Haifeng Tian

Sexually dimorphic expression of Foxl2 and Ftz‐F1 in chinese giant salamander Andrias Davidianus

Foxl2 and FTZ-F1 play a crucial role in the regulation of gonad development in fish and mammals, but studies of their function in amphibians are scarce. We isolated the full length of Foxl2 (adFoxl2) and Ftz-F1 (adFtz-f1) cDNA from the Chinese giant salamander Andrias davidianus and quantified its expression in various tissues and developing gonads. The adFoxl2 gene encodes 301aa including a conserved forkhead box, and the adFtz-f1 gene encodes 467aa containing an Ftz-F1 box. The amino acid sequences showed high homology with other amphibians. adFoxl2 expression was high in ovary, whereas adFtz-f1 was higher in testis, moderate in pituitary, ovary, and kidney; and low in the remaining tested tissues. Expression of adFoxl2 gradually increased from 1Y to 5Y in ovary, whereas adFtz-f1 expression gradually decreased in testis. In addition, adFoxl2 and adFtz-f1 were detected in granulosa cell in ovary and in spermatocytes in testis. The adFoxl2 transcription was inhibited in brain and ovary after treatment with methyltestosterone and with letrozole, whereas adFtz-f1 expression was upregulated. High-temperature suppressed the expression of adFxl2 in ovary and enhanced the transcription of adFtz-f1. These results suggest that adFoxl2 functioned in ovary differentiation, whereas adFtz-f1 played a role in testis development, which lays a foundation for study of the sex differentiation mechanism in A. davidianus.

Cloning, expression of, and evidence of positive selection for, the prolactin receptor gene in Chinese giant salamander (Andrias davidianus)

Prolactin receptor (PRLR) is a protein associated with reproduction in mammals and with osmoregulation in fish. In this study, the complete length of Chinese giant salamander Andrias davidianus prolactin receptor (AD-prlr) was cloned. Andrias davidianus prlr expression was high in the kidney, pituitary, and ovary and low in other examined tissues. The AD-prlr levels were higher in ovary than in testis, and increased in ovaries with age from 1 to 6 years. To determine effect of exogenous androgen and aromatase inhibitor on AD-prlr expression, methyltestosterone (MT) and letrozole (LE) were injected, resulting in decreased AD-prlr in both brain and ovary, with MT repressing prlr transcription more rapidly than did LE. The molecular evolution of prlr was assessed, and found to have undergone a complex evolution process. The obranch-site test detected four positively selected sites in ancestral lineages prior to the separation of mammals and birds. Fourteen sites underwent positive selection in ancestral lineages of birds and six were positively selected in amphibians. The site model showed that 16, 7, and 30 sites underwent positive selection in extant mammals, amphibians, and birds, respectively. The positively selected sites in amphibians were located outside the transmembrane domain, with four in the extracellular and three in the intracellular domain, indicating that the transmembrane region might be conserved and essential for protein function. Our findings provide a basis for further studies of AD-prlr function and molecular evolution in Chinese giant salamander. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 707-719, 2015.

Molecular cloning, characterization and evolutionary analysis of vitellogenin in Chinese giant salamander Andrias davidianus

Abstract Vitellogenin (VTG) is the major precursor of yolk proteins and could transport many components into oocytes, and hence it was thought to be a potential marker of reproduction of oviparious animal. Andrias davidianus is a critically endangered amphibian species, of which the wild population decreased dramatically and the artificial breeding has been successfully established. In this study, a full-length VTG cDNA was cloned and characterized from A. davidianus (designated as AdVTG) using 5’ and 3’ rapid amplification cDNA ends and RT-PCR. The AdVTG cDNA is 5,418 base pair in length and it encodes 1,805 amino acid long protein with typical quadripartite domains and several conserved cleavage sites. The tissue expression pattern indicated that AdVTG is mainly expressed in liver in one-year old individual. The threedimensional model of AdVTG constructed by homology modelling revealed the presence of conserved N-terminal β-sheet and lipid-binding cavity. Moreover, the phylogenetic analysis of VTG in metazoa was carried out, which revealed that Anuraspecific gene duplication occurred after the divergence of Caudata. All these results would improve the understanding of the physiological roles of AdVTG in A. davidianus and contribute to elucidating the evolution of VTG in vertebrates.

Comparative transcriptome reveal the potential adaptive evolutionary genes in Andrias davidianus

To search the evidence of molecular evolution mechanism for aquatic and cave habitat in Andrias davidianus, the evolution analysis was carried out among several species transcriptome data. The transcriptome data of Notophthalmus viridescens, Xenopus tropicalis, Cynops pyrrhogaster, Hynobius chinensis and A. davidianus were obtained from the Genbank and reassembled except Xenopus tropicalis. The BLAST search of transcriptome data obtained 1244 single-copy orthologous genes among five species. A phylogenetic tree showed A. davidianus to have the closest relationship to H. chinensis. Fourteen positively selected genes were detected in A. davidianus and N. vridescens group and fifteen in A. davidianus and H. chinensis group. Five genes were shared in the both groups which involved in the immune system, suggesting that A. davidianus adaptation to an aquatic and cave environment required rapid evolution of the immune system compared to N. viridescens and H. chinensis.

Characterization and function of the T-box 1 gene in Chinese giant salamander Andrias davidianus

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx1 was knocked down by tbx1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh, cyp26a, dmrt1, and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.

