Mouse models of disease illuminate pathophysiology but are limited in the ability to identify how distinct cells in a complex microenvironment are reprogrammed during disease or treatment. This project establishes the initial tool in a set of mouse resources that will allow unprecedented specificity in mapping cell-specific transcription in the in vivo microenvironment. A mouse model will be created in which a real-time RNA tagging process is directed to a single cell type so that transcripts generated by those cells can be pulled out of complex RNA mixtures prepared from whole organs or tissues. As a result, genetic information corresponding to dynamic cell-cell interactions will be retained and global transcription measured from the cells of interest. In the proposed project, this technology will be applied to follow fibroblast transcription in vivo. Pathogenetic processes such as fibrosis are highly dependent on interactions between fibroblasts and surrounding cells in the microenvironment Current approaches to identify interactions between fibroblasts and the microenvironment cannot capture active transcription in specific cells within tissues during pathogenesis and generally destroy the microenvironment in the process of analysis. We hypothesize that a RNA metabolic labeling strategy can be embodied in a transgenic mouse to capture global transcription within specific cells of interest (e.g. fibroblasts) in vivo without dissecting or destroying the microenvironment. By expressing a YFP/uracil phosphoribosyl transferase (UPRT) gene specifically in mouse fibroblasts, only fibroblasts will be able to incorporate the UPRT substrate thiouracil into RNA during a TU-pulse. This mouse model will be validated by demonstrating fibroblast reprogramming in response to the profibrotic agent bleomycin.
We aim to: 1. Establish a specific fibroblast-RNA tagging system in vitro and compare its ability to capture bleomycin-induced fibroblast reprogramming to standard approaches, 2. Generate a fibroblast-specific YFP/UPRT transgenic mouse, and 3. Measure changes in fibroblast transcription within lungs during fibrosis induced by intratracheal bleomycin and within kidneys for comparison. In addition to direct effects on fibroblasts this agent significantly affects epithelial cells and inflammation in vivo. We expect that this mouse model enables unprecedented specificity in mapping transcription in the fibrotic microenvironment. Fibroblast- specific transcription in intact tissues will be measured for the first time. The mouse will be quite useful in preclinical research in general, but will have particular utility in research tied to pulmonary or hepatic fibrosis, cardiac remodeling, cancer-stromal interactions, emphysema and scleroderma. Completion of the proposed work thereby initiates a useful strategy to dissect pathogenic signaling that plays a role in organ fibrosis by adding the option of in vivo cell-specific labeling to a broad range of preclinical models. Our approach will allow us to identify novel fibroblast-specific genes that will serve as targets for the development of more effective anti-fibrotic therapies.
Diseases such as liver cirrhosis, pulmonary fibrosis and scleroderma are examples of fibrotic diseases, in which fibroblasts produce excessive extracellular proteins such as collagen and cause tissue stiffening and dysfunction. There are currently no effective therapeutic approaches for the treatment of fibrosis and no known agents that can halt or reverse its progression. It is known that fibrosis is a complex process, involving interactions of fibroblasts with surrounding cells, however current methods cannot characterize these interactions in living animals. Our project develops a mouse model in which the specific molecular activities of fibroblasts can be measured in living tissue for the first time. This model should accelerate the development of therapies for fibrosis.