TY - CHAP
T1 - Using zebrafish for studying Rho GTPases signaling in vivo
AU - Zhu, Shizhen
AU - Low, Boon Chuan
PY - 2012
Y1 - 2012
N2 - 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.
AB - 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.
KW - In situ hybridization
KW - Microinjection
KW - Mopholino oligos
KW - Rho small GTPase
KW - Small molecule inhibitor
KW - Western blot
KW - Zebrafish
KW - mRNA rescue
UR - http://www.scopus.com/inward/record.url?scp=84855905231&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84855905231&partnerID=8YFLogxK
U2 - 10.1007/978-1-61779-442-1_21
DO - 10.1007/978-1-61779-442-1_21
M3 - Chapter
C2 - 22144284
AN - SCOPUS:84855905231
SN - 9781617794414
T3 - Methods in Molecular Biology
SP - 321
EP - 337
BT - Rho GTPases
A2 - Rivero, Francisco
ER -