PUBLICATION
Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos
- Authors
- Wühr, M., Obholzer, N.D., Megason, S.G., Detrich, H.W., and Mitchison, T.J.
- ID
- ZDB-PUB-110520-26
- Date
- 2011
- Source
- Methods in cell biology 101: 1-18 (Chapter)
- Registered Authors
- Detrich, H. William, Megason, Sean, Obholzer, Nikolaus
- Keywords
- cytoskeletal dynamics, fluorescent protein, live imaging, microfilament, microtubule, zebrafish embryo
- MeSH Terms
-
- Actins/metabolism
- Animals
- Microtubules/metabolism*
- Animals, Genetically Modified
- Humans
- Cytoskeleton/metabolism*
- Microscopy, Fluorescence/methods
- Zebrafish/embryology*
- Zebrafish/genetics
- Zebrafish/metabolism
- PubMed
- 21550437 Full text @ Meth. Cell. Biol.
Citation
Wühr, M., Obholzer, N.D., Megason, S.G., Detrich, H.W., and Mitchison, T.J. (2011) Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos. Methods in cell biology. 101:1-18.
Abstract
The large and transparent cells of cleavage-stage zebrafish embryos provide unique opportunities to study cell division and cytoskeletal dynamics in very large animal cells. Here, we summarize recent progress, from our laboratories and others, on live imaging of the microtubule and actin cytoskeletons during zebrafish embryonic cleavage. First, we present simple protocols for extending the breeding competence of zebrafish mating ensembles throughout the day, which ensures a steady supply of embryos in early cleavage, and for mounting these embryos for imaging. Second, we describe a transgenic zebrafish line [Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1] that expresses the green fluorescent protein (GFP)-labeled microtubule-binding part of ensconsin (EMTB-3GFP). We demonstrate that the microtubule-based structures of the early cell cycles can be imaged live, with single microtubule resolution and with high contrast, in this line. Microtubules are much more easily visualized using this tagged binding protein rather than directly labeled tubulin (injected Alexa-647-labeled tubulin), presumably due to lower background from probe molecules not attached to microtubules. Third, we illustrate live imaging of the actin cytoskeleton by injection of the actin-binding fragment of utrophin fused to GFP. Fourth, we compare epifluorescence-, spinning-disc-, laser-scanning-, and two-photon-microscopic modalities for live imaging of the microtubule cytoskeleton in early embryos of our EMTB-3GFP-expressing transgenic line. Finally, we discuss future applications and extensions of our methods.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping