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Pleiotropic functions of chordin gene causing drastic morphological changes in ornamental goldfish

Breeders and fanciers have established many peculiar morphological phenotypes in ornamental goldfish. Among them, the twin-tail and dorsal-finless phenotypes have particularly intrigued early and recent researchers, as equivalent morphologies are extremely rare in nature. These two mutated phenotypes appeared almost simultaneously within a short time frame and were fixed in several strains. However, little is known about how these two different mutations could have co-occurred during such a short time period. Here, we demonstrate that the chordin gene, a key factor in dorsal–ventral patterning, is responsible not only for the twin-tail phenotype but also for the dorsal-finless phenotype. Our F2 backcrossing and functional analyses revealed that the penetrance/expressivity of the dorsal-finless phenotype can be suppressed by the wild-type allele of chdS. Based on these findings, we propose that chdSwt may have masked the expression of the dorsal-finless phenotype, acting as a capacitor buffering gene to allow accumulation of genetic mutations. Once this gene lost its original function in the twin-tail goldfish lineages, the dorsal-finless phenotype could be highly expressed. Thus, this study experimentally demonstrates that the rapid genetic fixation of morphological mutations during a short domestication time period may be related to the robustness of embryonic developmental mechanisms.

www.nature.com/scientificreports/ Here, we conducted genetic and functional analyses in the dorsal-finless phenotype focusing on the chdS locus. Since all of the investigated modern dorsal-finless goldfish strains share the twin-tail phenotype and chdS mutated genotype (chdS E127X/E127X ), it is reasonable to suspect that the chdS locus may be related not only with the bifurcated caudal fin but also with the dorsal-finless phenotype 6,7,11 . To examine this possibility, we conducted genotyping and phenotyping of backcrossed F2 progenies derived from the hybrids of the single-tail common goldfish (wild-type) and the Ranchu strain ( Fig. 1a-d) 6,11 . Moreover, we performed microinjection of chdS wt mRNA into the progenies derived from Ranchu strain parents (Fig. 1e). Based on our results, we conclude that the chdS gene is indeed responsible for both the twin-tail and dorsal-finless phenotypes, suggesting that these two phenotypes are causally related to each other. These findings provide intriguing insights into how a highly conserved body architecture can be drastically changed during a short time period.
The F1 progenies were then backcrossed with the Ranchu strain to generate F2 progenies (Fig. 1d, Supplementary Fig. S1). Two clutches of F2 progenies were obtained and designated "2020-0511-06RA" and "2021-0406-01RA. From 116 individuals in these two clutches of F2 progenies, we successfully obtained phenotype and genotype data. Notably, the F2 progenies exhibited variations in their caudal and dorsal fins ( Fig. 2a-h). For each F2 individual, either a single or bifurcated caudal fin was observed, consistent with our previous report ( Fig. 2a--h) 11 . The dorsal-finless phenotypes were also varied, as shown in Fig. 2a-h. Most of the F2 progenies either completely lacked dorsal fin rays, similar to the Ranchu strain (Fig. 1b), or the animals had a wild-type-like dorsal fin (Fig. 2a,b,g,h). Nevertheless, some intermediate phenotypes were observed (Fig. 2c-f). For example, some individuals showed a rudimentary dorsal fin, while others had only a few dorsal fin rays (Fig. 2c-f).
To examine the influence of chdS wt and chdS E127X alleles on the dorsal fin phenotype, we counted the numbers of dorsal fin rays in the F2 backcross population and genotyped the chdS locus in 116 progenies (Fig. 2i,j; Supplementary Tables S1 and S2); the two clutches respectively contributed 67 and 49 individuals to the total. Our analyses of these F2 progenies clearly indicated that the chdS wt allele influences the dorsal-finless phenotype ( Fig. 2a-f, Supplementary Tables S1 and S2). In both clutches, most of the individuals with a chdS E127X/E127X genotype exhibited bifurcated anal and caudal fins, although many still exhibited single anal and caudal fins (Supplementary Tables S3 and S4). These findings were again consistent with our previous report 11 .
