The Tbx1 enhancer is dependent on Shh signaling in a temporo–spatial fashion. (a,b) X-gal staining of wild-type (Shh+/+) and Shh mutant (Shh−/−/) embryos harboring the 12.8-kb Tbx1–lacZ transgene at E9.25 showed relatively normal lacZ expression in the pharyngeal arches and head mesenchyme (hm) of Shh mutant embryos compared with wild-type embryos. (c,d) LacZ expression in wild-type and Shh mutant embryos at E9.5. (e,f) Higher magnification photographs of c and d focusing on pharyngeal arches show down-regulation of lacZ expression in the head mesenchyme and first (I), third (III), and fourth (IV) pharyngeal arches of Shh mutant embryos compared with wild-type embryos. lacZ expression in the second pharyngeal arch (II) was also down-regulated, but expression was still detected in the mesoderm of the second arch of Shh mutant embryos. ht, heart.