Apoptosis or programmed cell death (PCD) has been documented to be a key biological mechanism regulating the development and homeostasis of many multicellular organisms and tissues, yet virtually nothing is known about this important biological process in salivary gland biogenesis. The hypothesis of this proposal is that specific cell deletion via apoptosis is a component of the normal development of the salivary gland. The goal of this application is to initiate the investigation of the role of PCD in salivary gland biogenesis. The proposed studies are limited to establishing a temporal and spatial quantitation of apoptosis utilizing the hamster submandibular gland (SMG) from distinct stages of postnatal development. To address this novel and yet unexplored issue, state-of-the-art methodologies will be used. The following specific aims are proposed: 1) Determining the occurrence of apoptosis during postnatal development of the hamster SMG with quantitative flow cytometry. A population of cells undergoing PCD will be identified by bivariate DNA labeling and multiparameter flow cytometry. Salivary cells will be characterized in terms of their cytokinetic parameters (including cell loss) and their total DNA, RNA and protein content. 2) Detecting apoptotic cells in the developing SMG in situ with enzymologically-based assays and acridine orange (AO) staining. Enzymological staining of apoptotic cells involves commercially available kits (Trevigen Apoptotic Cell System, TACS). Labeling is done with either Klenow (known as ISEL) or with terminal deoxynucleotidyl transferase (TdT), referred to as TUNEL. The principle of each assay is based on the apoptotic feature of DNA fragmentation; double stranded breaks in genomic DNA are utilized for enzymatic modification and the latter is coupled to either a chromogenic or fluorescent secondary detection system. In addition, cells undergoing PCD will be also detected by staining with a vital dye, acridine orange (AO). Both the enzymatic and AO labeling of PCD will be evaluated by light microscopy and/or confocal imaging and correlated with the developmental stage-specific expression of cell-restricted salivary protein markers.
