Transcriptomic Characterization of Key Factors and Signaling Pathways for the Regeneration of Partially Hepatectomized Liver in Zebrafish.

Liver regeneration induced by partial hepatectomy (PHx) has attracted intensive research interests due to the great significance for liver resection and transplantation. The zebrafish (Danio rerio) is an excellent model to study liver regeneration. In the fish subjected to PHx (the tip of the ventral lobe was resected), the lost liver mass could be fully regenerated in seven days. However, the regulatory mechanisms underlying the liver regeneration remain largely unknown. In this study, gene expression profiles during the regeneration of PHx-treated liver were explored by RNA sequencing (RNA-seq). The genes responsive to the injury of PHx treatment were identified and classified into different clusters based on the expression profiles. Representative gene ontology (GO) enrichments for the early responsive genes included hormone activity, ribosome biogenesis and rRNA processing, etc., while the late responsive genes were enriched in biological processes such as glutathione metabolic process, antioxidant activity and cellular detoxification. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments were also identified for the differentially expressed genes (DEGs) between the time-series samples and the sham controls. The proteasome was overrepresented by the up-regulated genes at all of the sampling time points. Inhibiting proteasome activity by the application of MG132 to the fish enhanced the expression of Pcna (proliferating cell nuclear antigen), an indicator of hepatocyte proliferation after PHx. Our data provide novel insights into the molecular mechanisms underlying the regeneration of PHx-treated liver.

Biochemical, histological and transcriptomic analyses for the immunological organs provide insights into heat stress-induced disease susceptibility in Largemouth Bass.

Aquaculture of Largemouth Bass (LMB, Micropterus salmoides), an economically important species, is badly affected by the outbreak of bacterial diseases in summer. However, the mechanisms underlying heat-induced disease susceptibility remain largely unknown. In this study, after exposure to 34 °C for 1, 7 and 14 d, the head kidney, spleen and blood of LMB were sampled for biochemical and histological assays to explore the effects of heat exposure on the oxidative and immunological indices. Compared to the controls maintained at 28 °C, chronic heat exposure (34 °C for 14 d) induced oxidative stress, caused cell apoptosis and decreased expression of the immunological genes in the head kidney and spleen tissues; and attenuated the blood immunological indices. Consistent with the impaired immunological functions, chronic heat exposure predisposed LMB to Aeromonas hydrophila infection and significantly (p < 0.001) increased tissue bacterial load. Furthermore, the effects of chronic heat exposure (heat), A. hydrophila infection (infection) and heat exposure followed by A. hydrophila infection (heat + infection) on gene expression in the head kidney and spleen of LMB were characterized by RNA sequencing. The results indicated that chronic heat exposure facilitated the bacteria-elicited changes in expression of the genes involved in a couple of metabolic and signaling pathways in both tissues. Upon heat + infection, the pathways involved in energy production and nutrients biosynthesis were enhanced, whereas those associated with the host cell functions such as cell-cell interactions and cell signaling were depressed. Our data provide new insights into the mechanisms underlying heat-induced disease susceptibility in LMB.

Evidence for Adverse Effects on Liver Development and Regeneration in Zebrafish by Decabromodiphenyl Ethane.

Decabromodiphenyl ethane (DBDPE), a ubiquitous emerging pollutant, could be enriched in the liver of organisms, but its effects and mechanisms on liver development and regeneration remain largely unknown. In the present study, we first investigated the adverse effects on liver development and found decreased area and intensity of fluorescence in transgenic zebrafish larvae exposed to DBDPE; further results in wild-type zebrafish larvae revealed a possible mechanism involving disturbed MAPK/Fox O signaling pathways and cell cycle arrest as indicated by decreased transcription of growth arrest and DNA-damage-inducible beta a (gadd45ba). Subsequently, an obstructed recovery process of liver tissue after partial hepatectomy was characterized by the changing profiles of ventral lobe-to-intestine ratio in transgenic female adults upon DBDPE exposure; further results confirmed the adverse effects on liver regeneration by the alterations of the hepatic somatic index and proliferating cell nuclear antigen expression in wild-type female adults and also pointed out a potential role of a disturbed signaling pathway involving cell cycles and glycerolipid metabolism. Our results not only provided novel evidence for the hepatotoxicity and underlying mechanism of DBDPE but also were indicative of subsequent ecological and health risk assessment.

