Identification and expression profiles of Fox transcription factors in the Yesso scallop (Patinopecten yessoensis)

Gene 733 (2020) 144387

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Research paper

Identification and expression profiles of Fox transcription factors in the Yesso scallop (Patinopecten yessoensis)

T

Shaoxuan Wua,1, Yang Zhanga,1, Yajuan Lia, Huilan Weia, Zhenyi Guoa, Shi Wanga,b, ⁎ Lingling Zhanga,c, , Zhenmin Baoa,c a

MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, 5 Yushan Road, Qingdao 266003, China Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, Shandong, China c Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, Shandong, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Patinopecten yessoensis Fox family Fox cluster Early development Adult tissue Gonad development

The forkhead box (Fox) gene family is a family of transcription factors that play important roles in a variety of biological processes in vertebrates, including early development and cell proliferation and differentiation. However, at present, studies on the mollusk Fox family are relatively lacking. In the present study, the Fox gene family of the Yesso scallop (Patinopecten yessoensis) was systematically identified. In addition, the expression profiles of the Fox gene family in early development and adult tissues were analyzed. The results showed that there were 26 Fox genes in P. yessoensis. Of the 26 genes, 24 belonged to 20 subfamilies. The Fox genes belonging to the I, Q1, R and S subfamilies were absent in P. yessoensis. The other 2 genes formed 2 independent clades with the Fox genes of other mollusks and protostomes. They might be new members of the Fox family and were named FoxY and FoxZ. P. yessoensis contained a FoxC-FoxL1 gene cluster similar in structure to that of Branchiostoma floridae, suggesting that the cluster might already exist in the ancestors of bilaterally symmetrical animals. The gene expression analysis of Fox showed that most of the genes were continuously expressed in multiple stages of early development, suggesting that Fox genes might be widely involved in the regulation of embryo and larval development of P. yessoensis. Nine Fox genes were specifically expressed in certain tissues, such as the nerve ganglia, foot, ovary, testis, and gills. For the 9 genes that were differentially expressed between the testis and ovary, their expression levels were analyzed during the 4 developmental stages of gonads. The results showed that FoxL2, FoxE and FoxY were highly expressed in the ovary during all developmental stages, while FoxZ was highly expressed in the testis during all developmental stages. The results suggested that these genes might play an important role in sex maintenance or gametogenesis. The present study could provide a reference for evolutionary and functional studies of the Fox family in metazoans.

1. Introduction Members of the forkhead box (Fox) protein family are transcription factors that play important roles in the regulation of animal development (Coffer and Burgering, 2004). The Fox family proteins contain a relatively conserved DNA binding domain (DBD), namely, the forkhead (FH) domain. This domain consists of approximately 100 amino acids and includes 3 α-helices (H1, H2 and H3), 3 β-sheets (S1, S2 and S3) and 2 wing-shaped rings (W1 and W2) (Gajiwala and Burley, 2000).

Since Weigel et al. cloned the first FH gene fkh from the fruit fly (Drosophila melanogaster) in 1989 (Weigel et al., 1989), more than 2000 members of the Fox gene family have been identified. These Fox genes are divided into 24 subfamilies (A-S) (Fritzenwanker et al., 2014). The evolution and functions of the Fox gene family have been extensively studied in vertebrates. The Fox genes have been systematically identified in a number of species including humans (Homo sapiens) (Jackson et al., 2010; Benayoun et al., 2011), mice (Mus musculus) (Jackson et al., 2010), amphioxus (Branchistoma floridae) (Yu

Abbreviations: BMP, bone morphogenic protein; ChIP-seq, chromatin immunoprecipitation-sequencing; DV, dorsal–ventral; DBD, DNA binding domain; FH, forkhead; FHA, forkhead associated domain; Fox, forkhead box; NJ, neighbor-joining; ORF, open reading frame; RC, raw count; RNAi, RNA interference; TGF-beta, transforming growth factor-beta ⁎ Corresponding author at: Ocean University of China, 5 Yushan Road, Qingdao 266003, China. E-mail address: [email protected] (L. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.gene.2020.144387 Received 16 November 2019; Received in revised form 14 January 2020; Accepted 18 January 2020 Available online 20 January 2020 0378-1119/ © 2020 Elsevier B.V. All rights reserved.

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and genome using BLAST. To screen for Fox genes in P. yessoensis, the e value was set to 1e-5.

et al., 2008), and tilapia (Oreochromis niloticus) (Yuan et al., 2014). Studies have shown that the Fox genes play important roles in vertebrate embryo development and organogenesis. For example, Foxb1 is essential for the development of brain tissues (Aldea et al., 2015). Foxc1 and Foxc2 are closely related to the formation of the kidneys and urethra and participate in somitogenesis and cardiovascular and eye development (Maximilian et al., 2006; Boyle and Seaver, 2010; Rho and McClay, 2011). Foxe3 is essential for the development and differentiation of the crystalline lens (Brownell et al., 2015). Foxf1 is involved in the formation of the lung and intestine (Mahlapuu et al., 2001). In vertebrates, Fox genes are also involved in sex determination and gonadal development regulation. For example, FoxL2 plays a key role in female sex determination and ovarian differentiation (Wang et al., 2007; Boulanger et al., 2014). FoxA3 is involved in the growth of testicular germ cells and the maintenance of male fertility (Behr et al., 2007). FoxO1 participates in the proliferation, self-renewal and meiosis of spermatogonial stem cells (Goertz et al., 2011). Mollusca is the second largest phylum in the animal kingdom after Arthropoda. Mollusks are widely distributed, diverse in morphology, and represent an important group of lophotrochozoans (Brusca and Brusca, 2003). Compared to other animal groups, the Fox genes in mollusks are rarely studied. Moreover, most studies target individual Fox family members. FoxL2 has been the most studied and has been identified in species such as the Yesso scallop (Patinopecten yessoensis), Pacific oyster (Crassostrea gigas), Zhikong scallop (Chlamys farreri), and pearl oyster (Pinctada fucata). It is considered to be a key gene in ovarian differentiation and development in mollusks (Liu et al., 2012; Zhang et al., 2014; Teaniniuraitemoana et al., 2015; Li et al., 2016; Liu et al., 2016). To date, systematic identification of the Fox family has only been reported in C. gigas and the limpet (Lottia gigantea). Researchers have found that the Fox family in the above 2 species is composed of 20 subfamilies. Both species lack FoxI, Q1, R and S, while each has a newly identified member of the Y subfamily (Yang et al., 2014). Such findings provide new information for the investigation of the evolution of the Fox family. To comprehensively study the evolutionary process and the functions of the Fox family genes, it is necessary to conduct research on other representative mollusk species. P. yessoensis is a type of marine shellfish with important economic value. It is mainly distributed in China, Japan, Russia and South Korea. According to the statistics of the World Food and Agriculture Organization (FAO), the yield of P. yessoensis reached 223,216 tons in 2016. Recently, whole-genome sequencing of P. yessoensis was completed. It was found that the P. yessoensis genome displays many ancient features and is a good model for studying evolution (Wang et al., 2017). The present study selected P. yessoensis as the research object, systematically identified the Fox family genes in P. yessoensis based on the genomic and transcriptomic data, and analyzed the structure of the Fox family members and their expression levels in early development and adult tissues. In addition, the present study specifically analyzed the expression levels of 9 sex-differential genes in the testis and ovary at various developmental stages. The present study contributed to an indepth understanding of the evolution of the Fox gene family and the biological functions of the Fox genes in scallops.

