresses, including hypoxia (Ryu et al., 2012). For example, coelomocytes were
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recognized as immune cells because of their ability to respond to various environmental stresses, such as temperature and UVradiation (Matranga et al., 2005, 2006). The number of red spherule cells, a minor immune cell group, was increased in sea urchins exposed to different physical and chemical stresses (Glinski and Jarosz, 2000; Matranga et al., 2000), suggesting that sea urchin immune cells might be novel cellular biosensors of environmental stress (Pinsino et al., 2008). However, the molecular mechanisms underlying the response of immune cells to hypoxia in sea urchins are not fully understood. The aim of this study was to investigate the number of red cells and cell death in the immune cells of the sea urchin in response to hypoxia. cDNA microarray analysis was also performed to identify the genes differentially expressed in sea urchin immune cells under hypoxic conditions. Data from both the laboratory and the field revealed that RNA helicase and GATA-4/5/6 were hypoxiarepressed genes, suggesting they might act as biomarkers of the stress response to hypoxia in sea urchins.
2. Materials and methods 2.1. Sea urchin culture Strongylocentrotus nudus used in this study were collected directly from the coast of Geoje, South Korea, and were cultured at 18 1C, pH 8.45 and 33.72 (‰), and fed seaweed in the culturing room of the Korea Institute of Ocean Science and Technology. The diameter of the sea urchins used in this study was 7–10 cm. Hypoxic (DO ¼1.6 mg O2/L), normal (DO¼ 7.6 mg O2/L), and intermediate (DO ¼ 4.7 mg O2/L) environments were maintained using an aquacontroller (Neptune systems, Water Management Technologies Inc., USA). The levels of DO were measured using an oxygen probe with Unidata Starlog data loggers (cat# 7422A, Unidata Pty. Ltd., Australia) every 6 h. Adult sea urchins were incubated for 0, 6, 12, or 24 h in a 50-L water bath filled with filtered sea water (FSW). Twentyfour sea urchins were used in each treatment, with six harvested at per time point (0 h, 6 h, 12 h, 24 h). In the field, chambers containing fifteen sea urchins and 100 g seaweed were settled in normoxic (1 m) and hypoxic regions (9 m). After settling, sea urchins were cultured for seven days and then harvested. Environmental factors such as DO, temperature, salinity, and pH were measured daily. 2.2. Color change in sea urchin immune cells After sampling, the peristomial membrane around the mouth of the sea urchin was removed using scissors, and the immune cells were collected using 50-mL syringes. The collected immune cells were divided among the wells of a 12-well plate, and the colors of the immune cells in each treatment condition were compared.
2.3. Cell death assay Hypoxia-induced cell death was measured using a LIVE/DEAD Viability/Cytotoxicity kit (Invitrogen, CA, USA). Sea urchins were exposed to hypoxia (DO¼ 1.6 mg O2/L) and normoxia (DO¼ 7.6 mg O2/L) for 0, 6, 12, and 24 h in a 50L water bath. Immune cells ( 106 cells) were centrifuged at 1000g for 5 min and then resuspended in 200 μL PBS. Next, 5 μL 2 mM EthD-1 and 3 μL 4 mM calcein AM were added, after which the cells were vortexed and incubated at room temperature for 30 min. The immune cells were then washed with D-PBS and observed under the UV filter of a fluorescent microscope (Carl Zeiss, Axioplan II, Welwyn Garden, Germany). Viable and non-viable cells were colored green and red, respectively.
2.4. Microarrays Expression profiling was performed using the Strongylocentrotus purpuratus 135k microarray (NimbleGen Inc; http://www.nimblegen.com/). The 135k microarray was designed from 89,602 UniGenes clustered from 141,833 expressed sequence tags (ESTs) and 44,561 cDNAs available at NCBI (http://www.ncbi.nlm. nih.gov/). Three 60-nucleotide (nt)-long probes were designed along each gene, starting 60 bp prior to the stop codon and in 30-bp increments, such that the three probes covered 120 bp of the 30 region of the gene. A total of 133,244 probes were designed, with a mean probe size of 60 nt and melting temperatures of 75– 85 1C. Selection markers including gfp, gus, hyg, bar, and kan were also included. Random GC probes (38,000) were included to monitor hybridization efficiency, and four corner fiducial controls were included to assist with overlaying the grid on the images. The microarray analyses were repeated three times. A RevertAid H ( ) first strand cDNA synthesis kit (Fermentas, Ontario, Canada) was used to synthesize double-stranded DNA, and the synthesized cDNA was cleaned utilizing a MinElute reaction kit (Fermentas). For microarray hybridization, 10 μg DNA was mixed with 19.5 μL 2 hybridization buffer and 39 μL distilled water (DW). Hybridization was performed in a MAUI chamber at 42 1C for 18 h. After hybridization, the samples were washed twice and scanned using a GenePix Scanner 4000B (Axon, CA, USA).
2.5. Quantitative real-time PCR Total RNA was isolated from the immune cells of sea urchins, and cDNA synthesis was performed using the Reverse Transcription System (Promega, Madison, WI, USA). Actin was used as the housekeeping gene, and quantitative real-time PCR (qRT-PCR) was performed to assess the expression of six different genes identified in the microarray analyses. The primers used in qRT-PCR are summarized in Table S1. Differences in gene expression were quantified using the delta CT method. The qRT-PCR reactions were performed using the following conditions: 94 1C for 5 min, followed by 40 cycles at 94 1C for 20 s, 55 1C for 20 s, and 72 1C for 15 s. A reaction volume of 20 μL was used for the PCR amplifications, and the reactions were performed by mixing 2 μL 10 reaction buffer, 2 μL 2.5 mM dNTPs, 0.5 μL Taq DNA polymerase, 5 μL cDNA, and 1 μL SYBR.
Fig. 1. A schematic diagram to show how to prepare the sea urchin coelmocytes.
