Time-course analysis of MES degeneration in a live palate slice. MEE cells of 14.5 dpc palate shelves were labeled with CCFSE (bright signal) before contact. Three hours after contact, 200 μm transverse slices were produced. Selected individual slices were cultured, and micrographs of same slices were taken at 3, 6, 12 and 24 hours. At the end, cultured slices were processed for cell death detection (TUNEL positive, red). Note the accumulation of labeled cells in epithelial triangles at 3 and 6 hours of culture (blue arrows). At 6 and 12 hours of culture, the fragmented MES was obvious. At the end of culture (24 hours), the remaining labeled cells were detected as dying cells and none was clearly detected in the mesenchyme. These experiments were repeated more than three times at least in triplicate for each condition. Scale bar: 100μ m.

Activation of basal lamina degradation by MEE cell death stimuli. Individual palate shelves were cultured without contact in the presence of retinoic acid (RA; a MEE cell death activator) or staurosporine (a broad-spectrum cell death activator). At the end of culture (10 hours), cell death (green) and laminin (red) were detected in the same palate slice by TUNEL and specific immunohistochemistry, respectively. RA induced extensive cell death in the MEE and rugae (r) of isolated shelves, but basal lamina degraded only in the MEE apoptotic region (arrows). Treatment with z-VAD blocked RA-induced cell death and basal lamina remained intact. Generalized induction of epithelial cell death with staurosporine activated the degradation of the basal lamina underlying the dying MEE cells (arrowheads), whereas basal lamina underlying the MEE adjacent epithelium (left from the arrow) was unaffected. These experiments were repeated more than three times at least in triplicate for each condition. Scale bar: 100 μm.

Relationship between cell death and basal lamina degradation. Shelves of developing palates were put in contact and cultured in the presence of either a caspase inhibitor (z-VAD) or a metalloproteinase inhibitor (MMI). At the end of culture (24 hours), cell death (green) and laminin (red) were detected in the same palate slice by TUNEL and specific immunohistochemistry, respectively. After 12 hours in culture, control palates show an advanced MEE cell death and basal lamina degradation. At the end of culture, MEE and basal lamina completely disappeared. However, z-VAD treatment inhibited cell death but basal lamina remained intact. Application of 10 μM BB3103 (MMI) did not alter the apoptotic fate of MEE cells but, as expected, inhibited basal lamina degradation (arrowhead). These experiments were repeated more than three times with at least a triplicate for each condition. v, blood vessel. Scale bar: 50μ m.

Analysis of periderm cell fate. Periderm cells were labeled with CCFSE as described in the Materials and methods, and palates were cultured for 8 hours. Samples were analyzed every 2 hours. At the beginning of culture (2 hours), periderm cells were confined to the MES middle line between the two basal MEE; cell death was not detected at this time. As fusion proceeded (4, 6 and 8 hours), accumulation of labeled cells occurred at the apex of the MES, constituting a large proportion of epithelial triangle cells. Most labeled cells died within the epithelial triangles (yellow cells; see also Fig. 5). As noted here, basal MEE cells appeared to die in situ within the epithelial pearls (arrows at 8 hours; compare with Fig. 1). Arrowheads indicate autofluorescent erythrocytes. These experiments were repeated more than three times at least in triplicate for each condition. Scale bar: 100 μm.

Effect of inhibition of periderm cell migration on cell death and fusion. After periderm cell labeling (green), palate shelves were put in contact and cultured in the presence or absence of 6 μM cytochalasin D for 10 hours. (A) When cytochalasin D was included in the medium, palate morphology showed the lack of epithelial triangles (et) and weak shelf adhesion. To detect cell death, palates were either stained in whole-mount with Acridine Orange (C; bright spots) or slices processed for the TUNEL technique (B; red). (B) Periderm cells of control palates died within epithelial triangles (yellow; see also Fig. 3), whereas those from cytochalasin D-treated palates did not reach the oral and nasal closures and did not die (green cells; arrowheads). (C) Specific reduction in cell death was observed in the mes of cytochalasin D-treated palates with a minimum effect in rugae (r). These experiments were repeated more than three times at least in triplicate for each condition. Scale bars: in A, 100 μm for A; in C, 500 μm for C.

Effect of periderm cell removal on basal MEE cell viability and shelf fusion. Periderm cells were removed by washing the MEE region after controlled trypsin digestion performed on 14.5 dpc palate shelves before contact. Control palates were also treated with trypsin but washing was not performed. (A) Treated shelves were put in contact and fusion was analyzed 24 hours later by standard Hematoxylin-Eosin staining (HE). Although MES degenerated, proper fusion between `denuded' palate shelves did not occur. Note the absence of epithelial triangles and marked reduction in MES thickness when compared with a control sample (brackets). (B) Isolated halves were cultured for 10 hours and cell death analyzed with the TUNEL technique. More dying MEE cells were detected in `denuded' palates (i.e. without periderm cells). These experiments were repeated more than three times at least in triplicate for each condition. Scale bar: 100 μm.