Embryonic and postembryonic development of the ornamental twin-tail goldfish.

BACKGROUND: Twin-tail ornamental goldfish have "bifurcated median fins," a peculiar morphology known to be caused by a mutation in the chdA gene. However, several ambiguities regarding the development of the phenotype remain due to a paucity of detailed observations covering the entire developmental timeframe. RESULTS: Here, we report a detailed comparative description of embryonic and postembryonic development for two representative twin-tail ornamental goldfish strains and single-tail common goldfish. Our observations reveal a polymorphic developmental process for bifurcated median fins; disrupted axial skeletal development at early larval stages; and modified bilateral location of the pelvic fin. CONCLUSIONS: Variations in development of bifurcated median fins and disrupted axial skeletal patterns reflect how artificial selection for adult morphological features influenced molecular developmental mechanisms during the domestication of twin-tail ornamental goldfish. The polymorphic appearance of bifurcated median fins also implies that, unlike previously proposed hypotheses, the development of these structures is controlled by molecular mechanisms independent of those acting on the pelvic fin. Our present findings will facilitate further study of how modifications of preexisting developmental systems may contribute to novel morphological features. Developmental Dynamics 248:251-283, 2019. © 2019 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Craniofacial development of hagfishes and the evolution of vertebrates.

Cyclostomes, the living jawless vertebrates including hagfishes and lampreys, represent the most basal lineage of vertebrates. Although the monophyly of cyclostomes has been supported by recent molecular analyses, the phenotypic traits of hagfishes, especially the lack of some vertebrate-defining features and the reported endodermal origin of the adenohypophysis, have been interpreted as hagfishes exhibiting a more ancestral state than those of all other vertebrates. Furthermore, the adult anatomy of hagfishes cannot be compared easily with that of lampreys. Here we describe the craniofacial development of a series of staged hagfish embryos, which shows that their adenohypophysis arises ectodermally, consistent with the molecular phylogenetic data. This finding also allowed us to identify a pan-cyclostome pattern, one not shared by jawed vertebrates. Comparative analyses indicated that many of the hagfish-specific traits can be explained by changes secondarily introduced into the hagfish lineage. We also propose a possibility that the pan-cyclostome pattern may reflect the ancestral programme for the craniofacial development of all living vertebrates.

Evolutionary developmental transition from median to paired morphology of vertebrate fins: Perspectives from twin-tail goldfish.

Vertebrate morphology has been evolutionarily modified by natural and/or artificial selection. The morphological variation of goldfish is a representative example. In particular, the twin-tail strain of ornamental goldfish shows highly diverged anal and caudal fin morphology: bifurcated anal and caudal fins. Recent molecular developmental genetics research revealed that a stop codon mutation in one of the two recently duplicated chordin genes is important for the highly diverged fin morphology of twin-tail goldfish. However, some issues still need to be discussed in the context of evolutionary developmental biology (evo-devo). For example, the bifurcated anal and caudal fins of twin-tail goldfish provided early researchers with insights into the origin of paired fins (pectoral and pelvic fins), but no subsequent researchers have discussed this topic. In addition, although the fossil jawless vertebrate species Euphanerops is also known to have had a bifurcated anal fin, how the bifurcated anal fin of twin-tail goldfish is related to that of fossil jawless vertebrate species has never been investigated. In this review, we present an overview of the early anatomical and embryological studies of twin-tail goldfish. Moreover, based on the similarity of embryonic features between the secondarily bifurcated competent stripe in twin-tail goldfish and the trunk bilateral competent stripes in conventional gnathostomes, we hypothesized that they share the same molecular developmental mechanisms. We also postulate that the bifurcated anal fin of Euphanerops might be caused by the same type of modification of dorsal-ventral patterning that occurs in the twin-tail goldfish, unlike the previously suggested evolutionary process that required the co-option of paired fin developmental mechanisms. Understanding the molecular developmental genetics of twin-tail goldfish allows us to further investigate the evolutionary developmental mechanisms of the origin of paired fins.

An alternative evolutionary pathway for the twin-tail goldfish via szl gene mutation.

