Identification, characterization and functional analysis of regulatory region of nanos gene from half-smooth tongue sole (Cynoglossus semilaevis)

Accepted Manuscript Identification, characterization and functional analysis of regulatory region of nanos gene from half-smooth tongue sole (Cynoglossus semilaevis)

Jinqiang Huang, Yongjuan Li, Changwei Shao, Na Wang, Songlin Chen PII: DOI: Reference:

S0378-1119(17)30212-3 doi: 10.1016/j.gene.2017.03.033 GENE 41839

To appear in:

Gene

Received date: Revised date: Accepted date:

6 January 2017 20 March 2017 24 March 2017

Please cite this article as: Jinqiang Huang, Yongjuan Li, Changwei Shao, Na Wang, Songlin Chen , Identification, characterization and functional analysis of regulatory region of nanos gene from half-smooth tongue sole (Cynoglossus semilaevis). The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi: 10.1016/j.gene.2017.03.033

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ACCEPTED MANUSCRIPT Identification, characterization and functional analysis of regulatory region of nanos gene from half-smooth tongue sole (Cynoglossus semilaevis)

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,

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a

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Jinqiang Huang a,b,c, Yongjuan Li c, Changwei Shao a,b, Na Wang a,b, Songlin Chen a,b*

b

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Qingdao 266071, China

Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao

College of Animal Science and Technology, Gansu Agricultural University, Lanzhou

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c

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National Laboratory for Marine Science and Technology, Qingdao, 266237, China

Prof. Dr. Songlin Chen

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*Correspondence:

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730070, China

Yellow Sea Fisheries Research Institute

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Chinese Academy of Fishery Sciences

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Nanjing Road 106 Qingdao 266071 China Tel: 0086-532-85844606 Fax: 0086-532-85811514 E-mail:[email protected]

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ACCEPTED MANUSCRIPT Abstract The nanos gene encodes an RNA-binding zinc finger protein, which is required in the development and maintenance of germ cells. However, there is very limited information about nanos in flatfish, which impedes its application in fish breeding. In this study, we report the molecular cloning, characterization and functional analysis of

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the 3′-untranslated region of the nanos gene (Csnanos) from half-smooth tongue sole

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(Cynoglossus semilaevis), which is an economically important flatfish in China. The

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1233-bp cDNA sequence, 1709-bp genomic sequence and flanking sequences (2.8-kb 5′- and 1.6-kb 3′-flanking regions) of Csnanos were cloned and characterized.

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Sequence analysis revealed that CsNanos shares low homology with Nanos in other species, but the zinc finger domain of CsNanos is highly similar. Phylogenetic

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analysis indicated that CsNanos belongs to the Nanos2 subfamily. Csnanos expression was widely detected in various tissues, but the expression level was higher in testis

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and ovary. During early development and sex differentiation, Csnanos expression

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exhibited a clear sexually dimorphic pattern, suggesting its different roles in the migration and differentiation of primordial germ cells (PGCs). Higher expression

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levels of Csnanos mRNA in normal females and males than in neomales indicated that the nanos gene may play key roles in maintaining the differentiation of gonad.

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Moreover, medaka PGCs were successfully labeled by the microinjection of synthesized mRNA consisting of green fluorescence protein and the 3′-untranslated region of Csnanos. These findings provide new insights into nanos gene expression and function, and lay the foundation for further study of PGC development and applications in tongue sole breeding. Keywords: tongue sole, nanos, dimorphic expression, primordial germ cells, visualization 2

ACCEPTED MANUSCRIPT 1. Introduction Half-smooth tongue sole (Cynoglossus semilaevis) is a popular and valuable marine flatfish in China. The growth rate and pattern of this species show significant sexual dimorphism, with females growing faster and reaching two to three times the size of males. The phenomenon of sex reversal in genetic females is also common in

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natural and aquaculture environments. Therefore, monosex cultivation of half-smooth

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tongue sole has significant benefits for aquaculture. In most multicellular organisms,

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germ cells are involved in the transfer of genetic and epigenetic information to future generations (Saitou and Yamaji, 2012). Germ cells undergo two significantly different

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developmental phases, primordial germ cell (PGC) formation/migration and gamete formation (Castrillon et al., 2000). PGCs are established at the earliest stages of

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embryo development; they are involved in the formation of gonads and play a vital role in gonadal differentiation in fish (Siegfried et al., 2008; Braat et al., 1999). Study

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of germ cells helps to demonstrate the mechanism of sex determination and

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differentiation from a different perspective. Although important information about all-female stocks and sex determination in this species has been obtained, information

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on the expression of genes related to gonad development, by which we can understand the developmental patterns of germ cells, is still limited. Study of germ cell

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development of half-smooth tongue sole during embryogenesis and the larval/juvenile stages is useful to understand and control fish sex and reproductive function. As one of several germline-specific molecular markers that have been identified (Nagasawa et al., 2013; Li et al., 2015), nanos encodes an evolutionarily conserved RNA binding protein containing two consecutive Cys–Cys–His–Cys (CCHC)-type zinc-finger motifs in the C-terminal region (Curtis et al., 1997). Since nanos was first identified as a maternal effect gene in fruit fly (Wang et al., 1991), it has been found 3

ACCEPTED MANUSCRIPT that the Nanos protein is required for formation of the abdomen, the determination and migration of PGCs, and the maintenance of germ stem cells in fruit fly (Kobayashi et al., 1996; Wang and Lin, 2004). To date, nanos-related genes have been cloned and studied in several vertebrates, in addition to diverse insect species and other invertebrates. In mice, three nanos homologs (nanos1–3) have been found.

