Fig. 6. Dynamic regulation of her5 expression within the MH domain. (A-O) Comparison of her5 expression (revealed by in situ hybridisation, blue staining in C-E,G,I,K, red staining in M,O) and GFP protein distribution (direct visualization under fluorescence microscopy, green in A,B; or revealed by anti-GFP immunocytochemistry, brown staining in D or green staining in F,H,J,L,N,O) in her5PAC:egfp embryos at the stages indicated. (A-D) Whole-mount views (A, dorsal, anterior leftwards; B,C, lateral, anterior leftwards; D, dorsal view of a hemi-neural plate, anterior upwards); E-O are sagittal sections, all views focus on the MH domain and are oriented anterior towards the left. The MHB is indicated by a red arrow at all stages where it is morphologically visible. (E-L) Bright field (top panels) and fluorescent (bottom panels) views of the same sections; M-O are red, green or double fluorescent views of the same section. Note the dramatic difference in the extent of her5 transcripts (C) and GFP protein (A,B) along the AP axis at 24 hpf. Because egfp transcription faithfully reproduces her5 expression in her5PAC:egfp embryos (Fig. 3), whereas GFP protein is stable, this demonstrates that her5 expression is lost from progeny cells over time. This process is progressive (D-L) and sequential: it involves first a restriction of her5 expression in the posterior aspect of the MH domain (blue and brown arrows indicate the limits of her5 RNA and GFP protein staining, respectively, in D; blue dots indicate the posterior limit of her5 transcription. Note that the two limits coincide anteriorly but differ by one or two cell rows posteriorly). At three somites, her5 restriction begins in ventral and lateral aspects of the mesencephalon (black arrows in E,G), and continues after 16 somites (I) along the dorsal midline (blue arrows in E,G indicate maintained dorsal expression of her5 prior to that stage). Note that in M-O, the final her5 expression domain is located in the center of the GFP-positive territory, demonstrating that her5 expression gets restricted in a converging manner towards the MHB. (P) Resulting model for the regulation of her5 expression and the progression of neurogenesis between 70% epiboly (a), 90% epiboly (b) and 30 somites (c,d) in the MH domain [combined from the present data and data from Geling et al. (Geling et al., 2003)]. her5 expression at 70% epiboly (blue), traced using GFP protein in her5PAC:egfp embryos, is the entire MH anlage (green lines and labeling, 45-50 cell rows at 30 somites). Between 70 and 90% epiboly (b), her5 expression is lost from progeny cells posteriorly (compare green lines and blue). At 90% epiboly, her5 expression is adjacent to the first anterior neurogenesis sites: the ventrocaudal cluster (vcc, pink, precursor of the nucleus of the medial longitudinal fascicle, nMLF) and future motor and sensory neurons of r2 (orange) (see Geling et al., 2003). At 30 somites (c), her5 expression has been dramatically lost upon cell divisions and is restricted to three to five cell rows at the MHB. Correlatively (d), neurogenesis (revealed by zcoe2 expression) (Bally-Cuif et al., 1998), still adjacent and non-overlapping with her5 expression (compared c with d), progressed towards the MHB (red arrows) (embryo with the same orientation as in c, focus on the basal plate).

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