Characterization and expression of dax1 during embryonic and gonad development in the carp (Cyprinus carpio)

Fish stocks

Carp (C. carpio) were obtained from the Henan Academy of Fishery Science (Zhengzhou, Henan Province, China) and maintained at the Genetic Laboratory (Henan Normal University, Xinxiang Henan Province, China) in flow-through water tanks at 25±2°C under natural photoperiod for an initial acclimation period. Carp were fed Artemia nauplii twice a day for the duration of the experiment. No adult fish or larvae died during the experiment. Embryos were obtained by natural spawning and cultured in embryo medium following standard procedures. Staging of embryos was carried out according to Shaolian [14]. Carp embryos and larvae at 0, 5, 10, 15, 20, 25, 30, 40, 50, 65, and 80 days post hatching (dph) were sacrificed by briefly dipping them in ice-cold water as per animal ethics guidelines. As the gonad was impossible to isolate from larvae at 5 dph to 30 dph, the trunk region was sampled by removing most of the trunk muscle and head for total RNA isolation. As gonads are maintained in an undifferentiated state until 80 dph, they were isolated from 40 dph juvenile fish without separation of the sexes. After 80 dph, gonads and other tissues were selected from males and females separately. From 80 dph onwards, gonad was identified based upon gonadal histology. Such a strategy has been utilized previously for a similar ontogenic study in catfish [15]. Fifteen biological samples (n=15) were taken at each time point. Various tissues (ovary, testis, brain, kidney, liver, and heart) were collected from 80 dph larvae and from adult fish (n=5). The adult fish used for the tissue distribution analysis were sampled during the spawning phase in April of 2014. All tissue samples collected were snap frozen in liquid nitrogen and stored briefly at −80°C until use.

Preparation of total RNA

A total RNA extraction kit from RNAiso reagent (TaKaRa Bio, Shiga, Japan) was used to extract RNA. Approximately 20 mg of tissue were homogenized using the mortar containing liquid nitrogen. For the expression profile study, first-strand cDNA synthesis was carried out with PrimeScript reverse transcriptase (TaKaRa), using 1 μg of total RNA isolated from various stages of embryos, larvae, and different tissues.

Cloning the full-length cDNA of dax1

A 586 bp dax1 fragment was amplified from ovary by RT-PCR using degenerate primers (Table 1). Then, the rapid-amplification of cDNA ends (RACE) technique was used to obtain the 5′- and 3′-cDNA ends of dax1 using the SMART RACE Kit (TaKaRa) according to the manufacturer’s instructions and using the primers (Table 1) designed according to the cloned cDNA fragment sequence. Amplification conditions were: 95°C for 3 min, followed by 30 cycles at 94°C (30 s), 56°C (30 s) and 72°C (60 s), ending with an extension at 72°C for 10 min. PCR products were separated by electrophoresis on 1% agarose gels and visualized by scanning with a UV imaging system (Bio-Rad, Hercules, CA, USA).

Protein alignment and phylogenetic analysis

The resulting sequences were confirmed by the BLASTx program on the NCBI Blast Server (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). The deduced amino acid sequence of dax1 was aligned with those of other vertebrate members taken from GenBank, using ClustalW multiple alignment program software (http://www.ebi.ac.uk/Tools/clustalw2/index.html). A phylogenetic tree was constructed by the neighbor-joining algorithms in the MEGA6 program.

Fluorescent real-time quantitative reverse transcription (QRT)-PCR

Expression profiles of dax1 were investigated using a QRT-PCR assay on an ABI 7500 real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) with a SYBR green fluorescent label. Data were normalized to β-actin expression to account for differences among samples. The primers used are shown in Table 2. PCR amplification was performed as follows: 95°C for 10 s, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed using the 7500 system SDS Software v 1.4 (Applied Biosystems) and the 2^−ΔΔCt method was used to analyze the expression level of dax1. Negative controls were included in all runs to confirm the absence of DNA contamination.

