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

: 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/EBP α 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.


Introduction
In mammals, the forkhead box (fox) family has been reported to consist of over 40 members, including foxa-q. Forkhead box O (foxo) is a subgroup of the forkhead box transcription factor family [1]. It is the evolutionarily conserved transcription factor that was first identified in Drosophila melanogaster in 1989 [2,3]. foxo plays a major role in various cellular processes, such as cell differentiation and growth, apoptosis, proliferation, oxidative stress, and DNA damage repair [4,5]. foxo is regulated by two signalling pathways. The first is the classical insulin signaling pathway, which is regulated by phosphatidylinositol-3-kinase (PI3K) and protein kinase B (protein kinase B, AKT) in the presence of growth factors. The second pathway is c-Jun N-terminal kinase (c-Jun N-terminal kinase, JNK), which acts during oxidative stress [4,6]. Four subtypes of foxo have been identified in mammals: foxo1, foxo3, foxo4, and foxo6, while in bony fish, seven subtypes, foxo1a, foxo1b, foxo3a, foxo3b, foxo4, foxo6a, and foxo6b, are well known [7,8]. In zebrafish, foxo2 and foxo5 have also been reported [5]. However, foxo2 is a paralogue of foxo3, while foxo5 is specifically in zebrafish and is also called foxo3b.
Foxo family members are characterized by the presence of a 100-amino acid conserved DNA binding domain [9]. This domain exists in several different transcription factors and determines cell fate during the early stage of embryogenesis [10]. The most frequently reported members are foxo1 and foxo3. According to a previous transcriptome analysis [11], foxo1 is believed to play a role in spermatogenesis and spermatogonia differentiation in mammals [12]; for example, foxo1 regulates the maintenance of male spermatogonia stem cells in mice (Mus musculus) [13]. In fish, foxo1a is reported to function in the insulin-related pathway and growth of rainbow trout (Oncorhynchus mykiss), grass carp (Ctenopharyngodon idella) [14,15], and spotted seabass (Lateolabrax maculatus) [16]. foxo3 is a key effector of cell death in mammals [17]. foxo3 can inhibit follicle growth initiation and control reproductive potential. In the orange-spotted grouper (Epinephelus coioides), foxo3a and foxo3b proteins exist in ovarian germ cells and follicular cells, which may be involved in follicular formation [18]. In zebrafish, foxo4, foxo6a, and foxo6b are reported to function in the developing brain and under the regulation of PI3-kinase signalling [19].
The Chinese tongue sole exhibits obvious sexual growth dimorphism, wherein females can grow two to four times larger than males, so the study of sex differentiation has great potential for application in aquaculture. Many fox members have been reported to function in sex determination and differentiation, such as the classical foxl2 and foxl3 [20]. Due to their important role in sex differentiation and growth, foxo genes were selected for an in-depth analysis of the Chinese tongue sole. In this study, the conserved domain, phylogenetic tree, and conserved motifs of six Csfoxo members were analyzed. Their expression patterns in gonads at different developmental stages were also studied. The transcriptional regulation of Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4 was then analyzed. Finally, the siRNA-mediated knockdown of Csfoxo1a, Csfoxo3a, and Csfoxo3b and its effect were investigated. These results may improve our understanding of the role of foxo genes in the Chinese tongue sole.

Identification and Analysis of Csfoxo Genes
Six Csfoxo genes were identified in the tongue sole genome. Their gene IDs, chromosome localizations, amino acid numbers, molecular weights (MWs), and isoelectric points (pI) were analyzed (Table 1 and Supplementary Figure S1). The ORF sequences of these genes varied from 1872 to 2178 bp in length and encoded 623-725 amino acids. The predicted MWs were 66.52-78.07 kDa with pI values ranging from 4.79-6.78. As shown in Figure 1, all the proteins share a forkhead domain and contain low-complexity regions (LCRS) with little variation among different members.

