Tg(col2a1aR2:KalTA4) enables stable and transient oncogene expression in the developing notochord. (A,B) Lateral view (A) and transverse histology section (stained with H&E) (B) of 5 dpf zebrafish embryos transgenic for the transgene col2a1aR2:KalTA4 and crossed with stable UAS:Kaede (visible as green fluorescence in A). Note expression in the notochord, craniofacial cartilage, otic vesicle and pectoral fins; the prominent green heart (white arrowhead, A) indicates the myl7:EGFP transgenesis marker associated with col2a1aR2:KalTA4. The developing notochord shows that the large vacuolated cells take up the vast majority of the notochord volume and are rimmed by a thin layer of sheath cells (B). (C,D) Expression of stable UAS:EGFP-HRASV12 (detectable by green fluorescence of the fusion protein, C) by col2a1aR2:KalTA4causes invasive and widespread notochord hyperplasia (black arrowheads, D) and overgrowth of other cartilage tissue (i.e. otic vesicle, black asterisk in C); the white arrowhead indicates the myl7:EGFP transgenesis marker (A). (E-G) col2a1aR2 provides a potent driver for transient notochord expression. (E) Injection of UAS:EGFP into the one-cell-stage embryos with either twhh:Gal4 (F) or col2a1aR2:KalTA4 (G) to visualize the notochord mosaicism resulting from random integration of UAS:EGFP by Tol2 transposase. While injections into twhh:Gal4 result in highly patchy EGFP expression (F, green fluorescence; n=33/56), col2a1aR2:KalTA4 more consistently drives EGFP expression throughout the notochord (G, green fluorescence; n=25/47); n indicates representative EGFP-expressing embryos in an injected representative clutch. Scale bars: 500 μm in A,C,F,G; 200 μm in B,D. See also Fig. S1.
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