William Ka Fai Tse

Effect of osmotic shrinkage and hormones on the expression of Na+/H+ exchanger-1, Na+/K+/2Cl- cotransporter and Na+/K+ -ATPase in gill pavement cells of freshwater adapted Japanese eel, Anguilla japonica.

SUMMARY It is well-known that gill epithelial cells are important in fish osmoregulation. However, studies on the effect of osmotic stress on the direct cellular responses of the gill epithelial cells are limited. In this paper, we aimed to determine the effects of osmotic hypertonicity, hormones and cellular signaling molecules on the expression of ion transporters in the cultured primary freshwater pavement cells (PVCs), prepared from freshwater-adapted eels (Anguilla japonica). Our data demonstrated that the hypertonic (500 mOsmol l–1) treatment of the isolated PVCs induced cell shrinkage, followed by regulatory volume increase (RVI). Application of blockers (i.e. ouabain, bumetanide and EIPA) demonstrated that Na+/K+-ATPase, Na+/K+/2Cl– cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) were involved in RVI. Western blot analysis of the hypertonic-treated cells revealed a significant induction of NHE-1, NKCC and, α and β subunits of Na+/K+-ATPase. In nonshrunken cultured PVCs, we found that dexamethasone and dibutyryl cAMP treatments significantly stimulated the expression levels of the three ion transporters. Both prolactin and insulin-like growth factor-1, can only induce the expression of NKCC. The effect of thyroid hormone (T3) and dibutyryl cGMP was negligible. In this study, the induction of ion transporter expression was found to be post-transcriptionally regulated as no significant change in mRNA levels was detected. This observation implies that the regulation is rapid and is probably induced via nongenomic actions.

Transcriptomic and iTRAQ proteomic approaches reveal novel short-term hyperosmotic stress responsive proteins in the gill of the Japanese eel (Anguilla japonica).

UNLABELLED Osmoregulation is critical for the survival of fishes that migrate between freshwater (FW) and seawater (SW). The eel, as a catadromous fish, has been studied for decades to reveal the mechanisms of osmoregulation. These studies, however, have been limited by the lack of a genomic database to decipher the mechanism of osmoregulation at a molecular level. In this study, using high-throughput transcriptomic and proteomic technologies, we have provided the first genome-wide study to identify hyperosmotic responsive proteins in the gills of the Japanese eel. Deep sequencing using the 454 platform produced over 660,000 reads with a mean length of 385 bp. For the proteomic study, we collected gill samples from three different treatment groups of fish that had fully adapted to FW/SW or were transferred from FW to SW for 6h. The respective group of gill proteins were extracted and labeled using an isobaric tag for relative and absolute quantitation (iTRAQ) using LTQ-Orbitrap, a high resolution mass spectrometer. Among the 1519 proteins identified from the gill samples, 96 proteins were differentially expressed between FW and SW adapted fish. Nineteen hyperosmotic responsive proteins were detected (10 up-regulated and 9 down-regulated proteins) after 6h post FW to SW transfer. BIOLOGICAL SIGNIFICANCE The study has provided the most comprehensive, targeted investigation of eel gill proteins to date, and shown the powerfulness of combining transcriptomic and proteomic approaches to provide molecular insights of osmoregulation mechanisms in a non-model organism, eel.

Tissue-specific transcriptome assemblies of the marine medaka Oryzias melastigma and comparative analysis with the freshwater medaka Oryzias latipes

