Bottom-up reconstitution of BMP morphogenetic pattern formation

描述(申请人提供)：组织的形态形成是多细胞发育的一个基本方面，形态失调与发育障碍和癌症有关。最近的工作表明，形态发生图案化比最初想象的要复杂得多，多个时空反馈和细胞外形态调控因子影响形态发生梯度的形成和解释。因此，理解形态形成模式电路将需要在胚胎中难以实现的遗传控制和定量分析水平。在这里，我们提出了一种补充的、综合的方法来解决这个问题，在这个方法中，我们将自下而上、逐段地重建形态形成梯度。具体地说，我们将在空间扩展的单层细胞培养中重建梯度形成，使用被设计为分泌和响应形态发生蛋白(BMP)的细胞，BMP在胚胎模式中至关重要，并与许多疾病(特别是肌肉骨骼系统的疾病)有关。利用定量时间推移显微镜，我们将在单个细胞水平上监测梯度形成的时空动态。该系统将提供一个平台，系统地测量调节BMP梯度的因素的影响，包括Glypicans、分泌型抑制物和受体。最后，我们将重建一个穿梭电路，以支持诸如梯度锐化和基因剂量稳健性等关键功能。通过这些重建，我们将能够确定该电路的充分性，并理解使发展模式的基本特征成为可能的设计原则。我们预计，这种自下而上的合成方法将广泛有助于理解其他形态原形成模式的基础，以及疾病状态下形态生成系统的错误调节。这种方法在再生医学疗法的设计中也将是有用的。职业发展活动将补充这项研究计划，使申请者能够成为一名独立的调查员。

DESCRIPTION (provided by applicant): The patterning of tissues by morphogens is a fundamental aspect of multicellular development, and morphogen misregulation has been implicated in developmental disorders and cancer. Recent work suggests that morphogenetic patterning is more complex than originally envisioned, with multiple spatiotemporal feedbacks and extracellular morphogen modulators impacting the formation and interpretation of morphogen gradients. Understanding morphogen patterning circuits thus will require levels of genetic control and quantitative analysis difficult to achieve in embryos. Here, we propose a complementary, synthetic approach to this problem in which we will reconstitute morphogen gradient formation from the bottom up, piece by piece. Specifically, we will reconstitute gradient formation in spatially extended monolayer cell cultures using cells engineered to secrete and respond to the morphogen bone morphogenetic protein (BMP), which is critical in embryonic patterning and has been implicated in many disorders (especially those of the musculoskeletal system). Using quantitative time-lapse microscopy, we will monitor the spatiotemporal dynamics of gradient formation at the level of individual cells. This system will provide a platform for systematically measuring the effects of factors that modulate BMP gradients, including glypicans, secreted inhibitors, and receptors. Finally, we will reconstruct a shuttling circuit proposed to underlie key functional capabilities such as gradient sharpening and gene dosage robustness. Through these reconstructions we will be able to determine the sufficiency of this circuit and understand the design principles that enable basic features of developmental patterning. We expect that this bottom-up, synthetic approach will be broadly useful in understanding the foundations of pattern formation by other morphogens and the misregulation of morphogen systems in disease states. This approach also will be useful in the design of regenerative medicine therapies. Career development activities will complement this research program in enabling the applicant to become an independent investigator.