The twin-tail of ornamental goldfish provides unique evolutionary evidence that the highly conserved midline localization of axial skeleton components can be changed by artificial selection. This morphological change is known to be caused by a nonsense mutation in one of the recently duplicated chordin genes, which are key players in dorsal-ventral (DV) patterning. Since all of the multiple twin-tail ornamental goldfish strains share the same mutation, it is reasonable to presume that this mutation occurred only once in domesticated goldfish. However, zebrafish with mutated szl gene (another DV patterning-related gene) also exhibit twin-tail morphology and higher viability than dino/chordin-mutant zebrafish. This observation raises the question of whether the szl gene mutation could also reproduce the twin-tail morphology in goldfish. Here we show that goldfish have at least two subfunctionalized szl genes, designated szlA and szlB, and depletion of these genes in single-fin goldfish was able to reproduce the bifurcated caudal fin found in twin-tail ornamental goldfish. Interestingly, several phenotypes were observed in szlA-depleted fish, while low expressivity of the twin-tail phenotype was observed in szlB-depleted goldfish. Thus, even though szl gene mutations may produce twin-tail goldfish, these szl gene mutations might not be favorable for selection in domestic breeding. These results highlight the uniqueness and rarity of mutations that are able to cause large-scale morphological changes, such as a bifurcated axial skeleton, with high viability and expressivity in natural and domesticated populations.

A novel allele of the goldfish chdB gene: Functional evaluation and evolutionary considerations.

The twin tail of ornamental goldfish is known to be caused by a nonsense mutation in one chordin paralogue gene. Our previous molecular studies in goldfish revealed that the ancestral chordin gene was duplicated, creating the chdA and chdB genes, and the subsequent introduction of a stop codon allele in the chdA gene ( chdA E127X ) caused the twin-tail morphology. The chdA E127X allele was positively selected by breeders, and the allele was genetically fixed in the ornamental twin-tail goldfish population. However, little is known about the evolutionary history of the chdB paralogue, begging the question: are there the functionally distinct alleles at the chdB locus, and if so, how did they evolve? To address these questions, we conducted molecular sequencing of the chdB gene from five different goldfish strains and discovered two alleles at the chdB gene locus; the two alleles are designated chdB 1 and chdB 2 . The chdB 1 allele is the major allele and was found in all investigated goldfish strains, whereas the chdB 2 allele is minor, having only been found in one twin-tail strain. Genetic analyses further suggested that these two alleles are functionally different with regard to survivability ( chdB 1 > chdB 2 ). These results led us to presume that in contrast to the chdA locus, the chdB locus has tended to be eliminated from the population. We also discuss how the chdB 2 allele was retained in the goldfish population, despite its disadvantageous function. This study provides empirical evidence of the long-term retention of a disadvantageous allele under domesticated conditions.

Evolution of the Vertebrate Cranium: Viewed from Hagfish Developmental Studies.

Our knowledge of vertebrate cranium evolution has relied largely on the study of gnathostomes. Recent evolutionary and developmental studies of cyclostomes have shed new light on the history of the vertebrate skull. The recent ability to obtain embryos of the hagfish, Eptatretus burgeri, has enabled new studies which have suggested an embryonic morphological pattern (the "cyclostome pattern") of craniofacial development. This pattern is shared by cyclostomes, but not by modern jawed vertebrates. Because this pattern of embryonic head development is thought to be present in some stem gnathostomes (ostracoderms), it is possible that the cyclostome pattern represents the vertebrate ancestral pattern. The study of cyclostomes may thus lead to an understanding of the most ancestral basis of craniofacial development. In this review, we summarize the development of the hagfish chondrocranium in light of the cyclostome pattern, present an updated comparison of the cyclostome chondrocranium, and discuss several aspects of the evolution and development of the vertebrate skull.

Postembryonic staging of wild-type goldfish, with brief reference to skeletal systems.

BACKGROUND: Artificial selection of postembryonic features is known to have established morphological variation in goldfish (Carassius auratus). Although previous studies have suggested that goldfish and zebrafish are almost directly comparable at the embryonic level, little is known at the postembryonic level. RESULTS: Here, we categorized the postembryonic developmental process in the wild-type goldfish into 11 different stages. We also report certain differences between the postembryonic developmental processes of goldfish and zebrafish, especially in the skeletal systems (scales and median fin skeletons), suggesting that postembryonic development underwent evolutionary divergence in these two teleost species. CONCLUSIONS: Our postembryonic staging system of wild-type goldfish paves the way for careful and appropriate comparison with other teleost species. The staging system will also facilitate comparative ontogenic analyses between wild-type and mutant goldfish strains, allowing us to closely study the relationship between artificial selection and molecular developmental mechanisms in vertebrates.

Embryonic development of goldfish (Carassius auratus): a model for the study of evolutionary change in developmental mechanisms by artificial selection.

