Fig. 5

Fgf8 signalling negatively regulates pax3/7 expression. In situ mRNA hybridisation for pax3, pax7 or immunodetection of Pax3/7 in ace mutants or embryos injected with fgf8 MO or vehicle, followed by treatment with cyclopamine or vehicle and analyzed at 15 s (A, dorsal flatmount anterior to top), 18 s (B, lateral wholemount dorsal to left), 24 hpf (C) or 26 hpf (D, lateral confocal stacks of midbody somites, dorsal to top, anterior to left). (A) Both pax3 and pax7 mRNA are up-regulated in ace mutants, which were identified by their midhindbrain pattern defects (brackets). Note pax3/7 increase in the anterior of somites (arrowheads, boxes are enlarged), relative to neural tube. (B) Pax3 and pax7 up-regulation in all somites after fgf8 MO injection. (C) Pax3/7-containing nuclei are increased in number by fgf8 MO, cyclopamine or both. Fgf8 MO particularly increases staining at the horizontal myoseptum, in contrast to cyclopamine which increases cells on the somite borders (arrows). Note the lack of change in intensity of immunofluorescence and the lack of significant neural crest migration in both control and treated posterior somites. Insets show pax3 mRNA up-regulation persisting to 33 hpf. (D) Ace mutant embryos (identified by cerebellar morphology) have more weak Pax3/7 immunoreactive nuclei than siblings. Note clustering around the horizontal myoseptum (arrows) and more extensive neural crest migration in these older embryos compared to panel C. (E) Quantification of Pax3/7-expressing cells per somite after treatments indicated. Data represent mean ± standard deviation of counts from four somites in at least 4–7 embryos. Asterisks indicate significant difference from control, P < 0.001.