PUBLICATION

A GAL4-inducible transgenic toolkit for the in vivo modulation of Rho GTPase activity in zebrafish

Authors
Hanovice, N.J., McMains, E., Gross, J.M.
ID
ZDB-PUB-160424-8
Date
2016
Source
Developmental dynamics : an official publication of the American Association of Anatomists   245(8): 844-53 (Journal)
Registered Authors
Gross, Jeffrey, Hanovice, Nick
Keywords
Cdc42, GAL4/UAS, Rac1, RhoA, zebrafish
MeSH Terms
  • Animals
  • Animals, Genetically Modified
  • DNA-Binding Proteins/genetics
  • DNA-Binding Proteins/metabolism*
  • Transcription Factors/genetics
  • Transcription Factors/metabolism*
  • Zebrafish
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/metabolism*
  • cdc42 GTP-Binding Protein/genetics
  • cdc42 GTP-Binding Protein/metabolism
  • rac1 GTP-Binding Protein/genetics
  • rac1 GTP-Binding Protein/metabolism
  • rho GTP-Binding Proteins/genetics
  • rho GTP-Binding Proteins/metabolism*
  • rhoA GTP-Binding Protein/genetics
  • rhoA GTP-Binding Protein/metabolism
PubMed
27105927 Full text @ Dev. Dyn.
Abstract
Rho GTPases are small monomeric G-proteins that play key roles in many cellular processes. Due to their widespread expression and broad functions, analyses of Rho GTPase function during late development require tissue-specific modulation of activity. The GAL4/UAS system provides an excellent tool for investigating the function of Rho GTPases in vivo. With this in mind, we created a transgenic toolkit enabling spatial and temporal modulation of Rho GTPase activity in zebrafish.
Transgenic constructs were assembled driving dominant-negative, constitutively-active, and wild-type versions of Cdc42, RhoA and Rac1 under 10XUAS control. The self-cleaving viral peptide F2A was utilized to allow bicistronic expression of fluorescent reporter and Rho GTPase. Global heat shock of hsp70l:gal4(+) transgenic embryos confirmed GAL4-specific construct expression. Western blot analysis indicated myc-tagged Rho GTPases were expressed only in the presence of GAL4. Construct expression was confined to proper cells when combined with pou4f3:gal4 or ptf1a:gal4. Finally, transgene expression resulted in reproducible defects in lens formation indicating that the transgenes are functional in vivo.
We generated and validated ten transgenic lines, creating a versatile toolkit for the temporal-spatial modulation of Cdc42, RhoA and Rac1 activity in vivo. These lines will enable systematic analysis of Rho GTPase function in any tissue of interest. This article is protected by copyright. All rights reserved.
Errata / Notes
Corrigendum.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping