Mori T et al. (2009), Identification of a novel uromodulin-like gene ...

XB-ART-57334

PLoS One 2009 Jun 16;46:e5936. doi: 10.1371/journal.pone.0005936.

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Identification of a novel uromodulin-like gene related to predator-induced bulgy morph in anuran tadpoles by functional microarray analysis.

Mori T , Kawachi H , Imai C , Sugiyama M , Kurata Y , Kishida O , Nishimura K .

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Tadpoles of the anuran species Rana pirica can undergo predator-specific morphological responses. Exposure to a predation threat by larvae of the salamander Hynobius retardatus results in formation of a bulgy body (bulgy morph) with a higher tail. The tadpoles revert to a normal phenotype upon removal of the larval salamander threat. Although predator-induced phenotypic plasticity is of major interest to evolutionary ecologists, the molecular and physiological mechanisms that control this response have yet to be elucidated. In a previous study, we identified various genes that are expressed in the skin of the bulgy morph. However, it proved difficult to determine which of these were key genes in the control of gene expression associated with the bulgy phenotype. Here, we show that a novel gene plays an important role in the phenotypic plasticity producing the bulgy morph. A functional microarray analysis using facial tissue samples of control and bulgy morph tadpoles identified candidate functional genes for predator-specific morphological responses. A larger functional microarray was prepared than in the previous study and used to analyze mRNAs extracted from facial and brain tissues of tadpoles from induction-reversion experiments. We found that a novel uromodulin-like gene, which we name here pirica, was up-regulated and that keratin genes were down-regulated as the period of exposure to larval salamanders increased. Pirica consists of a 1296 bp open reading frame, which is putatively translated into a protein of 432 amino acids. The protein contains a zona pellucida domain similar to that of proteins that function to control water permeability. We found that the gene was expressed in the superficial epidermis of the tadpole skin.

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Species referenced: Xenopus
Genes referenced: gpi sst thdl18 umod.2 umod.3

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	Figure 1. Comparison of tadpoles with a normal phenotype and a bulgy morph phenotype induced by predatory larval salamanders. The normal tadpole is on the left, the bulgy morph tadpole on the right.
	Figure 2. Experimental design used to produce control, bulgy morph and reversion type tadpoles for the functional microarray analysis. One group of tadpoles (Ex1) was placed with a larval salamander for 4 days to induce formation of the bulgy morph phenotype; the predator was then removed and the tadpoles were allowed to revert to the normal phenotype for 4 days. This group is termed â8 days-out tadpolesâ. A second group of tadpoles (Ex2) was placed with the predator for the full 8 days. The control group was not exposed to a predator. Tadpoles from the Ex2 groups were sampled at 6 hours, 4 days and 8 days, those from the control group at 0 hour, 4 days, and 8 days. The tissues used for RNA extraction for the functional microarray analysis are indicated by the squares on the tadpoles. The symbols (1) to (4) indicate the comparative design of the microarray analysis, which was performed in triplicate with a dye swap experiment using cDNA microarray spotted onto different locations of the slide glass triplicates.
	Figure 3. Identification of candidate gene for monitoring up- and down- regulation of genes in the transcriptome of tadpoles using functional microarray analysis. (a) Cluster analysis of microarray data using tissue samples from tadpoles with induced bulgy morph phenotype and those reverting to normal from this phenotype. A Pearson correlation analysis was performed on clusters of up-regulated and down-regulated genes in the two tadpole groups. The columns headed 6 hours, 4 days, 8 days, and 8 days-out were calculated as (Ex2 6 hours/Cont 0 hour), (Ex2 4 days/Cont 4 days), (Ex2 8 days/Cont 8 days), and (Ex1 8 days-out/Ex2 8 days), respectively. Red indicates increased expression compared to median levels of the three replicates with dye swap, whereas green indicates decreased expression. The sequence data for type I-keratin (356), type I-keratin (393), type II-keratin (1018), and the novel uromodulin-like gene, pirica, were deposited in the DNA bank with accession numbers AB374263, AB374264, AB374265, and AB374266, respectively. (b) Relative levels of expression of keratin genes in the microarray analysis. Vertical bars represent standard errors, and the summary of the pairwise comparisons by Dunnett's T3 test is described in (c). The notations â€˜Sâ€™ and â€˜Nâ€™ respectively indicate â€˜significantâ€™ and â€˜not significantâ€™ at the family error rate of 5%. (d) Relative levels of expression of the pirica fragment in the microarray analysis. Vertical bars represent standard errors, and the same letters â€œaâ€ and â€œbâ€ indicate no significant differences by Dunnett's T3 multiple comparison post hoc test (p<0.05).
	Figure 5. In situ hybridization (ISH) analysis of control and bulgy morph tadpoles. (a) Tadpoles were longitudinally sectioned (5 Âµm) at the frontal side (1), the ventral side (2), and the dorsal side (3). (b) ISH analysis of bulgy tadpoles with an anti-sense probe for type-1 collagen expression. Arrowhead indicates type-1 collagen signal. (c) ISH of bulgy tadpoles with an anti-sense probe for pirica expression. Arrowhead indicates the signal for pirica expression. (d) Pirica gene expression with an anti-sense probe at the frontal side (1), ventral side (2), and dorsal side (3) of a bulgy morph tadpole. (e) ISH of pirica gene expression using a sense probe. (f) ISH of pirica gene expression in a control tadpole using an anti-sense probe. (g) ISH of pirica gene expression in a control tadpole using a sense probe.
	igure 4. Alignment of the deduced amino acid sequences of the pirica gene with those of homologous/related genes. Conserved residues in all sequences are highlighted: identical, similar and unrelated residues are indicated by dark, light and white backgrounds, respectively. The red box represents the zona pellucida domain. Potential GPI-anchor residues predicted by GPI-SOM (fttp://gpi.unibe.ch/) are represented by black letters with blue background, while those suggested from sequence similarity are highlighted in light blue. Residues underlined in red represent the transmembrane domain predicted by the TMpred program (http://www.ch.embnet.org/). Letters edged in yellow represent the predicted proteolytic cleavage site. All relevant sequences of the uromodulin (THP), GP-2, and XL18 have been registered in the DDBJ/EMBL/GenBank databases with accession numbers XL18 (AAC59871), rTHP (AAB33313), hTHP (P07911), and hGP-2 (AAB19240), respectively.
	Figure 4. Alignment of the deduced amino acid sequences of the pirica gene with those of homologous/related genes.Conserved residues in all sequences are highlighted: identical, similar and unrelated residues are indicated by dark, light and white backgrounds, respectively. The red box represents the zona pellucida domain. Potential GPI-anchor residues predicted by GPI-SOM (fttp://gpi.unibe.ch/) are represented by black letters with blue background, while those suggested from sequence similarity are highlighted in light blue. Residues underlined in red represent the transmembrane domain predicted by the TMpred program (http://www.ch.embnet.org/). Letters edged in yellow represent the predicted proteolytic cleavage site. All relevant sequences of the uromodulin (THP), GP-2, and XL18 have been registered in the DDBJ/EMBL/GenBank databases with accession numbers XL18 (AAC59871), rTHP (AAB33313), hTHP (P07911), and hGP-2 (AAB19240), respectively.

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