LIANG Shaoshuai, MA Xiaoshi, HAN Tiantian, YANG Dandan, and ZHANG Zhifeng

Key Laboratory of Marine Genetics and Breeding of Ministry of Education,College of Marine Life Sciences,Ocean University of China,Qingdao266003,P. R. China

Expression Pattern of Chlamys farreri sox2 in Eggs, Embryos and Larvae of Various Stages

LIANG Shaoshuai, MA Xiaoshi, HAN Tiantian, YANG Dandan, and ZHANG Zhifeng*

Key Laboratory of Marine Genetics and Breeding of Ministry of Education,College of Marine Life Sciences,Ocean University of China,Qingdao266003,P. R. China

The SOX2 protein is an important transcription factor functioning during the early development of animals. In this study, we isolated a full-length cDNA sequence of scallopChlamys farreri sox2,Cf-sox2which was 2194 bp in length with a 981 bp open reading frame encoding 327 amino acids. With real-time PCR analysis, it was detected thatCf-sox2was expressed in unfertilized oocytes, fertilized eggs and all the tested embryos and larvae. The expression level increased significantly (P ＜0.01) in embryos from 2-cell to blastula, and then decreased significantly (P ＜0.01) and reached the minimum in umbo larva. Moreover, location of theCfsox2expression was revealed using whole mountin situhybridization technique. Positive hybridization signal could be detected in the central region of unfertilized oocytes and fertilized eggs, and then strong signals dispersed throughout the embryos from 2-cell to gastrula. During larval development, the signals were concentrated and strong signals were restricted to 4 regions of viscera mass in veliger larva. In umbo larva, weak signals could be detected in regions where presumptive visceral and pedal ganglia may be formed. The expression pattern ofCf-sox2during embryogenesis was similar to that of mammalsox2, which implied that Cf-SOX2 may participate in the regulation of early development ofC. farreri.

Sox2; early development; development of nervous system;Chlamys farreri

1 Introduction

The SOX (SRY-related HMG-box) protein, as a transcription factor, was first identified in mammals (Gubbayet al., 1990). Till present, more than 30 members of SOX family have been identified in animals, which generally contain a conserved HMG (high-mobility group) domain of 79 amino acids and play several roles such as cell pluripotency (Avilionet al., 2003), sex determination (Bishopet al., 2000; Chaboissieret al., 2004; Koopman, 2005), and neurogenesis (Bylundet al., 2003; Grahamet al., 2003; Sandberget al., 2005). SOX2 belongs to the SOXB1 subgroup of SOX family, containing a polyglycine domain in N-terminal region, a Ser-rich domain in C-terminal region, and a group B motif behind HMG-box (Bowleset al., 2000).

A full-length cDNA clone ofsox2was first isolated from the fetal brain cDNA library of human being (Homo sapiens) (Stevanovicet al., 1994), and then it was identified and studied in other animals such as mouse (Mus musculus) (Collignonet al., 1996; Yuanet al., 1995), ovine (Ovis aries) (Payenet al., 1997) and chicken (Gallus gallus) (Uwanoghoet al., 1995). It has been reportedthatsox2may play roles in embryogenesis and neurogenesis in vertebrates such asM. musculus(Avilionet al., 2003; Keramariet al., 2010; Pan and Schultz, 2011),G. gallus(Grahamet al., 2003; Rexet al., 1997) andXenopus laevis(Mizusekiet al., 1998). During early development ofM. musculus,sox2expresses in unfertilized oocytes, fertilized eggs and ICM (inner cell mass) of blastula (Keramariet al., 2010; Pan and Schultz, 2011), primitive (Avilionet al., 2003) and extraembryonic ectoderms (Collignonet al., 1996), as well as uncommitted stem cells and precursor cells of developing central nervous system (CNS) (Liet al., 1998; Zapponeet al., 2000). Furthermore, Avilionet al. (2003) found thatM.musculusembryos withsox2-null homozygous are normal until blastocyst stage before embryo implantation, but begin to die shortly after embryo implantation. Such pattern revealed thatMm-sox2is essential for the embryonic development. Moreover,sox2also expresses in the anterior region of presumptive neuroectoderm, neural tube and throughout nervous system (Avilionet al., 2003). Deletion of a neural cell-specific enhancer sequence ofsox2affects the proliferation of neural precursor cells and the generation of neurons in adult mouse neurogenic region, suggesting that SOX2 plays an important role inM.musculusneural development (Ferriet al., 2004).