BACKGROUND: Highly divergent morphology among the different goldfish strains (Carassius auratus) may make it a suitable model for investigating how artificial selection has altered developmental mechanisms. Here we describe the embryological development of the common goldfish (the single fin Wakin), which retains the ancestral morphology of this species. RESULTS: We divided goldfish embryonic development into seven periods consisting of 34 stages, using previously reported developmental indices of zebrafish and goldfish. Although several differences were identified in terms of their yolk size, epiboly process, pigmentation patterns, and development rate, our results indicate that the embryonic features of these two teleost species are highly similar in their overall morphology from the zygote to hatching stage. CONCLUSIONS: These results provide an opportunity for further study of the evolutionary relationship between domestication and development, through applying well-established zebrafish molecular biological resources to goldfish embryos.

Exploring the origin of a unique mutant allele in twin-tail goldfish using CRISPR/Cas9 mutants.

Artificial selection has been widely applied to genetically fix rare phenotypic features in ornamental domesticated animals. For many of these animals, the mutated loci and alleles underlying rare phenotypes are known. However, few studies have explored whether these rare genetic mutations might have been fixed due to competition among related mutated alleles or if the fixation occurred due to contingent stochastic events. Here, we performed genetic crossing with twin-tail ornamental goldfish and CRISPR/Cas9-mutated goldfish to investigate why only a single mutated allele-chdS with a E127X stop codon (also called chdAE127X)-gives rise to the twin-tail phenotype in the modern domesticated goldfish population. Two closely related chdS mutants were generated with CRISPR/Cas9 and compared with the E127X allele in F2 and F3 generations. Both of the CRISPR/Cas9-generated alleles were equivalent to the E127X allele in terms of penetrance/expressivity of the twin-tail phenotype and viability of carriers. These findings indicate that multiple truncating mutations could have produced viable twin-tail goldfish. Therefore, the absence of polymorphic alleles for the twin-tail phenotype in modern goldfish likely stems from stochastic elimination or a lack of competing alleles in the common ancestor. Our study is the first experimental comparison of a singular domestication-derived allele with CRISPR/Cas9-generated alleles to understand how genetic fixation of a unique genotype and phenotype may have occurred. Thus, our work may provide a conceptual framework for future investigations of rare evolutionary events in domesticated animals.

Non-parsimonious evolution of hagfish Dlx genes.

BACKGROUND: The number of members of the Dlx gene family increased during the two rounds of whole-genome duplication that occurred in the common ancestor of the vertebrates. Because the Dlx genes are involved in the development of the cranial skeleton, brain, and sensory organs, their expression patterns have been analysed in various organisms in the context of evolutionary developmental biology. Six Dlx genes have been isolated in the lampreys, a group of living jawless vertebrates (cyclostomes), and their expression patterns analysed. However, little is known about the Dlx genes in the hagfish, the other cyclostome group, mainly because the embryological analysis of this animal is difficult. RESULTS: To identify the hagfish Dlx genes and describe their expression patterns, we cloned the cDNA from embryos of the Japanese inshore hagfish Eptatretus burgeri. Our results show that the hagfish has at least six Dlx genes and one pseudogene. In a phylogenetic analysis, the hagfish Dlx genes and those of the lampreys tended to be excluded from the clade of the gnathostome Dlx genes. In several cases, the lamprey Dlx genes clustered with the clade consisting of two hagfish genes, suggesting that independent gene duplications have occurred in the hagfish lineage. Analysis of the expression of these genes showed distinctive overlapping expression patterns in the cranial mesenchymal cells and the inner ear. CONCLUSIONS: Independent duplication, pseudogenization, and loss of the Dlx genes probably occurred in the hagfish lineage after its split from the other vertebrate lineages. This pattern is reminiscent of the non-parsimonious evolution of its morphological traits, including its inner ear and vertebrae, which indicate that this group is an early-branching lineage that diverged before those characters evolved.

Late development of hagfish vertebral elements.

It has been demonstrated recently that hagfishes, one of two groups of extant jawless vertebrates, have cartilaginous vertebral elements. Embryological and gene expression analyses have also shown that this group of animals develops a sclerotome, the potential primordium of the axial skeleton. However, it has not been shown unequivocally that the hagfish sclerotome truly differentiates into cartilage, because access to late-stage embryos and information about the cartilaginous extracellular matrix (ECM) are lacking for these animals. Here we investigated the expression patterns of the biglycan/decorin (BGN/DCN) gene in the inshore hagfish, Eptatretus burgeri. The homologue of this gene encodes the major noncollagenous component of the cartilaginous ECM among gnathostomes. We clearly identified the expression of this gene in adult vertebral tissues and in embryonic mesenchymal cells on the ventral aspect of the notochord. Taking into account that the sclerotome in the gnathostomes expresses BGN/DCN gene during the chondrogenesis, it is highly expected the hagfish BGN/DCN-positive mesenchymal cells are derived from the sclerotomes. We propose that hagfishes and gnathostomes share conserved developmental mechanisms not only in their somite differentiation, but also in chondrogenesis of their vertebral elements.