BackgroundThe marine medaka Oryzias melastigma has been demonstrated as a novel model for marine ecotoxicological studies. However, the lack of genome and transcriptome reference has largely restricted the use of O. melastigma in the assessment of in vivo molecular responses to environmental stresses and the analysis of biological toxicity in the marine environment. Although O. melastigma is believed to be phylogenetically closely related to Oryzias latipes, the divergence between these two species is still largely unknown. Using Illumina high-throughput RNA sequencing followed by de novo assembly and comprehensive gene annotation, we provided transcriptomic resources for the brain, liver, ovary and testis of O. melastigma. We also investigated the possible extent of divergence between O. melastigma and O. latipes at the transcriptome level.ResultsMore than 14,000 transcripts across brain, liver, ovary and testis in marine medaka were annotated, of which 5880 transcripts were orthologous between O. melastigma and O. latipes. Tissue-enriched genes were identified in O. melastigma, and Gene Ontology analysis demonstrated the functional specificity of the annotated genes in respective tissue. Lastly, the identification of marine medaka-enriched transcripts suggested the necessity of generating transcriptome dataset of O. melastigma.ConclusionsOrthologous transcripts between O. melastigma and O. latipes, tissue-enriched genes and O. melastigma-enriched transcripts were identified. Genome-wide expression studies of marine medaka require an assembled transcriptome, and this sequencing effort has generated a valuable resource of coding DNA for a non-model species. This transcriptome resource will aid future studies assessing in vivo molecular responses to environmental stresses and those analyzing biological toxicity in the marine environment.

The cloning of eel osmotic stress transcription factor and the regulation of its expression in primary gill cell culture

SUMMARY In the present study, we aimed to clone an osmotic stress transcriptional factor (Ostf) from gill cells of Japanese eels. In addition, we measured its expression in Percoll™-gradient-isolated gill chloride (CC) and pavement (PVC) cells and determined the regulation of its expression in primary gill cell culture. Using degenerative primers and RACE techniques, we cloned a cDNA of 615bp, encompassing the coding sequence of Ostf (204 amino acids). The cloned Ostf1 DNA sequence shared 84% DNA homology with the Ostf1 of tilapia. In general, the basal Ostf expression level was found to be significantly higher in CCs than in PVCs. In the direct transfer of fish from freshwater to seawater, a significant but transient induction of Ostf mRNA in CCs and PVCs was measured after 6h of acclimation. Compared with gill CCs, the level of induction measured at PVCs was lower. In the seawater-to-freshwater transfer, no significant change in Ostf transcript levels was detected in either CCs or PVCs. To decipher the regulatory mechanism of Ostf expression, we conducted experiments using primary gill cell culture to specifically address the involvement of two putative osmosensors (i.e. intracellular ion strength/macromolecular crowding and cytoskeleton) in the regulation of Ostf expression. Hypertonic treatment using impermeable solutes (i.e. NaCl, 500mOsmoll–1) induced Ostf mRNA expression in 6h, but no noticeable effect was measured using permeable solute (i.e. urea, 500mOsmoll–1). The induction was transcriptionally regulated and was abolished by the addition of organic osmolytes (i.e. betaine, inositol or taurine) into the culture media. Addition of colchicine (an inhibitor of microtubule polymerization) to hypertonic (with added NaCl, 500mOsmoll–1) cells reduced Ostf mRNA expression, suggesting that an increase in intracellular ionic strength and the integrity of the cytoskeleton are involved in the activation of Ostf mRNA expression in the cells. Collectively, the results of this study reveal, for the first time, the differential expression of Ostf in isolated CCs and PVCs. The resulting knowledge can shed light on how Ostf participates in hyperosmotic adaptation in fish gills.

Requirement for frzb and fzd7a in cranial neural crest convergence and extension mechanisms during zebrafish palate and jaw morphogenesis