Currently, SOX2 is concerned specially due to its role in iPSCs (induced pluripotent stem cells). Takahashi andYamanaka (2006, 2007) found that iPSCs can be generated from mouse fibroblast by simultaneous introduction of four genes,oct3/4,sox2,c-mycandklf4. Human iPSCs from human fibroblast can also be generated through a similar approach (Takahashi and Yamanaka, 2007). Furthermore,sox2is approved participating iPSCs formation inH. sapiens. Yuet al. (2007) found thatsox2,oct4,nanogandlin28are sufficient to reprogramH. sapienssomatic cells into pluripotent stem cells, and the removal ofsox2from reprogramming combinations will eliminate the appearance of iPSCs.

Althoughsox2has been revealed to be essential during early development and plays a role in iPSCs in several vertebrates, its expression and role in invertebrates are not clear. The scallop (Chlamys farreri) is an important commercial marine bivalve in China. In this study, we cloned a full-length cDNA sequence ofsox2and profiled its expression inC. farreriembryos and larvae, aiming to reveal the expression pattern ofsox2inC. farreriduring early development and understand its function in bivalves.

2 Materials and Methods

2.1 Animal and Sampling

Healthy male and female scallop (mean shell height 6.08 cm ± 0.71 cm) were purchased from NanShan Market, Qingdao, China. After cleaned, the scallop were induced to release gametes by drying in shade and then stimulating with UV-irradiated seawater (21℃ ± 1℃). Artificial fertilization was conducted and fertilized eggs were placed in filtered, UV-irradiated seawater at 21℃ ± 1℃. Hatched trochophores were reared in aerated seawater, renewed twice a day. Unfertilized oocytes, fertilized eggs, embryos (2-cell, 4-cell, 8-cell, 16-cell, blastula, gastrula), and larvae (trochophore, veliger and umbo larva) were collected. One part were fixed in 4% paraformaldehyde (PFA) in 0.1 mol L-1phosphate buffer (pH 7.4) at 4℃ for 16 h, and then dehydrated in a series of methanol solutions (70%, 85%, 95%, 100%) and stored in 100% methanol at -20℃ forin situhybridization. The veliger larvae and umbo larvae were relaxed by MgCl2before fixation. Another part were frozen immediately in liquid nitrogen and stored at -80℃ for total RNA extraction.

2.2 Isolation of Full-Length cDNA

A target cDNA fragment of 755 bp was retrieved fromC. farreritranscriptome by comparing with several SOX2 protein sequences of other species in GenBank. Specific primers (Table 1), including sense primers S1-3’, S2-3’and reverse primers R1-5’ and R2-5’, were designed based on the fragment to conduct 3’ and 5’ RACE (rapid amplification of cDNA ends) ofsox2by SMARTTMRACE cDNA amplification kit (Clontech, Moutain View, USA) according to the manufacturer’s instructions. The nested-PCR was performed to get 5’ RACE fragment; the specific primer R1-5’ and primer UPM were employed for the first round PCR with cDNA of testis at proliferative phase as template; primer NUP and specific primer R2-5’for the second round PCR with the first round PCR product as template. For 3’ RACE, the nested-PCR was also performed： the primer S1-3’ and primer UPM were used for the first round with cDNA of testis at proliferative phase as template; and the primer S2-3’ and primer NUP with first round PCR product as template for the second round. PCR condition was as following： 94℃ 5 min, followed by 29 cycles of 94℃ 30 s, 68℃ 30 s, 72℃3 min, and 72℃ 10 min for a final extension.

The PCR products were gel-purified and inserted into pMD18-T simple vector (Takara Bio Inc., Otsu, Japan), then transformed intoE. coliDH5α competent cells. Positive clones were selected and sequenced. A full-length sequence was assembled using DNASTAR.

Table 1 Sequence of the primers used in experiment

2.3 Sequence Analysis

Similarity searches were performed with the BLAST program at NCBI (http：//www.ncbi.nlm.nih.gov/blast). Multiple alignments were analyzed using the CLUSTAL X software. A phylogenetic tree was constructed using MEGA-4.0 with 1000 bootstrap trials.

