Aquatic environments exhibit wide temporal and spatial variations in oxygen levels compared to terrestrial environments. Fish are an excellent model for elucidating the underlying mechanisms of hypoxia adaptation. Over the past decade, several hypoxia-related proteins have been reported to act in concert to convey oxygen change information to downstream signaling effectors. Some signaling pathways, such as redox status, AMPK, MAPK and IGF/PI3K/Akt, are known to play a central role in hypoxia adaptation. These networks regulate oxygen-sensitive transcription factors which, in turn, affect the expression of hypoxia adaptation-related genes. This review summarizes current insights into hypoxia adaptation-related proteins and signaling pathways in fish.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 1266 growth performance (El-Sayed and Kawanna, 2008;Yan and Wang, 2010; Chen et al., 2011): (1) initial mass (g), wet mass of fish at the beginning of culture; (2) final mass (g), wet mass of fish at the final of culture; (3) daily mass gain (DMG), calculated as (final mass-initial mass)/culture days; (4) coefficient of variation (CV) for final mass, calculated as mean standard deviation of the final mass/mean final mass; (5) feed conversion ratio, calculated as feed consumption/mass gain; and (6) feed intake, calculated as 100×feed consumption/[days×(final mass+initial mass)/2)]. Quantitative PCRTotal RNAs were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and miRNAs were extracted using the miRNeasy kit (Qiagen, Gaithersburg, MD, USA). Real-time PCR for IGF-1 was performed using SYBR Green PCR mixture (Takara Bio, Otsu, Shiga, Japan) in a MyiQ5 Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Gene expression was normalized relative to the housekeeping gene (β-actin). The primer sequences are as follows: IGF-1, 5′-GTCTGTGGAGAGCGAGGCTTT-3′ (forward), 5′-CACGTGAC -CGCCTTGCA-3′ (reverse); β-actin, 5′-GTCCCTGTACGC-CTCTGGTCG-3′ (forward), 5′-GCCGGACTCATCGTACTCCTG-3′ (reverse). The relative amount of miRNA was detected using stemloop PCR, and U6 RNA was detected as the internal control. Relative gene or miRNA expression was determined using the comparative C t method, which is also referred to the 2 -ΔΔCt method (Chen et al., 2005;Schmittgen and Livak, 2008). Silencing of miR-206 in vivo using the antagomir methodThe antagomirs used in the study are single-stranded RNAs, which consist of 21-23 nucleotides modified as follows: antagomir-133a, CsAsGCUGGUUGAAGGGGACCsAs As As-Chol-3′; antagomir-206, CsCsACACACACUUCCUUACAUUs CsC sAs-Chol-3′; and mismatch antagomir, CsAsCGGUUCCAGGCACUGUsG sUs AsChol-3′. All nucleotides are 2′-OMe-modified; the 's' represents a phosphorothioate linkage; 'Chol' represents cholesterol linked through a hydroxyprolinol linkage. They were deprotected, desalted and purified by high-performance liquid chromatography. Antagomir was dissolved in phosphate-buffered saline before injection. Tilapia weighing approximately 1g received a 0.02ml tail-vein injection of saline or antagomir at a dose of 60mgkg -1 body mass twice a week. Tissues were harvested, snap-frozen and stored at -80°C.Cell culture and 3′-UTR luciferase reporter assay HEK 293T cells were obtained from the American Type Culture Collection (Rockville, MD, USA). They were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100Uml -1 penicillin, 100μgml -1 streptomycin and 250ngml -1 amphotericin B, and maintained at 37°C in a humidified 5% CO 2 incubator.To generate the 3′ UTR luciferase reporter construct, the full length of the 3′ UTR from IGF-1 was cloned into the downstream of the firefly luciferase gene in pGL3-control vector (Promega, Madison, WI, USA). The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla...

SUMMARY

SummaryMicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in skin color determination in fish has not been completely determined. Here, we identified that 13 miRNAs are differentially expressed between red and white skin. The analysis of miRNA spatial and temporal expression patterns suggests that miR-429 is a potential regulator of skin pigmentation. miR-429 silencing results in an obvious change in skin pigmentation. Bioinformatics analysis and a luciferase reporter assay show that miR-429 directly regulates expression of Foxd3 by targeting its 39-untranslated (39-UTR) region. miR-429 silencing leads to a substantial increase in the expression of Foxd3 in vivo, thereby repressing the transcription of MITF and its downstream genes, such as TYR, TYRP1 or TYRP2. These findings would provide a novel insight into the determination of skin color in fish.

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The Nile tilapia (Oreochromis niloticus; Cichlidae) is an economically important species in aquaculture and occupies a prominent position in the aquaculture industry. MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression involved in diverse biological and metabolic processes. To increase the repertoire of miRNAs characterized in tilapia, we used the Illumina/Solexa sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the different developmental stages of tilapia. Bioinformatic analyses suggest that 197 conserved and 27 novel miRNAs are expressed in tilapia. Sequence alignments indicate that all tested miRNAs and miRNAs* are highly conserved across many species. In addition, we characterized the tissue expression patterns of five miRNAs using real-time quantitative PCR. We found that miR-1/206, miR-7/9, and miR-122 is abundantly expressed in muscle, brain, and liver, respectively, implying a potential role in the regulation of tissue differentiation or the maintenance of tissue identity. Overall, our results expand the number of tilapia miRNAs, and the discovery of miRNAs in tilapia genome contributes to a better understanding the role of miRNAs in regulating diverse biological processes.