The chdS E127X/E127X progenies could be categorized as having wild-type and mutant dorsal fins based on the number of the dorsal fin rays present. Our previous research showed that most wild-type goldfish have a total  mRNA microinjection rescue. To definitively test whether the chdS locus influences the expression of the mutation phenotype in the dorsal fin and the other median fins, we designed a rescue experiment in Ranchu strain progenies. In this experiment, we performed chdS mRNA microinjection, which could rescue the twin-tail phenotype in our previous study 11 . Microinjections of chdS wt mRNA were made into the fertilized eggs derived from Ranchu parents (Methods) (Fig. 1e). The experiment was repeated four times in independent clutches (#2022-0315-01RARA, #2022-0315-04RARA, #2022-0321-01RARA, #2022-0321-03RARA), allowing us to examine the phenotypes in a total of 1474 hatched larvae at 3 days post-fertilization (dpf) (Supplementary Table S5). The examinations focused on the morphologies of the median fin fold and its derivatives ( Fig. 3a-n; Supplementary Fig. S2).
Our rescue experiments demonstrated that chdS wt mRNA microinjected into early-stage larvae caused the expression of different phenotypes than those seen in the control larvae (non-injected or water-injected larvae), in terms of the median fin fold at pre-and post-cloacal levels ( Fig. 3a-h). Moreover, these phenotypic variations of www.nature.com/scientificreports/ the median fin fold were related to the dorsal fin morphology at the late larval stage ( Fig. 3i-n). All of the control hatched larvae exhibited a relatively uniform ventralized phenotype (e.g., bifurcated and/or disrupted fin folds at the ventral side and enlarged blood island), as described in chordin-depleted teleost species, in addition to a reduced dorsal fin fold from the caudal to mid-trunk level (equivalent of yolk extension region) (Fig. 3a,b) 11,[17][18][19] .
On the other hand, the mRNA-injected larvae exhibited various phenotypes ( Fig. 3c-h). A number of the hatched larvae exhibited rescue of the median fin fold phenotype at both dorsal and ventral sides after microinjection of the mRNA. This rescue was consistent with our previous mRNA rescue experiment, although several hatched larvae exhibited malformations in the heart and the epithelial tissues at the anterior regions (Fig. 3g,h) 11,20 . www.nature.com/scientificreports/ Notably, the level of rescue in the hatched larvae appeared to vary in terms of the dorsal fin fold phenotype ( Fig. 3c-h). Based on the dorsal fin morphology, we categorized the control and mRNA-injected larvae into three different groups as follows: (i) no dorsal fin fold at the level of yolk and post-yolk levels (group 1; Fig. 3a,b), (ii) partial dorsal fin fold at the level of yolk and post-yolk levels (group 2; Fig. 3c,d), and (iii) a wild-type equivalent phenotype of complete dorsal fin fold at the yolk and post-yolk levels (group 3; Fig. 3e-h) (for further descriptions of each group, see Methods). The morphological observations of these categorized larvae at the late larval stage (Fig. 3i-n) were entirely consistent with the early larval phenotypes (Fig. 3a-h). More specifically, at the late larval stages, dorsal fin rays were not observed in group 1 individuals, dorsal fin rays were missing at several body levels in group 2 individuals, and dorsal fin rays were completely developed in the group 3 individuals (Fig. 3i-n) 16,21 . This concordance of phenotypes suggested that chdS wt mRNA at the early embryonic developmental stage influences the eventual skeletal morphology of the dorsal fin ( Fig. 3a-n).
To analyze the result of the mRNA microinjection rescue experiment in further detail, we compared the larvae within each clutch. The comparison showed a significant reduction of group 1 individuals among mRNA-injected embryos ( Fig. 3o-r). Although the reactivity to the mRNA varied between clutches, we obtained similar results in the four independent experiments (Fig. 3o-r). Based on the consistent results from the mRNA microinjection rescue experiments and the F2 segregant analyses, we conclude that it is highly probable that the dorsal-finless phenotype and its related mutated genes are under the strong influence of the presence/absence of chdS wt allele. To our knowledge, this is the first study to identify the responsible gene of the dorsal-finless phenotype in ornamental goldfish by applying not only genetic linkage analyses but also functional analyses in goldfish embryos.

