Using Zebrafish for Studying Rho GTPases Signaling In Vivo
Rho small GTPases play pivotal roles in a variety of dynamic cellular processes including cytoskeleton rearrangement, cell migration, cell proliferation, cell survival, and gene regulation. However, their functions in vivo are much less understood. Recently, the zebrafish, Danio rerio has emerged as a powerful model organism for developmental and genetic studies. Zebrafish embryos have many unique characteristics, such as optical transparency, external fertilization and development, and amenability for various molecular manipulations including morpholino oligo-mediated gene knockdown, mRNA or DNA overexpression-induced gain of function or rescue, in situ hybridization (ISH) with riboprobes for gene expression, western blot for protein analysis, small-molecule inhibition on signaling pathways, and bioimaging for tracking of molecular events. Taking many of such advantages, we have demonstrated the role of rhoA small GTPase in the control of gastrulation cell movements and cell survival during early zebrafish embryogenesis, linking RhoA functions to at least the noncanonical Wnt, Mek/Erk, and Bcl2 signaling nodes in vivo. Here, we describe the use of such techniques, including gene knockdown by morpholino oligo, functional rescue by mRNA overexpression, microinjection, ISH, western blot analysis and pharmacological inhibition of signaling pathways by small molecule inhibitors, with special considerations on their merits, potential drawbacks, and adaptation which could pave the way to our better understanding of the roles of various classes of small GTPases in regulating cell dynamics and development in vivo.
Key wordsRho small GTPase Zebrafish Mopholino oligos mRNA rescue Microinjection In situ hybridization Western blot Small molecule inhibitor
This work was supported in part by the Mechanobiology Institute, National University of Singapore, co-funded by the National Research Foundation and the Ministry of Education, Singapore, and also from a grant from the Biomedical Research Council of Singapore, and a fellowship from the Friends for Life.
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