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Although nanos1 contains a similar zinc-finger motif to other nanos-class genes, it is

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not detected in PGCs; in addition, nanos1-deficient mice develop to term without any

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detectable abnormalities and are fertile (Haraguchi et al., 2003). Nanos1 functions mainly in the chromatid bodies, as well as in the cytoplasm (Yokota and Onohara,

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2013). Nanos2 is specifically expressed in male gonads and plays a key role in the sexual differentiation of germ cells by ensuring a male fate while repressing a female

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one; it can also promote male germ cell self-renewal by the suppression of meiosis (Suzuki and Saga, 2008). Nanos2 is also expressed in the spermatogonial stem cells

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and functions as an intrinsic factor for the stem cell population during

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spermatogenesis (Saga, 2010). In contrast, nanos3 expression continues throughout the migration stages from the stage of formation of PGCs; the loss of this protein

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results in a germ cell-less phenotype in both sexes. Nanos2 has distinct and irreplaceable functions compared with nanos3 during male germ cell development

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(Tsuda and Suzuki, 2007). In teleosts, study of the nanos gene has mainly focused on model fishes such as zebrafish and medaka. Four different nanos genes were identified in the genome of medaka based on similarity and syntenic analyses. In contrast to a previous study on mice, nanos2 expression was detected in oogonia and spermatogonia of adult gonads, but not at early stages of sex differentiation (Aoki et al., 2009), while nanos1a was expressed in the somatic cells surrounding oocytes and spermatocytes. In zebrafish, 4

ACCEPTED MANUSCRIPT three nanos homologs have been found. Nanos1 is expressed in early-stage oocytes in the adult female germline; young female nanos1 mutants contain oocytes, but fail to maintain oocyte production (Draper et al., 2007). In contrast, nanos2 is expressed in potential germline stem cells in the adult gonads of both males and females. Finally, nanos3 is required for the continued production of oocytes and the maintenance of

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germline stem cells (Beer and Draper, 2013). In the olive flounder, nanos3 was

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consistently expressed during embryogenesis (Li et al., 2015), while the expression of

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Atlantic cod nanos3 was detected from the first cleavage division and ceased during early somitogenesis (Presslauer et al., 2012). During embryogenesis, the regulation of

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gene expression via the 3′-untranslated region (UTR) is essential for the discrimination of the germ cell lineage from somatic cells (Suzuki et al., 2010), so the

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nanos genes have also been exploited to visualize germ cells in zebrafish (Saito et al., 2006), common carp (Kawakami et al., 2011), medaka (Kurokawa et al., 2006),

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Japanese eel (Saito et al., 2011) and olive flounder (Li et al., 2015) by injecting green

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fluorescent protein (GFP)-zfnos1-3′-UTR mRNA. In this study, we cloned the half-smooth tongue sole’s nanos-related gene

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(Csnanos) and characterized the cDNA, genomic and flanking sequences of it. We also studied Csnanos mRNA expression levels in different tissues and embryonic

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stages. The sex-linked differential expression of Csnanos was detected during the early developmental and sex differentiation stages. The function of Csnanos 3′-UTR was confirmed by microinjecting synthesized mRNA into fertilized medaka eggs. The obtained results should promote the development of monosex half-smooth tongue sole by genetic breeding techniques.

2. Materials and methods 5

ACCEPTED MANUSCRIPT 2.1. Fish and samples All fish and embryos used in this study were collected from a commercial farm in Haiyang, Shandong Province, China. The fish were anesthetized with MS-222 before organ or tissue sampling. The heart, liver, gill, skin, blood, kidney, intestine, brain, spleen, muscle, pituitary and gonads were excised from 1-year-old tongue sole,

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snap-frozen in liquid nitrogen and stored at −80 °C until further analysis. The

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embryos, larval samples (4 to 66 days post-hatching, dph) and gonads (80 to 150 dph) from different developmental stages were collected in liquid nitrogen and stored at

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−80 °C until RNA extraction. The sex reversal of tongue sole was induced with a

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temperature of 28 °C as previously described (Deng et al., 2009). All of the experiments were carried out in accordance with the protocol approved by the Animal

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Care and Use Committee of the Chinese Academy of Fishery Sciences.

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2.2. Physiological sex and genetic sex identification

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By analyzing the expression of gonad-specific genes (dmrt1 and cyp19a1a), the physiological sex of each tongue sole was determined (Deng et al., 2009). PCR-based

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genetic sex identification was carried out with sex-linked SSR primers (CseF-SSR1 and SChen-1) (Chen et al., 2012). Fish that produced a 206-bp band were regarded as

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genetic males and those with two DNA bands of 206 and 218 bp as genetic females. Sex-reversed females with the male sexual phenotype were considered as neomales.

2.3. Cloning full-length cDNA and genomic DNA sequences of the Csnanos gene Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. First-strand cDNA was synthesized using the Transcript First-strand cDNA Synthesis Kit (TaKaRa, Dalian, 6

ACCEPTED MANUSCRIPT China) with 1 µg of total RNA. In accordance with the partial sequence of Csnanos from whole-genome and transcriptome sequencing (Chen et al., 2014), the primers Nanos-mid-A and Nanos-mid-S (Table 1) were designed and used to amplify the cDNA fragment of the Csnanos gene. Based on the 431-bp cDNA fragment obtained by amplification, the RACE-specific primers Nanos-5′-GSP and Nanos-3′-GSP (Table

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1) were designed. In accordance with the manufacturer’s protocol, 5′- and 3′-RACE

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were used to isolate the full-length cDNA sequence using the BD SMART™ RACE

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cDNA Amplification Kit (Clontech, Mountain View, CA, USA). The PCR conditions and cloning of amplification products were as previously described (Huang et al.,

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2014). The obtained sequences were assembled using the Vector NTI software package (Invitrogen, Carlsbad, CA, USA) and then subjected to bioinformatic

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analysis. The primers Nanos-DNA-S and Nanos-DNA-A (Table 1) were designed to amplify the genomic sequence. The 5′- and 3′-flanking sequences of Csnanos were

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amplified by using the primers shown in Table 1, which were designed in accordance

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with the partial sequence of Csnanos by whole-genome sequencing and following the genome walking kit instructions (TaKaRa, Dalian, China). All of the purified

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sequenced.

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fragments were cloned into the pMD-18T (TaKaRa, Dalian, China) vector and

2.4. Sequence analysis and alignment Using the online tool BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), protein sequences homologous to CsNanos were identified. Phylogenetic analysis was performed using Mega7 software by bootstrap analysis of 1000 replicates using the neighbor-joining method (Tamura et al., 2011). The deduced amino acid sequences of the zinc-finger domain of CsNanos were aligned using the Clustal W program. 7

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2.5. Quantitative real-time PCR (qRT-PCR) of the Csnanos gene Because of the small size of fish at 4 to 66 dph, it is difficult to dissect the gonads from them, so we divided each body trunk into several parts and used the expression of gonad-specific genes (dmrt1 and cyp19a1a) to identify and confirm the

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putative gonad parts (4 to 66 dph). The gonad sex differentiation and the gonadal

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stages of adult fish were determined as previously described (Liang et al., 2012; Chen

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et al., 2010; Song et al., 2009). The expression of Csnanos during different stages of gonadal development was analyzed by qRT-PCR. Three samples (n=3) were collected

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for RNA extraction at each stage. For tongue sole at the embryo developmental stage, each sample contained a pool of 30 embryos; for tongue sole at 4 to 66 dph, each

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sample contained six gonad parts; for tongue sole at 67 to 150 dph, each sample contained three gonads; and for adult tongue sole, each sample contained one gonad.

