Depletion of stearoyl-CoA desaturase (scd) leads to fatty liver disease and defective mating behavior in zebrafish.

Stearyl coenzyme A desaturase (SCD), also known as delta-9 desaturase, catalyzes the rate-limiting step in the formation of monounsaturated fatty acids. In mammals, depletion or inhibition of SCD activity generally leads to a decrease in triglycerides and cholesteryl esters. However, the endogenous role of scd in teleost fish remains unknown. Here, we generated a zebrafish scd mutant (scd-/-) to elucidate the role of scd in lipid metabolism and sexual development. Gas chromatography-mass spectrometry (GC-MS) showed that the scd-/- mutants had increased levels of saturated fatty acids C16:0 and C18:0, and decreased levels of monounsaturated fatty acids C16:1 and C18:1. The mutant fish displayed a short stature and an enlarged abdomen during development. Unlike Scd-/- mammals, the scd-/- zebrafish showed significantly increased fat accumulation in the whole body, especially in the liver, leading to hepatic mitochondrial dysfunction and severe cell apoptosis. Mechanistically, srebf1, a gene encoding a transcriptional activator related to adipogenesis, acc1 and acaca, genes involved in fatty acid synthesis, and dgat2, a key gene involved in triglyceride synthesis, were significantly upregulated in mutant livers to activate fatty acid biosynthesis and adipogenesis. The scd-/- males exhibited defective natural mating behavior due to defective genital papillae but possessed functional mature sperm. All defects in the scd-/- mutants could be rescued by ubiquitous transgenic overexpression of scd. In conclusion, our study demonstrates that scd is indispensable for maintaining lipid homeostasis and development of secondary sexual characteristics in zebrafish.

Deficiency of lrp4 in zebrafish and human LRP4 mutation induce aberrant activation of Jagged-Notch signaling in fin and limb development.

Low-density lipoprotein receptor-related protein 4 (LRP4) is a multi-functional protein implicated in bone, kidney and neurological diseases including Cenani-Lenz syndactyly (CLS), sclerosteosis, osteoporosis, congenital myasthenic syndrome and myasthenia gravis. Why different LRP4 mutation alleles cause distinct and even contrasting disease phenotypes remain unclear. Herein, we utilized the zebrafish model to search for pathways affected by a deficiency of LRP4. The lrp4 knockdown in zebrafish embryos exhibits cyst formations at fin structures and the caudal vein plexus, malformed pectoral fins, defective bone formation and compromised kidney morphogenesis; which partially phenocopied the human LRP4 mutations and were reminiscent of phenotypes resulting form a perturbed Notch signaling pathway. We discovered that the Lrp4-deficient zebrafish manifested increased Notch outputs in addition to enhanced Wnt signaling, with the expression of Notch ligand jagged1b being significantly elevated at the fin structures. To examine conservatism of signaling mechanisms, the effect of LRP4 missense mutations and siRNA knockdowns, including a novel missense mutation c.1117C > T (p.R373W) of LRP4, were tested in mammalian kidney and osteoblast cells. The results showed that LRP4 suppressed both Wnt/ß-Catenin and Notch signaling pathways, and these activities were perturbed either by LRP4 missense mutations or by a knockdown of LRP4. Our finding underscore that LRP4 is required for limiting Jagged-Notch signaling throughout the fin/limb and kidney development, whose perturbation representing a novel mechanism for LRP4-related diseases. Moreover, our study reveals an evolutionarily conserved relationship between LRP4 and Jagged-Notch signaling, which may shed light on how the Notch signaling is fine-tuned during fin/limb development.

[Development of resources, technologies and services at the China Zebrafish Resource Center].

With the rapid growth of the Chinese zebrafish community, there is an increasing demand for various types of zebrafish-related resources and technologies. The China Zebrafish Resource Center (CZRC, web: http://zfish.cn) was established at the Institute of Hydrobiology (IHB), Chinese Academy of Sciences (CAS) in 2012. Till now, CZRC has built the largest zebrafish aquaculture unit in China, organized a resource bank containing more than 1200 zebrafish lines and more than 10 000 frozen sperm samples, among which over 200 mutant and transgenic lines were generated by CZRC. CZRC has established several technical supporting platforms, such as the zebrafish husbandry and health control program of international standard, a high-efficient gene manipulation technology platform, and a stable and efficient sperm cryopreservation technology platform. The main task of CZRC is to provide different types of services to zebrafish investigators in China and worldwide, such as resource services (e.g. zebrafish lines), technical services (e.g. gene knockout) and transgenic services, consultancy services (e.g. zebrafish husbandry and health consultation), and conference services [e.g. holding regular technical training courses and biennale Chinese Zebrafish Principal Investigator Meeting (CZPM)]. After five years' development, CZRC is now recognized as one of the three major resource centers in the global zebrafish community.

