Fig. 6. Her5 activates cell proliferation within the MH domain, but this process is in itself insufficient to account for the regulation of ngn1 expression. (A,F) her5 expression (in situ hybridization, blue staining) and density of cells in M phase (brown anti-phosphoH3 immunostaining) (in number of positive cells per cell row) in the anterior neural plate at the 3-somite stage (in territories schematized in A) in hsp-her5 transgenics (C), their wild-type siblings (B) (both heat-shocked), wild-type (D) and MO^her5-injected embryos (E). A is a schematic representation of the neural plate in B,C; B-E are flat-mounted views of the anterior neural plate (B,C) or the endogenous her5 domain (D,E), anterior to the top. White arrow in B,C, the endogenous domain of her5 expression (territories A + B); box in D,E indicates territory A. Proliferation is enhanced in territory A compared to other neural plate domains in wild-type embryos (brown arrows in B,D). Her5 is sufficient to increase proliferation within the MH domain upon ectopic expression (territories A-D) (F, left panel), and is necessary for the increased level of proliferation of territory A (F, right panel). (G-J) Expression of the cyclin-dependent kinase inhibitor-encoding gene p27^Xic1-a is downregulated by Her5 within the IZ. Expression of p27^Xic1-a and pax2.1, as indicated (bottom left, color-coded), in hsp-her5 transgenic embryos after heat-shock (H) and MO^her5-injected embryos (J) compared to their wild-type siblings (G,I) at the 3-somite stage. In the vcc and IZ, p27^Xic1-a expression is strikingly similar to that of ngn1 (e.g. Fig. 3D). p27^Xic1-a expression is down-regulated within the neural plate following ectopic her5 expression (arrow in H), and is activated across the IZ when Her5 activity is blocked (white arrows in J). Concomitantly in the latter case, p27^Xic1-a expression is partially reduced in the vcc area, a phenomenon at present unexplained but independent of cell migration (A.G. and L.B.-C., unpublished data). (K-N) The direct inhibition of cell proliferation does not affect IZ formation and her5 expression. Expression of ngn1 or her5 (blue in situ hybridization staining) and anti-phosphoH3 immunostaining (brown nuclei) at the 3-somite stage in embryos treated with the cell proliferation inhibitor aphidicolin at the onset of neurogenesis (L,N) compared to mock-treated siblings (K,M). Although phosphoH3 staining is virtually abolished upon aphidicolin treatment, both IZ size (bracket in K,L) and her5 expression appear normal. Aphi, aphidicholin-treated embryo; hs, heat-shocked embryo; IZ, intervening zone; tg, transgenic; vcc, ventrocaudal cluster.