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.

A Long Polymorphic GT Microsatellite within a Gene Promoter Mediates Non-Imprinted Allele-Specific DNA Methylation of a CpG Island in a Goldfish Inter-Strain Hybrid.

It is now widely accepted that allele-specific DNA methylation (ASM) commonly occurs at non-imprinted loci. Most of the non-imprinted ASM regions observed both within and outside of the CpG island show a strong correlation with DNA polymorphisms. However, what polymorphic cis-acting elements mediate non-imprinted ASM of the CpG island remains unclear. In this study, we investigated the impact of polymorphic GT microsatellites within the gene promoter on non-imprinted ASM of the local CpG island in goldfish. We generated various goldfish heterozygotes, in which the length of GT microsatellites or some non-repetitive sequences in the promoter of no tail alleles was different. By examining the methylation status of the downstream CpG island in these heterozygotes, we found that polymorphisms of a long GT microsatellite can lead to the ASM of the downstream CpG island during oogenesis and embryogenesis, polymorphisms of short GT microsatellites and non-repetitive sequences in the promoter exhibited no significant effect on the methylation of the CpG island. We also observed that the ASM of the CpG island was associated with allele-specific expression in heterozygous embryos. These results suggest that a long polymorphic GT microsatellite within a gene promoter mediates non-imprinted ASM of the local CpG island in a goldfish inter-strain hybrid.

Persistence and Transcription of Paternal mtDNA Dependent on the Delivery Strategy Rather than Mitochondria Source in Fish Embryos.

BACKGROUND/AIMS: Mitochondria (MT) and mitochondrial DNA (mtDNA) show maternal inheritance in most eukaryotic organisms; the sperm mtDNA is usually delivered to the egg during fertilization and then rapidly eliminated to avoid heteroplasmy, which can affect embryogenesis. In our previous study, fertilization-delivered sperm mtDNA exhibited late elimination and transcriptional quiescence in cyprinid fish embryos. However, the mechanisms underlying elimination and transcriptional quiescence of paternal mtDNA are unclear. METHODS: Goldfish and zebrafish were used to investigate the fate of mtDNAs with different parental origins delivered by fertilization or microinjection in embryos. Goldfish MT from heart, liver and spermatozoa were microinjected into zebrafish zygotes, respectively. Specific PCR primers were designed so that the amplicons have different sizes to characterize goldfish and zebrafish cytb genes or their cDNAs. RESULTS: The MT injection-delivered paternal mtDNA from sperm, as well as those from the heart and liver, was capable of persistence and transcription until birth, in contrast to the disappearance and transcriptional quiescence at the heartbeat stage of fertilization-delivered sperm mtDNA. In addition, the exogenous MT-injected zebrafish embryos have normal morphology during embryonic development. CONCLUSIONS: The fate of paternal mtDNA in fishes is dependent on the delivery strategy rather than the MT source, suggesting that the presence of sperm factor(s) is responsible for elimination and transcriptional quiescence of fertilization-delivered sperm mtDNA. These findings provide insights into the mechanisms underlying paternal mtDNA fate and heteroplasmy in cyprinid fishes.

The chromosomal constitution of fish hybrid lineage revealed by 5S rDNA FISH.

BACKGROUND: The establishment of the bisexual fertile fish hybrid lineage including the allodiploid and allotetraploid hybrids, from interspecific hybridization of red crucian carp (Carassius auratus red var. 2n = 100, 2n = AA) (♀) × common carp (Cyprinus carpio L. 2n = 100, 2n = BB) (♂), provided a good platform to investigate genetic relationship between the parents and their hybrid progenies. RESULTS: The chromosomal inheritance of diploid and allotetraploid hybrid progenies in successive generations, was studied by applying 5S rDNA fluorescence in situ hybridization. Signals of 5S rDNA distinguished the chromosomal constitution of common carp (B-genome) from red crucian carp (A-genome), in which two strong signals were observed on the first submetacentric chromosome, while no major signal was found in common carp. After fish hybridization, one strong signal of 5S rDNA was detected in the same locus on the chromosome of diploid hybrids. As expected, two strong signals were observed in 4nF3 tetraploid hybrids offspring and it is worth mentioning that two strong signals were detected in a separating bivalent of a primary spermatocyte in 4nF3. Furthermore, the mitosis of heterozygous chromosomes was shown normal and stable with blastular tissue histological studies. CONCLUSIONS: We revealed that 5S rDNA signal can be applied to discern A-genome from B-genome, and that 5S rDNA bearing chromosomes can be stably passed down in successive generations. Our work provided a significant method in fish breeding and this is important for studies in fish evolutionary biology.