Transcriptome Analysis Reveals the Molecular Basis of Overfeeding-Induced Diabetes in Zebrafish.

Diabetes has gradually become a serious disease that threatens human health. It can induce various complications, and the pathogenesis of diabetes is quite complex and not yet fully elucidated. The zebrafish has been widely acknowledged as a useful model for investigating the mechanisms underlying the pathogenesis and therapeutic interventions of diabetes. However, the molecular basis of zebrafish diabetes induced by overfeeding remains unknown. In this study, a zebrafish diabetes model was established by overfeeding, and the molecular basis of zebrafish diabetes induced by overfeeding was explored. Compared with the control group, the body length, body weight, and condition factor index of zebrafish increased significantly after four weeks of overfeeding. There was a significant elevation in the fasting blood glucose level, accompanied by a large number of lipid droplets accumulated within the liver. The levels of triglycerides and cholesterol in both the serum and liver exhibited a statistically significant increase. Transcriptome sequencing was employed to investigate changes in the livers of overfed zebrafish. The number of up-regulated and down-regulated differentially expressed genes (DEGs) was 1582 and 2404, respectively, in the livers of overfed zebrafish. The DEGs were subjected to KEGG and GO enrichment analyses, and the hub signaling pathways and hub DEGs were identified. The results demonstrate that sixteen genes within the signal pathway associated with fatty acid metabolism were found to be significantly up-regulated. Specifically, these genes were found to mainly participate in fatty acid transport, fatty acid oxidation, and ketogenesis. Furthermore, thirteen genes that play a crucial role in glucose metabolism, particularly in the pathways of glycolysis and glycogenesis, were significantly down-regulated in the livers of overfed zebrafish. These results indicate insulin resistance and inhibition of glucose entry into liver cells in the livers of overfed zebrafish. These findings elucidate the underlying molecular basis of zebrafish diabetes induced by overfeeding and provide a model for further investigation of the pathogenesis and therapeutics of diabetes.

Dysfunction of Prkcaa Links Social Behavior Defects with Disturbed Circadian Rhythm in Zebrafish.

Protein kinase Cα (PKCα/PRKCA) is a crucial regulator of circadian rhythm and is associated with human mental illnesses such as autism spectrum disorders and schizophrenia. However, the roles of PRKCA in modulating animal social behavior and the underlying mechanisms remain to be explored. Here we report the generation and characterization of prkcaa-deficient zebrafish (Danio rerio). The results of behavioral tests indicate that a deficiency in Prkcaa led to anxiety-like behavior and impaired social preference in zebrafish. RNA-sequencing analyses revealed the significant effects of the prkcaa mutation on the expression of the morning-preferring circadian genes. The representatives are the immediate early genes, including egr2a, egr4, fosaa, fosab and npas4a. The downregulation of these genes at night was attenuated by Prkcaa dysfunction. Consistently, the mutants demonstrated reversed day-night locomotor rhythm, which are more active at night than in the morning. Our data show the roles of PRKCA in regulating animal social interactions and link the social behavior defects with a disturbed circadian rhythm.

Effects of Nutritionally Induced Obesity on Metabolic Pathways of Zebrafish.

Human obesity has become a global epidemic that can lead to many metabolic diseases, including insulin resistance, type 2 diabetes, dyslipidemia, hypertension and nonalcoholic fatty liver. The development of obesity is closely associated with excess food intake and energy imbalance, family history, lifestyle, psychology and other factors, but molecular mechanisms underlying the induction and development of obesity remain to be intensively studied under a variety of internal and external pathogenesis conditions. In this study, we generated two obesity models of zebrafish that were treated with a high-fat diet (HFD) or an overfeeding diet (DIO). Both HFD and DIO zebrafish exhibited higher levels of lipid accumulation, fat distribution, microvascular steatosis and ectopic accumulation of lipid droplets in liver and muscle than normal diet (NOD) fish. The comparison of transcriptome sequencing data for the livers of HFD, DIO and NOD groups identified common and specific genes and signaling pathways that are potentially associated with zebrafish obesity induced by HFD and/or DIO. These findings provide clues for further understanding the mechanisms of obesity development and preventing nutritionally induced obesity through targeting the common signaling pathways and biological processes.

