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Fig. 3 Fgf8 expression in early midbrain and anterior hindbrain development. (A-D) Double ISH with Fgf8 (blue) and Krox20 (red, fluorescent) of wild-type embryo at tailbud stage (A,B) and 5 somites (C,D). At tailbud stage, Fgf8 expression extends throughout the anterior hindbrain incl. rhombomere 4 (r4) posteriorly. At 5 somites, expression of Fgf8 is detected at the MHB, in r1, r4 and in ventral r2 (see also Fig. 2H). (E-J) Double ISH with Fgf8 (blue) and Pax2.1 (red, fluorescent) at 90% epiboly (E-H) and 6 somites (I,J). At 90% epiboly, the Fgf8 expression domain is located posterior to the Pax2.1 domain with very little overlap (E,F; higher magnification: G,H), while at 6 somites the Fgf8 domain at the MHB is completely included in the Pax2.1 expression domain (visible as quenching of the fluorescent Pax2.1 signal). Embryos in C-J are flat mounted, A,C,E,G and I show bright field, B,D,F,H and J show fluorescent images of the same embryos.