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First published online March 20, 2009
doi: 10.1242/10.1242/dev.028167

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Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth

Center for Oral Biology and Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

View larger version (107K): [in this window] [in a new window]   Fig. 1. Partial inactivation of Shh in the developing palate in K14-Cre;Shhc/n mutant mice. (A,B) Frontal sections of E17.5 K14-Cre;Shhc/+ (A) and K14-Cre;Shhc/n (B) mutant mouse heads showing cleft palate in the K14-Cre;Shhc/n mutant. Arrowheads point to palatal shelves, and the asterisk in B marks the cleft between the bilateral palatal shelves in the mutant. (C,D) At E13.5, the percentage of BrdU-labeled cells are reduced in the palatal epithelium and mesenchyme in some K14-Cre;Shhc/n mutant embryos (D) in comparison with K14-Cre;Shhc/+ littermates (C). White arrows point to the first molar tooth buds in which cell proliferation is also reduced in the K14-Cre;Shhc/n mutant embryo in comparison with K14-Cre;Shhc/+ littermate. (E,F) Ptch1 mRNA expression is reduced in the oral epithelium and palatal mesenchyme in K14-Cre;Shhc/n mutant embryos (F) in comparison with K14-Cre;Shhc/+ littermates (E). (G,H) Gli1 mRNA expression was also downregulated in the oral epithelium and palatal mesenchyme in the K14-Cre;Shhc/n mutant embryos (H) in comparison with K14-Cre;Shhc/+ littermates (G) at E13.5. BrdU staining and mRNA signals were detected in blue. Black arrows in E-H point to the maxillary molar tooth buds. oc, mandibular ossification center; p, palatal shelf; t, tongue.

View larger version (119K): [in this window] [in a new window]   Fig. 2. Osr2-IresCre-mediated inactivation of Smo efficiently blocks Hedgehog signaling in the palatal mesenchyme by E13. (A) Whole-mount X-gal staining of an E11.5 Osr2-IresCre;R26R embryo showing specific Cre-mediated activation of lacZ expression in the primordia of the primary (arrowheads) and secondary (arrows) palate. Asterisks mark the sites of fusion between the medial nasal and the maxillary processes. (B) X-gal stained frontal section of E12.5 Osr2-IresCre;R26R embryo showing β-galactosidase activity throughout the palatal mesenchyme but absent in the palatal epithelium (arrowheads). (C,D) By E13.0, Ptch1 mRNA expression was dramatically downregulated in the palatal mesenchyme and persisted in the palatal epithelium (arrowhead) in the Osr2-IresCre;Smoc/c embryo (D) in comparison with control littermate (C). (E,F) Gli1 mRNA expression was also dramatically downregulated in the palatal mesenchyme and persisted in the palatal epithelium (arrowhead) in the Osr2-IresCre;Smoc/c mutant embryo (F) in comparison with the control littermate (E) by E13.0. lnp, lateral nasal process; max, maxillary process; mnp, medial nasal process; p, palatal shelf; t, tongue. Scale bar: 100 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 3. Osr2-IresCre;Smoc/c mutant mice exhibit cleft palate and open eyelids at birth. (A,B) Neonatal control (A) and Osr2-IresCre;Smoc/c mutant (B) mice showing open eyelids (arrow) at birth in the mutant. (C,D) Frontal sections of E17.5 control (C) and Osr2-IresCre;Smoc/c mutant (D) embryos showing cleft palate in the mutant. (E,F) Skeletal preparations of control (E) and Osr2-IresCre;Smoc/c mutant (F) neonatal mice showing widely separate palatine bones (arrows) in the mutant. The insets show high magnification views of the tympanic rings (arrowheads) in the Osr2-IresCre;Smo+/c (E) and Osr2-IresCre;Smoc/c mutant (F) skeletons. Bones are stained red and cartilage blue. The asterisk in F marks the presphenoid bone of the cranial base, which is hidden under the palatine bones from the oral view of the skeleton in the control mouse in E but fully exposed in the Osr2-IresCre;Smoc/c mutant skeleton due to cleft palate. n, nasal septum; p, palatal shelf; t, tongue.

View larger version (133K): [in this window] [in a new window]   Fig. 4. Histological analyses of palate development in the control and Osr2-IresCre;Smoc/c mutant embryos. (A,B) HE-stained frontal sections of E12.5 Osr2-IresCre;Smo+/c (A) and Osr2-IresCre;Smoc/c mutant (B) embryos showed comparable initial outgrowth of palatal shelves. (C,D) At E13.5, the palatal shelves of the Osr2-IresCre;Smoc/c mutant embryo (D) appeared slightly retarded distally in comparison with the Osr2-IresCre;Smo+/c embryo (C). (E,F) At E14.5, whereas the palatal shelves had elevated and initiated fusion at the midline in the control embryo (E), the palatal shelves of the Osr2-IresCre;Smoc/c mutant embryo (F) appeared severely retarded and failed to contact each other. (G,H) Some Osr2-IresCre;Smoc/c mutant embryos exhibited tissue protrusion into the nasopharynx, shown in H (marked by an asterisk), which was never observed in control embryos. p, palatal shelf; t, tongue.