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Total RNA was extracted and reverse-transcribed as described above. Samples were

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run in triplicate for PCR and diluted cDNA was used as the template for qRT-PCR reactions. qRT-PCR was then performed using specific primers (Nanos-RT-A,

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Nanos-RT-S; Actin-RT-A, Actin-RT-S) (Table 1) on an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems, Foster City, USA). Reactions were run in a

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volume of 20 L for SYBR Premix Ex Taq (TaKaRa, Dalian, China) and β-actin was used as an internal reference (Li et al., 2010). Each PCR cycle included 95 °C for 10 s, and 40 cycles at 95 °C for 5 s and 60 °C for 34 s. Then, a dissociation curve was added to check amplification specificity. To further analyze the differences in expression of Csnanos at different developmental stages, relative expression levels of Csnanos mRNA were analyzed and calculated using the 2−ΔΔCt method and 7500 system SDS software (Applied Biosystems, Foster City, USA). 8

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2.6. Preparation of GFP-Csnanos 3'-UTR mRNA and microinjection The GFP-Csnanos 3′-UTR vector was constructed with the 3′-UTR of Csnanos, which was amplified using primers (Nanos3U-A, Nanos3U-S) (Table 1) and the plasmid of GFP-Drnos 3′-UTR (Lin et al., 2012). Capped mRNAs were synthesized

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from linearized plasmids using the mMessage machine SP6 kit (Ambion, Austin, TX).

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GFP-Csnanos 3′-UTR mRNA (200 ng/L, 0.05% phenol red) was microinjected into

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one-cell-stage fertilized medaka eggs with a glass capillary needle. Injected embryos were cultured in Holtfreter’s solution at 28 °C and used for the fluorescence analysis

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under a fluorescence microscope (Nikon, Japan).

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2.7. Statistical analysis

The data were log-transformed to normalize the distribution and statistically

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analyzed using SPSS 22.0 software (SPSS, IL, USA). One-way ANOVA followed by

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Duncan’s multiple comparison tests was performed to analyze the differences in Csnanos expression among different developmental stages or different tissues.

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Two-way ANOVA followed by Duncan’s multiple comparison tests was performed to analyze the differences in Csnanos expression between males and females during the

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same and different developmental stages. Student’s t-test was used to detect differences between different sexes at the same stage. Differences were considered significant at a p-value of < 0.05.

3. Results 3.1. Isolation and characterization of the Csnanos gene A 431-bp fragment of Csnanos was obtained using specific primers, and the 9

ACCEPTED MANUSCRIPT remaining unknown regions were cloned through 3′- and 5′-RACE. A full-length 1233-bp Csnanos cDNA sequence was produced by overlapping the sequences of all fragments, which consisted of a 5′-UTR of 267 bp, an open reading frame of 564 bp encoding 188 amino acids, and a 3′-UTR of 375 bp (Fig. 1). A 2.8-kb 5′-flanking region and a 1.6-kb 3′-flanking region of half-smooth tongue sole were obtained by

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genome walking and PCR amplification (GenBank ID: KY392596). The 5′-flanking

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sequence contained a 526-bp intron and a promoter sequence. Several transcription

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factor binding sites in the 5'-flanking sequence of the Csnanos gene were predicted using the tools TFSEARCH and Alibaba 2.1, for example, SRY, Oct-1, Sox-5, CREB,

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GATA, AP-1, C/EBP, Sp-1, c-Myc, HNF and NKX-2. The Brinker, Zen and DRE transcription factor binding sites were also identified (Fig. S1).

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A phylogenetic tree was constructed using the amino acid sequences of nanos gene analogues from half-smooth tongue sole and other species. The CsNanos family

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was divided into two branches based on the evolutionary relationships; one consisted

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of Nanos1, and the other consisted of Nanos2 and Nanos3. CsNanos belonged to the Nanos2 subfamily (Fig. 2). The CsNanos amino acid sequence was aligned with other

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typical Nanos sequences available in GenBank using the BLASTP program. The results showed that CsNanos shared low homology with other species. In the Nanos2

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cluster, the homology of representative species with half-smooth tongue sole, from high to low, were as follows: European seabass (46.1%), medaka (41.3%), zebrafish (38.4%), Nile tilapia (37%), human (36.2%) and mouse (33.1%). Upon comparing CsNanos with other Nanos homologs, the homology levels were lower, such as for Nanos of Atlantic salmon (27.3%), Nanos of common carp (31.4%), Nanos1 (32.7%) and Nanos3 (32.7%) of zebrafish, and Nanos1a (23.4%), Nanos1b (26.7%) and Nanos3 (28.9%) of medaka. However, the zinc finger domain of CsNanos shared 10

ACCEPTED MANUSCRIPT higher homology with those of other species; the homology levels for Japanese pufferfish and Europe bass were both 72.2%, while that for Nile tilapia was 70.4% (Fig. 3).

3.2. Csnanos expression in different tissues and different developmental stages

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The Csnanos mRNA expression levels in 12 different tissues of 1-year-old

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tongue sole were examined by qRT-PCR. It was found that Csnanos

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was widely expressed in various tissues, and the expression level was highest in the testes, followed by the ovaries. The sampled fish had ovaries at stage III of

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development and testes at stage Ⅳ. Expression was higher in brain than in other nongonadal tissues, including liver, blood, skin, gill, kidney, intestine, spleen, heart

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and muscle (Fig. 4). During embryonic development, the expression levels of Csnanos persisted at different stages. Nanos transcript was present at a steady and

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high level between the four-cell and multi-cell stages, until the somite stage, followed

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by an apparent decrease in subsequent stages. It reached a nearly undetectable level from the time when the heart started to beat until 1 dph (Fig. 5). The expression of

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Csnanos mRNA was also detected in 2-year-old males, females and neomales. The ovaries of two-year-old sampled fish were at stages Ⅳ–Ⅴ of development, while testes

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were at stage Ⅴ. Csnanos mRNA was found to be expressed at a higher level in the females and males than in neomales (Fig. 6).

