Cuervo, R and Covarrubias, L. Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis. Development 131: 15-24

Departament of Developmental Genetics and Molecular Physiology, Universidad Nacional Autónoma de México, Ciudad de México, Mexico City, Mexico
Development (Impact Factor: 6.46). 02/2004; 131(1):15-24. DOI: 10.1242/dev.00907
Source: PubMed


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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    • "There are three processes thought to be involved: apoptosis (Cuervo et al. 2002; Nawshad , 2008), epithelial–mesenchymal transformation (EMT) (Fitchett & Hay, 1989; Martinez-Alvarez et al. 2000; Nawshad , 2008) and migration of the epithelial cells to adjacent epithelia (Cuervo & Covarrubias, 2004; Jin & Ding, 2006; Xu et al. 2006). However, mouse studies in which permanent genetic labels were inserted into the epithelium do not support the idea that EMT occurs in vivo (Cuervo & Covarrubias, 2004; Jin & Ding, 2006; Iwata et al. 2014). Regardless of the mode of seam degradation, from a clinical point of view, the failure to remove the epithelium leads to congenital midline cysts of the hard palate (Epstein's pearls) (Kitamura, 1966, 1989; Saunders, 1972) and perhaps submucous clefts. "
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    ABSTRACT: It is essential to complete palate closure at the correct time during fetal development, otherwise a serious malformation, cleft palate, will ensue. The steps in palate formation in humans take place between the 7th and 12th week and consist of outgrowth of palatal shelves from the paired maxillary prominences, reorientation of the shelves from vertical to horizontal, apposition of the medial surfaces, formation of a bilayered seam, degradation of the seam and bridging of mesenchyme. However, in the soft palate, the mechanism of closure is unclear. In previous studies it is possible to find support for both fusion and the alternative mechanism of merging. Here we densely sample the late embryonic-early fetal period between 54 and 74 days post-conception to determine the timing and mechanism of soft palate closure. We found the epithelial seam extends throughout the soft palates of 57-day specimens. Cytokeratin antibody staining detected the medial edge epithelium and distinguished clearly that cells in the midline retained their epithelial character. Compared with the hard palate, the epithelium is more rapidly degraded in the soft palate and only persists in the most posterior regions at 64 days. Our results are consistent with the soft palate following a developmentally more rapid program of fusion than the hard palate. Importantly, the two regions of the palate appear to be independently regulated and have their own internal clocks regulating the timing of seam removal. Considering data from human genetic and mouse studies, distinct anterior-posterior signaling mechanisms are likely to be at play in the human fetal palate. © 2015 Anatomical Society.
    Journal of Anatomy 08/2015; 227(4). DOI:10.1111/joa.12365 · 2.10 Impact Factor
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    • "Another location where little mesenchymal migration takes place is in the midline of the recently fused mammalian secondary palate. Mouse organ culture experiments demonstrating fusion between cultured palatal shelves revealed no mesenchymal cell migration between the two tissues, despite epithelial cells migrating across the fusion zone (Cuervo and Covarrubias, 2004; Jin and Ding, 2006). "
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    ABSTRACT: The assembly of the upper jaw is a pivotal moment in the embryonic development of amniotes. The upper jaw forms from the fusion of the maxillary, medial nasal, and lateral nasal prominences, resulting in an intact upper lip/beak and nasal cavities; together called the primary palate. Due to the risk of craniofacial clefting, this process requires a balance of proper facial prominence shape and positioning, whilst still accommodating the vast phenotypic diversity of adult amniotes. As such, variation in craniofacial ontogeny is not tolerated beyond certain bounds. We have placed primary palatogenesis of amniotes into two categories, depending on whether the nasal and oral cavities remain connected throughout ontogeny or not. The transient separation of these two structures occurs in mammals and crocodilians, while remaining connected in birds, turtles and squamates. In the latter group, the craniofacial prominences fuse around a persistent choanal groove that connects the two cavities. Subsequently, select lineages within both categories develop a secondary palate that either completely or partially separates oral and nasal cavities in adults. Here we review the shared, early developmental events, and highlight the points at which development diverges in both primary and secondary palate formation. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Developmental Dynamics 08/2015; DOI:10.1002/dvdy.24338 · 2.38 Impact Factor
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    • "Previous studies have revealed that cleft of the secondary palate originates from a failure of signaling molecules and their receptors to control palatal shelf growth, elevation, and fusion involving palatal mesenchyme and epithelium [5], [6]. In the fusion process, most studies have focused on the mechanisms responsible for the disappearance of the MES; there still remains considerable disagreement regarding the fate of the MES, such as : (A) apoptosis in the MES [7], (B) migration of the MEE resulting in loss of MES [8], and/or (C) epithelial-mesenchymal transformation of MES [9], [10], [11]. On the other hand, before the process of disappearance of the MES, epithelial adhesion of the MEE by each opposing palatal shelf is required. "
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    ABSTRACT: Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at various developmental stages before, during, and after palate fusion using GeneChip® microarrays. Ceacam1 was one of the highly up-regulated genes during palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was present in prefusion palatal epithelium and was degraded during fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1 (-/-)) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1 (-/-) mice. TGFβ3 expression, apoptosis, and cell proliferation in palatal epithelium were not affected in the palate of Ceacam1(-/-)mice. However, CEACAM1 expression was retained in the remaining MEE of TGFβ-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion.
    PLoS ONE 04/2013; 8(4):e61653. DOI:10.1371/journal.pone.0061653 · 3.23 Impact Factor
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