Goldfish strains and maintenance

Wild-type and twin-tail strain goldfish (duplicated caudal fin Wakin, Ryukin, Oranda, Redcap Oranda, Black telescope, Red telescope, Perl scale and Ranchu) were purchased from an aquarium fish agency and breeder in Taiwan. To avoid confusion derived from differences between the goldfish nomenclature systems used by breeders and researchers6,10,31, this study defined goldfish individuals with a slender body and a single fin as wild type. The nomenclature of the twin-tail goldfish strains primarily followed that of Smartt6.

Anatomical and histological analyses of axial skeletons

Cleaned skeletal samples were prepared by fixing juvenile goldfish specimens in 10% formalin. The specimens were subsequently washed in 70% ethanol, stained first with alcian blue staining solution (0.1% alcian blue in 99.8% ethanol:acetic acid=4:1) and then with alizarin red solution (0.1% alizarin red in 95% ethanol), and finally cleared using ScaleA2 (ref. 32). For histological analysis, Bouin-fixed juvenile goldfish specimens were embedded in paraffin, sectioned to 5 μm using a slicer (RM2245, Leica), and stained with Alcian blue, hematoxylin and eosin. The cleaned skeletal and histological samples were examined using stereo and standard microscopes (SZX16 and BX43, Olympus). Caudal axial skeletal elements were identified and described on the basis of earlier reports1,2,3,4,7,9. The research followed internationally recognized guidelines. We received ethical approval from Institutional Animal Care & Utilization Committee, Academia Sinica.

Goldfish embryos

Sperm was extracted from multiple male goldfish and preserved in Modified Kurokura’s extender 2 solution at 4 °C33. Eggs were squeezed from mature female goldfish onto Teflon-coated dishes. Artificial fertilization was performed using dry methods. Fertilized eggs were placed in 9 cm Petri dishes or 3 l aquarium tanks containing tap water (22–24 °C). Serial ID numbers were provided for male and female individuals used for artificial fertilization. Petri dishes containing approximately 50–100 individuals were incubated at 24 °C until observation and harvesting. Embryonic stages were determined using the goldfish staging table before harvesting13. Gastrula, segmentation, pharyngular and hatching stages were determined on the basis of blastopore closure, somite number, otic vesicle closure and pectoral fin shape, respectively13.

Molecular cloning and sequencing

Homologues of chdA, chdB, eve1, sizzled, bmp4 and krox20 were isolated from wild-type goldfish embryo cDNA using PCR. Total RNA was extracted from wild-type gastrula stage embryos using TRIzol Reagent (Ambion). Degenerate and specific PCR primers were designed on the basis of conserved amino-acid sequences and RNA-seq data. Multiple amino-acid alignments were used to identify conserved regions. Total RNA was also subjected to RNA-seq analysis by the Beijing Genomics Institute (BGI, Shenzhen, China). The insert size of the sequencing library for RNA-seq was 200-bp, and the 90-bp pair-end raw reads were generated using an Illumina HiSeq2000. The zebrafish chordin, eve1, sizzled, bmp4 and krox20 sequences were used as queries to BLAST search (blastn and tblastn) against the RNA-seq data for primer design. Primers used to clone goldfish genes are listed in Supplementary Table 2. PCR fragments amplified using these primers were isolated and purified, and then ligated into vectors using the TOPO TA Cloning Kit Dual Promoter (Invitrogen), T&A Cloning Vector Kit (Yeastern Biotech) or pGEM-T Easy Vector system (Promega). The resulting vectors were used to transform Escherichia coli DH5α. Over 10 clones were picked from each population for sequencing. The sequenced cDNA fragments were used as backbones to obtain the almost-complete sequences by PCR, with specific primers and the GeneRacer kit (Invitrogen), according to the manufacturer’s protocol (Supplementary Table 2). The isolated genes were identified by generating multiple amino-acid alignments of goldfish and orthologous genes using CLUSTALW34. The phylogenetic relationships of goldfish chordin genes were investigated by reconstructing maximum likelihood trees using MEGA5 (ref. 35).

To identify candidate loci in the chdA and chdB genes responsible for the twin-tail goldfish phenotype, the complete coding regions were isolated and sequenced from wild-type and twin-tail goldfish embryonic cDNA (Supplementary Table 2). Single-nucleotide polymorphisms were examined by visual inspection; this revealed a substitution at the first nucleotide of the codon encoding the 127th amino acid of chdA in twin-tail goldfish, which causes the stop codon mutation (designated as chdAE127X). DNA fragments containing the mutation site were amplified from the genomic DNA of wild-type and twin-tail goldfish individuals by PCR, using a specific pair of primers (chda-f5 and chd-r5 in Supplementary Table 2). These PCR amplicons were sequenced directly using an ABI 3730. The goldfish RNA-seq data and chdA, chdB, eve1, sizzled, bmp4 and krox20 sequences have been deposited in NCBI under accession numbers DRA001224, and AB874473 to AB874479, respectively.

Backcross lines

In 2012, wild-type male goldfish (chdAwt/wt) were independently mated with two twin-tail goldfish females (Oranda and Ryukin strains; chdAE127X/E127X) (Supplementary Fig. 5). Two F 1 hybrid strains derived from the two crosses were maintained in aquaria (200–1,000 l) at the Yilan Marine Station, ICOB, Academia Sinica, and the Aquaculture Breeding Institute, Hualien, Taiwan. In 2013, two F 1 hybrid individuals (WO, the wild type × Oranda strain, and WR, the wild type × Ryukin strain; chdAwt/E127X) were mated with twin-tail goldfish individuals (chdAE127X/E127X) to obtain two backcross strains (OR × WO and RY × WR). The pedigree diagram is shown in Supplementary Fig. 5.