3.3. Sexually dimorphic expression of Csnanos during early development and sex differentiation The characteristics of Csnanos expression were determined by qRT-PCR during early development and sex differentiation in males and females. During early 11

ACCEPTED MANUSCRIPT development, the Csnanos expression level was higher in males than in females from 4 to 8 d, but these levels were nearly the same at 16 d; however, this level was higher in females than in males throughout the subsequent developmental period (from 26 to 66 d). Moreover, the lower expression level in females persisted from 16 to 56 d, but that in males persisted until 66 d. Then, a marked increase in nanos mRNA expression

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was observed in both sexes during sex differentiation; a significant difference of

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nanos expression level was also observed between females and males from 66 to 150

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d (Fig. 7).

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3.4. Regulatory activity of GFP-Csnanos 3'-UTR mRNA in medaka PGCs To confirm that the 3′-UTR of tongue nanos can be used to visualize PGCs,

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GFP-Csnanos 3′-UTR mRNA was injected into fertilized medaka eggs. The results showed that GFP fluorescence was observed in somatic cells before embryo stage 17

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and did not differentiate between somatic cells and PGCs until stage 25, as

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embryogenesis developed. GFP fluorescence was stronger in PGCs in the presumptive gonad region as the fluorescence of somatic cells gradually decreased. The

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fluorescence signal aligned bilaterally along the trunk from stage 25 to 29 (Fig. 8). The GFP signals were round with a large size and distributed uniformly throughout

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the entire cell, which are typical characteristics of PGCs. Approximately 88% of injected embryos (n=158) showed PGC-specific GFP expression. These results confirmed that the chimeric mRNA can be used to visualize the migration route of PGCs in medaka by microinjection of GFP-Csnanos 3′-UTR mRNA during embryogenesis.

4. Discussion 12

ACCEPTED MANUSCRIPT Although Nanos proteins were shown to be essential to the development of germ cells, studies on their function and expression have focused on mice and flies, while such information for flatfish is limited. In this study, a full-length 1233-bp cDNA sequence of the Csnanos gene was obtained, which contained a 375-bp 3′-UTR. In mice, the 3′-UTR of the nanos2 gene is 900 bp, and the number of germ cells in

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seminiferous tubules in a mouse line without the 3′-UTR was decreased compared

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with that in normal mice. These results suggested that the level of Nanos2 protein

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could regulate spermatogenesis through the nanos2 3′-UTR (Tsuda et al., 2006). In previous studies, the 3′-UTR was identified in all studied nanos genes and was

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shown to be necessary for mRNA localization, translation or directing the specific expression of the protein in the PGCs in fruit fly (Gavis et al., 1996), frog (Zhou and

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King, 1996), zebrafish (Koprunner et al., 2001) and Chinese sturgeon (Ye et al., 2012). This showed that the function of the 3′-UTR of the nanos gene is conserved among

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different species. Based on the phylogenetic and homology analyses, two branches of

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the Nanos family were identified and CsNanos was clustered with Nanos2. The levels of homology among the various amino acid sequences of the nanos gene were low,

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but the zinc-finger motif of the nanos gene was highly conserved, implying its functional significance.

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Tissue distribution analysis showed that Csnanos mRNA was preferentially expressed in the testes and ovaries, which implied the importance of the nanos gene in gonad development. In addition, higher expression was observed in the brain, but expression levels were very low in other tissues. The expression of the nanos homolog of Spinibarbus caldwelli was previously found to be restricted to the gonads, with no signal in other somatic tissues (Min et al., 2012). In contrast, in Chinese sturgeon, nanos1 mRNAs were detected in various tissues. In medaka, the expression 13

ACCEPTED MANUSCRIPT of nanos1a and nanos1b was observed in the brain (Aoki et al., 2009). In dairy goat, it was highly expressed in the pancreas and spleen (Yao et al., 2014). These results may indicate that the nanos gene influences multiple tissues in different species. In mouse, the nanos gene is expressed in adult brain; however, no significant neural defects were observed in nanos1-deficient mice (Haraguchi et al., 2003). Therefore, further studies

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are required to confirm the function of the nanos gene in somatic tissue.

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The expression of nanos mRNA was examined continuously during

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embryogenesis. Expression levels were highest from the four-cell to multi-cell stages and dropped rapidly at the gastrula stage. Expression was barely detectable until the

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stages when the heart started to beat and at hatching. Similar expression patterns of nanos mRNA were found in zebrafish (Dosch et al., 2015), silkworm (Zhao et al.,

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2008) and lancelet (Wu et al., 2011). The findings indicated that nanos mRNA is a maternally supplied transcript, similar to vasa mRNA, but also suggested that the

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expression patterns of Nanos protein are relatively similar among species, although

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the protein sequences themselves show some differences among species, apart from in particular conserved regions.

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To understand the role of the nanos gene in early gonadal differentiation, its expression patterns in males and females were examined during early development

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and gonadal differentiation by using sex-linked microsatellite markers and genes with gonad-specific expression to determine the sex and distinguish the gonads. Similar to the vasa gene of half-smooth tongue sole, the expression level of the nanos gene showed a trend of first decreasing and then increasing with the development of juvenile fish in both sexes. This indicated that nanos exhibited a switch from maternal to de novo expression. Moreover, the expression level of the nanos gene was higher in males than in females during early embryonic development, but expression 14

ACCEPTED MANUSCRIPT levels were higher in females than in males from 26 d. The expression differences can be caused by the different methods of proliferation of PGCs in male and female (Morinaga et al., 2004; Morinaga et al., 2007). The first clear sex-related differences could be observed at 66 d, according to the results of tissue biopsies obtained at our laboratory. Sex differentiation has begun at that time, after which females are

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easily discriminated by the expression of nanos. Therefore, the sex differentiation of

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half-smooth tongue sole is ongoing at 66 d and sex can also be distinguished by the

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dimorphic expression of the nanos gene. An increase of the expression levels of the nanos gene was associated with PGCs, suggesting that the period from 56 to 66 d is

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crucial for sex differentiation and that the nanos gene plays an important role in sex differentiation. In Nile tilapia and turbot, the correspondence between the gene

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expression profiles and histological sex was also confirmed by identifying the first sex-associated genetic cues during the early stages of gonad development (Robledo et

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al., 2015; Kobayashi et al., 2003).