In situ hybridization

Embryos were fixed overnight in 4% paraformaldehyde (PFA) at 4°C and stored in methanol at −20°C until use. Gonad specimens were dissected and pre-fixed for 20 h in 4% PFA at 4°C, placed in 30% sucrose in PBS overnight at 4°C, and finally embedded in Tek OCT (Leica, Wetzlar, Germany). Sections (20 μm thick) were obtained using a cryostat and collected on Superfrost plus slides. In situ hybridization (ISH) for dax1 was performed on carp embryos and gonad specimens using a DIG-labeled dax1 riboprobe. A dax1 cDNA fragment was obtained by RT-PCR (primers shown in Table 2) and was cloned into the Pspt18 vector (Roche, Basel, Switzerland). Digoxigenin-labeled RNA probes were synthesized from XbaI linearized dax1 cDNA using T7 RNA polymerase from an RNA labeling kit (Roche). The probes were detected with an alkaline phosphatase (AP)-conjugated anti-DIG antibody and visualized with BM Purple AP substrate (Roche, Basel, Switzerland). Specimens were photographed under Normaski optics on a Zeiss Axio microscope system (Carl Zeiss, Oberkochen, Germany).

Statistical analysis

Data were analyzed using SPSS version 13 and presented as mean±standard error. All parameters were tested for normality using the Shapiro-Wilk test. Statistical differences between groups were analyzed using one-way analysis of variance (ANOVA). Differences were considered statistically significant when p<0.05.

Molecular cloning of carp dax1

The full length dax1 cDNA obtained by RACE was 1228 bp long, with a 52 bp 5′-untranslated region (UTR), a 384 bp 3′-UTR, and a 792 bp open reading frame (ORF), which encoded a protein of 264 amino acid residues (Figure 1).

Figure 1:

Nucleotide and amino acid sequences of carp dax1 cDNA (GenBank Accession No. KF703999).

The translated amino acid sequence is shown in standard one-letter code below the nucleotide sequence.

Protein alignment and phylogenetic analysis

The amino acid sequence of the putative carp dax1 was compared with dax1 orthologues in zebrafish, tilapia, frog, alligator, chick, mouse, and human (Figure 2). Multiple sequence alignment revealed that the deduced carp dax1 protein sequence shared the highest identity (94.5%) with that of Carassius auratus ssp. High sequence identities with dax1 in other teleosts, alligator, and chicken (>60%) were also found, but identities were lower when compared to mammals (<60%) (Table 3). In addition, the predicted carp dax1 contained only the second and fourth of the four LXXLL motifs found in mammals, although the core of the AF-2 carboxy-terminal ligand binding-like motif was conserved. Based on the alignment results, a phylogenetic tree of dax1 proteins in vertebrates was constructed, and this demonstrated that carp dax1 clusters with dax1s from other teleost fish. The teleost grouping was also separate from the other vertebrate classes, including mammals, birds, reptiles, and amphibians (Figure 3).

Figure 2:

Alignment of the amino acid sequences of carp dax1 with those of other vertebrates.

GenBank accession numbers are given in the legend of Figure 3. Clustalw2 was used to make this figure.

Figure 3:

Phylogenetic analysis of carp dax1 and other known dax1s in vertebrates.

The scale bar indicates 0.05 expected amino acid substitutions per site.

Tissue-specific expression of dax1 in juvenile (80 dph) and adult carp

The mRNA expression patterns of dax1 in tissues including the brain, kidney, liver, heart, and gonad of both juvenile (80 dph) and adult carp were determined using QRT-PCR analysis. In adult female carp, dax1 was expressed predominantly in ovary, liver, and brain. In adult males, dax1 mRNA levels were higher in testis and heart, and a sexually dimorphic pattern was also observed in these tissues, with expression levels being 11.5-fold and 6.32-fold higher than that in the ovary and heart of females, respectively. However, dax1 expression was higher in the liver of adult females when compared to adult males (Figure 4A).

Figure 4:

QRT-PCR analysis of dax1 from various tissues of carp.

