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Fig. 5 Misexpression of ngn1 induces ectopic neurons. (A) Ventral view of 3-somite stage, ngn1 RNA-injected embryo showing ectopic isl-1 expression in the non-neural ectoderm of the yolk sac (arrowheads). (B) Transverse section through yolk sac of ngn1 RNA-injected embryo showing ectopic isl-1-expressing cell in the yolk sac ectoderm. (C,D) ngn1 RNA-injected embryo (C), and uninjected control (D), at the 18- somite stage hybridised to isl-1 probe. Ventral view of injected embryo (C) shows the ectopic isl-1-expressing cells in the yolk sac (arrowhead) forming cell clusters at this stage. Control embryo (D) is shown in lateral view. (E-G) ngn1-injected embryos analysed for isl-1 expression at the 18-somite stage. (E) Lateral view of the head with an expanded trigeminal ganglion (arrows, tg; compare to D); (F,G; lateral views, dorsal up). Optical sections at the level of the surface ectoderm (F) and the midline of the neural tube of the same injected embryo. Position of frames along the anteroposterior axis is indicated in D. While injected embryos develop numerous isl-1- expressing cells in the surface ectoderm (arrowheads in F) the number of isl-1-expressing Rohon- Beard (rb in G) and motor neurons (arrow in G) in the neural tube is unaltered in injected embryos (compare with uninjected control in D). (H) Head of ngn1 RNA-injected 24h embryo immunohistochemically stained with antibody zn12 which recognises the L2/HNK1 epitope. Embryo (dorsal view onto head, anterior left) shows loss of an eye and concomitant unilateral ectopic formation of a ganglion-like structure (arrowhead) and unilateral expansion of the trigeminal ganglion (arrows). (I) Optical section through surface ectoderm at the level of the hindgut of ngn1 RNA-injected 24h embryo immunohistochemically stained with antibody zn12. Neurons (arrowheads) with processes which contact each other develop ectopically in the epidermis of injected embryos. (J, K) View onto yolk sac (J) and optical section through body axis (K) of embryos injected with β-gal RNA. Embryos were stained for β-gal activity (turquoise) and immunohistochemically with antibody zn12 (brown). Expression of β-gal did not cause the formation of ectopic neurons. Only the normal expression of the L2/HNK1 epitope could be detected in injected embryos (K, arrowheads). (L,M) ngn1-myc RNAinjected embryo (L) and uninjected control (M) at the 3-somite stage hybridised to isl-1 probe. The embryo shown in L was injected in both blastomeres at the two-cell stage. Ectopic isl-1-expressing cells are found lateral to, but not within the neural plate. Views onto the neural plate at the level of the posterior hind brain/anterior trunk, anterior up. The optical section through the embryo shown in L is taken from a deeper position than the section in M to show ectopic neurons (arrowheads) on both sides of the neural plate. The normal pattern of isl-1 expression in the neural plate is therefore out of focus in the injected embryo. (N,O) Embryos injected with ngn1-myc RNA in one blastomere at the two-cell stage and immunohistochemically stained with anti-myc antibody 9E10 at the 3-somite stage to visualise ectopic expression of Ngn1-MYC. View is dorsal with anterior up (N) and optical transverse section through neural plate with dorsal up (O). Ngn1-MYC is strongly expressed in the neural plate (arrowhead in O) in the injected side (in). Lack of immunostaining on the uninjected side (co) shows that the MYC immunoreactivity is specific. Abbreviations: e, eye; y, yolk; tg, trigeminal ganglion; rb; Rohon-Beard sensory neurons; in, injected side of embryo; co, control side of embryo. Scale bar represents 50 μm in A,C,D,L,M,N,O; 25 μm in E,F,GJ,K; and 12 μm in B,I.