An Engineered Gene Network for Multicellular Pattern Formation

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DESCRIPTION (provided by applicant): Highly differentiated multicellular organisms derive from morphologically symmetrical embryos with a single, discreet gene expression profile. This remarkable process is controlled by hierarchically organized networks of genetic regulatory elements which control the expression of the genes that drive morphological changes. These genetic networks frequently produce molecules which spread by diffusion or through cell surface signaling to regulate their own expression. Such systems enable multicellular patterning, the basis for the formation of higher order structures such as tissues and organs. Theoretical models have predicted that many patterns observed in biology can be generated by a genetic system composed of a short-ranging (local) self-activator and a long-ranging inhibitor. Intriguingly, the activator-inhibitor network is predicted to drive patterns of multicellular spots, stripes, oscillations and traveling waves under only slightly different kinetic parameters. Although many candidate gene networks have been proposed to use the activator- inhibitor strategy to regulate patterning, the complexity of biology has often precluded molecular-level validation. This proposal focuses on the construction of a synthetic, well-defined activator-inhibitor gene network capable of guiding pattern formation in populations of cells. The first step in this process is the construction of an autocatalytic component capable of guiding the radial propagation of a diffusible compound across a two-dimensional population of cells. This will be based on bacterial quorum sensing (cell-cell communication) systems. The autocatalytic propagation system will then be advanced to include an inhibitor component in order to dictate spot and stripe patterning within a community of growing cells. The inhibitor component selected will be based on an orthogonal quorum sensing system and will be freely membrane diffusible, enabling long range inhibition as compared to the less diffusible activator. The 'activated' state will be indicated by a fluorescent reporter gene and pattern formation will be monitored by time-lapse fluorescence microscopy under conditions compatible with cell growth. A computational reaction- diffusion model incorporating all engineered genetic components will be used to investigate the parameters affecting pattern formation. PUBLIC HEALTH RELEVANCE: During human development, groups of cells must function in concert to form the patterns which give rise to higher-order structures such as organs, and limbs. Errors in cellular pattern formation can result in a multitude of developmental defects as well as late onset diseases in adults. We aim to investigate the genetic mechanisms underlying these highly orchestrated pattern formation processes in an attempt to improve knowledge of natural and diseased cellular states.