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 epithelial-mesenchymal 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.
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ABSTRACT: Background: Retinoic acid (RA) is a key regulator of embryonic development and linked to several birth defects including cleft lip and palate (CLP). The aim was to investigate the effects of RA on proliferation and gene expression of human palatal keratinocytes (KCs) in vitro. Methods: KCs from children with and without CLP were cultured with 2 and 5 μM RA. Proliferation was measured by quantification of DNA after 2, 4, 6, and 8 days. In addition, we analysed the effects of RA on messenger RNA expression of genes for proliferation, differentiation, apoptosis, and RA receptors. Results: RA similarly inhibited proliferation of palatal KC from cleft and non-cleft subjects. The proliferation of KCs from cleft subjects was reduced to 59.8±13.4% (2 μM) and 41.5±14.0% (5 μM, Day 6), while that of cells from age-matched non-cleft subjects was reduced to 66.9±12.1% (2 μM) and 33.9±10.1% (5 μM). RA treatment reduced the expression of several of the investigated genes; the proliferating cell nuclear antigen (PCNA) was reduced in CLP KCs only. Keratins 10 and 16 were downregulated in keratinocytes from both cleft and non-cleft subjects. P63, a master regulator for epithelial differentiation, was only downregulated in KCs from cleft subjects, as was the RXRa receptor. Two P63 target genes (GJB6 and DLX5) were strongly downregulated by RA in all cell lines. None of the apoptosis genes was affected. Conclusion: Overall, RA similarly inhibits proliferation of palatal KCs from cleft and non-cleft subjects and reduces the expression of specific genes.The European Journal of Orthodontics 02/2014; · 1.08 Impact Factor
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ABSTRACT: Cleft palate is one of the most common human birth defects and is associated with multiple genetic and environmental risk factors. Although mutations in the genes encoding transforming growth factor beta (TGFβ) signaling molecules and interferon regulatory factor 6 (Irf6) have been identified as genetic risk factors for cleft palate, little is known about the relationship between TGFβ signaling and IRF6 activity during palate formation. Here, we show that TGFβ signaling regulates expression of Irf6 and the fate of the medial edge epithelium (MEE) during palatal fusion in mice. Haploinsufficiency of Irf6 in mice with basal epithelial-specific deletion of the TGFβ signaling mediator Smad4 (Smad4(fl/fl);K14-Cre;Irf6(+/R84C)) results in compromised p21 expression and MEE persistence, similar to observations in Tgfbr2(fl/fl);K14-Cre mice, although the secondary palate of Irf6(+/R84C) and Smad4(fl/fl);K14-Cre mice form normally. Furthermore, Smad4(fl/fl);K14-Cre;Irf6(+/R84C) mice show extra digits that are consistent with abnormal toe and nail phenotypes in individuals with Van der Woude and popliteal pterygium syndromes, suggesting that the TGFβ/SMAD4/IRF6 signaling cascade might be a well-conserved mechanism in regulating multiple organogenesis. Strikingly, overexpression of Irf6 rescued p21 expression and MEE degeneration in Tgfbr2(fl/fl);K14-Cre mice. Thus, IRF6 and SMAD4 synergistically regulate the fate of the MEE, and TGFβ-mediated Irf6 activity is responsible for MEE degeneration during palatal fusion in mice.Development 03/2013; 140(6):1220-30. · 6.60 Impact Factor
Article: Shaping organisms with apoptosis.[Show abstract] [Hide abstract]
ABSTRACT: Programmed cell death is an important process during development that serves to remove superfluous cells and tissues, such as larval organs during metamorphosis, supernumerary cells during nervous system development, muscle patterning and cardiac morphogenesis. Different kinds of cell death have been observed and were originally classified based on distinct morphological features: (1) type I programmed cell death (PCD) or apoptosis is recognized by cell rounding, DNA fragmentation, externalization of phosphatidyl serine, caspase activation and the absence of inflammatory reaction, (2) type II PCD or autophagy is characterized by the presence of large vacuoles and the fact that cells can recover until very late in the process and (3) necrosis is associated with an uncontrolled release of the intracellular content after cell swelling and rupture of the membrane, which commonly induces an inflammatory response. In this review, we will focus exclusively on developmental cell death by apoptosis and its role in tissue remodeling.Cell Death and Differentiation advance online publication, 1 March 2013; doi:10.1038/cdd.2013.11.Cell death and differentiation 03/2013; · 8.24 Impact Factor