Ontogeny of Shh expression in the craniofacial primordia. (A) At St. 10, Shh is expressed in the prosencephalon (pros) and in oral ectoderm and pharyngeal endoderm comprising the buccopharyngeal membrane (bm). (B) By St. 15 the prosencephalon has separated into the telencephalon (te) and the diencephalon (di), where Shh transcripts are localized. Neural crest cells have migrated as far as the dorsal aspect of the frontonasal primordium (arrowheads). rp, Rathke’s pouch. (C) At St. 17, Shh is induced in a new domain within the ventral telencephalon (arrow). mn, mandible; mes, mesencephalon; or, optic recess. (D) At St. 20, Shh is induced in a second new domain, ventral facial ectoderm (fe, arrow), which is separated from the telencephalon by intervening frontonasal neural crest (nc). The pharyngeal endodermal (pe) domain of Shh, first evident at St. 10, persists through embryogenesis. (E) These Shh domains are preserved from St. 20 onward. (F) Illustration of the sequential induction of Shh relative to craniofacial development and to age. The time points of cyclopamine administration are indicated by arrows and correspond to the stepwise induction of Shh in the craniofacial primordia. The units of measurement on the x axis correspond to Hamburger-Hamilton stages of avian development and span a period of approximately seven days. fnp, frontonasal process; mx, maxillary process. (G) Whole-mount in situ hybridization analyses at St. 10 show Shh transcripts in the medial prechordal plate (pcp), which will contribute to the ventral neural tube after neurulation. (H) Shh transcripts in the diencephalon are barely discernible through the facial ectoderm at St. 15 (arrow). ey, eye. (I) By St. 20, Shh is strongly expressed throughout the ectoderm of the stomodeum (arrow) and the maxillary primordia (J), which persists at St. 22. Scale bars: 100 μm (A); 200 μm (B–E).