Spatial patterning of branching morphogenesis

描述（由申请人提供）：乳腺的树状结构是由分支形态发生产生的，这是一个分支位点起始和小管侵袭的重复过程。分支是由正负调节因子之间的相互作用控制的，其中任何一种的缺陷都可能引起从增生到恶性生长的异常。我们的长期目标是描述这些积极和消极的信号是如何在组织内的空间整合，以确定哪些细胞分支，从而定义分支模式。我们开发了一种基于光刻的三维器官型培养模型，该模型概括了乳腺上皮管的结构，实现了对组织几何形状和微环境的微米分辨率控制，并提供了生理学相关背景下的定量数据。工程管道执行一系列完整的形态发生事件，可以通过计算预测。使用这种培养模型，我们已经证明分支的位置部分取决于转化生长因子(TGF)-21的浓度分布，TGF -21是一种自分泌抑制形态因子。此外，我们发现位于分支位置的细胞在形态发生过程中调节间充质标记物的表达。基于这些初步和已发表的数据，我们提出：1-研究乳腺上皮管感知和转导的TGF21浓度谱的特征。2-确定在形态发生过程中差异表达的间充质标记，以及这些标记是否必要和/或足以确定分支的位置。我们将进一步测试间充质基因表达模式是否受TGF21抑制谱的调节。3-通过确定细胞外基质是否改变分支模式、TGF21抑制浓度谱或间质标记物的新表达，开始剖析微环境的物理特性如何调节分支。这些研究将提供对控制分支位置的局部线索和基因表达变化的深入了解。

DESCRIPTION (provided by applicant): The tree-like architecture of the mammary gland is generated by branching morphogenesis, a reiterative process of branch site initiation and tubule invasion from a pre-existing epithelial structure. Branching is controlled by the interplay between positive and negative regulators, defects in either of which can give rise to aberrancies ranging from hyperplasia to malignant growth. Our long term goal is to delineate how these positive and negative signals are integrated spatially within the tissue to determine which cells branch, and thereby define the branching pattern. We have developed a lithography-based three-dimensional organotypic culture model that recapitulates the architecture of mammary epithelial ducts, enables micrometer-resolution control of tissue geometry and microenvironment, and provides quantitative data in a physiologically relevant context. The engineered ducts execute a complete series of morphogenetic events that can be predicted computationally. Using this culture model, we have shown that the position of branching is determined in part by the concentration profile of transforming growth factor (TGF)-21, an autocrine inhibitory morphogen. Furthermore, we have found that cells located in positions that branch up- regulate the expression of mesenchymal markers during morphogenesis. Based on these preliminary and published data, we propose: 1- To investigate the features of the TGF21 concentration profile perceived and transduced by mammary epithelial ducts. 2- To determine the mesenchymal markers differentially expressed during morphogenesis, and whether these are necessary and/or sufficient to define position of branching. We will further test whether the pattern of mesenchymal gene expression is regulated by the TGF21 inhibitory profile. 3- To begin to dissect how branching is regulated by the physical properties of the microenvironment, by determining whether the extracellular matrix alters branching pattern, TGF21 inhibitory concentration profile, or neo-expression of mesenchymal markers. These studies will provide insight into the local cues and gene expression changes that govern position of branching. PROJECT RELEVANCE: Cells integrate information from stimulatory and inhibitory signals during branching morphogenesis to develop into the tree-like structure of the mammary gland; disruption or misregulation of these signals can lead to neoplastic growths and eventual development of frank tumors. Here we present studies aimed at understanding how mammary epithelial cells perceive inhibitory signals and translate them into patterned differences in gene expression during branching morphogenesis.