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Fig. 3. Her5 acts by blocking the formation of a proneural cluster across the IZ. (A-D) Expression of components of the lateral inhibition machinery (e.g. delA, notch1a) is induced across the IZ upon injection of MO^her5 (B,D, arrows) compared with non-injected controls (A,C) (brackets indicate IZ). All views are whole-mount in situ hybridisation for the markers indicated (bottom left), dorsal views of the MH area in flat-mounted embryos at the three-somite stage. (E-G) Induced expression of ngn1 across the IZ upon injection of MO^her5 (arrows in E) exhibits at the three-somite stage a salt-and-pepper pattern similar to that of anterior (vcc) or posterior (r2) proneural clusters. This is followed by the development, at the 18-somite stage, of zn12-positive differentiated neurons across the IZ (G, compare with F; brown immunocytochemistry staining). Arrow in F indicates the IZ, which is labelled in blue for her5 expression. (Geling et al., 2003). (H) The number of ectopic zn12-positive neurons (right panel) differentiating across the IZ in the absence of Her5 activity [G, blue box (calculated as black box in G minus black box in F)] is lower than the number of ngn1-positive cells (left panel) initially induced across the IZ at the three-somite stage (Fig. 1C, blue box). (I,J) Fate of MH cells expressing ngn1 at the onset of neurogenesis in wild-type embryos (I) and across the IZ in MO^her5-injected embryos (J). Descendants of early ngn1-positive cells are revealed by their retention of GFP protein at the 20-somites stage in the –3.4ngn1:gfp transgenic line (green staining), while differentiated neurons are positive for the zn12 antigen (red staining). Note in the overlay (right panels) that several green cells are negative for zn12 in both cases (green arrows). (K-M) The ngn1-positive domain induced across the IZ in the absence of Her5 activity is sensitive to lateral inhibition. The number of strongly ngn1-positive cells in the IZ (brackets) at the three-somite stage, induced by lack of Her5 expression, is reduced upon forced expression of NICD (L, compare with K) and increased upon expression of Delta^Stu (M, compare with K). It follows similar dynamics as ngn1 expression in adjacent anterior (vcc) and posterior (r2) proneural clusters (arrowheads).

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