Phenotyping of backcross segregants

The backcross segregants were phenotyped at both the late embryonic and larval stages. In addition, mutant segregants and wild-type individuals were maintained separately, to prevent the loss of segregants to competition. Initially, embryos at 2–3 dpf were pre-categorized based on morphology at the post-cloacal levels (Supplementary Figs 1 and 5) into three types: wild-type; weakly-ventralized; and bifurcated fin fold. At 9–10 dpf, these pre-categorized segregants were further categorized into three types: wild-type (Wt) larvae from wild-type embryos; single caudal fin (Vent) larvae from weakly-ventralized or bifurcated caudal fin fold embryos; and bifurcated caudal fin (Twin) larvae from weakly ventralized or bifurcated fin fold embryos (Supplementary Fig. 5). The embryonic phenotypes were defined on the basis of earlier descriptions of zebrafish dino mutants17,19, and the larval phenotypes on the basis of the presence or absence of duplicated caudal skeletons. All backcross embryos and larvae were observed under stereomicroscopy (SZX16 and SZ16; Olympus). In total, 942 segregants were phenotyped at the embryonic stage and 670 of these segregants were phenotyped at larval stages.

Genotyping of chdA in backcross segregants

A total of 670 phenotyped specimens were genotyped by restriction digestion of genomic PCR products. PCR primers were designed to amplify the region containing both the twin-tail chdAE127X allele and the closely linked AvaI restriction enzyme site (Fig. 2b and chda-f5 and chd-r5 in Supplementary Table 2). PCR fragments amplified by these specific primers were digested by AvaI, and separated on 2% agarose gels. Genotypes were determined on the basis of the resulting band patterns.

Injection of mRNA

To generate constructs for transcription, the coding regions of chdAwt, chdAE127X or chdB were amplified by PCR and cloned into the pCS2+ vector36. These constructs were digested with NotI, and used as templates to synthesize capped mRNA with the mMESSAGE mMACHINE SP6 Kit, according to the manufacturer’s instructions (Ambion Inc.). The synthesized mRNA transcripts were purified using Quick Spin Columns for purification of radiolabelled RNA (Roche), and resuspended in nuclease-free water. A microinjector (Eppendorf Femtojet; Eppendorf) was used to inject mRNA into the centre of the yolk of 1–2 cell stage fertilized eggs, maintained on Petri dishes in 2 nl of 0.2 M KCl. Phenol red (Sigma) was added as an indicator at a final concentration of 0.05%. The injected embryos were incubated at 24 °C. Three independent rescue experiments were performed by injecting chdAwt into twin-tail goldfish embryos (Supplementary Fig. 6). Control embryo phenotypes were examined for all three experiments. All control embryos derived from two of three clutches possessed bifurcated fin folds (Supplementary Fig. 6). At 2 days after injection, embryos were classified by morphological inspection into the following three categories: weakly-ventralized, bifurcated fin fold and dorsalized. Categorization of the weakly-ventralized and bifurcated fin fold was based on the post-cloacal morphology (Supplementary Fig. 1). Embryos lacking part (or all of) the caudal fins were defined as dorsalized, with reference to descriptions of zebrafish mutant phenotypes19,37.

In situ hybridization

Digoxigenin-labelled anti-sense RNA probes were produced using PCR product as template, and the SP6/T7 RNA polymerase Riboprobe Combination System (Promega), in accordance with the manufacturer’s instructions. The probes were subsequently purified using mini Quick Spin RNA Columns (Roche). Primer sets used for the amplification of the PCR fragments are listed in Supplementary Table 2. For eve1, sizzled, bmp4 and krox20, the PCR product used to generate the probe corresponds to the full-length coding region. To avoid non-specific hybridization resulting from the high similarity of the chdA and chdB transcripts, anti-sense probes against these chordin genes were synthesized from the sequences encompassing the 3′ untranslated regions.

Whole-mount in situ hybridization was performed as previously described38, with minor modifications. Goldfish embryos were fixed with 4% paraformaldehyde in PBS overnight. Gastrula stage embryos were fixed and then dechorionated using forceps. Segmentation and pharyngeal stage embryos were dechorionated before fixation through pronase treatment. After fixation and dechorionation, embryos were dehydrated with methanol. Dehydrated embryos were re-hydrated with PBT and re-fixed with 4% paraformaldehyde in PBS. Embryos were subsequently treated with Proteinase K for 20 min, and then re-fixed again. Pre-hybridization and hybridization were performed at 65 °C for a period between 1 h and overnight. The samples were washed sequentially with 66% formamide/2 × SSCT at 65 °C for 30 min, 33% formamide/2 × SSCT at 65 °C for 30 min, 2 × SSCT at 65 °C for 15 min, and 0.2 SSCT at 65 °C for 30 min (this last wash was repeated twice). The samples were then incubated in blocking solution (PBS, 10% heat-inactivated goat serum (Roche), 0.1% Tween-20) for 1 h, before being incubated with a 1/4,000–1/8,000 volume of anti-digoxigenin-AP Fab fragments (Roche) at room temperature for 4 h or at 4 °C overnight. Samples were washed four times with blocking solution at room temperature for 25 min each. Signals were detected using BCIP/NBT Color Development Substrate (Promega). The reaction was stopped by washing the samples with PBS. To ensure accurate comparison of gene expression levels, the wild-type and twin-tail goldfish embryos were treated under identical conditions.