2.2. Analysis of the structure of the Fox genes The exon and intron positions in each gene were determined based on the genomic and transcriptomic data of P. yessoensis using the open reading frame (ORF) finder (http://www.ncbi.nlm.nih.gov/gorf/gorf. html). The conserved domains of the Fox proteins were predicted using SMART (http://smart.embl.de/), and the structure of the Fox genes was visualized using the online software IBS (http://ibs.biocuckoo.org). The isoelectric points and molecular weights of the Fox proteins were predicted using Compute pI/Mw (http://web.expasy.org/compute_pi/). 2.3. Phylogenetic analysis The Fox protein sequences of various species were downloaded from the National Center for Biotechnology Information (NCBI) (http:// www.ncbi.nlm.nih.gov) and UniProt database (specific gene names and registration numbers were shown in the Supplementary Table S1), including H. sapiens, D. rerio, C. intestinalis, acorn worm (Saccoglossus kowalevskii), S. purpuratus, D. melanogaster, C. elegans, L. gigantea, C. gigas, N. vectensis, hydra (Hydra vulgaris), brewer's yeast (Saccharomyces cerevisiae), purple scallop (Argopecten purpuratus) (Li et al., 2018a), and P. fucata (Takeuchi et al., 2012). The FH domain of each Fox protein was predicted using the online software CDD (https://www.ncbi.nlm. nih.gov/Structure/cdd/wrpsb.cgi). The protein sequences of the FH domains were retrieved, and multiple sequence alignments were performed using ClustalW software (Thompsom et al., 1994). The phylogenetic tree of the FH domains of various species was constructed using the software MEGA 7.0 (Kumar et al., 1994). The neighbor-joining (NJ) method was selected as the tree-making method. The genetic distance was calculated using the p-distance method, and the bootstrap value was set to 1000. 2.4. Structural analysis of the FoxC-FoxF-FoxL1-FoxQ1 gene cluster By reviewing the literature (Yang et al., 2014) and the NCBI database, the chromosomes or scaffolds where the FoxC, FoxL1, FoxF and FoxQ1 genes of H. sapiens, B. floridae, D. melanogaster, C. teleta, and C. gigas were located were identified. In addition, the order and spacing of the genes were determined. The order and spacing of FoxC, FoxL1 and FoxF were determined based on the genome of P. yessoensis (Wang et al., 2017). The collinearity of the FoxC-FoxL1-FoxF-FoxQ1 genes was analyzed based on the evolutionary relationship of the species. 2.5. Expression levels and differential analysis of the Fox genes The expression levels of the Fox genes of P. yessoensis during early development, in adult tissues and at different gonad developmental stages were analyzed based on the transcriptome data obtained previously by our laboratory. The stages of early development include the embryonic (multicell, blastula, gastrula) and larval (trochophore larvae, D-type larvae, pediveliger larvae, juvenile) stages. For each stage, more than 1000 embryos/larvae were pooled for RNA-seq. Adult tissues include the foot, smooth muscle, striated muscle, eye, mantle, gill, hepatopancreas, kidney, hemocytes, nerve ganglia, ovary, and testis. For each tissue, 2 to 3 biological replicates were obtained. Testes and ovaries at resting, proliferative, growing and maturation stages were collected, with 3 samples for each stage. All these data is available under the NCBI Bioproject ID of PRJNA259405, PRJNA423107 and PRJNA516336 (Wang et al., 2017; Zhang et al., 2018; Li et al., 2019). The analysis procedure was as follows. The raw data was filtered to obtain high-quality data. The high-quality reads were aligned against the P. yessoensis genome (Wang et al., 2017) using the STAR software package (Dobin et al., 2013). The raw count (RC) of each gene was

2. Materials and methods 2.1. Identification of the Fox genes The Fox genes of P. yessoensis were identified by homologous sequence alignment (the accession numbers are shown in Table 1). First, the sequences of Fox proteins from human (H. sapiens), Zebrafish (Danio rerio), ascidian (Ciona intestinalis), sea urchin (Strongylocentrotus purpuratus), fruit fly (D. melanogaster), nematode (Caenorhabditis elegans), C. gigas and sea anemone (Nematostella vectensis) were downloaded from the UniProt database (http://www.uniprot.org). Subsequently, these Fox proteins were aligned with the P. yessoensis transcriptomes 2

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Table 1 Structural characteristics of the Fox genes in P. yessoensis. Gene name

Exon No.

Gene length (bp)

Protein length (aa)

Molecular weight (kDa)

PI

Accession number

FoxA FoxB FoxAB FoxC FoxD FoxE FoxF FoxG FoxHa FoxHb FoxJ1 FoxJ2/3 FoxK FoxL1 FoxL2 FoxM FoxN1/4 FoxN2/3 FoxO FoxP FoxQ2a FoxQ2b FoxQ2c1 FoxQ2c2 FoxY FoxZ

1 1 1 1 1 1 2 1 1 1 2 10 10 1 2 7 6 8 2 14 1 2 1 1 3 11

1979 1451 1815 2215 2555 1971 23,784 2216 1605 1800 5025 28,147 16,654 2073 17,457 9498 12,136 53,532 29,862 43,725 1983 6231 2317 2496 7426 16,109

429 311 341 515 457 467 410 425 534 599 452 453 739 413 368 1014 498 485 655 955 475 445 260 251 452 590

47.54 35.24 37.88 56.54 50.47 51.99 44.83 46.57 59.20 67.16 50.04 48.89 80.24 46.13 41.97 110.64 54.64 54.70 72.27 106.29 53.71 49.92 29.88 28.14 51.13 67.60