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2.6. Statistical analysis
3.2. Hypoxia-induced immune cell death
All analyses were performed using GraphPad Prism statistical software (GraphPad). One-way ANOVA was used for statistical analyses. Data are presented as means7SE.
Next, we assessed the effects of hypoxia on the survival of sea urchin immune cells using a LIVE/DEAD Viability/Cytotoxicity kit. This kit uses two different fluorescence dyes: calcein AM and ethidium homodimer (EthD-1). Calcein AM exhibits green fluorescence when bound to viable cells, whereas EthD-1 fluoresces red when bound to non-viable cells. As such, viable and non-viable cells can be differentiated using fluorescence analysis. The experiment was performed by exposing the immune cells to DO levels of 7.6 and 1.6 mg O2/L for 0, 12, and 24 h. Most of the immune cells exposed to normoxia (DO¼ 7.6 mg O2/L) fluoresced green, whereas under hypoxia (DO¼1.6 mg O2/L), the proportion of red cells increased rapidly over time: 40 percent of cells stained red after 24 h of hypoxia (Fig. 3A and B). This suggests that hypoxia induces death of immune cells in the sea urchin.
3. Results 3.1. Hypoxia affects the cellular composition of immune cells Sampling was performed 0, 6, 12, and 24 h after the sea urchins were transferred to chambers in which the DO levels had been adjusted to 7.6, 4.7, or 1.6 mg O2/L (Fig. 1). Although the immune cells exposed to 4.7 mg O2/L DO had a noticeable difference in the number of red spherule cells after 24 h, a significant increase in the number of red spherule cells was observed in urchins treated with 1.6 mg O2/L DO (Fig. 2A and B). Furthermore, under depleted oxygen conditions (DO ¼1.6 mg O2/L), the protein and mRNA levels of HSP70 and GR, as molecular indicators of the response to hypoxia, were increased, respectively, suggesting that sea urchin immune cells responded effectively to hypoxic conditions (Fig. 2C and D). Taken together, these data suggest that 1.6 mg O2/L could be used as an experimental hypoxic DO level to significantly increase the number of red spherule immune cells.
3.3. Identification of genes modulated by hypoxic conditions We used DNA microarrays to investigate gene expression patterns in sea urchin immune cells to elucidate the mechanisms regulating the response of sea urchins to hypoxic stress. Immune cells were sampled 12 and 24 h after exposure to hypoxic conditions. To interpret and organize the data, the differentially
Fig. 2. Hypoxia significantly increased red spherule cells. (A) Sea urchin immune cells were exposed to three different concentrations of the dissolved oxygen (DO), 7.6, 4.7, and 1.6 mg O2/L. The population of red spherule cells were dramatically increased in response to hypoxia (DO¼ 1.6 mg O2/L). (B) The ratio of red spherule cells vs. total cells in treatment of different concentrations of oxygen. (C) Expression level of HSP70 was induced with 6 h hypoxia time course experiment (DO ¼ 1.6 mg O2/L). (D) An increase of glutathione reductase (GR), an antioxidant gene, was caused by hypoxia. The results are means 7 s.d, n ¼3 experiments.
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Fig. 3. Hypoxia-induced cell death of immune cells. (A) Cell death of the immune cells was measured using the LIVE/DEAD Viability/Cytotoxicity kit. The experiment was carried out by treating immune cells with DO ¼ 7.6 mg O2/L and DO ¼ 1.6 mg O2/L for 6, 12 and 24 h exposure times and tested for their viability. (B) The quantification of green or red fluorescence intensity by spectrometer. The green-fluorescent (530 nm) indicates live-cell population and the red-fluorescent (585 nm) does dead-cell population.
Fig. 4. Identification of hypoxia-modulated genes using cDNA microarray. (A) cDNA Microarray assay was carried out in order to identify the differentially expressed genes in response to hypoxia. The genes were classified into six different functional processes using COG analysis (B).
expressed cDNAs were sequenced and grouped according to their temporal expression pattern using a hierarchical clustering algorithm. Fig. 4A shows that the expression of many genes changed over time after treatment with hypoxia. Among these, we were interested in genes which exhibited a 4two-fold difference in expression compared with the non-hypoxia group. Clusters of Orthologous Groups (COG) analysis revealed that many of the differentially expressed genes shared functions such as posttranslational modification, signal transduction, cell defense, cell cycle,
transcription, and RNA processing and modification (Fig. 4B and Table S2). For example, among the 39 genes related to signal transduction, 27 that play roles in post-translational modification and twelve that regulate RNA processing were decreased in expression in hypoxia-treated sea urchins. In addition, of the twenty genes related to transcriptional regulation, five that play roles in cell cycle and five that regulate defense were also reduced in expression (Fig. 3B and Table S2). Among these, the genes closely associated with cell growth are shown in Table S3.
