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Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.

Salivary gland (SG) development involves dynamic epithelial-mesenchymal interactions resulting in the formation of highly branched epithelial structures that produce and secrete saliva. The SG epithelium differentiates into saliva-producing terminal buds, i.e., acini, and transporting ducts. Most studies on the salivary gland have focused on branching morphogenesis; however, acinar cell differentiation underlying the determination of serous or mucous salivary glands is unclear. The objective of this study was to identify the mesenchymal signaling molecules involved in the epithelial differentiation of the salivary gland type as serous or mucous. Salivary glands undergoing stage-specific development, including the parotid gland (PG) and the sublingual gland (SLG) at embryonic day 14.5 (E14.5) were dissected. The glands were treated with dispase II to separate the epithelium and the mesenchyme. RNA from mesenchyme was processed for microarray analysis. Thereafter, microarray data were analyzed to identify putative candidate molecules involved in salivary gland differentiation and confirmed via quantitative reverse transcription polymerase chain reaction. The microarray analysis revealed the expression of 31,873 genes in the PG and SLG mesenchyme. Of the expressed genes 21,026 genes were found to be equally expressed (Fold change 1.000) in both PG and SLG mesenchyme. The numbers of genes expressed over onefold in the PG and SLG mesenchyme were found to be 5247 and 5600 respectively. On limiting the fold-change cut off value over 1.5 folds, only 214 and 137 genes were expressed over 1.5 folds in the PG and the SLG mesenchyme respectively. Our findings suggest that differential expression patterns of the mesenchymal signaling molecules are involved in fate determination of the salivary acinar cell types during mouse embryogenesis. In the near future, functional evaluation of the candidate genes will be performed using gain- and loss-of-function mutation studies during in vitro organ cultivation. [ABSTRACT FROM AUTHOR]