Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man

Zebrafish were used as a model to study haemostasis, a vertebrate function of paramount importance. A limitation of the zebrafish model is the difficulty in assaying small amounts of blood to detect coagulation mutants. We report the use of a rapid total coagulation activity (TCA) assay to screen for coagulation defects in individual adult zebrafish. We screened the TCA in 1000 gynogenetic half-tetrad diploids derived from 86 clutches. Each clutch was from a single F1 female offspring of males mutagenized with ethylnitrosourea (ENU). We found 30-50% defective zebrafish among six clutches, consistent with a heritable defect. The assay developed here provided a rapid screen to detect overall coagulation defects. However, because of the limited amounts of plasma, we could not detect defects in specific pathways. Therefore, a novel, ultra-sensitive kinetic method was developed to identify specific pathway defects. To test whether the kinetic assay could be used as a screening tool, 1500 Florida wild-type zebrafish pairs were analysed for naturally occurring coagulation defects. We detected 30 fish with extrinsic pathway defects, but with intact common and intrinsic pathways. We conclude that it is now possible to identify specific coagulation pathway defects in zebrafish.     

Oligodendrocyte development and myelination in GFP-transgenic zebrafish

Green fluorescent protein (GFP) transgenic zebrafish technology has been employed to directly visualize and analyze dynamic developmental processes, such as cell migration and morphogenesis. Stable transgenic zebrafish that express GFP in oligodendrocytes can be a valuable tool to visualize complex myelination processes in vivo, as well as to conduct rapid mutagenesis screens for defective myelination mutants. We investigated whether two myelin gene promoters, the zebrafish P0 promoter and the mouse proteolipid protein (PLP) promoter, drive GFP expression in zebrafish oligodendrocytes. Transiently, both promoters drive enhanced GFP (EGFP) expression in morphologically identifiable oligodendrocytes, premyelinating oligodendrocytes, and possible oligodendrocyte precursors. We have established a stable transgenic zebrafish line, tg(plp:EGFP) zebrafish, at the F1 generation, which expresses enhanced GFP (EGFP) driven by the mouse PLP promoter. In this transgenic line, EGFP-expressing cells are visually detectable around 24-hr postfertilization (hpf), and later at 54 hpf, these cells start exhibiting the clear morphologic characteristics of oligodendrocytes. Shortly afterward, EGFP-expressing oligodendrocytes establish a ventral dominant distribution pattern throughout the central nervous system. This transgenic zebrafish line is likely to serve as a useful tool, in which normal myelination as well as abnormal myelination can be recorded under time-lapse confocal microscopy. Furthermore, it has the potential to greatly facilitate mutagenesis screening for novel dysmyelinating mutants.     

Identification of a zebrafish model of muscular dystrophy

1. Large-scale mutagenic screens of the zebrafish genome have identified a number of different classes of mutations that disrupt skeletal muscle formation. Of particular interest and relevance to human health is a class of recessive lethal mutations in which muscle differentiation occurs normally, but is followed by tissue-specific degeneration reminiscent of human muscular dystrophies. 2. We have shown that one member of this class of mutations, sapje (sap), results from mutations within the zebrafish orthologue of the human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology by disrupting the link between the actin cytoskeleton and the extracellular matrix in skeletal muscle cells. 3. We have found that the progressive muscle degeneration phenotype of sapje-mutant zebrafish embryos is caused by the failure of somitic muscle attachments at the embryonic myotendinous junction (MTJ). 4. Although a role for dystrophin at the MTJ has been postulated previously and MTJ structural abnormalities have been identified in the dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure is thus far lacking. Therefore, the sapjre mutation may provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases. In the present review, we discuss this finding in light of previously postulated models of dystrophin function.     

Flies and fish: birds of a feather

The identification of specific clock-containing structures has been a major endeavour of the circadian field for many years. This has lead to the identification of many key components of the circadian system, including the suprachiasmatic nucleus in mammals, and the eyes and pineal glands in lower vertebrates. However, the idea that these structures represent the only clocks in animals has been challenged by the discovery of peripheral pacemakers in most organs and tissues, and even a number of cell lines. In Drosophila, and vertebrates such as the zebrafish, these peripheral clocks appear to be highly autonomous, being set directly by the environmental light/dark cycle. However, a hierarchy of clocks may still exist in mammals. In this review, we examine some of the current views regarding peripheral clocks, their organization and how they are entrained.     

Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 µM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24–48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.     

乙醇对斑马鱼胚胎发育中tenascin-c基因表达的影响

目的 探讨tenascin-c(tnc)基因在斑马鱼胚胎异常发育过程中表达的变化,从而揭示该基因在斑马鱼胚胎发育中可能存在的作用.方法 不同水平乙醇(100、200、300、400、500 mmol/L)持续处理斑马鱼胚胎,制备斑马鱼异常发育模型,收集受精后24、48 h斑马鱼胚胎并固定,用原位杂交和反转录聚合酶链反应(RT-PCR)方法观察tnc在斑马鱼异常发育时表达的变化.组间比较采用SPSS 11.5软件进行统计学处理.结果 200 mmol/L乙醇处理的斑马鱼胚胎在受精72 h其他系统未发现发育异常,而心脏已出现畸形.100、200 mmol/L乙醇处理后,RT-PCR结果示tnc在斑马鱼胚胎发育中的表达上调,而原位杂交信号变化不明显;300 mmol/L及以上乙醇处理的胚胎,原位杂交结果示tnc在体节处表达下降;100～400 mmol/L乙醇处理的胚胎,在受精48 h tnc在斑马鱼心脏部位均有表达.结论 tnc在低水平乙醇处理时表达即上调,对胚胎正常发育有保护作用;在心脏异常发育时表达,促进心脏的正常发育;tnc在病理状态下的表达模式不仅在脊椎动物中具有保守性,而且在胚胎和成体时期也具有保守性.     

Discovery and characterization of three novel synuclein genes in zebrafish.

The neuronal protein alpha-synuclein has been linked to the pathogenesis of synucleinopathies, a collection of neurodegenerative disorders, including Parkinson's disease. alpha-Synuclein belongs to a family of synuclein genes that includes beta- and gamma-synuclein. However, despite being associated with several fatal human neurodegenerative diseases, little is known about the normal function of synucleins. Here we report the cloning and characterization of three synucleins from zebrafish, sncga, sncgb, and sncb. The sequences of these zebrafish synucleins are very similar to those of the human proteins. We used whole-mount in situ hybridization to analyze their spatial and temporal expression patterns during development. sncgb was expressed exclusively in the notochord, while sncga and sncb were expressed strongly in the nervous system. Our identification of synuclein genes in zebrafish and the characterization of their expression patterns will facilitate future experiments aimed at assessing their functions in normal physiology as well as their role in pathophysiology. Developmental Dynamics 237:2490-2495, 2008. (c) 2008 Wiley-Liss, Inc.     

Zebrafish: a genetic model for hemostasis and thrombosis.

Here we review the zebrafish hemostatic system, its relevance to mammalian hemostasis, and its efficacy as a vertebrate genetic model to further the understanding of hemostasis and thrombosis.