The dnd RNA Identifies Germ Cell Origin and Migration in Olive Flounder (Paralichthys olivaceus)

The present study obtained a germ cell-specific marker dead end (dnd) in olive flounder (Paralichthys olivaceus) named Podnd. The tissue-specific expressions of Podnd transcripts were present in testis and ovary but were not detectable in other somatic tissues detected. SISH showed that Podnd expressed only in germ cells at different developmental stages but not in surrounding somatic cells. The expression of Podnd during embryonic development at 16 different stages revealed that the relative expression of Podnd transcript fluctuated at a high level in the cleavage stages, gradually decreased through subsequent development, and reached the lowest at late gastrula stage till it was nearly undetectable. The Podnd transcripts localization and migration were similar to zebrafish. Further research on the specification migration mechanism of PGCs and the role of germ cell during gonadal development in olive flounder would improve our understanding of germline development.


Introduction
Primordial germ cells (PGCs) are a small population of cells segregated from other cell lineages during embryogenesis. Germline development originates from the formation of PGCs, which migrate from the position where they are specified toward the presumptive genital ridge [1]. Germ cells whose unique role is to transmit genetic information to the next generations can only be detected after gastrula stage by histological methods, until germ cell-specific marker emerged, expressed specifically in PGCs, such as dead end (dnd) [2], vasa [3], and nanos [4]. The PGCs development has been attracting more attention, as the specific markers were applied to track PGCs origination and migration during embryogenesis.
Dead end (dnd) is a vertebrate germ cell-specific marker that was first identified as a gene encoding an RNA-binding protein essential for zebrafish PGC migration and survival [2]. Similarly, knockdown of the dnd gene in mouse, Xenopus, also caused PGCs loss and abnormal migration. The dnd mRNA expression showed sex-specific, such as femalespecific, expression in Xenopus [5] and bisexual expression in chicken [6], medaka [7], and turbot [8][9][10]. In addition, the development of PGCs and their role in normal gonadal development and differentiation are important in mouse, chicken [6], and fishes [11][12][13]. The different expression pattern of dnd between male and female among organisms may suggest its diverse function during evolution.
It has been reported that germplasm specification from cleavage stage to presumptive genital ridge region is different among teleost fishes. In zebrafish, at cleavage stage, the vasa, dnd mRNA was localized to the edge of cleavage furrows [14,15], while, in medaka, the vasa mRNA localization function was lost [16]. Furthermore, the migration path of PGCs toward the genital ridge appeared to be varied by the position specified for teleost [17]. In zebrafish [18], PGCs actively 2 BioMed Research International migrated by attraction towards an intermediate target and then moved posterior-ward along the border of the trunk mesoderm to settle at the junction of the yolk extension, whereas, in ukigori [19], PGCs migrated in the dorsal direction and from both sides seemed to be mingled on the endoderm and were redistributed to both sides of the gut. Recently, as artificial breeding technology breakthrough, research on germ cell development and differentiation in marine fishes has received increasing research focus.
Olive flounder (Paralichthys olivaceus) is an economically important marine flatfish species for aquaculture and females grow faster than males [20]; however, limited information on germ cell origination, migration, and differentiation has been reported. So, germ cell markers are needed to identify and to research on germline development for sex control. The aims of this study were (1) to isolate and identify a germ cell-specific marker dnd-like gene (Podnd) in olive flounder; (2) to investigate its tissue-specific and embryonic expression levels by real-time quantitative PCR (qRT-PCR); (3) to detect Podnd expression pattern in gonadal germ cells for both sexes; and (4) to trace the germ cell lineage origination and migration during embryogenesis by whole mount in situ hybridization (WISH).

