Temporally-controlled site-specific recombination in zebrafish

Abstract

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER(T2). Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.

Figure 1. Cre-mediated recombination in the red-to-green reporter line.

(a) Scheme of the recombination event. In the absence of Cre the EF1α promoter drives the expression of DsRed2 but changes to EGFP after successful Cre-mediated recombination. (b) Embryos of the red-to-green reporter line show strong DsRed2 and no EGFP fluorescence. (c) Maternal contribution of the Tg(hsp70:EGFP-Cre) allele results in complete loss of DsRed2 and ubiquitous EGFP expression in double transgenic embryos. (d) Paternal contribution of the Tg(hsp70:EGFP-Cre) allele leads to strong DsRed2 and mosaic EGFP expression in double transgenic embryos. (e) Paternal contribution of the Tg(hsp70:EGFP-Cre) allele and brief heat induction at mid-gastrulation stages results in reduced DsRed2 and strong ubiquitous EGFP expression in double transgenic embryos. (f) Embryos of the Tg(hsp70:EGFP-Cre) line show only weak EGFP fluorescence after brief heat induction at mid-gastrulation stages. b–f Lateral views of live 24 hpf embryos bearing different transgenes. Scale bar, 125 µm.

Figure 2. Ligand-dependent Cre-mediated recombination.

(a) Scheme of the ligand-dependent recombination event in cells of the red-to-green reporter line. The chimeric CreER^T2 recombinase is retained in the cytoplasm in the absence of the ligand. After administration of TAM which is converted to the active ligand 4-OHT, CreER^T2 translocates to the nucleus, where it catalyzes the recombination event. (b) Expression of CreER^T2 in the diencephalon of the Tg(pax2a:CreER^T2)^#19 line at early segmentation stages revealed by in situ hybridization. (c) EGFP expression in the diencephalon of double transgenic embryos at 24 hpf bearing the red-to-green reporter and the Tg(pax2a:CreER^T2)^#19 alleles after TAM treatment at mid-gastrulation stages. (d) Expression of CreER^T2 in rhombomere 3 and 5 of the Tg(pax2a:CreER^T2)^#45 line at early segmentation stages revealed by in situ hybridization. (e) EGFP expression in rhombomere 3 and 5 of double transgenic embryos at 24 hpf bearing the red-to-green reporter and the Tg(pax2a:CreER^T2)^#45 alleles after TAM treatment at mid-gastrulation stages. Abbreviations: f, forebrain; h, hindbrain; m, midbrain. Scale bar, 50 µm.

Figure 3. Kinetics of ligand-dependent Cre-mediated recombination.

(a) Expression of CreER^T2 in the Tg(pax2a:CreER^T2)^#19 line at the 12-somite stage revealed by in situ hybridization. (b) Control embryos treated with DMSO never show any EGFP. (c) Immunofluorescence staining with antibodies to EGFP is detectable 4 hours after application of TAM and expanded further after 6 hours. (d) Onset of EGFP expression by immunofluorescence staining is detected after 2 hours and expanded further after 4 and 6 hours after application of 4-OHT. a–d Dorsal views of double transgenic embryos at 12-, 16-, 20 and 24-somite stage (15, 17, 19 and 21 hpf). Abbreviations: f, forebrain; e, eye anlage; h, hours; m, midbrain. Scale bar, 30 µm.

Figure 4. Dose-dependent recombination in the Tg(pax2a:CreER^T2)^#19 and Tg(pax2a:CreER^T2)^#45 lines by TAM.

(a) Double transgenic embryos bearing the red-to-green reporter and the Tg(pax2a:CreER^T2)^#19 alleles show strong EGFP expression in the diencephalon after application of 5 µM TAM at mid-gastrulation stages. Application of 0.5 µM TAM or 0.05 µM TAM at the same stage, results in reduced EGFP expression or single EGFP-positive cells, respectively. (b) Double transgenic embryos bearing the red-to-green reporter and the Tg(pax2a:CreER^T2)^#45 alleles show strong EGFP expression in rhombomere 3 and 5 after application of 5 µM TAM at mid-gastrulation stages. Application of 0.5 µM TAM at the same stage, results in single EGFP-positive cells. a, b Dorsal views of double transgenic embryos at 24 hpf. Scale bar, 30 µm.