Discussion
In this study, we show that the chdS gene is responsible not only for the twin-tail phenotype but also for the dorsal-finless phenotype, suggesting that the chdS gene has pleiotropic functions that influence the formation of mutated morphologies in all median fins. Based on our results, we can posit an evolutionary process of the dorsal-finless phenotype. (1) Before the genetic fixation of the twin-tail phenotype, the penetrance/expressivity of the dorsal-finless phenotype was suppressed due to the chdS wt (magenta circle in Fig. 4). (2) After the genetic fixation of the twin-tail phenotype, the penetrance/expressivity of the dorsal-finless phenotype was increased due to the absence of chdS wt (magenta lines in Fig. 4). (3) In certain lineages of goldfish (called the "Ranchu group" in Kon and his colleagues 6 ), the dorsal-finless phenotype was intensively selected by breeders and fanciers (magenta triangle and bold magenta line in Fig. 4). (4) Finally, the appearance of Ranchu-group (dorsal-finless and twin-tail phenotype) goldfish was recorded in an early painting due to its common presence in the breeding population of ornamental goldfish (magenta square in Fig. 4; see Supplementary information). It seems that this hypothetical evolutionary process is completely consistent with the early report by Matsui 22 , which describes the occasional appearance of dorsal fin malformations in the Ryukin strain. This report states that although the majority of progenies of this strain show a complete dorsal fin phenotype, 51 of 1222 exhibited a "rudimentary dorsal finned phenotype", suggesting that the dorsal-finless phenotype can occur in goldfish with the chdS E127X/E127X genotype. However, such a low penetrance/expressivity of the dorsal-finless phenotype suggests that some additional mutated alleles should be accumulated before stable expression of the dorsal-finless phenotype can occur.
Although our study does not hint at the additional genetic mutations necessary for the dorsal-finless phenotype, our F2 segregant analyses provides important insights into how the chdS gene is related with those other www.nature.com/scientificreports/ loci in terms of phenotype expression. The fact that several F2 progenies with chdS wt/E127X genotype expressed the dorsal-finless phenotype indicates that there is at least one other responsible locus for the dorsal-finless phenotype independent of the chdS gene (Fig. 2i,j: Supplementary Fig. S2a-d). However, our results from the F2 segregant analyses indicated that the majority of mutated alleles responsible for the dorsal-finless phenotype are under the influence of chdS gene. Thus, the chdS gene appears to be epistatic to most genes responsible for the dorsal-finless phenotype. Taking into consideration the difficulty of detecting epistatic effects in complex human phenotypes using GWAS studies as well as the complicated molecular networks of chordin-related genes, it may be extraordinarily difficult to unambiguously identify the genes responsible for the dorsal-finless phenotype with simple application of a GWAS design on the domesticated population of goldfish 6,7,23 .
In fact, there is still no consensus about the responsible genes for the dorsal-finless phenotype, even though two GWAS reports have been published on the dorsal-finless phenotype by two independent research groups 6,7 . One group identified lrp6 gene as a candidate gene for the dorsal-finless phenotype, while the other group suggested a relationship exists between the mutated phenotype and the dhfr gene 6,7 . These inconsistent findings cannot be adequately explained at the level of the molecular mechanism due to a paucity of studies on these genes in goldfish. However, our study suggests that the inconsistent findings from these GWAS reports might be at least partially due to the complex epistatic relationships between the gene regulatory network and the chordin gene 18,[24][25][26][27][28] . Our study also strongly indicates that further investigations will be required to determine how the early embryonic developmental process relates to the simultaneous occurrence of the twin-tail and dorsal-finless phenotypes [25][26][27] .