Hagfish embryology with reference to the evolution of the neural crest.

Hagfish, which lack both jaws and vertebrae, have long been the subject of intense interest owing to their position at a crucial point in the evolutionary transition to a truly vertebrate body plan. However, unlike the comparatively well characterized vertebrate agnathan lamprey, little is known about hagfish development. The inability to analyse hagfish at early embryonic stages has frustrated attempts to resolve questions with important phylogenetic implications, including fundamental ones relating to the emergence of the neural crest. Here we report the obtainment of multiple pharyngula-stage embryos of the hagfish species Eptatretus burgeri and our preliminary analyses of their early development. We present histological evidence of putative neural crest cells, which appear as delaminated cells that migrate along pathways corresponding to neural crest cells in fish and amphibians. Molecular cloning studies further revealed the expression of several regulatory genes, including cognates of Pax6, Pax3/7, SoxEa and Sox9, suggesting that the hagfish neural crest is specified by molecular mechanisms that are general to vertebrates. We propose that the neural crest emerged as a population of de-epithelialized migratory cells in a common vertebrate ancestor, and suggest that the possibility of classical and molecular embryology in hagfish opens up new approaches to clarifying the evolutionary history of vertebrates.

Development of the chondrocranium in hagfishes, with special reference to the early evolution of vertebrates.

Recent molecular phylogenetic analyses have shown that the modern jawless vertebrates, hagfishes and lampreys, are more closely related to each other than to the other vertebrates, constituting a monophyletic group, the cyclostomes. In terms of their developmental morphology as well, it is possible to identify an embryonic pattern in hagfish embryos that is common to cyclostomes but not shared by jawed vertebrate embryos. On the basis of this pan-cyclostome embryonic pattern, we describe the developmental sequence of the chondrocranium and associated structures in the hagfish species Eptatretus burgeri and E. atami. Our aim was to establish homologies of the skeletal elements among cyclostomes by comparison of the developmental patterns with a lamprey, Lethenteron reissneri, to characterize further the cyclostome morphotype and its diversification in early vertebrate evolution. We show that the hagfish and lamprey chondrocrania can be compared perfectly at the level of modules corresponding to the craniofacial primordia constituting the cyclostome morphotype. In the adult anatomy, however, there are many instances in which homology cannot be established at the level of single skeletal elements, mainly because of the apparently highly apomorphic nature of the hagfish cranium. Even at the craniofacial modular level, the chondrocrania of cyclostomes and those of jawed vertebrates display very few primary homologies and are therefore very difficult to compare. We also discuss the problem of the homology of a neurocranial element, the trabecula.

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.

Overview of the transcriptome profiles identified in hagfish, shark, and bichir: current issues arising from some nonmodel vertebrate taxa.

Because of their crucial phylogenetic positions, hagfishes, sharks, and bichirs are recognized as key taxa in our understanding of vertebrate evolution. The expression patterns of the regulatory genes involved in developmental patterning have been analyzed in the context of evolutionary developmental studies. However, in a survey of public sequence databases, we found that the large-scale sequence data for these taxa are still limited. To address this deficit, we used conventional Sanger DNA sequencing and a next-generation sequencing technology based on 454 GS FLX sequencing to obtain expressed sequence tags (ESTs) of the Japanese inshore hagfish (Eptatretus burgeri; 161,482 ESTs), cloudy catshark (Scyliorhinus torazame; 165,819 ESTs), and gray bichir (Polypterus senegalus; 34,336 ESTs). We deposited the ESTs in a newly constructed database, designated the "Vertebrate TimeCapsule." The ESTs include sequences from genes that can be effectively used in evolutionary developmental studies; for instance, several encode cartilaginous extracellular matrix proteins, which are central to an understanding of the ways in which evolutionary processes affected the skeletal elements, whereas others encode regulatory genes involved in craniofacial development and early embryogenesis. Here, we discuss how hagfishes, sharks, and bichirs contribute to our understanding of vertebrate evolution, we review the current status of the publicly available sequence data for these three taxa, and we introduce our EST projects and newly developed database.

Expression pattern of two collagen type 2 alpha1 genes in the Japanese inshore hagfish (Eptatretus burgeri) with special reference to the evolution of cartilaginous tissue.