Fish and Embryo Collection.
Mature olive flounder used in this study was obtained from Oriental Ocean Sci-Tech Co., Ltd. (Shandong Province, China). Tissue samples from ten fish of 42.50 cm-45.23 cm total length and 1.23 kg-1.54 kg body weight (mean ± standard deviation) were rapidly excised after anesthesia with 0.05% solution of ethyl 3-aminobenzoate methanesulfonate (Sigma-Aldrich, Shanghai, China). The tissue samples included heart, brain, liver, gut, stomach, kidney, spleen, muscle, gill, testis, and ovary. Fertilized eggs were obtained by artificial insemination and cultured at 16 ∘ C ± 0.5 ∘ C in fresh seawater at Shenghang Sci-Tech Co., Ltd. (Weihai, Shandong Province, China).
Tissue and embryos samples were stored in liquid nitrogen for RNA extraction. For WISH, embryos were fixed in 4% paraformaldehyde (Sigma-Aldrich) in PBS overnight and stored at −20 ∘ C in PBS with 50% formamide (Sangon, Shanghai, China). For section in situ hybridization (SISH), ovary samples were fixed in 4% paraformaldehyde in PBS overnight and embedded in paraffin after dehydration in a series of ethanol baths and clearance by xylene.

cDNA Cloning of Olive Flounder dnd (Podnd) Homologue.
Total RNA was extracted from the ovary of olive flounder using RNA fast 200 (Fastagen Biotech, Shanghai, China), according to the manufacturer's instructions, and all total RNA was dissolved in 25 L RNase-free water. The first-strand cDNAs were synthesized using a TransScript one-step gDNA removal and cDNA synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer's instructions. The cDNA fragment of the Podnd gene was amplified by RT-PCR with primers (Podnd F1 and Podnd R1) designed according to the highly conserved regions of dnd homologues Subsequently, 5 and 3 rapid amplification of cDNA ends (RACE) were performed to clone the full-length coding sequence with a SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) according to the manual. The gene-specific primers for 5 -RACE (Podnd-5RACE-1 and Podnd-5RACE-2) and 3 -RACE (Podnd-3RACE-1 and Podnd-3RACE-2) were also showed in Table 1. PCR was performed using denaturation at 94 ∘ C for 5 minutes; 35 cycles of amplification at 94 ∘ C for 30 seconds, 62 ∘ C for 45 seconds, 72 ∘ C for 2 minutes; and an additional elongation at 72 ∘ C for 5 minutes. Each PCR product was separated by electrophoresis on 1% agarose gel. The fragments were purified and subcloned into pMD TM 18-T Vector (TaKaRa, Dalian, China) for sequencing. The positive clones were obtained by antibiotic selection and the inserts were sequenced at Sangon (Shanghai, China). Sequences were analysed by the Lasergene software package (DNAStar, Madison, USA) and examined against the GenBank database.

Phylogenetic Analysis.
A homology search of the nucleotide and deduced amino acid sequences of Podnd were confirmed using the National Center for Biotechnology Information Website (http://www.ncbi.nlm.nih.gov/). The deduced amino acid sequences were aligned using the AlignX program in Vector NTI suite 11.5 software package. The phylogenetic tree was constructed by Mega 6 software with neighborjoining method. The branching reliability was tested via bootstrap analysis of 1000 replicates.

In Situ Hybridization and Histology. Localization of
Podnd mRNA was analyzed by WISH and paraffin sections of ovary, as previously described by Lin et al. [21]. Sense and antisense Podnd probes were individually synthesized using the DIG RNA Labeling Kit (SP6/T7) (Roche, Mannheim, Germany) following the manufacture's instruction. The 724 bp dnd PCR product (529-1253) was subcloned into pGEM-T Easy Vector (Promega) and transcribed in vitro to obtain the probes. Paraffin sections of 1-day larva hybridized with Podnd antisense probe were applied to identify the precise location of PGCs. Meanwhile, for SISH, paraffin sections (7 m) were cut, dewaxed by xylene, and rehydrated through an ethanol series. Before hybridization, sections were washed with PBST, refixed with 4% paraformaldehyde, and permeabilized with 0.2 M HCl, 10 g/mL proteinase K (Sangon). The other procedures for washes and detection of DIG were similar to the WISH.
For histology, the ovaries were fixed in Bouin's fixative for 24 h, dehydrated in an ascending ethanol series, cleared with xylene, and embedded in paraffin, according to standard paraffin-embedding methods. Tissues were cut into 7 m thick sections and stained with hematoxylin and eosin (H&E). For WISH, SISH, and histology, the photographs were taken using a Nikon E50i microscope with Nikon DS-Fi imaging system.