Careful consideration of the embryonic proximity of the dorsal and caudal fins and their modular relationship may provide a deeper understanding of how these two phenotypes may have simultaneously occurred 12,16,21,29,30 . Molecular developmental studies in zebrafish revealed that the mesenchymal cells of the dorsal-and caudal fins are derived from the different levels of somite, but the epithelial cells are connected to each other at the embryonic stage; the epithelial cells form the fin fold and its primordia 31,32 . Based on the close embryonic proximity of epithelial cells, it is naturally presumed that both dorsal-and caudal fins are under the influence of the same molecular mechanisms. In fact, this interpretation allows us to explain why the majority of our F2 backcross progenies consistently exhibited mutant phenotypes in dorsal, anal, and caudal fins (Supplementary Tables S1,  S2). Moreover, previous reports on szl-depleted goldfish are consistent with this interpretation 18,33 . Depletion of szl gene expression in goldfish embryos leads to the dorsal-finless phenotype, which is related to the chordin gene 33 . Although the detailed molecular developmental mechanisms of the dorsal fin disappearance are still unknown, it is certain that the depletion of this gene disrupts dorsal-ventral patterning to generate the dorsalfinless phenotype in the goldfish 33 . Thus, it is reasonable to assume that the morphogenesis processes of the dorsal and caudal fin are under the regulation of the same dorsal-ventral patterning molecular developmental mechanism (which involves the chordin genes) at the early embryonic stage, even though these fins are separate structures at juvenile and adult stages.
We can rephrase the aforementioned assumption as follows. The chdS wt allele might have a buffering capacitor function that allows the dorsal-ventral patterning mechanisms to accumulate genetic mutations [25][26][27] . This assumption allows us to answer why the twin-tail and the dorsal-finless phenotype might appear quasi-simultaneously during the goldfish domestication history. The accumulation of responsible mutations for the dorsalfinless phenotype might have been masked by the chdS wt allele, and the effects of these mutations could have been unmasked specifically in the twin-tail goldfish lineages. Of course, our present study cannot completely reject the proposition that all of the responsible mutated alleles of the dorsal-finless phenotype had appeared after the fixation of chdS E127XE127X in the goldfish population. Nevertheless, an evolutionary scenario that includes a buffering capacitor function of the chdS gene seems more plausible than a scenario in which de novo genetic mutations for the dorsal-finless phenotype appeared in the lineage of the Ranchu group, as the first scenario could easily explain why the fixation process of the dorsal-finless phenotype in the twin-tail ornamental goldfish as so rapid (Fig. 4).
Researchers have investigated how masked genetic variations (or cryptic genetic variations) contribute to the expression of phenotypes upon genetic perturbation in several vertebrate species, including teleost species [34][35][36][37] . For example, the effects of chaperone protein HSP90 have been investigated in zebrafish and cavefish 34,35 . Moreover, the mef2ca gene was also examined in terms of its buffering function in the context of dermal skeleton morphogenesis 36 . Unlike these experimentally derived phenotypic variations, the phenotypic variations in goldfish were established through a process of artificial selection for the stabilization of visually classifiable strains (for example, Ryukin and Ranchu strains) after the fixation of the chdS E127X allele 11 . Thus, it is reasonable to assume that the ornamental morphologies of established goldfish strains are the consequence of the canalization of morphogenesis [38][39][40] . More specifically, the stable expression of both the twin-tail and dorsal-finless phenotypes has been canalized in the lineage of the Ranchu strain, but not in Ryukin strain. This fact would imply that the various dorsal-finless phenotype-associated candidate genes identified by GWAS analyses might actually contribute to the phenotypic canalization, rather than acting as decisive factors for the dorsal-finless phenotype 6,7 . We hope that future studies may combine the GWAS approach with molecular developmental genetics methodologies to better reveal the relationships between drastic morphological changes, developmental robustness, and genetic variations in different ornamental goldfish strains.

Methods
Goldfish strains. Goldfish were purchased from an aquarium fish agency in Taiwan. Two strains were used in this research, the single tail common goldfish (wild-type) and the Ranchu strain. To avoid confusion about the definition of "wild-type, " which may arise from differences in goldfish nomenclature systems used by breeders and researchers, this study defined goldfish individuals with a slender body and a single fin as having a wildtype phenotype (Fig. 1a) 12  www.nature.com/scientificreports/ (Fig. 1b). The F0 parents were genotyped by PCR and restriction enzyme digestion as described in a previous paper 11 . The wild-type goldfish parents were homozygous for the chdS wt allele, and dorsal-finless goldfish were homozygous for the chdS E127X allele. By crossing these parental strains, F1 individuals were obtained (Fig. 1c). These F1 individuals were subsequently used to obtain F2 segregants. The research was performed in accordance with internationally recognized guidelines and ARRIVE guidelines. Ethical approval was from the Institutional Animal Care & Utilization Committee of Academia Sinica, Taiwan (Protocol ID: 19-11-1351).
