Morphogen Gradients in Microfluidic Cultures

描述（由申请人提供）：该项目的长期目标是了解大脑皮层和其他组织在发育过程中形态梯度是如何形成的。形态发生梯度是动物发育的基础，而形态发生缺陷是人类出生缺陷和皮质畸形的主要原因。尽管如此，关于形态梯度作用的机制仍然存在巨大的争议，这限制了我们对这些人类疾病的理解。在很大程度上，这场争论围绕着一个问题——无法区分不依赖于细胞间交流的形态发生活动（“经典”模式）和依赖于细胞间交流的形态发生活动。迄今为止，对这一问题的深入研究依赖于传统的分离细胞培养，这在实验效率和自然形态梯度模型方面都受到限制。然而，微流体培养装置有潜力解决这些限制。这种微型装置产生精确和连续的生物分子梯度，具有不同的剖面到细胞上，是专为延时显微镜设计的。这些特征应该为建模和研究形态梯度提供了优于传统文化的生物学和实践优势。皮层前体细胞（CPC）的初步研究证实了该系统的前景，但也确定了需要优化的设备设计特征，以回答驱动该提议的基本问题-正常皮层中哪些CPC反应仅由细胞外形态素浓度决定，哪些不是？

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how morphogen gradients pattern the cerebral cortex and other tissues during development. Morphogen gradients are fundamental to animal development, and morphogen defects are primary causes of human birth defects and malformations of the cortex. Nonetheless, tremendous controversy remains about the mechanisms by which morphogen gradients act, which limits our understanding of these human disorders. For the most part, this controversy revolves around a single issue - the inability to distinguish morphogen activities that do not depend on cell-cell communication (the "classical" model) from those that do. To date, insight into this issue has relied on heroic studies using traditional dissociated cell cultures, which are limited both in terms of experimental efficiency and as models of natural morphogen gradients. However, a microfluidic culture device has the potential to address these limitations. This microscale device generates precise and continuous biomolecular gradients with different profiles onto cells, and is designed for time-lapse microscopy. These features should provide several biological and practical advantages over traditional cultures for modeling and studying morphogen gradients. Preliminary studies with cortical precursor cells (CPCs) confirm the promise of this system, but have also identified device design features that need to be optimized in order to answer the basic question driving this proposal - which CPC responses in the normal cortex are determined solely by extracellular morphogen concentration, and which are not? The goals of this R21 proposal are to fabricate optimized microfluidic devices for culturing CPCs (Aim 1) and to develop real time assays with single cell resolution in order to efficiently study CPC responses as functions of morphogen concentration, gradient profile, cell density, and time (Aim 2). To achieve these goals, a multidisciplinary team with primary expertise in bioengineering (Noo Li Jeon, the developer of the original gradient-generating device), morphogen gradients (Arthur Lander) and cortical development (Edwin Monuki) has been assembled. If successful, this proposal should not only advance our understanding of cortical development and malformations, and of morphogen gradients in general, but should also provide a versatile microfluidic tool with a wide range of basic and clinical applications.