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First published online 3 August 2006
doi: 10.1242/dev.02520

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Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion

Department of Molecular, Cellular and Craniofacial Biology and Birth Defects Center, University of Louisville, Louisville, KY 40202, USA.

View larger version (32K): [in a new window]   Fig. 1. Lateral and anteroposterior (AP) migration of MEE seam cells during in vitro palate fusion. (A,D) Diagrams showing the [Rosa26C57BL/6] chimeric pair culture of palate and the section positions for panels (B,C,E), as indicated by the lines. (B) Cross section through the middle region of a [Rosa26C57BL/6] chimeric palate pair. The palate shelves were dissected from E14.0 mouse embryos and cultured oral side up for18 hours then stained for ß-gal. ß-gal-positive cells (blue) are present in the nasal epithelium region of the C57BL/6 half of the pair (arrowhead). (C) Cross section through the posterior region of the [Rosa26C57BL/6] chimeric palate pair shown in B. A low number of ß-gal-positive cells (blue) were detected in the medial edge epithelium region of the C57BL/6 half of the pair (arrowhead), even though the two palate shelves were still separated in this posterior region. (E) A [Rosa26C57BL/6] chimeric palate pair from an E14.0 mouse embryo cultured for 18 hours and then stained with ß-gal and sectioned as indicated in D. Note the extension of the ß-gal-positive cells (blue) from the middle joint region (arrow) to the posterior separated region (arrowhead). Scale bars: 50 µm.

View larger version (68K): [in a new window]   Fig. 2. In situ hybridization analysis shows epithelial-specific expression of the Cre transgene in the developing palate of [K14-Cre; R26R] embryos. (A) Diagram showing mouse secondary palates at E14.5 and the positions of sections shown in C,D,E. (B) In situ hybridization showing high expression of the Cre transgene in the entire palatal epithelium (arrowheads) at E13.5. (C,E) In situ hybridization showing that the Cre transgene expression domain covers almost the entire palatal epithelium in the anterior (C) and posterior (E) regions of the palate at E14.5. (D) In situ hybridization showing that the Cre transgene is expressed only in the oral side of palatal epithelium (arrowheads) and the triangular region (arrow) in the middle region of the palate at E14.5. Scale bars: 50 µm.

View larger version (82K): [in a new window]   Fig. 3. ß-gal staining pattern in [K14-Cre; R26R] embryos demonstrates the occurrence of epithelial-mesenchymal transdifferentiation during and after seam degeneration. (A) ß-gal staining in the anterior region of the palate at early E14.5, where the two shelves have just made contact. Note that the MEE cells were strongly labeled with ß-gal (arrowhead), but no signal was present in mesenchymal cells. (B) ß-gal staining in the middle region of the palate at early E14.5, where seam degeneration has just been initiated. Some epithelial-like ß-gal-positive cells have dissociated from the midline and migrated into the mesenchymal region (arrowhead). (C,D) ß-gal staining in the middle region of the palate at late E14.5, when seam degeneration is advanced. Both clump-like blue cells (arrowhead in C,D) and typical mesenchymal-looking blue cells (arrow in C,D) were observed in the mesenchymal region of the palate. (E) ß-gal staining in the fully fused palate at E15.5, showing that a high portion of the mesenchymal cells were ß-gal positive (arrow). Scale bars: 100 µm in A; 50 µm B-E.

View larger version (69K): [in a new window]   Fig. 4. Hematoxylin and eosin staining reveals complete palate fusion and seam degeneration in Apaf1 mutant embryos. (A,D) The MEE seam (arrowhead) forms normally in both wild-type and Apaf1 mutant embryos at E14.5. (B,E) The MEE seam undergoes degeneration in both wild-type and Apaf1 mutant embryos at E15.5 to establish the mesenchyme confluence cross the midline (arrowheads). (C,F) At E16.5, both wild-type and Apaf1 mutant embryos form a continuous palate with no sign of seam cells in the midline area (arrowhead).

View larger version (129K): [in a new window]   Fig. 5. Hematoxylin and eosin staining and TUNEL assay reveal abnormal triangular areas and a lack of apoptosis in the Apaf1 mutant palate. (A) An E15.5 wild-type palate showing a normal triangular area (arrow). (B) An E15.5 Apaf1 mutant palate containing an enlarged and highly compacted triangular area (arrow). (C) Apoptotic cells were visualized by TUNEL assay in the triangular area of the wild-type palate at E15.5 (arrow). (D) No TUNEL-positive cells were detected in the triangular area of the Apaf1 mutant palate at E15.5 (arrow).

View larger version (13K): [in a new window]   Fig. 6. Schematic illustration of the MEE seam cell migration routes during palate fusion based on in vitro palate culture data.

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