A novel method to identify hub pathways of rheumatoid arthritis based on differential pathway networks.

The aim of the current study was to identify hub pathways of rheumatoid arthritis (RA) using a novel method based on differential pathway network (DPN) analysis. The present study proposed a DPN where protein­protein interaction (PPI) network was integrated with pathway­pathway interactions. Pathway data was obtained from background PPI network and the Reactome pathway database. Subsequently, pathway interactions were extracted from the pathway data by building randomized gene­gene interactions and a weight value was assigned to each pathway interaction using Spearman correlation coefficient (SCC) to identify differential pathway interactions. Differential pathway interactions were visualized using Cytoscape to construct a DPN. Topological analysis was conducted to identify hub pathways that possessed the top 5% degree distribution of DPN. Modules of DPN were mined according to ClusterONE. A total of 855 pathways were selected to build pathway interactions. By filtrating pathway interactions of weight values >0.7, a DPN with 312 nodes and 791 edges was obtained. Topological degree analysis revealed 15 hub pathways, such as heparan sulfate/heparin­glycosaminoglycan (HS­GAG) degradation, HS­GAG metabolism and keratan sulfate degradation for RA based on DPN. Furthermore, hub pathways were also important in modules, which validated the significance of hub pathways. In conclusion, the proposed method is a computationally efficient way to identify hub pathways of RA, which identified 15 hub pathways that may be potential biomarkers and provide insight to future investigation and treatment of RA.

[A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos].

Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, including the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions.

Serum Levels of IL-6 and TNF-α May Correlate with Activity and Severity of Rheumatoid Arthritis.

BACKGROUND We aimed to investigate the association of rheumatoid arthritis (RA) with interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) through a meta-analysis. MATERIAL AND METHODS The case-control studies that investigated the association between RA and serum levels of IL-6 and TNF-α were retrieved strictly according to the inclusion and exclusion criteria. The statistical analysis was performed using STATA statistical software (Version 12.0, Stata Corporation, College Station, TX, USA). RESULTS Fourteen studies were enrolled in our meta-analysis, with a total of 890 patients with RA and 441 healthy people as the controls. The results of this meta-analysis revealed that the serum IL-6 and TNF-α levels of RA patients were significantly higher than in the controls, and this difference was statistically significant (IL-6: SMD=2.40, 95% CI=1.57~3.24, P<0.001; TNF-α: SMD=1.93, 95% CI=1.23~2.64, P<0.001). According to ethnic subgroup analysis, the serum IL-6 and TNF-α levels of RA patients were also significantly higher compared with the controls in Asians and Caucasians (IL-6: Asians: SMD=3.64, 95% CI=2.16~5.12, P<0.001; Caucasians: SMD=0.75, 95% CI=0.47~1.02, P<0.001; TNF-α: Asians: SMD=2.74, 95%CI=1.58~3.91, P<0.001; Caucasians: SMD=0.81, 95% CI=0.50~1.11, P<0.001). CONCLUSIONS IL-6 and TNF-α may play crucial roles in the activity and severity of RA.

Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos.

DNA double-strand break (DSB) repair is of considerable importance for genomic integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are considered as two major mechanistically distinct pathways involved in repairing DSBs. In recent years, another DSB repair pathway, namely, microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ is generally believed to utilize an alternative mechanism to repair DSBs when NHEJ and other mechanisms fail. In this study, we utilized zebrafish as an in vivo model to study DSB repair and demonstrated that efficient MMEJ repair occurred in the zebrafish genome when DSBs were induced using TALEN (transcription activator-like effector nuclease) or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technologies. The wide existence of MMEJ repair events in zebrafish embryos was further demonstrated via the injection of several in vitro-designed exogenous MMEJ reporters. Interestingly, the inhibition of endogenous ligase 4 activity significantly increased MMEJ frequency, and the inhibition of ligase 3 activity severely decreased MMEJ activity. These results suggest that MMEJ in zebrafish is dependent on ligase 3 but independent of ligase 4. This study will enhance our understanding of the mechanisms of MMEJ in vivo and facilitate inducing desirable mutations via DSB-induced repair.

Transcriptional Activity and DNA Methylation Dynamics of the Gal4/UAS System in Zebrafish.

The Gal4/upstream activating sequence (UAS) system is a powerful genetic tool for the temporal and spatial expression of target genes. In this study, the dynamic activity of the Gal4/UAS system was monitored in zebrafish throughout the entire lifespan and during germline transmission, using an optimized Gal4/UAS, KalTA4/4xUAS, which is driven by two muscle-specific regulatory sequences. We found that UAS-linked gene expression was transcriptionally amplified by Gal4/UAS during early developmental stages and that the amplification effects tended to weaken during later stages and even disappear in subsequent generations. In the F2 generation, the transcription of a UAS-linked enhanced green fluorescent protein (EGFP) reporter was transcriptionally silent from 16 days post-fertilization (dpf) into adulthood, yet offspring of this generation showed reactivation of the EGFP reporter in some strains. We further show that the transcriptional silencing and reactivation of UAS-driven EGFP correlated with the DNA methylation levels of the UAS regulatory sequences. Notably, asymmetric DNA methylation of the 4xUAS occurred in oocytes and sperm. Moreover, the paternal and maternal 4xUAS sequences underwent different DNA methylation dynamics after fertilization. Our study suggests that the Gal4/UAS system may represent a powerful tool for tracing the DNA methylation dynamics of paternal and maternal loci during zebrafish development and that UAS-specific DNA methylation should be seriously considered when the Gal4/UAS system is applied in zebrafish.

Tissue-specific derepression of TCF/LEF controls the activity of the Wnt/ß-catenin pathway.

Upon stimulation by Wnt ligands, the canonical Wnt/ß-catenin signalling pathway results in the stabilization of ß-catenin and its translocation into the nucleus to form transcriptionally active complexes with sequence-specific DNA-binding T-cell factor/lymphoid enhancer factor (TCF/LEF) family proteins. In the absence of nuclear ß-catenin, TCF proteins act as transcriptional repressors by binding to Groucho/Transducin-Like Enhancer of split (TLE) proteins that function as co-repressors by interacting with histone deacetylases whose activity leads to the generation of transcriptionally silent chromatin. Here we show that the transcription factor Ladybird homeobox 2 (Lbx2) positively controls the Wnt/ß-catenin signalling pathway in the posterior lateral and ventral mesoderm of the zebrafish embryo at the gastrula stage, by directly interfering with the binding of Groucho/TLE to TCF, thereby preventing formation of transcription repressor complexes. These findings reveal a novel level of regulation of the canonical Wnt/ß-catenin signalling pathway occurring in the nucleus and involving tissue-specific derepression of TCF by Lbx2.

Cross-species cloning: influence of cytoplasmic factors on development.

It is widely accepted that the crosstalk between naive nucleus and maternal factors deposited in the egg cytoplasm before zygotic genome activation is crucial for early development. This crosstalk may also exert some influence on later development. It is interesting to clarify the relative roles of the zygotic genome and the cytoplasmic factors in development. Cross-species nuclear transfer (NT) between two distantly related species provides a unique system to study the relative role and crosstalk between egg cytoplasm and zygotic nucleus in development. In this review, we will summarize the recent progress of cross-species NT, with emphasis on the cross-species NT in fish and the influence of cytoplasmic factors on development. Finally, we conclude that the developmental process and its evolution should be interpreted in a systemic way, rather than in a way that solely focuses on the role of the nuclear genome.

Double transgenesis of humanized fat1 and fat2 genes promotes omega-3 polyunsaturated fatty acids synthesis in a zebrafish model.