Topical drug delivery strategies for enhancing drug effectiveness by skin barriers, drug delivery systems and individualized dosing.

Topical drug delivery is widely used in various diseases because of the advantages of not passing through the gastrointestinal tract, avoiding gastrointestinal irritation and hepatic first-pass effect, and reaching the lesion directly to reduce unnecessary adverse reactions. The skin helps the organism to defend itself against a huge majority of external aggressions and is one of the most important lines of defense of the body. However, the skin's strong barrier ability is also a huge obstacle to the effectiveness of topical medications. Allowing the bioactive, composition in a drug to pass through the stratum corneum barrier as needed to reach the target site is the most essential need for the bioactive, composition to exert its therapeutic effect. The state of the skin barrier, the choice of delivery system for the bioactive, composition, and individualized disease detection and dosing planning influence the effectiveness of topical medications. Nowadays, enhancing transdermal absorption of topically applied drugs is the hottest research area. However, enhancing transdermal absorption of drugs is not the first choice to improve the effectiveness of all drugs. Excessive transdermal absorption enhances topical drug accumulation at non-target sites and the occurrence of adverse reactions. This paper introduces topical drug delivery strategies to improve drug effectiveness from three perspectives: skin barrier, drug delivery system and individualized drug delivery, describes the current status and shortcomings of topical drug research, and provides new directions and ideas for topical drug research.

Understanding the Function and Mechanism of Zebrafish Tmem39b in Regulating Cold Resistance.

Autophagy and endoplasmic reticulum (ER) stress response are among the key pathways regulating cold resistance of fish through eliminating damaged cellular components and facilitating the restoration of cell homeostasis upon exposure to acute cold stress. The transmembrane protein 39A (TMEM39A) was reported to regulate both autophagy and ER stress response, but its vertebrate-specific paralog, the transmembrane protein 39B (TMEM39B), has not been characterized. In the current study, we generate tmem39b-knockout zebrafish lines and characterize their survival ability under acute cold stress. We observed that the dysfunction of Tmem39b remarkably decreased the cold resilience of both the larval and adult zebrafish. Gene transcription in the larvae exposed to cold stress and rewarming were characterized by RNA sequencing (RNA-seq) to explore the mechanisms underlying functions of Tmem39b in regulating cold resistance. The results indicate that the deficiency of Tmem39b attenuates the up-regulation of both cold- and rewarming-induced genes. The cold-induced transcription factor genes bif1.2, fosab, and egr1, and the rewarming-activated immune genes c3a.3, il11a, and sting1 are the representatives influenced by Tmem39b dysfunction. However, the loss of tmem39b has little effect on the transcription of the ER stress response- and autophagy-related genes. The measurements of the phosphorylated H2A histone family member X (at Ser 139, abbreviated as γH2AX) demonstrate that zebrafish Tmem39b protects the cells against DNA damage caused by exposure to the cold-warming stress and facilitates tissue damage repair during the recovery phase. The gene modules underlying the functions of Tmem39b in zebrafish are highly enriched in biological processes associated with immune response. The dysfunction of Tmem39b also attenuates the up-regulation of tissue C-reactive protein (CRP) content upon rewarming. Together, our data shed new light on the function and mechanism of Tmem39b in regulating the cold resistance of fish.

Mitochondria Dysfunction and Cell Apoptosis Limit Resistance of Nile Tilapia (Oreochromis niloticus) to Lethal Cold Stress.

