Regulation of thymic epithelial cell differentiation

描述(申请人提供)：我们正在研究控制胸腺上皮细胞(TECs)分化的分子机制，TECs是促进胸腺内T细胞发育所必需的。最初的TEC分化依赖于Foxn1的功能，它编码一个叉头转录因子基因，为研究Foxn1的功能提供了独特的资源。FOXN1纯合子缺失小鼠(裸鼠)在功能上是无菌的，在非常早期就阻止了TEC的分化。利用基因打靶技术，我们已经为Foxn1产生了一个新的等位基因，表示为Foxn1/Delta。该等位基因编码一种截短的蛋白质，其中DNA结合域和激活域是完整的，但N-末端结构域的一部分被删除。成年Foxn1Delta/Delta小鼠的胸腺较小，胸腺细胞发育存在特定缺陷，包括胸腺细胞数量下降40倍，胸腺细胞发育存在特定缺陷。带有一个Delta和一个零等位基因的Foxn1Delta/nu小鼠有更严重的缺陷。这些缺陷与Kit和IL-7信号突变的小鼠相似，Kit-I的RT-PCR显示Foxn1 Delta/Delta胸腺几乎完全缺乏Kit-I消息。令人惊讶的是，Foxn1Delta/Delta和Foxn1Delta/nu表型是胸腺特有的，皮肤和毛发发育明显正常，确定了Foxn1 N末端的胸腺特有活动，这在以前是未知的功能。因此，Foxn1Delta/Delta、Foxn1Delta/nu和Foxn1nu/nu构成了Foxn1基因的等位基因序列，为研究Foxn1的功能提供了独特的资源。我们针对Foxn1基因提出了4个特异的等位基因序列，为研究Foxn1的功能提供了独特的资源。我们提出了4个特定的目的来检验这一假设，即Foxn1Delta突变编码了一个亚型等位基因，允许正常启动TEC分化和胸腺器官发生，但扰乱了随后TEC分化所需的胸腺特异功能域。测试狐狸1号的假设？突变编码一个亚型等位基因，允许TEC分化和胸腺器官发生的正常启动，但扰乱随后TEC分化所需的胸腺特异功能域。我们的目标是：1)确定在Foxn1Delta/Delta和Foxn1Delta/nu胚胎中胸腺发育受到影响的最早阶段；2)确定Foxn1Delta/Delta和Foxn1Delta/nu小鼠在胎儿和出生后发育过程中特定的胸腺细胞和TEC缺陷；3)测试Kit-L和IL7信号通路是否作用于Foxn1下游；以及4)确定Foxn1 N端域是否作为胸腺特异的蛋白-蛋白相互作用结构域。通过这些方法，我们将首次能够研究Foxn1在胸腺器官发生初期以外的TEC发育和功能中的作用。此外，我们对N-末端结构域新功能的鉴定可能对其他叉头家族转录因子的功能结构域也有意义。

DESCRIPTION (provided by the applicant): We are studying the molecular mechanisms that control the differentiation of thymic epithelial cells (TECs), which are required to promote T-cell development within the thymus. Initial TEC differentiation depends on the function of Foxn1, which encodes a forkhead transcription factor gene, providing a unique resource to investigate Foxn1 function. Foxn1 homozygous null mice (nude mice) are functionally athymic, arresting TEC differentiation at a very early stage. Using gene targeting we have generated a new allele for Foxn1, denoted Foxn1/delta. This allele encodes a truncated protein in which the DNA binding and activation domains are intact, but a portion of the N-terminal domain is deleted. Adult Foxn1delta/delta mice have small thymi with specific defects in thymocyte development, including a 40-fold drop in thymocyte number and specific defects in thymocyte development. Foxn1delta/nu mice, with one delta and one null allele, have even more severe defects. These defects are similar to those seen in mice with mutations in Kit and IL-7 signaling, and RT-PCR for kit ligand (kit-I) showed a near total absence of kit-I message in Foxn1delta/delta thymus. Surprisingly, the Foxn1delta/delta and Foxn1delta/nu phenotypes are thymus-specific, with apparently normal skin and hair development, identifying a thymus-specific activity for the Foxn1 N-terminus, which previously had no known function. Foxn1delta/delta, Foxn1delta/nu, and Foxn1nu/nu therefore constitute an allelic series for the Foxn1 gene, providing a unique resource to investigate Foxn1 function. We propose 4 specific aims to allelic series for the Foxn1 gene, providing a unique resource to investigate Foxn1 function. We propose 4 specific aims to test the hypothesis that the Foxn1delta mutation encodes a hypomorphic allele that allows normal initiation of TEC differentiation and thymus organogenesis, but disrupts a thymus-specific functional domain required for subsequent TEC differentiation. test the hypothesis that the Foxn1? mutation encodes a hypomorphic allele that allows normal initiation of TEC differentiation and thymus organogenesis, but disrupts a thymus-specific functional domain required for subsequent TEC differentiation. Our aims are :1) to determine the earliest stage at which thymus development is affected in Foxn1delta/delta and Foxn1delta/nu embryos; 2) to identify the specific thymocyte and TEC defects during fetal and post-natal development in Foxn1delta/delta and Foxn1delta/nu mice; 3) to test whether the Kit-l andIL7 signaling pathways act downstream of Foxn1; and 4) to determine whether the Foxn1 N-terminal domain acts as a thymus-specific protein-protein interaction domain. Through these approaches, we will for the first time be able to study the role of Foxn1 in TEC development and function beyond the initial stages of thymus organogenesis. Further, our identification of a new function for the N-terminal domain may have implications for functional domains in other forkhead family transcription factors.