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First published online October 31, 2008
Journal of Experimental Biology 211, 3636-3649 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.022160
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A microarray-based transcriptomic time-course of hyper- and hypo-osmotic stress signaling events in the euryhaline fish Gillichthys mirabilis: osmosensors to effectors

Tyler G. Evans* and George N. Somero

Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950, USA


Figure 1
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  		Fig. 1. (A) Functional classification of all annotated genes that differed significantly from control values (N=168; ANOVA P<0.05) during hyper-(filled bars) and hypo-osmotic stress (open bars) in G. mirabilis gill tissue. Designations were based upon an assigned putative role during osmotic stress as determined by data contained within the Gene Ontology database, Uniprot and the primary literature. (B) Principal components analysis (PCA) illustrating gene expression differences between hyper- and hypo-osmotic stressed G. mirabilis gill tissue. PCA was performed on conditions (hyperosmotic stress shown as filled symbols; hypo-osmotic stress shown as open symbols; time=0 and controls shown as gray symbols) using all genes that differed significantly from control values (ANOVA P<0.05) during osmotic stress. Clustering indicates similar expression profiles among conditions (x-axis PCA component 1, 49.81% of variance; y-axis PCA component 2, 18.73% of variance).

 

Figure 2
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  		Fig. 2. Expression profiles of possible novel osmosensors in gill tissue of G. mirabilis. (A) FK506 binding protein 51 (FKBP-51) mRNA. (B) Translationally controlled tumor protein (TCTP) mRNA. Data points were plotted as the natural log of the ratio of Cy5 (experimental and control) to Cy3 (reference) divided by the mean ratio of Cy5 (control) to Cy3 (reference) for all control time points. The natural log of the ratio generated for control fish was then subtracted from all hyperosmotic (filled symbols, solid line), hypo-osmotic (open symbols, broken line) and control values at each time point (0, 1, 2, 4, 12 h post-exposure). Therefore, the expression of the control data point used in the microarray ANOVA is represented as 0 at each time point, and time=0 values have a degree of handling stress incorporated. A Tukey's post hoc test was performed to determine at which time points each gene differed significantly from controls. Filled stars denote significant expression during hyperosmotic stress. Open stars denote significant expression during hypo-osmotic stress. Values are shown ± s.e.m. P<0.05.

 

Figure 3
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  		Fig. 3. Expression profiles of genes participating in insulin-like growth factor signaling cascades identified during osmotic stress in G. mirabilis gill tissue. (A) Insulin receptor substrate 2 mRNA (IRS-2; Gm_43o05). (B) Insulin-like growth factor binding protein 1 precursor mRNA (IGFBP-1 precursor; Gm_CC11). (C) Insulin-like growth factor binding protein 1 mRNA (IGFBP-1; Gm_CF01). (D) Prolactin receptor precursor mRNA (PRL-R precursor). (E) Suppressor of cytokine signaling 3 mRNA (SOSC-3). (F) Somatostatin receptor 5 mRNA (SS-5). Data points were plotted and labeled as described previously. In cases where a single gene was represented by multiple significant features (those proceeded by a clone ID above), the feature with the strongest P-value was illustrated. Filled stars denote significant expression during hyperosmotic stress. Open stars denote significant expression during hypo-osmotic stress. Values are shown ± s.e.m. P<0.05.

 

Figure 4
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  		Fig. 4. Expression profiles of signal transduction genes expressed during osmotic stress in G. mirabilis gill tissue. (A) Serum and glucocorticoid regulated kinase isoform 1 mRNA (SGK-1; Gm_11g08). (B) Protein kinase C {Delta} mRNA (PKC-{Delta}). (C) Lipid phosphate phosphatase 2 mRNA (LPP-2). (D) Ras-guanyl releasing protein 1 mRNA (Ras-GRP-1). Data points were plotted and labeled as described previously. (E) Phosphorylated protein kinase C (PKC) substrate protein expression as determined by western blot analysis. Relative intensity was calculated by normalizing intensities against a standard protein loaded on each gel. The resulting value was then averaged from each of the three fish at each treatment (hyperosmotic stress shown as filled bars; hypo-osmotic stress shown as open bars; control and time=0 shown as gray bars) and time point (1, 2, 4, 12 h post-exposure) to yield the mean relative intensity values illustrated. Filled stars denote significant expression during hyperosmotic stress. Open stars denote significant expression during hypo-osmotic stress. Values are shown ± s.e.m. P<0.05.

