Profiling Active Protein Expression in Salivary Gland Using Multiplex Methods

The molecular mechanisms governing salivary gland morphogenesis and differentiation are little understood. However, understanding the basic molecular pathways controlling morphogenesis and differentiation in mammalian organ development is a first step towards understanding pathogenesis and developing therapeutic strategies for disease. Objectives: We are developing an in situ proteomics atlas of developing salivary glands to identify the spatio-temporal expression profile of active signaling molecules using high-throughput methods that allow quantification of image data at a subcellular resolution. Methods: Proteins and phosphoproteins are being profiled in developing murine submandibular and sublingual salivary glands from embryonic day 12 (E12) through adult in a tissue microarray (TMA) format. The method relies upon high-specificity antibodies that are directly conjugated to fluorophores of the cyanine dye family. For each round of immunostaining, antibodies are hybridized to the TMAs, images acquired, and then the dyes are inactivated. The process is repeated such that multiple protein targets can be imaged within a single TMA. Novel image analysis and quantification methods are used to assign signals to individual cells with subcellular resolution. Results: A publically-available atlas is under construction to facilitate lineage tracking and characterization of signaling pathways in developing salivary glands. Conclusion: Signaling pathways controlling morphogenesis and differentiation within specific cell types have been identified. Additionally, the probes and methods developed during these studies will be applied to disease-relevant samples in the future.

Funded by NIH RC1-DE020402; DE0192444; University at Albany, SUNY; and GE Global Research.