Evolution of trefoil factor(s): genetic and spatio-temporal expression of trefoil factor 2 in the chicken (Gallus gallus domesticus)

Abstract

Trefoil factors are essential healing initiators participating in mucosal reconstitution and tissue morphogenesis, especially on the surfaces of the gastrointestinal tract. This family has been cloned and characterized predominantly from mammals and amphibians. Avian species ingest stone and grit to help digest food, which may expose their gut to severe physical conditions. To further the understanding of the function of the TFF gene family across species, we undertook this research to clone, sequence, and characterize the spatio-temporal expression patterns of chicken TFF2 (ChTFF2) cDNA. Bioinformatics analysis of the promoter region and deduced amino acid sequence demonstrated that ChTFF2 contained unique characteristics; specifically the chicken promoter has multiple start sites and the protein contains a series of Lys-Lys-Val repeats. Unlike mammals, where TFF2 is detected primarily in the stomach, and occasionally in the proximal duodenum, chicken TFF2 transcripts are found throughout the gastrointestinal tract, with major expression sites in the glandular and muscular stomach as well as evident expression in the colon, small intestine, cecal tonsil and crop. Temporal analysis of intestinal ChTFF2 transcripts by quantitative RT-PCR showed high levels in embryos and a trend of constant expression during embryonic and post-hatch development, with a reduction occurring around hatch. Phylogenetic analysis highlighted the conservation of TFF proteins and functional divergence of trefoil domains, which suggest a transitional role in the bird during evolution.

Figure 1. Sequence and analysis of ChTFF2.

A: Strategy of amplying and sequencing full length ChTFF2 cDNA. B: RLM-RACE products were electrophoresed in a 2% agarose gel. Lane 1: 3′ RACE product; lane 2: water control; lane 3: 5′ RACE product; lane 4: water control. D: Intron-exon localization of ChTFF2 at chromosome. C: Representative electrophoresis result from EcoRI digestion of purified plasmids inserted with 5′ RACE product indicates alternative start site with shorter product in lane 2 as compared with lane 1 and 3; alternative transcription starting site was confirmed by sequencing determination. The ORFs are gray. E: Potential regulatory sequences in the promoter of the ChTFF2 as compared with human; nucleotides are numbered with +1 being the A of the ATG initiation codon; target sequences for the underlined transcription factors (motif) are in bold. F: Full-length ChTFF2 cDNA sequence and deduced amino acids.

Figure 2. Sequence alignment.

A: sequence alignments for chicken TFF2 cDNA with human and mouse. B: Amino acid sequence alignment and analysis for chicken with predicted sequences of other avian species and human and mouse; domains are indicated below aligned sequences. PTD: trefoil domain (P domain); locations of conserved Cys bonds and secondary structural element: α-helixes, β-sheets and loops are indicated.

Figure 3. Spatio-temporal expression of TFF2 transcripts in chicken.

Representative amplification plots of quantitative PCR (duplicates) for measuring ChTFF2 (A) and 18S (B) in the small intestine; water was used as a control. Quantification of ChTFF2 transcripts in embryonic (E) and post-hatch (H) ages in duodenum (C), jejunum (D) and ileum (E) using RT-qPCR; values were expressed in mean ± SE, n = 5 to 8. The expression of ChTFF2 (F) and 18S (G) in different tissues; forty ng of cDNA were amplified for 33 cycles using a forward primer from RT-PCR1and a reverse primer from RT-PCR2; reverse transcriptase was used (+) or omitted (−) for each tissue and all the RT-PCR products were examined by electrophoresis through a 3% agarose gel in TBE.

Figure 4. Phylogenetic tree of TFF2.

Forty five TFF2 proteins and related structure were aligned by using ClustalW, and the neighbor-joining tree derived by the same program was shown. A. carolinensis: anole lizard. G.gallus: chicken. P. hamadryas: baboon. A. melanoleuca: giant panda. G. gorilla: gorilla. P. pygmaeus: orangutan. A. platyrhynchos: duck. H. sapiens: human. P. troglodytes: chimpanzee. B. maxima: toad. L. africana: elephant. P. vampyrus: megabat. B. Taurus: cow. M. domestica: opossum. R. norvegicus: rat. C. intestinalis: vase tunicate. M. eugenii: wallaby. S. scrofa: pig. C. jacchus: common marmoset. M. gallopavo: turkey. S. tridecemlineatus: squirrel. C. lupus familiaris: dog. M. lucifugus: microbat. T. belangeri: tree shrew. C. porcellus: guinea pig. M. mulatta: macaque. T. guttata: zebra finch. D. ordii: kangaroo rat. M. murinus: mouse. T. syrichta: tarsier. E. caballus: horse. O. aries: sheep. T. truncatus: dolphin. E. telfairi: lesser hedgehog tenrec. O. garnettii: bushbaby. X. laevis: African clawed frog. F. catus: cat. O. princeps: pika.

Figure 5. Phylogenetic tree of trefoil domains of TFF2.

TD1 (red) and TD2 (black) were extracted from a range of animals as listed in Figure 4, aligned by using ClustalW, and analyzed with the neighbor-joining method.