Fig. 2

Non-neural ectoderm contributes to the pineal organ. (A-C) Small patches of cells (white arrows) were labelled using uncaging of fluorescein (Fluo, red) at bud stage. Embryos were fixed immediately and subjected to in situ hybridisation for irx1b (blue) to demonstrate precision of labelling (anterior is leftwards). A, n=6; B, n=4; C, n=7. (D-F) Lateral views (anterior is leftwards) of the diencephalon of embryos labelled as in A-C, incubated for 24 h and labelled for expression of otx5 (red) marking the definitive pineal organ (red arrowheads). Cells labelled by uncaging (blue) in the otx5-positive pineal organ are labelled in E and F. D, n=11; E, n=11; E, n=19/23. (G-I) Sequential images of time-lapse recording of an embryo from late bud to the 26-somite stage in which Kaede-expressing cells have been converted to red fluorescence in the ectoderm flanking the diencephalon. The trajectories of three representative cells (blue, red and green arrowheads in G) are shown in red, green and blue in H,I. (J) Dorsal view showing converted cells in the pineal organ at 22 hpf. See also Movies 1 and 2. (K) Lateral view, anterior towards the left, showing converted cells in the ectoderm and pineal organ, but not in the brain proper, at 30 hpf. Four out of 12 embryos had no converted cells in the brain proper; all four of those showed converted cells in the pineal organ. E, eye; L, lens; T, telencephalon. Dotted lines in J and K outline the pineal organ. Scale bars: 50 µm; bar in A applies to A-C,G-I; bar in D applies to D-F,J,K.