Epithelial bridging of the primary palate: II. In vitro model mimics in vivo behavior

Previously, Forbes et al. [J Craniofac Genet Dev. Biol, 9:271-284, 1989] and Millicovsky et al. [Am J Anat 164:29-44, 1982], demonstrated that some of the epithelial cells of the primary palate formed extensive projections, bridging the medial and lateral nasal prominences. These connections are thought to aide in the fusion process by facilitating union of the prominences, a process known as secondary fusion [Millicovsky et al., 1982]. In order to study the epithelial cell and its behavior more closely an in vitro model was established [Gibson et al.: J Craniofac Genet Dev Biol, 1989], where epithelial cells in culture were shown to produce many of the morphologic characteristics observed in vivo. In the present study, an in vitro model is discussed which reproduces the epithelial projections observed in vivo. Epithelial cells, previously characterized, were obtained from the primary palate of 13-day-old rat embryos and sub-cultured as explants. Comparisons were made with the epithelial bridging observed in vivo of two species of animals. The results indicated sub-cultured epithelium as isolated cells, at either low or high density, rarely formed bridges. Primary cultures of epithelial explants also infrequently formed projections. However, sub-cultures of epithelial explants, plated as small clusters of cells with intervening spaces between cell groups, demonstrated extensive epithelial bridging. Epithelial projections did not form from cells that were directly attached to the plastic culture dish; only superficial, elevated cells formed projections. Significantly, the connections that occurred between explants did not attach to the plastic substratum. Instead, they appeared as line connections suspended by the medium. With time, the number of projections increased and epithelial cells could be seen along the projections forming an epithelial bridge. This study established a model of epithelial bridging in vitro for analysis of a process which has been shown to be an integral part of primary palate fusion.