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3.4. Analysis of gene expression ratios using quantitative real-time PCR Interestingly, cDNA microarray analysis revealed widespread suppression of gene expression after exposure to hypoxia (Fig. 4B). Therefore, we next assessed the changes in expression of hypoxia-repressed genes after different exposure times. Specifi cally, we selected downregulated genes with functional relevance to hypoxia, including CDP, GATA, RNA helicase, DCP2, and c-type lectin. Interestingly, there were three distinct expression patterns observed in the presence of hypoxia in terms of hypoxic exposure time and the concentration of DO. Specifically, the expression levels of c-type lectin, GATA-4/5/6, and DCP were reduced, compared with the control group, when sea urchins were chronically exposed to hypoxic (DO¼1.6 mg O2/L), but not to non-hypoxic conditions (DO¼4.7 mg O2/L), regardless of a 6 or 12 h exposure time (Fig. 5A). In particular, expression decreased gradually over the 12 h of exposure to hypoxia. In contrast, the response of RNA helicase to hypoxia was dependent on the exposure time to hypoxia, but not the DO concentration (Fig. 5B). In addition, the expression of CDP1 and spindle pole body genes was decreased in response to both hypoxic exposure time and DO; a more significant reduction was seen in the expression of spindle pole body genes compared with CDP1 (Fig. 5C). 3.5. RNA helicase and GATA-4/5/6 could be biomarkers of hypoxia exposure in on-site samples To determine whether the observations after hypoxic stress in sea urchins in a laboratory setting could be applied to the field, urchins were cultured in hypoxic seawater for 30 days. The expression of hypoxia-modulated genes was then analyzed using cDNA microarrays. For the purpose of this study, it was first necessary to define the environmental seawater conditions
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necessary to induce a hypoxic response. DO observations along the Jangmok-myeon coast of South Korea over the preceding ten years revealed that hypoxic conditions were maintained at the bottom of the sea in July, whereas the upper seawater levels were not hypoxic (Fig. 6A). Furthermore, additional environmental parameters, such as temperature, salinity, and pH, which can affect physiological processes in marine organisms, were consistent across different sea depths (Fig. 6A). Therefore, sea urchins were cultured in chambers placed in hypoxic or non-hypoxic areas of the sea (Fig. 6A). qRT-PCR revealed that the levels of RNA helicase and GATA were decreased under hypoxic compared with normoxic conditions (Fig. 6B), whereas the expression of other hypoxia-suppressed genes was unchanged. This data suggests that RNA helicase and GATA might be useful biomarkers of hypoxiainduced stress in the sea urchin S. nudus.
4. Discussion Because of the current expansion of hypoxic water masses due to global warming and air pollution, the development of a suitable biomarker for hypoxia is required for the successful management of targets to avoid hypoxia-derived loss of biodiversity in coastal waters. Recent studies revealed that immune cells, which are comprised of granular cells, including white and red spherule cells and vibratile cells, act as immune effector cells in the sea urchin. After stimulation by different physical and chemical stresses, they secrete a number of specific molecules such as lectins, perforins, lysosomal enzymes, and profilins (Matranga, 1996; Matranga et al., 2005). However, the molecular mechanisms that explain this property of immune cells in the presence of hypoxia are not fully understood. In the current study, we observed physiological changes, such as a significant increase in the number of red spherule cells and induction of cell death in sea urchin immune
Fig. 5. Quantitative real time RT-PCR (qRT-PCR) analysis of the hypoxia-regulated genes. (A, B, C) Expression patterns of genes related with defense mechanism, cell cycle control, transcription, and RNA processing and modification, which are C-type lectin, spindle pole body, CDP1, GATA-4/5/6, RNA helicase, and DCP2 were determined by qRTPCR. Experiments were performed three times and the data are presented as the mean 7s.d. n, nn and nnn indicate significant differences from control sample at p o0.05, p o 0.01, and p o 0.001, respectively (one-way ANOVA and Tukey's post hoc t-test).
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Fig. 6. Expression patterns of hypoxia-modulated genes in field condition. A figure showing the settlement of sea urchin in the Jangmok-myeon coast are exposed to the hypoxia conditions and environmental parameters in there (A). (B) The levels of RNA helicase and GATA mRNAs were decreased in response to hypoxia zone. The relative concentrations of hypoxia related genes were calculated after normalization to the actin gene. Data bars represent mean 7 SE. *p o 0.05.
cells in response to hypoxia, as well as molecular changes in gene expression. These observations are consistent with those of previous studies (Glinski and Jarosz, 2000; Matranga et al., 2000, 2005). Unexpectedly, the predominant consequence of gene expression in this model system was a widespread repression of gene expression. Interestingly, some of the repressed genes could participate in the regulatory mechanisms of gene expression such as gene transcription and signal transduction (Table S2). This fact suggests that the down-regulation of these types of genes might lead to a widespread repression of gene expression in sea urchin exposed to hypoxia. In addition, among the suppressed genes, CCAAT displacement protein (CDP) belongs to a novel family of homeodomain proteins and represses apoptosis regulators (Aufiero et al., 1994, Zhai et al., 2012). GATA is a transcription factor that regulates many cellular pathways and also plays a role in apoptosis (Suzuki and Evans, 2004). RNA helicase plays a role in the motor-rearrangement of RNA secondary structures and is downregulated by environmental stresses (Owttrim, 2006). Another RNA processing regulator, DCP2, is an important part of the mRNA decapping complex that removes the 50 cap of mRNAs and is closely related to mRNA stability (Piccirillo et al., 2003). In addition, hypoxia also altered the expression of c-type lectin, which plays a role in cellular defense and the cell cycle. The mRNA levels of these genes were significantly decreased in immune cells under hypoxic conditions. In particular, the reduction in the expression of GATA and RNA-helicase observed in sea urchins from the field was lower than what observed for sea urchins exposed to hypoxia in the tanks. The variation in their expression levels between the field and tanks might be occurred due to the difference of environmental contents in two places. For example, in the case of tank experiment environmental
parameters such as DO, salinity, temperature and pH were maintained at a constant levels during the experimental period, but not in the field. In addition, Hsp70 protein levels were shown to increase after hypoxia (Fig. 2). However, no hsp70 gene expression levels were described in the identification of hypoxia-modulated genes using cDNA microarray because it was omitted in the processing of sorting the data with respect to insignificant p value (p 40.05). Furthermore, its mRNA levels were less than two fold compared with control group. Altogether, these data suggest that sea urchins might protect themselves from stress by increasing their populations of red spherule cells by activating different genes involved in the cellular defense mechanism. Although previous reports have described changes in the expression of specific genes in sea urchins in response to hypoxia, this is the first report to assess in overall gene expression changes in sea urchin immune cells. In addition, consistent with previous studies (Azad et al., 2008; Shimizu et al., 1996), in vitro assays to quantify cell death in hypoxia-treated immune cells revealed that a large proportion of them underwent apoptosis, which can be used as a defense mechanism against environmental chemicals, and physiological and mechanical stresses. However, the exact mechanism by which immune cells undergo cell death, in spite of an increased number of red spherule cells, remains unclear. As such, a more comprehensive investigation is needed to further assess this phenomenon. The current study also assessed changes in the expression of hypoxia-regulated genes using cDNA microarrays, which revealed a widespread decrease in the expression of genes related to a variety of cellular processes, such as RNA processing, transcription, cell cycle, and cellular defense. Among these, the expression of RNA helicase and GATA-4/5/6 were also modulated in field
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experiments, suggesting that these genes might be candidate biomarkers for assessing the hypoxic response in sea urchins.
5. Conclusions This study used cDNA microarrays to profile hypoxia-modulated genes in sea urchin immune cells. Our data suggest that sea urchins might protect themselves from hypoxic conditions by increasing the number of immune cells (red spherule cells) and inducing the expression of genes involved in cellular defense mechanisms. Knowledge of gene expression patterns in response to hypoxia could provide crucial clues to elucidate the molecular regulation of the blooming events caused by hypoxic stress in coastal areas. Nevertheless, further studies using more conventional biochemical and physiological approaches will be needed to interpret the complex hypoxic responses in sea urchins.
Acknowledgments This research study was carried out within the coordinated research Project no. PE99193 supported financially by the Korea Institute of Ocean Science & Technology.
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Gracey, A.Y., Troll, J.V., Somero, G.N., 2001. Hypoxia-induced gene expression profiling in the euryoxic fish Gillichthys mirabilis. Proc. Natl. Acad. Sci. USA 98, 1993–1998. Hibino, T., Loza-Coll, M., Messier, C., Majeske, A.J., Cohen, A.H., Terwilliger, D.P., Buckley, K.M., Brockton, V., Nair, S.V., Berney, K., Fugmann, S.D., Anderson, M.K., Pancer, Z., Cameron, R.A., Smith, L.C., Rast, J.P., 2006. The immune gene repertoire encoded in the purple sea urchin genome. Dev. Biol. 300, 349–365. Holm, K., Hernroth, B., Thorndyke, M., 2008. Coelomocyte numbers and expression of HSP70 in wounded sea stars during hypoxia. Cell Tissue Res. 334, 319–325. Hood, L., Fernandez, N., 2008. Gene regulatory networks and embryonic specification. Proc. Natl. Acad. Sci. USA 105, 5951–5952. Matranga, V., 1996. Molecular aspects of immune reactions in Echinodermata. Prog. Mol. Subcell. Biol. 15, 235–247. Matranga, V., Pinsino, A., Celi, M., Di Bella, G., Natoli, A., 2006. Impacts of UV-B radiation on short-term cultures of sea urchin coelomocytes. Mar. Biol. 149, 25–34. Matranga, V., Pinsino, A., Celi, M., Natoli, A., Bonaventura, R., Schröder, H., Müller, W., 2005. Monitoring chemical and physical stress using sea urchin immune cells. Prog. Mol. Subcell. Biol. 39, 85–110. Matranga, V., Toia, G., Bonaventura, R., Müller, W.E., 2000. Cellular and biochemical responses to environmental and experimentally induced stress in sea urchin coelomocytes. Cell Stress Chaperones 5, 113–120. Morales, J., Mulner-Lorillon, O., Cosson, B., Morin, E., Belle, R., Bradham, C.A., Beane, W.S., Cormier, P., 2006. Translational control genes in the sea urchin genome. Dev. Biol. 300, 293–307. Owttrim, G.W., 2006. RNA helicases and abiotic stress. Nucleic Acids Res. 34, 3220–3230. Padilla, P.A., Roth, M.B., 2001. Oxygen deprivation causes suspended animation in the zebrafish embryo. Proc. Natl. Acad. Sci. USA 98, 7331–7335. Piccirillo, C., Khanna, R., Kiledjian, M., 2003. Functional characterization of the mammalian mRNA decapping enzyme hDcp2. RNA 9, 1138–1147. Pinsino, A., Della Torre, C., Sammarini, V., Bonaventura, R., Amato, E., Matranga, V., 2008. Sea urchin coelomocytes as a novel cellular biosensor of environmental stress: a field study in the Tremiti Island Marine Protected Area, Southern Adriatic Sea, Italy. Cell Biol. Toxicol. 24, 541–552. Ryu, T.K., Lee, G., Rhee, Y., Park, H.S., Chang, M., Lee, S., Lee, J., Lee, T.K., 2012. Identification of nickel response genes in abnormal early developments of sea urchin by differential display polymerase chain reaction. Ecotoxicol. Environ. Saf. 84, 18–24. Shimizu, S., Enuchi, Y., Karniike, W., Itoh, Y., Hasegawa, J.I., Yamabe, K., Otsuki, Y., Matsuda, H., Tsujimoto, Y., 1996. Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-xL. Cancer Res. 56, 2161–2166. Siikavuopio, S.I., Dale, T., Mortensen, A., Foss, A., 2007. Effects of hypoxia on feed intake and gonad growth in the green sea urchin, Strongylocentrotus droebachiensis. Aquaculture 266, 112–116. Suzuki, Y.J., Evans, T., 2004. Regulation of cardiac myocyte apoptosis by the GATA-4 transcription factor. Life Sci. 74, 1829–1838. Vaquer-Sunyer, R., Duarte, C.M., 2008. Thresholds of hypoxia for marine biodiversity. Proc. Natl. Acad. Sci. USA 105, 15452–15457. Zhai, Z., Ha, N., Papagiannouli, F., Hamacher-Brady, A., Brady, N., Sorge, S., Bezdan, D., Lohmann, I., 2012. Antagonistic regulation of apoptosis and differentiation by the cut transcription factor represents a tumor-suppressing mechanism in Drosophila. PLoS Genet. 8, e1002582.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Hypoxia-modulated gene expression profiling in sea urchin (Strongylocentrotus nudus) immune cells Sung-Suk Suh a, Jinik Hwang a, Mirye Park a, So Yun Park a, Tae Kwon Ryu b, Sukchan Lee c, Taek-Kyun Lee a,n a
South Sea Environment Research Department, Korea Institute of Ocean Science and Technology, Geoje 656-830, Republic of Korea Accident Prevention and Assessment Division, Yuseong-gu, Daejeon, 305-343, Republic of Korea c Department of Genetic Engineering, SungKyunKwan University, Suwon 440-746, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 13 January 2014 Received in revised form 7 August 2014 Accepted 11 August 2014 Available online 24 August 2014