Inability of Nile tilapia (Oreochromis niloticus) to withstand cold stress represents a major economic concern, which restricts the culture area, limits the growing period and even results in mass mortality in cold seasons. However, the cellular and molecular mechanisms determining cold susceptibility of Nile tilapia remain largely unknown. In this study, we characterized the ability of juvenile Nile tilapia to survive lethal cold stress (12 °C) and the median survival time (LT50) of the experimental fish under exposure to 12 °C cold stress was estimated as 3.14 d. After being exposed to 12 °C for 3 d, the survivors that lost equilibrium (LE) and those that swam normally (NO) were regarded as cold-sensitive and cold-tolerant, respectively. The untreated (Ctrl), NO and LE fish were subjected to histological, biochemical and gene expression analyses to explore the cellular and molecular events underlying cold susceptibility of Nile tilapia. Exposure of Nile tilapia to lethal cold stress caused systemic tissue structure changes, mitochondrial swelling and dysfunction, induction of apoptosis and endoplasmic reticulum (ER) stress-related genes and cell apoptosis. The extent of these adverse cellular and molecular events determines an individual's ability to survive cold stress. Our data indicate that mitochondria dysfunction and mitochondria-mediated cell apoptosis are the main factors limiting Nile tilapia's cold resistance.

Comparative Transcriptome Analysis Provides Novel Molecular Events for the Differentiation and Maturation of Hepatocytes during the Liver Development of Zebrafish.

The liver plays an essential role in multiple biological functions including metabolism, detoxification, digestion, coagulation, and homeostasis in vertebrates. The specification and differentiation of embryonic hepatoblasts, the proliferation of hepatocytes, and the hepatic tissue architecture are well documented, but molecular events governing the maturation of hepatocytes during liver development remain largely unclear. In this study, we performed a comparative transcriptome analysis of hepatocytes that were sorted by flow cytometry from developing zebrafish embryos at 60, 72, and 96 hpf. We identified 667 up-regulated and 3640 down-regulated genes in hepatocytes between 60 and 72 hpf, 606 up-regulated and 3924 down-regulated genes between 60 and 96 hpf, and 1693 up-regulated genes and 1508 down-regulated genes between 72 and 96 hpf. GO enrichment analysis revealed that key biological processes, cellular components, and molecular functions in hepatocytes between 60 to 72 hpf, such as cell cycle, DNA replication, DNA repair, RNA processing, and transcription regulation, are mainly associated with the proliferation of hepatocytes. In addition to biological processes, cellular components, and molecular functions for cell proliferation, molecular functions for carbohydrate metabolism were enriched in hepatocytes during 72 to 96 hpf. KEGG enrichment analysis identified key signaling pathways, such as cell cycle, RNA degradation, ubiquitin-mediated proteolysis, ErbB and Hedgehog signaling, basal transcription factors, Wnt signaling, and glycan degradation, which are closely associated with cell proliferation or carbohydrate metabolism in hepatocytes between 60 to 72 hpf. Newly enriched signaling pathways in hepatocytes during 72 to 96 hpf include metabolisms of pyrimidine, purine, nicotinate and nicotinamide, caffeine, glycine, serine and threonine, ABC transporters, and p53 signaling that function in metabolisms of lipid, protein and energy, cellular secretion, or detoxification, indicating the functional maturation of hepatocytes between 72 to 96 hpf. These findings provide novel clues for further understanding the functional differentiation and maturation of hepatocytes during liver development.

Transcriptomic Profiling Revealed Signaling Pathways Associated with the Spawning of Female Zebrafish under Cold Stress.

