Spinyhead Croaker Germ Cells Gene dnd Visualizes Primordial Germ Cells in Medaka

Highlights Cldnd was specifically expressed in adult gonads. Cldnd RNA was enriched in spermatogonia and early oocytes and decreased with gametogenesis. Gfp-Cldnd 3′ UTR mRNA could identify primordial germ cells in medaka. Abstract Spinyhead croaker (Collichthys lucidus) is an economically important fish suffering from population decline caused by overfishing and habitat destruction. Researches on the development of primordial germ cell (PGC) and reproduction biology were an emergency for the long-term conservation of the involved species. Dead end (dnd) gene plays an indispensable role in PGC specification, maintenance, and development. In the current study, we report the cloning and expression patterns of dnd in C. lucidus (Cldnd). RT-PCR analysis revealed that Cldnd was specifically expressed in both sexual gonads. In the ovary, Cldnd RNA was uniformly distributed in the oocytes and abundant in oogonia, and gradually decreased with oogenesis. A similar expression pattern was also detected in testis. Dual fluorescent in situ hybridization of Cldnd and Clvasa demonstrated that they almost had the same distribution except in oocytes at stage I, in which the vasa RNA aggregated into some particles. Furthermore, Cldnd 3′ UTR was sufficient to guide the Green Fluorescent Protein (GFP) specifically and stably expressed in the PGCs of medaka. These findings offer insight into that Cldnd is an evolutionarily conserved germline-specific gene and even a potential candidate for PGC manipulation in C. lucidus.


Introduction
Primordial germ cells (PGCs) are the germline precursor which are specified from somatic cells and migrate into the genital ridges, ensuring population and intergenerational inheritance of genetic information. The specification, migration, proliferation, and differentiation of PGCs are precisely regulated by a sophisticated molecular network. As well, it has been demonstrated that a number of functional genes play critical roles in the development of PGCs [1,2]. Among these, dead end (dnd) gene attaches attention not only for its indispensable role in PGC development, but also for its successful application to prepare germ cell-deficient recipients for germ cell transplantation [3].
It is proven that dnd is essential for primordial germ cell (PGC) specification, maintenance, and development. In mice, dnd mutation causes germ cell loss or testicular germ cell tumors [5]. Dnd knockdown leads to abnormal migration and loss of PGCs in frogs [6] and zebrafish [4,15,16]. Especially in medaka, dnd is a specifier that can abolish or increase the PGC in a dosage-dependent manner [1]. Dnd is of great importance in the capability of the PGCs fate protection from somatic differentiation [16,17]. Dnd has been broadly used to produce PGC-deficiency individuals for the application of surrogate broodstock technology (SBT) in some teleosts, such as medaka [18], Atlantic cod (Gadus morhua) [19], olive flounder (Paralichthys olivaceus) [20], carp [21,22], grouper [11], salmon (Salmo salar) [23] and rainbow trout (Oncorhynchus mykiss) [24]. The SBT [18,[25][26][27] is recognized as a promising approach for preventing endangered species or expanding valuable fish species from suffering population decline. Furthermore, the fact that rescue of dnd crispant embryos enables inherited sterile offspring production benefits the actual application of sterile fish as an approach to avoid genetic introgression in the aquaculture industry [28].
Spinyhead croaker (Collichthys lucidus) is an economically important fish in the east coastal areas and estuaries of China. Due to its delicious taste and high nutritional value, it is popular among other fish species [29]. The market-oriented demand that ascends gradually pushes up the price of C. lucidus and leads to its population decline due to overfishing [30]. Therefore, large-scale artificial reproduction is imperative to compensate for the limited natural reproductive rate. Nevertheless, research on C. lucidus is mainly limited to morphology, immune-related gene identification, population genetics, genome assembly, and transcriptome assembly with few reports on sex determination [29,[31][32][33][34][35][36]. In the previous study, C. lucidus germ cells marker gene vasa was isolated by us [37]. In the present study, another gem cells gene dnd(Cldnd) was cloned, and ClDnd protein was highly conserved across species. Cldnd was only expressed in the germ cells of both sexual. Furthermore, Cldnd 3 UTR was sufficient to guide the Green Fluorescent Protein (GFP) specifically and stably expressed in the PGCs in medaka, suggesting that Cldnd is an evolutionarily conserved germline-specific gene and even a potential candidate for PGC manipulation in C. lucidus.

Fish and Sampling
C. lucidus were sampled from the East China Sea in Ningde, Fujian province, China. For the total RNA extraction, tissue samples from the kidney, gill, intestines, brain, heart, ovary and testis were immediately collected and flash-frozen in liquid nitrogen. Gonad tissues were fixed for the frozen section. The O. latipes was kept at 26 • C in a 14-h light/10-h dark cycle and its embryos were maintained and staged as described [38]. All experiments were conducted in strict accordance with the guidance of the Committee for Laboratory Animal Research at Shanghai Ocean University.

