New Isocoumarins from a Cold‐Adapted Fungal Strain Mucor sp. and Their Developmental Toxicity to Zebrafish Embryos

Three new isocoumarin derivatives, mucorisocoumarins A–C ( 1 – 3 , resp.), together with seven known compounds, 4 – 10 , were isolated from the cold‐adapted fungal strain Mucor sp. (No. XJ07027‐5). The structures of the new compounds were identified by detailed IR, MS, and 1D‐ and 2D‐NMR analyses. It was noteworthy that compounds 1, 2, 4 , and 5 were successfully resolved by chiral HPLC, indicating that 1 – 7 should exist as enantiomers. In an embryonic developmental toxicity assay using a zebrafish model, compound 3 produced developmental abnormalities in the zebrafish embryos. This is the first report of isocoumarins with developmental toxicity to zebrafish embryos.     

Identification of the F1F0 mitochondrial ATPase as a target for modulating skin pigmentation by screening a tagged triazine library in zebrafish

A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a dramatic increase in the pigmentation of early stage zebrafish embryos. This compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. Oligomycin, a small molecule known to inhibit the mitochondrial ATPase, competed with PPA for its cellular target in melanocytes. In addition, PPA was shown to alter the membrane potential of mitochondria, consistent with inhibition of the mitochondrial ATPase. Thus, PPA has been successfully used as a chemical probe in a forward chemical genetic approach to establish a link between the phenotype and the protein. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells.     

Induction of reversible hemolytic anemia in living zebrafish using a novel small molecule

We used zebrafish to screen and identify small molecules that affect the process of vertebrate hematopoietic development. Zebrafish embryos were exposed to a library of 5000 synthetic compounds and screened for defects in primitive erythropoiesis. Here, we present the characterization of hemolytic anemia induced in zebrafish by the small molecule 5115318 (3-[5-methyl-furan 2-yl]-propionic acid N'-phenyl-hydrazide). This compound is capable of generating hemoglobin aggregates and Heinz bodies in red cells in vivo only. The induced anemia is reversible and treated fish recover in about 4 days. This study shows the feasibility of using zebrafish to screen for small molecules that can modulate the specific process of erythropoiesis.     

Clinically Approved Iron Chelators Influence Zebrafish Mortality,Hatching Morphology and Cardiac Function

Iron chelation therapy using iron (III) specific chelators such as desferrioxamine (DFO, Desferal), deferasirox (Exjade or ICL-670), and deferiprone (Ferriprox or L1) are the current standard of care for the treatment of iron overload. Although each chelator is capable of promoting some degree of iron excretion, these chelators are also associated with a wide range of well documented toxicities. However, there is currently very limited data available on their effects in developing embryos. In this study, we took advantage of the rapid development and transparency of the zebrafish embryo, Danio rerio to assess and compare the toxicity of iron chelators. All three iron chelators described above were delivered to zebrafish embryos by direct soaking and their effects on mortality, hatching and developmental morphology were monitored for 96 hpf. To determine whether toxicity was specific to embryos, we examined the effects of chelator exposure via intra peritoneal injection on the cardiac function and gene expression in adult zebrafish. Chelators varied significantly in their effects on embryo mortality, hatching and morphology. While none of the embryos or adults exposed to DFO were negatively affected, ICL -treated embryos and adults differed significantly from controls, and L1 exerted toxic effects in embryos alone. ICL-670 significantly increased the mortality of embryos treated with doses of 0.25 mM or higher and also affected embryo morphology, causing curvature of larvae treated with concentrations above 0.5 mM. ICL-670 exposure (10 µL of 0.1 mM injection) also significantly increased the heart rate and cardiac output of adult zebrafish. While L1 exposure did not cause toxicity in adults, it did cause morphological defects in embryos at 0.5 mM. This study provides first evidence on iron chelator toxicity in early development and will help to guide our approach on better understanding the mechanism of iron chelator toxicity.     

Dichloroacetate-induced developmental toxicity and production of reactive oxygen species in zebrafish embryos

Dichloroacetate (DCA) is one of the toxic by products that are formed during the chlorine disinfection process of drinking water. In this study, the developmental toxicity of DCA has been determined in zebrafish (Danio rerio) embryos. Embryos were exposed to different concentrations (4, 8, 16, and 32 mM) of the compound at the 4 h postfertilization (hpf) stage of development, and were observed for different developmental toxic effects at 8, 24, 32, 55, 80, and 144 hpf. Exposure of embryos to 8-32 mM of DCA resulted in significant increases in the heart rate and blood flow of the 55 and 80 hpf embryos that turned into significant decreases at the 144 hpf time point. At 144 hpf, malformations of mouth structure, notochord bending, yolk sac edema and behavioral effects including perturbed swimming and feeding behaviors were also observed. DCA was also found to produce time- and concentration-dependent increases in embryonic levels of superoxide anion (O2*-) and nitric oxide (NO), at various stages of development. The results of the study suggest that DCA-induced developmental toxic effects in zebrafish embryos are associated with production of reactive oxygen species in those embryos.     

Antibiotic Toxicity and Absorption in Zebrafish Using Liquid Chromatography-Tandem Mass Spectrometry

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10–1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD₅₀ values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.     

