An Engineered Gene Network for Multicellular Pattern Formation

描述（由申请人提供）：高度分化的多细胞生物体来源于形态对称的胚胎，具有单一、离散的基因表达谱。这个非凡的过程是由遗传调控元件的分层组织网络控制的，这些遗传调控元件控制着驱动形态变化的基因的表达。这些遗传网络经常产生通过扩散或通过细胞表面信号传导传播的分子，以调节其自身的表达。这样的系统能够实现多细胞模式化，这是形成更高级结构如组织和器官的基础。理论模型预测，生物学中观察到的许多模式可以由一个由短程（局部）自激活剂和长程抑制剂组成的遗传系统产生。有趣的是，激活剂-抑制剂网络被预测为在仅略微不同的动力学参数下驱动多细胞斑点、条纹、振荡和行波的模式。虽然许多候选基因网络已被提出使用激活剂-抑制剂策略来调节模式，但生物学的复杂性通常排除了分子水平的验证。该提案的重点是构建一个合成的，定义明确的激活剂-抑制剂基因网络，能够指导细胞群体中的模式形成。该过程的第一步是构建能够引导可扩散化合物径向传播穿过二维细胞群的自催化组分。这将基于细菌群体感应（细胞-细胞通信）系统。然后将自催化繁殖系统推进到包括抑制剂组分，以便在生长细胞的群落内指示斑点和条纹图案化。所选择的抑制剂组分将基于正交群体感应系统，并且将是自由膜扩散的，与扩散性较低的活化剂相比，能够实现长程抑制。“激活”状态将由荧光报告基因指示，并且模式形成将在与细胞生长相容的条件下通过延时荧光显微镜监测。一个计算反应扩散模型，包括所有工程遗传成分将被用来调查影响格局形成的参数。公共卫生相关性：在人类发育过程中，细胞群必须协同工作，以形成产生器官和四肢等高级结构的模式。细胞模式形成中的错误可能导致成人中的多种发育缺陷以及迟发性疾病。我们的目标是调查这些高度协调的模式形成过程的遗传机制，试图提高自然和患病细胞状态的知识。

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.