RNA Isolation and cDNA Synthesis
Total RNA was isolated from the adult tissues mentioned above by using TRIzol ® Reagent (Invitrogen, Carlsbad, CA, USA), and the integrity of the RNA was detected by 1% agarose gel electrophoresis with ethidium bromide (EB) staining. The first-strand cDNA was synthesized with 1 µg total RNA and oligo dT 18 primers following the manufacturer's Life 2022, 12, 1226 3 of 11 manual for the M-MLV reverse transcription kit (Takara, Shiga, Japan). Additionally, using BD SMART ® RACEs kit (Clontech, Beijing, China), two cDNA libraries from the ovary and testis were constructed for the full-length sequence amplification.

Molecular Cloning and Analysis of C. lucidus dnd Gene
To identify the full-length sequence of Cldnd, the classic RACEs approach was performed. Briefly, according to the conserved amino acid sequences (WEFRLMM and MAKKVLVE) of Dnd protein, a pair of degenerate primers were designed to explore the corresponding fragment of Cldnd ( Figure 1A). Subsequently, specific RACEs primers were designed based on the above fragment to amplify the 5 and 3 cDNA ends of Cldnd by using BD SMART ® RACEs kit (Clontech). All the primers used in the present research were listed in Table 1. Finally, the full-length mRNA sequence of Cldnd was assembled via DNAMAN and aligned to the Dnd protein across the examined species by Vector NTI Suite 11.0 (Invitrogen). Dnd proteins in vertebrates, from teleosts, amphibians to mammals, were achieved from NCBI (https://www.ncbi.nlm.nih.gov/protein) to investigate ClDnd conservatism in evolution. Afterwards, the phylogenetic tree was constructed by the neighbor-joining method based on Mega 7.0 package.

RT-PCR
To confirm the tissue distribution of Cldnd, a pair of gene-specific primers targeting to its coding sequence (CDS) together with β-actin as an internal control was designed and RT-PCR was carried out by using Ex-Taq ® (Takara, Beijing, China). PCR program was performed in a 25 µL reaction system for 28 cycles: denaturation at 95 • C for 20 s, annealing at 58 • C for 20 s, and extension at 72 • C for 1 min.

Cryosection and In Situ Hybridization
Spinyhead croaker gonads were fixed with 4% paraformaldehyde, and then were sliced into 6 µm. To investigate the subcellular location of Cldnd RNA, in situ hybridization (SISH) and dual fluorescence in situ hybridization (FISH), which could hybridize labeled probes with nucleic acids in cells or tissues, were performed as described [39,40]. Plasmid (pT-Cldnd) was constructed by inserting CDS of Cldnd into the pGEM-T vector. Then, pT-Cldnd was linearized for sense and antisense RNA probes from SP6 polymerase by using FITC RNA Labeling Kit (Roche, Basel, Switzerland). Clvasa probe labeled by digoxigenin (DIG) was prepared as described previously [37].

Microscopy
Observation and photography were carried out on Nikon SMZ25 and Ni-E microscope with Nikon Ds-Ri2 camera (Nikon, Tokyo, Japan) as described [39].

RT-PCR
To confirm the tissue distribution of Cldnd, a pair of gene-specific primers targeting to its coding sequence (CDS) together with β-actin as an internal control was designed and RT-PCR was carried out by using Ex-Taq ® (Takara, Beijing, China). PCR program was performed in a 25 μL reaction system for 28 cycles: denaturation at 95 °C for 20 s, annealing at 58 °C for 20 s, and extension at 72 °C for 1 min.

Cryosection and In Situ Hybridization
Spinyhead croaker gonads were fixed with 4% paraformaldehyde, and then were sliced into 6 μm. To investigate the subcellular location of Cldnd RNA, in situ hybridization (SISH) and dual fluorescence in situ hybridization (FISH), which could hybridize labeled probes with nucleic acids in cells or tissues, were performed as described [39,40]. Plasmid (pT-Cldnd) was constructed by inserting CDS of Cldnd into the pGEM-T vector. Then, pT-Cldnd was linearized for sense and antisense RNA probes from SP6 polymerase

Cloning and Characterization of Cldnd
By the approach combining degenerate PCR with RACEs, a 1377 bp Cldnd complementary (c) DNA (GenBank accession No. MK547285) in full-length containing a 32 bp 5 UTR, a 244 bp 3 UTR and a 1098 bp CDS that encodes 366 amino acids was obtained ( Figure S1). Multiple alignment analysis showed that ClDnd had a high identity with Dnd orthologs across species, ranging from 97.2% in large yellow croaker to 79.6% in medaka ( Figure 1A). In addition, by the blast to the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), it was found that the deduced ClDnd protein carried six conserved domains or motifs, including N-terminal region (NR), RNA recognition motif (RRM) and four C-terminal regions (CR1-4), which were typically present in Dnd protein (Figures 1A and S1).
The molecular phylogenetic tree demonstrated that Dnd proteins formed two distinct clades, which were the fish clade and the other vertebrates', and ClDnd was the closest with that of the large yellow croaker in the fish sub-clade ( Figure 1B).

