Programmed cell death is not a necessary prerequisite for fusion of the fetal mouse palate.

Abstract

It has been widely accepted that programmed cell death (PCD) is an essential event in palatogenesis and that its failure can result in cleft palate, one of the most common birth defects in the human. However, some conflicting results have been reported concerning the timing of cell death occurring in the fusing palate and therefore the role of PCD in palatal fusion is controversial. In order to clarify whether cell death is indispensable for mammalian palatogenesis, we cultivated the palates of day-13 mouse fetuses in vitro and prevented cell death by treating them with the inhibitors of caspases-1 and -3 or with aurintricarboxylic acid which inhibits the activity of caspaseactivated DNase. Even when cell death was almost completely inhibited, palatal fusion took place successfully. Histological examination revealed that in the absence of apoptotic cell death, the medial edge epithelia of opposing palatal shelves adhered to each other and subsequently, the midline epithelial seam was disrupted and disappeared to bring about mesenchymal confluence across the palate. It seems that cell death is not a necessary prerequisite for palatal fusion but it may help to efficiently eliminate unnecessary cells which failed to migrate or differentiate properly.

Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation

“…Except for rats and mutant mice, Swiss-albino mice (SwissWebster, ICR, CD-1) have been most generally used to investigate the mechanisms underlying mammalian palatogenesis (Shuler et al, 1991(Shuler et al, , 1992Griffith and Hay, 1992;Martínez-Álvarez et al, 2000;Cuervo et al, 2002;Cuervo and Covarrubias, 2004;Takahara et al, 2004). Since we obtained similar results using CD-1 mice (Charles River Japan Inc.), the ability of MEE cells to differentiate independently of palatal shelf contact and midline seam formation appears to be common at least in Swissalbino mice.…”

“…In the true sense of anoikis, cell death in the MEE does not necessarily mean a genetically determined cell destiny but rather occurs accidentally during MEE cell differentiation, although it must be 'programmed' because of being predictable spatially and temporally. This concept of MEE cell death is further supported by our recent evidence that the midline epithelial seam of the fusing mouse plates can disappear in the absence of MEE cell death (Takahara et al, 2004).…”

“…Although cytokeratin-positive MEE cells always disappeared from the midline of the fusing palate by 36 hours of culture, the complete disruption of the basement membrane in the fusing palate was delayed probably due to the influence of amniotic fluid (Fig. 6H-J) when compared with that in culture without amniotic fluid (Takahara et al, 2004), which appeared to simulate the palatal fusion process in utero (Ferguson, 1988). TUNEL-positive MEE cells were observed in the oral and nasal epithelia and in the mesenchyme near the midline (Fig.…”

“…The left or right palatal shelf was further removed from each maxilla and was used as the single palatal shelf. The explants were cultured according to the suspension culture method (Shiota et al, 1990;Takahara et al, 2004). Briefly, five to seven explants were put into a bottle with BGJb medium (Invitrogen) containing 1% penicillin/ streptomycin (Invitrogen) (1ml/explant) and flushed for 2 min with a gas-mixture of 95% O 2 /5%CO 2 or 95% air/5%CO 2 , and sealed air-tight.…”

“…Sections taken at every 25 µm were stained with hematoxylin and eosin for routine histology. Other sections were examined by dual immunofluorescent staining for cytokeratin with mouse monoclonal antibody, clone AE1+AE3 (DAKO), and for type IV collagen with rabbit polyclonal antibody (LSL, Tokyo, Japan), or by the combination of immunofluorescence and the TUNEL method (Gavrieli et al, 1992), as previously described (Mori et al, 1994;Takahara et al, 2004). Immunoproducts were visualized with a secondary antibody conjugated with FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine isothiocyanate).…”