9.02 9.76 6.58 8.45 6.36 6.83 7.99 8.63 6.32 6.73 4.88 5.57 9.29 6.47 9.23 9.02 5.41 5.33 5.32 6.36 6.16 6.55 8.38 9.40 5.92 5.89

XM_021506116.1 XM_021501945.1 XM_021501937.1 XM_021491292.1 XM_021489550.1 XM_021523183.1 XM_021502333.1 XM_021508115.1 MN510858 MN510859 XM_021495384.1 XM_021518958.1 XM_021506021.1 XM_021491290.1 XM_021497746.1 XM_021521584.1 XM_021515873.1 XM_021511290.1 XM_021521691.1 XM_021507629.1 XM_021487993.1 XM_021515362.1 XM_021492744.1 XM_021504913.1 XM_021498763.1 XM_021497738.1

has the largest cDNA (3705 bp in length) (Yang et al., 2014). All 26 Fox genes contain the FH domain, while certain genes also contain other domains (Fig. 1). For example, the C-terminus of FoxA and FoxO contains the HNF_C and FOXO-TAD domains, respectively. FoxK and FoxP contain the FH associated domain (FHA) and FOXP-CC domains, respectively. The FH domain of the 26 Fox genes of P. yessoensis was analyzed. The results showed that the FH domain is 89 amino acids in length. It consists of 3 relatively conserved α-helices (H1, H2 and H3), 3 β-sheets (S1, S2 and S3), and 2 less-conserved winged rings (W1 and W2) (Fig. 2a).

computed using HTseq software, and the expression of each gene (Transcripts Per Million, TPM) was calculated (Wagner et al., 2012). Differential gene expression analysis was performed using SPSS 16.0 software. The differences in gene expression between various developmental stages of gonads were analyzed using the independent samples T test. A P value of < 0.05 was considered statistically significant.

3. Results and discussion 3.1. Identification of the Fox family genes of P. yessoensis and analysis of their sequences and structural characteristics

3.2. Phylogenetic analysis of the Fox gene family of P. yessoensis The present study identified 26 Fox genes in the genome of P. yessoensis. Similar numbers of Fox genes have been identified in the lophotrochozoans C. gigas (25 Fox genes) (Yang et al., 2014) and C. teleta (25 Fox genes) (Yang et al., 2014), the echinoderm S. Purpuratus (22 Fox genes) (Tu et al., 2006), and the hemichordate S. kowalevskii (23 Fox genes) (Fritzenwanker et al., 2014). However, the number of Fox genes in P. yessoensis was significantly less than that in vertebrates such as D. rerio (67 Fox genes) (Yuan et al., 2014), O. niloticus (65 Fox genes) (Yuan et al., 2014), M. musculus (44 Fox genes) (Jackson et al., 2010), and H. sapiens (50 Fox genes) (Jackson et al., 2010). These findings indicated that there may be over 20 Fox genes in bilaterally symmetrical animal ancestors. The increased number of Fox genes in vertebrates is related to the occurrence of whole-genome duplication. After analyzing the genome and transcriptome of P. yessoensis and identifying the intron–exon boundaries by the GT-AG principle, the structures of the 26 Fox genes were obtained (Table 1). It was found that 50% (13) of the 26 Fox genes of P. yessoensis have a single exon. These 13 genes all have a length of < 2.5 kb. The remaining 13 genes all contain introns and are 5.0–53.5 kb in length. In addition, the number of introns ranges from 1 to 13. Of the 13 Fox genes, 9 have intron insertions in the FH domain. Compared with the differences in gene length, the protein length differences are smaller. The 26 Fox proteins have lengths between 251 and 1014 aa, while the molecular weights of the Fox proteins are between 28.14 and 110.64 kDa. The smallest Fox protein is FoxQ2c2, while the largest is FoxM. Similarly, in C. gigas, FoxQ2c has the smallest cDNA (825 bp in length), and FoxM

To determine the subfamilies to which these 26 Fox genes belong, we constructed a phylogenetic tree using the FH domains of the Fox proteins from various phyla of animals (Fig. 2b). The results showed that the 26 Fox genes of P. yessoensis were clustered into 22 independent clades with the Fox genes of other animals. Among the 24 Fox gene subfamilies that are identified in bilaterally symmetrical animals, 20 subfamilies were found in P. yessoensis; the FoxI, FoxQ1, FoxR and FoxS subfamilies were absent. The lack of FoxR and FoxS in P. yessoensis was consistent with expectations because these 2 genes were only found in vertebrates (Song et al., 2015) and were absent in C. elegans, B. floridae, and C. gigas (Yang et al., 2014; Mazet et al., 2003). The phenomenon of lacking FoxI and FoxQ1 exists not only in P. yessoensis but also in C. gigas. However, these 2 genes are present in C. teleta (Yang et al., 2014). The above findings indicate that FoxI and FoxQ1 are not lost in all protostomes. It is possible that these 2 genes are only lost in mollusks or bivalves. P. yessoensis has one Fox gene in most subfamilies. The Fox gene first clustered with the mollusks C. gigas and L. gigantean and then with other animals. The FoxQ2 and FoxH subfamilies expanded in P. yessoensis. Four FoxQ2 and 2 FoxH subfamily members were identified in P. yessoensis, which was consistent with C. gigas. Such findings indicated that the expansion of these 2 subfamilies might be common in mollusks. The cnidarians Hydra magnipapillata and Clytia hemisphaerica carry 3 and 2 FoxQ2 genes, respectively (Chevalier et al., 2006; Chapman et al., 2010). The hemichordate S. kowalevskii has 3 FoxQ2 genes, the cephalochordate B. 3

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Fig. 1. The structure of the Fox family genes in P. yessoensis. The pink boxes represent the coding regions, while the orange boxes represent the FH domains. The double horizontal lines represent introns, and the double oblique lines indicate that the scale is not proportional. The numbers indicate the nucleotide lengths of the exons or introns. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4

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Fig. 2. (A) The comparison results of the FH domains between the Fox family genes of P. yessoensis. The darker the color is, the higher the degree of sequence conservation. H1-3 represent 3 α-helix structures, S1-3 represent 3 β-sheet structures, and W1-2 represent 2 winged ring structures; (B) Phylogenetic analysis of the Fox gene family. The phylogenetic tree was built using the NJ method and MEGA 7.0 software. Hs: H. sapiens; Dr: D. rerio; Ci: C. intestinalis; Sk: S. kowalevskii; Sp: S. purpuratus; Dm: D. melanogaster; Ce: C. elegans; Lg: L. gigantea; Cg: C. gigas; Py: P. yessoensis; Nv: N. vectensis; Hv: H. vulgaris; Ap: A. purpuratus; Pf: P. fucata; Ob: O. bocki; Lu: L. unguis; Ct: C. teleta; Cs: C. sculpturatus; Sm: S. morsitans; Sc: S. cerevisiae. The registration numbers of the protein sequences used in the phylogenetic tree are shown in Table S1. 5