2.4 RNA Isolation and cDNA Synthesis

Total RNA was isolated with guanidine thiocyanate method. The RNA was subjected to DNase treatment with DNase I (Takara Bio. Co. Ltd., Dalian, China). Quality and quantity of RNA were assessed through agarose gel electrophoresis and spectrophotometry at 260 and 280 nm (NanoVue, GE Healthcare, Piscataway, NJ, USA). Firststrand cDNA was synthesized using Prime-Script RT reagent Kit (Takara Bio. Co. Ltd., Dalian, China) following manufacturer’s instructions. The reaction was performed at 37℃ for 3 h, which was terminated by heating at 85℃for 5 s. The cDNA mix was 10 times diluted and stored at -20℃ for subsequent quantitative real-time PCR.

2.5 Quantitative Real-Time PCR (qRT-PCR)

The qRT-PCR was carried out in a total volume of 20 μL using ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and SYBR Green Master Mix (Takara Bio. Co. Ltd., Dalian, China) follow-ing the manufactures’ instructions. Two special primers,sox2-F 5’-GAGGTGCTTGACTATTGGAGAC-3’ andsox2-R 5’-CCGAGAGACTGTTGTAACTGAG-3’ were designed based on the full-length sequence ofC. farreri sox2for amplifying a 285 bp fragment using the sample cDNA as template andef-1α(elongation factor 1 alpha) of scallop was used as a reference gene (Zhouet al., 2012). A melting curve analysis was performed to confirm that only one PCR product was amplified. Data were analyzed by ABI 7500 system SDS software version 1.4 (Applied Biosystems). The 2-ΔΔCtmethod was used to determine the mRNA abundance.

All data were presented as mean ± SEM (n= 3). Differences were tested using one-way analysis of variance followed by the least significant difference test (SPSS software version 18.0; SPSS Inc., Chicago, IL, USA) with a significant level set atP＜ 0.01.

2.6In situHybridization

2.6.1 Probe synthesis

A fragment of 555 bp was amplified using two specific primers, sense 5’-CCGGAATTCGCACATTTATTGGAA CATTC-3’ and antisense 5’-CCCAAGCTTCATGTTTCT TTTACATGTCC-3’, based on the full-length sequence ofC. farrerisox2cDNA. DIG-labeled RNA sense and antisense probes were synthesized using a DIG RNA Labeling Kit (SP6/T7, Roche) according to the instructions.

2.6.2 Whole mountin situhybridization

Whole mountin situhybridization was conducted as described with slight modifications (Fenget al., 2011). The samples were digested with protease K of 2 μg mL-1, and the umbo larvae were sonicated in PBST for 10 s before digested for 45 min. TheC. farreri sox2RNA probe was employed as the hybridization probe. Observation and digital images were taken with a Nikon E80i microscope.

3 Results

3.1 Sequence and Characterization ofCf-sox2

Fig.1 Nucleotide sequence ofChlamys farreri sox2cDNA and its deduced amino acid sequence. Start (ATG) and stop (TAA) codons are indicated with asterisks. The HMG-box is underlined. The group B motif is shaded. The DNA-binding sites are boxed. The polyadenylation signal AATAAA is double underlined.

Fig.2 Sequence comparison of SOX2 protein from different species. a, multiple alignment of the deduced amino acid sequence with other known SOX2s. The HMG-box has a single overline; the group B motif has a double overline; the Serrich regions and glycine repeats, which are characteristics of vertebrates, are boxed in solid and dotted line, respectively. b, phylogenetic relationships among SOX2 from all species. Numbers in the branches represent bootstrap values (percentage) with 1000 replicates. The scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per site.

Two fragments, 530 bp and 1950 bp respectively, wereisolated. The assembled full-length cDNA was 2194 bp (KF836755), which consisted of an open reading frame of 981 bp encoding a protein of 327 amino acids. The PI (isoelectric point) of the deduced protein was 9.71, and the molecular mass was 36 kD. A 259 bp 5’ untranslated region (UTR) and a 954 bp 3’UTR bounded the ORF (Fig.1).

Homology analysis revealed that the deduced amino acid sequence showed a high similarity with other known SOX2s. For example, it was 71% similar to that of oyster (Crassostrea gigas), 72% to that of pearl oyster (Pinctada fucata), 51% to that of chicken (G. gallus) and human (H. sapiens). Multiple alignment analysis indicated that the deduced amino acid sequence contained a conserved domain HMG-box and a group B motif which were presented in all eukaryote SOX2, while lacked poly-glycine domain in N-terminal region and Ser-rich domain in C-terminal region (Fig.2a). A phylogenetic tree based on amino acid alignment was constructed (Fig.2b), which indicated thatC. farreriSOX2 was clustered primarily with those ofC. gigasandP. fucata.

