Demonstration The Vertebrate-like Thyroid Hormone Signaling Pathway In Amphioxus

The thyroid-liver axis is present in all the vertebrates including lamprey. Amphioxus, a primitive chordate, has been shown to possess a thyroid-like organ, endostyle, as well a liver-like organ, hepatic caecum, suggesting the presence of a vertebrate-like thyroid-liver axis in this animal. However, a definitive link between these organs remains to be established. This study mainly dealt with the demonstration of the presence in amphioxus of a signaling flow from the endostyle to the hepatic caecum.The cDNA of BbC/EBPα/βobtained contained an open reading frame (ORF) of 240 bp encoding a deduced polypeptide of 80 amino acids with a molecular weight of 9.3 kDa. Sequence comparison revealed that the polypeptide of BbC/EBPα/βshared 94.9% identity to B.floridae C/EBP and was 51.9-54.4% and 51.9-59.7% identical to the vertebrate C/EBPa and C/EBPβ, respectively. It was clear that BbC/EBPa/p was closely equally similar to C/EBPαand C/EBPβin sequence (therefore named C/EBPα/β), suggesting that BbC/EBPα/βmay be the archetype of vertebrate C/EBPa and C/EBPβ. This was also supported by the phylogenetic tree constructed which showed that C/EBPa and C/EBPP were each grouped together, forming two separated clades, while BbC/EBPα/βwas located at the base of both C/EBPa and C/EBPP clades. qRT-PCR showed that BbC/EBPα/βtranscript was most abundant in the hepatic caecum, which strengthens the notion that the hepatic caecum is homologous to the vertebrate liver. The BbTR cDNA obtained was 1317 bp long with an ORF of 1290 bp encoding a deduced protein of 430 amino acids with amolecular size of about 49 kDa. Sequence comparison revealed that BbTR shared 94% identity to B. floridae TR peptide. BbTR was found to be widely expressed in all the tissues examined, including the hepatic caecum, but it was abundantly expressed in the endostyle. In agreement, in situ hybridization histochemistry also revealed that BbTR was mainly expressed in the endostyle and gill. In the endostyle, BbTR was predominantly expressed in the zones 5a and 5b of the endostyle and at a lower level present in the zones 6 and 1 of the tissue. The zones 5 and 6 had been shown to be the iodine-binding center, and therefore our results showed that in the endostyle, the cells expressing BbTR were basically restricted in the iodine-binding regions.Both in vivo and in vitro assays showed that the expression of BbC/EBPα/βin the hepatic caecum was significantly increased following the exposure to T3,T4 or TRIAC. but compared with T3 and TRIAC, T4 acts less effective. And IOP, a deiodinase inhibitor, was able to inhibit T4 action, whereas T3 and TRIAC actions were not affected by IOP. These results suggested that BbC/EBPα/βexpression was mainly induced by T3 synthesized through deiodination of T4 in B. belcheri, as in vertebrates.To test if T3, T4 and TRIAC bind to BbTR, a fluorescence spectroscopy was performed. both T3 and TRIAC were able to bind to BbTR, with the TRIAC-BbTR binding being much stronger. In contrast, T4 did not bind to BbTR. At last, we confirm that the endostyle abound in thyroxine (T4) and triiodothyronine (T3), just like the thyroid of vertebrate. And now we know the expression of BbC/EBPα/βis mediated through interaction between T3/TRIAC-TR. Altogether these results suggest the presence in amphioxus of a signaling flow from the endostyle to the hepatic caecum.We have also studied the possible immune role of the yolk protein phosvitin(Pv), derived from vitellogenin of zebrafish. Firstly, we sythesized recombinant Pv, which was identified by MALDI/TOF MS analysis. Then we tested the antibacterial activity of purified Pv and found that Pv have strong antimicrobial activity to both Gram-negative bacterium Escherichia coli (E. coli) and Gram-positive bacterium Staphylococcus aureus (S. aureus). In addition, scanning electron microscope (SEM) showed that both E. coli and S. aureus were damaged by treatment with purified Pv.To test if His-Pv can bind to microbes, FITC-labeled His-Pv was incubated with E. coli and S. aureus. It was found that His-Pv is able to bind both E. coli and S. aureus. To better understand the mechanisms of binding activity, an enzyme-linked immunosorbent assay (ELISA) was carried out to investigate what molecules on the microbial surfaces are recognized by His-Pv, and it was found that His-Pv had a significantly stronger affinity to the immobilized ligands including LPS from Gram-negative bacteria, LTA from Gram-positive bacteria, PGN from both Gram-positive and Gram-negative bacteria than to BSA. These results indicated that His-Pv was a multivalent pattern recognition receptor. Iron binding assay indicated that His-Pv was not able to bind iron, and iron failed to decrease the antimicrobial activity of His-Pv. These reveal that iron 'robbing'is not involved in His-Pv's bacteriostatic activity. Analysis of the content of Pv at the different periods of embryonic development by ELISA showed that the Pv concentration decreases with development. but until 12 hours, the concentration of Pv within the embryo remains enough for antimicrobial activity. This denoted that at the early stage of embryo development, Pv is possibly a immuodefence player. To search for the "active core" of Pv, two assay were performed, N-terminal truncation assay and point mutation assay. It was found that when N- terminal 194 amino acid residues were removed, the remaining 55 amino acid residues still have antibacterial activity. Preliminary point mutation approach results showed that positively charged amino acid residues of Pv seems to play a key role. In summary, the present study demonstrates that Pv plays an immunedefense role in the zebrafish embryo development, rather than just a nutrients protein. The antibacterial active center of Pv is at the C-terminal, with positively charged amino acid residues and hydrophobic amino acid residues more crucial for the antimicrobial activity.