Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish

Asakawa, K., Suster, M.L., Mizusawa, K., Nagayoshi, S., Kotani, T., Urasaki, A., Kishimoto, Y., Hibi, M., and Kawakami, K.
Proceedings of the National Academy of Sciences of the United States of America   105(4): 1255-1260 (Journal)
Registered Authors
Hibi, Masahiko, Kawakami, Koichi, Kishimoto, Yasuyuki, Suster, Maximiliano
targeted gene expression, Gal4-UAS, tetanus toxin, touch response, interneuron
MeSH Terms
  • 5' Untranslated Regions/genetics
  • Animals
  • Animals, Genetically Modified
  • Crosses, Genetic
  • DNA Transposable Elements/genetics
  • DNA Transposable Elements/physiology*
  • DNA-Binding Proteins
  • Enhancer Elements, Genetic/genetics*
  • Gene Expression Regulation, Developmental/genetics
  • Gene Targeting*
  • Genes, Reporter
  • Green Fluorescent Proteins/genetics
  • HSP70 Heat-Shock Proteins/genetics
  • Nerve Net/physiology*
  • Neural Inhibition/genetics
  • Organ Specificity/genetics
  • Saccharomyces cerevisiae Proteins/biosynthesis
  • Saccharomyces cerevisiae Proteins/genetics*
  • Saccharomyces cerevisiae Proteins/metabolism
  • Touch/genetics
  • Touch/physiology
  • Trans-Activators/genetics
  • Transcription Factors/biosynthesis
  • Transcription Factors/genetics*
  • Transcription Factors/metabolism
  • Zebrafish/embryology
  • Zebrafish/genetics*
18202183 Full text @ Proc. Natl. Acad. Sci. USA
Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.
Genes / Markers
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Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes