Quantitative single cell analysis of cell population dynamics during submandibular salivary gland development and differentiation

Abstract

SummaryEpithelial 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.

The Wnt-Myb pathway suppresses KIT expression to control the timing of salivary proacinar differentiation and duct formation

“…In cytodifferentiation processes, aquaporin 5 (AQP5), an early marker of proacinar cells (Larsen et al, 2011;Nelson et al, 2013), is expressed at E15 (the canalicular stage) after the onset of the ductal differentiation markers keratin 7 and 19 (KRT7/19) (Nelson et al, 2013). Parotid secretory protein (PSP; also known as BPIFA2), which is expressed transiently in developing AQP5-positive secretory proacinar cells (Ball et al, 2003), and mucin 10 (MUC10; also known as PROL1), which is expressed in developing proacini and mature mucous acinar cells (Melnick et al, 2001), are first detected at E17 (the terminal bud stage).…”

Opportunities and Challenges in Implementation of Multiparameter Single Cell Analysis Platforms for Clinical Translation

“…Mutually exclusive phosphorylation patterns of the two canonical substrates of mTORC1 (S6K1 and 4E‐BP1) were observed in individual cells, large regions of most tumors, and in distinct cell lineages, demonstrating differential pathway activation. In the research setting, MxIF has also been applied to evaluate signaling pathway changes in mouse salivary glands,26 estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 colocalization in breast cancer,27 and other single‐cell analyses 28, 29…”

“…Multiplex protein expression (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) …”

Endothelial cell regulation of salivary gland epithelial patterning

“…We examined the productal marker K19 together with Kit, a tyrosine kinase expressed by proacinar cells in developing salivary glands (Nelson et al, 2013;Lombaert et al, 2013;Matsumoto et al, 2016). In whole organ explant cultures, VEGFR signaling and vasculature development were manipulated using the pharmacological VEGFR2 inhibitors ZM 323881 and SU 5416.…”