Fig. 1. Ectopic Bmp4 expression in the palatal mesenchyme rescued the cleft palate in Msx1^–/– mice. (A-C) Expression of the human Bmp4 transgene (arrows) was detected in the anterior palatal mesenchyme of E12.5 (A) and E13.5 (B) Msx1-Bmp4 transgenic embryos using a transgene-specific probe, as described previously (Zhao et al., 2000). No signals were detected in an E13.5 wild-type control palate using the same transgene-specific probe (C). (D) A coronal section through an E14.5 wild-type embryonic head showing fusion of the palatal shelves. (E) An E14.5 Msx1^–/– embryo showing cleft secondary palate (arrows). (F) A coronal section of an E14.5 Msx1^–/–/Tg embryo showing fusion of the palatal shelves. (G,H) A wild-type newborn mouse displaying a closed palate (G) when compared with the cleft palate (arrows) shown in an Msx1^–/– newborn (H). (I) Rescue of cleft palate in an Msx1^–/–/Tg newborn mouse. (Insert) Rugae did not fuse at the midline (arrow) of the palate in a three-month-old Msx1^–/–/Tg mouse. (J-L) Skeletal staining showing cleft palate in a newborn Msx1^–/– mouse (K) and the rescue of cleft palate in a newborn Msx1^–/–/Tg mouse (L), when compared with a newborn wild type mouse (J). The sphenoid bone (white arrow in K), which could be directly viewed in the Msx1 mutant, was not apparent in the Msx1^–/–/Tg mouse (L). The sharp appearance of the premaxilla (black arrow in K) found in Msx1 mutant was also seen in a Msx1^–/–/Tg mouse (L), when compared with the normal rounded morphology of premaxilla in a wild-type mouse (black arrow, J), indicating that the rescue seen in Msx1^–/–/Tg mouse was specific to the cleft palate phenotype. (M) Scanning electron microscopic image of the oral view of an E13.5 wild-type embryonic head showing the overall shape of the developing palate, the planes of sections shown in Figs 2, 3, 6, and the regions excised for the explantation studies shown in Figs 4, 5. The yellow line indicates cuts made during dissection to separate the anterior region (top to the line) from the posterior region (bottom from the line) of the palate in this study. The red line indicates the anterior section plane, and green line indicates the posterior section plane used in the section in situ hybridization studies and BrdU labeling experiments shown in Figs 2, 3, 6. The white-lined box labeled ‘A’ represents the anterior region of palatal tissue, and the black-lined box labeled ‘P’ represents the posterior region of palatal tissue used for the explantation experiments shown in Figs 4, 5. (N) A coronal section through the oral region of an E13.5 embryo indicating the precise location (boxed) of palatal shelf shown in Fig. 1A-C, Figs 2, 3, Fig. 6D-F. I, incisor; M, molar; T, tongue; NS, nasal septum; PS, palatal shelf. Scale bars: in D,E, 100 µm; in N, 300 µm.

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