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floridae has 3 (Yu et al., 2008), and S. purpuratus, O. niloticus and D. rerio only have one (Tu et al., 2006; Yuan et al., 2014). In contrast, FoxQ2 is not found in D. melanogaster and C. intestinalis (Ogasawara and Satou, 2003; Lee and Frasch, 2010). These findings suggest that multiple copies of the FoxQ2 subfamily may already be present in the metazoan ancestors, and they may be lost in ecdysozoans and some deuterostomes. The present study discovered 2 more Fox genes in P. yessoensis, which formed 2 new clades with the Fox genes from other animals. These 2 genes are named FoxY and FoxZ. FoxY clustered with the genes of other mollusks (such as P. fucata, C. gigas and L. gigantea) into 1 clade, demonstrating that FoxY may be a member of the Fox family specific to mollusks. Compared with FoxY, FoxZ is present not only in mollusks but also in other lophotrochozoans and arthropods. However, FoxZ has not been found in deuterostomes. It is speculated that FoxZ might be a subfamily unique to protostomes.

3.4. The expression levels of the Fox genes in the early development of P. yessoensis Fox genes play important roles during the early development of animals. These genes are involved in germ layer specialization, gastrulation, cell fate determination and morphogenesis (Magie et al., 2005). Shellfishes undergo embryonic and larval stages during early development. To understand the roles of the Fox genes during the early development of P. yessoensis, the present study analyzed the expression levels of the 26 Fox genes in 3 embryonic and 4 larval stages (Fig. 4a). The results showed that except for FoxO and FoxHa, the remaining 24 genes were all expressed during early development. Some of the genes (FoxJ1, FoxN2/3 and FoxQ2c2) were expressed in all embryonic and larval stages, some genes (FoxA, FoxAB, FoxL1 and FoxD) were continuously expressed in all stages except the multicellular stage, some genes were mainly expressed at a certain stage (FoxZ in the multicellular stage, FoxP and FoxHb in the blastula stage), and other genes were expressed in several consecutive stages. Such results demonstrated that the Fox family might be widely involved in the early development of P. yessoensis. Most Fox genes exhibited expression patterns similar to those in C. gigas (Yang et al., 2014). For example, FoxY was mainly expressed prior to the blastula stage, FoxN1/4 was mainly expressed before the trochophore stage, FoxM was mainly expressed from the blastula stage to the trochophore stage, FoxL2 was mainly expressed in trochophore larvae and D-type larvae, FoxG and FoxC was continuously expressed beginning with the gastrula stage and trochophore stage, respectively, while FoxB was expressed after the D-type larva stage. Therefore, it is speculated that these genes might have conservative functions in the early development of bivalves. The expression patterns of certain Fox genes were different between P. yessoensis and C. gigas during early development. For example, in P. yessoensis, FoxAB was continuously highly expressed from the blastula to the juvenile stage. However, in C. gigas, FoxAB was mainly expressed from the blastula to the D-type larva stage, followed by a significant decrease in expression. FoxO was expressed at an extremely low level during the early development of P. yessoensis (TPM < 1). In contrast, FoxO was highly expressed in C. gigas at multiple stages from trochophore larva to juvenile. Such expression patterns suggest that not all Fox genes have conserved

3.3. Evolution analysis of the FoxC-FoxF-FoxL1-FoxQ1 gene cluster FoxC, FoxL1, FoxF, and FoxQ1 exist as a cluster in the genome of B. floridae and vertebrates (Mazet et al., 2006; Wotton and Shimeld, 2006; Wotton et al., 2008). Since the conservativeness of this gene cluster may be related to mesoderm development (Shimeld et al., 2010b), it has received great attention. Fig. 3 shows the arrangement of these genes in the genomes of H. sapiens, B. floridae, D. melanogaster, C. teleta, C. gigas and P. yessoensis. The results showed that although both P. yessoensis and C. gigas lack FoxQ1, FoxC, FoxL1 and GDP-mannose 4,6-dehydratase (GMDS) are on the same scaffold and are arranged in the same order (which is similar to C. teleta). These results indicate that the FoxC-FoxL1 gene cluster is highly conserved among lophotrochozoans. In C. teleta, C. gigas and P. yessoensis, FoxF and the FoxC-FoxL1 gene cluster are located on different scaffolds. However, in P. yessoensis, the 2 scaffolds are located on the same chromosome, which is similar to the structure of the gene cluster of B. floridae (Shimeld et al., 2010a). These results suggest that the gene cluster has an ancient origin and might exist in the ancestors of bilaterally symmetrical animals.

Fig. 3. The FoxC-FoxF-FoxL1-FoxQ1 gene cluster. The colored boxes represent the genes, and the arrows below indicate the direction of gene transcription. The number above the horizontal line between the genes represents the number of genes in the interval, while the number below represents the distance between genes. // indicates that the number of interval genes is not provided because gene spacing exceeds 1 Mb. 6

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Fig. 4. The expression profile of Fox genes at various developmental stages of embryo and larvae (A) and in the adult tissues (B) of P. yessoensis.

