Inhibition of periderm removal in all-trans retinoic acid-induced cleft palate in mice

Abstract

Cleft palate is a common craniofacial birth defect. The aim of the present study was to investigate the effect of excess all-trans retinoic acid (atRA) on periderm removal and the disappearance of basal medial edge epithelial (MEE) cells during palatogenesis, particularly during the stage prior to contact. atRA (200 mg/kg) was administered to C57BL/6N mice at embryonic day (E) 12.0 by gavage. Fetal palates were processed and analyzed by histology and electron microscopy. Single palate shelf peridermal cells were removed and cultured in the presence of atRA (3 µM) only or in the presence of or the caspase inhibitor, Z-VAD (100 µM) only, for 48 h. Once cultured, morphological changes were analyzed by histological staining and electron microscopy. A TUNEL assay was used to detect apoptotic neurons. Paired palatal shelves with periderm removal were cultured in the presence of atRA (3 µM) only or in the presence of Z-VAD (100 µM) only for 48 h and analyzed by hematoxylin and eosin staining. At E14.5, medial edge epithelium periderm was retained in the atRA-treated palates but had been shed prior to contact in the control groups. In addition, atRA was revealed to disrupt the cell cycle in the periderm by downregulating p21. Furthermore, atRA inhibited apoptosis in the periderm and basal MEE cells; however, atRA exhibited no effect on basement membrane degradation in single palatal organ culture. Additionally, once paired palates were cultured for 48 h, all of the groups in which the periderm had been removed exhibited confluence of the embryonic palatal mesenchyme. The present results suggest that periderm removal is inhibited in atRA-induced cleft palate in mice and that removal of the periderm contributes to EPM confluence in vitro.

Keywords: all-trans retinoic acid; cleft palate; p21; periderm.

Figure 1.

Hematoxylin and eosin staining indicated the development of the palate shelf at E14.5 and E15.5 in the atRA-treated and control groups. (A) Palatal shelf contact was exhibited at E14.5 in the control group. (B) Palates were observed to be sliding off one another at E14.5 in the atRA-treated group. (C) Palate fusion at E15.5 was exhibited in the control group. (D) Development of cleft palate in the atRA-treated groups was observed at E15.5. Scale bar, 20 µm. Magnification, ×20. atRA, all-trans retinoic acid; E, embryonic day.

Figure 2.

Scanning electron microscopy and transmission electron microscopy of morphological changes in the MEE periderm in atRA-treated and control palates at E14.5. (A) Sloughing off of the periderm on the surface of the medial edge epithelium in the control groups (arrow). Scale bar, 1 mm. (B) The periderm on the surface of the medial edge epithelium was intact in atRA-treated palatal shelves (arrow). Scale bar, 1 mm. (C) Apoptotic peridermal cell (arrow) and loss of the junction between the peridermal and basal cells. Scale bar, 2 µm. (D) Tight junctions between peridermal cells and basal cells. Scale bar, 1 µm (A, B). atRA, all-trans retinoic acid; E, embryonic day; MEE, medial edge epithelial; P, peridermal cell; B, basal cell.

Figure 3.

Immunohistochemical staining for P21. (A) Protein expression levels of P21 in the peridermal cells of MEE in control palatal shelves and atRA-treated palates at E13.5, E14.5 and E15.5. Scale bar, 50 µm. (B) At E13.5, the protein expression levels of P21 in peridermal cells did not significantly differ between atRA-treated palates and control palatal shelves (P=0.64). At E14.5, P21 protein was expressed at a significantly decreased level in the atRA-treated palatal periderm in comparison to the control palatal periderm. At E15.5, the protein expression level of p21 was significantly increased in the atRA-treated palatal periderm compared with the control groups. Error bars represent the mean ± standard deviation. *P<0.05. Scale bar, 50 µm. Magnification, ×10. atRA, all-trans retinoic acid; MEE, medial edge epithelial; E, embryonic day.

Figure 4.

Hematoxylin and eosin staining, transmission electron microscopy and scanning electron microscopy observations of periderm removal in the single palatal shelf. (A) Once the periderm was removed, the medial edge epithelium exhibited a single layer of basal cells. Scale bar, 20 µm. (B) Scanning electron microscopy revealed sloughing of the peridermal cells. (C) Transmission electron microscopy revealed a single layer of basal cells with an intact basement membrane (arrow). B, basal cell.

Figure 5.

Immunohistochemistry staining of P21 and transmission electron microscopy observations of differences in the medial edge epithelium in a single palate culture for 48 h with or without periderm removal. (A) The medial edge epithelium disappeared in the control palates and the MEE cells were retarded despite periderm removal when they were cultured with atRA or Z-VAD. Scale bar, 20 µm. (B) The medial edge epithelium and basement disappeared in control palates despite periderm removal. In untreated periderm groups, basal and peridermal cells were intact in the presence of atRA or Z-VAD; however, in the groups with periderm removal, most of the basal cells remained on the surface of the palate and exhibited basement membrane degradation. Arrow in B indicates apoptotic cell. All of the experiments were repeated at least three times. Scale bar, 2 µm. Magnification, ×20. MEE, medial edge epithelial; atRA, all-trans retinoic acid.

Figure 6.

TUNEL assay in single palate cultures for 48 h with or without periderm removal. (A) TUNEL-positive cells were detected in the control groups with or without periderm removal. Following treatment with atRA or Z-VAD, the number of apoptotic cells decreased in the untreated periderm groups and groups with periderm removal. Scale bar, 20 µm. (B) The ratio of apoptotic cells was significantly higher in the control groups when compared with the atRA or Z-VAD-treated groups (P<0.05) irrespective of whether the periderm was removed. Error bars represent the mean ± standard deviation. *P<0.05. atRA, all-trans retinoic acid.

Figure 7.

Culture of paired palatal shelves for 48 h. Complete fusion of the palates occurred in the (A-C) untreated periderm groups and (D-F) in the groups with periderm removal. (A) No MES were observed and the palate fused completely in the control group with untreated periderm. (B) EPM was separated by the thick MES barrier in the untreated periderm group in the presence of atRA (3 µM). (C) In the presence of Z-VAD (100 µM), EPM was separated by the multilayer of the MES barrier in the untreated periderm groups. (D) The medial edge epithelium broke apart into epithelial islands in the groups with periderm removal. (E) After culturing the paired palates in the presence of atRA (3 µM), two cell layers of MES were observed in the untreated periderm groups. (F) The MES became disrupted (arrow) in the groups with periderm removal that were treated with Z-VAD (100 µM). Furthermore, the MES (arrow) was disrupted in all groups that were subjected to periderm removal (D, E and F). Scale bar, 20 µm. Magnification, ×20. atRA, all-trans retinoic acid; EPM, embryonic palatal mesenchyme; MES, midline epithelial seam.