Phylogenetic Tree Analysis
We performed a multiple alignment procedure, which showed the similarity of the six Foxo proteins is 53.1%. To study the phylogenetic relationships, an NJ tree was constructed based on the amino acid sequences of nine fish and two mammals. As shown in Figure 2, 53 Foxo proteins were divided into four clusters (Foxo1, Foxo3, Foxo4, and Foxo6), indicating the evolutionary relationship between foxo members in vertebrates. Only one copy was found in mammals, whereas multiple copies of Foxo1, Foxo3, and Foxo6 were identified in fish. It is worth noting that inside each of the clusters, the Foxo proteins in fish and mammals formed different subclusters.

Phylogenetic Tree Analysis
We performed a multiple alignment procedure, which showed the similarity of the six Foxo proteins is 53.1%. To study the phylogenetic relationships, an NJ tree was constructed based on the amino acid sequences of nine fish and two mammals. As shown in Figure 2, 53 Foxo proteins were divided into four clusters (Foxo1, Foxo3, Foxo4, and Foxo6), indicating the evolutionary relationship between foxo members in vertebrates. Only one copy was found in mammals, whereas multiple copies of Foxo1, Foxo3, and Foxo6 were identified in fish. It is worth noting that inside each of the clusters, the Foxo proteins in fish and mammals formed different subclusters.

Structure Analysis of Foxo
As shown in Figure 3, a total of 12 motifs were detected, and the signature of the 12 motifs are shown in Supplementary Figure S2. Motifs 1 and 2 are h served FH domains, and motif 9 is only found in Foxo3 and Foxo4 of bony fish of 50 amino acids. In fish, Foxo3 contains all motifs, while Foxo4 lacks motif 10 and Foxo6 lack motif 9. In mammals, the Foxo motifs differ among humans, mi (Supplementary Figure S3). In general, mammalian Foxo1 and Foxo3 lack mot lacks motif 8, and Foxo6 lacks motifs 8, 10, and 12. 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.

Structure Analysis of Foxo
As shown in Figure 3, a total of 12 motifs were detected, and the signature sequences of the 12 motifs are shown in Supplementary Figure S2. Motifs 1 and 2 are highly conserved FH domains, and motif 9 is only found in Foxo3 and Foxo4 of bony fish, consisting of 50 amino acids. In fish, Foxo3 contains all motifs, while Foxo4 lacks motif 10, and Foxo1 and Foxo6 lack motif 9. In mammals, the Foxo motifs differ among humans, mice, and rats (Supplementary Figure S3). In general, mammalian Foxo1 and Foxo3 lack motif 11, Foxo4 lacks motif 8, and Foxo6 lacks motifs 8, 10, and 12.

Analysis of Csfoxo Expression Patterns at Different Developmental Stages
As shown in Figure 4, the expression patterns of six Csfoxo members in the gonads at different developmental stages were analyzed. All Csfoxo members showed higher expression before 6 mph, and this expression was male-biased. However, the peak levels of different members occurred at different stages, e.g., foxo6-like at 40 dpf, foxo3a, foxo3b and foxo4 at 60 dpf, and foxo1a and foxo1a-like at 90 dpf. These data suggest their involvement in male differentiation and testicular development.

Analysis of Csfoxo Expression Patterns at Different Developmental Stages
As shown in Figure 4, the expression patterns of six Csfoxo members in the gonads at different developmental stages were analyzed. All Csfoxo members showed higher expression before 6 mph, and this expression was male-biased. However, the peak levels of different members occurred at different stages, e.g., foxo6-like at 40 dpf, foxo3a, foxo3b and foxo4 at 60 dpf, and foxo1a and foxo1a-like at 90 dpf. These data suggest their involvement in male differentiation and testicular development.