Collagen type 2 alpha1 (Col2A1) protein is a major component of the cartilaginous extracellular matrix (ECM) in vertebrates. Over the past two decades, the evolutionary origin of Col2A1 has been studied at the biochemical and molecular levels in extant jawless vertebrates (hagfishes and lampreys). Although these studies have contributed to our understanding of ECM protein evolution, the expression profile of the Col2A1 gene in hagfishes has not been fully described. We have performed molecular cloning and analyzed the gene expression pattern of the Col2A1 gene in the Japanese inshore hagfish (Eptatretus burgeri). We succeeded in isolating two Col2A1 genes, EbCol2A1A and EbCol2A1B, in which EbCol2A1A was expressed in the noncartilaginous connective tissues whereas EbCol2A1B was detected in some cartilaginous elements. Based on these results, we discuss the evolutionary history of Col2A1 genes in early vertebrates.

Primitive versus derived traits in the developmental program of the vertebrate head: views from cyclostome developmental studies.

Evolution can be viewed as a series of changes in the developmental program along the phylogenetic tree. To better understand the early evolution of the vertebrate skull, we can use the embryos of the cyclostome species as models. By comparing the cyclostome developmental patterns with those of gnathostomes, it becomes possible to distinguish the primitive and derived parts of the developmental program as taxon-specific traits. These traits are often recognizable as developmental constraints that define taxa by biasing the developmental trajectories within a certain limited range, resulting in morphological homologies in adults. These developmental constraints are distributed on the phylogenetic tree like the morphological character states of adult animals and are associated with specific regions of the tree. From this perspective, we emphasize the importance of considering gene expression and embryonic anatomy as the mechanistic bases that can result in homologous or nonhomologous morphological patterns at later developmental stages. Taking the acquisition of the jaw and trabecula cranii as examples, we demonstrate that a set of embryonic features can be coupled or decoupled during evolution and development. When they are coupled, they exert an ancestral developmental constraint that results in homologous morphological patterns, and when they are decoupled, the ancestral constraints tend to be abandoned, generating a new body plan. The heterotopy behind the specification of the oral domain is an example of decoupling, based on shifted tissue interactions. We also stress the importance of "developmental burden" in determining the sequential order of changes through evolution.

Developmental biology of hagfishes, with a report on newly obtained embryos of the Japanese inshore hagfish, Eptatretus burgeri.

The apparently primitive features of hagfishes are recognized as a crucial problem in the study of vertebrate evolution, although the monophyletic relationship between these animals and lampreys has been confirmed by large amounts of molecular data, including genome and EST sequences. To solve this problem requires knowledge of the developmental biology of hagfishes. We attempted to obtain embryos from the Japanese inshore hagfish ( Eptatretus burgeri ) and succeeded in preparing several nicely fixed embryos. Based on detailed histological observations and comparison of gene expression patterns with those of conventional vertebrates, we examined the developmental processes involved in some important morphological traits, including the neural crest, placode, pharyngeal arches, and others. Our data revealed that some apparently primitive morphological traits can be regarded as artifacts deriving mainly from fixation conditions. In addition, our long-term observations of live embryos revealed a slow developmental rate in this animal. In this review, we summarize recent developmental data from these hagfish embryos and discuss a plausible evolutionary scenario for vertebrate development, making comparisons with some old descriptions.

Hagfish and lamprey Hox genes reveal conservation of temporal colinearity in vertebrates.

Hox genes exert fundamental roles for proper regional specification along the main rostro-caudal axis of animal embryos. They are generally expressed in restricted spatial domains according to their position in the cluster (spatial colinearity)-a feature that is conserved across bilaterians. In jawed vertebrates (gnathostomes), the position in the cluster also determines the onset of expression of Hox genes (a feature known as whole-cluster temporal colinearity (WTC)), while in invertebrates this phenomenon is displayed as a subcluster-level temporal colinearity. However, little is known about the expression profile of Hox genes in jawless vertebrates (cyclostomes); therefore, the evolutionary origin of WTC, as seen in gnathostomes, remains a mystery. Here, we show that Hox genes in cyclostomes are expressed according to WTC during development. We investigated the Hox repertoire and Hox gene expression profiles in three different species-a hagfish, a lamprey and a shark-encompassing the two major groups of vertebrates, and found that these are expressed following a whole-cluster, temporally staggered pattern, indicating that WTC has been conserved during the past 500 million years despite drastically different genome evolution and morphological outputs between jawless and jawed vertebrates.