Toxicity Assessment of Iron Oxide Nanoparticles in Zebrafish (Danio rerio) Early Life Stages

Iron oxide nanoparticles have been explored recently for their beneficial applications in many biomedical areas, in environmental remediation, and in various industrial applications. However, potential risks have also been identified with the release of nanoparticles into the environment. To study the ecological effects of iron oxide nanoparticles on aquatic organisms, we used early life stages of the zebrafish (Danio rerio) to examine such effects on embryonic development in this species. The results showed that ≥10 mg/L of iron oxide nanoparticles instigated developmental toxicity in these embryos, causing mortality, hatching delay, and malformation. Moreover, an early life stage test using zebrafish embryos/larvae is also discussed and recommended in this study as an effective protocol for assessing the potential toxicity of nanoparticles. This study is one of the first on developmental toxicity in fish caused by iron oxide nanoparticles in aquatic environments. The results will contribute to the current understanding of the potential ecotoxicological effects of nanoparticles and support the sustainable development of nanotechnology.     

Glutathione transferase pi class 2 (GSTp2) protects against the cardiac deformities caused by exposure to PAHs but not PCB-126 in zebrafish embryos

Glutathione transferases (GSTs) are phase II enzymes that detoxify a wide range of toxicants and reactive intermediates. One such class of toxicants is the ubiquitous polycyclic aromatic hydrocarbons (PAHs). Certain PAHs are known to cause developmental cardiac toxicity in fish. Herein, we explored the role of GST pi class 2 (GSTp2) in PAH- and PCB-induced cardiac toxicity in zebrafish (Danio rerio) embryos. We measured expression of GSTp2 in embryos exposed to individual and co-exposures of the PAHs benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), and fluoranthene (FL) as well as 3,3',4,4',5-pentachlorobiphenyl (PCB-126). GSTp2 mRNA expression was induced by exposure to BkF, BaP, PCB-126, and BaP+FL and BkF+FL co-exposure. A splice junction morpholino was then used to knockdown GSTp2 in developing zebrafish. GSTp2 knockdown exacerbated the toxicity caused by co-exposures to BkF+FL and BaP+FL. However, GSTp2 knockdown did not affect PCB-126 toxicity. These results further suggest that pi class GSTs serve a protective function against the synergistic toxicity caused by PAHs in developing zebrafish.     

Knockdown of ribosomal protein S7 causes developmental abnormalities via p53 dependent and independent pathways in zebrafish

Ribosomal proteins (RPs), structural components of the ribosome involved in protein synthesis, are of significant importance in all organisms. Previous studies have suggested that some RPs may have other functions in addition to assembly of the ribosome. The small ribosomal subunits RPS7, has been reported to modulate the mdm2-p53 interaction. To further investigate the biological functions of RPS7, we used morpholino antisense oligonucleotides (MO) to specifically knockdown RPS7 in zebrafish. In RPS7-deficient embryos, p53 was activated, and its downstream target genes and biological events were induced, including apoptosis and cell cycle arrest. Hematopoiesis was also impaired seriously in RPS7-deficient embryos, which was confirmed by the hemoglobin O-dianisidine staining of blood cells, and the expression of scl, gata1 and α-E1 globin were abnormal. The matrix metalloproteinase (mmp) family genes were also activated in RPS7 morphants, indicating that improper cell migration might also cause development defects. Furthermore, simultaneously knockdown of the p53 protein by co-injecting a p53 MO could partially reverse the abnormal phenotype in the morphants. These results strengthen the hypothesis that specific ribosomal proteins regulate p53 and that their deficiency affects hematopoiesis. Moreover, our data implicate that RPS7 is a regulator of matrix metalloproteinase (mmp) family in zebrafish system. These specific functions of RPS7 may provide helpful clues to study the roles of RPs in human disease.     

Zebrafish Hsp70 is required for embryonic lens formation

Heat shock proteins (Hsps) were originally identified as proteins expressed after exposure of cells to environmental stress. Several Hsps were subsequently shown to play roles as molecular chaperones in normal intracellular protein folding and targeting events and to be expressed during discrete periods in the development of several embryonic tissues. However, only recently have studies begun to address the specific developmental consequences of inhibiting Hsp expression to determine whether these molecular chaperones are required for specific developmental events. We have previously shown that the heat-inducible zebrafish hsp70 gene is expressed during a distinct temporal window of embryonic lens formation at normal growth temperatures. In addition, a 1.5-kb fragment of the zebrafish hsp70 gene promoter is sufficient to direct expression of a gfp reporter gene to the lens, suggesting that the hsp70 gene is expressed as part of the normal lens development program. Here, we used microinjection of morpholino-modified antisense oligonucleotides (MOs) to reduce Hsp70 levels during zebrafish development and to show that Hsp70 is required for normal lens formation. Hsp70-MO-injected embryos exhibited a small-eye phenotype relative to wild-type and control-injected animals, with the phenotype discernable during the second day of development. Histological and immunological analysis revealed a small, underdeveloped lens. Numerous terminal deoxynucleotidyl transferase-mediated dUTP-fluoroscein nick-end labeling (TUNEL)-positive nuclei appeared in the lens of small-eye embryos after 48 hours postfertilization (hpf), whereas they were no longer apparent in untreated embryos by this age. Lenses transplanted from hsp70-MO-injected embryos into wild-type hosts failed to recover and retained the immature morphology characteristic of the small-eye phenotype, indicating that the lens phenotype is lens autonomous. Our data suggest that the lens defect in hsp70-MO-injected embryos is predominantly at the level of postmitotic lens fiber differentiation, a result supported by the appearance of mature lens organization in these embryos by 5 days postfertilization, once morpholino degradation or dilution has occurred.     

