Alk3/Alk3b and Smad5 Mediate BMP Signaling during Lymphatic Development in Zebrafish

Lymphatic vessels are essential to regulate interstitial fluid homeostasis and diverse immune responses. A number of crucial factors, such as VEGFC, SOX18, PROX1, FOX2C, and GJC2, have been implicated in differentiation and/or maintenance of lymphatic endothelial cells (LECs). In humans, dysregulation of these genes is known to cause lymphedema, a debilitating condition which adversely impacts the quality of life of affected individuals. However, there are no currently available pharmacological treatments for lymphedema, necessitating identification of additional factors modulating lymphatic development and function which can be targeted for therapy. In this report, we investigate the function of genes associated with Bone Morphogenetic Protein (BMP) signaling in lymphatic development using zebrafish embryos. The knock-down of BMP type II receptors, Bmpr2a and Bmpr2b, and type I receptors, Alk3 and Alk3b, as well as SMAD5, an essential cellular mediator of BMP signaling, led to distinct lymphatic defects in developing zebrafish. Therefore, it appears that each constituent of the BMP signaling pathway may have a unique function during lymphatic development. Taken together, our data demonstrate that BMP signaling is essential for normal lymphatic vessel development in zebrafish.     

Lymphatic biomarkers in primary melanomas as predictors of regional lymph node metastasis and patient outcomes

Recently developed lymphatic‐specific immunohistochemical markers can now be utilized to assess intratumoral and/or peritumoral lymphatic vessel density (LVD), to detect lymphatic vessel invasion (LVI) by melanoma cells and to identify lymphatic marker expression in melanoma cells themselves. We systematically reviewed the available evidence for the expression of lymphatic markers as predictors of regional node metastasis and survival in melanoma patients. The currently available evidence suggests that LVD (particularly in a peritumoral location) and LVI are predictors of sentinel node metastasis and poorer survival. Nevertheless, adherence to international guidelines in the conduct and reporting of the studies was generally poor, with wide methodologic variations and heterogeneous findings. Larger, carefully conducted and well‐reported studies that confirm these preliminary findings are required before it would be appropriate to recommend the routine application of costly and time‐consuming immunohistochemistry for lymphatic markers in the routine clinical assessment of primary cutaneous melanomas.     

The plaque protein myozap identified as a novel major component of adhering junctions in endothelia of the blood and the lymph vascular systems

Recently the protein myozap, a 54-kD polypeptide which is not a member of any of the known cytoskeletal and junctional protein multigene families, has been identified as a constituent of the plaques of the composite junctions in the intercalated disks connecting the cardiomyocytes of mammalian hearts. Using a set of novel, highly sensitive and specific antibodies we now report that myozap is also a major constituent of the cytoplasmic plaques of the adherens junctions (AJs) connecting the endothelial cells of the mammalian blood and lymph vascular systems, including the desmoplakin-containing complexus adhaerentes of the virgultar cells of lymph node sinus. In light and electron microscopic immunolocalization experiments we show that myozap colocalizes with several proteins of desmosomal plaques as well as with AJ-specific transmembrane molecules, including VE-cadherin. In biochemical analyses, rigorous immunoprecipitation experiments have revealed N-cadherin, desmoplakin, desmoglein-2, plakophilin-2, plakoglobin and plectin as very stably bound complex partners. We conclude that myozap is a general component of cell-cell junctions not only in the myocardium but also in diverse endothelia of the blood and lymph vascular systems of adult mammals, suggesting that this protein not only serves a specific role in the heart but also a broader set of functions in the vessel systems. We also propose to use myozap as an endothelial cell type marker in diagnoses.     

斑马鱼胚胎发育中细胞凋亡的研究进展

细胞凋亡是细胞在基因调控下发生的主动消亡过程,在脊椎动物胚胎发育过程中非常重要。斑马鱼作为一种十分理想的发育分子生物学研究模型,在有关细胞凋亡在诸如形态发生、性别分化等方面功能之活体在位研究中日益受到重视。目前,斑马鱼胚胎发育中主要凋亡通路研究已进行了不少工作,特别是caspase及其它凋亡调控基因在斑马鱼中已被成功克隆,通过转基因斑马鱼胚胎中胁迫诱导细胞凋亡并研究其信号通路以及斑马鱼胚胎形态发生的异常改变,为阐明这些凋亡调控基因与发育之间的关系提供了一个强有力的手段。     

Isolation of Complementary DNAs Coding for a Receptor for Activated C Kinase (RACK) from Zebrafish (Danio rerio) and Tilapia (Oreochromis niloticus): Constitutive Developmental and Tissue Expression

We have cloned and sequenced complementary DNAs coding for a receptor for activated C kinase (RACK) from two species of teleost fishes, zebrafish (Danio rerio) and tilapia (Oreochromis niloticus). The tilapia clone is 1063 nucleotides long, and the zebrafish clone is 1069 nucleotides long. Both clones contain an open reading frame coding for the complete RACK protein of 317 amino acids. Northern hybridization analysis using these clones as probes detected a 1.2-kb band, indicating that these are nearly full-length cDNA clones. In tilapia, RACK messenger RNA was expressed in all tissues examined. In situ hybridization detected the presence of mRNA for this RACK sequence in unfertilized eggs and embryos (development up to 24 hours) from zebrafish. Received July 13, 1998; accepted January 14, 1999     

