HCR staining of the indicated genes in control and Fgf4 mutant embryos at 6ss (A, A’, B, B’) and somite stage 24 (C, C’, D, D’). The probe (Fgf4rec) hybridizes only to sequences between the loxP sites in the Fgf4flox allele; hence it hybridizes to wildtype Fgf4 mRNA but not to mRNA transcribed from the Cre-deleted allele. Fgf4rec and Msgn1 patterns indicate that Fgf4 is expressed throughout the PSM in controls (bracket), with higher expression in the posterior midline (asterisk) (A’, C’). Fgf4 does not appear to be expressed in a gradient in the normal PSM (A, C). Fgf4 expression is absent in mutants in the PS and PSM at all examined stages (B, B’, D, D’). Representative embryos are shown; control, n = 4 for somite stage 6, n = 3 for somite 24; mutant, n = 4 for somite stage 6, n = 4 for somite 24. (E–G’) Colorimetric staining of Fgf4 expression using a full-length probe in wildtype embryos at E9.5, E11.5, and E12.5, showing persistent expression of Fgf4 expression through late development. All images are anterior to left. A-D’ are MIPs of confocal images. A-B’, dorsal view; C-D’, lateral view. E-G lateral views, E’-G’ dorsal views.