As one of the critical abiotic factors, temperature controls fish development and reproduction. However, the effects of low temperature on the transcriptional regulation of zebrafish reproduction remain largely unclear. In this study, the fecundity of zebrafish was examined after exposure to cold temperatures at 19.5 °C, 19 °C, 18.5 °C, or 18 °C. The temperature at 19 °C showed no significant influence on the fecundity of zebrafish, but temperature at 18.5 °C or 18 °C significantly blocked the spawning of females, suggesting the existence of a low temperature critical point for the spawning of zebrafish females. Based on these observations, the brains of anesthetized fish under cold stress at different cold temperatures were collected for high-throughput RNA-seq assays. Key genes, hub pathways and important biological processes responding to cold temperatures during the spawning of zebrafish were identified through bioinformatic analysis. The number of down-regulated and up-regulated genes during the temperature reduction from egg-spawning temperatures at 19.5 °C and 19 °C to non-spawning temperatures at 18.5 °C and 18 °C were 2588 and 2527 (fold change ≥ 1.5 and p-value ≤ 0.01), respectively. Venn analysis was performed to identify up- and down-regulated key genes. KEGG enrichment analysis indicated that the hub pathways overrepresented among down-regulated key genes included the GnRH signaling pathway, vascular smooth muscle contraction, C-type lectin receptor signaling pathway, phosphatidylinositol signaling system and insulin signaling pathway. GO enrichment analysis of down-regulated key genes revealed the most important biological processes inhibited under non-spawning temperatures at 18.5 °C and 18 °C were photoreceptor cell outer segment organization, circadian regulation of gene expression and photoreceptor cell maintenance. Furthermore, 99 hormone-related genes were found in the brain tissues of non-spawning and spawning groups, and GnRH signaling pathway and insulin signaling pathway were enriched from down-regulated genes related to hormones at 18.5 °C and 18 °C. Thus, these findings uncovered crucial hormone-related genes and signaling pathways controlling the spawning of female zebrafish under cold stress.

Bis (2-ethylhexyl)-2,3,4,5-tetrabromophthalate showed poor penetrability but increased the permeability of blood brain barrier: Evidences from in vitro and in vivo studies.

Bis(2ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH), a replacement for restricted flame retardants, has become ubiquitous in the environment. To reveal the neurotoxicity and underlying mechanism of TBPH, we first evaluated its penetrability through the blood-brain barrier (BBB) using hCMEC/D3 cells as in vitro model, and found TBPH had poor penetrability through BBB with a maximum Papp of 14.8 × 10-6 cms-1. Further study using transgenetic zebrafish (Tg flk1: EGFP) as in vivo model confirmed that TBPH could affect the BBB permeability, probably via affecting the transcription of genes encoding tight junction proteins. Finally, wild type zebrafish embryos/larvae were exposed to TBPH to evaluate the neurotoxicity. The neurodevelopment, neurotransmitters and locomotor activity of zebrafish larvae did not changed, which may be because TBPH can hardly cross the BBB to pose direct exposure to the central nervous system. However, the transcription of opsins genes and visual response to light stimulation in zebrafish larvae were inhibited, pointing to additional mechanism that may cause visual impairment indirectly. Above all, these results can help further understand the neurotoxicity and underlying mechanism by TBPH, and also pointed out potential risk of this chemical to aquatic organisms.

Functions of SMC2 in the Development of Zebrafish Liver.

SMC2 (structural maintenance of chromosomes 2) is the core subunit of condensins, which play a central role in chromosome organization and segregation. However, the functions of SMC2 in embryonic development remain poorly understood, due to the embryonic lethality of homozygous SMC2-/- mice. Herein, we explored the roles of SMC2 in the liver development of zebrafish. The depletion of SMC2, with the CRISPR/Cas9-dependent gene knockout approach, led to a small liver phenotype. The specification of hepatoblasts was unaffected. Mechanistically, extensive apoptosis occurred in the liver of SMC2 mutants, which was mainly associated with the activation of the p53-dependent apoptotic pathway. Moreover, an aberrant activation of a series of apoptotic pathways in SMC2 mutants was involved in the defective chromosome segregation and subsequent DNA damage. Therefore, our findings demonstrate that SMC2 is necessary for zebrafish liver development.

Generation of GCaMP6s-Expressing Zebrafish to Monitor Spatiotemporal Dynamics of Calcium Signaling Elicited by Heat Stress.