MicroRNA-20a is essential for normal embryogenesis by targeting vsx1 mRNA in fish.

MicroRNAs are major post-transcriptional regulators of gene expression and have essential roles in diverse developmental processes. In vertebrates, some regulatory genes play different roles at different developmental stages. These genes are initially transcribed in a wide embryonic region but restricted within distinct cell types at subsequent stages during development. Therefore, post-transcriptional regulation is required for the transition from one developmental stage to the next and the establishment of different cell identities. However, the regulation of many multiple functional genes at post-transcription level during development remains unknown. Here we show that miR-20a can target the mRNA of vsx1, a multiple functional gene, at the 3'-UTR and inhibit protein expression in both goldfish and zebrafish. The expression of miR-20a is initiated ubiquitously at late gastrula stage and exhibits a tissue-specific pattern in the developing retina. Inhibition of vsx1 3'-UTR mediated protein expression occurs when and where miR-20a is expressed. Decoying miR-20a resulted in severely impaired head, eye and trunk formation in association with excessive generation of vsx1 marked neurons in the spinal cord and defects of somites in the mesoderm region. These results demonstrate that miR-20a is essential for normal embryogenesis by restricting Vsx1 expression in goldfish and zebrafish, and that post-transcriptional regulation is an essential mechanism for Vsx1 playing different roles in diverse developmental processes.

Optimization protocol for storage of goldfish (Carassius auratus) embryos in chilled state.

A series of five experiments were conducted to explore suitable conditions for storing of goldfish embryos in a chilled state. The factors studied were embryo stage, storage temperature, physiological saline solutions and goldfish artificial coelomic fluid (GFACF) medium, antibiotics (penicillin and streptomycin), antioxidants (vitamin E, vitamin C), buffer (Hepes, Tris) and BSA (bovine serum albumin). First, goldfish embryos at eight developmental stages were incubated in aerated and dechlorinated tap water at 0 °C for 24 h. Result shows that early developmental stages were most sensitive to chilling. Heartbeat-stage goldfish embryos were chilled at 0, 4 or 8 °C for up to 72 h in water, and chilled storage was possible only for up to 18, 24 and 48 h at 0, 4 and 8 °C, respectively, without a decrease in viability. Chilling of goldfish embryos at 8 °C in GFACF medium and Dettlaff's solution instead of water and other physiological saline solutions prolonged their viability (p < 0.01). Nevertheless, viability of chilled embryos in GFACF medium was slightly, but non-significantly, higher than in Dettlaff's solution. Supplementation of the GFACF medium with antibiotics, Hepes or BSA increased the viability of chilled embryos, but the tested vitamin E analogue Trolox, vitamin C or Tris concentration had no effect on embryo viability. The outcome of this series of experiments shows that heartbeat-stage goldfish embryos could be chilled for 60 h in GFACF supplemented with 25 mm Hepes, 100 U/ml penicillin, 10 µg/l streptomycin and 1 g/l BSA in such a way that embryonic development does not proceed, and viability is not lost.

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.

HIRA is essential for the development of gibel carp.

HIRA is one of the chaperones of histone H3.3. Mutation of Hira results in embryonic lethality in mice, suggesting a critical role in embryogenesis. However, Hira-mutated Drosophila may survive to adults, indicating that it is dispensable in Drosophila development. The role of Hira in fish development is unknown. In this study we first investigated the expression of Hira during embryogenesis of gibel carp (Carassius auratus gibelio) by whole-mount in situ hybridization. We found that Hira signal appeared ubiquitously in the early embryos. After gastrulation, it appeared mainly along the anterior-posterior axis, including the tail bud. In hatching period, the signal was detected in head, heart, and the endoderm region on the back of yolk. Then by microinjection with morpholino-HIRA at the beginning of development, we observed delayed gastrulation and abnormal somitogenesis in gibel carp embryos. The HIRA morphants exhibited short trunk, limited yolk extension, and twisted tail. Most of the mutants died during embryogenesis or shortly after hatching. The rest of the HIRA morphants could survive to larvae but with severe defects in organogenesis. These data suggest that HIRA may be essential for the development of gibel carp, and this function is conserved in vertebrates.

