Date of Award

1-1-2015

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (v, 57 pages) : color illustrations

Dissertation/Thesis Chair

Melinda Larsen

Committee Members

Benjamin Szaro, Michael Gerdes

Keywords

Submandibular gland, Morphogenesis, Branching processes, Phosphorylation, Cellular signal transduction

Subject Categories

Cell Biology | Developmental Biology | Molecular Biology

Abstract

The development of submandibular salivary glands is complex and requires coordination of specific signaling events. Submandibular salivary glands originate as an epithelial invagination into the adjacent mesenchyme that leads to a single stalk and end bud; this end bud will go through a clefting process. Numerous rounds of clefting will lead to a fully developed salivary gland by this process, which is known as branching morphogenesis. As the gland undergoes morphogenesis, specific cues leading to differentiation of multiple cell types and even epithelial sub classes are required. By the later stages of development the glands are fully innervated, have an integrated vasculature, and maintain a myoepithelial population to pump the saliva out of the acinar cells and through the ducts. Within the early stage developing epithelium, there are two epithelial sub classes, which are the outer polarized cells and the inner polymorphic cells. We have found that within these two cell populations there are differences in localization and quantity of protein phosphorylation, leading us to hypothesize that regulation of activation of certain signaling pathways in a cell-type specific manner is critical for proper development. One of these critical pathways is the PI3K pathway. Phosphotidyl inositide signaling can affect many downstream signaling cascades and was previously found to be necessary for proper branching morphogenesis in submandibular salivary glands. Ex vivo embryonic glands cultured with a PI3K inhibitor, LY294002 , cleft significantly less than control glands. We hypothesize that there are essential PI3K signaling-mediated events that are restricted both in location and time to control salivary gland development. The primary tool used in this study is a novel multiplexed immunohistochemistry method that has been developed at General Electric Global Research Center. This is a histology-based approach that uses conjugated antibodies and a dye inactivation solution to facilitate overlapping of images from multiple rounds of immunohistochemistry. The algorithms register multiple images captured sequentially from the same sample and provide information regarding protein levels and on their subcellular localization. We have validated multiple probes to facilitate computational segmentation of cells into different cellular compartments, cell types, and epithelial subclasses. Using immunohistochemistry we have demonstrated that in the presence of the PI3K inhibitor, LY294002, downstream signaling proteins AKT and GSK-3β show a decrease in levels of phosphorylation along with a change in localization of the phosphorylated forms. This study sets the stage for further investigation into the function of PI3K-regulated signaling in salivary gland morphogenesis and differentiation.

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