Identification and Functional Analysis of foxo Genes in Chinese Tongue Sole (Cynoglossus semilaevis)

Abstract

The Chinese tongue sole (Cynoglossus semilaevis) is a traditional, precious fish in China. Due to the large growth difference between males and females, the investigation of their sex determination and differentiation mechanisms receives a great deal of attention. Forkhead Box O (FoxO) plays versatile roles in the regulation of sex differentiation and reproduction. Our recent transcriptomic analysis has shown that foxo genes may participate in the male differentiation and spermatogenesis of Chinese tongue sole. In this study, six Csfoxo members (Csfoxo1a, Csfoxo3a, Csfoxo3b, Csfoxo4, Csfoxo6-like, and Csfoxo1a-like) were identified. Phylogenetic analysis indicated that these six members were clustered into four groups corresponding to their denomination. The expression patterns of the gonads at different developmental stages were further analyzed. All members showed high levels of expression in the early stages (before 6 months post-hatching), and this expression was male-biased. In addition, promoter analysis found that the addition of C/EBPα and c-Jun transcription factors enhanced the transcriptional activities of Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4. The siRNA-mediated knockdown of the Csfoxo1a, Csfoxo3a, and Csfoxo3b genes in the testicular cell line of Chinese tongue sole affected the expression of genes related to sex differentiation and spermatogenesis. These results have broadened the understanding of foxo's function and provide valuable data for studying the male differentiation of tongue sole.

Keywords: Cynoglossus semilaevis; foxo; promoter activity; siRNA knockdown.

Figure 1

The conserved domain of the Csfoxo protein. Horizontal grey bars represent amino acid sequences with no predictive functional domains, while colored boxes represent regions with reliable predictive domains. Forkhead binding domain (yellow); Low-complexity region (pink).

Figure 2

Phylogenetic trees of foxo from nine fish, Chinese tongue sole (Csfoxo), medaka (Olfoxo), turbot (Smfoxo), zebrafish (Drfoxo), spotted gar (Lofoxo), Nile tilapia (Onfoxo), fugu (Trfoxo), pufferfish (Pffoxo), and Atlantic halibut (Hhfoxo), and two mammals, mice (Mmfoxo) and humans (Hsfoxo). Different colors in the circle represent different fox members (blue for foxo1, red for foxo6, green for foxo3, orange for foxo4), and light color for fish and dark color for mammal. Red pentagrams indicate fox members of Chinese tongue sole.

Figure 3

Structure and motif analysis of Foxo proteins in five fish species: Chinese tongue sole (Csfoxo), medaka (Olfoxo), turbot (Smfoxo), Nile tilapia (Onfoxo), and fugu (Trfoxo).

Figure 4

Expression patterns of Csfoxo in gonads at different developmental stages. (A) Expression patterns of Csfoxo1a at different developmental stages; (B) expression patterns of Csfoxo3a at different developmental stages; (C) expression patterns of Csfoxo3b at different developmental stages; (D) expression patterns of Csfoxo4 at different developmental stages; (E) expression patterns of Csfoxo6-like at different developmental stages; (F) expression patterns of Csfoxo1a-like at different developmental stages. The letters above each chart indicate significant differences, and the bars represent standard deviations.

Figure 5

Transcriptional activity of the Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4 promoters: (A) Csfoxo1a; (B) Csfoxo3a; (C) Csfoxo3b; (D) Csfoxo4. Results correspond to the addition of c-Jun and C/EBPα transcription factors, and the effect on the mutation of two transcription-factor-binding sites. Different letters indicate significant differences, and bars represent standard deviations.

Figure 6

Effects of the knockdown of the genes Csfoxo1a, Csfoxo3a, and Csfoxo3b in a testicular cell line. (A) The effect of the knockdown of three siRNAs on Csfoxo1a. (B) The effect of the knockdown of Csfoxo1a on other genes. (C) The effect of the knockdown of three siRNAs on Csfoxo3a. (D) The effect of the knockdown of Csfoxo3a on other genes. (E) The effect of the knockdown of three siRNAs on Csfoxo3b. (F) The effect of the knockdown of Csfoxo3b on other genes. * indicates significant differences.