Metamorphosis Of Japanese Flounder Research Articles

The differential expression and effects of Drosha on metamorphosis of Japanese flounder (paralichthys olivaceus)

Drosha is critical for producing mature microRNAs (miRNAs) from their precursor molecules and small interfering RNAs (siRNAs). Although Drosha has been well characterized in most vertebrate species, identifying the Japanese flounder Drosha has remained elusive. In this study, we cloned the Japanese flounder Drosha gene, which has 4,122 nucleotides (nt), including a 5’-untranslated region (UTR) of 14 nt, a 3’-UTR of 230 nt, and a 3,879 nt open reading frame (ORF) encodes a 1,292 amino acid polypeptide. Then, we analyzed the conservation and phylogenetic evolution of Drosha in some species. Real-time quantitative PCR revealed that Drosha mRNA is highly expressed in the brain, and a lower amount of mRNA was also found in muscle than in other tissues. Drosha plays a vital role in controlling flounder development and metabolism, and its mRNA levels were relatively high at 36 dph (days post-hatching) in the period of metamorphosis and growth of the Japanese flounder. Drosha expression showed in advance with a peak at 23 dph following TH (thyroid hormone) treatment. To further investigate the role of Drosha in metamorphosis, we performed siRNA knockdown of Drosha in flounder embryonic cells (FEC) cells. The result shows that the Drosha-specific siRNA significantly down-regulated Drosha mRNA and miR-1,133,17,214,181a,181b levels, while primary miRNA (pri-miRNA) levels were not significantly different from negative control (NC). These results suggest that Drosha plays a vital role in Japanese flounder development and metamorphosis through processing to produce mature miRNAs, providing essential information for further studying of the part of the Drosha gene in the Japanese flounder development.

MiR-430a regulates the development of left-right asymmetry by targeting sqt in the teleost.

microRNAs (miRNAs) are short, endogenous non-coding RNAs that contain approximately 18-22 nucleotides. miRNAs are involved in gene regulation by recognizing and binding the 3'UTR of target gene. In our previous data, miR-430 family showed significant differential expression modes through metamorphosis in Japanese flounder. It was speculated that miR-430a plays a key role in left-right patterning. We predicted the targets of miR-430a and gene ontology (GO) was performed. We speculated miR-430a is involved in the basal molecular function and organ development. In Japanese flounder, sqt as a target of miR-430a was enriched into heart development term. Sqt has been reported to participate in mesendoderm formation and organ development. Cardiac morphogenesis is the first asymmetric development process, which breaks left-right symmetry in bilateria. It was used as a marker to detect L-R asymmetric effects of miR-430a. Overexpression and suppression of miR-430a resulted in abnormal KV (Kupffer's vesicles) development and disordered in nodal-related expression with consequent cardiac laterality. Squint mRNA of Japanese flounder (Posqt) as a target of miR-430a was overexpressed and caused similar phenotype with miR-430a suppression group, such as longer cilia in KV and high range of clmc2 and spaw ectopic expression. Moreover, rescue experiments were performed and suggested that cardiac and KV defections, induced by overexpressing miR-430a, could be rescued by injecting Posqt mRNA. These results suggested that miR-430a regulates the development of left-right asymmetry by targeting sqt in the teleost.

The role of miR-92 in regulating early development and metamorphosis of Japanese flounder Paralichthys olivaceus.

MicroRNAs are a class of short non-coding RNAs that contain approximately 22 nucleotides and play a regulatory role in RNA silencing and translational repression. miR-92 belongs to the miR-17-92 family and has a regulatory effect on cell proliferation, apoptosis, and expression of proto-oncogenes and tumor suppressor genes. However, its function in flatfish is unclear. In this study, we used farmed Japanese flounder, Paralichthys olivaceus, and showed that gata5 is a target gene of miR-92. Experiments on miR-92 overexpression indicated that gata5 and sox17 were downregulated, while the transcription level of ntl increased. By contrast, depletion of miR-92 resulted in increased gata5 and sox17 levels and reduced ntl level. Moreover, thiourea treatment indicated that miR-92 may inhibit the metamorphic development of Japanese flounder. Our study suggests that miR-92 regulates the fate of endoderm and mesoderm by controlling gata5.