Omega-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential nutrients for human health. However, vertebrates, including humans, have lost the abilities to synthesize EPA and DHA de novo, majorly due to the genetic absence of delta-12 desaturase and omega-3 desaturase genes. Fishes, especially those naturally growing marine fish, are major dietary source of EPA and DHA. Because of the severe decline of marine fishery and the decrease in n-3 LC-PUFA content of farmed fishes, it is highly necessary to develop alternative sources of n-3 LC-PUFA. In the present study, we utilized transgenic technology to generate n-3 LC-PUFA-rich fish by using zebrafish as an animal model. Firstly, fat1 was proved to function efficiently in fish culture cells, which showed an effective conversion of n-6 PUFA to n-3 PUFA with the n-6/n-3 ratio that decreased from 7.7 to 1.1. Secondly, expression of fat1 in transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.8- and 2.4-fold, respectively. Third, co-expression of fat2, a fish codon-optimized delta-12 desaturase gene, and fat1 in fish culture cell significantly promoted n-3 PUFA synthesis with the decreased n-6/n-3 ratio from 7.7 to 0.7. Finally, co-expression of fat1 and fat2 in double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.7- and 2.8-fold, respectively. Overall, we generated two types of transgenic zebrafish rich in endogenous n-3 LC-PUFA, fat1 transgenic zebrafish and fat1/fat2 double transgenic zebrafish. Our results demonstrate that application of transgenic technology of humanized fat1 and fat2 in farmed fishes can largely improve the n-3 LC-PUFA production.

Transcriptional factors smad1 and smad9 act redundantly to mediate zebrafish ventral specification downstream of smad5.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that play crucial roles during embryonic development and cell fate determination. Nuclear transduction of BMP signals requires the receptor type Smad proteins, Smad1, Smad5, and Smad9. However, how these Smad proteins cooperate in vivo to regulate various developmental processes is largely unknown. In zebrafish, it was widely believed that the maternally expressed smad5 is essential for dorso-ventral (DV) patterning, and the zygotically transcribed smad1 is not required for normal DV axis establishment. In the present study, we have identified zygotically expressed smad9, which cooperates with smad1 downstream of smad5, to mediate zebrafish early DV patterning in a functional redundant manner. Although knockdown of smad1 or smad9 alone does not lead to visible dorsalization, double knockdown strongly dorsalizes zebrafish embryos, which cannot be efficiently rescued by smad5 overexpression, whereas the dorsalization induced by smad5 knockdown can be fully rescued by overexpression of smad1 or smad9. We have further revealed that the transcription initiations of smad1 and smad9 are repressed by each other, that they are direct transcriptional targets of Smad5, and that smad9, like smad1, is required for myelopoiesis. In conclusion, our study uncovers that smad1 and smad9 act redundantly to each other downstream of smad5 to mediate ventral specification and to regulate embryonic myelopoiesis.

[Nuclear transfer and reprogramming in fish].

As an important sub-field in the study of animal cloning, fish nuclear transfer was first established in the early 1960s by Chinese embryologists. Due to its advantages, zebrafish has become a unique animal model to study the mystery of reprogramming in nuclear transfer. This article summarizes the history and current situation in fish nuclear transfer technology and discusses the factors that may influence the development of the cloned embryos. A comprehensive understand-ing of the mechanism for epigenetic modification following nuclear transfer, such as genomic DNA methylation and histone acetylation and/or methylation, will likely increase the success rate and eventually lead to the future freedom of cloning technique.

Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation.

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.

Targeted expression in zebrafish primordial germ cells by Cre/loxP and Gal4/UAS systems.

In zebrafish and other vertebrates, primordial germ cells (PGCs) are a population of embryonic cells that give rise to sperm and eggs in adults. Any type of genetically manipulated lines have to be originated from the germ cells of the manipulated founders, thus it is of great importance to establish an effective technology for highly specific PGC-targeted gene manipulation in vertebrates. In the present study, we used the Cre/loxP recombinase system and Gal4/UAS transcription system for induction and regulation of mRFP (monomer red fluorescent protein) gene expression to achieve highly efficient PGC-targeted gene expression in zebrafish. First, we established two transgenic activator lines, Tg(kop:cre) and Tg(kop:KalTA4), to express the Cre recombinases and the Gal4 activator proteins in PGCs. Second, we generated two transgenic effector lines, Tg(kop:loxP-SV40-loxP-mRFP) and Tg(UAS:mRFP), which intrinsically showed transcriptional silence of mRFP. When Tg(kop:cre) females were crossed with Tg(kop:loxP-SV40-loxP-mRFP) males, the loxP flanked SV40 transcriptional stop sequence was 100 % removed from the germ cells of the transgenic hybrids. This led to massive production of PGC-specific mRFP transgenic line, Tg(kop:loxP-mRFP), from an mRFP silent transgenic line, Tg(kop:loxP-SV40-loxP-mRFP). When Tg(kop:KalTA4) females were crossed with Tg(UAS:mRFP) males, the hybrid embryos showed PGC specifically expressed mRFP from shield stage till 25 days post-fertilization (pf), indicating the high sensitivity, high efficiency, and long-lasting effect of the Gal4/UAS system. Real-time PCR analysis showed that the transcriptional amplification efficiency of the Gal4/UAS system in PGCs can be about 300 times higher than in 1-day-pf embryos. More importantly, when the UAS:mRFP-nos1 construct was directly injected into the Tg(kop:KalTA4) embryos, it was possible to specifically label the PGCs with high sensitivity, efficiency, and persistence. Therefore, we have established two targeted gene expression platforms in zebrafish PGCs, which allows us to further manipulate the PGCs of zebrafish at different levels.