hepatopancreas, which was consistent with the function of FoxF reported in the literature. FoxF has a conserved function in coordinating myogenesis (Andrikou et al., 2013). Knocking out Foxf1 and Foxf2 in mouse smooth muscle led to delayed gastric emptying (Herring et al., 2019). FoxZ was highly expressed in the testis of P. yessoensis. Because this gene is a unique member in protostomes that was newly identified in the present study, its role in the testis is still unclear. In section 3.6, we further analyze the expression of this gene in different developmental stages of the gonads. FoxG was expressed in the ganglion, foot and hepatopancreas of P. yessoensis. The highest FoxG expression was detected in the ganglion. Although there is no relevant report in shellfish, studies of other animals show that FoxG is expressed and plays an important role in the adult nervous system. For example, in the annelid Dinophilus gyrociliatus, FoxG is expressed in the anterior and posterior regions of the brain of female adults (Kerbl et al., 2016). In mice, Foxg1 is continuously highly expressed until adulthood (Shen et al., 2006). Recently, overexpression and knockdown studies have shown that Foxg1 promotes neurite elongation after the mitosis of neurons and maintains neural plasticity (Chiola et al., 2019; Yu et al., 2019). Nine genes exhibited tissue-specific expression: FoxQ2a, FoxB, FoxD, FoxC, FoxL2, FoxE, FoxQ2c2, FoxY and FoxL1. These genes were mainly expressed in 1–2 adult tissues. Among the 9 Fox genes, FoxQ2a was expressed in the ganglion and eye of P. yessoensis, suggesting that it might be involved in neuromodulation. FoxB was also highly expressed in the ganglion of P. yessoensis, which was consistent with the expression of FoxB in the nervous system of D. melanogaster (Häcker et al., 1992). In addition, FoxB, FoxD and FoxC were all highly expressed in the foot of P. yessoensis. Interestingly, FoxB and FoxD are coexpressed in many animals during early development. For example, in vertebrates, FoxB and FoxD are always expressed on the dorsal side (Gamse and Sive, 2001; Mazet and Shimeld, 2002; Pohl and Knochel, 2005). In contrast, these 2 genes are mainly expressed on the ventral side of S. kowalevskii, D. melanogaster (Häcker et al., 1992) and C. elegans (Tan et al., 1998; Nash et al., 2000; Sarafi-Reinach and Sengupta, 2000). FoxD and FoxB are locally expressed along the dorsal–ventral (DV) axis of bilateral animals. Such phenomenon may be related to the bone morphogenic protein (BMP)/transforming growth factor-beta (TGFbeta) signaling pathway (Fritzenwanker et al., 2014). Whether the simultaneous foot-specific expression of FoxB and FoxD in P. yessoensis is related to BMP signaling needs to be further investigated. In addition, the potential functions of FoxB and FoxD need to be explored too. FoxC was expressed in the testis and ovary of P. yessoensis. Such results were consistent with the results in C. gigas (Yang et al., 2014), indicating that

functions in the early development of bivalves. Comparison of the expression patterns of the Fox genes in the early development of P. yessoensis and C. gigas contributes to further understanding the roles of various Fox genes in the embryonic and larval development of shellfishes. 3.5. The expression levels of the Fox genes in the adult tissues of P. yessoensis In addition to participating in the regulation of early development, Fox transcription factors also play important roles in the function and homeostasis of adult tissues and organs (Benayoun et al., 2011; Lam et al., 2013; Genin et al., 2014). The present study analyzed the expression levels of 26 Fox genes in 12 adult tissues of P. yessoensis. The results showed that 5 of the 26 genes (FoxAB, FoxHa, FoxHb, FoxQ2b and FoxQ2c1) were expressed at extremely low levels in various adult tissues of P. yessoensis (TPM < 1). The expression patterns of the other 21 Fox genes in various tissues are shown in Fig. 4b. We found that 8 genes were widely expressed in the adult tissues of P. yessoensis, including FoxN2/3, FoxK, FoxJ1, FoxO, FoxN1/4, FoxP, FoxJ2/3 and FoxM. Such finding indicated that these genes might participate in the basic biological processes of adult P. yessoensis. The expression patterns of most Fox genes in P. yessoensis were consistent with those in C. gigas. Among these Fox genes, FoxJ1 and FoxM were highly expressed in the testis and gills of P. yessoensis. The testis is a gamete-producing tissue, and cell division is relatively robust in gills. Therefore, we speculated that FoxJ1 and FoxM might be involved in the regulation of meiosis and mitosis in P. yessoensis. The Fox genes have similar functions in mammals: FOXM1 is a major regulator of cell cycle progression and cell proliferation and is overexpressed in various tumor tissues (Nicolau-Neto et al., 2018). FOXJ1 has also been proven to be related to the development of various cancers, such as hepatocellular carcinoma (Chen et al., 2013), ovarian cancer (Siu et al., 2013), and gastric cancer (Wang et al., 2015). Knockdown of FoxJ1 inhibits cell proliferation and migration (Liu et al., 2019). Four Fox genes (FoxA, FoxF, FoxZ, and FoxG) were expressed in multiple tissues of P. yessoensis. In P. yessoensis, FoxA was expressed in multiple tissues including the hepatopancreas, ovary, eye, and mantle. FoxA expression was highest in the hepatopancreas, which might be related to the important role of FoxA in the digestive system. Recently, it has been reported that the FoxA gene of D. melanogaster maintains the stemness of intestinal stem cells and progenitor cells and is an essential gene for intestinal regeneration (Lan et al., 2018). In P. yessoensis, FoxF was mainly expressed in striated muscle, smooth muscle and the 7

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ovarian differentiation in O. niloticus (Wang et al., 2007). The above findings indicate that FoxL2 may be a conserved, key sex gene shared between protostomes and deuterostomes. In addition, FoxN2/3 and FoxE are highly expressed in the mature ovary of mud clam (Tegillarca granosa) (Chen et al., 2017), while FoxN2/3, FoxY and FoxQ2c are highly expressed in the mature ovary of C. gigas (Yang et al., 2014; Zhang et al., 2014; Chen et al., 2017). Such findings indicate that these genes might be involved in egg maturation in various bivalves. Fig. 6 illustrates the expression levels of 3 genes that were highly expressed in the testis (FoxZ, FoxJ1 and FoxC) in the 4 developmental stages of male and female gonads. Among the 3 genes, only FoxZ was significantly highly expressed in the testis at all 4 developmental stages, suggesting that this unique member of the protostomes might play an important role in the development of the testis in P. yessoensis and worthy of further study. FoxJ1 was significantly highly expressed only in mature testis, suggesting that it may be related to sperm metamorphosis. FoxC was significantly highly expressed in the testis at the resting and proliferative stages, suggesting that it might be involved in the regulation of spermatogonia or spermatocytes. Our results were consistent with the recent findings for FOXC2 in mice, showing that FOXC2 is the key regulatory factor for the self-renewal and differentiation of spermatogonial stem cells (Wei et al., 2018).

