A GAL4-inducible transgenic tool kit for the in vivo modulation of Rho GTPase activity in zebrafish.

Abstract

Rho GTPases are small monomeric G-proteins that play key roles in many cellular processes. Due to Rho GTPases' widespread expression and broad functions, analyses of their 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 tool kit 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 a 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 10 transgenic lines, creating a versatile tool kit 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. Developmental Dynamics 245:844-853, 2016. © 2016 Wiley Periodicals, Inc.