Hypoxia is an issue that affects ocean coastal waters worldwide. It has severe consequences for marine organisms, including death and rapid adaptive changes in metabolic organization. Although some aquatic animals are routinely exposed and resistant to severe environmental hypoxia, others such as sea urchins (Strongylocentrotus nudus) have a limited capacity to withstand this stress. In this study, hypoxia induced a significant increase in the number of red spherule cells among coelomocytes, which function as immune cells. This suggests that sea urchin immune cells could be used as a biological indicator of hypoxic stress. In the current study, we used cDNA microarrays to investigate the differential expression patterns of hypoxia-regulated genes to better understand the molecular mechanisms underlying the response of immune cells to hypoxia. Surprisingly, the predominant major effect of hypoxia was the widespread suppression of gene expression. In particular, the expression of RNA helicase and GATA-4/5/6 was decreased significantly in response to hypoxia, even in field conditions, suggesting that they could be utilized as sensitive bioindicators of hypoxic stress in the sea urchin. & 2014 Elsevier Inc. All rights reserved.
Keywords: Hypoxia Coelomocytes Strongylocentrotus nudus RNA helicase GATA-4/5/6
1. Introduction Rapid changes in dissolved oxygen (DO) levels in coastal waters have occurred over recent decades. The combined effects of the continued spread of coastal eutrophication and global warming have led to the widespread occurrence of hypoxia, a condition in which DO is below the level necessary to sustain organismal life, defined as o2 mg/l (Vaquer-Sunyer and Duarte, 2008). The area of oceanic hypoxic zones has increased continuously since 1960, with a current total area of 245,000 square kilometers worldwide (Diaz and Rosenberg, 2008). This has caused severe changes in marine ecosystems in the adjacent waters, which has resulted in the mass death of organisms including fishes, echinoderms, crustaceans, mollusks, and cnidarians (Vaquer-Sunyer and Duarte, 2008). Recently, molecular studies were performed to investigate the effects of hypoxia on aquatic organisms, including invertebrates and fish. Data revealed that the hypoxic response upregulated genes related to glycolysis, iron metabolism, amino acid metabolism and growth suppression, whereas genes related to
n
Corresponding author. Fax: þ 82 55 639 8539. E-mail address: [email protected] (T.-K. Lee).
http://dx.doi.org/10.1016/j.ecoenv.2014.08.011 0147-6513/& 2014 Elsevier Inc. All rights reserved.
translation were downregulated in aquatic organisms (Gracey et al., 2001; Padilla and Roth, 2001; Holm et al., 2008). In addition, a decrease in the expression of the GTP-binding protein Rab, which is relevant to cell growth and proliferation, and increases in HSP70 and glutathione reductase (GR) levels were observed in the embryos of zebrafish and sea stars exposed to hypoxia (Padilla and Roth, 2001; Holm et al., 2008). The consumption of green sea urchins was found to be decreased dramatically in oxygen-depleted environments, suggesting altered expression patterns of genes associated with translation (Morales et al., 2006; Siikavuopio et al., 2007). Nevertheless, limited studies have assessed the molecular aspects of the responses of marine organisms, including sea urchins, to hypoxia. Because they are suitable biological organisms for investigating the quality of seawater, sea urchins are becoming widely used to assess seawater pollution and the effects of marine environmental changes on aquatic organisms. For example, genome studies in purple sea urchins were performed recently (Hood and Fernandez, 2008; Hibino et al., 2006). Strongylocentrotus nudus belongs to the family Strongylocentrotidae and mostly exists on the coasts of South Korea. S. nudus is advantageous as a model organism for studies assessing the initial effects of external stresses, including hypoxia (Ryu et al., 2012). For example, coelomocytes were
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recognized as immune cells because of their ability to respond to various environmental stresses, such as temperature and UVradiation (Matranga et al., 2005, 2006). The number of red spherule cells, a minor immune cell group, was increased in sea urchins exposed to different physical and chemical stresses (Glinski and Jarosz, 2000; Matranga et al., 2000), suggesting that sea urchin immune cells might be novel cellular biosensors of environmental stress (Pinsino et al., 2008). However, the molecular mechanisms underlying the response of immune cells to hypoxia in sea urchins are not fully understood. The aim of this study was to investigate the number of red cells and cell death in the immune cells of the sea urchin in response to hypoxia. cDNA microarray analysis was also performed to identify the genes differentially expressed in sea urchin immune cells under hypoxic conditions. Data from both the laboratory and the field revealed that RNA helicase and GATA-4/5/6 were hypoxiarepressed genes, suggesting they might act as biomarkers of the stress response to hypoxia in sea urchins.