Graphene-based anticancer nanosystem and its biosafety evaluation using a zebrafish model.

In this paper, a facile strategy to develop graphene-based delivery nanosystems for effective drug loading and sustained drug release was proposed and validated. Specifically, biocompatible naphthalene-terminated PEG (NP) and anticancer drugs (curcumin or doxorubicin (DOX)) were simultaneously integrated onto oxidized graphene (GO), leading to self-assembled, nanosized complexes. It was found that the oxidation degree of GO had a significant impact on the drug-loading efficiency and the structural stability of nanosystems. Interestingly, the nanoassemblies resulted in more effective cellular entry of DOX in comparison with free DOX or DOX-loaded PEG-polyester micelles at equivalent DOX dose, as demonstrated by confocal microscopy studies. Moreover, the nanoassemblies not only exhibited a sustained drug release pattern without an initial burst release, but also significantly improved the stability of formulations which were resistant to drug leaking even in the presence of strong surfactants such as aromatic sodium benzenesulfonate (SBen) and aliphatic sodium dodecylsulfonate (SDS). In addition, the nanoassemblies without DOX loading showed negligible in vitro cytotoxicity, whereas DOX-loaded counterparts led to considerable toxicity against HeLa cells. The DOX-mediated cytotoxicity of the graphene-based formulation was around 20 folds lower than that of free DOX, most likely due to the slow DOX release from complexes. A zebrafish model was established to assess the in vivo safety profile of curcumin-loaded nanosystems. The results showed they were able to excrete from the zebrafish body rapidly and had nearly no influence on the zebrafish upgrowth. Those encouraging results may prompt the advance of graphene-based nanotherapeutics for biomedical applications.

[Estimating total nitrogen content in wetland vegetation based on measured reflectance spectra].

More and more urban wetlands have been supplied with reclaimed water. And monitoring the growth condition of large-area wetland vegetation is playing a very important role in wetland restoration and reconstruction. Recently, remote sensing technology has become an important tool for vegetation growth monitoring. The South Wetland in the Olympic Park, a typical wetland using reused water, was selected as the research area. The leaf reflectance spectra and were acquired for the main wetland plants reed (Phragmites australis) and cattail (Typha angustifolia) with an ASD FieldSpec 3 spectrometer (350 2 500 nm). The total nitrogen (TN) content of leaf samples was determined by Kjeldahl method subsequently. The research established univariate models involving simple ratio spectral index (SR) model and normalized difference spectral index (ND) model, as well as multivariate models including stepwise multiple linear regression (SMLR) model and partial least squares regression (PLSR) model. Moreover, the accuracy of all the models was tested through cross-validated coefficient of determination (R2(CV)) and cross-validated root mean square error (RMSE(CV)). The results showed that (1) comparing different types of wetland plants, the accuracy of all established prediction models using Phragmites australis reflectance spectra was higher than that using Typha angustifolia reflectance spectra. (2) compared with univariate techniques, multivariate regressions improved the estimation of TN concentration in leaves. (3) among the various investigated models, the accuracy of PLSR model was the highest (R2(CV) = 0.80, RMSE(CV) = 0.24). PLSR provided the most useful explorative tool for unraveling the relationship between spectral reflectance and TN consistence of leaves. The result would not only provide a scientific basis for remote sensing retrieval of biochemical variables of wetland vegetation, but also provide a strong scientific basis for the monitoring and management of urban wetlands using recycled water.