Rapid changes in the extracellular matrix accompany in vitro palatal shelf remodelling

Abstract

The sequence of events and the distribution of extracellular matrix (ECM) components was examined during mouse secondary palatal shelf elevation in an in vitro model using standard roller tube culture methods developed for the culture of early embryos. In this culture system, the morphological changes associated with remodelling and reorientation of the palatal shelves of gestational day 13 mouse fetuses were similar to those observed in vivo. However, in specimens explanted 24–30 h prior to reorientation in vivo, remodelling began rapidly after explantation, and significant reorientation was accomplished within 4 h. Midline contact between the shelves did not occur until after 18 h in vitro, concomitant with shelf growth. Therefore, in this in vitro model, events related to palatal shelf remodelling and reorientation can be distinguished from those related to shelf growth. We used this in vitro model to characterize the transient changes in ECM distribution and accumulation that occur concomitant with events in shelf remodelling. Our results show that, during rapid remodelling in vitro, the relative distributions of collagen III, fibronectin and hyaluronate, as visualized by immunofluorescent staining, decreased within specific regions of the mesenchymal compartment. In contrast, the distribution of collagen I within the mesenchyme increased, and the distribution of tenascin did not change significantly. All molecules examined, except tenascin, showed changes in distribution within the basement membrane. These patterns of distribution are similar to those observed during more gradual remodelling in vivo. During remodelling in vitro, the deposition of [³H]-glucosamine- and [³H]-proline-labelled components of the ECM, as visualized by autoradiography, was greatest during the first 3 h of culture. During this period, labelled ECM accumulated within specific regions of the mesenchyme and palatal epithelial basement membrane. Uptake was reduced dramatically during the subsequent 3 h in culture and was restricted mainly to the palatal epithelium and its underlying basement membrane. The in vitro system permitted the characterization of early events in shelf remodelling leading to reorientation. Results suggest that remodelling is accompanied by rapid, local accumulation of ECM in specific regions of the palatal shelf previously thought to play a role in the process.