Real-Time PCR.
Total RNA was extracted from embryos and adult tissues including heart, brain, liver, gut, stomach, kidney, spleen, muscle, gill, testis, and ovary. All assays were carried out in triplicate for each stage of embryo independently. The number of embryos in each triplicate was about 50. The biological replicates and technical replicates for each tissue were three independently. The expressions of Podnd in different tissues and embryonic samples were analyzed by qRT-PCR using SYBR Green detection method in an ABI 7300 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA).
The results were analyzed by comparative Ct method. Podnd expression was normalized against -actin, generating a ΔCt (Ct(Podnd) − Ct( -actin)). According to the equation, 2 −ΔΔCt , relative expression was calculated. During tissuespecific expression, gut was taken as a calibrator and during embryogenesis 16-cell stage was taken as a calibrator.

Statistical Analysis.
Statistical analysis was carried out using the software package SPSS 19.0 for Windows (SPSS Inc., Chicago, IL, USA), and data were expressed as mean ± SD ( < 0.05). qRT-PCR data was statistically analyzed by oneway ANOVA followed by a Tukey test, respectively. All assays were carried out in triplicate for each stage independently and < 0.05 denoted statistically significant difference.

Molecular Characterization of Podnd.
A partial cDNA fragment (176 bp) of dnd was isolated from Paralichthys olivaceus ovary. The full sequence of Podnd was obtained successfully by using 3 -and 5 -RACE. The full length of Podnd cDNA is 1446 bp, containing a 1178 bp open reading frame. It encodes a putative protein of 392 amino acids, an 18 bp 5 -untranslated region (UTR), and a 249 bp 3 -UTR. The nucleotide sequence of Podnd had been submitted to Gen-Bank (accession number: KP224455). The deduced amino acid sequences were aligned using the AlignX program in Vector NTI suite 11.5 software package (Figure 1(a)).

Phylogenetic Analysis.
A phylogenetic tree was constructed with full-length DND sequences by Mega 6 software, using neighbor-joining method with a bootstrap analysis of 1000 replicates ( Figure 1(b)). The deduced DND protein sequence was aligned with related protein sequence of other typical vertebrates (Figure 1(c)).

Podnd RNA Expression in Different Tissues and during
Embryogenesis by qRT-PCR. Podnd mRNA expression levels in different tissues including heart, brain, liver, intestine, kidney, spleen, muscle, gill, ovary, and testis of mature olive flounder by qRT-PCR were performed. Total RNA was extracted from tissues of healthy adults, each sample of different developmental stages during embryogenesis, and reversely transcribed. The results showed that tissue-specific expressions of Podnd transcripts were present in testis and ovary but were not detectable in other somatic tissues detected (Figure 2(a)). In addition, the expression of Podnd transcripts was much higher in the ovary compared with that in the testis. The expression of Podnd during embryonic development at 16 different stages revealed that the relative expression of Podnd transcript fluctuated at a high level in the cleavage stages (Figure 2(b)). The highest level presented at the 2-cell stage, gradually decreased through subsequent development, and reached the lowest at late gastrula stage till it was nearly undetectable.

Localization of Podnd RNA in Gonad by Tissue Section In
Situ Hybridization. Tissue section in situ hybridization was carried out to investigate the Podnd expression pattern of adult gonad. SISH showed that Podnd expressed only in germ cells at different developmental stages but not in surrounding somatic cells. In ovarian sections, the strongest signal was present in certain oogonia and oocytes at stage II (diffused in the ooplasm) whereas it was difficult to discern in oocytes at stage V (Figure 3(b)). In testis, obvious signal was present in spermatogonium, weaker in spermatocyte, and undetectable in spermatid and spermatozoon (Figure 3(d)). 1 120     (Figures 4(g1), 4(g2), and 5(g)), the Podnd-positive cells increased from one to two and the sister cells were located adjacent to each other at every positive area and then formed PGCs.
At the heart beating stage (Figures 4(m) and 5(l)), the Podnd-positive cells formed clusters above the hind gut in the presumptive genital ridge. Subsequently, at the hatching period (Figures 4(n), 4(c), and 5(m)), the dnd-positive cells formed two relatively compact clusters under the genital ridge, which were above the posterior end of the hind gut and below the somites on either side of the embryonic body. Then 1 day after hatching (Figure 4(o1)) and 2 days after hatching ( Figure 4(p)), the positive cells still aligned at the genital ridge (Figures 4(o2), 4(o4), and 5(n)), at the dorsal sides of the hind gut, ventral side of notochord.