The ability of organisms to quickly sense and transduce signals of environmental stresses is critical for their survival. Ca2+ is a versatile intracellular messenger involved in sensing a wide variety of stresses and regulating the subsequent cellular responses. So far, our understanding for calcium signaling was mostly obtained from ex vivo tissues and cultured cell lines, and the in vivo spatiotemporal dynamics of stress-triggered calcium signaling in a vertebrate remains to be characterized. Here, we describe the generation and characterization of a transgenic zebrafish line with ubiquitous expression of GCaMP6s, a genetically encoded calcium indicator (GECI). We developed a method to investigate the spatiotemporal patterns of Ca2+ events induced by heat stress. Exposure to heat stress elicited immediate and transient calcium signaling in developing zebrafish. Cells extensively distributed in the integument of the head and body trunk were the first batch of responders and different cell populations demonstrated distinct response patterns upon heat stress. Activity of the heat stress-induced calcium signaling peaked at 30 s and swiftly decreased to near the basal level at 120 s after the beginning of exposure. Inhibition of the heat-induced calcium signaling by LaCl3 and capsazepine and treatment with the inhibitors for CaMKII (Ca²2/calmodulin-dependent protein kinase II) and HSF1 (Heat shock factor 1) all significantly depressed the enhanced heat shock response (HSR). Together, we delineated the spatiotemporal dynamics of heat-induced calcium signaling and confirmed functions of the Ca2+-CaMKII-HSF1 pathway in regulating the HSR in zebrafish.

Characterization of Biological Pathways Regulating Acute Cold Resistance of Zebrafish.

Low temperature stress represents a major threat to the lives of both farmed and wild fish species. However, biological pathways determining the development of cold resistance in fish remain largely unknown. Zebrafish larvae at 96 hpf were exposed to lethal cold stress (10 °C) for different time periods to evaluate the adverse effects at organism, tissue and cell levels. Time series RNA sequencing (RNA-seq) experiments were performed to delineate the transcriptomic landscape of zebrafish larvae under cold stress and during the subsequent rewarming phase. The genes regulated by cold stress were characterized by progressively enhanced or decreased expression, whereas the genes associated with rewarming were characterized by rapid upregulation upon return to normal temperature (28 °C). Genes such as trib3, dusp5 and otud1 were identified as the representative molecular markers of cold-induced damages through network analysis. Biological pathways involved in cold stress responses were mined from the transcriptomic data and their functions in regulating cold resistance were validated using specific inhibitors. The autophagy, FoxO and MAPK (mitogen-activated protein kinase) signaling pathways were revealed to be survival pathways for enhancing cold resistance, while apoptosis and necroptosis were the death pathways responsible for cold-induced mortality. Functional mechanisms of the survival-enhancing factors Foxo1, ERK (extracellular signal-regulated kinase) and p38 MAPK were further characterized by inhibiting their activities upon cold stress and analyzing gene expression though RNA-seq. These factors were demonstrated to determine the cold resistance of zebrafish through regulating apoptosis and p53 signaling pathway. These findings have provided novel insights into the stress responses elicited by lethal cold and shed new light on the molecular mechanisms underlying cold resistance of fish.

Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus.

Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

Establishment and characterization of a cold-sensitive neural cell line from the brain of tilapia (Oreochromis niloticus).

The aquaculture of tilapia (Oreochromis sp.) is adversely affected by the sensitivity to cold stress. A large number of genes in tilapia were found to be regulated by cold stress, but their functions and mechanisms in cold tolerance remain largely unknown, partially due to the lack of a suitable in vitro model. An immortal neural cell line designated as tilapia brain neural (TBN) was established from brain tissue of the genetically improved farmed tilapia strain of Nile tilapia (Oreochromis niloticus). The TBN cells show a neuron-like morphology at low density and form a fibroblast-like monolayer at high density. Transcriptome profiling through RNA-sequencing revealed that a total of 15,011 genes were expressed in the TBN cells. The TBN cells express a wide array of marker genes for neural cells. A comparative analysis of the featured genes among the 17 cell clusters isolated from the subventricular zone of mouse brain revealed the highest transcriptome similarity between the TBN cells and the transient amplifying progenitors (TAPs). The TBN cells tolerate relatively high culture temperatures, and the highest growth rate was observed for the cells cultured at 32°C compared with those at 30°C, 28°C and 26°C. Nonetheless, this cell line is cold sensitive. Exposure of the cells to 16°C or lower temperatures significantly decreased cell confluences and induced apoptosis. The TBN cells were more sensitive to cold stress than the ZF4 cells (embryonic zebrafish fibroblasts). Moreover, the TBN cells can be efficiently transfected through electroporation. This study provides an invaluable research tool to understand the nature of cold sensitivity of tilapia and to dissect the function and mechanism of genes in regulating cold tolerance of fish.

Transcriptional Programs Underlying Cold Acclimation of Common Carp (Cyprinus carpio L.).

Properly regulated transcriptional responses to environmental perturbations are critical for the fitness of fish. Although gene expression profiles in the tissues of common carp upon cold stress were previously characterized, the transcriptional programs underlying cold acclimation are still not well known. In this study, the ability of three common carp strains including Hebao red carp (HB), Songpu mirror carp (SPM) and Yellow river carp (YR) to establish cold resistance after acclimation to a mild hypothermia stress at 18°C for 24 h was confirmed by measurements of the critical thermal minimums (CTMin). The gene expression profiles of the brain and the heart from these strains under both control and cold-acclimated conditions were characterized with RNA-sequencing. The data of the three common carp strains with different genetic background were combined in the differential gene expression analyses to balance the effects of genetic diversity on gene expression. Marked effects of tissue origins on the cold-induced transcriptional responses were revealed by comparing the differentially expressed gene (DEG) lists of the two tissues. Functional categories including spliceosome and RNA splicing were highly enriched in the DEGs of both tissues. However, steroid biosynthesis was specifically enriched in DEGs of the brain and response to unfolded protein was solely enriched in DEGs of the heart. Consistent with the up-regulation of the genes involved in cholesterol biosynthesis, total cholesterol content of the brain was significantly increased upon cold stress. Moreover, cold-induced alternative splicing (AS) events were explored and AS of the rbmx (RNA-binding motif protein, X chromosome) gene was confirmed by real-time quantitative PCR. Finally, a core set of cold responsive genes (CRGs) were defined by comparative transcriptomic analyses. Our data provide insights into the transcriptional programs underlying cold acclimation of common carp and offer valuable clues for further investigating the genetic determinants for cold resistance of farmed fish.

De novo transcriptome assembly and sex-biased gene expression in the gonads of Amur catfish (Silurus asotus).

Amur catfish is extensively distributed and cultured in Asian countries. Despite of economic importance, the genomic information of this species remains limited. A reference transcriptome of Amur catfish was assembled and the sex-biased gene expression in the gonads was characterized using RNA-sequencing. The assembled transcriptome of Amur catfish consisted of 74,840 transcripts. The N50, mean length and max length of transcripts are 1970, 1235 and 16,748 bp. Putative sex-specific transcripts were identified and sex-specific expression of the representative genes was verified by RT-PCR. Differential expression analysis identified 5401 ovary-biased and 5618 testis-biased genes. The ovary-biased genes were mainly enriched in pathways such as RNA transport and ribosome biogenesis in eukaryotes. The testis-biased genes were enriched in calcium signaling and cytokine-cytokine receptor interaction, etc. Our data provide a valuable genomic resource for further investigating the genetic basis of sex determination, sex differentiation and sexual dimorphism of catfish.

Dynamic Phosphoproteome Profiling of Zebrafish Embryonic Fibroblasts during Cold Acclimation.

Temperature affects almost all aspects of the fish life. To cope with low temperature, fish have evolved the ability of cold acclimation for survival. However, intracellular signaling events underlying cold acclimation in fish remain largely unknown. Here, the formation of cold acclimation in zebrafish embryonic fibroblasts (ZF4) is monitored and the phosphorylation events during the process are investigated through a large-scale quantitative phosphoproteomic approach. In total, 11 474 phosphorylation sites are identified on 4066 proteins and quantified 5772 phosphosites on 2519 proteins. Serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation accounted for 85.5%, 13.3%, and 1.2% of total phosphosites, respectively. Among all phosphosites, 702 phosphosites on 510 proteins show differential regulation during cold acclimation of ZF4 cells. These phosphosites are divided into six clusters according to their dynamic changes during cold exposure. Kinase-substrate prediction reveals that mitogen-activated protein kinase (MAPK) among the kinase groups is predominantly responsible for phosphorylation of these phosphosites. The differentially regulated phosphoproteins are functionally associated with various cellular processes such as regulation of actin cytoskeleton and MAPK signaling pathway. These data enrich the database of protein phosphorylation sites in zebrafish and provide key clues for the elucidation of intracellular signaling networks during cold acclimation of fish.