MicroRNA repertoire and comparative analysis of Andrias davidianus infected with ranavirus using deep sequencing

ABSTRACT Andrias davidianus is a large and economically important amphibian in China. Ranavirus infection causes serious losses in A. davidianus farming industry. MicroRNA mediated host‐pathogen interactions are important in antiviral defense. In this study, five small‐RNA libraries from ranavirus infected and non‐infected A. davidianus spleens were sequenced using high throughput sequencing. The miRNA expression pattern, potential functions, and target genes were investigated. In total, 1356 known and 431 novel miRNAs were discovered. GO and KEGG analysis revealed that certain miRNA target genes are associated with apoptotic, signal pathway, and immune response categories. Analysis identified 82 downregulated and 9 upregulated differentially expressed miRNAs, whose putative target genes are involved in pattern‐recognition receptor signaling pathways and immune response. These findings suggested miRNAs play key roles in A. davidianuss response to ranavirus and could provide a reference for further miRNA functional identification, leading to novel approaches to improve A. davidianus ranavirus resistance. HighlightsThe microRNA of Andrias davidianus and Andrias davidianus responding to ranavirus were sequenced.MiRNA repertoire of ranavirus infected and non‐infected A. davidianus was investigated.Differential expression miRNAs immune‐related were analyzed.

Identification and expression of cytochrome P450 genes in the Chinese giant salamander Andrias davidianus

Cytochrome P450 (cyp) genes play vital roles in biological processes, including in metabolism of endogenous and exogenous compounds. Exogenous hormone influences on gene expression that leads to disruption of gonad development have been studied in several species, but whether exogenous hormones affect cyp genes that show sexually dimorphic expression remains to be determined. Here, we identified and characterized cyp genes from the widely-cultured Chinese giant salamander Andrias davidianus. We obtained 20 cyp genes including 11 genes with complete sequences. Phylogenetic analyses supported the classification of cyp genes similar to other vertebrates. Expression profile of female and male salamanders showed multiple cyp genes to exhibit higher expression in ovary than in testis, including cyp26a, cyp19a, cyp1a1, cyp4v2, cyp3a24, cyp2c20, cyp2d14, cyp2d15, and cyp4b; while cyp11a, cyp2b11, cyp11b1, cyp4f22, cyp2j6, cyp2k1, cyp2k4, cyp1a5 was higher in testis than in ovary. Seven sex-biased genes were detected after aromatase inhibitor injection and with exposure to high water temperatures. Cyp2k1, cyp11b1, and cyp2j6 expression were increased, while cyp26a, cyp2c20, cyp4b genes were decreased with aromatase inhibition. In ovary, cyp26a and cyp2c20 expression were significantly reduced; whereas cyp2k1, cyp2j6, and cyp2k4 expression were significantly elevated with no changes in cyp11a and cyp11b1 expression after temperature treatment. These findings provide valuable information for further study of sex differentiation mechanisms and cyp gene evolution.

Identification and expression of forkhead box genes in the Chinese giant salamander Andrias davidianus

In the present study, 21 forkhead box (Fox) genes were identified in Andrias davidianus, including 13 full-length genes and eight partial sequences. Phylogenetic analysis showed that most were conserved in other investigated amphibians, whereas the Foxk1 gene was found exclusively in A. davidianus. Molecular evolution analysis indicated that most Fox genes underwent purifying selection, whereas two sites of the adFoxp4 gene showed positive selection and were located on the adFoxp4 protein surface. Expression profiles of all Fox genes identified were analysed in the hypothalamic-pituitary-gonad axis by reverse transcription-quantitative polymerase chain reaction. Eighteen genes exhibited sexually dimorphic expression (15 ovary-biased and three testis-biased genes), whereas two genes showed no difference between ovary and testis. Further investigation of 12 selected sexually dimorphic Fox genes showed changes in the expression profile of 11 genes in the ovary of larvae reared at high temperatures (28°C). The results of the present study provide information on Fox genes in an amphibian and suggest that they play key roles in sexual development and reproduction in A. davidianus.

Molecular cloning, characterization and evolutionary analysis of leptin gene in Chinese giant salamander, Andrias davidianus

Abstract Leptin is an important hormone possessing diverse physiological roles in mammals and teleosts. However, it has been characterized only in a few amphibian species, and its evolutions are still under debate. Here, the full length of the leptin (Adlep) cDNA of Chinese giant salamander (Andrias davidianus), an early diverging amphibian species, is characterized and according to the results of the primary sequence analysis, tertiary structure reconstruction and phylogenetic analysis is confirmed to be an ortholog of mammalian leptin. An intron was identified between the coding exons of A. davidianus leptin, which indicated that the leptin is present in the salamander genome and contains a conserved gene structure in vertebrates. Adlep is widely distributed but expression levels vary among different tissues, with highest expression levels in the muscle. Additionally, the leptin receptor and other genes were mapped to three known leptin signaling pathways, suggesting that the leptin signaling pathways are present in A. davidianus. Phylogenetic topology of leptins are consistent with the generally accepted evolutionary relationships of vertebrates, and multiple leptin members found in teleosts seem to be obtained through a Cluopeocephala-specific gene duplication event. Our results will lay a foundation for further investigations into the physiological roles of leptin in A. davidianus.

Characterization and tissue distribution of Lhx9 and Lhx9 α in Chinese giant salamander Andrias davidianus

Lhx9 is an LIM (named for the first three proteins in which the domain was found, Lin-11, Isl1 and Mec-3) homeodomain protein involved in development and differentiation of the gonad. In this study, we isolated the full-length Lhx9 and Lhx9 α from Andrias davidianus, detected the tissue distribution and analysed the methylation of the promoters. We identified Lhx9 of 1411 bp and Lhx9 α of 1153-bp length, differing in the 3′