Germ cells are not the primary factor for sexual fate determination in goldfish.

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.

Goldfish transposase Tgf2 presumably from recent horizontal transfer is active.

Hobo/Activator/Tam3 (hAT) superfamily transposons occur in plants and animals and play a role in genomic evolution. Certain hAT transposons are active and have been developed as incisive genetic tools. Active vertebrate elements are rarely discovered; however, Tgf2 transposon was recently discovered in goldfish (Carassius auratus). Here, we found that the endogenous Tgf2 element can transpose in goldfish genome. Seven different goldfish mRNA transcripts, encoding three lengths of Tgf2 transposase, were identified. Tgf2 transposase mRNA was detected in goldfish embryos, mainly in epithelial cells; levels were high in ovaries and mature eggs and in all adult tissues tested. Endogenous Tgf2 transposase mRNA is active in mature eggs and can mediate high rates of transposition (>30%) when injected with donor plasmids harboring a Tgf2 cis-element. When donor plasmid was coinjected with capped Tgf2 transposase mRNA, the insertion rate reached >90% at 1 yr. Nonautonomous copies of the Tgf2 transposon with large-fragment deletions and low levels of point mutations were also detected in common goldfish. Phylogenetic analysis indicates the taxonomic distribution of Tgf2 in goldfish is not due to vertical inheritance. We propose that the goldfish Tgf2 transposon originated by recent horizontal transfer and maintains a highly native activity.

Evolutionary history of c-myc in teleosts and characterization of the duplicated c-myca genes in goldfish embryos.

c-Myc plays an important role during embryogenesis in mammals, but little is known about its function during embryonic development in teleosts. In addition, the evolutionary history of c-myc gene in teleosts remains unclear, and depending on the species, a variable number of gene duplicates exist in teleosts. To gain new insight into c-myc genes in teleosts, the present study was designed to clarify the evolutionary history of c-myc gene(s) in teleosts and to subsequently characterize DNA methylation and early embryonic expression patterns in a cyprinid fish. Our results show that a duplication of c-myc gene occurred before or around the teleost radiation, as a result of the teleost-specific whole genome duplication giving rise to c-myca and c-mycb in teleosts and was followed by a loss of the c-mycb gene in the Gasterosteiforms and Tetraodontiforms. Our data also demonstrate that both c-myc genes previously identified in carp and goldfish are co-orthologs of the zebrafish c-myca. These results indicate the presence of additional c-myca duplication in Cyprininae. We were able to identify differences between the expression patterns of the two goldfish c-myca genes in oocytes and early embryos. These differences suggest a partial sub-functionalization of c-myca genes after duplication. Despite differences in transcription patterns, both of the c-myca genes displayed similar DNA methylation patterns during early development and in gametes. Together, our results clarify the evolutionary history of the c-myc gene in teleosts and provide new insight into the involvement of c-myc in early embryonic development in cyprinids.

Attempt at cloning high-quality goldfish breed 'Ranchu' by fin-cultured cell nuclear transplantation.

The viability of ornamental fish culture relies on the maintenance of high-quality breeds. To improve the profitability of culture operations we attempted to produce cloned fish from the somatic nucleus of the high-quality Japanese goldfish (Carassius auratus auratus) breed 'Ranchu'. We transplanted the nucleus of a cultured fin-cell from an adult Ranchu into the non-enucleated egg of the original goldfish breed 'Wakin'. Of the 2323 eggs we treated, 802 underwent cleavage, 321 reached the blastula stage, and 51 reached the gastrula stage. Two of the gastrulas developed until the hatching stage. A considerable number of nuclear transplants retained only the donor nucleus. Some of these had only a 2n nucleus derived from the same donor fish. Our results provide insights into the process of somatic cell nuclear transplantation in teleosts, and the cloning of Ranchu.

Embryonic fate after somatic cell nuclear transfer in non-enucleated goldfish oocytes is determined by first cleavages and DNA methylation patterns.

