Bottom-up reconstitution of BMP morphogenetic pattern formation

描述（由申请人提供）：形态发生素对组织的模式化是多细胞发育的基本方面，形态发生素失调与发育障碍和癌症有关。最近的工作表明，形态发生模式比最初设想的更复杂，多个时空反馈和细胞外形态发生调节剂影响形态发生梯度的形成和解释。因此，了解形态发生素的模式化回路需要一定水平的遗传控制和定量分析，这在胚胎中是很难实现的。在这里，我们提出了一个互补的，合成的方法来解决这个问题，我们将重建形态梯度形成从下而上，一块一块。具体来说，我们将重建梯度形成空间扩展单层细胞培养使用细胞工程分泌和响应的形态发生蛋白（BMP），这是至关重要的胚胎模式，并已牵连在许多疾病（特别是那些肌肉骨骼系统）。使用定量延时显微镜，我们将监测梯度形成的时空动态在单个细胞的水平。该系统将提供一个平台，用于系统地测量调节BMP梯度的因素的影响，包括磷脂酰肌醇蛋白聚糖，分泌抑制剂和受体。最后，我们将重建一个穿梭电路提出的关键功能的能力，如梯度锐化和基因剂量的鲁棒性。通过这些重建，我们将能够确定这个回路的充分性，并理解使发育模式的基本特征成为可能的设计原则。我们期望这种自下而上的合成方法将广泛用于理解其他形态发生素形成模式的基础以及疾病状态下形态发生素系统的失调。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一研究计划，使申请人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.