Regulation of convergence and extension by wnt-frizzled signaling is a common theme in embryogenesis. This study examines the functional requirements of frzb and fzd7a in convergence and extension mechanisms during craniofacial development. Using a morpholino knockdown approach, we found that frzb and fzd7a are dispensable for directed migration of the bilateral trabeculae, but necessary for the convergence and extension of the palatal elements, where the extension process is mediated by chondrocyte proliferation, morphologic change and intercalation. In contrast, frzb and fzd7a are required for convergence of the mandibular prominences, where knockdown of either frzb or fzd7a resulted in complete loss of lower jaw structures. Further, we found that bapx1 was specifically downregulated in the wnt9a/frzb/fzd7a morphants, while general neural crest markers were unaffected. In addition, expression of wnt9a and frzb was also absent in the edn-/- mutant. Notably, over-expression of bapx1 was sufficient to partially rescue mandibular elements in the wnt9a/frzb/fzd7a morphants, demonstrating genetic epistasis of bapx1 acting downstream of edn1 and wnt9a/frzb/fzd7a in lower jaw development. This study underscores the important role of wnt-frizzled signaling in convergence and extension in palate and craniofacial morphogenesis, distinct regulation of upper vs. lower jaw structures, and integration of wnt-frizzled with endothelin signaling to coordinate shaping of the facial form.

Early embryogenesis in zebrafish is affected by bisphenol A exposure

Summary Exposure of a developing embryo or fetus to endocrine disrupting chemicals (EDCs) has been hypothesized to increase the propensity of an individual to develop a disease or dysfunction in his/her later life. Although it is important to understand the effects of EDCs on early development in animals, sufficient information about these effects is not available thus far. This is probably because of the technical difficulties in tracing the continuous developmental changes at different stages of mammalian embryos. The zebrafish, an excellent model currently used in developmental biology, provides new insights to the field of toxicological studies. We used the standard whole-mount in situ hybridization screening protocol to determine the early developmental defects in zebrafish embryos exposed to the ubiquitous pollutant, bisphenol A (BPA). Three stages (60–75% epiboly, 8–10 somite, and prim-5) were selected for in situ screening of different molecular markers, whereas BPA exposure altered early dorsoventral (DV) patterning, segmentation, and brain development in zebrafish embryos within 24 hours of exposure.

Triclosan (TCS) exposure impairs lipid metabolism in zebrafish embryos

Triclosan (TCS) is an active antimicrobial ingredient used in many household products, such as skin creams and toothpaste. It is produced in high volumes, and humans are directly exposed to it and dispose it on a daily basis. TCS has been found to contaminate water worldwide. This study aimed to understand the potential developmental and metabolic abnormalities caused by TCS exposure by using zebrafish as the experimental model. Four developmental stages (70-85% epiboly, 10-12 somite, prim-5, and 5dpf) were selected to perform in situ hybridization staining to investigate the effects of TCS on dorsal ventral patterning, segmentation, brain development, and organ formation. Results showed, in terms of developmental toxicology, that neither phenotypic nor molecular changes were found after 5 days of 250μg/L TCS exposure. However, such dosage of TCS exposure resulted in lipid droplet accumulation in the yolk sac, which might due to the deregulated mRNA expression level of beta-oxidation transcripts. This study showed that 250μg/L TCS exposure does not affect normal embryogenesis or organogenesis; however, there are concerns regarding possible impairment of lipid metabolism.

Medaka osmotic stress transcription factor 1b (Ostf1b/TSC22D3-2) triggers hyperosmotic responses of different ion transporters in medaka gill and human embryonic kidney cells via the JNK signalling pathway.