FoxC might be involved in the regulation of gonadal development or gametogenesis in bivalves. Similarly, Foxc1 and Foxc2 are expressed in the gonads of both female and male mice, and Foxc1 has been proven to play an important role in follicle formation (Mattiske et al., 2006). FoxL2, FoxE, FoxQ2c2 and FoxY were highly expressed in the ovary of P. yessoensis. Such results were consistent with the results in C. gigas (Yang et al., 2014), suggesting that the roles of these genes might be conservative in bivalve ovary development. The role of FoxL2 in ovarian development and differentiation has been reported in a variety of shellfishes (Naimi et al., 2009; Liu et al., 2012; Teaniniuraitemoana et al., 2014). However, there is currently no report on the functions of FoxE, FoxQ2c2 and FoxY in the ovary of shellfish. FoxL1 was specifically expressed in the gill of P. yessoensis and moderately expressed in the gills of C. gigas (Yang et al., 2014), indicating that this gene might play a role in the gills of bivalves. 3.6. Analysis of the expression patterns of the gonadal development-related Fox genes in P. yessoensis In the analysis of adult tissues, the testis and ovary were mostly in the growth or maturation phase. To further analyze the expression patterns of the gonadal development-related Fox genes at different developmental stages, 9 genes that were significantly differentially expressed between the testis and the ovary were selected for study. Fig. 5 shows the expression levels of 6 genes that were highly expressed in the ovary (FoxL2, FoxE, FoxY, FoxN2/3, FoxP and FoxQ2c2) in the 4 developmental stages of male and female gonads. Three of the 6 genes (FoxL2, FoxE, and FoxY) were expressed at a significantly higher level in the ovary than in the testis at all 4 developmental stages, indicating that these genes might play an important role in ovary maintenance and development in P. yessoensis. FoxN2/3 was significantly highly expressed only in the ovary during the growth and maturation stages, indicating that this gene is mainly involved in egg maturation. FoxP was highly expressed in mature ovary, while FoxQ2c2 was highly expressed in growing ovary. However, in the resting phase, the expression levels of both genes were significantly higher in the testis in comparison to the ovary. Such an expression pattern was unexpected and needs to be further investigated. Among the above 6 genes, FoxL2 has been the most studied. Previous studies show that FoxL2 is a key gene for ovarian differentiation and maintenance in P. yessoensis (Li et al., 2018b; Li et al., 2016). In addition, this gene is a sex-determining gene in goats (Boulanger et al., 2014) and plays a decisive role in

4. Conclusions The present study identified the Fox gene family members in P. yessoensis through omics screening and discovered 2 new members, FoxY and FoxZ. Systematic analysis of the expression levels of Fox transcription factors in early development, adult tissues and gonads at different developmental stages primarily revealed the expression patterns of 26 Fox genes in P. yessoensis, which provides basic information for the in-depth study of the evolution of the Fox family and the functions of Fox family members in shellfish. In subsequent studies, different transcription factors can be localized by in situ hybridization, and their roles in early development should be analyzed. In terms of the genes that exhibited different expression patterns in adult tissues in the present study, their functions in specific tissues can be investigated by means of chromatin immunoprecipitation-sequencing (ChIP-seq), RNA interference (RNAi), and gene editing.

Fig. 5. The expression profile of 6 Fox genes (which were highly expressed in the ovary) in P. yessoensis testis and ovary at different developmental stages. Significant differences: * P < 0.05, ** P < 0.01, *** P < 0.001. 8

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Fig. 6. The expression profile of 3 Fox genes (which were highly expressed in the testis) in P. yessoensis testis and ovary at different developmental stages. Significant differences: * P < 0.05, ** P < 0.01, *** P < 0.001.

CRediT authorship contribution statement

neocortical pyramids stimulates dendrite elongation Via Hes1 and pCreb1 upregulation. Cereb. Cortex 29, 1006–1019. Coffer, P.J., Burgering, B.M.T., 2004. Forkhead-box transcription factors and their role in the immune system. Nat. Rev. Immunol. 4, 889–899. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., Gingeras, T.R., 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. Fritzenwanker, J.H., Gerhart, J., Freeman, R.M., Lowe, C.J., 2014. The Fox/Forkhead transcription factor family of the hemichordate Saccoglossus kowalevskii. EvoDevo 5, 17. Gajiwala, K.S., Burley, S.K., 2000. Winged helix proteins. Curr. Opin. Struct. Biol. 10, 110–116. Gamse, J.T., Sive, H., 2001. Early anteroposterior division of the presumptive neurectoderm in Xenopus. Mech. Dev. 104, 21–36. Genin, E.C., Nicolas, C., Renaud, V., Laurent, N., Brigitte, M., 2014. Concise review: Forkhead pathway in the control of adult neurogenesis. Stem Cells 32, 1398–1407. Goertz, M.J., Wu, Z., Gallardo, T.D., Hamra, F.K., Castrillon, D.H., 2011. Foxo1 is required in mouse spermatogonial stem cells for their maintenance and the initiation of spermatogenesis. J. Clin. Invest. 121, 3456. Häcker, U., Grossniklaus, U., Gehring, W.J., Jäckle, H., 1992. Developmentally regulated Drosophila gene family encoding the fork head domain. Proc. Natl. Acad. Sci. 89, 8754–8758. Herring, B.P., Hoggatt, A.M., Gupta, A., Wo, J.M., 2019. Gastroparesis is associated with decreased FOXF1 and FOXF2 in humans, and loss of FOXF1 and FOXF2 results in gastroparesis in mice. Neurogastroenterol. Motil. 31, e13528. Jackson, B.C., Carpenter, C., Nebert, D.W., Vasiliou, V., 2010. Update of human and mouse forkhead box (FOX) gene families. Hum. Genomics 4, 345. Kerbl, A., Martín-Durán, J.M., Worsaae, K., Hejnol, A., 2016. Molecular regionalization in the compact brain of the meiofaunal annelid Dinophilus gyrociliatus (Dinophilidae). EvoDevo 7, 20. Kumar, S., Tamura, K., Nei, M., 1994. MEGA: Molecular Evolutionary Genetics Analysis software for microcomputers. Comput. Appl. Biosci. 10, 189–191. Lam, E.W.F., Brosens, J.J., Gomes, A.R., Koo, C.Y., 2013. Forkhead box proteins: Tuning forks for transcriptional harmony. Nat. Rev. Cancer 13, 482. Lan, Q., Cao, M., Kollipara, R.K., Rosa, J.B., Kittler, R., Jiang, H., 2018. FoxA transcription factor Fork head maintains the intestinal stem/progenitor cell identities in Drosophila. Dev. Biol. 433, 324–343. Lee, H.H., Frasch, M., 2010. Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns. Dev. Dyn. 229, 357–366. Li, C., Liu, X., Liu, B., Ma, B., Liu, F., Liu, G., Shi, Q., Wang, C., 2018a. Draft genome of the Peruvian scallop Argopecten purpuratus. GigaScience 7, giy031. Li, R., Zhang, L., Li, W., 2018b. FOXL2 and DMRT1L are yin and yang genes for determining timing of sex differentiation in the bivalve mollusk Patinopecten yessoensis. Front. Physiol. 9, 1166. Li, Y., Zhang, L., Li, R., Zhang, M., Li, Y., Wang, H., Wang, S., Bao, Z., 2019. Systematic identification and validation of the reference genes from 60 RNA-Seq libraries in the scallop Mizuhopecten yessoensis. BMC Genomics 20, 288. Li, Y., Zhang, L., Sun, Y., Ma, X., Wang, J., Li, R., Zhang, M., Wang, S., Hu, X., Bao, Z., 2016. Transcriptome sequencing and comparative analysis of ovary and testis identifies potential key sex-related genes and pathways in scallop Patinopecten yessoensis. Mar. Biotechnol. 18, 453–465. Liu, L., Zhang, P., Shao, Y., Quan, F., Li, H., 2019. Knockdown of FOXJ1 inhibits the proliferation, migration, invasion, and glycolysis in laryngeal squamous cell carcinoma cells. J. Cell. Biochem. 120, 15874–15882. Liu, X.L., Zhang, Z.F., Shao, M.Y., Liu, J.G., Muhammad, F., 2012. Sexually dimorphic expression of foxl2 during gametogenesis in scallop Chlamys farreri, conserved with vertebrates. Dev. Genes. Evol. 222, 279–286. Liu, X.L., Li, Y., Liu, J.G., Cui, L.B., Zhang, Z.F., 2016. Gonadogenesis in scallop Chlamys farreri and Cf-foxl2 expression pattern during gonadal sex differentiation. Aquac. Res. 47, 1605–1611. Magie, C.R., Pang, K., Martindale, M.Q., 2005. Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis. Dev. Genes. Evol. 215, 618–630. Mahlapuu, M., Enerback, S., Carlsson, P., 2001. Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development 128, 2397. Mattiske, D., Kume, T., Hogan, B.L., 2006. The mouse forkhead gene Foxc1 is required for primordial germ cell migration and antral follicle development. Dev. Biol. 290,

