The extracellular matrix (ECM) regulates cell behavior by influencing cell proliferation, survival, shape, migration and differentiation. Far from being a static structure, the ECM is constantly undergoing remodeling – i.e. assembly and degradation – particularly during the normal processes of development, differentiation and wound repair. When misregulated, this can contribute to disease. ECM assembly is regulated by the 3D environment and the cellular tension that is transmitted through integrins. Degradation is controlled by complex proteolytic cascades, and misregulation of these results in ECM damage that is a common component of many diseases. Tissue engineering strives to replace damaged tissues with stem cells seeded on synthetic structures designed to mimic the ECM and thus restore the normal control of cell function. Stem cell self-renewal and differentiation is influenced by the 3D environment within the stem cell niche. For tissue-engineering strategies to be successful, the intimate dynamic relationship between cells and the ECM must be understood to ensure appropriate cell behavior.

Embryonic tissues may provide clues about mechanisms required for tissue reassembly and regeneration, but few studies have utilized primary embryonic tissue to study tissue assembly. To test the capacity of tissue fragments to regenerate, we cultured fragments of embryonic day 13 (E13) mouse submandibular gland (SMG) epithelium and found that fragments as small as a quarter-bud retain the ability to branch. Further, we found that completely dissociated SMG epithelial cells self-organize into structures that undergo significant branching. Investigation into the mechanisms involved in tissue self-assembly demonstrated that inhibition of beta(1) integrin prevents cell aggregation, while inhibition of E-cadherin hinders aggregate compaction. Immunostaining showed that the cellular architecture and expression patterns of E-cadherin, beta-catenin, and actin in the reassembled aggregates mirror those seen in intact glands. Adding SMG mesenchymal cells to the epithelial cell cultures facilitates branching and morphological differentiation. Quantitative real-time RT-PCR indicated that the aggregates express the differentiation markers aquaporin-5 (AQP5), prolactin-inducible protein (PIP), and SMG protein C (SMGC). Together, these data show that dissociated SMG epithelial cells self-organize and undergo branching morphogenesis to form tissues with structural features and differentiation markers characteristic of the intact gland. These findings provide insights into self-assembly and branching that will facilitate future regeneration strategies in the salivary gland and other organs.

Summary: Epithelial organ morphogenesis involves reciprocal interactions between epithelial and mesenchymal cell types to balance progenitor cell retention and expansion with cell differentiation for evolution of tissue architecture. Underlying submandibular salivary gland branching morphogenesis is the regulated proliferation and differentiation of perhaps several progenitor cell populations, which have not been characterized throughout development, and yet are critical for understanding organ development, regeneration, and disease. Here we applied a serial multiplexed fluorescent immunohistochemistry technology to map the progressive refinement of the epithelial and mesenchymal cell populations throughout development from embryonic day 14 through postnatal day 20. Using computational single cell analysis methods, we simultaneously mapped the evolving temporal and spatial location of epithelial cells expressing subsets of differentiation and progenitor markers throughout salivary gland development. We mapped epithelial cell differentiation markers, including aquaporin 5, PSP, SABPA, and mucin 10 (acinar cells); cytokeratin 7 (ductal cells); and smooth muscle α-actin (myoepithelial cells) and epithelial progenitor cell markers, cytokeratin 5 and c-kit. We used pairwise correlation and visual mapping of the cells in multiplexed images to quantify the number of single- and double-positive cells expressing these differentiation and progenitor markers at each developmental stage. We identified smooth muscle α-actin as a putative early myoepithelial progenitor marker that is expressed in cytokeratin 5-negative cells. Additionally, our results reveal dynamic expansion and redistributions of c-kit- and K5-positive progenitor cell populations throughout development and in postnatal glands. The data suggest that there are temporally and spatially discreet progenitor populations that contribute to salivary gland development and homeostasis.