2. Materials and methods 2.1. Sea urchin culture Strongylocentrotus nudus used in this study were collected directly from the coast of Geoje, South Korea, and were cultured at 18 1C, pH 8.45 and 33.72 (‰), and fed seaweed in the culturing room of the Korea Institute of Ocean Science and Technology. The diameter of the sea urchins used in this study was 7–10 cm. Hypoxic (DO ¼1.6 mg O2/L), normal (DO¼ 7.6 mg O2/L), and intermediate (DO ¼ 4.7 mg O2/L) environments were maintained using an aquacontroller (Neptune systems, Water Management Technologies Inc., USA). The levels of DO were measured using an oxygen probe with Unidata Starlog data loggers (cat# 7422A, Unidata Pty. Ltd., Australia) every 6 h. Adult sea urchins were incubated for 0, 6, 12, or 24 h in a 50-L water bath filled with filtered sea water (FSW). Twentyfour sea urchins were used in each treatment, with six harvested at per time point (0 h, 6 h, 12 h, 24 h). In the field, chambers containing fifteen sea urchins and 100 g seaweed were settled in normoxic (1 m) and hypoxic regions (9 m). After settling, sea urchins were cultured for seven days and then harvested. Environmental factors such as DO, temperature, salinity, and pH were measured daily. 2.2. Color change in sea urchin immune cells After sampling, the peristomial membrane around the mouth of the sea urchin was removed using scissors, and the immune cells were collected using 50-mL syringes. The collected immune cells were divided among the wells of a 12-well plate, and the colors of the immune cells in each treatment condition were compared.
2.3. Cell death assay Hypoxia-induced cell death was measured using a LIVE/DEAD Viability/Cytotoxicity kit (Invitrogen, CA, USA). Sea urchins were exposed to hypoxia (DO¼ 1.6 mg O2/L) and normoxia (DO¼ 7.6 mg O2/L) for 0, 6, 12, and 24 h in a 50L water bath. Immune cells ( 106 cells) were centrifuged at 1000g for 5 min and then resuspended in 200 μL PBS. Next, 5 μL 2 mM EthD-1 and 3 μL 4 mM calcein AM were added, after which the cells were vortexed and incubated at room temperature for 30 min. The immune cells were then washed with D-PBS and observed under the UV filter of a fluorescent microscope (Carl Zeiss, Axioplan II, Welwyn Garden, Germany). Viable and non-viable cells were colored green and red, respectively.
2.4. Microarrays Expression profiling was performed using the Strongylocentrotus purpuratus 135k microarray (NimbleGen Inc; http://www.nimblegen.com/). The 135k microarray was designed from 89,602 UniGenes clustered from 141,833 expressed sequence tags (ESTs) and 44,561 cDNAs available at NCBI (http://www.ncbi.nlm. nih.gov/). Three 60-nucleotide (nt)-long probes were designed along each gene, starting 60 bp prior to the stop codon and in 30-bp increments, such that the three probes covered 120 bp of the 30 region of the gene. A total of 133,244 probes were designed, with a mean probe size of 60 nt and melting temperatures of 75– 85 1C. Selection markers including gfp, gus, hyg, bar, and kan were also included. Random GC probes (38,000) were included to monitor hybridization efficiency, and four corner fiducial controls were included to assist with overlaying the grid on the images. The microarray analyses were repeated three times. A RevertAid H ( ) first strand cDNA synthesis kit (Fermentas, Ontario, Canada) was used to synthesize double-stranded DNA, and the synthesized cDNA was cleaned utilizing a MinElute reaction kit (Fermentas). For microarray hybridization, 10 μg DNA was mixed with 19.5 μL 2 hybridization buffer and 39 μL distilled water (DW). Hybridization was performed in a MAUI chamber at 42 1C for 18 h. After hybridization, the samples were washed twice and scanned using a GenePix Scanner 4000B (Axon, CA, USA).
2.5. Quantitative real-time PCR Total RNA was isolated from the immune cells of sea urchins, and cDNA synthesis was performed using the Reverse Transcription System (Promega, Madison, WI, USA). Actin was used as the housekeeping gene, and quantitative real-time PCR (qRT-PCR) was performed to assess the expression of six different genes identified in the microarray analyses. The primers used in qRT-PCR are summarized in Table S1. Differences in gene expression were quantified using the delta CT method. The qRT-PCR reactions were performed using the following conditions: 94 1C for 5 min, followed by 40 cycles at 94 1C for 20 s, 55 1C for 20 s, and 72 1C for 15 s. A reaction volume of 20 μL was used for the PCR amplifications, and the reactions were performed by mixing 2 μL 10 reaction buffer, 2 μL 2.5 mM dNTPs, 0.5 μL Taq DNA polymerase, 5 μL cDNA, and 1 μL SYBR.
Fig. 1. A schematic diagram to show how to prepare the sea urchin coelmocytes.
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2.6. Statistical analysis
3.2. Hypoxia-induced immune cell death
All analyses were performed using GraphPad Prism statistical software (GraphPad). One-way ANOVA was used for statistical analyses. Data are presented as means7SE.
Next, we assessed the effects of hypoxia on the survival of sea urchin immune cells using a LIVE/DEAD Viability/Cytotoxicity kit. This kit uses two different fluorescence dyes: calcein AM and ethidium homodimer (EthD-1). Calcein AM exhibits green fluorescence when bound to viable cells, whereas EthD-1 fluoresces red when bound to non-viable cells. As such, viable and non-viable cells can be differentiated using fluorescence analysis. The experiment was performed by exposing the immune cells to DO levels of 7.6 and 1.6 mg O2/L for 0, 12, and 24 h. Most of the immune cells exposed to normoxia (DO¼ 7.6 mg O2/L) fluoresced green, whereas under hypoxia (DO¼1.6 mg O2/L), the proportion of red cells increased rapidly over time: 40 percent of cells stained red after 24 h of hypoxia (Fig. 3A and B). This suggests that hypoxia induces death of immune cells in the sea urchin.