 

Figure 5
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  		Fig. 5. Expression profiles of genes participating in mitogen activated protein kinase (MAPK) signaling cascades during osmotic stress in G. mirabilis gill tissue. (A) Receptor interacting serine/threonine protein kinase 2 mRNA (RIPK-2). (B) MAPK phosphatase 1 mRNA (MKP-1). (C) MAPK phosphatase 8 mRNA (MKP-8). Data points were plotted and labeled as described previously. (D) Phosphorylated Erk1/Erk 2 protein expression as determined by western blot analysis. Relative intensity and significance values were calculated as described previously. Filled stars denote significant expression during hyperosmotic stress. Open stars denote significant expression during hypo-osmotic stress. Values are shown ± s.e.m. P<0.05.

 

Figure 6
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  		Fig. 6. Expression profiles of genes regulating ion homeostasis (A,B) and organic osmolyte production (C) during osmotic stress in G. mirabilis gill tissue. (A) Sodium/potassium transporting ATPase {alpha}-1 chain precursor mRNA (Na+, K+-ATPase-{alpha} precursor). (B) Sodium channel protein type IX {alpha} subunit mRNA (Na+ channel XI {alpha}). (C) Osmolyte producing enzymes during hyperosmotic stress. Cysteine sulfinic acid decarboxylase mRNA (CSAD; Gm_49c21) catalyzes the rate limiting step in the formation of taurine (triangular symbols). Glutamate decarboxylase mRNA (GluDC) catalyzes the formation of GABA (circular symbols). Inositol monophosphatase mRNA (IMPase) catalyzes the formation of inositol (diamond-shaped symbols). Data points were plotted and labeled as described previously. Filled stars denote significant expression during hyperosmotic stress. Open stars denote significant expression during hypo-osmotic stress. Values are shown ± s.e.m. P<0.05. The expressions of ion homeostasis and organic osmolyte genes not illustrated are described in Table S1C,D, respectively, in supplementary material.

 

Figure 7
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  		Fig. 7. Expression profiles of cytoskeleton associated genes during osmotic stress in G. mirabilis gill tissue. (A) Rho-GTPase activating protein 7 mRNA (Rho-GAP 7). (B) Rho-GTPase activating protein 8 mRNA (Rho-GAP 8). (C) Scinderin mRNA. (D) {alpha}-actin mRNA ({alpha}-actin). Data points were plotted and labeled as described previously. Filled stars denote significant expression during hyperosmotic stress. Open stars denote significant expression during hypo-osmotic stress. Values are shown ± s.e.m. P<0.05. The expressions of cytoskeleton genes not illustrated are described in Table S1E in supplementary material.

 

Figure 8
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  		Fig. 8. Expression profiles of cell cycle associated genes during osmotic stress in G. mirabilis gill tissue. (A) Cyclin dependent kinase inhibitor 1B mRNA (CDK inhibitor-1B; Gm_39c13). Data points were plotted and labeled as described previously. The expressions of cell cycle genes not illustrated are described in Table S1I in supplementary material. (B) Phosphorylated (Phospho) histone H3 protein expression as determined by western blot analysis. Relative intensity and significance values were calculated as described previously. Filled stars denote significant expression during hyperosmotic stress. Open stars denote significant expression during hypo-osmotic stress. Values are shown ± s.e.m. P<0.05.

 

Figure 9
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  		Fig. 9. Expression profiles of proteolysis associated genes during osmotic stress in G. mirabilis gill tissue. (A) RING-finger protein mRNA (Gm_44a16). Data points were plotted and labeled as described previously. The expressions of proteolysis genes not illustrated are described in Table S1N in supplementary material. (B) Ubiquitin conjugated protein expression as determined by western blot analysis. Relative intensity and significance values were calculated as described previously. Filled stars denote significant expression during hyperosmotic stress. Open stars denote significant expression during hypo-osmotic stress. Values are shown ± s.e.m. P<0.05.

 

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© The Company of Biologists Ltd 2008