Co-Expression of Neighboring Genes in the Zebrafish (Danio rerio) Genome

Neighboring genes in the eukaryotic genome have a tendency to express concurrently, and the proximity of two adjacent genes is often considered a possible explanation for their co-expression behavior. However, the actual contribution of the physical distance between two genes to their co-expression behavior has yet to be defined. To further investigate this issue, we studied the co-expression of neighboring genes in zebrafish, which has a compact genome and has experienced a whole genome duplication event. Our analysis shows that the proportion of highly co-expressed neighboring pairs (Pearson’s correlation coefficient R>0.7) is low (0.24% ~ 0.67%); however, it is still significantly higher than that of random pairs. In particular, the statistical result implies that the co-expression tendency of neighboring pairs is negatively correlated with their physical distance. Our findings therefore suggest that physical distance may play an important role in the co-expression of neighboring genes. Possible mechanisms related to the neighboring genes’ co-expression are also discussed.     

An Association between Insulin Resistance and Neurodegeneration in Zebrafish Larval Model (Danio rerio)

Background: Type 2 diabetes mellitus has recently been identified as a mediator of neurodegeneration. However, the molecular mechanisms have not been clearly elucidated. We aimed to investigate insulin resistance associated with neurodegenerative events in zebrafish larvae. Methods: Larvae aged 72 h-post-fertilization (hpf) were induced to insulin resistance by immersion in 250 nM insulin and were then reinduced with 100 nM insulin at 96 hpf. This model was validated by a glucose levels assay, qPCR analysis of selected genes (akt, pepck, zglut3 and claudin-5a) and Oil Red-O (ORO) staining of the yolk sac for lipid distribution. The association of insulin resistance and neurodegeneration was validated by malondialdehyde (MDA), glutathione (GSH) assays, and by integrating next-generation sequencing with database for annotation, visualization and integrated discovery (DAVID). Results: There was a significant increase in glucose levels at 180 min in the insulin-resistant group. However, it decreased at 400 min after the re-challenge. Insulin-signaling mediators, akt and pepck, were showed significantly downregulated up to 400 min after insulin immersion (p < 0.05). Meanwhile, claudin-5a assessed blood–brain barrier (BBB) integrity and showed significant deterioration after 400 min of post-insulin immersion. ORO staining remarked the increase in yolk sac size in the insulin-resistant group. After the confirmation of insulin resistance, MDA levels increased significantly in the insulin-resistant group compared to the control group in the following parameters. Furthermore, dysregulated MAPK- and Wnt/Ca²⁺-signaling pathways were observed in the insulin-resistant group, disrupting energy metabolism and causing BBB injury. Conclusions: We conclude that the insulin-resistant zebrafish larvae alter the metabolic physiology associated with neurodegeneration.     

Localization of BDNF and Calretinin in Olfactory Epithelium and Taste Buds of Zebrafish (Danio rerio)

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and it is involved in several fundamental functions in the central and peripheral nervous systems, and in sensory organs. BDNF regulates the chemosensory systems of mammals and is consistently expressed in those organs. In zebrafish, the key role of BDNF in the biology of the hair cells of the inner ear and lateral line system has recently been demonstrated. However, only some information is available about its occurrence in the olfactory epithelium, taste buds, and cutaneous isolated chemosensory cells. Therefore, this study was undertaken to analyze the involvement of BDNF in the chemosensory organs of zebrafish during the larval and adult stages. To identify cells displaying BDNF, we compared the cellular pattern of BDNF-displaying cells with those immunoreactive for calretinin and S100 protein. Our results demonstrate the localization of BDNF in the sensory part of the olfactory epithelium, mainly in the ciliated olfactory sensory neurons in larvae and adult zebrafish. Intense immunoreaction for BDNF was also observed in the chemosensory cells of oral and cutaneous taste buds. Moreover, a subpopulation of olfactory sensory neurons and chemosensory cells of olfactory rosette and taste bud, respectively, showed marked immunopositivity for calcium-binding protein S100 and calretinin. These results demonstrate the possible role of BDNF in the development and maintenance of olfactory sensory neurons and sensory cells in the olfactory epithelium and taste organs of zebrafish during all stages of development.     

