Craniofacial Developmental Dynamics

National Institute Of Dental & Craniofacial Research

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Abstract

This project is focused primarily on determining the mechanisms of morphogenesis of salivary glands and other organs. We are addressing the following major questions: 1. How do embryonic salivary glands and other branched organs generate their characteristic branched architectures during the process of branching morphogenesis? Specifically, how is the formation of clefts, buds, and ducts mediated and coordinated at molecular and biophysical levels? How can we facilitate bioengineering for organ replacement - particularly of salivary glands - by understanding branching morphogenesis and by promoting specific steps of this process? 2. What are the contributions of selective, local regulation of organ-specific gene expression, cell adhesion, embryonic cell sorting, extracellular matrix, integrins, signal transduction, and local cell migration to organ branching morphogenesis and organ specification? Branching morphogenesis of developing organs requires coordinated but still relatively poorly understood changes in gene expression, epithelial cell-cell adhesion, cell-matrix adhesion, and cell motility. We had previously performed single-cell and bulk RNA transcriptomic analyses on embryonic submandibular versus parotid salivary glands to characterize their molecular identities at the very early stage of bud initiation. A key finding was the surprising degree of difference in gene expression patterns between these glands quite early in development, indicating gland specificity at even this early single-bud stage. Mesenchymal cells formed separate, well-defined clusters specific to each gland. For example, there were substantial differences between molecular markers and tissue localization of neuronal and muscle-related cells between these two glands. There were both distinct and overlapping patterns of gene expression in the epithelial cells of these two glands. In preliminary studies, recombination experiments in which the mesenchymal tissues of these two types of gland were swapped showed significant alterations in the epithelial gene expression patterns of each gland. The specific alterations in recombined tissues are currently being characterized in depth. Our previous studies identified highly dynamic interactions of salivary gland epithelial cells with their surrounding basement membrane during organ development. The epithelial cells translocate most actively when in contact with the basement membrane. In order to define global patterns of cell movement and their contributions to cleft and bud formation in early salivary gland development, the movements of virtually every epithelial cell in an embryonic gland were carefully tracked in comparison to the location of the basement membrane by time-lapse multiphoton confocal microscopy. These analyses revealed that the outer layer of epithelial cells interacting with the basement membrane functions as a relatively stable sheet of cells. For mitosis of cells in this layer, the cells lose their attachment to the basement membrane and divide primarily in the interior of the gland. Importantly, however, all dividing cells eventually insert back into the outer layer of epithelial cells. The mechanism appears to be cell sorting due to differential adhesion due to the low levels of E-cadherin in the outer cells, which is maintained after cell division; these cells are then eventually sorted out to the bud surface. The net effect of this pattern of cell division is to generate strong population pressure within this outer cell layer compared to cells deeper in the gland, which in combination with cell adhesion to the basement membrane drives cleft and bud formation by tissue and basement membrane folding. This process could be characterized by simple mathematical modeling that emphasized the importance of tissue free energy, which could be tested by a series of experimental analyses altering integrin- and cadherin-based cell surface interactions. The mechanism was summarized in a model in which a combination of decreased cell-cell adhesion and the maintenance of strong cell-matrix adhesion of peripheral epithelial cells to the basement membrane drives the expansion and mechanical folding of the surface epithelial sheet. Single-cell RNA sequencing and single-molecule RNA fluorescence in situ hybridization revealed that the outer, surface-located epithelial bud cells have distinct transcriptional features. Besides decreased E-cadherin transcription, there are elevations in certain integrin genes and genes involved in cell proliferation. In order to recapitulate cleft and bud formation in a synthetic cell system, we applied a method for producing a basement membrane around a cell spheroid. A low concentration of the basement membrane extract Matrigel permitted various cells to form tight spheroids surrounded by a cell-assembled basement membrane. Using this system plus targeted suppression of specific genes using a CRISPR/Cas9 lentiviral system, we were able to demonstrate successful reconstitution of budding morphogenesis. Experimental suppression of expression of E-cadherin plus induction of basement membrane formation in 3D spheroid cultures of engineered cancer epithelial cells that normally do not form buds resulted in robust budding morphogenesis. Suppressing integrin expression and/or function, changing the composition of the experimentally assembled basement membrane, or increasing its thickness inhibited this reconstitution of budding. Thus, budding morphogenesis in this organ system is based on a fundamental self-organizing mechanism based on preferential cell-matrix adhesion versus cell-cell adhesion. This mechanism can explain how stratified epithelia can undergo budding morphogenesis without external physical inputs, and it permits tissue engineering of the early budding stage of branching morphogenesis. These studies are beginning to elucidate the complex mechanisms that underlie the cell and tissue dynamics involved in craniofacial organ development, particularly of salivary glands. Understanding these underlying morphogenetic mechanisms during embryonic development should promote more effective tissue engineering for restoration of damaged organ function.