Multidimensional analysis of cell lineages

 描述：干细胞在发育过程中具有显著的自我更新和分化能力。此外，成体的自我更新和分化也在损伤反应和整体组织维持中发挥重要作用。在过去的二十年中，人们做出了相当大的努力来开发和应用基因表达谱分析的方法，这些方法现在被用来生成整个发育过程中不同组织和成人中基因表达的图谱。相比之下，在开发同时允许多基因分析、原位操作单细胞而对发育或完全分化和老化组织的干扰最小的工具方面取得的进展少得多。这些工具是必不可少的，以确定组织是如何维持在成年生活，并了解细胞如何有时可以进入一个异常的路径，导致疾病。用单细胞分辨率捕获、干扰和分析成人和衰老组织中原位基因表达的影响所面临的主要挑战源于：（1）标记和可视化非活性组织中特定细胞谱系的技术限制，（2）干扰特定细胞群体的非侵入性工具的需要，以及（3）细胞的非侵入性。 一个可靠的系统，以确定在成人和衰老组织中的基因表达的开始的可用性。为了开始解决这些问题，我们一直在使用三种技术的组合，使我们能够通过完整复杂组织中的颜色代码报告细胞谱系的时间出生顺序;使用组合光谱条形码原位杂交以单细胞分辨率可视化几个基因的表达;最后使用RNA干扰去除特定细胞谱系内的基因。在这项提案中，我们将优化和扩大这些技术在其他细胞类型（包括成年细胞和衰老细胞）中的应用范围，并使其更容易被最终用户使用。

 DESCRIPTION: Stem cells have the remarkable ability to self-renew and differentiate during development. In addition, adult self-renewal and differentiation also play an essential role in injury response and overall tissue maintenance. In the past two decades, a considerable effort was made to develop and apply methods of gene expression profiling which are now used to generate atlases of gene expression in different tissues throughout development and in adults. In contrast, considerably less progress has been achieved to develop tools that simultaneously allow multi-gene profiling, manipulation of single cells in situ with minimal perturbation in developing or fully differentiated and aging tissues. Such tools are indispensable to determine how tissues are maintained during the adult life and understand how cells can sometimes enter in an abnormal path that leads to disease. The main challenges posed to capture, interfere and analyze the effects of gene expression in situ in adult and aging tissues with single cell resolution stem from: (1) technical limitations to label and visualize specific cell lineages in inact tissues, (2) the need of non-invasive tools to interfere with specific cell populations and (3) the availability of a reliable system to determine the onset of gene expression in adult and aging tissues. To begin addressing these issues, we have been using a combination of three technologies that allow us to report the temporal birth order of cell lineages by a color-code in intact complex tissues; visualize the expression of several genes with single cell resolution using a combinatorial spectral barcoding in situ hybridization; and finally remove genes within specific cell lineages using RNA interference. In this proposal, we will optimize and expand the range of applications of these technologies to other cell types, which include adult and senescing cells, and make it more accessible to the end-user.