In Vitro Analysis of Palatal Shelf Elevation During Secondary Palate Formation

Abstract

Palatal shelf elevation is an essential morphogenetic process during secondary palate formation. It has been proposed that shelf elevation results from an intrinsic elevating force and is regulated by extrinsic factors that are associated with development of other orofacial structures. Although dynamic palate culture is a common in vitro approach for studying shelf elevation, it requires the tongue or the tongue and mandible to be removed before culture, which prevents any determination of the role of the extrinsic factors in regulating shelf elevation. We showed that ex vivo removal of the tongue and mandible from unfixed embryonic heads led to spontaneous shelf movements that were more pronounced at late E13.5 and early E14.5 than those of E12.5 and early E13.5, suggesting that the strength of the elevating force increases over time during palate development. We further used a suspension culture technique to analyze palatal shelf movement in an intact oral cavity by culturing the orofacial portion of embryonic heads that include the maxilla, palatal shelves, mandible, and tongue (MPMT). MPMT explants were cultured in the serum-free medium with slow rotation for 24-48 hr. The palatal shelves successfully elevated during culture and displayed intermediate morphologies that closely resemble those of in vivo shelf elevation. We demonstrate that the tongue and mandible facilitate shelf medial movement/growth during shelf elevation and further suggest that the interaction of the palatal shelves and tongue could be one of the extrinsic factors that regulate the elevation process.The mammalian secondary palate is formed by fusion of paired palatal shelves that arise from the maxillary process. The palatal shelves are wedge-shaped tissues that initially grow in a vertical direction along the lateral sides of the tongue. At a specific time of development, the vertical shelves undergo an elevation process to become horizontally oriented and move into a position superior to the tongue in the oral cavity. After elevation, continual shelf growth results in contact and adhesion of medial edge epithelial (MEE) cells to form a midline epithelial seam (MES) between the opposing shelves. Degeneration of the MES through increased MEE cell death, cell

Three-dimensional reconstruction of systematic histological sections: application to observations on palatal shelf elevation

“…Currently, the understanding of the mechanism of shelf elevation is mainly based on histomorphological studies from 2D sections and various in vitro studies. 6 , 7 , 9 , 24 , 31 – 33 However, conventional 2D histology provides only planar information of the PS and surrounding tissues. Furthermore, there were no definite sites or anatomical landmarks of the anterior, middle (the presumptive hard palate), and posterior (the presumptive soft palate) regions of the palate, which were inconsistent in various articles and might give rise to misinterpretations and the loss of crucial shelf morphological change during palatal elevation along the AP axis.…”

FACEts of mechanical regulation in the morphogenesis of craniofacial structures

“…Conversely, forces generated by HA hydration within the palatal tissue may help overcome the initial blockade by the tongue during normal palatogenesis, allowing the palate to displace the tongue dorsally in a timely manner; although this force is not required for the eventual shelf elevation. 178 , 179

Fig. 4 Mechanical regulation of palatal shelf elevation and fusion.

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Transcriptome sequencing analysis of the role of β-catenin in F-actin reorganization in embryonic palatal mesenchymal cells

“…Palatal shelf elevation results from an endogenous factor that remodels mesenchyme, but the source of the factor remains unknown (9)(10)(11). A recent study reported that actin cytoskeleton aligned substantially in palatal mesenchymal cells of elevating PSs (9).…”