The development and maintenance of epithelial organs on a cellular level is reliant upon concerted networks of biochemical signaling pathways, which transmit environmental cues such as growth factor and calcium concentrations to the level of gene expression in the cell. One example is basal keratinocytes, which have important proliferative and differentiation roles in the body. The precise regulation of these behaviors establishes the spatial axis of differentiated cells that makes up the skin. Both proliferative and differentiation cell behaviors have been linked to Ras/ERK MAP kinase pathway signaling; however, the precise mechanisms through which the same pathway regulates both responses remain unclear. Moreover, with an increasing body of work suggesting that epidermal progenitor cells may someday be used to culture patient-specific skin grafts in a dish, an understanding of single-cell signaling dynamics becomes increasingly important in dissecting the emergent tissue level behavior. Our lab has introduced live reporters of ERK activity into primary mouse keratinocytes and found that ERK demonstrates rich, complex endogenous dynamics. The objective of this proposal is to understand if and how Ras/ERK pathway dynamics in keratinocytes are read out at the level of cellular behavior, resulting in either proliferation or differentiation. I will use a combination of live cell reporter imaging, single-cell imaging of transcriptional dynamics, and engineering of synthetic reporter genes to dissect how ERK activity dynamics feed into the ERK-downstream gene expression program. To control physiologically relevant ERK behavior, I will use a combination of organotypic cell culture and optogenetic inputs. These approaches will be used in concert to probe input-output functions of ERK dynamics to gene expression, to test the hypothesis that different dynamic ERK behaviors are read out in different gene expression programs. A successful completion of the proposed work will constitute the first detailed dissection of ERK dynamics in a primary cell context as well as mapping of these dynamics to outputs in the form of physiologically relevant gene expression and cell responses. A comprehensive understanding of live signaling dynamics in differentiating, proliferating skin will likely have a broad impact on tissue engineering, regenerative medicine, and a deeper fundamental understanding of epithelial organs.
Live cell reporters have given researchers an unprecedented look into the dynamic behaviors of signaling pathways, but the consequences of dynamic signaling in a physiological context remain a mystery. Our findings that basal keratinocytes demonstrate a rich mileu of ERK dynamics suggest that previously determined ERK-responsive gene programs in these cells might be determined by dynamics and not by the absolute level of signal. The experiments proposed here will be the first dissection of the dynamics-to-gene expression circuit in any primary cell type, and will yield insights into the regulation of keratinocyte differentiation, maintenance, and proliferation.
