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Fig. 4 Fgf8 functions in the heart primordium. (A,B) Misexpression of fgf8 in ace embryos by RNA injection. Localization of lacZ co-injected cells with an antibody to β-gal (brown). fgf8 RNA injection can restore gata4 (A), but not nkx2.5 (B) expression in mutant embryos. (C,D) Expression of XFD in wild-type embryos by RNA injection. Localization of lacZ co-injected cells with an antibody to β-gal. XFD RNA injection suppresses gata4 (C) and nkx2.5 (D) expression in wild-type embryos. (E,F) Implantation of a Fgf8-soaked bead (arrow) after gastrulation can rescue nkx2.5 expression in ace embryos (E), while PBS bead gives no effect (F). (G) Implantation of a Fgf8-soaked bead (arrow) after gastrulation can caudally extend (dashed line) the endogenous nkx2.5 expression domain in wild-type embryos. (H) Model of Fgf8 function in cardiogenesis. All embryos are oriented with anterior to the left; A,B,E-G are flat mounted; C,D are dorsal views of whole embryos. nkx2.5 and gata4 were detected by in situ hybridization. (I-P) Fgfr inhibitor treatments. (I-K) 10-somite stage, (L,M) 15-somite stage, (N) 18- somite stage, (O,P) 26 hpf. (I-N) nkx2.5 and myoD expression detected by ISH; dorsal view of whole embryo, anterior to the left. (O,P) Double staining with MF20 and S46 antibodies, lateral view, anterior to the left. (I-N) Embryos have been treated with SU5402 from the 1-somite stage onwards for 2 hours (J,M) or continuously (K,N). (I,L) Non-treated control embryos. nkx2.5 expression is absent after short exposure to the inhibitor at the 10-somite stage (J), but begins to recover at the 15-somite stage (M). Continuous treatment results in permanent loss of nkx2.5 expression (K,N). (O,P) Embryos that have been continuously treated with SU5402 from the 18-somite stage onwards show no ventricular tissue (P); compare with the untreated control (O). Arrow marks the ventricle, arrowheads point to the MF20-positive somites. a, atrium; v, ventricle.