(A), image of adult carp. (B), image of juvenile carp. G, gonad; H, heart; L, liver; K, kidney and B, brain. The values were calibrated with the internal control β-actin, *indicate statistically significant differences between the female and male (p<0.05), **indicate statistically significant differences between the female and male (p<0.01).

Expression of dax1 was highest in the liver, followed by the ovary, heart, brain, and kidney, of juvenile female carp. In juvenile male carp, the highest level of expression was observed in the liver and a relatively high level of expression was also found in the testis. The expression levels of dax1 in the gonad (p<0.05) and liver (p<0.01) of juvenile male carp were significantly higher than in females. A comparatively low level of expression was observed in the kidney of both sexes without any dimorphism (Figure 4B).

Expression of dax1 during developmental stages

To investigate dax1 gene expression during embryogenesis and gonadal differentiation, samples were collected from the blastocyst stage until 80 dph. Expression of dax1 was detected from the beginning of the blastula stage and there were no significant changes observed from the gastrula stage to 50 dph. Thereafter, expression of dax1 increased marginally over that at the gastrula stage, beginning at 65 dph, and it was significantly higher in the testis at 80 dph compared to the gastrula stage (p<0.05). However, the expression of dax1 in the ovary was stable at 80 dph, and was 9.8 times higher in the testis than that in the ovary at this time. Moreover, dax1 expression at the gastrula stage and at 65 dph were higher than at other stages, but these differences were not significant (Figure 5).

Figure 5:

The mRNA expressions of dax1 measured by QRT-PCR from blastocyst to 80 dph.

The values were calibrated with the internal control β-actin. Each value is expressed as mean±SEM (n=15 for each value). *indicate statistically significant differences (p<0.05).

In situ hybridization

To determine the cell types that expressed dax1 mRNA in gonads, expression of dax1 in tissue sections was analyzed by in situ hybridization. In oocytes, dax1 expression was detected in the cytoplasm. In the testis, dax1 mRNA was detected in the cytoplasm of germline and somatic cells, especially within spermatocytes (Figure 6). Whole-mount in situ hybridization revealed that dax1 was widely expressed in many embryonic stages (Figure 7), and strongly expressed in the rostral diencephalon between the eye fields at hatching. There were also bilateral dax1-expressing structures located in the dorsal part of the trunk, emerging as two clusters of cells on either side of the body midline. Later, at about 5 dph, de novo expression of dax1 in the hindbrain was observed. Another intriguing ISH result was that dax1 expression was detected in the liver at 5 dph (Figure 7).

Figure 6:

In situ hybridization of dax1 in gonad of carp.

(A), image of in situ hybridization of dax1 in ovary of juveniles; arrowheads indicate dax1 positive cytoplasm of provitellogenic oocytes. (B), dax1 in testis of juveniles; arrowheads indicate dax1 positive cytoplasm of germ cell, include spermatocyte. (C), dax1 in ovary of adult carp; arrowheads indicate dax1 positive cytoplasm of oocyte. (D), dax1 in testis of adult carp; arrowheads indicate dax1 positive cytoplasm of cytoplasm of germcell. SC, spermatocytes; SP, spermatid; SZ, spermatozoa; Y, yolks; N, nucleus; C, cytoplasm; PO, provitellogenic oocytes; VO, vitellogenic oocytes.

Figure 7:

Expression analysis of dax1 by whole-mount in situ hybridization. (A) blastula stage (2.5 hpf); (B) gastrulae stage (7 hpf); (C) neurula stage (13 hpf); (D) tailbud formed stage (24 hpf) lateral view, hybrid signals focused on nerviduct; (E) hatched larva stage (54 hpf), ventral view, arrowheads indicate the rostral diencephalon of carp; (F) hatched larva stage (54 hpf), dorsal view, arrow heads indicate dax1 positive two clusters of cells symmetric to the embryo body; (G) 5d post hatching, lateral view, L, liver; H, hindbrain. Arrow head shows obvious hybrid signals.