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First published online 21 March 2007
doi: 10.1242/dev.02837

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Multiple functions of Snail family genes during palate development in mice

The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA.

View larger version (87K): [in this window] [in a new window]   Fig. 1. The Snai1 and Snai2 genes are essential for palate development. (A,B) Scanning electron micrographs showing cleft secondary palate at E17.5 in Snai1+/- Snai2-/- mutant embryos. (C,D) Coronal sections at E15 showing the fused secondary palate in control embryos (C), and the adjacent, but unfused palatal shelves in Snai1+/- Snai2-/- mutant embryos (D). (E-H) Expression of the Snai1 gene in the developing palate of E13.5 and 14.5 embryos. Note the expression in the palatal shelf mesenchyme at each stage and the strong expression adjacent to the MES at E14.5 (inset). (I,J) Snai2 expression, detected by ß-galactosidase activity of the Snai2lacZ allele, showing widespread expression in the palatal shelf mesenchyme and nasal/oral epithelia. Note the increase in ß-galactosidase activity in mice with deletion of a single Snai1 allele (J). ns, nasal septum; ps, palatal shelf; t, tongue; tb, tooth bud. Scale bar: 100 µm in C,D,I,J; 200 µm in E-H.

View larger version (96K): [in this window] [in a new window]   Fig. 2. Expression of the Tgfb3 and Irf6 genes is unaffected in Snai2-/- and Snai1+/- Snai2-/- palates at E14.5. (A-C) In situ hybridization showing expression of Tgfb3 along the MES of control embryos and clear expression at the adjacent but unfused MEE of mutant embryos. (D-F) The Irf6 gene is expressed throughout the oral and nasal epithelia in both control and mutant embryos. ps, palatal shelf. Scale bar: 200 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 3. Snai1+/- Snai2-/- palatal shelves display reduced apoptosis and defective periderm migration. (A,B) Scanning electron micrographs of the MEE of control (A) and Snai1+/- Snai2-/- mutant (B) embryos at E14.5. Samples were dissected just before palatal shelf fusion, when shelves were in close approximation. Note the appearance of lamellipodia on the MEE of both control and mutant embryos. (C-F) TUNEL staining showing apoptosis along the MES (E) and in the epithelial triangles (C,E) of control embryos at E14.5, whereas mutant embryos (D,F) lack any detectable cell death along the MEE. Arrows, epithelial triangles; arrowheads, MES. (G,H) Keratin 6 immunofluorescence marking periderm cells at the palate fusion point in control embryos at E14.5. Note the concentration of periderm cells in the epithelial triangles in the control embryo, with occasional remaining cells in the MES (G). Mutant palates, in contrast, show the presence of periderm cells at the junction of the adjacent shelves without the formation of epithelial triangles (H). Scale bar: 50 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Cleft palate and craniofacial abnormalities in Snai1/2-dko mice. (A,B) Whole-mount in situ hybridization for Snai1 of E9.5 control and Snai1-cko embryos showing effective deletion of Snai1 by Wnt1-Cre in the neural crest. Note the absence of Snai1 expression in the first branchial arch (arrow in B) in the mutant embryo versus control. (C,D) Histology of the cleft palate in Snai1/2-dko embryos, showing the vertically oriented palatal shelves in mutant embryos. (E,F) Skulls of Snai1/2-dko mice are smaller, dome-shaped, and have a shortened mandible (arrow). (G,H) Ventral view of the mandible of control (G) and mutant (H) neonates. Note the overall shorter length, missing rostral Meckel's cartilage and midline fusion (arrow) in the mutant. (I,J) An enlarged frontal foramen (asterisk) is apparent in the Snai1/2-dko neonate. Scale bar: 200 µm in A,B; 100 µm in C,D; 2 mm in E,F; 1 mm in G-J.

View larger version (165K): [in this window] [in a new window]   Fig. 5. Expression of markers of palate development in Snai1/2-dko embryos. (A-C) At E13.5, Pax9 is expressed in a dynamic pattern, transitioning from the lateral to medial aspects of the palatal shelf. Both control and mutant embryos show similar expression of an intermediate pattern (A,B), while E14.5 mutant shelves show the full transition to medial expression in the posterior palate (C). (D-F) Msx1 expression at E13.5 is normally restricted to the anterior palatal shelf mesenchyme. Expression of Msx1 appears unaltered in mutant shelves at E13.5 and in the vertically oriented E14.5 shelves. (G-I) Shh expression at E13.5 is localized to the thickened oral epithelium lateral to the vertical palatal shelf. Mutant embryos display the proper pattern at both stages, but the expression level appears somewhat reduced. (J-L) At E13.5 Osr1 is expressed in discrete domains in the lateral palatal shelf mesenchyme (arrows). As with Shh, note the proper patterning, but reduced expression, in mutant embryos. ps, palatal shelf; t, tongue; tb, tooth bud. Scale bar: 200 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 6. Similar levels of cell proliferation in control and Snai1/2-dko palatal shelves. Phospho-histone H3 immunostaining of E13.5 (A-D) and E14.5 (E-H) palates showing a similar number of mitotic cells in control (A,B,E,F) and Snai1/2-dko (C,D,G,H) embryos. Note the vertical positioning of the mutant palate shelf at E14.5 (H). Scale bar: 200 µm in A,C,E,G; 50 µm in B,D,F,H.

View larger version (118K): [in this window] [in a new window]   Fig. 7. Defects in Meckel's cartilage extension in Snai1/2-dko embryos. Alcian Blue staining of control and mutant embryos at E14.5, showing the dramatically reduced extension of the Meckel's cartilage. (A,B) Lateral views. (C,D) Ventral views. Scale bar: 1 mm.