Identification and Differential Expression of MicroRNAs during Metamorphosis of the Japanese Flounder (Paralichthys olivaceus)

BackgroundMicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20–25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA.Methodology/Principal FindingsSolexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis.Conclusions/SignificanceWe identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.

Differential expression of <I>HSP</I>90 genes and its relation with thyroid hormone during metamorphosis in Japanese flounder(<I>Paralichthys olivaceus</I>)

HSP90 proteins are major chaperones that play pivotal roles in controlling multiple regulatory pathways such as hormone signaling,cell differentiation,cell proliferation and apoptosis,morphogenesis and morphological evolution,metamorphosis and stress defense.Using real-time fluorescent quantitative RT-PCR approach,expression profiles of HSP90 genes were examined during larval development and in adult tissues in Japanese flounder,Paralichthys olivaceus.In adult tissues,HSP90α was mainly expressed with higher transcript levels in skeletal muscle,intestine and stomach;but HSP90β exhibited higher mRNA levels in brain,spleen and kidney.During metamorphosis,HSP90α transcript increased distinctly with the highest mRNA levels at the metamorphosis climax;whereas HSP90β mRNA levels did not change.Due to the important role of thyroid hormone(TH)for the flounder metamorphosis,the transcriptional regulation of HSP90 genes by TH was evaluated using exogenous TH and thiourea(TU)treatment.Compared with untreated control,HSP90α transcript increased significantly in larvae sampled 8 and 13 days after TH treatment,and TU-treated larvae exhibited lower HSP90α mRNA levels.In contrast,the transcript levels of HSP90β did not vary after TH and TU treatment.Overall,these results demonstrate that HSP90α transcription is up-regulated by thyroid hormone and plays an important role during metamorphosis in Japanese flounder.

Expression of insulin-like growth factor I receptors at mRNA and protein levels during metamorphosis of Japanese flounder ( Paralichthys olivaceus)

Insulin-like growth factor I (IGF-I) is an important regulator of fish growth and development, and its biological actions are initiated by binding to IGF-I receptor (IGF-IR). Our previous study has revealed that IGF-I could play an important role during metamorphosis of Japanese flounder, Paralichthys olivaceus. The analysis of IGF-IR expression thus helps further elucidate the IGF-I regulation of metamorphic processes. In this study, the spatial–temporal expression of two distinct IGF-IR mRNAs was investigated by real-time RT-PCR. The spatial distribution of two IGF-IR mRNAs in adult tissues is largely overlapped, but they exhibit distinct temporal expression patterns during larval development. A remarkable decrease in IGF-IR-2 mRNA was detected during metamorphosis. In contrast, a significant increase in IGF-IR-1 mRNA was determined from pre-metamorphosis to metamorphic completion. These indicate that they may play different function roles during the flounder metamorphosis. The levels and localization of IGF-IR proteins during larval development were further studied by Western blotting and immunohistochemistry. Immunoreactive IGF-IRs were detected throughout larval development, and the IGF-IR proteins displayed a relatively abundant expression during metamorphosis. Moreover, the IGF-IR proteins appeared in key tissues, such as thickened skin beneath the migrating eye, developing intestine, gills and kidney during metamorphosis. These results further suggest that the IGF-I system may be involved in metamorphic development of Japanese flounder.

Cloning and stage-specific expression of CK-M1 gene during metamorphosis of Japanese flounder, Paralichthys olivaceus

The symmetrical body of flatfish larvae changes dramatically into an asymmetrical form after metamorphosis. The molecular mechanisms responsible for this change are poorly understood. As an initial step to clarify these mechanisms, we used representational difference analysis of cDNA for the identification of genes active during metamorphosis in the Japanese flounder, Paralichthys olicaceus. One of the up-regulated genes was identified as creatine kinase muscle type 1 (CK-M1). Sequence analysis of CK-M1 revealed that it spanned 1 708 bp and encoded a protein of 382 amino acids. The overall amino acid sequence of the CK-M1 was highly conserved with those of other organisms. CK-M1 was expressed in adult fish tissues, including skeletal muscle, intestine and gill. Whole mount in-situ hybridization showed that the enhanced expression of CK-M1 expanded from the head to the whole body of larvae as metamorphosis progressed. Quantitative analysis revealed stage-specific high expression of CK-M1 during metamorphosis. The expression level of CK-M1 increased initially and peaked at metamorphosis, decreased afterward, and finally returned to the pre-metamorphosis level. This stage-specific expression pattern suggested strongly that CK-M1 was related to metamorphosis in the Japanese flounder. Its specific role in metamorphosis requires further study.

