Fig. S1. Tissue specificity of Cre-mediated activation of lacZ expression in the developing craniofacial complex of Osr2-IresCre;R26R embryos at E12.5. (A-C) X-gal staining on frontal sections from the anterior (A), middle (B) and posterior (C) palate regions is shown. β-galactosidase activity was detected throughout the anteroposterior axis of the developing palatal mesenchyme, indicating strong Cre activity from the onset of palatal outgrowth. In addition, β-galactosidase activity was detected in a subset of the nasal mesenchyme, in a subset of mesenchymal cells underlying the epithelium in the developing tongue, in the developing eyelid epithelium and mesenchyme (arrows in B,C), in the lateral maxillary and mandibular mesenchyme, and in the mandibular mesenchyme flanking the base of the developing tongue. No β-galactosidase activity was detected in the developing brain. Some β-galactosidase activity was also detected in the thin mesenchymal layer surrounding the developing brain at this stage (arrowheads in B,C). br, brain; e, eye; man, mandible; max, maxilla; n, nasal septum; p, palatal shelf; t, tongue.

Fig. S2. Highly specific downregulation of expression of Ptch1, Gli1 and Fgf10 in the developing eyelid and palate in E13 Osr2-IresCre;Smo^c/c mutant embryos. (A,B) Compared with the control embryo (A), the Osr2-IresCre;Smo^c/c mutant embryo (B) had a dramatic downregulation of Ptch1 expression in the palatal mesenchyme and in the eyelid epithelium and mesenchyme. By contrast, expression of Ptch1 in the ventral diencephalon (arrowheads in A,B) and in the mandibular osteoblasts next to the Meckel’s cartilage was not significantly different in the control and Osr2-IresCre;Smo^c/c mutant littermates. Insets in A and B show higher magnification views of Ptch1 expression in the developing upper eyelids (arrows) of control and Osr2-IresCre;Smo^c/c mutant embryos, respectively. (C,D) Similar to that of Ptch1, expression of Gli1 was also highly specifically downregulated in the developing palatal mesenchyme and eyelid tissues (arrows) in Osr2-IresCre;Smo^c/c mutant embryos (D), compared with control littermates (C). (E,F) Fgf10 expression was downregulated in the developing palatal and eyelid mesenchyme in Osr2-IresCre;Smo^c/c mutant embryos (F), in comparison with the control littermates (E). Expression of Fgf10 in the control embryos (E) overlapped with that of Ptch1 (A) and of Gli1 (C) in the developing palatal shelves and eyelids. By contrast, strong Fgf10 expression in the mesenchyme behind the developing eyes (arrows in E and F) and in the mandibular mesenchyme surrounding the Meckel’s cartilage was unaffected in the Osr2-IresCre;Smo^c/c mutant embryos (F), compared with the control littermates (E). Insets in E and F show higher magnification views of the Fgf10 expression in the developing upper eyelids (arrowheads) of control and Osr2-IresCre;Smo^c/c mutant littermates, respectively. e, eye; mc, Meckel’s cartilage; p, palatal shelves; t, tongue.

Fig. S3. Comparison of Bmp2 and Bmp4 mRNA expression in the craniofacial tissues in the Osr2-IresCre;Smo^c/+ and Osr2-IresCre;Smo^c/c mutant embryos. (A-D) Both Bmp2 (A,B) and Bmp4 (C,D) are expressed abundantly in multiple tissues in the craniofacial region, including in the medial nasal, lateral nasal, maxillary and mandibular processes. Limited expression of Bmp2 and Bmp4 mRNA was detected in the medial edge palatal mesenchyme in the control embryos. Bmp2 expression in the palatal mesenchyme was downregulated whereas Bmp4 expression in the anterior palatal mesenchyme was expanded in the Osr2-IresCre;Smo^c/c mutant embryos (B,D), compared with control littermates (A,C). Arrowheads point to the medial edge of the anterior palatal shelves. man, mandible; max, maxilla; n, nasal septum; p, palatal shelf; t, tongue.