3.2 Temporal Expression ofCf-sox2During Early Development Stages

The abundance ofCf-sox2transcript increased significantly (P ＜0.01) from unfertilized oocytes, fertilized eggs to blastula, and reached the maximum in blastula. Then it decreased significantly (P ＜0.01) after blastula and touched the minimum in umbo larva (Fig.3). Before and after fertilization, a significant difference (P ＜0.01) ofCfsox2expression was found. The abundance was 0.82-fold higher in fertilized eggs than in unfertilized oocytes. During the cleavage, the expression difference was pronounced further, 2.57-fold higher in blastula than in 4-cells embryo. After that, the abundance ofCf-sox2transcript dropped significantly (P ＜0.01) to 55.3% (in gastrula), 21.0% (in trochophore), 14.5% (in veliger) and 4.2% (in umbo larva) of that in blastula, respectively.

Fig.3 Expression ofCf-sox2detected by qRT-PCR inChlamys farreriduring early development. The expression level of umbo larva is set as 1.00 to calibrate the relative levels in other embryos and larvae. Values are the mean ± SEM (n=3). Different letters (a-h) indicate statistically significant differences (P ＜0.01).

3.3 Location ofCf-sox2in Embryos and Larvae

TheCf-sox2was detected in all samples examined. However, its distribution mode was different. In the unfertilized oocytes and fertilized eggs, positive signals were presented in the central region (Figs.4A and 4B), and then became obvious gradually and filled throughout embryos until the gastrula (Figs.4C-4G). In trochophore, strong hybridization signals were restricted in 4 regions, and the distribution feature was maintained in the veliger larva, in which the signals were located in visceral mass (Fig.4I). Thereafter, most signals disappeared in the umbo larva. Only faint signals were detected around the location where the presumptive visceral ganglia and pedal ganglia may be formed (Fig.4J).

4 Discussion

In the present study, a full-length cDNA ofCf-sox2was cloned and characterized. The deduced amino acid sequence contained a highly conserved HMG-box of the SOX family and a group B motif of the SOXB subfamily. Nevertheless, the poly-glycine domain in N-terminal region and the Ser-rich domain in C-terminal region of vertebrate SOX2 (Bowleset al., 2000) were not found in Cf-SOX2 (Fig.2a), as in oyster Cg-SOX2 (EKC24855.1) and pearl oyster Pf-SOX2 (AGS18764.1). Therefore, we speculated that the absence of poly-glycine domain and Serrich domain may be a characteristic of SOX2 in mollusk or invertebrates. Comparatively, the Cf-SOX2 sequence in N-terminal region was more divergent than that in C-terminal region, which is similar with other known SOX2 in invertebrates, and is unlike SOX2 in vertebrates. In vertebrates, it has been proved that the C-terminal region was important for transactivation (Kamachiet al., 1998) and activation of thefgf4enhancer in mouse (Yuanet al., 1995). Thus, we deduced that the C-terminal region of Cf-SOX2 might also play a role in transactivation.

We found thatCf-sox2was expressed in unfertilized oocyte (Figs.3, 4A) although the expression level was significantly lower than that in fertilized egg and other embryos (Fig.3). This indicated that theCf-sox2transcript is maternally inherited inC. farreri. Similar expression characteristic ofsox2has been described inM. musculus(Keramariet al., 2010; Pan and Schultz, 2011). However, the maternal heredity ofsox2seems to be different among fishes. In zebrafish (Danio rerio) (Okudaet al., 2006) and goldfish (Carassius auratus) (Marandelet al., 2012), the transcript ofsox2is first detected in gastrula; while in medaka (Oryzias oryziaslatipes) it is first detected in blastula (Cuiet al., 2011).

Fig.4 Location ofCf-sox2in embryos and larvae of scallop. The positive signals are in blue or dark blue (A-J), whereas controls are not stained (A0-J0). A, unfertilized egg; B, fertilized oocyte; C, 2-cell embryo; D, 4-cell embryo; E, 8-cell embryo; F, blastula; G, gastrula; H, trochophore; I, veliger larva; J, umbo larva. Sclar bar, 10 μm.

During the cleavage, the expression level ofCf-sox2increased significantly from 4-cell embryo to 16-cell embryo, and peaked in blastula (Fig.3). Furthermore, theCfsox2signals dispersed and filled in the embryos before hatching. The expression characteristic inC. farreriis similar to that of theMm-sox2in mouse cleavage embryos, but different from that of other fish embryos, such asD. rerioandC. auratus(Marandelet al., 2012; Okudaet al., 2006). InM. musculus,sox2mRNA and protein are detectable from 2-cell to 8-cell, and then becomes abundant in blastocyst (Keramariet al., 2010; Pan and Schultz, 2011). However,sox2expression can not be detected in cleavage embryos and blastula inD. rerioandC. auratusuntil gastrula (Marandelet al., 2012; Okudaet al., 2006), or only weak expression can be detected inO. oryziaslatipesblastula (Cuiet al., 2011). It is interesting thatsox2transcript presents variant expression characteristic among these species, especially among fishes. The expression difference betweenC. farreri, amniotes and fish may be partly explained by the diversity of species. The reference genes used insox2relative expression level detection are different among those fishes. InO. oryziaslatipes,β-actinwas applied as the reference gene, whileluciferaseandef-1α were applied as reference genes inD. rerioandC. auratusrespectively (Cuiet al., 2011; Marandelet al., 2012; Okudaet al., 2006). Maybe the different reference genes arouse the distinction in the detection ofsox2relative expression level. Although,sox2initial expression during cleavage stages is different among these fishes, but in all three fishes,sox2is expressed consistently at 24 h and 48 h after fertilization. Based on the consistency ofsox2expression during the cleavage embryos between the scallopC.farreriand the mouseM. musculus, we deduced that theCf-sox2may participate in the regulation of embryogenesis inC. farreri.

Sox2plays an important role in the maintenance of cell pluripotency, CNS development in vertebrates. InM. musculus,sox2mRNA is present persistently in the ICM, the epiblast and extraembryonic ectoderm of blastocyst,and then it becomes restricted in chorion, the presumptive neuroectoderm in the anterior of mid-late-streak embryo, chorion, headfolds and neural tube at 8.5-dpc (days postcoitum). After that, it is located throughout the nervous system, sensory placodes, branchial arches and gut by 9.5-dpc (Avilionet al., 2003). InX. laevis,sox2is first detected in the presumptive dorsal side of gastrula, and then restricted to CNS through early development (Mizusekiet al., 1998). InD. rerio, the expression ofsox2is also restricted in the early neuroectoderm and is very low in the posterior CNS (Okudaet al., 2006). In this study, the distribution ofCf-sox2transcript became restricted from diffusion in the cleavage embryos and blastula to regional concentration in larvae (Figs.4H-4J). InC. farreritrochophore, the four regions of hybridization signals were observed in the top and middle parts of the larva. In veliger larva, the anti-sense hybridization signals were presented in 4 regions of visceral mass (Fig.4I). After that, in umbo larva, the positive signals restricted to the locations where the presumptive visceral ganglia and pedal ganglia may be formed, although the expression level ofCf-sox2was low. We found the distribution characteristic ofsox2inC. farreriduring early embryonic development is similar to that in vertebrates, implying Cf-SOX2 plays some roles during the early development, which is similar to that in vertebrates. However, the located regions ofCf-sox2were different in the scallopC. farrerifrom vertebrates. The difference may be caused by different morphological structures between the scallop and vertebrates.

In conclusion, we isolated a full-length cDNA, 2194 bp in length. Multiple alignment and phylogenetic analysis showed that it issox2of scallop (C. farreri). The expression pattern ofCf-sox2in unfertilized oocyte, fertilized egg, embryos and larvae suggested thatCf-sox2is maternally expressed, and may participate in the early development ofC. farreri. Further studies are necessary to clarify the exact function of Cf-SOX2, which can optimize the culture condition ofC. farreriembryosin vitro.

Acknowledgements

This work was supported by the National High Technology Research and Development Program of China (863 Program) (2012AA10A402).

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(Edited by Qiu Yantao)

(Received December 12, 2013; revised March 31, 2014; accepted May 21, 2015)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

* Corresponding author. Tel： 0086-532-82031647 E-mail： zzfp107@ouc.edu.cn