Shaoxuan Wu: Writing - original draft, Formal analysis. Yang Zhang: Writing - original draft, Formal analysis. Yajuan Li: Formal analysis. Huilan Wei: Investigation. Zhenyi Guo: Investigation. Shi Wang: Writing - review & editing. Lingling Zhang: Conceptualization, Writing - original draft, Writing - review & editing. Zhenmin Bao: Conceptualization. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This research was funded by the National Key Research and Development Project (2018YFD0900206) and the National Natural Science Foundation of China (U1706203). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2020.144387. References Aldea, D., Leon, A., Bertrand, S., Escriva, H., 2015. Expression of Fox genes in the cephalochordate Branchiostoma lanceolatum. Front. Ecol. Evol. 3, 80. Andrikou, C., Iovene, E., Rizzo, F., Oliveri, P., Arnone, M.I., 2013. Myogenesis in the sea urchin embryo: The molecular fingerprint of the myoblast precursors. Evodevo 4, 33. Behr, R., Sackett, S.D., Bochkis, I.M., Le, P.P., Kaestner, K.H., 2007. Impaired male fertility and atrophy of seminiferous tubules caused by haploinsufficiency for Foxa3. Dev. Biol. 306, 636–645. Benayoun, B.A., Caburet, S., Veitia, R.A., 2011. Forkhead transcription factors: Key players in health and disease. Trends Genet. 27, 224–232. Boulanger, L., Pannetier, M., Gall, L., Allais-Bonnet, A., Elzaiat, M., Le Bourhis, D., Daniel, N., Richard, C., Cotinot, C., Ghyselinck, N.B., 2014. FOXL2 is a female sex-determining gene in the goat. Curr. Biol. 24, 404–408. Boyle, M.J., Seaver, E.C., 2010. Expression of FoxA and GATA transcription factors correlates with regionalized gut development in two lophotrochozoan marine worms: Chaetopterus (Annelida) and Themiste lageniformis (Sipuncula). EvoDevo 1, 2. Brownell, I., Dirksen, M., Jamrich, M., 2015. Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation. Genesis 27, 81–93. Brusca, R.C., Brusca, G.J., 2003. Invertebrates. Quart. Rev. Biol. 100, 669–677. Chapman, J.A., Kirkness, E.F., Oleg, S., Hampson, S.E., Therese, M., Thomas, W., Thomas, R., Balasubramanian, P.G., Jon, B., Dana, B., 2010. The dynamic genome of Hydra. Nature 464, 592–596. Chen, H., Xiao, G., Chai, X., Lin, X., Fang, J., Teng, S., 2017. Transcriptome analysis of sex-related genes in the blood clam Tegillarca granosa. PLoS One 12, e0184584. Chen, H.W., Huang, X.D., Li, H.C., He, S., Ni, R.Z., Chen, C.H., Peng, C., Wu, G., Wang, G.H., Wang, Y.Y., 2013. Expression of FOXJ1 in hepatocellular carcinoma: correlation with patients' prognosis and tumor cell proliferation. Mol. Carcinog. 52, 647–659. Chevalier, S., Martin, A., Leclère, L., Amiel, A., Houliston, E., 2006. Polarised expression of FoxB and FoxQ2 genes during development of the hydrozoan Clytia hemisphaerica. Dev. Genes. Evol. 216, 709–720. Chiola, S., Do, M.D., Centrone, L., Mallamaci, A., 2019. Foxg1 overexpression in