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Sex reversal of half-smooth tongue sole is common in both wild and aquaculture environments, which causes considerable loss to producers. The expression levels of

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neomales obtained by heat treatment were determined and compared with those of normal fish; we found that the expression levels of the nanos gene were highest in

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females, followed by those in males, with those of neomales being the lowest. These results differ from those for the vasa gene, the expression level of which in neomales was much closer to that in females, rather than that in males. Researchers have generally believed that the direction of differentiation of germ cells is determined by the expression of sex cell genes and the regulation of somatic cells. The nanos2 gene is endogenously expressed in germ cells and plays a key role in sex determination in mouse. The level of transcription of specific genes in males was found to be decreased 15

ACCEPTED MANUSCRIPT if nanos2 was knocked down, but females developed into males when the nanos2 gene was overexpressed. However, the expression of the nanos gene in neomales was low, which appeared to indicate that the function of nanos differs between mouse and tongue sole. Further study is needed to determine whether the incidence of sex reversal can be reduced by regulating the expression of the nanos gene in females.

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Transcription of the nanos gene was shown to be essential to multiple functions

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during the development of organisms. The nanos promoter has been used in

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the construction of a vector to drive transgene expression in germ cells and somatic cells. Its function in germline stem cells was also confirmed in early studies (Van

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Doren et al., 1998; Chen et al., 2003). In addition, a 1-kb sequence in the upstream region of the transcription start site of the promoter and 250 bp in the 5′-UTR were

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shown to be important for expression of the reporter gene in germ cells (Tracey et al., 2000). In transgenic flies, a short fragment of the promoter sequence (nucleotides

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−108 to −90) was effective for expressing GFP in germ cells (Okada et al., 2004). In

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this study, a promoter sequence of the nanos gene was obtained by genome walking and PCR amplification. Some common transcription factor binding sites in the

DRE,

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5′-flanking sequence were predicted using online tools, including Brinker, Zen and which

were

specific

to

fruit

fly

nanos

gene.

These

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results laid the foundations for the future construction of expression vectors. Besides the regulatory function of the promoter region, the function of the 3′-UTR of the nanos gene in germ cell development was confirmed in mouse (Suzuki et al., 2010), fruit fly (Rangan et al., 2008), frog (Lai et al., 2012) and fish (Škugor et al., 2014). In zebrafish, a vector constructed using GFP and the 3′-UTR of nanos1 was used to label PGCs in zebrafish or other fish (Saito et al., 2006). Recently, the visualization of PGCs in common carp embryos using GFP fused to the 3′-UTR of the 16

ACCEPTED MANUSCRIPT common carp or zebrafish nanos-related gene was studied. The results demonstrated that the injection of artificial GFP mRNA fused with the autologous nanos-3′-UTR in common carp was more effective for visualization than injection of the exogenous nanos-3′-UTR (Kawakami et al., 2011). In this study, GFP-fused Csnanos 3′-UTR mRNA was constructed and injected into medaka embryos; the results confirmed the

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site-directed expression function of Csnanos 3′-UTR.

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In conclusion, in the present study, we cloned and characterized the sequences of

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Csnanos. The expression patterns of nanos during different developmental stages and different tissues of half-smooth tongue sole were analyzed and a unique sex-related

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differential expression pattern during early development and sex differentiation was identified. The chimeric GFP-Csnanos 3′-UTR mRNA could be used to label medaka

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PGCs by microinjection into fertilized eggs. These findings confirmed the potential of Csnanos as a molecular marker of germ cells and lay the foundation for further study

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of PGC development and applications in tongue sole breeding.

Acknowledgments

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This study was supported by National Nature Science Foundation of China

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(31530078) and Taishan Scholar Climbing Project Fund of Shandong of China.

Conflict of Interest The authors declare that there are no conflicts of interest.

References Aoki Y, Nakamura S, Ishikawa Y, Tanaka M. Expression and syntenic analyses of four nanos genes in medaka. Zoolog Sci. 2009, 26(2): 112–118. 17

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ACCEPTED MANUSCRIPT major). Int J Biol Sci. 2012, 8(6): 882–890. Min SU, ChenYZ, Bo-Yan LV, Tang LH. Expression of nanos homolog gene in germ cells of spinibarbus caldwelli. Biotechnology. 2012, 22(4): 28–32. Morinaga C, Saito D, Nakamura S, Sasaki T, Asakawa S, Shimizu N, Mitani H, Furutani-Seiki M, Tanaka M, Kondoh H. The hotei mutation of medaka in the

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transcription through E-box motifs in the PF4 gene in megakaryocytes. Blood. 2004, 104(7): 2027–2034. Presslauer C, Nagasawa K, Fernandes JM, Babiak I. Expression of vasa and nanos3 during primordial germ cell formation and migration in Atlantic cod (Gadus morhua L.). Theriogenology. 2012, 78(6): 1262–1277. Rangan P, DeGennaro M, Lehmann R. Regulating gene expression in the Drosophila germ line. Cold Spring Harb Symp Quant Biol. 2008, 73: 1–8. 21

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Yamaha E. The mechanism for primordial germ-cell migration is conserved between Japanese eel and zebrafish. PLoS One. 2011, 6(9): e24460.

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Saitou M, Yamaji M. Primordial germ cells in mice. Cold Spring Harb Perspect Biol. 2012, 4(11): 59–66.

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Siegfried KR, Nüsslein-Volhard C. Germ line control of female sex determination in

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zebrafish. Dev Biol. 2008, 324(2): 277–287. Škugor A, Slanchev K, Torgersen JS, Tveiten H, Andersen Ø. Conserved mechanisms

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Song HX, Weng YZ, Liu ZG, Fang YQ. A histological study on spermatogensis in Cynoglossus semilaevis. Journal of Oceanography in Taiwan Strait. 2009, 28(1): 19–24. Suzuki A, Saga Y. Nanos2 suppresses meiosis and promotes male germ cell differentiation. Genes Dev. 2008, 22(4): 430–435. Suzuki A, Tsuda M, Saga Y. Functional redundancy among Nanos proteins and a distinct role of Nanos2 during male germ cell development. Development. 2007, 22

ACCEPTED MANUSCRIPT 134(1): 77–83. Suzuki H, Saba R, Sada A, Saga Y. The nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos. PLoS One. 2010, 5(2): e9300. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance,

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and maximum parsimony methods. Mol Biol Evol. 2011, 28(10): 2731–2739.

