FIGURE SUMMARY
Title

Temporally-controlled site-specific recombination in zebrafish

Authors
Hans, S., Kaslin, J., Freudenreich, D., and Brand, M.
Source
Full text @ PLoS One
Fig. 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.
Fig. 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 CreERT2 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, CreERT2 translocates to the nucleus, where it catalyzes the recombination event. (b) Expression of CreERT2 in the diencephalon of the Tg(pax2a:CreERT2)#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:CreERT2)#19 alleles after TAM treatment at mid-gastrulation stages. (d) Expression of CreERT2 in rhombomere 3 and 5 of the Tg(pax2a:CreERT2)#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:CreERT2)#45 alleles after TAM treatment at mid-gastrulation stages. Abbreviations: f, forebrain; h, hindbrain; m, midbrain. Scale bar, 50 μm.
Fig. 3

Kinetics of ligand-dependent Cre-mediated recombination.

(a) Expression of CreERT2 in the Tg(pax2a:CreERT2)#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.
Fig. 4

Dose-dependent recombination in the Tg(pax2a:CreERT2)#19 and Tg(pax2a:CreERT2)#45 lines by TAM.

(a) Double transgenic embryos bearing the red-to-green reporter and the Tg(pax2a:CreERT2)#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:CreERT2)#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.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ PLoS One
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