Characterization of thyroid hormones and thyroid hormone receptors during the early development of Pacific bluefin tuna ( Thunnus orientalis)

We studied the profiles of 3,5,3′- l-triiodothyronine (T 3), thyroxine (T 4), and thyroid hormone receptors (TRs) in Pacific bluefin tuna ( Thunnus orientalis) during embryonic and post-embryonic development. Both T 3 and T 4 were detected in embryos just before hatching, and it was found that the levels of both were increased in postflexion fish. The thyroid follicles were increased in both size and number in postflexion fish compared with preflexion fish. A TRβ cDNA clone was generated by RACE. Two TRα cDNA clones were also partially identified and analyzed by real-time RT-PCR in this study. The TR mRNA levels in embryos were determined, and these were found to be lower than those in preflexion fish. Therefore, we considered that thyroid hormones function during early post-embryonic development as well as during embryonic development. Moreover, there was a peak in the TR mRNA level during postflexion stages, as seen during metamorphosis in Japanese flounder and Japanese conger eel. It is possible that thyroid hormones control the early development of scombrid fish through TRs, as they do for Pluronectiformes and Anguilliformes.

Changes in growth performance and proximate composition in Japanese flounder during metamorphosis

Morphological changes are described in Japanese flounder Paralichthys olivaceus larvae and juveniles with emphasis on growth during the period of metamorphosis. Ontogenetic changes in condition factor and lipid, glycogen and protein levels were also analysed to determine the utilization of stored energy. Fish grew from 6·6 to 20·3 mm LT(1·15 to 84·4 mg in mass, M) during the period from 11 to 40 days after hatching (DAH) at 19·3° C. Per cent specific growth rate per day (%G day−1) for wet mass was lowest during the metamorphic phase (21–30 DAH) compared to pre‐(11–20 DAH) and post‐metamorphic (31–40 DAH) phases. When LT and M were expressed as , the b value was highest during the pre‐metamorphic phase and lowest during the metamorphic phase. These findings indicate that the developmental changes that occur during metamorphosis of Japanese flounder are closely related to the growth pattern. Moisture, lipid and glycogen contents were also at the lowest level during metamorphosis compared to pre‐ and post‐metamorphosis, which suggest that Japanese flounder use their energy reserves to accomplish metamorphosis due to an apparent decline in feeding during this period.

Asymmetrical development of bones and soft tissues during eye migration of metamorphosing Japanese flounder, Paralichthys olivaceus.

The symmetrical body of flatfish larvae dramatically changes into an asymmetrical form after metamorphosis. Eye migration results in the most significant asymmetrical development seen in any vertebrate. To understand the mechanisms involved in eye migration, bone and cartilage formation was observed during metamorphosis in laboratory-reared Japanese flounder, Paralichthys olivaceus, by using whole-body samples and histological sections. Most of the hard tissues of the cranium (parasphenoid, trabecular cartilage, supraorbital canal, and supraorbital bar) exist symmetrically in the larval period before metamorphosis and develop by twisting in the same direction as that in which the eye migrates. An increase in skin thickness beneath the eye was observed only on the blind side at the beginning of eye migration; this was the first definitive difference between the right and left sides of the body. The pseudomesial bar, a peculiar bone present only in flatfishes, developed from this thick skin and grew dorsad. Novel sac-like structures were found and named retrorbital vesicles. The retrorbital vesicle of the blind side grew larger and faster than that of the ocular side when the right eye moved most dramatically, whereas no difference was observed between the volume of right and left connective tissue in the head. The asymmetrical presence and growth of the pseudomesial bar together with inflation of the retrorbital vesicle on the blind side may be responsible for right eye migration during metamorphosis in the Japanese flounder.