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First published online August 3, 2006
Journal of Experimental Biology 209, 3257-3265 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02352

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Regulation of osmotic stress transcription factor 1 (Ostf1) in tilapia (Oreochromis mossambicus) gill epithelium during salinity stress

Diego F. Fiol, Stephanie Y. Chan and Dietmar Kültz^*

Physiological Genomics Group, Department of Animal Science, University of California, Davis, One Shields Avenue, Meyer Hall, Davis, CA 95616, USA

View larger version (17K): [in a new window]   Fig. 1. Ostf1 mRNA response to hyperosmotic stress and dexamethasone (DEX) in EGCPC. (A) Primary cultures were exposed to isosmotic medium or different hyperosmotic media in the absence (black bars) or with the addition of 1 µmol l-1 DEX (grey bars). After 2 h treatment, cells were collected and mRNA quantified by qPCR. Relative Ostf1 mRNA abundances with respect to control (isosmotic medium, no DEX) and normalized to ß-actin content are shown. Values are means ± s.e.m. (N=3). Asterisks indicate significant differences (P<0.05) relative to the control. (B) Primary cultures were exposed to isosmotic medium (open squares) or 600 mOsm kg-1 hyperosmotic medium (black triangles) prepared by addition of NaCl. At the indicated times cells were collected and mRNA quantified by qPCR. Relative Ostf1 mRNA abundances with respect to control (time zero) and normalized to ß-actin content are shown. Values are means ± s.e.m. Asterisks indicate significant differences (P<0.05) relative to the control. N=3.

View larger version (16K): [in a new window]   Fig. 2. Ostf1 mRNA response to different hyperosmotic media in EGCPC. Osmolality was increased from 300 to 600 mOsmol kg-1 by addition of the indicated compounds. After 2 h, cells were collected and Ostf1 mRNA quantified by qPCR. Relative mRNA abundance with respect to control (isosmotic medium) and normalized to ß-actin content are depicted. Values are means ± s.e.m. (N=3 experiments). Asterisks indicate significant differences (P<0.05) relative to the control.

View larger version (33K): [in a new window]   Fig. 3. Identification of intronic sequences of tilapia Ostf1. (A) Possible exon-exon junctions were determined through an analysis of synteny between Tetraodron nigroviridis, Fugu rubripes and Danio rerio genomic sequences based on homology to full-length tilapia Ostf1 mRNA (GenBank AY679524). Blast hits link regions of high similarity between Ostf1 mRNA and the three genomic sequences identified using BLAST. Gray boxes along the genomic representations indicate transcripts predicted using the Ensembl bioinformatics tool. Tilapia exon-exon junctions, predicted from the conserved gene structure of Ostf1 paralogs, are indicated by black arrows over tilapia Ostf1 mRNA. Lengths of predicted introns 1/2 and 2/3 are indicated in base pairs for each genomic representation. (B) Detail of exon 2-exon 3 junction region in tilapia Ostf1 mRNA. The arrow indicates the predicted junction site. PCR primers designed to amplify intron 2/3 are shown. Bold letters in the primer sequences correspond to conserved 3' acceptor and 5' donor consensus sites. (C) Genomic sequence of tilapia Ostf1 intron 2/3 (lowercase), flanked by exons (uppercase). Arrows indicate the position of PCR primers used for amplification. Numbers indicate the sequence position in Ostf1 mRNA and relative intron length (between parentheses).

View larger version (33K): [in a new window]   Fig. 4. Validation and specificity of qPCR primers and reactions. (A) Proposed partial intron/exon structure for tilapia Ostf1. Exons (boxes, E1-3) and introns (lines) are depicted. Location of qPCR primers for the mature (mF and mR) and nascent (hnF and hnR) mRNAs are indicated. (B,C) Gel electrophoresis of the qPCR products (B) and dissociation curve profiles (C). (D,E) Standard curves of qPCR reactions for Ostf1 mRNA (D) and hnRNA (E) obtained with serial dilutions of cDNA. The equation of the linear correlation and the correlation coefficient (R) are indicated.

View larger version (22K): [in a new window]   Fig. 5. Analysis of Ostf1 mRNA transcription rates. Intact fish (A) or EGCPC (B) were exposed to hyperosmotic stress (freshwater to seawater transfer for intact fish, and from 300 to 600 mOsmol kg-1 exposure for EGCPC). At the indicated times samples were collected and Ostf1 mature (Ostf1) and nascent (hnOstf1) mRNAs were quantified by qPCR. Relative mRNA abundance with respect to control (time 0) and normalized to ß-actin content are depicted. Values are means ± s.e.m., N=3. Asterisks indicate significant differences between Ostf1 and hnOstf1 (P<0.05).

View larger version (12K): [in a new window]   Fig. 6. Stability of Ostf1 transcripts. EGCPC were pre-incubated for 2 h with 5 µg ml-1 actinomycin D in isosmotic medium (300 mOsmol kg-1). Treatments were initiated at time 0 for isosmotic controls (black squares) and cells exposed to hyperosmotic stress (600 mOsmol kg-1, NaCl; open circles). Ostf1 mRNA levels were determined by quantitative real-time PCR and normalized to 18S RNA. Remaining mRNA relative to time 0 is shown on a logarithmic scale. Asterisks indicate significant differences between pairs (P<0.05). Values are means ± s.e.m., N=4.

View larger version (4K): [in a new window]   Fig. 7. Localization of adenine-uridine rich elements (ARE) in the 3' untranslated region of tilapia Ostf1 mRNA. Localization of Ostf1 open reading frame (ORF) and ARE are indicated (in base pairs) along full-length tilapia Ostf1 cDNA (AY679524).