Cell-type specific assembly of apical extracellular matrices

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Project Summary/Abstract The development and function of tubular organs, including the lungs, intestines, and inner ear, depends in part on an apical extracellular matrix (aECM) that lines the organ interior. aECMs contain many components that are shared across organisms such as mucins, collagens, and zona pellucida domain (ZP) proteins. Matrix proteins are often arranged into distinct layers or spatial domains within an organ; however, these structures are often destroyed by fixation, limiting the ability to study how they are formed. This proposal uses the well characterized and highly tractable model of the developing C. elegans vulva to understand how aECM is specified and assembled. Due to its transparent body and the ease of fluorescently tagging proteins, C. elegans is an ideal system to study aECM in a live organism. Many of the aECM domains in the vulva are cell type specific, allowing us to directly connect the assembly of matrix proteins to the biology of the cells they coat. This study focuses on two ZP matrix proteins, LET-653 and NOAH-1, that localize to the matrix of specific vulva cells. Like ZP proteins in some other systems, such as the mammalian inner ear, the localization of LET-653 and NOAH-1 is not well explained by their transcription patterns. ZP proteins require protease cleavage to properly localize and must also bind surface factors at their destination site. The identity of these ZP protein partners is unknown. Aim 1 will dissect the relative contributions of each cell type to the expression, processing and export, and matrix localization of ZP proteins. It will also identify the transcription factors required for cell type specific matrix fate. Aim 2 will identify candidate ZP protein partners with differential expression between cell types by single nucleus RNA sequencing (snRNA-Seq) and test their requirement for ZP matrix assembly. Ultimately, these experiments will uncover a gene regulatory network that specifies apical matrix identity and identify ZP protein partners that could be applicable to many systems. This project is designed to provide training in epithelial and matrix biology, as well as relevant techniques in single cell transcriptomics, genetics, and microscopy. It is sponsored by Dr. Meera Sundaram, an expert in matrix biology and C. elegans vulva development, and co-sponsored by Dr. John Murray, an expert in gene regulatory networks and single cell technologies in C. elegans. The research will be conducted at the University of Pennsylvania, an institution with a collaborative and stimulating intellectual environment as well as first-rate facilities and resources to conduct biomedical research. The proposed training plan incorporates many professional development opportunities available through the university, sponsors, and C. elegans community. At the conclusion of the proposed research and professional activities, the applicant will be prepared for a career incorporating teaching undergraduates and running her own laboratory.