Fig. 4. The distribution of GFP protein in her5PAC:egfp embryos reveals the fate of endodermal and neuroectodermal cells expressing her5 at gastrulation. GFP protein in her5PAC:egfp embryos was observed on live specimen (J) or revealed by immunocytochemistry (A-I, brown DAB staining; and K-Q, green FITC staining) at the stages indicated (bottom left of each panel). (H-J) Whole-mount views: (H,J) dorsal views, anterior leftwards; (I) lateral view, anterior leftwards. (K-Q) Sagittal sections, anterior leftwards. In K,L,O-Q, the top and bottom panels are bright-field and fluorescent views, respectively, of the same sections that were each processed for in situ hybridisation (top panels, blue staining, probes indicated in the bottom right-hand corner) and immunocytochemistry against GFP protein (bottom panels). (M,N) High magnifications of levels equivalent to those boxed in K and L, respectively (red arrows indicate rhombomere boundaries). Overlay pictures of the in situ hybridisation staining (revealed using Fast Red, red fluorescence) and GFP immunocytochemistry (FITC staining). The cytoplasm of cells doubly positive for GFP protein and for the in situ hybridisation marker (hoxa2 or krox20, respectively) appears yellow. The descendants of endodermal her5-expressing cells distribute to the entire AP and mediolateral extent of the pharynx (A; cross-section at hindbrain level in B, bottom). At 12 somites, the descendants of neural her5-expressing cells distribute over a broad domain at the level of the MH (A; cross-section at forebrain level in B, top). Neural crest cells that exit the MH are also GFP-positive (C, note a dorsal stream and a stream caudal to the eyes, and cross-section at forebrain level in D). In E-J, arrows indicate the midbrain-hindbrain boundary; note that GFP protein distributes posterior to this level (i.e. to metencephalic derivatives) until 24 hpf, and encompasses r2 (K,O,Q; blue and green arrowheads to the anterior limit of hoxa2 expression; green arrows to GFP-positive cells in r2), with a minor contribution to r3 and r4 before the 20-somite stage (L,P; white brackets indicate r3 and r5, green arrow in L indicates GFP cells in r4; green arrowhead in P indicates the posterior limit of GFP extension at the r2/r3 boundary). At 10 somites, GFP-positive cells in r2 and r3 co-express hoxa2 and krox20, respectively (yellow arrows in M,N). e, endoderm; hg, hatching gland; MH, midbrain-hindbrain domain; MHB, midbrain-hindbrain boundary; nc, neural crests streams.