Discovery of Osmotic Sensitive Transcription Factors in Fish Intestine Via a Transcriptomic Approach

Discovery of Osmotic Sensitive Transcription Factors in Fish Intestine Via a Transcriptomic Approach

Wong et al. BMC Genomics 2014, 15:1134 http://www.biomedcentral.com/1471-2164/15/1134 RESEARCH ARTICLE Open Access Discovery of osmotic sensitive transcription factors in fish intestine via a transcriptomic approach Marty Kwok-Shing Wong1*, Haruka Ozaki2, Yutaka Suzuki2, Wataru Iwasaki1,2,3 and Yoshio Takei1 Abstract Background: Teleost intestine is crucial for seawater acclimation by sensing osmolality of imbibed seawater and regulating drinking and water/ion absorption. Regulatory genes for transforming intestinal function have not been identified. A transcriptomic approach was used to search for such genes in the intestine of euryhaline medaka. Results: Quantitative RNA-seq by Illumina Hi-Seq Sequencing method was performed to analyze intestinal gene expression 0 h, 1 h, 3 h, 1 d, and 7 d after seawater transfer. Gene ontology (GO) enrichment results showed that cell adhesion, signal transduction, and protein phosphorylation gene categories were augmented soon after transfer, indicating a rapid reorganization of cellular components and functions. Among >50 transiently up-regulated transcription factors selected via co-expression correlation and GO selection, five transcription factors, including CEBPB and CEBPD, were confirmed by quantitative PCR to be specific to hyperosmotic stress, while others were also up-regulated after freshwater control transfer, including some well-known osmotic-stress transcription factors such as SGK1 and TSC22D3/ Ostf1. Protein interaction networks suggest a high degree of overlapping among the signaling of transcription factors that respond to osmotic and general stresses, which sheds light on the interpretation of their roles during hyperosmotic stress and emergency. Conclusions: Since cortisol is an important hormone for seawater acclimation as well as for general stress in teleosts, emergency and osmotic challenges could have been evolved in parallel and resulted in the overlapped signaling networks. Our results revealed important interactions among transcription factors and offer a multifactorial perspective of genes involved in seawater acclimation. Keywords: Transcriptome, Fish osmoregulation, Intestine, Seawater acclimation, Transcription factors, CEBPB, CEBPD, SGK1, TSC22D3 Background to maintain body fluid homeostasis in both FW and SW Osmoregulation is an important topic in fish physiology. [2,3]. SW teleosts drink copiously and the gastrointes- Bony fishes maintain their body fluid osmolality approxi- tinal tract is responsible for water absorption to com- mately one-third that of seawater (SW) and therefore pensate for the water loss by osmosis [4,5]. Although the they constantly lose water and gain ions in SW but gain intestine is an internal organ, its lumen directly contacts en- water and lose ions in fresh water (FW). Osmoregulation vironmental water upon drinking in teleost fishes. Osmosen- consumes a high proportion of daily energy expenditure sing in fish is accomplished by a combination of sensors in in teleosts as they either actively excrete excess ions in the central nervous system and peripheral osmoregulatory SW or take up ions in FW against the respective con- epithelia such as gill, nasal cavity, and intestine [6]. A reflex centration gradients [1]. The gills, kidney, and intestine inhibition in drinking was demonstrated in eel intestine in − are major osmoregulatory organs and play different roles response to Cl ions (but not Na+) in ingested fluid, indicat- − ing the presence of a Cl specific sensor in eel intestine [7]. * Correspondence: [email protected] Euryhaline fishes that are able to acclimate in both 1Atmosphere and Ocean Research Institute, The University of Tokyo, Tokyo, Japan FW and SW transform their intestines dramatically to Full list of author information is available at the end of the article fulfill the appropriate osmoregulatory roles. When the © 2014 Wong et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wong et al. BMC Genomics 2014, 15:1134 Page 2 of 13 http://www.biomedcentral.com/1471-2164/15/1134 eel is transferred from FW to SW, the intestinal wall could initiate subsequent intestinal transformations and decreases in thickness, and the anterior intestine be- lead to altered function from FW to SW. came highly vascularized through angiogenesis within + − 1–2 days [8]. Monovalent ions (Na ,Cl)intheim- Results bibed SW are actively absorbed while divalent ions Illumina sequencing and reference gene mapping 2+ 2+ 2− (Ca ,Mg,SO4) are precipitated to decrease Illumina 101 bp paired end sequencing were performed luminal fluid osmolality [5,9]. The composition of on all 25 intestine samples collected from medaka after epithelial transporters is also reorganized extensively. 