Comparison of the genomic locus and organization of <i>X. tropicalis</i> and human TIMP2.

<p><i>X. tropicalis,</i> chicken, mouse and human TIMP2 genomic organizations were obtained from NCBI database. The lines represent the genomic sequences and the boxes with arrowed lines on top represent transcribed regions and the direction of the transcription. The lines and boxes were not drawn to the same scale for better visualization. The TIMP2 gene in all four species has the same intron/exon organization with 5 exons total as shown for <i>X. tropicalis</i> TIMP2 (not shown). Note that mouse and human TIMP2 loci are identical. In the chicken, CNTNAP1 gene is present on a different chromosome (shown in a darker shade).</p

There are two <i>X. laevis</i> TIMP2 genes that are highly homologous to the TIMP2 gene in other vertebrates.

<p>A) Nucleotide sequence comparison of <i>X. laevis</i> TIMP2A and TIMP2B. Dot (.): identical sequence; dash (-):a gap introduced for better alignment. Arrowed lines indicate the locations of the primers used to distinguish the two isoforms in RT-PCR analysis. B) Comparison of the protein sequences of putative <i>X. laevis</i> TIMP2A (XlTIMP2A, AAH74452), <i>X. laevis</i> TIMP2B (XlTIMP2B, NP_001087748), and <i>X. tropicalis</i> TIMP2 (XtTIMP2, NP_001015760) with their ortholog from chick (ChTIMP2, NP_989629), mouse (mTIMP2, P25785) and human (HuTIMP2, NP_003246). The conserved cysteine sequences are in bold and the signal peptide sequence is underlined. Dot (.): identical sequence; dash (-): a gap introduced for better alignment.</p

T3-inducktion of TIMP2 expression in different tissues of premetamorphic tadpoles.

<p>Tadpoles at premetamorphic stage 54 were treated with 10 nM T3 at 18°C and collected every other day for isolating total RNA from different tissues. The RNA was analyzed by qRT-PCR analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036707#pone-0036707-g005" target="_blank">Fig. 5</a>. *: p<0.05 when compared to the group without T3 treatment.</p

The gastrointestinal tract of a premetamorphic <i>X. laevis</i> (A) <i>and X. tropicalis</i> (B) tadpoles.

<p>Scale bars (yellow lines): 1 mm.</p

ST3 is upregulated while IFABP is downregulated during <i>X. tropicalis</i> intestinal metamorphosis.

<p>Total RNA were extracted from intestines of tadpoles at the indicated stages and analyzed by qRT-PCR. The expression levels for ST3 or IFABP were normalized against the control gene rpl8 and represented as means with with standard deviations in arbitrary units.</p

Homology of X. laevis TIMP2 with the ortholog of other species.

<p>GenBank accession number for nucleotide sequences: HuTIMP2: NM_003255; mTIMP2: X62622; ChTIMP2: NM_204298. The homology among the nucleotide sequences (coding sequences) was on grey background.</p

Reductions in the length of typhlosole and the small intestine of premetamorphic <i>X. laevis</i> and <i>X. tropicalis</i> tadpoles upon T3 treatment.

<p>Stage 54 <i>X. laevis</i> or <i>tropicalis</i> tadpoles were treated with 10 nM T3 at 18°C (<i>Xenopus laevis</i> tadpoles) or 25°C (<i>Xenopus tropicalis</i> tadpoles) for the indicated days and the intestine was isolated. The length of the typhlosole and the small intestine were measured. Note that the lengths of the intestine was reduced upon T3 treatment and that after 4–5 days of treatment, the typhlosole was no longer identifiable due to metamorphic changes in the intestine, resembling that at the climax of metamorphosis. The faster changes for <i>X. tropicalis</i> tadpoles were in part due to the higher temperature at which the animals needed to be reared.</p

T3 induces intestinal remodeling in premetamorphic <i>X. tropicalis</i> tadpoles.

<p>Stages 54 <i>X. tropicalis</i> tadpoles were treated with 10 nM T3 at 25°C. The intestine was isolated from the tadpoles at day 0, 1, 3, and 5, respectively. The intestine was fixed, sectioned, and stained with MGPY as in Fig. 2. A–D: a cross-section of the intestine after 0, 1, 3, 5 days of T3 treatment, respectively. E–H: the enlarged image of the boxed area for the corresponding tissues in A–D, respectively. Note that after 3 days of T3 treatment, the MGPY staining became weaker in larval epithelium as the cells undergo apoptosis. At the same time, the newly formed, proliferating adult epithelial islets were strongly stained by MGPY (arrows). The connective tissue (CT) and muscles (Mu) increased dramatically during metamorphosis. Ep: epithelium; Lu: lumen; Ty: typhlosole. Scale bars: 50 µm.</p

Co-regulation of TIMP2A and TIMP2B expression during intestinal metamorphosis.

<p>A) Validation of primer specificity for <i>X. laevis</i> TIMP2A or TIMP2B. The TIMP2A and TIMP2B cDNA were cloned into pCR2.1 vector, sequenced and serially diluted to serve as template DNA for PCR. Note that each primer pair specifically amplified only the intended TIMP2 gene. B) Total RNA was isolated from intestines of tadpoles during <i>X. laevis</i> metamorphosis and subjected to One-Step RT-PCR with a pair of primers recognizing both TIMP2 genes (TIMP2), specific for either TIMP2A or TIMP2B, respectively. The expression of the house-keeping gene rpL8 was similarly determined as a control.</p

TIMP2 expression during <i>X. laevis</i> development.

<p>Total RNA was isolated from whole animals during <i>X. laevis</i> development and subjected to One-Step RT-PCR analysis with a pair of primers amplifying both TIMP2A and TIMP2B (TIMP2) or stromelysin-3 (ST3). The expression of the house-keeping gene histone H4 was similarly determined as a control.</p