Reducing the variability in nuclear transfer outcome requires a better understanding of its cellular and epigenetic determinants, in order to ensure safer fish regeneration from cryobanked somatic material. In this work, clones from goldfish were obtained using cryopreserved fin cells as donor and non-enucleated oocytes as recipients. We showed that the high variability of clones survival was not correlated to spawn quality. Clones were then characterized for their first cleavages pattern in relation to their developmental fate up to hatching. The first cell cycle duration was increased in clones with abnormal first cleavage, and symmetric first two cleavages increased clone probability to reach later on 24 h- and hatching-stages. At 24 h-stage, 24% of the clones were diploids and from donor genetic origin only. However, ploidy and genetic origin did not determine clones morphological quality. DNA methylation reprogramming in the promoter region of pou2, nanog, and notail marker genes was highly variable, but clones with the nicest morphologies displayed the best DNA methylation reprogramming. To conclude, non-enucleated oocytes did allow authentic clones production. The first two cell cycles were a critical determinant of the clone ability to reach hatching-stage, and DNA methylation reprogramming significantly influenced clones morphological quality.

Optimization of somatic cell injection in the perspective of nuclear transfer in goldfish.

BACKGROUND: Nuclear transfer has the potential to become one strategy for fish genetic resources management, by allowing fish reconstruction from cryopreserved somatic cells. Survival rates after nuclear transfer are still low however. The part played by unsuitable handling conditions is often questioned, but the different steps in the procedure are difficult to address separately. In this work led on goldfish (Carassius auratus), the step of somatic cells injection was explored. Non-enucleated metaphase II oocytes were used as a template to explore the toxicity of the injection medium, to estimate the best location where the cell should be injected, and to assess the delay necessary between cell injection and oocyte activation. RESULTS: Trout coelomic fluid was the most suitable medium to maintain freshly spawned oocytes at the metaphase II stage during oocyte manipulation. Oocytes were then injected with several media to test their toxicity on embryo development after fertilization. Trout coelomic fluid was the least toxic medium after injection, and the smallest injected volume (10 pL) allowed the same hatching rates as the non injected controls (84.8% +/- 23). In somatic cell transfer experiments using non enucleated metaphase II oocytes as recipient, cell plasma membrane was ruptured within one minute after injection. Cell injection at the top of the animal pole in the oocyte allowed higher development rates than cell injection deeper within the oocyte (respectively 59% and 23% at mid-blastula stage). Embryo development rates were also higher when oocyte activation was delayed for 30 min after cell injection than when activation was induced without delay (respectively 72% and 48% at mid-blastula stage). CONCLUSIONS: The best ability of goldfish oocytes to sustain embryo development was obtained when the carrier medium was trout coelomic fluid, when the cell was injected close to the animal pole, and when oocyte activation was induced 30 min after somatic cell injection. Although the experiments were not designed to produce characterized clones, application of these parameters to somatic cell nuclear transfer experiments in enucleated metaphase II oocytes is expected to improve the quality of the reconstructed embryos.

Embryonic and developmental toxicity of the ionic liquid 1-methyl-3-octylimidazolium bromide on goldfish.

The embryonic developmental toxicity of the ionic liquid (IL) 1-methyl-3-octylimidazolium bromide ([C(8)mim]Br) on the goldfish Carassius auratus was evaluated in this study. First, the 72 h 50% lethal concentrations (72 h-LC(50)) for [C(8)mim]Br in goldfish embryos at the stages of cleavage, early gastrula, closure of blastopore, and heart beating were determined by preliminary acute toxicity tests. After that, fish embryos in different developmental stages (cleavage, early gastrula, closure of blastopore, and heart beating) were exposed to 10.4, 20.8, 41.6, and 104 mg/L of [C(8)mim]Br until their hatching stage. The results of the acute toxicity tests showed that 72 h-LC(50) values at the early cleavage, early gastrula, closure of blastopore, and heart beating stages of development were 208.96, 187.1, 245.03, and 298.33 mg/L, respectively. In the subchronic tests, [C(8)mim]Br exposure prolonged the duration of embryo dechorionation and decreased the hatching rates of the treated embryos compared to control embryos. In addition, [C(8)mim]Br treatment also caused remarkable increases of embryonic malformation and mortality ratio in most treatment groups. Finally, we also found that the embryonic and developmental toxicity of [C(8)mim]Br on fish embryos was dose-response and developmental stage-specific. These results indicate that [C(8)mim]Br has toxic effects on the early embryonic development of goldfish, and the risk to aquatic ecosystem by ILs leaking into the water body must be evaluated in the future.

Histone H2A has a novel variant in fish oocytes.

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.