Cldnd RNA Is Specifically Expressed in Germ Cells
As described above, dnd is a gene which is specifically expressed in germ cells [41]. The RT-PCR analysis of seven distinct organs in C. lucidus revealed that only in the testis and ovary, bands corresponding to Cldnd RNA were shown, but absent in other somatic tissues such as the brain, kidney, liver, gill, and intestines ( Figure 2A). Furthermore, SISH was carried out to investigate the subcellular localization of Cldnd RNA in the gonads. In the ovary, Cldnd signals were intensely expressed in oocyte I and were easily detected in oocyte II. As oogenesis progressed, the signals in stage III oocytes weakened and eventually vanished ( Figure 2B). In the testis, Cldnd RNA was abundant in spermatogonia, then reduced markedly in primary spermatocytes and was faint in secondary spermatocytes, and finally disappeared in spermatozoon and sperm ( Figures 2C and 3A). On the contrary, the sense probe was unable to detect any signal (data not shown). According to the findings, Cldnd RNA was only found in male and female germ cells.  In several species, including C. lucidus, vasa is referred to as the best-characterized germ cell marker [37,42]. In this study, Clvasa was selected to explore the Cldnd RNA expression patterns. By FISH, Cldnd and Clvasa RNA showed the same expression pattern in the testis ( Figure 3A-F). In the ovary, Cldnd RNA was as weak as Clvasa in oogonia, but it was uniformly distributed in the cytoplasm of oocyte I, while Clvasa aggregated into some particles ( Figure 4A-C). In several species, including C. lucidus, vasa is referred to as the best-characterized germ cell marker [37,42]. In this study, Clvasa was selected to explore the Cldnd RNA expression patterns. By FISH, Cldnd and Clvasa RNA showed the same expression pattern in the testis ( Figure 3A-F). In the ovary, Cldnd RNA was as weak as Clvasa in oogonia, but it was uniformly distributed in the cytoplasm of oocyte I, while Clvasa aggregated into some particles ( Figure 4A-C).  In several species, including C. lucidus, vasa is referred to as the best-characterized germ cell marker [37,42]. In this study, Clvasa was selected to explore the Cldnd RNA expression patterns. By FISH, Cldnd and Clvasa RNA showed the same expression pattern in the testis ( Figure 3A-F). In the ovary, Cldnd RNA was as weak as Clvasa in oogonia, but it was uniformly distributed in the cytoplasm of oocyte I, while Clvasa aggregated into some particles ( Figure 4A-C).

Cldnd 3′ UTR Enables GFP to Express in the PGCs of Medaka Stably
There is a potential mechanism that enables foreign proteins to be stably and specifically expressed in germ cells under the protection of the 3′ UTR that is isolated from some germplasm-specific genes, of which the 3′ UTR of nanos1 from zebrafish is the first candidate in fish, and has been widely verified and used for PGCs visualization in diverse spe-

Cldnd 3 UTR Enables GFP to Express in the PGCs of Medaka Stably
There is a potential mechanism that enables foreign proteins to be stably and specifically expressed in germ cells under the protection of the 3 UTR that is isolated from Life 2022, 12, 1226 7 of 11 some germplasm-specific genes, of which the 3 UTR of nanos1 from zebrafish is the first candidate in fish, and has been widely verified and used for PGCs visualization in diverse species [43]. In order to confirm whether Cldnd 3 UTR was responsible for the localization of mRNA and labeled PGCs, gfp-Cldnd 3 UTR mRNA was injected into fertilized medaka embryos with rfp-Drnos1 3 UTR mRNA which could effectively visualize PGCs [43]. At the early stage of embryogenesis, the GFP signals were distributed all over the cells (data not shown). Until the mid-gastrula stage, it turned into some intense particles and accumulated on the peripheral margin on both sides of the embryonic shield (body) (Figure 5B). At the somitogenesis stage, cells with positive GFP signal migrated dorsally aligned bilaterally along the trunk at the two sides of the embryonic body ( Figure 5F,J). Finally, these cells migrated along the embryo body to the genital ridge, the position where gonads formed under the interaction of germ cells and mesoderm ( Figure 5N). Meanwhile, the RFP signal ( Figure 5C,G,K,O) displayed a similar expression pattern as GFP, suggesting that gfp-Cldnd 3 UTR mRNA could label the PGCs of medaka ( Figure 5D,H,L,P). When observed at high magnification, most of the PGCs with GFP signal was positive for RFP expression as well ( Figure 5M -P ).