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Gene 733 (2020) 144387

S. Wu, et al.

Teaniniuraitemoana, V., Huvet, A., Levy, P., Gaertnermazouni, N., Gueguen, Y., Le, G.M., 2015. Molecular signatures discriminating the male and the female sexual pathways in the pearl oyster Pinctada margaritifera. PLoS One 10, e0122819. Teaniniuraitemoana, V., Huvet, A., Levy, P., Klopp, C., Lhuillier, E., Gaertner-Mazouni, N., Gueguen, Y., Moullac, G.L., 2014. Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes. BMC Genomics 15, 491. Thompsom, J., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Tu, Q., Brown, C.T., Davidson, E.H., Oliveri, P., 2006. Sea urchin Forkhead gene family: Phylogeny and embryonic expression. Dev. Biol. 300, 49–62. Wagner, G.P., Kin, K., Lynch, V.J., 2012. Measurement of mRNA abundance using RNAseq data: RPKM measure is inconsistent among samples. Theory Biosci 131, 281–285. Wang, D.S., Kobayashi, T., Zhou, L.Y., Paul-Prasanth, B., Ijiri, S., Sakai, F., et al., 2007. Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1. Mol. Endocrinol. 21, 712–725. Wang, J., Cai, X., Xia, L., Zhou, J., Xin, J., Liu, M., Shang, X., Liu, J., Li, X., Chen, Z., 2015. Decreased expression of FOXJ1 is a potential prognostic predictor for progression and poor survival of gastric cancer. Ann. Surg. Oncol. 22, 685–692. Wang, S., Zhang, J., Jiao, W., Li, J., Xun, X., Sun, Y., Guo, X., Huan, P., Dong, B., Zhang, L., 2017. Scallop genome provides insights into evolution of bilaterian karyotype and development. Nat. Ecol. Evol. 1, 120. Wei, C., Lin, H., Cui, S., 2018. The forkhead transcription factor FOXC2 is required for maintaining murine spermatogonial stem cells. Stem Cells Dev. 27, 624–636. Weigel, D., Jürgens, G., Küttner, F., Seifert, E., Jäckle, H., 1989. The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo. Cell 57, 645–658. Wotton, K.R., Mazet, F., Shimeld, S.M., 2008. Expression of FoxC, FoxF, FoxL1, and FoxQ1 genes in the dogfish Scyliorhinus canicula defines ancient and derived roles for Fox genes in vertebrate development. Dev. Dyn. 237, 1590–1603. Wotton, K.R., Shimeld, S.M., 2006. Comparative genomics of vertebrate Fox cluster loci. BMC Genomics 7, 271. Yang, M., Xu, F., Liu, J., Que, H., Li, L., Zhang, G., 2014. Phylogeny of forkhead genes in three spiralians and their expression in Pacific oyster Crassostrea gigas. Chin. J. Oceanol. Limnol. Yu, B., Liu, J., Su, M., Wang, C., Chen, H., Zhao, C., 2019. Disruption of Foxg1 impairs neural plasticity leading to social and cognitive behavioral defects. Mol. Brain. Yu, J.K., Mazet, F., Chen, Y.T., Huang, S.W., Jung, K.C., Shimeld, S.M., 2008. The Fox genes of Branchiostoma floridae. Dev. Genes. Evol. 218, 629–638. Yuan, J., Tao, W., Cheng, Y., Huang, B., Wang, D., 2014. Genome-wide identification, phylogeny, and gonadal expression of fox genes in Nile tilapia Oreochromis niloticus. Fish Physiol. Biochem. 40, 1239. Zhang, M., Wang, Y., Li, Y., Li, W., Li, R., Xie, X., Wang, S., Hu, X., Zhang, L., Bao, Z., 2018. Identification and characterization of neuropeptides by transcriptome and proteome analyses in a bivalve mollusc Patinopecten yessoensis. Front. Genet. 9, 197. Zhang, N., Xu, F., Guo, X., 2014. Genomic analysis of the Pacific oyster (Crassostrea gigas) reveals possible conservation of vertebrate sex determination in a mollusc. G3: Genes Genomes Genet. 4, 2207–2217.

447–458. Maximilian, S., Antje, R.S., Cornelia, D., Walter, K.C., 2006. Temporal and spatial expression patterns of FoxN genes in Xenopus laevis embryos. Int. J. Dev. Biol. 50, 429–434. Mazet, F., Amemiya, C.T., Shimeld, S.M., 2006. An ancient Fox gene cluster in bilaterian animals. Curr. Biol. CB 16, R314–R316. Mazet, F., Shimeld, S.M., 2002. The evolution of chordate neural segmentation. Dev. Biol. 251, 258–270. Mazet, F., Yu, J.K., Liberles, D.A., Holland, L.Z., Shimeld, S.M., 2003. Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria. Gene 316, 79–89. Naimi, A., Martinez, A.S., Specq, M.L., Diss, B., Mathieu, M., Sourdaine, P., 2009. Molecular cloning and gene expression of Cg-Foxl2 during the development and the adult gametogenetic cycle in the oyster Crassostrea gigas. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 154, 134–142. Nash, B., Colavita, A., Zheng, H., Roy, P.J., Culotti, J.G., 2000. The forkhead transcription factor UNC-130 is required for the graded spatial expression of the UNC-129 TGFbeta guidance factor in C. elegans. Genes Dev. 14, 2486–2500. Nicolau-Neto, P., Palumbo, A., De, M.M., Esposito, F., De, A.S.T., Fusco, A., Nasciutti, L.E., Meireles, D.C.N., Ribeiro Pinto, L.F., 2018. UBE2C is a transcriptional target of the cell cycle regulator FOXM1. Genes 9, 188. Ogasawara, M., Satou, Y., 2003. Expression of FoxE and FoxQ genes in the endostyle of Ciona intestinalis. Dev. Genes. Evol. 213, 416–419. Pohl, B.S., Knochel, W., 2005. Of Fox and Frogs: Fox (fork head/winged helix) transcription factors in Xenopus development. Gene 344, 21–32. Rho, H.K., McClay, D.R., 2011. The control of foxN2/3 expression in sea urchin embryos and its function in the skeletogenic gene regulatory network. Development 138, 937–945. Sarafi-Reinach, T.R., Sengupta, P., 2000. The forkhead domain gene unc-130 generates chemosensory neuron diversity in C. elegans. Genes Dev. 14, 2472–2485. Shen, L., Nam, H.S., Song, P., Moore, H., Anderson, S.A., 2006. FoxG1 haploinsufficiency results in impaired neurogenesis in the postnatal hippocampus and contextual memory deficits. Hippocampus 16, 875–890. Shimeld, S.M., Boyle, M.J., Brunet, T., Luke, G.N., Seaver, E.C., 2010a. Clustered Fox genes in lophotrochozoans and the evolution of the bilaterian Fox gene cluster. Dev. Biol. 340, 234–248. Shimeld, S.M., Degnan, B., Luke, G.N., 2010b. Evolutionary genomics of the Fox genes: Origin of gene families and the ancestry of gene clusters. Genomics 95, 256–260. Siu, M.K., Wong, E.S.Y., Kong, D.S.H., Chan, H.Y., Jiang, L., Wong, O.G., et al., 2013. Stem cell transcription factor NANOG controls cell migration and invasion via dysregulation of E-cadherin and FoxJ1 and contributes to adverse clinical outcome in ovarian cancers. Oncogene 32, 3500–3509. Song, J., Li, Z., Tong, X., Chen, C., Chen, M., Meng, G., et al., 2015. Genome-wide identification and characterization of Fox genes in the silkworm Bombyx mori. Funct. Integr. Genomics 15 (5), 511–522. Takeuchi, T., Kawashima, T., Koyanagi, R., Gyoja, F., Tanaka, M., Ikuta, T., Shoguchi, E., Fujiwara, M., Shinzato, C., Hisata, K., 2012. Draft genome of the pearl oyster Pinctada fucata: A platform for understanding bivalve biology. DNA Res. 19, 117–130. Tan, P.B., Lackner, M.R., Kim, S.K., 1998. MAP kinase signaling specificity mediated by the LIN-1 Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction. Cell 93, 569–580.

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