Tissue and lymphoid defects induced by Birc5 deletion in thymic epithelial cells.

 描述（由申请人提供）：像所有的血细胞一样，T淋巴细胞在整个生命过程中都会丢失，必须不断补充。这个过程涉及诱导多能祖细胞/干细胞离开血流并进入胸腺，随后是一系列独特的诱导和支持事件，导致多个功能性T谱系。由于胸腺的淋巴成分是短暂的，胸腺的持久特性显然是由其稳定的基质成分建立的。胸腺中的基质细胞主要是上皮细胞，其他非造血细胞（主要是成纤维细胞）和一些非淋巴造血细胞（树突状细胞，巨噬细胞）的贡献较小。胸腺基质细胞的表征一直存在问题，因为物理分离方法（机械破碎、酶消化、细胞分选等）高度偏向于某些细胞类型，并且进一步导致感兴趣的细胞的深刻变化。因此，胸腺基质生物学是非常了解。我们设计了一种计算方法，使我们能够使用从完整组织区域（通过显微切割分离）提取的RNA或该组织的相应淋巴成分（通过细胞分选以原始状态分离）原位去卷积基质转录组。使用信息学方法，我们花了几年时间描述基质生物学，并试图识别基质细胞中的关键调控基因和回路。在这个建议中，我们表明，生存素，Birc 5基因的产物和IAP家族的caspase抑制蛋白的成员，是胸腺上皮基质细胞（TEC）所必需的。TEC中生存素的条件性缺失（使用Foxn 1 [Cre]和floxed Birc 5）导致非常小的胸腺，缺乏皮质/髓质区室化，并呈现大的囊性结构。在这些条件下，类淋巴细胞发育严重受损，DN：DP比率明显增加，但所有阶段的细胞显著减少，导致外周T淋巴细胞减少症。使用角蛋白-14启动子驱动的生存素转基因对条件性生存素缺陷进行遗传互补，使胸腺大小、组织和功能恢复正常，为生存素在TEC中的基本功能提供了经典的遗传证据。在此应用中，我们建议进一步表征TEC中生存素缺失诱导的缺陷，并分析那些保留的TEC和发展的淋巴细胞之间的上下文关系。我们还建议确定生存素是否仅对分化的TEC的稳态维持很重要，或者它是否也/仅在出生后TEC干细胞中或在胎儿器官形成期间起作用。最后，我们将以存活素为工具，探讨胸腺在胚胎中是如何组织和分化的，以及在成人中是如何维持的。

 DESCRIPTION (provided by applicant): Like all blood cells, T lymphocytes are lost throughout life, and must be continuously replaced. This process involves inducing multipotent progenitor/stem cells to leave the bloodstream and enter the thymus, followed by a unique series of inductive and supportive events that result in multiple functional T lineages. Since the lymphoid components of the thymus are transient, the durable identity of the thymus is obviously established by its stable stromal components. Stromal cells in the thymus are primarily of epithelial lineages, with a lesser contribution by other non-hematopoietic cells (mostly fibroblasts) and some non-lymphoid hematopoietic cells (dendritic cells, macrophages). Characterization of thymic stromal cells has been problematic, since physical isolation methods (mechanical disruption, enzymatic digestion, cell sorting, etc.) are highly biased towards certain cell types, and further, result in profound changes in the cells of interest. Consequently, thymic stromal biology is very poorly understood. We have devised a computational approach that allows us to deconvolve the stromal transcriptome in situ, using RNA extracted from intact tissue regions (isolated by microdissection), or the corresponding lymphoid components of that tissue (isolated in a pristine state by cell sorting). Using informatic approaches, we have spent the last several years characterizing stromal biology, and attempting to identify critical regulatoy genes and circuits in stromal cells. In this proposal, we show that survivin, the product of the Birc5 gene and a member of the IAP family of caspase inhibitory proteins, is essential for thymic epithelial stromal cells (TEC). Conditional deletion of survivin in TEC (using Foxn1[Cre] and floxed Birc5) results in a very small thymus that lacks cortical/medullary compartmentalization and presents with large cystic structures. Lymphoid development is profoundly impaired under these conditions, with an apparent increase in the DN:DP ratio but dramatically fewer cells at all stages, leading to peripheral T lymphopenia. Genetic complementation of conditional survivin deficiency, using a keratin-14 promoter driven survivin transgene, restored thymic size, organization, and function to normal, providing classical genetic evidence for the essential function of survivin in TEC. In this application, we propose to further characterize the defects induced by survivin deletion in TEC, and to analyze the contextual relationship between those TEC that do remain, and the lymphoid cells that do develop. We also propose to determine whether survivin is important solely for the homeostatic maintenance of differentiated TEC, or whether it also/only plays a role in post-natal TEC stem cells, or during fetal organogenesis. Finally, we will use survivin as a tool to probe how the thymus is organized and differentiated in the embryo, and maintained in the adult.