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First published online 26 November 2003
doi: 10.1242/dev.00907

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Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis

Departament of Developmental Genetics and Molecular Physiology, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, México

View larger version (87K): [in a new window]   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.

View larger version (76K): [in a new window]   Fig. 2. 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: 100 µm.

View larger version (82K): [in a new window]   Fig. 4. 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.

View larger version (71K): [in a new window]   Fig. 3. 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: 50 µm.

View larger version (112K): [in a new window]   Fig. 5. 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.

View larger version (85K): [in a new window]   Fig. 6. 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.

View larger version (58K): [in a new window]   Fig. 7. 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.

View larger version (66K): [in a new window]   Fig. 8. Schematic representation of palate shelf fusion. (A) Initially, shelves approach each other at the time the periderm cells (yellow cells) overlying the basal MEE cells (white cells) emit filopodia. (B) First contact and adhesion occurs between periderm cells; proteoglycans appear to be important at this stage. Adhesion becomes stronger as periderm cells move up and down (arrows) the MES (bracket) forming the epithelial triangles (et). (C) Basal MEE cells of each shelf intercalate (convergent extension) resulting in a single epithelial layer. (D) MES breaks up and epithelial pearls (ep) form; periderm and MEE cells start to die within epithelial triangles and epithelial pearls, respectively (red cells). (E) MES, which is composed of periderm and basal MEE cells, essentially degenerates by cell death; dying cells activate basal lamina degradation (cataptosis; broken orange line). (F) Fusion is complete without a major mesenchymal cell movement across the midline; some oral and nasal epithelial cells do move across the middle line (doubleheaded arrows). Pink cells represent mesenchymal cells. Orange lines represent basal lamina.

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