The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

Abstract

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.

Figure 1. The structures of full-length Gal4, Gal4-VP16 and Gal4FF

The structures of the full-length Gal4 (top), Gal4-VP16 (middle) and Gal4FF (bottom) are shown. The DNA-binding domain of Gal4 (DBD, 1–147 a.a,) is shown in blue. Transcription activation domains of Gal4 (AD I: 148–196 a.a. and AD II: 768–881 a.a.), a transcriptional activation domain from VP16 (VP16) and two transcription activation modules (FF) are shown in red. The bar corresponds to 100 a.a.

Figure 2. The Tol2 transposable element and constructs

(A) The structure of the full-length Tol2 transposable element. The Tol2 sequence is shown as a thick black line. Thin lines and dotted lines indicate exons and introns of a gene for the transposase. (B) The structures of the enhancer trap construct T2KhspGFF and the gene trap construct T2KSAGFF. Tol2 sequences are shown in thick black lines. Arrows indicate the coding sequence for Gal4FF (red) and the zebrafish hsp70 promoter (orange). SA indicates a splice acceptor from the rabbit β-globin gene. (C) The structures of UAS constructs. Tol2 sequences are shown in thick black lines. Arrows indicate the EGFP (green), mRFP1 (magenta) and TeTxLC:CFP fusion (gray and blue) genes. Scale bars indicate 500 bp.

Figure 3. The Gal4FF gene trap and enhancer trap screens

(A) A scheme for the Gal4FF gene trap and enhancer trap screens. A plasmid DNA containing a Gal4FF construct and the transposase mRNA are coinjected into fertilized eggs. The Gal4FF construct is excised from the plasmid and integrated in the genome during embryonic development. The excision event is detected by PCR (excision assay). An outline of the excision assay is shown in the box. The gel shows a typical result of the excision assay. Injected fish are raised and mated with the reporter fish homozygous for the UAS:GFP insertion. The resulting F1 embryos are analyzed with a fluorescence stereomicroscope. F1 embryos expressing GFP are collected and raised for further studies. (B) Specific GFP expression patterns observed in the Gal4FF;UAS:GFP double transgenic embryos and larvae. (C) GFP or RFP expression patterns observed in embryos carrying the 213A-Gal4FF insertion and the UAS:GFP reporter (left), or carrying the 213A-Gal4FF insertion and the UAS:RFP insertion (right).

Figure 4. Molecular characterization of the SAGFF73A insertion

(A) GFP expression in the SAGFF73A;UAS:GFP double transgenic embryo. (B) Southern blot analysis of F1 fish used to raise F2 fish. The F1 fish carried 6 insertions of T2KSAGFF. (C) Southern blot analysis of F2. An arrow indicates a common band, which is responsible for the expression pattern. The fish on lane 5 (arrowhead) was used to raise F3. (D) Southern blot analysis of F3. Two fish (lanes 3 and 6) carried a single insertion (arrow). (E) Results of inverse PCR. PCR products were amplified that contained 5’ (left) and 3’ (right) ends of Tol2 and surrounding genomic sequences. (F) 8-bp target site duplication was created adjacent to the SAGFF73A insertion. (G) Analysis of the genomic sequence by using the ensemble database. The T2KSAGFF construct was integrated within the zfand5b gene. (H) RT-PCR analysis of the SAGFF73A gene trap line. Arrowheads indicate positions and orientations of primers used for RT-PCR. (I) The results of the RT-PCR analysis. The fusion transcript of the zfand5b and Gal4FF (f1-r1) was only amplified from heterozygous SAGFF73A embryos (het). A normal zfand5b transcript (f1-r2) was reduced in heterozygous embryos (het) in comparison to wild type (wt). SD: the splice donor of the first exon of zfand5b. SA: the splice acceptor on the T2KSAGFF construct.

Figure 5. Targeted inhibition and visualization of neural circuits by the UAS:TeTxLC:CFP fish

(A) Generation of Gal4FF;UAS:TeTxLC:CFP double transgenic embryos. Gal4FF;UAS:GFP double transgenic fish and the UAS:TeTxLC:CFP fish are crossed. The CFP-positive embryos are selected with a fluorescence stereomicroscope. (B–D) The CFP fluorescence detected in the hspGGFF27A;UAS:TeTxLC:CFP embryo (B), the SAGFF31B;UAS:TeTxLC:CFP embryo (C) and the SAGFF36B;UAS:TeTxLC:CFP embryo (E) at 48 hpf. (E–G) Immunohistochemistry using anti-GFP antibody detected TeTxLC:CFP expression (green) in the hindbrain interneurons (E, hspGGFF27A, 48 hpf), subpopulations of spinal interneurons (F, SAGFF31B, 48 hpf) and Rohon-Beard sensory neurons (G, SAGFF36B, 30 hpf). The boxes with yellow dotted line on the left panels show the approximate position of right panels. Anti-Hb9 and zn-12 antibodies (magenta) detect motor neurons and Rohon-Beard sensory neurons, respectively. Arrowheads indicate cells co-labeled with both of the antibodies (F and G). The bars indicate 20 µm (E, F and G). (H) Touch response assay. A tactile stimulus is applied by a needle to the wild-type, hspGGFF27A;UAS:TeTxLC:CFP, SAGFF31B;UAS:TeTxLC:CFP and SAGFF36B;UAS:TeTxLC:CFP embryos at 48 hpf.