A Defined Zebrafish Line for High-Throughput Genetics and Genomics: NHGRI-1

Substantial intrastrain variation at the nucleotide level complicates molecular and genetic studies in zebrafish, such as the use of CRISPRs or morpholinos to inactivate genes. In the absence of robust inbred zebrafish lines, we generated NHGRI-1, a healthy and fecund strain derived from founder parents we sequenced to a depth of ∼50×. Within this strain, we have identified the majority of the genome that matches the reference sequence and documented most of the variants. This strain has utility for many reasons, but in particular it will be useful for any researcher who needs to know the exact sequence (with all variants) of a particular genomic region or who wants to be able to robustly map sequences back to a genome with all possible variants defined.     

外源性胰岛素对斑马鱼血糖及其转运的影响

斑马鱼（Danio rerio）在糖负荷状态下表现出持续高血糖现象。与对照组（仅腹腔注射灭菌去离子水）相比,葡萄糖组（仅腹腔注射葡萄糖）血浆胰岛素水平无显著差异,胰岛素基因表达显著上调,肝胰脏葡萄糖转运蛋白（glucose transporters,GLUTs）基因表达无显著差异,说明斑马鱼自身胰岛素分泌不足和葡萄糖转运迟缓是导致其在糖负荷状态下持续高血糖的原因。为了观察外源性胰岛素对斑马鱼血糖及其在体内转运的影响,设计低（1.25 IU/kg）、中（12.5 IU/kg）、高（125 IU/kg）3个浓度的胰岛素,分别与葡萄糖溶液（0.1 g/mL）共注射斑马鱼并观察其血糖变化。结果表明,低剂量胰岛素能有效促进斑马鱼血糖的降低,且能直观反映糖负荷后血糖的变化情况,为最适注射浓度。此外,研究显示斑马鱼血糖变化不受性别影响。在胰岛素最适注射浓度下,与葡萄糖组相比,胰岛素组（葡萄糖与胰岛素共注射）可以显著减少斑马鱼血糖恢复到正常水平的时间,进一步分析发现,斑马鱼血浆胰岛素水平增加,肝胰脏葡萄糖转运蛋白基因表达显著上调,但胰岛素基因表达却被显著抑制。综上所述,胰岛素分泌不足和葡萄糖转运迟缓是造成斑马鱼持续高血糖的原因;外源性胰岛素能够促进糖负荷状态下斑马鱼血糖的降低,但是具有反馈抑制斑马鱼肝胰脏胰岛素基因表达的作用。     

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.     

Visualizing Compound Distribution during Zebrafish Embryo Development: The Effects of Lipophilicity and DMSO

The predictability of the zebrafish embryo model is highly influenced by internal exposure of the embryo/larva. As compound uptake is likely to be influenced by factors such as lipophilicity, solvent use, and chorion presence, this article focuses on investigating their effects on compound distribution within the zebrafish embryo. To visualize compound uptake and distribution, zebrafish embryos were exposed for 96 hr, starting at 4 hr postfertilization, to water‐soluble dyes: Schiff's reagent (logP –4.63), Giemsa stain (logP –0.77), Van Gierson stain (logP 1.64), Cresyl fast violet (logP 3.5), Eosine Y (logP 4.8), Sudan III (logP 7.5), and Oil red O (logP 9.81), with and without 1% dimethyl‐sulfoxide (DMSO). Three additional compounds were used to analytically determine the uptake and distribution: Acyclovir (logP –1.56), Zidovudine (logP 0.05), and Metoprolol Tartrate Salt (logP 1.8). Examinations were performed every 24 hr. Both methods (visualization and specific analysis) showed that exposure to higher logP values results in higher compound uptake. Specific analysis showed that for lipophilic compounds >90% of compound is taken up by the embryo. For hydrophilic compounds, >90% of compound within the complete egg could not be associated to embryo or chorion and is probably distributed into the perivitelline space. Overall, internal exposure analyses on at least two occasions (i.e., before and after hatching) is crucial for interpretation of zebrafish embryotoxicity data, especially for compounds with extreme logP values. DMSO did not affect exposure when examined with the visualization method, however, this method might be not sensitive enough to draw hard conclusions.     

Binding of Hyaluronan to the Native Lymphatic Vessel Endothelial Receptor LYVE-1 Is Critically Dependent on Receptor Clustering and Hyaluronan Organization

The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely on in vitro studies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HA in vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposed in vivo functions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-association and/or HA multimerization. In particular, we demonstrate the prerequisite of a critical LYVE-1 threshold density and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addition, we show that cross-linking of biotinylated HA in streptavidin multimers or supramolecular complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 cross-linking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte trafficking in vivo.     