Promoter Activity Analysis
As shown in Figure 5, the promoter regions of the Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4 genes were cloned to explore promoter activity. The promoter activity of pGL3-Csfoxo1a, 3a, 3b, and 4 was significantly higher than that of pGL3-basic, indicating that the Csfoxo promoter had a positive effect on Csfoxo gene expression. The activity of pGL3-Csfoxo1, 3a, 3b, and 4 was significantly promoted by adding C/EBPα and c-Jun transcription factors, suggesting that C/EBPα and c-Jun could positively regulate foxo genes. This activity was closely related to the specific site. When the C/EBPα and c-Jun binding sites were mutated (muCsfoxo1a-C/EBPα, muCsfoxo3a-C/EBPα, muCsfoxo3b-C/EBPα, muCsfoxo4-C/EBPα, muCsfoxo1a-c-Jun, mufoxo3a-c-Jun, mufoxo3b-c-Jun, and mufoxo4-c-Jun), the levels of activity were significantly decreased.

Promoter Activity Analysis
As shown in Figure 5, the promoter regions of the Csfoxo1a, Csfoxo3a, Csfoxo3b, and Csfoxo4 genes were cloned to explore promoter activity. The promoter activity of pGL3-Csfoxo1a, 3a, 3b, and 4 was significantly higher than that of pGL3-basic, indicating that the Csfoxo promoter had a positive effect on Csfoxo gene expression. The activity of pGL3-Csfoxo1, 3a, 3b, and 4 was significantly promoted by adding C/EBPα and c-Jun transcription factors, suggesting that C/EBPα and c-Jun could positively regulate foxo genes. This activity was closely related to the specific site. When the C/EBPα and c-Jun binding sites were mutated (muCsfoxo1a-C/EBPα, muCsfoxo3a-C/EBPα, muCsfoxo3b-C/EBPα, muCsfoxo4-C/EBPα, muCsfoxo1a-c-Jun, mufoxo3a-c-Jun, mufoxo3b-c-Jun, and mufoxo4c-Jun), the levels of activity were significantly decreased.

Csfoxo Knockdown and the Effect on Other Genes
For Csfoxo1a, 3a, and 3b, three siRNAs were designed for each gene. The knockdown effect was examined, and the siRNAs with the most obvious effect (siRNA1 for Csfoxo1a, siRNA1 for Csfoxo3a, and siRNA3 for Csfoxo3b) were selected ( Figure 6A,C,E). After the

Discussion
foxo is involved in a wide range of biological processes, including cell differentiation, metabolism, tumor inhibition, cell cycle arrest, protection from stress, and cell death [21][22][23]. However, the functionality of foxo genes has not been systematically studied in the

