Fig. 1. Analysis of MEE cell fate using different approaches. (A) Left lane (T/L) shows the cell death and basal lamina degradation patterns during palate shelf fusion in vitro (0-24 hours). Cell death detection by TUNEL (green) and laminin immunohistochemistry (red) were performed on the same slice. Lamininspecific immunohistochemistry detected basal lamina as well as blood vessels (V). Three hours after shelf contact, MES and basal lamina were unaltered. By 6 hours, concomitant cell death and basal lamina fragmentation indicate that MES degradation had begun. By 12 hours, MES degradation was very advanced, showing many dying cells within the epithelial triangles and few dying in the MES within the epithelial pearls (arrowhead) as indicated by the surrounding basal lamina. No MEE cells (alive or dead) and laminin were detected in the region surrounding the MES at the end of culture (24 hours). MEE cells of palates of equivalent stages were labeled with CCFSE or Ad-lacZ (LacZ in figure) before contact and their fate was analyzed after 3, 6, 12 and 24 hours in anterior and posterior palatal regions. Epithelial pearls were evident at 6 and 12 hours after contact (arrowheads) using both labeling protocols. At the end of culture (24 hours) no labeled cells were detected in the MES. At no time was the presence of labeled cells evident in the mesenchyme compartment. By joining one wild-type shelf with one from the EGFP mouse strain, we produced chimeric palates (WTEGFP). At no time were EGFP-positive cells detected in the wild-type mesenchyme compartment of these chimeric palates. Interestingly, chimeric palates showed intercalation of MEE cells (asterisks), at the time abundant apoptotic bodies were detected (arrows). After 24 hours of culture, fusion was complete and no mesenchymal cell migration was detected between halves. et, epithelial triangles; mes, medial epithelial seam. (B) Although no EGFP-positive cells were detected in the wild-type mesenchyme compartment of chimaeric palates, EGFP-positive cells (green) were detected in the wild-type oral and nasal epithelia. (C) CCFSE-labeled cells are not detected in the mesenchyme compartment when cell death is inhibited by z-VAD. Scale bar: 100 µm.