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Tracey WD, Ning X, Klingler M, Kramer SG, Gergen JP. Quantitative analysis of

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gene function in the Drosophila embryo. Genetics. 2000, 154(1): 273–284. Tsuda M, Kiso M, Saga Y. Implication of nanos2 3′UTR in the expression and

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function of nanos2. Mech Dev. 2006, 123(6): 440–449. Tsuda M, Sasaoka Y, Kiso M, Abe K, Haraguchi S, Kobayashi S, Saga Y. Conserved

MA

role of Nanos proteins in germ cell development. Science. 2003, 301(5637): 1239–1241.

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Van Doren M, Williamson AL, Lehmann R. Regulation of zygotic gene expression in

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Drosophila primordial germ cells. Curr Biol. 1998, 8(4): 243–246. Wang C, Lehmann R. Nanos is the localized posterior determinant in Drosophila. Cell.

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differentiation. Science. 2004, 303(5666): 2016–2019. Wu HR, Chen YT, Su YH, Luo YJ, Holland LZ, Yu JK. Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae. Dev Biol. 2011, 353(1): 147–159. Yao X, Tang F, Yu M, Zhu H, Chu Z, Li M, Liu W, Hua J, Peng S. Expression profile of Nanos2 gene in dairy goat and its inhibitory effect on Stra8 during meiosis. Cell Prolif. 2014, 47(5): 396–405. 23

ACCEPTED MANUSCRIPT Ye H, Chen X, Wei Q, Zhou L, Liu T, Gui J, Li C, Cao H. Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis. Gene. 2012, 511(2): 285–292. Yokota S, Onohara Y. Expression and localization of nanos1 in spermatogenic cells during spermatogenesis in rat. Cellbio. 2013, 02(1): 1–10.

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Zhao G, Chen K, Yao Q, Wang W, Wang Y, Mu R, Chen H, Yang H, Zhou H. The

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nanos gene of Bombyx mori and its expression patterns in developmental

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embryos and larvae tissues. Gene Expr Patterns. 2008, 8(4): 254–260. Zhou Y, King ML. Localization of Xcat-2 RNA, a putative germ plasm component, to

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the mitochondrial cloud in Xenopus stage I oocytes. Development. 1996, 122(9):

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CE

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D

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2947–2953.

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ACCEPTED MANUSCRIPT Tables Table 1 Primers and their sequences used for PCR. Primer sequences (5' to3')

Usage

CseF-SSR1

GAGGCCGACAGGATCGTAC

Genetic sex identification

SChen-1

TACGACGTACTCCGGTGGTTTT

Genetic sex identification

P450a-A

CAGGTAGGAGGTTGCTGGGTA

Gonad identification

P450a-S

CAGGAGGAAGAACTTGGGATTT

Dmrt1-A

GGTGAGGATGTGACCCAGTGT

Dmrt1-S

ACGGGCTGAAATCGCAAG

Nanos-mid-A

CGGCGTGTGTGTGCGTAAT

Nanos-mid-S

TGGCGTGACTACATGGACCTG

Nanos-5' GSP

GTGGTGAAGTTGGAGAAGGTGACGGAGC

5' RACE PCR

Nanos-3' GSP

GCTCCGTCACCTTCTCCAACTTCACCAC

3' RACE PCR

Nanos-DNA-A

TTGGAAAAAATGTAAACCTGCCTT

DNA fragment PCR

Nanos-DNA-S

GTGATTACACTGGATGACTGGCA

DNA fragment PCR

Nanos-5'-A1

AAGTTGGAGAAGGTGACGGAGC

Genome Walking

Nanos-5'-A2

TTCAGCAGGGACCATCAGTAGG

Genome Walking

Nanos-5'-A3

GAATAGCGTGCCAGTCATCCAGTGT

Genome Walking

Nanos-5′F-A

TTAGGGCACGCATTGTCAGTC

5' flanking region clone

Nanos-5′F-S

CTCACCTACGACAAGTCTTTCCG

5' flanking region clone

Nanos-3'-A1

CACTGGATGACTGGCACGCTAT

Genome Walking

Nanos-3'-A2

CACGGTTCCCTACTGATGGTCC

Genome Walking

Nanos-3'-A3

CCGTCACCTTCTCCAACTTCAC

Genome Walking

Nanos-RT-A

GGTTGTCGGATGAACTGGTGC

qRT-PCR

Nanos-RT-S

CCTACTGATGGTCCCTGCTGAA

qRT-PCR

Actin-RT-A

GCTGTGCTGTCCCTGTA

qRT-PCR

Actin-RT-S

GAGTAGCCACGCTCTGTC

qRT-PCR

Nanos3U-A

GGGGTACCATATGCTTTGTTATCATCAATTTTA

3' UTR clone

Nanos3U-S

CCGCTCGAGGTGACGTCAAAGTTCAGTTTTTA

3' UTR clone

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Primer

AC

CE

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D

MA

NU

SC

RI

Gonad identification

25

Gonad identification Gonad identification Partial fragment clone Partial fragment clone

ACCEPTED MANUSCRIPT Figure Captions

Fig. 1 Genomic sequence and predicted amino acid sequence of Csnanos. Exons and introns are indicated in uppercase and lowercase, respectively. The 18 amino acid

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N-terminal domain and the 53 amino acid CCHC zinc-finger domains are shaded in gray and the conserved histidine and cysteine residues are marked in yellow. Initiation

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codon, termination codon and poly-A tail are shaded in bold. The stop codon is

NU

SC

indicated by an asterisk.

Fig. 2 Phylogenetic analysis of Nanos orthologs using the neighbor-joining method.

MA

Bootstrap values of 1000 replicates (> 80%) are indicated next to the branches. The sequences used for alignment and phylogenetic analysis are as follows: Drosophila

D

melanogaster (DmNanos, AAA28715); Homo sapiens (HsNanos1, NP_955631;

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HsNanos2, NP_001025032; HsNanos3, NP_001092092); Mus musculus (MmNanos1, NP_848508; MmNanos2, NP_918953; MmNanos3, NP_918948); Danio rerio

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(DrNanos1, AAL15474; DrNanos2, DAA64468; DrNanos3, NP_571953); Oryzias latipes (OlNanos1a, NP_001116380; OlNanos1b, NP_001153941; OlNanos2,

AC

NP_001153919; OlNanos3, NP_001116300); Cynoglossus semilaevis (CsNanos); Oreochromis niloticus (OnNanos, XP_005448912); Takifugu rubripes (TrNanos, XP_003968060); Dicentrarchus labrax (DlNanos, CBN81978).