Apo-14 is a fish-specific apolipoprotein and its biological function remains unknown. In this study, CagApo-14 was cloned from gibel carp (Carassius auratus gibelio) and its expression pattern was investigated during embryogenesis and early larval development. The CagApo-14 transcript and its protein product were firstly localized in the yolk syncytial layer at a high level during embryogenesis, and then found to be restricted to the digestive system including liver and intestine in later embryos and early larvae. Immunofluorescence staining in larvae and adults indicated that Cag Apo-14 protein was predominantly synthesized in and excreted from sinusoidal endothelial cells of liver tissue. Morpholino knockdown of Cag Apo-14 resulted in severe disruption of digestive organs including liver, intestine, pancreas and swim bladder. Moreover, yolk lipid transportation and utilization were severely affected in the Cag Apo-14 morphants. Overall, this data indicates that Cag Apo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.

Insertional mutagenesis in ChordinA induced by endogenous ΔTgf2 transposon leads to bifurcation of axial skeletal systems in grass goldfish.

The grass goldfish appeared early in the evolutionary history of goldfish, and shows heritable stability in the development of the caudal fin. The twin-tail phenotype is extremely rare, however, some twin-tail individuals were produced in the process of breeding for ornamental value. From mutations in the twin-tail goldfish genome, we identified two kinds of Tgf2 transposons. One type was completely sequenced Tgf2 and the other type was ΔTgf2, which had 858 bp missing. We speculate that the bifurcation of the axial skeletal system in goldfish may be caused by an endogenous ΔTgf2 insertion mutation in Chordin A, as ΔTgf2 has no transposition activity and blocks the expression of Chordin A. The twin-tail showed doubled caudal fin and accumulation of red blood cells in the tail. In addition, in situ hybridization revealed that ventral embryonic tissue markers (eve1, sizzled, and bmp4) were more widely and strongly expressed in the twin-tail than in the wild-type embryos during the gastrula stage, and bmp4 showed bifurcated expression patterns in the posterior region of the twin-tail embryos. These results provide new insights into the artificial breeding of genetically stable twin-tail grass goldfish families.

Morphological differences in embryos of goldfish (Carassius auratus auratus) under different incubation temperatures.

The goldfish (Carassius auratus auratus) is a useful species for embryonic micromanipulations because of its large egg size and wide temperature tolerance. Here, we describe in detail the rate of development and morphological characteristics of goldfish embryos incubated at temperatures between 10 °C and 30 °C. The cleavage speed increased rapidly as temperature increased. Synchronized cell divisions occurred at 131 min intervals at 10 °C, at 33 min intervals at 20 °C, and at 19 min intervals at 30 °C during the cleavage period. The rate of hatched abnormal embryos significantly increased at temperatures of 26 °C and above, while there was no change in the number of abnormal embryos at temperatures less than 24 °C. Moreover, the blastomeres around the center of the blastodisc rose in the direction of the animal pole at temperatures less than 14 °C. At the lower temperatures, clusters of maternally-supplied germplasm were visualized both at the ends of the first three cleavage furrows and at the border between the lower and upper tiers at the 16- to 32-cell stage, with injection of artificial mRNA and vasa in situ hybridization. This study showed that temperature affects not only developmental speed but also the shape of the blastodisc and the distribution of maternally-supplied materials in the blastodisc. By controlling the temperature, it is possible for researchers to prepare many stages of embryos and shapes of the blastodisc from a single batch of eggs.

Ectopic Six3 expression in the dragon eye goldfish.

For goldfish (Carassius auratus), there are many varieties with different eye phenotypes due to artificial selection and adaptive evolution. Dragon eye is a variant eye characterized by a large-size eyeball protruding out of the socket similar to the eye of dragon in Chinese legends. In this study, anatomical structure of the goldfish dragon eye was compared with that of the common eye, and a stretching of the retina was observed in the enlarged dragon eye. Moreover, the homeobox-containing transcription factor Six3 cDNAs were cloned from the two types of goldfish, and the expression patterns were analyzed in both normal eye and dragon eye goldfish. No amino acid sequence differences were observed between the two deduced peptides, and the expression pattern of Six3 protein in dragon eye is quite similar to common eye during embryogenesis, but from 2 days after hatching, ectopic Six3 expression began to occur in the dragon eye, especially in the outer nuclear layer cells. With eye development, more predominant Six3 distribution was detected in the outer nuclear layer cells of dragon eye than that of normal eye, and fewer cell-layers in outer nuclear layer were observed in dragon eye retina than in normal eye retina. The highlight of this study is that higher Six3 expression occurs in dragon eye goldfish than in normal eye goldfish during retinal development of larvae.