Discussion
foxo is involved in a wide range of biological processes, including cell differentiation, metabolism, tumor inhibition, cell cycle arrest, protection from stress, and cell death [21][22][23]. However, the functionality of foxo genes has not been systematically studied in the Chinese tongue sole. In this study, we first focused our attention on Csfoxo based on our previous transcriptomic analysis [11]. We identified six foxo members in the Chinese tongue sole: foxo1a, 1a-like, 3a, 3b, 4, and 6-like. In comparison to seven members present in bony fish, one copy of foxo6 seems to be missing in tongue sole [6]. The Csfoxo members share the conserved FH binding domains of "winged helix" or "forkhead box", which can bind to B-DNA as monomers [6]. A low-complexity region (LCR) is found in all Csfoxo members; it may participate in flexible binding, and its position in the protein may determine its binding properties and specific function [24]. Structural and phylogenetic tree analysis indicate that Foxo is highly conserved in vertebrates. It is interesting that all six members showed high expression before 6 mph and were male-biased, suggesting their role in male differentiation and testicular development. However, their peak levels appeared at different stages, e.g., foxo6-like appeared at 40 dpf, foxo3b and foxo4 at 60 dpf, foxo1a and foxo1a-like at 90 dpf, and fox3a at 6 mpf. Whether they play a sequential role in male differentiation and testicular development requires further investigation.
CCAAT/enhancer-binding protein α (C/EBPα) is an evolutionarily conserved transcription factor in vertebrates that plays a role in cell growth and differentiation [25]. In Chinese tongue sole, C/EBPα was reported to be involved in early gonadal differentiation and to act as a negative regulator of the male-determining gene dmrt1 [26]. c-Jun is a transcriptional activator that plays an important role in cell proliferation and differentiation [27]. Based on our data, C/EBPα and c-Jun enhanced the promoter activity of all foxo members. However, the interaction of foxo with other sex-related genes (especially dmrt1) requires further investigation.
To investigate the effects of foxo genes, several growth-and sex-related genes were selected, including igf1, sox9a, wtla, tesk1, and neurl3. As a well-known growth-related gene, igf is also reported to play a role in sex differentiation. In tilapia, igf1 is distributed in the gonads at early stages [28,29], and it may be involved in the regulation of growth and differentiation [30,31]. In mammals, sox9 plays a role in cell differentiation and male differentiation [32]. In fish species, such as rainbow trout and zebrafish, sox9a showed higher expression in the testis [33,34]. Wilms tumor gene, wt1a, has multiple pivotal roles in gonads. In adult medaka, the wt1a gene is highly expressed in Sertoli cells of the testis and is required for PGC maintenance [35]. In tongue sole, the alternative splicing of wt1a has been suggested to play a role in gonadal differentiation [36]. Tesk1 plays a role in spermatogenesis in mice, especially during early spermatogenesis [37]. In Chinese tongue sole, tesk1 and neurl3 are closely related to spermatogenesis [38]. It is worth noting that the knockdown of foxo1a, foxo3a, and foxo3b led to the downregulation of all the abovementioned genes, suggesting the positive regulation of foxo members in male differentiation, testis development, and spermatogenesis. In future studies, in vivo trials are still required for the in-depth investigation of the mechanisms of foxo genes in these processes.

Phylogenetic Tree and Structural Characterization
A phylogenetic tree was constructed based on the protein sequences of nine teleost fish and two mammals using MEGA v7.0. The adjacency (NJ) method and 1000 bootstrap replications were used. All protein sequences were downloaded from the NCBI database, and the information is shown in Supplementary Table S1. The conserved motifs were analyzed using MEME (5.4.1) (http://meme-suite.org/tools/meme https://www.ncbi. nlm.nih.gov/ (accessed on 20 May 2022)) [39]. The number of motifs identified was 12, and other parameters were set to default values to obtain conservative motifs.

Experimental Animals and Ethics Approval
The Chinese tongue sole used in this experiment were from the breeding stock of Chinese tongue sole of Weizhuo aquatic company (Tangshan, China). MS-222 (20 mg/L solubilized in seawater, treated for 5 min) was used for anesthesia to minimize suffering before the gonads were dissected. Different developmental stages were selected, including 40 days post-hatching (dpf), 60 dpf, 90 dpf, 6 months post-hatching (mpf), 1.5 years posthatching (ypf), and 3 ypf. For each stage, three male and three female individuals were examined. The gonads were quickly removed, placed into RNA preservation solution, and stored at −80 • C until RNA extraction was performed. The study was conducted under the inspection of the committee at the Yellow Sea Fisheries Research Institute (Approval number, YSFRI-2022035).

siRNA-Mediated Knockdown of Csfoxo in Testicular Cells
The siRNAs of Csfoxo1a, Csfoxo3a, and Csfoxo3b were synthesized by Sangon (Shanghai, China). Three siRNAs targeting different sites were designed for each gene, and the siRNA with the best knockdown effect was selected for further study. siRNA interference transfection was performed in a tongue sole testicular cell line (predominantly Sertoli cells) from our laboratory using a CP Reagent transfection kit (Ribobio, Guangzhou, China). According to the previously established experimental protocol, the Csfoxo1a, Csfoxo3a, and Csfoxo3b siRNA transfection and negative control (NC) transfection were performed in triplicate [41]. The cell status and fluorescence of cy3-transfected cells were examined 48 h after transfection. qPCR was used to detect the knockdown effect and the expression profiles of sex-related genes, such as sox9a and tesk1.