THE ROLE OF NOTCH1 IN THYMIC EPITHELIAL CELLS

描述(申请人提供)：Notch信号通路在胚胎发育中起着多个关键作用，包括细胞类型指定、分化和模式形成。 在胸腺中，Notch信号调节T细胞谱系决定和胸腺细胞分化的关键阶段。通过基于胎儿胸腺器官培养(FTOC)的检测，Notch信号也与调节胎儿TEC的分化有关，尽管Notch信号在TEC中的作用尚未得到直接证实。在毛囊发育中，Notch1是Foxn1的直接下游靶基因，Foxn1是在裸鼠体内突变的基因，也是调节胎儿和出生后胸腺TEC分化的多个阶段的关键转录因子。我们最近发现Notch1在与Foxn1和Plet-1共同标记的胎儿TEC子集中特异表达，Plet-1是TEC的前体标记。使用Foxn1Cre等位基因缺失TEC中的Notch1会导致TEC前体标记Plet-1、Cld3/4和UEA-1在胎儿期耗尽，并在出生后1个月的胸腺中出现大的无上皮区。T细胞的特性没有改变，胸腺细胞的数量和分化令人惊讶地正常。这些表型与已发表的Kremen1Wnt抑制物缺失的表型惊人地相似，这导致Wnt信号的轻微上调。WNT信号也与TEC的分化有关，尽管证据相互矛盾。由于Notch信号在其他系统中已被证明能够直接抑制典型的Wnt信号通路，TEC特异性Notch缺失和Wnt上调之间的相似性表明Notch1可能在一定程度上调节TEC中的Wnt信号。在这个方案中，我们将在这些初步观察的基础上，研究Notch信号在TEC中在胎儿和出生后胸腺中的作用。我们将确定TEC特异性Notch1突变胸腺中无上皮区的个体发育，并确定这种表型如何随着年龄的增长而进展。我们将确定这种改变的间质结构是否会影响胸腺细胞分化的特定方面，包括胸腺细胞分化和输出的后期阶段。最后， 我们将测试这些表型是否部分或全部是由于TEC中规范的Wnt信号通路的Notch1调节所致。这些实验将提供必要和关键的信息，验证我们的初步假设，即Notch1在TEC前体细胞中发挥作用，调节自我更新和分化之间的平衡，部分是通过调节对Wnt信号的反应。

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.