Synthetic morphogenesis to recapitulate multicellular airway branching patterns

点击翻译按钮获取中文摘要

Abstract The bronchial network of the human lung is a tree-like structure comprising over 20 generations of dichotomous branching; yet, the signaling basis for how this elaborate network is patterned has remained an enduring mystery. This represents not only a fundamental knowledge gap in developmental biology, but also a limiting factor for developing regenerative therapies to counter lung disease. While there are several plausible hypotheses as to how this patterning mechanism could operate, testing them has proven beyond the limits of classical gene knock- out experiments and other traditional reverse engineering approaches due to the complex signaling crosstalk found in situ. In this proposal, I will unify a classic experimental model in lung development (mesenchyme-free culture of distal lung epithelium) with state-of-the-art synthetic cell-cell signaling tools in order to map the design space for branch-patterning mechanisms. Working in a state-of-the-art Biological Design Center with a team of experts in mammalian synthetic biology and lung development, I will employ a “build-to-understand” approach wherein I construct synthetic cell populations that can either communicate with ex vivo tissues using endogenous signaling networks, or communicate with other synthetic cells using signaling pathways orthogonal to any found in nature. I will use these engineered cells to recapitulate an activation/repression feedback cycle which is thought to be vital in lung branching morphogenesis. By manipulating cell-cell communication, I will be able to isolate the fundamental design principles that govern how activation and repression signals between cells can manifest in higher- order structures. Furthermore, by decoupling specific signaling axes from their larger developmental context, and by performing high-resolution, time-lapse imaging of cell fate, I will be uniquely positioned to interrogate tissue pattern- ing mechanisms with unprecedented control. I hypothesize that reciprocal activation and repression between two cell types can give rise to a broad range of multicellular patterning outcomes depending on additional feedback loops and initial conditions. To test this hypothesis, I will explore the how the morphology and topol- ogy of multicellular patterns can be tuned by manipulating the signaling interactions between them. My overarching hypothesis is based on the predictions of previous computational models of branching morphogenesis via reaction- diffusion patterning, so I will use those predictions, and this theoretical framework, to guide my experimental designs. To assess whether synthetic signaling by engineered cells could also be a tractable approach for generating regen- erative lung tissue, I will further interrogate a 3D in vitro model where cell-cell signaling occurs exclusively through synthetic morphogens and receptors. Taken together, these studies will provide fundamental insights into how complex anatomical structures can be encoded in relatively simple signaling schemes which are executed locally between cells. Analysis of the resulting branch patterns is also expected to inspire a new paradigm for har- nessing synthetic cell-cell signaling to guide and direct the morphogenesis of therapeutically relevant cell types into tissue-specific architectures for regenerative medicine.