One Host-Multiple Applications: Zebrafish (Danio rerio) as Promising Model for Studying Human Cancers and Pathogenic Diseases

In recent years, zebrafish (ZF) has been increasingly applied as a model in human disease studies, with a particular focus on cancer. A number of advantages make it an attractive alternative for mice widely used so far. Due to the many advantages of zebrafish, modifications can be based on different mechanisms and the induction of human disease can take different forms depending on the research goal. Genetic manipulation, tumor transplantation, or injection of the pathogen are only a few examples of using ZF as a model. Most of the studies are conducted in order to understand the disease mechanism, monitor disease progression, test new or alternative therapies, and select the best treatment. The transplantation of cancer cells derived from patients enables the development of personalized medicine. To better mimic a patient’s body environment, immune-deficient models (SCID) have been developed. A lower immune response is mostly generated by genetic manipulation but also by irradiation or dexamethasone treatment. For many studies, using SCID provides a better chance to avoid cancer cell rejection. In this review, we describe the main directions of using ZF in research, explain why and how zebrafish can be used as a model, what kind of limitations will be met and how to overcome them. We collected recent achievements in this field, indicating promising perspectives for the future.     

Apex Resection in Zebrafish (Danio rerio) as a Model of Heart Regeneration: A Video-Assisted Guide

Ischemic heart disease is one of the leading causes of deaths worldwide. A major hindrance to resolving this challenge lies in the mammalian hearts inability to regenerate after injury. In contrast, zebrafish retain a regenerative capacity of the heart throughout their lifetimes. Apex resection (AR) is a popular zebrafish model for studying heart regeneration, and entails resecting 10–20% of the heart in the apex region, whereafter the regeneration process is monitored until the heart is fully regenerated within 60 days. Despite this popularity, video tutorials describing this technique in detail are lacking. In this paper we visualize and describe the entire AR procedure including anaesthesia, surgery, and recovery. In addition, we show that the concentration and duration of anaesthesia are important parameters to consider, to balance sufficient levels of sedation and minimizing mortality. Moreover, we provide examples of how zebrafish heart regeneration can be assessed both in 2D (immunohistochemistry of heart sections) and 3D (analyses of whole, tissue cleared hearts using multiphoton imaging). In summary, this paper aims to aid beginners in establishing and conducting the AR model in their laboratory, but also to spur further interest in improving the model and its evaluation.     

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.     

Brain Proteome and Behavioural Analysis in Wild Type,BDNF+/− and BDNF−/− Adult Zebrafish (Danio rerio) Exposed to Two Different Temperatures

Experimental evidence suggests that environmental stress conditions can alter the expression of BDNF and that the expression of this neurotrophin influences behavioural responses in mammalian models. It has been recently demonstrated that exposure to 34 °C for 21 days alters the brain proteome and behaviour in zebrafish. The aim of this work was to investigate the role of BDNF in the nervous system of adult zebrafish under control and heat treatment conditions. For this purpose, zebrafish from three different genotypes (wild type, heterozygous BDNF^+/− and knock out BDNF^−/−) were kept for 21 days at 26 °C or 34 °C and then euthanized for brain molecular analyses or subjected to behavioural tests (Y-maze test, novel tank test, light and dark test, social preference test, mirror biting test) for assessing behavioural aspects such as boldness, anxiety, social preference, aggressive behaviour, interest for the novel environment and exploration. qRT-PCR analysis showed the reduction of gene expression of BDNF and its receptors after heat treatment in wild type zebrafish. Moreover, proteomic analysis and behavioural tests showed genotype- and temperature-dependent effects on brain proteome and behavioural responding. Overall, the absent expression of BDNF in KO alters (1) the brain proteome by reducing the expression of proteins involved in synapse functioning and neurotransmitter-mediated transduction; (2) the behaviour, which can be interpreted as bolder and less anxious and (3) the cellular and behavioural response to thermal treatment.     

Effects of Cadmium Exposure on Gut Villi in Danio rerio

In aquatic organisms, cadmium exposure occurs from ovum to death and the route of absorption is particularly wide, being represented by skin, gills and gastrointestinal tract, through which contaminated water and/or preys are ingested. It is known that cadmium interferes with the gut; however, less information is available on cadmium effects on an important component of the gut, namely goblet cells, specialized in mucus synthesis. In the present work, we studied the effects of two sublethal cadmium concentrations on the gut mucosa of Danio rerio. Particular attention was paid to changes in the distribution of glycan residues, and in metallothionein expression in intestinal cells. The results show that cadmium interferes with gut mucosa and goblet cells features. The effects are dose- and site-dependent, the anterior gut being more markedly affected than the midgut. Cadmium modifies the presence and/or distribution of glycans in the brush border and cytoplasm of enterocytes and in the goblet cells’ cytoplasm and alters the metallothionein expression and localization. The results suggest a significant interference of cadmium with mucosal efficiency, representing a health risk for the organism in direct contact with contamination and indirectly for the trophic chain.     

Tryptophan Hydroxylase 2 Deficiency Modifies the Effects of Fluoxetine and Pargyline on the Behavior, 5-HT- and BDNF-Systems in the Brain of Zebrafish (Danio rerio)

The mechanisms of resistance to antidepressant drugs is a key and still unresolved problem of psychopharmacology. Serotonin (5-HT) and brain-derived neurotrophic factor (BDNF) play a key role in the therapeutic effect of many antidepressants. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in 5-HT synthesis in the brain. We used zebrafish (Danio rerio) as a promising model organism in order to elucidate the effect of TPH2 deficiency caused by p-chlorophenylalanine (pCPA) on the alterations in behavior and expression of 5-HT-related (Tph2, Slc6a4b, Mao, Htr1aa, Htr2aa) and BDNF-related (Creb, Bdnf, Ntrk2a, Ngfra) genes in the brain after prolonged treatment with two antidepressants, inhibitors of 5-HT reuptake (fluoxetine) and oxidation (pargyline). In one experiment, zebrafish were treated for 72 h with 0.2 mg/L fluoxetine, 2 mg/L pCPA, or the drugs combination. In another experiment, zebrafish were treated for 72 h with 0.5 mg/L pargyline, 2 mg/L pCPA, or the drugs combination. Behavior was studied in the novel tank diving test, mRNA levels were assayed by qPCR, 5-HT and its metabolite concentrations were measured by HPLC. The effects of interaction between pCPA and the drugs on zebrafish behavior were observed: pCPA attenuated “surface dwelling” induced by the drugs. Fluoxetine decreased mRNA levels of Tph2 and Htr2aa genes, while pargyline decreased mRNA levels of Slc6a4b and Htr1aa genes. Pargyline reduced Creb, Bdnf and Ntrk2a genes mRNA concentration only in the zebrafish treated with pCPA. The results show that the disruption of the TPH2 function can cause a refractory to antidepressant treatment.     

人血管生成抑制素基因的克隆、表达及纯化

目的 构建携带人血管生成抑制素基因的原核表达载体,诱导表达具有活性的血管生成抑制素。方法 用PCR法从人纤溶酶原cDNA中扩增出Kringle 1-3基因,克隆入pET21a(+)载体中,在大肠杆菌BL 21中表达,表达产物经亲和层析纯化。结果 所构建的原核表达载体为高效表达系统,所表达的产物经层析纯化后可获得重组人血管生成抑制素。结论 可获得大量纯化人血管生成抑制素,为进一步临床应用奠定基础。     

耐辐射奇异球菌R12海藻糖合成酶基因的克隆与表达

耐辐射奇异球菌(Deinococcus wulumuqiensis)R12在环境胁迫下具有合成海藻糖的能力。采用PCR方法从耐辐射奇异球菌R12基因组中分离得到分子量约1 700bp的海藻糖合成酶基因,为验证其功能而在大肠杆菌中进行了表达,经IPTG诱导表达出约66kDa的目的蛋白。经酶活检测发现,目的蛋白能够催化麦芽糖一步合成海藻糖。以30%麦芽糖为底物反应1h,进行海藻糖转化实验,结果表明,重组海藻糖合成酶的最适反应温度为35℃、最适反应pH值为8.0,转化率达67%,具有较高的应用价值。     

生长抑素(SS)基因在大肠杆菌pT7ZZ表达系统中的克隆与表达

目的 构建和筛选既能稳定、高效表达SS融合蛋白，又能使这种融合蛋白保持良好的SS抗原性和高水溶性等特点的重组质粒。方法 用 BamH I/Xho I (B/X）和 BamH I/EcoR I（B/E）双酶切，将含有SS基因的片段由pThioHis A中切出，然后分别克隆到 pT7ZZa中的相应酶切位点，得到pSSB/X（短尾）和pSS-B/E(长尾)两个重组质粒，用常规表达技术在大肠杆菌中对其进行表达。结果 pSS-B/X和pSS-B/E两个重组质粒在宿主菌BL21（DE3）中均获得表达，pSS-B/X获得高效表达后融合蛋白占菌体总蛋白的30．6％。两个表达菌经超声裂解后电泳发现，SS－B/X－ZZ和SS－B/E－ZZ这两种融合蛋白基本为可溶性蛋白，沉淀中含量极低。二者均具有良好的抗原性，结论pSS-B/X重组质粒很有希望成为SS基因疫苗的候选质粒。     

RsrA-CFP融合蛋白的克隆表达及圆二色性分析

以天蓝色链霉菌M145基因组作模板,扩增σR(sigR)的anti-sigR因子基因rsrA,得到了333 bp基因;以质粒pECFP-N1作模板,扩增青色荧光蛋白基因cfp,得到了744 bp基因.将两个基因融合串联,构建rsrA/cf p/pET-28a(+)融合表达质粒.利用荧光检测技术,确定大肠杆菌BL21 (DE3)在OD600为0.5时,用1 mmol·L-1 IPTG进行诱导能大量表达融合蛋白RsrA-CFP.用镍柱亲和层析分离纯化RsrA-CFP蛋白,并用SDS-PAGE鉴定其分子量为40 kD左右.利用圆二色性初步测定RsrA-CFP蛋白的CD值,经CDNN软件分析得到:螺旋结构占32.2％、平行结构占8.0％、反向平行结构占9.3％、β折叠占16.7％、无规则卷曲占34.4％,为RsrA蛋白的功能研究奠定了基础.     

Phylogenetic and Expression Analysis of Fos Transcription Factors in Zebrafish

Members of the FOS protein family regulate gene expression responses to a multitude of extracellular signals and are dysregulated in several pathological states. Whilst mouse genetic models have provided key insights into the tissue-specific functions of these proteins in vivo, little is known about their roles during early vertebrate embryonic development. This study examined the potential of using zebrafish as a model for such studies and, more broadly, for investigating the mechanisms regulating the functions of Fos proteins in vivo. Through phylogenetic and sequence analysis, we identified six zebrafish FOS orthologues, fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2, which show high conservation in key regulatory domains and post-translational modification sites compared to their equivalent human proteins. During embryogenesis, zebrafish fos genes exhibit both overlapping and distinct spatiotemporal patterns of expression in specific cell types and tissues. Most fos genes are also expressed in a variety of adult zebrafish tissues. As in humans, we also found that expression of zebrafish FOS orthologs is induced by oncogenic BRAF-ERK signalling in zebrafish melanomas. These findings suggest that zebrafish represent an alternate model to mice for investigating the regulation and functions of Fos proteins in vertebrate embryonic and adult tissues, and cancer.     

中国人MBL基因编码区的克隆与原核表达

目的克隆中国人MBL基因编码区序列,并在原核细胞中表达。方法提取肝总RNA,RT-PCR扩增MBL编码区序列,并克隆到pGEM-T-easy载体上,再亚克隆到pET-30(a)上。诱导表达重组MBL,并通过SDS-PAGE检测表达情况,表达蛋白经初步纯化后,用ELISA及点杂交试验检测其免疫原性。结果克隆得到长747bp的MBL编码区序列,与GenBank中的MBL序列同源性在99%以上。重组MBL相对分子质量约为36000,以包涵体形式存在,表达量占菌体总蛋白量的20%左右,表达蛋白经初步纯化,纯度可达95%以上。ELISA检测显示重组MBLA490值与阴性对照差异有显著意义(10倍以上),点杂交检测结果为阳性。结论已成功克隆中国人MBL基因的编码区序列,并在大肠杆菌中表达,表达产物与天然MBL具有相似的免疫原性。