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Fig. 3 Zebrabow-based lineage tracing to visualize lineage restriction patterns at the MHB. Multicolor-labeling of midbrain and hindbrain cells using the otx2b:CreERT2 and fgf8a:CreERT2 knock-in driver lines. (A) Schematic representation of CreERT2 -mediated recombination strategy using the zebrabow transgenic responder fish. In cells expressing CreERT2, 4-OH-tamoxifen (4-OHT) induces recombination between either the two lox2272 sites (marked by spotted triangles) or the two loxp sites (marked by a triangle), which results in the stochastic labeling of cells due to the expression of CFP (cyan fluorescent protein) or YFP in the recombined cells. All non-recombined cells express only RFP (red fluorescent protein). In a cell with multiple copies of RFP, CFP and YFP, CreERT2 -mediated stochastic recombination events lead to the formation of clones marked by different colors. Embryos obtained by crossing Cre driver fish (either otx2b or fgf8a) with the zebrabow responder line were treated with 4-OH-tamoxifen, either at 6 hpf (1 μM) or 24 hpf (10 μM) for 12 h; these embryos were live-imaged at 48 hpf. (B-B″) otx2b:CreERT2 embryos treated with 4-OHT at 6 hpf show effective recombination in the midbrain region but also a few recombined cells in the hindbrain region (arrowheads). (C-C″) otx2b:CreERT2 embryos treated with 4-OHT at 24 hpf show recombined cells only in the midbrain. (D-D″) Embryos of the fgf8a:CreERT2 knock-in driver line treated with 4-OHT at 6 hpf show effective recombination in the hindbrain with the exception of a few recombined cells in the midbrain (arrowheads). The above-described phenotype was consistently observed in multiple embryos, and the total number of embryos (n) analyzed for each condition are: otx2b:CreERT2 (4-OHT at 6 hpf, n=9; at 24 hpf, n=3); fgf8a:CreERT2 (4-OHT at 6 hpf, n=6). Scale bars: 20 µm.