Fig. 2. Mis-expression of eng3 results in an anterior shift of the position of the DMB. Mis-expression of 500 pg eng3 mRNA in one cell at the two blastomere stage (A-D). The broken line indicates the midline; the upper side is the control side (con) and the lower side is the injected side (inj). At 1 ss, pax6.1 expression is strongly diminished in the injected side (black arrowheads). The expression of otx2 is not altered. To localize the experimental side, lacZ mRNA was co-injected and detected with a secondary, FITC-coupled antibody (B). At 26hpf, efna4 is suppressed at the injected side (C). The mis-expression of eng3 results in an altered formation (n=36, out of 84) or in a loss of the posterior commissure (n=23, out of 84; D, white arrowheads). To follow the fate of small cell clones in eng3-mRNA-injected embryos, caged fluorescein was co-injected with lacZ-mRNA in control embryos or together with eng3-mRNA (E-J). For this experiment, a microscope was used that was provided with an nitrogen laser unit (E) and a grid (raster size 0.05 mm). At shield stage, the embryos were orientated dorsal side up with the shield to the left. The fluorescein dye was uncaged with a laser beam (365 nm) at the position of the grid: x-axis, –4; y-axis, –3 (F). A picture of a living embryo at shield stage was superimposed on a dark field picture to visualize the clone, and a grid was overlaid. `sh' marks the position of the shield. At 15 ss, the cell clones are visible in the eye or in the posterior diencephalon visualized by a superimposed pseudo lateral picture of the same living wild-type embryo with a darkfield-fluorescence picture (G). In situ hybridization for pax6.1 expression on the same embryo shows that the clone is located in the pax6.1-positive domain (encircled by red dots, H). Small cell clones that were uncaged in embryos injected with 500 pg eng3-mRNA are located in the pax6.1-negative region, suggesting a transformation of cell identity (I,J). (K-M) Injection of different amounts of eng3-mRNA (300 pg and 600 pg, indicated by the red triangle below) show concentration-dependent reduction of pax6.1 expression and of the size of the eye anlage (arrowheads). After the injection of 600 pg eng3-mRNA, the forebrain expression of pax6.1 is completely abolished and the eye anlage was not detectable (M). DMB, diencephalic-mesencephalic boundary; eye, eye anlage; hb, hindbrain; mb, midbrain; PC, posterior commissure; sh, shield.

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