Imaging Cilia in Zebrafish

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Methods in cell biology (Impact Factor: 1.42). 01/2010; 97:415-35. DOI: 10.1016/S0091-679X(10)97022-2
Source: PubMed

ABSTRACT Research focused on cilia as extremely important cellular organelles has flourished in recent years. A thorough understanding of cilia regulation and function is critical, as disruptions of cilia structure and/or function have been linked to numerous human diseases and disorders. The tropical freshwater zebrafish is an excellent model organism in which to study cilia structure and function. We can readily image cilia and their motility in embryonic structures including Kupffer's vesicle during somite stages and the pronephros from 1 day postfertilization onward. Here, we describe how to image cilia by whole-mount immunofluorescence, transverse cryosection/immunohistochemistry, and transmission electron microscopy. We also describe how to obtain videos of cilia motility in living embryos.

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    • "Regardless of technique—mRNA, morpholino, or peptide nucleic acid injection—the primary purpose is to demonstrate the appropriate change in protein level. If an antibody is available for the protein of interest, for determination of morpholino efficacy either a western blot from zebrafish protein fry extracts or whole mount immunohistochemistry staining can be performed [39] [40]. If an antibody is unavailable, a transgenic or coinjected mRNA with 5 í® í° UTR of the gene of interest upstream of a marker or epitope tag (e.g., hemagglutinin or GFP) could be used to assess the level of knockdown. "
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    ABSTRACT: Data for genes relevant to glomerular filtration barrier function or proteinuria is continually increasing in an era of microarrays, genome-wide association studies, and quantitative trait locus analysis. Researchers are limited by published literature searches to select the most relevant genes to investigate. High-throughput cell cultures and other in vitro systems ultimately need to demonstrate proof in an in vivo model. Generating mammalian models for the genes of interest is costly and time intensive, and yields only a small number of test subjects. These models also have many pitfalls such as possible embryonic mortality and failure to generate phenotypes or generate nonkidney specific phenotypes. Here we describe an in vivo zebrafish model as a simple vertebrate screening system to identify genes relevant to glomerular filtration barrier function. Using our technology, we are able to screen entirely novel genes in 4-6 weeks in hundreds of live test subjects at a fraction of the cost of a mammalian model. Our system produces consistent and reliable evidence for gene relevance in glomerular kidney disease; the results then provide merit for further analysis in mammalian models.
    09/2013; 2013(3):658270. DOI:10.1155/2013/658270
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    • "Embryos were prepared for flat-mounting by dissecting the yolk away from the embryo proper with a pair of fine forceps; the embryos were then mounted between two glass coverslips. Immunofluorescence detection of acetylated tubulin in the whole-mount embryos was performed according to a published protocol (Jaffe et al., 2010). High-speed video microscopy analysis has been described in detail in a recent publication (Pathak et al., 2011). "
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    ABSTRACT: In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified.
    Molecular biology of the cell 06/2012; 23(16):3069-78. DOI:10.1091/mbc.E12-01-0019 · 4.47 Impact Factor
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    • "Gene knockdown can be efficiently achieved with morpholino antisense oligonucleotides (with appropriate controls to verify specificity and detect off-target effects), so both forward and reverse genetic tools are available. Combined with high-quality in vivo imaging (Jaffe et al., 2010; Wallingford, 2010a, 2010b, 2010c), these features render frog and fish attractive models for the study of cilia in vertebrates. "
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    ABSTRACT: Most mammalian cell types have the potential to assemble at least one cilium. Immotile cilia participate in numerous sensing processes, while motile cilia are involved in cell motility and movement of extracellular fluid. The functional importance of cilia and flagella is highlighted by the growing list of diseases due to cilia defects. These ciliopathies are marked by an amazing diversity of clinical manifestations and an often complex genetic aetiology. To understand these pathologies, a precise comprehension of the biology of cilia and flagella is required. These organelles are remarkably well conserved throughout eukaryotic evolution. In this review, we describe the strengths of various model organisms to decipher diverse aspects of cilia and flagella biology: molecular composition, mode of assembly, sensing and motility mechanisms and functions. Pioneering studies carried out in the green alga Chlamydomonas established the link between cilia and several genetic diseases. Moreover, multicellular organisms such as mouse, zebrafish, Xenopus, Caenorhabditis elegans or Drosophila, and protists such as Paramecium, Tetrahymena and Trypanosoma or Leishmania each bring specific advantages to the study of cilium biology. For example, the function of genes involved in primary ciliary dyskinesia (due to defects in ciliary motility) can be efficiently assessed in trypanosomes.
    Biology of the Cell 03/2011; 103(3):109-30. DOI:10.1042/BC20100104 · 3.51 Impact Factor
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