0 h, 1 h, 3 h, 1d, and 7 d after SW transfer (N = 5) The An upregulation of mucosal Na-K-2Cl cotranspoter sequenced reads ranged from 17.3 to 60.5 million reads (NKCC2/ SLC12A1) remarkably increases the ion ab- with average 37.9 million reads in each sample (Table 1). sorption rate, which is facilitated by the serosal Na-K- The read were mapped against the annotated genome + ATPase and Na -bicarbonate exchanger [10]. The of Medaka. More than 83% of the sequences were rapid and efficient ion absorption mechanism in tele- successfully mapped sequences, indicating the data ost intestine is unique in vertebrates [11]. The SW sets are representative. teleost intestine is an absorptive epithelium that is similar to the thick ascending loop of Henle in mam- Gene ontology analysis malian nephron, and thus was often used as a com- GO terms enriched in the 1 h-after-SW transfer group parative model for the study of kidney tubules, are listed in Table 2. According to biological process, especially in the study of transporter mechanisms and protein phosphorylation, regulation of DNA-dependent cellular regulation in response to volume and salt transcription, cell adhesion, and signal transduction stresses [12]. Several hormones such as cortisol, pro- were highly ranked in GO enrichment. According to lactin, growth hormone, atrial natriuretic peptide, ar- molecular function, protein binding, protein tyrosine ginine vasotocin, guanylin, and vasoactive intestinal kinase activity, protein serine/threonine kinase activity, peptide regulate transepithelial ion transport [13-20]. and actin binding were enriched. According to cellular However, the transcription factors that govern hor- component, significant enrichment in GO terms was mone actions, cell proliferation, apoptosis, angiogen- found in integrin complex and cytoskeleton. esis, transporter metabolism etc., are unclear. Transcriptomic approaches have been used to inves- tigate the dynamics osmoregulatory organ function Transiently upregulated genes of teleosts [21-24]. However, microarray- or pyrose- The intestinal transcription-related genes that are in- quencing-based transcriptomic reads do not provide volved in the SW acclimation were screened from the sufficient depth and coverage for the detection and genes with early transient increase in expression, which quantitation of low expression genes, which may result is defined as genes with significant increases in gene p in a biased discovery towards high expression genes. expression (one-way ANOVA, Tukey; < 0.05) in 1 h In the present study, the objective was to discover the and/or 3 h post-transfer groups compared to those of 0 h, transcription factors that are responsive to the SW 1d, and 7d. The candidates genes were further filtered to re- challenge in medaka intestine. Illumina HiSeq Sequen- move low expression genes (<20 RPM at 1 h or 3 h post- cing was selected to provide a deep coverage of iden- transfer). Genes with significant correlation (Pearson r > 0.8) tified genes as the large number of reads allows in expression with a known osmotic transcription factor quantification of gene expression by mapping to refer- (TSC22D3) among the early transient increase genes ence genome, and is usually sufficient to detect most were selected for further analysis (Additional file 1: expressed gene even at low expression level [25], Table S1). The selected genes were further filtered by which is an ideal choice at a cost-performance per- GO annotation to obtain transcription-related genes spective. Medaka was used owing to the relative com- and 57 genes were further analyzed by real-time PCR. pleteness of the genome data, which can streamline the RNA-seq analysis and guarantee high accuracy and Real-time PCR reliability. Medaka is also a euryhaline species that can To confirm the adequacy of the SW transfer stimulus on survive a direct FW to 50% SW (ca. twice hypertonic the medaka intestine, SLC12A1/NKCC2 and AQP1 were to plasma) transfer [26]. We combined physiological used as reference genes since these transporters are and bioinformatic approaches in the experimental de- well-acknowledged to change in teleost intestine after sign, in which FW medaka was challenged by 50% SW SW challenges [27-29]. The real-time PCR results of transfer and time-dependent changes in intestinal SLC12A1 and AQP1 showed a gradual increase and transcriptomewereanalyzedbyRNA-seq.Wefocused

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