View larger version (85K): [in this window] [in a new window]   Fig. 5. Osr2-IresCre;Smoc/c mutant embryos exhibited defects in palatal shelf growth at E13.5. Frontal sections through the palatal regions of BrdU-labeled Osr2-IresCre;Smo+/c (A,D) and Osr2-IresCre;Smoc/c mutant (B,E) embryos were stained with anti-BrdU antibody. The labeled cell nuclei were stained blue. The white line in each panel divided each image of the palatal shelf to medial and lateral halves for the calculation of the percentage of BrdU-labeled nuclei in those regions separately. A and B show typical sections through the middle of the anterior half of the palatal shelves, whereas D and E are from the posterior third of the palatal shelves. (C,F) Comparison of the percentage of BrdU-labeled cells in the anterior (C) and posterior (F) regions of the developing palate in the Osr2-IresCre;Smo+/c (control) and Osr2-IresCre;Smoc/c (mutant) embryos. Standard deviation values were used for the error bars. An asterisk denotes a significant reduction in the percentage of BrdU-labeled cells in the mutant (P<0.05). p, palatal shelf.

View larger version (142K): [in this window] [in a new window]   Fig. 6. Comparison of Ccnd1 protein and Ccnd2 mRNA expression in the developing palate in control and Osr2-IresCre;Smoc/c mutant embryos at E13.5. (A,B) Ccnd1 immunostaining (shown in brown) in frontal sections of control (A) and Osr2-IresCre;Smoc/c mutant (B) embryos. Ccnd1 expression in the tooth bud epithelium (arrows) served as internal controls because Osr2-IresCre was not expressed in this tissue. Arrowheads point to the palatal epithelium, which appeared thinner in the Osr2-IresCre;Smoc/c mutant embryos than in the control littermates. (C,D) In situ hybridization detection of Ccnd2 mRNA (in blue) in frontal sections of control (C) and Osr2-IresCre;Smoc/c mutant (D) embryos. p, palatal shelf.

View larger version (102K): [in this window] [in a new window]   Fig. 7. Comparison of Bmp2, Bmp4, Msx1 and Shox2 mRNA expression in the anterior palate of Osr2-IresCre;Smo+/c and Osr2-IresCre;Smoc/c mutant embryos at E13.5. (A-F) In comparison with the control embryos, Bmp2 mRNA was downregulated whereas Bmp4 and Msx1 mRNAs were upregulated in the mutant anterior palatal mesenchyme. Arrowheads point to differences in expression in the control (A,C,E) and mutant (B,D,F) palatal shelves. (G,H) Shox2 mRNA expression was unaltered in the Osr2-IresCre;Smoc/c mutant palate. p, palatal shelf; t, tongue.

View larger version (100K): [in this window] [in a new window]   Fig. 8. Foxf1 and Foxf2 mRNA expression in the developing palate were downregulated in the Osr2-IresCre;Smoc/c mutant embryos. (A) At E13.5, Foxf1a mRNA was expressed in the anterior palatal mesenchyme underlying the lateral and medial edge epithelium (arrow) in the control embryos. (B) Foxf1a mRNA expression was specifically downregulated in the palatal mesenchyme but its expression in the tooth mesenchyme was not affected in the Osr2-IresCre;Smoc/c mutant embryos. Asterisks in A and B mark the maxillary first molar tooth buds. (C,D) At E13.5, Foxf1 mRNA expression in the posterior palate was restricted to more lateral and proximal cells in the control embryo (C) and this domain of expression (arrows in C and D) was also downregulated in the Osr2-IresCre;Smoc/c mutant embryos (D). (E-H) Foxf2 mRNA expression exhibited a lateral-to-medial gradient in both the anterior and posterior palate in the control embryos (E,G) and was downregulated in both the anterior and posterior palatal mesenchyme in the Osr2-IresCre;Smoc/c mutant embryos (F,H). E and F show anterior palates; G and H, posterior. p, palatal shelf; t, tongue.

View larger version (124K): [in this window] [in a new window]   Fig. 9. Comparison of expression of Osr2, Pax9 and Fgf10 in the developing palate of the control and Osr2-IresCre;Smoc/c mutant embryos. (A) At E13.5, Osr2 mRNA was strongly expressed in the mesenchyme lingual to the first molar tooth buds and exhibited a lateral-to-medial gradient in the palatal mesenchyme in control embryos. (B) In the Osr2-IresCre;Smoc/c mutant embryos at E13.5, Osr2 mRNA expression was specifically downregulated in the palatal mesenchyme but the domain of expression in the lingual tooth mesenchyme was unaffected. (C,D) Pax9 mRNA expression in the developing palate and tooth mesenchyme was not affected in the Osr2-IresCre;Smoc/c mutant embryos. (E,F) Fgf10 expression in the anterior palatal mesenchyme (arrows) was significantly downregulated in the Osr2-IresCre;Smoc/c mutant embryos (F) in comparison with the control embryos (E). m, mandibular first molar tooth bud; p, palatal shelf.

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