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

Abstract

During mammalian development, a pair of shelves fuses to form the secondary palate, a process that requires the adhesion of the medial edge epithelial tissue (MEE) of each shelf and the degeneration of the resulting medial epithelial seam (MES). It has been reported that epithelialmesenchymal transformation (EMT) occurs during shelf fusion and is considered a fundamental process for MES degeneration. We recently found that cell death is a necessary process for shelf fusion. These findings uncovered the relevance of cell death in MES degeneration; however, they do not discard the participation of other processes. In the present work, we focus on the evaluation of the processes that could contribute to palate shelf fusion. We tested EMT by traditional labeling of MEE cells with a dye, by infection of MEE with an adenovirus carrying the lacZ gene, and by fusing wild-type shelves with the ones from EGFP-expressing mouse embryos. Fate of MEE labeled cells was followed by culturing whole palates, or by a novel slice culture system that allows individual cells to be followed during the fusion process. Very few labeled cells were found in the mesenchyme compartment, and almost all were undergoing cell death. Inhibition of metalloproteinases prevented basal lamina degradation without affecting MES degeneration and MEE cell death. Remarkably, independently of shelf fusion,activation of cell death promoted the degradation of the basal lamina underlying the MEE (`cataptosis'). Finally, by specific labeling of periderm cells (i.e. the superficial cells that cover the basal epithelium), we observed that epithelial triangles at oral and nasal ends of the epithelial seam do not appear to result from MEE cell migration but rather from periderm cell migration. Inhibition of migration or removal of these periderm cells suggests that they have a transient function controlling MEE cell adhesion and survival, and ultimately die within the epithelial triangles. We conclude that MES degeneration occurs almost uniquely by cell death, and for the first time we show that this process can activate basal lamina degradation during a developmental process.

Development of the upper lip: Morphogenetic and molecular mechanisms

“…In contrast to the few studies of the lip fusion process, the fate of the medial edge epithelial (MEE) cells of the secondary palatal shelves, which form the midline epithelial seam upon palatal shelf adhesion, has been studied extensively although considerable disagreement still exists. 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).…”

“…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).…”

“…The confusion may have arisen due in part to species differences (e.g., chick vs. mouse and human) in facial morphogenesis and in part to lack of synthesis of the fragmentary and often incomplete information gained from individual studies. Moreover, whereas it has been widely accepted that epithelial-mesenchymal transformation (EMT) of the epithelial seam is the major mechanism for both lip and palate fusion (Fitchett and Hay, 1989;Shuler et al, 1991Shuler et al, , 1992Griffith and Hay, 1992;Hay, 1995Hay, , 2005Sun et al, 2000;Cox, 2004;Nawshad et al, 2004), recent studies have challenged this theory and demonstrated that the palatal epithelial seam gradually regresses by programmed cell death rather than by EMT (Cuervo and Covarrubias, 2004;Vaziri Sani et al, 2005). At the molecular level, recent studies in chick and mice have identified specific roles for several major signaling pathways, including Bmp, Fgf, and Shh signaling pathways in midfacial morphogenesis (Hu and Helms, 1999;Trumpp et al, 1999;Ashique et al, 2002;Trokovic et al, 2003;Jeong et al, 2004;Liu et al, 2005b).…”

Jag2‐Notch1 signaling regulates oral epithelial differentiation and palate development

“…There is strong evidence that disruption of the palatal epithelial seam during palatal fusion results at least in part from apoptosis (Cecconi et al, 1998;Cuervo and Covarrubias, 2004;Martinez-Alvarez et al, 2004;Vaziri Sani et al, 2005). We carried out terminal deoxynucleotidyl transferasemediated deoxyuridinetriphosphate nick end-labeling (TUNEL) analysis to investigate the mechanism involved in the disruption of the epithelial seam between the palate and tongue in the Jag2 ⌬DSL/⌬DSL mutants and found that the palate-tongue seam contained numerous apoptotic cells, similar to the wild-type palate-palate seam during palatal fusion (Fig.…”

“…The process of palatal fusion has been studied extensively, but there is still disagreement over the relative contributions of epithelial-mesenchymal transformation versus apoptosis to the disappearance of the epithelial seam (Fitchett and Hay, 1989;Cuervo and Covarrubias, 2004;reviewed in Nawshad et al, 2004). Those favoring epithelial-mesenchymal transformation as the major mechanism propose that apoptosis is restricted to the periderm layer and suggest that this is important for the initial adhesion of the palatal shelf epithelia.…”