Fig. 3 Alignment of the amino acid sequences of the Nanos zinc-finger domain. Identical residues are in the same color, the arrowheads indicate the two conserved CCHC zinc-finger motifs. Homologues of the Nanos are arranged into three clusters: 26

ACCEPTED MANUSCRIPT Nanos1 (above the blue line), Nanos2 (between the blue and red line) and Nanos3 (below the red line). The amino acid identities of CsNanos with other Nanos are shown on the right. Cs (Cynoglossus semilaevis), On (Oreochromis niloticus), Ol (Oryzias latipes), Mm (Mus musculus), Hs (Homo sapiens), Tr (Takifugu rubripes), Dl

RI

PT

(Dicentrarchus labrax), Dm (Drosophila melanogaster), Dr (Danio rerio).

SC

Fig. 4 The relative expression levels of Csnanos in adult tissues. The data represent the mean gene expression levels normalized by β-actin expression ± the standard

NU

error of mean (SEM) from three separate individuals performed in triplicate. Different

MA

letters indicated statistical significance (p < 0.05), which were calculated by one-way

D

ANOVA followed by Duncan′s multiple range tests.

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Fig. 5 Expression pattern of Csnanos during embryosgenesis. The data were shown as mean ± SEM (n = 3). Each sample consisted of a pool of 30 embryos, a pool of 30

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larvae (1 dph). Different letters indicated statistical significance (p < 0.05).

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Fig. 6 The relative expression of Csnanos mRNA in the gonads of neomales, normal males and females. Data represent the mean ± SEM (n = 3). Different letters indicated statistical significance (p < 0.05).

Fig. 7 The expression profile of Csnanos at different developmental stages of the gonads. The data represent mean ± SEM (n = 3). Each sample consisted of a pool of 6 body trunks (4–66 dph), a pool of 3 gonads (80–150 dph). Different letters indicated statistical significance (p < 0.05), which were calculated by two-way ANOVA 27

ACCEPTED MANUSCRIPT followed by Duncan′s multiple range tests. Statistical significance between sexes (*P < 0.05; **P < 0.01) were shown as asterisks at each sampling time after a Student’s t-test.

Fig. 8 Visualization GFP-labeled PGCs in medaka embryos by microinjection of

PT

GFP-Csnanos 3'UTR mRNA. PGCs are denoted by white arrows. (A–D) embryos of

RI

stage 17, stage 25, stage 29, and stage30 were observed under light and fluorescence

AC

CE

PT E

D

MA

NU

SC

microscopy.

28

ACCEPTED MANUSCRIPT

CE

PT E

D

MA

NU

SC

RI

PT

ACATGGGcgccttcacactattaaaacaactacgcgctccacgatttcagtgtgtgcgat tgatgcgagtcagcagtttgattgaccactcagactgtcagctaccgcctcatccacgca gtgacctgtcattgctatggaccgcactgatagtcgtgcccgtagacagccaggtacata aaacagtttattttcaattaagcagtgtagaatggaaaagtaggacaaaaactccaattt gcatgacgcgggtcaaaggctaaaattgctttgcaacagaacgtaatgcgggacaggtgg tcactctacctctgctgaaaaggttgccgacacctgtcgtcattgttggcgccacagtgt ctgctcggctctttaacaatgtctcaaggcgcttggcagctacagacggtcagtgcattt ttgcatgatgactgacgaagtgaactggtcttagttctgtagaattctgtaacattgtta aagggctgacctgacactcaacctgcagtcCTCATGGTTTAAGTATAATGTCCATAGATA AAACTCGTAAGACTTTGTGATCATAAGAAGAGTCGGTGATTACACTGGATGACTGGCACG CTATTCGAAAAGAAATCAAAGAAAAGAACCAAAATCCAGATGGGGTGTTTAGTGGGGGAC AAACCTCCCATTGATCTCTGAACATTGTCGCAAATGCATCGCTTTACCGATGTCTTCAAA M T A M TAGTACGAGCAGACGCAAAGACGCACAAATTTGCGCACGTCCTCTGACAAATGACAGCCA Q R D V H G S L L M V P A E G D C F D V TGCAAAGAGACGTCCACGGTTCCCTACTGATGGTCCCTGCTGAAGGAGACTGTTTCGACG W R D Y M D L S R L L Q G L R V R N Q V TGTGGCGTGACTACATGGACCTGAGCAGACTGCTGCAGGGCCTGCGCGTCAGGAACCAGG E R T D G D G P K Q E E L M S P S P F S TGGAGCGCACGGACGGTGATGGACCGAAACAAGAAGAACTGATGTCACCATCACCTTTTT S L W S P I R A P S P S P T S P H P P P CATCCCTGTGGAGTCCCATCCGAGCTCCGTCACCTTCTCCAACTTCACCACATCCTCCTC P P P S F F Q T E Y N K H S E G T S S S CTCCTCCTCCTTCATTTTTTCAGACCGAATACAACAAACACTCTGAAGGCACCAGTTCAT D N L S D H S Y S A S S D Y C R F C K H CCGACAACCTGTCTGATCACAGCTACAGCGCCTCCTCCGACTACTGTCGCTTCTGCAAAC N R E S P N V Y M S H R L K G M D G R V ACAACCGCGAGTCTCCAAACGTTTACATGTCTCACAGATTGAAGGGCATGGACGGCAGAG T C P I L R K Y T C V I C G A S G D Y A TCACCTGTCCCATCCTGAGGAAGTACACCTGTGTCATCTGTGGAGCCAGCGGCGATTACG H T R R Y C P R T Q R R G A K A V T S K CACACACACGCCGCTACTGTCCACGGACGCAGCGGCGAGGGGCTAAGGCTGTGACGTCAA F S F * AGTTCAGTTTTTAGTAGATGGATGTTGATGATTTACCAATGTGTTTACATGATTAAGTTC GACATGTAGTTCTGCAAGTCATGTTTGTGACTGCCGTTTGAAAATGTCATTCGTTTTAAG GAGTTCTAACTGTATATATATATATTTGACCAAAACAAAAATAAAACACGAGTTAAGTCA AAGTTTTTTTTATCTATTCTCGTTTTATTGTAACAAAAAGAGCTGTTTTAGAAATGTCTT CTATTAGTTATATGCTGTATTCTTTAAAACGAACGTTAGCTTTGGTTAACAAATAACGAT AAAATACTTATTCTTTCACAGTTAACAACAAAAGGCAGGTTTACATTTTTTCCAAGAAAT GGCATAAAATTGATGATAACAAAGCATATAAAAAAAAAAAAAAAAAAAAAAAAAAA

