Molecular cloning and functional characterization of two forms of Pax8 in the rainbow trout, Oncorhynchus mykiss

Abstract

We have identified two distinct Pax8 (a and b) mRNAs from the thyroid gland of the rainbow trout (Oncorhynchus mykiss), which seemed to be generated by alternative splicing. Both Pax8a and Pax8b proteins were predicted to possess the paired domain, octapeptide, and partial homeodomain, while Pax8b lacked the carboxy-terminal portion due to an insertion in the coding region of the mRNA. RT-PCR analysis showed each of Pax8a and Pax8b mRNAs to be abundantly expressed in the thyroid and kidney. In situ hybridization histochemistry further detected the expression of Pax8 mRNA in the epithelial cells of the thyroid follicles of the adult trout and in the thyroid primordial cells of the embryo. The functional properties of Pax8a and Pax8b were investigated by dual luciferase assay. The transcriptional regulation by the rat thyroid peroxidase (TPO) promoter was found to be increased by Pax8a, but not by Pax8b. Pax8a further showed synergistic transcriptional activity with rat Nkx2-1 for the human TPO upstream region including the enhancer and promoter. On the other hand, Pax8b decreased the synergistic activity of Pax8a and Nkx2-1. Electrophoretic mobility shift assay additionally indicated that not only Pax8a but also Pax8b can bind to the TPO promoter and enhancer, implying that the inhibitory effect of Pax8b might result from the lack of the functional carboxy-terminal portion. Collectively, the results suggest that for the trout thyroid gland, Pax8a may directly increase TPO gene expression in cooperation with Nkx2-1 while Pax8b may work as a non-activating competitor for the TPO transcription.

Keywords: Nkx2-1; Pax8; Rainbow trout; TTF1; Thyroid gland; Thyroid transcription factor 1.

Figure 1

Nucleotide sequences encompassing the coding regions of Pax8a and Pax8b cDNAs from the rainbow trout (AB828387, AB828388), and their deduced amino acid sequences. Thick lines above the Pax8a amino acid sequence indicates, from N- to C-terminus, the predicted paired domain, octapeptide, and truncated homeodomain. Gaps marked by hyphens (−) are inserted to optimize homology. Vertical lines represent the nucleotide identity between the Pax8a and Pax8b cDNAs, and bold fonts highlight the nucleotides in two inserted/deleted regions. Nucleotides are numbered from 5′ to 3′, beginning with the first base in the coding region. The termination codons are denoted with the asterisk. Rt: Rainbow trout.

Figure 2

Comparison of amino acid sequences of Pax8 proteins from the rainbow trout (AB828387, AB828388), zebrafish (ENSDART00000029014), Xenopus laevis (AF179301), mouse (ENSMUST00000028355), and human (ENST00000429538; (Poleev et al., 1995). These sequences are aligned using Clustal W (Thompson et al., 1994). Gaps marked by hyphens (−) are inserted to optimize homology. Asterisks denote identical amino acid residues, while colons and periods indicate amino acid residues of strongly similar properties, scoring > 0.5 in the Gonnet PAM 250 matrix, and of weakly similar properties, scoring =< 0.5, respectively. The trout Pax8a and Pax8b share three highly conserved domains with Pax8 from other species, depicted by boxes: i.e. the paired domain, octapeptide, and partial homeodomain. The positions of introns in the human Pax8 gene are indicated by numbered arrowheads (Poleev et al., 1995). The transactivation region and adjacent inhibitory domain of mouse Pax8 are indicated, respectively, by single and double underlining (Dorfler and Busslinger, 1996). Rt: Rainbow trout.

Figure 3

Phylogenetic tree of the Pax2/5/8 proteins from the rainbow trout (AB828387, AB828388), zebrafish (BC066690, BC162214, ENSDART00000076986, ENSDART00000029014), Xenopus laevis (AF179300, NM001085768, AF179301), chicken (AB026496, AB004249), mouse (ENSMUST00000174490, ENSMUST00000014174, ENSMUST00000028355), and human (ENST00000428433, ENST00000358127, ENST00000429538), obtained by the neighbour joining method (Saitou and Nei, 1987). The length of each branch is shown in the units of the number of amino acid differences per residue, and the numbers around the interior branches are bootstrap probabilities (percent; 10,000 replicates). The topology has confirmed that trout Pax8a and Pax8b belong to the cluster containing zebrafish, Xenopus, mouse, and human Pax8 proteins.

Figure 4

Tissue distribution of Pax8 mRNAs in the rainbow trout, determined by RT-PCR analysis. Total RNA (10 μg) from various trout tissues were reverse-transcribed and amplified by PCR with specific Pax8 primers. A distinctive band for Pax8a mRNA was detected in the thyroid tissue, including the ventral aorta, and kidney, and faintly in the testis and ovary. The Pax8b mRNA was expressed in the thyroid tissue and kidney. The expression of EF1-α mRNA, an endogenous control, was seen in all the tissues examined. Rt: Rainbow trout.

Figure 5

In situ hybridization histochemistry for Pax8 mRNA in the thyroid gland of the adult trout (A) and in the developing thyroid of the trout embryo (C, D). A: Follicular cells of the thyroid gland showed hybridization signals with the DIG-labelled antisense Pax8 cRNA probe. B: Control section to (A). No signal was seen by hybridization with the sense probe. C: Ventral view of the hybridized 15 dpf embryo showed the signal (arrow) in the pharyngeal region, as a single patch close to the heart tube (ht). The location of the signal appears to correspond to that of the thyroid primordium in the zebrafish embryo (Porazzi et al., 2009). ph, pharynx. D: A transverse section revealed that the signal (arrow) originated from the thyroid primordial cells near the developing ventral aorta (va). The scale bar for A and B = 25 μm; C = 250 μm; D = 20 μm.

Figure 6

Transactivation potential of rainbow trout Pax8a and Pax8b for the 713-bp rat TPO gene promoter (A) and for the 6.3-kbp human TPO gene upstream region (B). A: The rat TPO promoter was transactivated by trout Pax8a, but not by Pax8b. B: The trout Pax8a and rat Nkx2-1 showed a remarkable synergy on the transactivation from the human TPO upstream region. On the other hand, trout Pax8b had no synergistic effect with rat Nkx2-1, and inhibited the synergy of Pax8a and Nkx2-1. Values are expressed as fold of activation above that obtained with the control vector, and represent the average of six independent experiments. Each bar depicts the mean value ± SEM. Rt: Rainbow trout.

Figure 7

Binding of rainbow trout Pax8a, Pax8b, and rat Nkx2-1 to oligonucleotide CT (A) or oligo B (B). EMSA was performed with His-tag fused recombinant transcription factor proteins, and FAM-labelled oligonucleotide CT (A) or oligo B (B) as probes. The incubation with rainbow trout Pax8a (lane 1), Pax8b (lane 3), or rat Nkx2-1 (B, lane 5) resulted in shifted bands. The binding specificity was assessed in the absence (−) and presence (+) of a 100-fold molar excess of non-labelled oligonucleotide CT (A) or oligo B (B) as competitors (lanes 2, 4, and 6), and further by using the Tag polypeptide produced from the expression vector pET32a (A, lane 5; B, lane 7). Rt: Rainbow trout.