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

Abstract

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.

Fig. 1.

The gene trap and enhancer trap constructs and the UAS reporter system. The Tol2 vector sequences are shown as thick black lines. (A) The structures of T2KhspGGFF, T2KhspGGFF, and T2KSAGFF. (B) The UAS:GFP reporter fish carries a single-copy insertion of T2KUASGFP within a gene encoding a homolog of Nedd4-binding protein 1. Blue boxes indicate exons. (C) The UAS:RFP reporter fish carries a single-copy insertion of T2ZUASRFP within a gene encoding a solute carrier protein homolog. Blue boxes indicate exons. (D and E) The hspGGFF1B embryos before (D) and after (E) heat shock. (F and G) The hspGGFF1B;UAS:GFP embryos before (F) and after (G) heat shock. (H and I) The hspGGFF1B;UAS:RFP embryos before (H) and after (I) heat shock.

Fig. 2.

GFP and RFP expression in the hspGGFF15A enhancer trap line. (A) The structure of the hspGGFF15A insertion. T2KhspGGFF is integrated in the first exon of the skib gene. (B) GFP expression in the hspGGFF15A;UAS:GFP embryo at 24 hpf. (C) RFP expression in the hspGGFF15A;UAS:RFP embryo at 24 hpf. (D) Whole-mount in situ hybridization of the hspGGFF15A embryo at 24 hpf using the GGFF probe. (E) Whole-mount in situ hybridization of a wild-type embryo at 24 hpf and the skib probe.

Fig. 3.

Abnormal touch response phenotypes in the UAS:TeTxLC double transgenic embryos. (A) The UAS:TeTxLC effector fish carries a single-copy insertion of T2MUASTeTxLC in the myosin heavy chain gene. A blue box indicates an exon. (B) The touch response behavior of a wild-type embryo at 48 hpf. (C) GFP expression in the hspGGFF27A;UAS:GFP embryo at 24 hpf and the touch response behavior of the hspGGFF27A;UAS:TeTxLC embryo at 48 hpf. (D) GFP expression in the SAGFF31B;UAS:GFP embryo at 24 hpf and the touch response behavior of the SAGFF31B;UAS:TeTxLC embryo at 48 hpf. (E) GFP expression in the SAGFF36B;UAS:GFP embryo at 24 hpf and the touch response behavior of the SAGFF36B;UAS:TeTxLC embryo at 48 hpf.

Fig. 4.

Expression of TeTxLC:CFP in double transgenic embryos. (A) The UAS:TeTxLC:CFP fish carries a single-copy insertion of T2SUASTeTxLCCFP within the CSPP1 gene. Blue boxes indicate exons. (B–G) Lateral views of the trunk of double transgenic embryos immunostained with the anti-GFP antibody (green) and the anti-Hb9 (B–E) or the zn-12 (F and G) antibody (red). Arrowheads indicate costaining with anti-GFP and anti-Hb9 (E) or anti-GFP and zn-12 (G). Anterior is to the left, and dorsal is to the top. (Scale bars: 50 μm.) (B and C) The hspGGFF27A;UAS:TeTxLC:CFP embryo at 48 hpf. The anti-GFP antibody detects the TeTxLC:CFP fusion protein but does not detect the GGFF protein in this condition (data not shown). Axons of descending hindbrain interneurons were strongly stained (green). The anti-Hb9 antibody detected spinal motor neurons (red). (D and E) The SAGFF31B;UAS:TeTxLC:CFP embryo at 48 hpf. The anti-GFP antibody detected spinal interneurons and motor neurons (green). (F and G) The SAGFF36B;UAS:TeTxLC:CFP embryo at 30 hpf. Both the anti-GFP and zn-12 antibodies detected Rohon-Beard neurons (green and red).