3. Results 3.1. Hypoxia affects the cellular composition of immune cells Sampling was performed 0, 6, 12, and 24 h after the sea urchins were transferred to chambers in which the DO levels had been adjusted to 7.6, 4.7, or 1.6 mg O2/L (Fig. 1). Although the immune cells exposed to 4.7 mg O2/L DO had a noticeable difference in the number of red spherule cells after 24 h, a significant increase in the number of red spherule cells was observed in urchins treated with 1.6 mg O2/L DO (Fig. 2A and B). Furthermore, under depleted oxygen conditions (DO ¼1.6 mg O2/L), the protein and mRNA levels of HSP70 and GR, as molecular indicators of the response to hypoxia, were increased, respectively, suggesting that sea urchin immune cells responded effectively to hypoxic conditions (Fig. 2C and D). Taken together, these data suggest that 1.6 mg O2/L could be used as an experimental hypoxic DO level to significantly increase the number of red spherule immune cells.
3.3. Identification of genes modulated by hypoxic conditions We used DNA microarrays to investigate gene expression patterns in sea urchin immune cells to elucidate the mechanisms regulating the response of sea urchins to hypoxic stress. Immune cells were sampled 12 and 24 h after exposure to hypoxic conditions. To interpret and organize the data, the differentially
Fig. 2. Hypoxia significantly increased red spherule cells. (A) Sea urchin immune cells were exposed to three different concentrations of the dissolved oxygen (DO), 7.6, 4.7, and 1.6 mg O2/L. The population of red spherule cells were dramatically increased in response to hypoxia (DO¼ 1.6 mg O2/L). (B) The ratio of red spherule cells vs. total cells in treatment of different concentrations of oxygen. (C) Expression level of HSP70 was induced with 6 h hypoxia time course experiment (DO ¼ 1.6 mg O2/L). (D) An increase of glutathione reductase (GR), an antioxidant gene, was caused by hypoxia. The results are means 7 s.d, n ¼3 experiments.
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Fig. 3. Hypoxia-induced cell death of immune cells. (A) Cell death of the immune cells was measured using the LIVE/DEAD Viability/Cytotoxicity kit. The experiment was carried out by treating immune cells with DO ¼ 7.6 mg O2/L and DO ¼ 1.6 mg O2/L for 6, 12 and 24 h exposure times and tested for their viability. (B) The quantification of green or red fluorescence intensity by spectrometer. The green-fluorescent (530 nm) indicates live-cell population and the red-fluorescent (585 nm) does dead-cell population.
Fig. 4. Identification of hypoxia-modulated genes using cDNA microarray. (A) cDNA Microarray assay was carried out in order to identify the differentially expressed genes in response to hypoxia. The genes were classified into six different functional processes using COG analysis (B).
expressed cDNAs were sequenced and grouped according to their temporal expression pattern using a hierarchical clustering algorithm. Fig. 4A shows that the expression of many genes changed over time after treatment with hypoxia. Among these, we were interested in genes which exhibited a 4two-fold difference in expression compared with the non-hypoxia group. Clusters of Orthologous Groups (COG) analysis revealed that many of the differentially expressed genes shared functions such as posttranslational modification, signal transduction, cell defense, cell cycle,
transcription, and RNA processing and modification (Fig. 4B and Table S2). For example, among the 39 genes related to signal transduction, 27 that play roles in post-translational modification and twelve that regulate RNA processing were decreased in expression in hypoxia-treated sea urchins. In addition, of the twenty genes related to transcriptional regulation, five that play roles in cell cycle and five that regulate defense were also reduced in expression (Fig. 3B and Table S2). Among these, the genes closely associated with cell growth are shown in Table S3.
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3.4. Analysis of gene expression ratios using quantitative real-time PCR Interestingly, cDNA microarray analysis revealed widespread suppression of gene expression after exposure to hypoxia (Fig. 4B). Therefore, we next assessed the changes in expression of hypoxia-repressed genes after different exposure times. Specifi cally, we selected downregulated genes with functional relevance to hypoxia, including CDP, GATA, RNA helicase, DCP2, and c-type lectin. Interestingly, there were three distinct expression patterns observed in the presence of hypoxia in terms of hypoxic exposure time and the concentration of DO. Specifically, the expression levels of c-type lectin, GATA-4/5/6, and DCP were reduced, compared with the control group, when sea urchins were chronically exposed to hypoxic (DO¼1.6 mg O2/L), but not to non-hypoxic conditions (DO¼4.7 mg O2/L), regardless of a 6 or 12 h exposure time (Fig. 5A). In particular, expression decreased gradually over the 12 h of exposure to hypoxia. In contrast, the response of RNA helicase to hypoxia was dependent on the exposure time to hypoxia, but not the DO concentration (Fig. 5B). In addition, the expression of CDP1 and spindle pole body genes was decreased in response to both hypoxic exposure time and DO; a more significant reduction was seen in the expression of spindle pole body genes compared with CDP1 (Fig. 5C). 3.5. RNA helicase and GATA-4/5/6 could be biomarkers of hypoxia exposure in on-site samples To determine whether the observations after hypoxic stress in sea urchins in a laboratory setting could be applied to the field, urchins were cultured in hypoxic seawater for 30 days. The expression of hypoxia-modulated genes was then analyzed using cDNA microarrays. For the purpose of this study, it was first necessary to define the environmental seawater conditions
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necessary to induce a hypoxic response. DO observations along the Jangmok-myeon coast of South Korea over the preceding ten years revealed that hypoxic conditions were maintained at the bottom of the sea in July, whereas the upper seawater levels were not hypoxic (Fig. 6A). Furthermore, additional environmental parameters, such as temperature, salinity, and pH, which can affect physiological processes in marine organisms, were consistent across different sea depths (Fig. 6A). Therefore, sea urchins were cultured in chambers placed in hypoxic or non-hypoxic areas of the sea (Fig. 6A). qRT-PCR revealed that the levels of RNA helicase and GATA were decreased under hypoxic compared with normoxic conditions (Fig. 6B), whereas the expression of other hypoxia-suppressed genes was unchanged. This data suggests that RNA helicase and GATA might be useful biomarkers of hypoxiainduced stress in the sea urchin S. nudus.
4. Discussion Because of the current expansion of hypoxic water masses due to global warming and air pollution, the development of a suitable biomarker for hypoxia is required for the successful management of targets to avoid hypoxia-derived loss of biodiversity in coastal waters. Recent studies revealed that immune cells, which are comprised of granular cells, including white and red spherule cells and vibratile cells, act as immune effector cells in the sea urchin. After stimulation by different physical and chemical stresses, they secrete a number of specific molecules such as lectins, perforins, lysosomal enzymes, and profilins (Matranga, 1996; Matranga et al., 2005). However, the molecular mechanisms that explain this property of immune cells in the presence of hypoxia are not fully understood. In the current study, we observed physiological changes, such as a significant increase in the number of red spherule cells and induction of cell death in sea urchin immune
Fig. 5. Quantitative real time RT-PCR (qRT-PCR) analysis of the hypoxia-regulated genes. (A, B, C) Expression patterns of genes related with defense mechanism, cell cycle control, transcription, and RNA processing and modification, which are C-type lectin, spindle pole body, CDP1, GATA-4/5/6, RNA helicase, and DCP2 were determined by qRTPCR. Experiments were performed three times and the data are presented as the mean 7s.d. n, nn and nnn indicate significant differences from control sample at p o0.05, p o 0.01, and p o 0.001, respectively (one-way ANOVA and Tukey's post hoc t-test).
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Fig. 6. Expression patterns of hypoxia-modulated genes in field condition. A figure showing the settlement of sea urchin in the Jangmok-myeon coast are exposed to the hypoxia conditions and environmental parameters in there (A). (B) The levels of RNA helicase and GATA mRNAs were decreased in response to hypoxia zone. The relative concentrations of hypoxia related genes were calculated after normalization to the actin gene. Data bars represent mean 7 SE. *p o 0.05.
cells in response to hypoxia, as well as molecular changes in gene expression. These observations are consistent with those of previous studies (Glinski and Jarosz, 2000; Matranga et al., 2000, 2005). Unexpectedly, the predominant consequence of gene expression in this model system was a widespread repression of gene expression. Interestingly, some of the repressed genes could participate in the regulatory mechanisms of gene expression such as gene transcription and signal transduction (Table S2). This fact suggests that the down-regulation of these types of genes might lead to a widespread repression of gene expression in sea urchin exposed to hypoxia. In addition, among the suppressed genes, CCAAT displacement protein (CDP) belongs to a novel family of homeodomain proteins and represses apoptosis regulators (Aufiero et al., 1994, Zhai et al., 2012). GATA is a transcription factor that regulates many cellular pathways and also plays a role in apoptosis (Suzuki and Evans, 2004). RNA helicase plays a role in the motor-rearrangement of RNA secondary structures and is downregulated by environmental stresses (Owttrim, 2006). Another RNA processing regulator, DCP2, is an important part of the mRNA decapping complex that removes the 50 cap of mRNAs and is closely related to mRNA stability (Piccirillo et al., 2003). In addition, hypoxia also altered the expression of c-type lectin, which plays a role in cellular defense and the cell cycle. The mRNA levels of these genes were significantly decreased in immune cells under hypoxic conditions. In particular, the reduction in the expression of GATA and RNA-helicase observed in sea urchins from the field was lower than what observed for sea urchins exposed to hypoxia in the tanks. The variation in their expression levels between the field and tanks might be occurred due to the difference of environmental contents in two places. For example, in the case of tank experiment environmental
parameters such as DO, salinity, temperature and pH were maintained at a constant levels during the experimental period, but not in the field. In addition, Hsp70 protein levels were shown to increase after hypoxia (Fig. 2). However, no hsp70 gene expression levels were described in the identification of hypoxia-modulated genes using cDNA microarray because it was omitted in the processing of sorting the data with respect to insignificant p value (p 40.05). Furthermore, its mRNA levels were less than two fold compared with control group. Altogether, these data suggest that sea urchins might protect themselves from stress by increasing their populations of red spherule cells by activating different genes involved in the cellular defense mechanism. Although previous reports have described changes in the expression of specific genes in sea urchins in response to hypoxia, this is the first report to assess in overall gene expression changes in sea urchin immune cells. In addition, consistent with previous studies (Azad et al., 2008; Shimizu et al., 1996), in vitro assays to quantify cell death in hypoxia-treated immune cells revealed that a large proportion of them underwent apoptosis, which can be used as a defense mechanism against environmental chemicals, and physiological and mechanical stresses. However, the exact mechanism by which immune cells undergo cell death, in spite of an increased number of red spherule cells, remains unclear. As such, a more comprehensive investigation is needed to further assess this phenomenon. The current study also assessed changes in the expression of hypoxia-regulated genes using cDNA microarrays, which revealed a widespread decrease in the expression of genes related to a variety of cellular processes, such as RNA processing, transcription, cell cycle, and cellular defense. Among these, the expression of RNA helicase and GATA-4/5/6 were also modulated in field
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experiments, suggesting that these genes might be candidate biomarkers for assessing the hypoxic response in sea urchins.
5. Conclusions This study used cDNA microarrays to profile hypoxia-modulated genes in sea urchin immune cells. Our data suggest that sea urchins might protect themselves from hypoxic conditions by increasing the number of immune cells (red spherule cells) and inducing the expression of genes involved in cellular defense mechanisms. Knowledge of gene expression patterns in response to hypoxia could provide crucial clues to elucidate the molecular regulation of the blooming events caused by hypoxic stress in coastal areas. Nevertheless, further studies using more conventional biochemical and physiological approaches will be needed to interpret the complex hypoxic responses in sea urchins.
Acknowledgments This research study was carried out within the coordinated research Project no. PE99193 supported financially by the Korea Institute of Ocean Science & Technology.
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