AC

1 61 121 181 241 301 361 421 481 541 601 661 1 721 5 781 25 841 45 901 65 961 85 1021 105 1081 125 1141 145 1201 165 1261 185 1321 1381 1441 1501 1561 1621 1681

Fig. 1 Genomic sequence and predicted amino acid sequence of Csnanos. Exons and introns are indicated in uppercase and lowercase, respectively. The 18 amino acid N-terminal domain and the 53 amino acid CCHC zinc-finger domains are shaded in gray and the conserved histidine and cysteine residues are marked in yellow. Initiation codon, termination codon and poly-A tail are shaded in bold. The stop codon is indicated by an asterisk. 29

ACCEPTED MANUSCRIPT

96

DrNanos3 OlNanos3 HsNanos3

100

Nanos 3

MmNanos3

85

OmNanos2

PT

DrNanos2

CsNanos

Nanos 2

RI

TrNanos2

82

DlNanos2

OlNanos2

DrNanos1

NU

89

SC

OnNanos2

OlNanos1b

OlNanos1a

MA

100

MmNanos1 HsNanos1

D

0.05

Nanos 1

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Fig. 2 Phylogenetic analysis of Nanos orthologs using the neighbor-joining method. Bootstrap values of 1000 replicates (> 80%) are indicated next to the branches. The

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sequences used for alignment and phylogenetic analysis are as follows: Drosophila melanogaster (DmNanos, AAA28715); Homo sapiens (HsNanos1, NP_955631;

AC

HsNanos2, NP_001025032; HsNanos3, NP_001092092); Mus musculus (MmNanos1, NP_848508; MmNanos2, NP_918953; MmNanos3, NP_918948); Danio rerio (DrNanos1, AAL15474; DrNanos2, DAA64468; DrNanos3, NP_571953); Oryzias latipes (OlNanos1a, NP_001116380; OlNanos1b, NP_001153941; OlNanos2, NP_001153919; OlNanos3, NP_001116300); Cynoglossus semilaevis (CsNanos); Oreochromis niloticus (OnNanos, XP_005448912); Takifugu rubripes (TrNanos, XP_003968060); Dicentrarchus labrax (DlNanos, CBN81978).

30

Identity 61.1 % 61.1 % 55.6 % 59.3 % 55.6 % 72.2 % 70.4 % 66.7 % 72.2 % 59.3 % 63.0 % 100 % 63.0 % 64.8 % 61.1 % 61.1 % 57.4 % 59.3 % 53.7 %

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 3 Alignment of the amino acid sequences of the Nanos zinc-finger domain.

MA

Identical residues are in the same color, the arrowheads indicate the two conserved CCHC zinc-finger motifs. Homologues of the Nanos are arranged into three clusters:

D

Nanos1 (above the blue line), Nanos2 (between the blue and red line) and Nanos3

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(below the red line). The amino acid identities of CsNanos with other Nanos are shown on the right. Cs (Cynoglossus semilaevis), On (Oreochromis niloticus), Ol

CE

(Oryzias latipes), Mm (Mus musculus), Hs (Homo sapiens), Tr (Takifugu rubripes), Dl

AC

(Dicentrarchus labrax), Dm (Drosophila melanogaster), Dr (Danio rerio).

31

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 4 The relative expression levels of Csnanos in adult tissues. The data represent

D

the mean gene expression levels normalized by β-actin expression ± the standard

PT E

error of mean (SEM) from three separate individuals performed in triplicate. Different letters indicated statistical significance (p < 0.05), which were calculated by one-way

AC

CE

ANOVA followed by Duncan′s multiple range tests.

32

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 5 Expression pattern of Csnanos during embryosgenesis. The data were shown as

PT E

D

mean ± SEM (n = 3). Each sample consisted of a pool of 30 embryos, a pool of 30

AC

CE

larvae (1 dph). Different letters indicated statistical significance (p < 0.05).

33

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 6 The relative expression of Csnanos mRNA in the gonads of neomales, normal

AC

CE

PT E

D

statistical significance (p < 0.05).

MA

males and females. Data represent the mean ± SEM (n = 3). Different letters indicated

34

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

D

Fig. 7 The expression profile of Csnanos at different developmental stages of the

PT E

gonads. The data represent mean ± SEM (n = 3). Each sample consisted of a pool of 6 body trunks (4–66 dph), a pool of 3 gonads (80–150 dph). Different letters indicated

CE

statistical significance (p < 0.05), which were calculated by two-way ANOVA

AC

followed by Duncan′s multiple range tests. Statistical significance between sexes (*P < 0.05; **P < 0.01) were shown as asterisks at each sampling time after a Student’s t-test.

35

ACCEPTED MANUSCRIPT

White light A

Fluorescence light

White light

A1

A

C B

C11 B

T P

Stage 29

Stage 17 C

I R

C S U

N A

C1

B

Fluorescence light

B1

D D

D D11

Stage 30

Stage 25

D E

M

T P E

C C

A

Fig. 8 Visualization GFP-labeled PGCs in medaka embryos by microinjection of GFP-Csnanos 3'UTR mRNA. PGCs are denoted by white arrows. (A–D) embryos of stage 17, stage 25, stage 29, and stage30 were observed under light and fluorescence microscopy.

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Abbreviations list

T P

PGC, primordial germ cell; CCHC, Cys–Cys–His–Cys; UTR, untranslated region; GFP, green fluorescent protein; dph, days post-hatching;

I R

RACE, rapid amplification of cDNA ends; qRT-PCR, quantitative real-time PCR.

C S U

N A

D E

M

T P E

C C

A

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Highlights

T P

I R

• We cloned and characterized the cDNA sequence, genomic sequence and flanking sequence of nanos gene from Cynoglossus

C S U

semilaevis.

• Sexually dimorphic expression of nanos was identified during early development and sex differentiation.

N A

• Nanos were expressed at higher levels in normal females and males than in neomals.

M

• The chimeric GFP-nanos 3'UTR mRNA could be used to label PGCs.

D E

T P E

C C

A

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