Medial Edge Epithelial Cell Fate during Palatal Fusion

Abstract

To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial-mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed beta-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor beta(3) (TGF-beta(3)) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-beta(3) null and wild-type mice. We show that MEE cell death in TGF-beta(3) null palates is greatly reduced at the time of fusion, revealing that TGF-beta(3) has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-beta(3) null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration.

Development of the upper lip: Morphogenetic and molecular mechanisms

“…TEM and cell biological studies have provided clear evidence of apoptosis of at least a portion of the MEE cells (Glucksmann, 1965;Saunders, 1966;DeAngelis and Nalbandian, 1968;Smiley and Dixon, 1968;Shapiro and Sweney, 1969;Smiley and Koch, 1975;Mori et al, 1994;Taniguchi et al, 1995;Cuervo et al, 2002;Cuervo and Covarrubias, 2004). Others, however, reported that the midline epithelial seam cells looked healthy at the TEM level and found evidence of transdifferentiation of MEE cells into mesenchymal cells by using various cell labeling techniques (Fitchett and Hay, 1989;Shuler et al, 1991Shuler et al, , 1992Griffith and Hay, 1992;Sun et al, 1998;Martinez-Alvarez et al, 2000;Nawshad et al, 2004). Because large numbers of apoptotic cells in the fusing epithelial seam were only observed in palatal explant cultures (Martinez-Alvarez et al, 2000;Cuervo et al, 2002;Cuervo and Covarrubias, 2004), Nawshad et al (2004) and Hay (2005) , 1997).…”

“…Others, however, reported that the midline epithelial seam cells looked healthy at the TEM level and found evidence of transdifferentiation of MEE cells into mesenchymal cells by using various cell labeling techniques (Fitchett and Hay, 1989;Shuler et al, 1991Shuler et al, , 1992Griffith and Hay, 1992;Sun et al, 1998;Martinez-Alvarez et al, 2000;Nawshad et al, 2004). Because large numbers of apoptotic cells in the fusing epithelial seam were only observed in palatal explant cultures (Martinez-Alvarez et al, 2000;Cuervo et al, 2002;Cuervo and Covarrubias, 2004), Nawshad et al (2004) and Hay (2005) , 1997). However, the loxP-flanked transcription STOP cassette prevents the lacZ gene from being transcribed in the R26R mice (Soriano, 1999).…”

Jag2‐Notch1 signaling regulates oral epithelial differentiation and palate development

“…Targeted disruption of Tgf␤3 in mice causes cleft palate (Kaartinen et al, 1995;Proetzel et al, 1995). Palatal shelves in Tgf␤3 Ϫ/Ϫ mutant mouse embryos grow, elevate, and approach normally but fail to adhere properly (Proetzel et al, 1995;Martinez-Alvarez et al, 2000). Compared with normal developing palatal shelves, the Tgf␤3 Ϫ/Ϫ mutant palatal shelves had reduced MEE periderm bulging and filopodial activity as well as reduced CSPG coat on the MEE (Taya et al, 1999;Martinez-Alvarez et al, 2000;Gato et al, 2002).…”

“…Palatal shelves in Tgf␤3 Ϫ/Ϫ mutant mouse embryos grow, elevate, and approach normally but fail to adhere properly (Proetzel et al, 1995;Martinez-Alvarez et al, 2000). Compared with normal developing palatal shelves, the Tgf␤3 Ϫ/Ϫ mutant palatal shelves had reduced MEE periderm bulging and filopodial activity as well as reduced CSPG coat on the MEE (Taya et al, 1999;Martinez-Alvarez et al, 2000;Gato et al, 2002). Addition of Tgf␤3 to cultures of Tgf␤3 Ϫ/Ϫ mutant mouse palatal shelf explants or chick palatal shelf explants, which normally do not express Tgf␤3 and do not fuse, stimulates secretion of CSPG and causes palatal shelf fusion in vitro (Gato et al, 2002).…”

A specific requirement for PDGF-C in palate formation and PDGFR-α signaling

“…3n,p) 3,4 . In the palatal shelf of Pdgfc À/À embryos, the number of proliferating bulging MEE cells was reduced markedly, and the mesenchyme immediately subjacent to the MEE was depleted of cells ( Fig.…”