斑马鱼胚胎细胞和中国仓鼠卵巢细胞远缘杂交融合条件的优化

以斑马鱼胚胎细胞(ZEM-2s)和中国仓鼠卵巢细胞(CHO-k1)为实验材料,采用聚乙二醇(PEG)作为促融剂,从PEG的相对分子质量、浓度、作用温度和时间等方面进行单因子实验,以期寻找两种细胞融合的最佳条件。实验结果表明,融合的最适条件是融合温度为37℃,浓度为40%,分子量为2 000的PEG处理斑马鱼胚胎细胞和中国仓鼠卵巢细胞100sec,平均融合率高达25.3%,与未加入PEG的细胞相比,最佳条件下处理的两种细胞融合现象明显(p<0.05),表明该条件下的处理能够显著促进细胞的融合。     

A Single Method for Cryofixation and Correlative Light, Electron Microscopy and Tomography of Zebrafish Embryos

The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples.     

人EGFR显性负性突变体对胃癌细胞体内成瘤及淋巴结转移的影响

为了研究人表皮生长因子显性负性突变体(dominant negative mutant of EGFR, dnEGFR)对胃癌细胞体内成瘤及淋巴结转移的影响,用目的质粒pEGFPN1 dnEGFR,空质粒载体pEGFP N1分别转染胃癌细胞NCI-N87.筛选出稳定转染株,实验共分3组：NCI-N87细胞未转染组（untreated NCI-N87,UN组）,NCI-N87细胞pEGFP-N1转染组（EN组）;NCI-N87细胞pEGFPN1-dnEGFR转染组（DN组）.将3组细胞接种于裸鼠右后足垫,6周后测量移植瘤大小及对应腹股沟转移淋巴结数目,HE染色验证,real-time PCR和Western 印迹检测3组细胞中AKT1、MAPK3 mRNA和蛋白的表达改变.结果发现,DN组移植瘤较UN、EN组明显缩小（P<0.05）,且右腹股沟转移淋巴结数目较UN、EN组减少（P<0.05）.DN组细胞中,AKT1、MAPK3 mRNA和蛋白水平较UN、EN组降低（P<0.05）.提示pEGFPN1-dnEGFR可抑制裸鼠体内胃癌细胞成瘤及淋巴结转移,AKT1及MAPK3信号通路可能参与其中.     

大型哺乳动物的趋势监测:青海野牛沟和甘肃阿克塞国际狩猎场的案例

大型野生动物种群数量估算的理想条件是使用数学模型以及严格的实验设计来选择样本。可是,野外条件状况往往违背数学模型假设前提,不可能随机地选择样本。于是,计算的结果不但不可靠,而且很可能没有意义。就野生动物管理来说,不需要获得一个准确的种群数量,只需一个长期的数量趋势,就足以指导相关管理工作。在中国大型哺乳动物长期监测还没有纳入常规。本文报道了位于青海省野牛沟和甘肃省阿克塞县两个野生有蹄类动物种群数量的长期趋势监测项目。我们这些年里一直用相同的方法持续监测,并明确了监测数值结果包含有不确定性。尽管存在不确定性,仍可以发现监测地点野生有蹄类动物种群变化趋势,这些结果可以帮助野生动物管理者据此变化及时作出相应管理计划。     

Insights from Zebrafish and Mouse Models on the Activity and Safety of Ar-Turmerone as a Potential Drug Candidate for the Treatment of Epilepsy

In a previous study, we uncovered the anticonvulsant properties of turmeric oil and its sesquiterpenoids (ar-turmerone, α-, β-turmerone and α-atlantone) in both zebrafish and mouse models of chemically-induced seizures using pentylenetetrazole (PTZ). In this follow-up study, we aimed at evaluating the anticonvulsant activity of ar-turmerone further. A more in-depth anticonvulsant evaluation of ar-turmerone was therefore carried out in the i.v. PTZ and 6-Hz mouse models. The potential toxic effects of ar-turmerone were evaluated using the beam walking test to assess mouse motor function and balance. In addition, determination of the concentration-time profile of ar-turmerone was carried out for a more extended evaluation of its bioavailability in the mouse brain. Ar-turmerone displayed anticonvulsant properties in both acute seizure models in mice and modulated the expression patterns of two seizure-related genes (c-fos and brain-derived neurotrophic factor [bdnf]) in zebrafish. Importantly, no effects on motor function and balance were observed in mice after treatment with ar-turmerone even after administering a dose 500-fold higher than the effective dose in the 6-Hz model. In addition, quantification of its concentration in mouse brains revealed rapid absorption after i.p. administration, capacity to cross the BBB and long-term brain residence. Hence, our results provide additional information on the anticonvulsant properties of ar-turmerone and support further evaluation towards elucidating its mechanism of action, bioavailability, toxicity and potential clinical application.     

血管内皮生长因子及其受体在斑马鱼胚胎血管发育中的作用

血管内皮生长因子(vascular endothelial growth factor,VEGF)是一种多功能的细胞因子,其主要作用是促进血管内皮细胞增殖和增加血管通透性,是肿瘤及正常组织血管生成的中心调控因素。以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。斑马鱼作为一种重要的模式生物,被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。文章对斑马鱼作为心血管系统研究模型的优势及其血管研究方法做一阐述,重点对斑马鱼VEGF及其受体的最新研究进展做了介绍,并展望了其发展前景。     

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.