Discussion
In the present study, we isolated and characterized a dnd homolog gene Podnd. The Podnd contains RNA recognition motif (RRM) and five conserved regions (NR, CR1-4), with high identity to other DND proteins. N-terminal RNA recognition motive (RRM) and C-terminal ATPase activity within DND mRNA were found very important in PGC development and survival [22,23]. Phylogenetic trees based on DND amino acid sequences showed that all fish species formed a distinct group separated from those of other vertebrates, and a significant homology was obtained with another flatfish species turbot.
The tissue specificity and SISH revealed that expression of Podnd transcripts was exclusively restricted in oogenetic and spermatogenetic but not in somatic cells. The expression level of ovary was much higher than that of testis. The difference in expression between female and male suggested that Podnd may play different role in oogenesis and spermatogenesis. In the ovary, Podnd expression showed dynamic localization pattern. The signal diffused evenly in the ooplasm in oogonia, stage I and III oocytes, with the strongest signal in stage II, and can not be detected in stages IV and V. The signal should be still there in stage IV and V oocytes, since it is present at the 2-cell stage unless the zygotic genome is already activated at the 2-cell stage. Quantitative RT-PCR of sorted oocytes will allow the transcript abundance to be determined whether the oocytes are too opaque/yolky or the signal is too "diluted" in the large oocyte volume to be visualized in situ. These analyses suggest that the olive flounder, like zebrafish, may use a maternal mode of germ cell specification.
Podnd RNA expression by qRT-PCR during embryogenesis revealed that the strongest expression of Podnd occurred at two-cell stage and then gradually decreased to the lowest at late gastrula stage till it was nearly undetectable. However, the number of PGCs signals increased with the embryo development. Similar expression pattern was also revealed in zebrafish [2] and medaka [7] by RT-PCR. These phenomena may be due to the somatic cell proliferation speed which is quicker than that of PGCs.
In this study, as revealed by WISH during cleavage stages, Podnd mRNA already presented at the two-cell stage and aggregated at both edges of the first, second, and third cleavage furrows at two-eight cell cleavage stages. It was maternal so it was transcribed in oocytes, maternally inherited, and then localized to the furrows. At blastula stage, the number of Podnd-positive cells increased from 4 to 8 and the sister cells were located adjacent to each other. Similar results were reported in zebrafish [2] and turbot [8] marked with dnd. Meanwhile, the Podnd mRNA location pattern also verified its maternal origination.
In olive flounder, the migration pattern of PGCs detected by WISH with Podnd mRNA was summarized in Figure 5. Generally, the PGCs movement was similar to that reported in zebrafish but was not identical. PGCs moved dorsally at early gastrula stage, then scattered in anterior of the trunk mesoderm, and appeared to align along anterior of the body axes from being randomly oriented. It seems that there are intermediate targets within the anterior of trunk mesoderm as reported in zebrafish [24]. Detailed information is required by visualizing the actual movement of PGCs around the anterior of trunk during gastrulation. In the final step, PGCs migrated posterior-ward accompanied with outward along body axes, then aggregated together, respectively, and finally settled at the genital ridge above the hind gut, which was along the inner layer of the lateral plate mesoderm. It appeared slightly in more posterior position than that in the zebrafish [18]. The majority of studies about germ cell marker localization during gametogenesis compared with embryogenesis are limited. Thus, comprehensive comparative studies between species are necessary in the future for better understanding of the properties of PGCs.

Conclusion
The present study identified dnd as a germ cell-specific marker and stage-specific expression pattern was observed during gametogenesis in olive flounder. It seems that the origin and migration of PGCs during embryonic development   are mainly conserved among teleosts. Further research on the specification migration mechanism of PGCs and the role of germ cell during gonadal development in olive flounder would improve our understanding of germline development.