Discussion
In this study, we obtain full-length of Cldnd cDNA and analyze its RNA expression in adult gonads. In addition, SISH and dual-color FISH demonstrate that Cldnd RNA is exclusively expressed in the germ cells of both sexes and shows a similar expression pat-

Discussion
In this study, we obtain full-length of Cldnd cDNA and analyze its RNA expression in adult gonads. In addition, SISH and dual-color FISH demonstrate that Cldnd RNA is exclusively expressed in the germ cells of both sexes and shows a similar expression pattern with Clvasa. Surprisingly, medaka PGCs can be visualized by the injection of gfp-Cldnd 3 UTR mRNA and dnd is identified as the second germline marker gene in C. lucidus.
Dnd protein multiple alignment analysis manifests that ClDnd protein shows high identity to others, especially large yellow croaker. Moreover, ClDnd protein possesses the conserved motifs and domains (NR, RRM and CR1-4) similar to many examined fishes. RRM has been reported to be able to determine the subcellular localization of Dnd [44]. In zebrafish, the active site of Dnd protein exists in the C terminal domain which is required for the survival of PGCs [45]. Based on phylogenetic tree analysis of the Dnd protein, two main clades are found in vertebrates, and C. lucidus Dnd homologue is the closest to that of large yellow croaker in the fish sub-clade.
In most vertebrates, dnd homologues are expressed only in both ovary and testis, except frog dnd, which is specific to the ovary [6]. Notably, in some teleost fish, the expression levels of dnd are higher in the ovary than testis [46][47][48]. However, in adults, the results of RT-PCR demonstrate that Cldnd RNA is exclusive and displays a similar expression level in both sexual gonads, which is consistent with the expression pattern in medaka [8] and zebrafish [4].
Dnd RNA as a marker for germ cells of both sexes has been identified in many teleost fish. Based on our previous reports in C. lucidus, we have identified germ cells marker gene vasa which is significant for germ cell development during gamogenesis. In the present study, SISH was performed to reveal the subcellular localization of Cldnd RNA, whose phenomenon is in keeping with zebrafish [4], medaka [8], turbot [13] and carp [21,22], suggesting that the Cldnd RNA expression is germ cell-specific. Moreover, dual color FISH is used to compare precisely the RNA expression patterns between Cldnd and Clvasa. The results of co-localization show that there is almost no difference between Cldnd and Clvasa in the testis, which is in line with the expression pattern in summer flounder (Paralichthys dentatus) [49]. In the ovary, Cldnd RNA is always uniformly distributed in the cytoplasm of germ cells, which differs from the specific expression of Clvasa in oocytes of stage I. These results indicate that Cldnd plays an important role in germ cell development.
Numerous studies have reported that the fusion of the gfp/rfp reporter gene and 3 UTR of several germline-specific genes is very stable in PGCs but gradually degradative in somatic cells [50]. What more convincing explanation made for this biological characteristic is that the Dnd protein interacts with microRNA (miR-430) to protect gonadal-specific genes from degradation [1,13,51]. For example, hub 3 UTR contains four noncanonical binding sites of miR-430 which is also a target of global miRNA-mediate repression. Interestingly, the function of germ-plasm specific genes is conserved widely across fish species. For instance, 3 UTR of zebrafish nanos1 can not only visualize its own PGCs but most of the examined fish, such as medaka, Chinese sturgeon, and salmon [1,9,39,52]. In this study, gfp-Cldnd 3 UTR mRNA and rfp-Drnos1 3 UTR mRNA are co-injected into medaka embryos and show a similar expression pattern in the PGCs during embryogenesis, which means that gfp-Cldnd 3 UTR mRNA successfully marks medaka PGCs. Cldnd 3 UTR is found in existence of several noncanonical miR-430 binding sites (-UCGUGAAA-) which may be responsible for the characteristic that eliminates the gfp-Cldnd 3 UTR mRNA from somatic cells. Thus, Cldnd 3 UTR is conserved for PGCs specific visualization.
In summary, these results manifest that dnd is an evolutionarily conserved germ cells marker and plays a vital role in the reproductive development of C. lucidus. The sequence structure, expression pattern, and function of 3 UTR of Cldnd are highly similar to that in the examined species. These findings not only provide another germ cell maker but also benefit the functional and practical research on Cldnd and conservation of the C. lucidus population.