Single-Molecule Microscopy Reveals Membrane Microdomain Organization of Cells in a Living Vertebrate

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.     

Zebrafish on a Chip: A Novel Platform for Real-Time Monitoring of Drug-Induced Developmental Toxicity

Pharmaceutical safety testing requires a cheap, fast and highly efficient platform for real-time evaluation of drug toxicity and secondary effects. In this study, we have developed a microfluidic system for phenotype-based evaluation of toxic and teratogenic effects of drugs using zebrafish (Danio rerio) embryos and larvae as the model organism. The microfluidic chip is composed of two independent functional units, enabling the assessment of zebrafish embryos and larvae. Each unit consists of a fluidic concentration gradient generator and a row of seven culture chambers to accommodate zebrafish. To test the accuracy of this new chip platform, we examined the toxicity and teratogenicity of an anti-asthmatic agent-aminophylline (Apl) on 210 embryos and 210 larvae (10 individuals per chamber). The effect of Apl on zebrafish embryonic development was quantitatively assessed by recording a series of physiological indicators such as heart rate, survival rate, body length and hatch rate. Most importantly, a new index called clonic convulsion rate, combined with mortality was used to evaluate the toxicities of Apl on zebrafish larvae. We found that Apl can induce deformity and cardiovascular toxicity in both zebrafish embryos and larvae. This microdevice is a multiplexed testing apparatus that allows for the examination of indexes beyond toxicity and teratogenicity at the sub-organ and cellular levels and provides a potentially cost-effective and rapid pharmaceutical safety assessment tool.     

水平回转培养对斑马鱼血管发育的影响

随着空间生命科学的发展, 微重力对生命体的影响已成为科学家们日益关注的问题。多数研究表明, 微重力对生物体胚胎早期发育有着重要影响, 而血管系统作为胚胎最早行使功能的系统备受关注。目前关于微重力对血管发育影响的研究大多来自对离体培养细胞的体外回转模拟实验, 在体实验相对较少。文章利用斑马鱼作为模式动物, 在体探究水平回转培养环境下斑马鱼胚胎早期发育及水平回转培养对其血管系统发育的影响。在斑马鱼受精后24 h(24 hpf)时进行水平回转培养处理, 至36 hpf时收集胚胎, 通过体视显微镜观察斑马鱼表型变化, 通过半定量RT-PCR、qPCR及全胚原位杂交等手段对比水平回转培养环境下与对照组血管相关因子的表达情况, 并通过BrdU掺入及TUNEL法进行斑马鱼全胚细胞凋亡及增殖检测。结果表明, 在90 r/min水平回转培养环境下, 斑马鱼死亡数量没有差异, 24 hpf时破壳数量显著降低(10.3±0.41 vs. 0.0, P<0.05), 心率显著加快(223.5±2.32 vs. 185.0±3.23, P<0.05), 黑色素明显增加, 动静脉发育紊乱, 且在120 r/min转速下, 胚胎血管特异性表达因子flk1、flt4及 ephrinB2的表达均显著降低, 斑马鱼细胞凋亡明显增加, 而细胞增殖无显著差异, 提示水平回转培养对斑马鱼胚胎发育特别是早期血管发育具有重要影响。     

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).     

Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp^y11 mutant embryos or cardia bifida phenotypes in fau^tm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.     

The effect of butenolide on behavioral and morphological changes in two marine fouling species, the barnacle Balanus amphitrite and the bryozoan Bugula neritina

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC50=0.82 μg ml(-1)) and B. neritina (EC50=3 μg ml(-1)). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml(-1) and 10 μg ml(-1), butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.     

斑马鱼胚胎和肿瘤细胞评价细胞毒药物活性比较研究*

目的:比较斑马鱼胚胎和肿瘤细胞作为药物筛选模型的优缺点.方法:采用MTT法检测顺铂、紫杉醇、阿霉素、5-氟尿嘧啶四种药物对HL-60和Hela细胞的增殖影响;同时,观察药物对斑马鱼胚胎发育的影响.结果:阿霉素、顺铂及紫杉醇作用于HL-60及Hela细胞的IC50均显著高于作用于斑马鱼胚胎的LD50;而5-FU作用于肿瘤细胞和斑马鱼胚胎的结果与其它药物相反;四种抗肿瘤药物对斑马鱼胚胎的生长发育均有致畸作用.结论:斑马鱼胚胎作为细胞毒类药物筛选模型,对于抗微管类药物较为敏感,但对于抗代谢药敏感性较肿瘤细胞差.