THE ROLE OF NOTCH1 IN THYMIC EPITHELIAL CELLS

描述（由申请人提供）：Notch信号通路在胚胎发育中起着多种关键作用，包括细胞类型特化、分化和模式化。 在胸腺中，Notch信号调节T谱系决定和胸腺细胞分化的关键阶段。在基于胎儿胸腺器官培养（FTOC）的测定中，Notch信号传导也涉及调节胎儿TEC分化，尽管TEC中的Notch信号传导没有被直接证实。在毛囊发育中，Notch 1是Foxn 1的直接下游靶点，Foxn 1是裸鼠中突变的基因，也是调节胎儿和出生后胸腺中TEC分化多个阶段的关键转录因子。我们最近发现Notch 1在胎儿TEC的一个亚组中特异性表达，该亚组与Foxn 1和Plet-1（TEC祖细胞标记物）共标记。使用Foxn 1Cre等位基因在TEC中缺失Notch 1导致TEC祖细胞标志物Plet-1、Cld 3/4和UEA-1在胎儿阶段耗尽，并且在出生后1个月的胸腺中存在大的无上皮区。T细胞系的特化没有改变，胸腺细胞的数量和分化也令人惊讶地正常。这些表型与Kremen 1 Wnt抑制剂缺失的已发表表型惊人相似，这导致Wnt信号传导的轻度上调。Wnt信号传导也与TEC分化有关，尽管证据相互矛盾。由于Notch信号传导已在其他系统中被证明能够直接抑制经典Wnt信号传导途径，TEC特异性Notch缺失和Wnt上调之间的相似性表明Notch 1可能部分地调节TEC中的Wnt信号传导。在这个建议中，我们将建立在这些初步的观察，以调查的作用，Notch信号在TEC在胎儿和出生后的胸腺。我们将确定TEC特异性Notch 1突变体胸腺中无上皮区的个体发育，并确定这种表型如何随着衰老而进展。我们将确定这种改变的基质结构是否影响胸腺细胞分化的特定方面，包括胸腺细胞分化和出口的后期阶段。最后， 我们将测试这些表型中的一些或全部是否是由于TECs中经典Wnt信号通路的Notch 1调节。这些实验将提供必要和关键的信息，将测试我们的初步假设，Notch 1在TEC祖细胞中的作用，以调节自我更新和分化之间的平衡，部分通过调节对Wnt信号的响应。

DESCRIPTION (provided by applicant): The Notch signaling pathway plays multiple critical roles in embryonic development, including cell type specification, differentiation, and patterning. In the thymus, Notch signaling regulates key stages of T lineage determination and thymocyte differentiation. Notch signaling has also been implicated in regulating fetal TEC differentiation i a fetal thymic organ culture (FTOC)-based assay, although Notch signaling in TECs was not directly proven. In hair follicle development, Notch1 is a direct downstream target of Foxn1, which is the gene mutated in nude mice and is also a key transcription factor regulating multiple stages in TEC differentiation in both the fetal and postnatal thymus. We recently discovered that Notch1 is specifically expressed in a subset of fetal TECs that co-label with Foxn1 and Plet-1, a TEC progenitor marker. Deletion of Notch1 in TECs using a Foxn1Cre allele results in depletion of the TEC progenitor markers Plet-1, Cld3/4, and UEA-1 during fetal stages and the presence of large epithelial-free zones in the 1-month postnatal thymus. T-lineage specification is unaltered, and thymocyte numbers and differentiation are surprisingly normal. These phenotypes are strikingly similar to the published phenotype for deletion of the Kremen1 Wnt inhibitor, which results in a mild up-regulation of Wnt signaling. Wnt signaling has also been implicated in TEC differentiation, although the evidence has been conflicting. As Notch signaling has been demonstrated in other systems to be capable of directly inhibiting the canonical Wnt signaling pathway, the similarity between TEC-specific Notch deletion and Wnt up-regulation suggests that Notch1 may be acting in part to modulate Wnt signaling in TECs. In this proposal, we will build on these preliminary observations to investigate the role of Notch signaling in TECs in the fetal and postnatal thymus. We will determine the ontogeny of the epithelial-free zones in the TEC-specific Notch1 mutant thymus, and determine how this phenotype progresses with aging. We will determine whether this altered stromal structure affects specific aspects of thymocyte differentiation, including later stages of thymocyte differentiation and export. Finally, we will test whether some or all of these phenotypes are due to Notch1 modulation of the canonical Wnt signaling pathway in TECs. These experiments will provide essential and critical information that will test our preliminary hypothesis that Notch1 acts in TEC progenitors to regulate the balance between self-renewal and differentiation, in part by modulating the response to Wnt signaling.