Eukaryotic cells undergo rapid regulatory processes to maintain cellular homeostasis upon osmotic stress. In fishes, gill epithelial cells play main roles in these processes. Although osmoregulatory functions of fish gills have been well studied, little is known about the underlying mechanisms, particularly the hypertonic-induced signalling pathways during osmotic stress. This study reports for the first time on the osmo-sensing signal cascade that related to the medaka osmotic stress transcription factor 1 (Ostf1), a hypertonic induced immediate early gene, under hypertonic stress. Quantitative real-time PCR showed the rapid increase of Ostf1 in gill after transfer of medaka from fresh water to 50% seawater; particularly Ostf1b whose mRNA expression increased to 4 folds at 0.5h and reached to 10 folds at 6h after the transfer. The in vivo knockdown of Ostf1b profoundly inhibited SEK and JNK phosphorylation, but not p38 and ERK phosphorylation in the medaka gill tissue. To further investigate the possible role of Ostf1b in the JNK pathway, Ostf1b was ectopically expressed in HEK293 cells. Results indicated that Ostf1b is a downstream target of SEK and JNK and exerts a positive feedback loop on the JNK signalling pathway via activation of GCK and/or MLK3 proteins. Additionally, MAPK inhibitors experiments suggested that activation of the JNK pathway by hypertonicity is involved in the maintenance of Ostf1b stability, which in turn provides continuous stimulation of GCK for JNK phosphorylation. Lastly, changes in transcription levels of different water/ion transporters were found in knockdown or ecoptic over-expression of Ostf1b in medaka gills and human embryonic kidney cells, suggesting the role of Ostf1b in modulation of critical water channel/ion transporters during osmotic stress.

Transcriptomic responses of corpuscle of Stannius gland of Japanese eels (Anguilla japonica) to Changes in Water Salinity

Physiological studies of a unique endocrine gland in fish, named corpuscles of Stannius (CS), described a Ca2+-regulatory function for this gland mediated by stanniocalcin-1, a hypocalcemic polypeptide hormone. However, to date, the endocrine functions of the glands have not been completely elucidated. We hypothesized that other unidentified active principles in the glands are involved in the regulation of plasma ion (Na+, Ca2+) and/or blood pressure. In this study, transcriptome sequencing of CS glands was performed using Japanese eels (Anguilla japonica) adapted to freshwater (FW) or seawater (SW) to reveal the presence and differential expression of genes encoding proteins related to the ion-osmoregulatory and pressor functions. We acquired a total of 14.1 Mb and 12.1 Mb quality-trimmed reads from the CS glands collected from FW and SW adapted eels, respectively. The de novo assembly resulted in 9254 annotated genes. Among them, 475 genes were differentially expressed with 357 up- and 118 down-regulated in the SW group. Gene ontology analysis further demonstrated the presence of natriuresis and pressor related genes. In summary, ours is the first study using high-throughput sequencing to identify gene targets that could explain the physiological importance of the CS glands.

iTRAQ-based quantitative proteomic analysis reveals acute hypo-osmotic responsive proteins in the gills of the Japanese eel (Anguilla japonica).

UNLABELLED Osmoregulation in fish has been a classical research topic for several decades. Salmon and eels are the widely used model animals because of their wide distribution in different geographical locations and spawning migration between fresh- and salt-water habitats. Numerous fish osmoregulatory hormones and ion transporters were identified for their essential roles in acclimation and adaptation to waters of different salinities. Because of the lack of a genomic database, the scope of most studies, however, is very limited. Recently, our group reported the first high-throughput transcriptomic and proteomic studies to identify hyperosmotic-responsive genes/proteins in gills of Japanese eels. In this study, we aimed to decipher changes in hypo-osmotic-responsive proteins in fish acclimating from seawater (SW) to freshwater (FW) conditions. We collected gill samples from SW-adapted and SW-to-FW-acclimating fish. The respective gill proteins were extracted and labeled using isobaric tags for relative and absolute quantitation (iTRAQ) and analyzed using a high-resolution mass spectrometer. In the short-term transfer from SW to FW, 51 hypo-responsive proteins were detected, and 24 unique hypo-osmotic-responsive proteins were identified (15 up-regulated and nine down-regulated proteins). Our data support the use of an omics approach to facilitate the application of functional genomics in non-model organisms. BIOLOGICAL SIGNIFICANCE By combining transcriptomic and proteomic approaches, the study has provided the most comprehensive, targeted investigation of eel gill hypo-osmotic responsive proteins that provides molecular insights of osmoregulation mechanisms in a non-model organism, eel. This article is part of a Special Issue entitled: Proteomics of non-model organisms.