Artificial fertilization. The procedure for artificial fertilization was based on our previous report 30 . In the spawning season (March to June), sperm were taken from males and preserved in Modified Kurokura's extender 2 solution at 4 °C 41 . Eggs were squeezed out from mature females into Teflon-coated dishes. Artificial fertilization was performed using dry methods. The fertilized eggs were spread onto 9-cm Petri dishes, containing tap water (approx. 24 °C).
Genotyping. The genotype of chdS locus was examined by PCR and restriction enzyme digestion as previously reported 11 . PCR primers were designed to amplify the region containing both the chdS E127X allele and the closely linked AvaI restriction enzyme site. 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.
mRNA microinjection rescue experiment. The pCS2 + plasmid vector containing the coding regions of the chdS wt sequence were used to synthesize mRNA for the microinjection rescue experiments 42 . The sequence containing the coding region of chdS wt was amplified by PCR reaction, using a forward primer Sp6 (5′-ATT TAG GTG ACA CTA TAG A-3′) and reverse primer M13R (5′-TCA CAC AGG AAA CAG CTA TGAC-3′). The PCR product was cleaned with a GeneMark DNA Clean/Extraction kit before being used as the DNA template to synthesize capped mRNA. The mRNA synthesis was performed with the mMESSAGE mMACHINE SP6 kit, according to the manufacturer's instructions (Ambion Inc.). Then, the synthesized mRNA transcripts were purified using a Monarch RNA Cleanup kit and resuspended in nuclease-free water. The microinjection mixture contained 1 μl Phenol Red (Sigma), 1 μl of 2 M KCl, 4.7 μl nuclease-free water, and 3.3 μl synthesized mRNA (300 ng/μl) in a total of 10 μl. Phenol red (Sigma) was used as an indicator at a final concentration of 0.05%. Injection needles were prepared from borosilicate glass filaments (Sutter instrument, BF100-50-10) using a micropipette puller (Model P-1000, Sutter Instrument). A microinjector (Eppendorf Femtojet; Eppendorf) was used to inject mRNA into the center of the yolk of fertilized eggs at the 1-2 cell stage, which were maintained on Petri dishes. Each embryo was injected with 2 μl of the mRNA mixture containing a total of 250 pg mRNA. The injected embryos were then incubated at 24 °C until phenotyping at 3 dpf. Four independent rescue experiments were performed by injecting chdS wt mRNA into the embryos derived from Ranchu strain parents. At 3 dpf, embryos were categorized into three groups (described below). These categorized 3 dpf larvae were maintained for more than one month and assessed for their phenotype at hatching. To examine the potential influence of the mechanical contact with the injection needle, nuclease-free water was injected into the embryos as a control. As expected, mechanical contact did not cause significant changes to the morphology of the larvae during the developmental process.
Phenotype analysis. The F2 segregants were phenotyped at both late embryonic (3 dpf) and post-embryonic stages. The embryos and larvae were observed under stereomicroscopy. The 3 dpf embryos were categorized into three groups (group 1, group 2 and group 3) based on the position of rescued fin fold shape and its relative position to yolk; the position of dorsal fin rays at the juvenile stage is known to be related with that of the cloaca 16,21 . The criteria for these categories were as follows: group 1 individuals had no dorsal fin fold at the level of yolk; group 2 had dorsal fin fold partially at the level of yolk; and group 3 had a complete dorsal fin fold at the yolk and post-yolk levels. The phenotyping of late larvae and juveniles was conducted based on the numbers of dorsal fin rays. Maintenance of larvae followed previous reports 12,21 . The progenies from Pelvic fin ray stage to juvenile stage were fixed with PFA (4 wt% of paraformaldehyde in 1 × PBS solution) overnight, washed in 70% ethanol, and stained with alizarin red solution (0.02% alizarin red in 70% ethanol). Alizarin red-stained specimens were washed with 70% ethanol to reduce background. Phenotypic observations in larvae and stained specimens were made under stereomicroscopy (SZX16 and SZ16, Olympus). Images were acquired using a stereomicroscope system with digital microscope camera (SZX16 with DP80; Olympus). All data comparisons were made with the R statistical computing package of RStudio (Build 554, version 2022.07.1 + 554).