Molecular mechanisms and epigenetic signatures that specify thymus fate

描述（由申请人提供）：尽管胸腺在T细胞发育中起着关键作用，但在胎儿发育过程中指定初始胸腺命运所需的机制尚不清楚。了解这些机制将有助于在培养中产生功能胸腺器官的策略的发展，并使老化的、受影响的胸腺恢复活力。小鼠胚胎发育期间，胸腺命运在E9.5和E11之间的第3咽囊（pp）腹侧内胚层中被指定；这个眼袋的背域产生甲状旁腺。由于第三pp必须被分成两种不同的细胞命运，因此必须有适当的机制来确保这两个器官都得到适当的指定。我们的实验室和其他人之前的研究表明，BMP2/4和FGF8/10信号与胸腺的命运有关，而SHH信号是甲状旁腺命运所必需的。这两种信号通路似乎是相反的；然而，它们相互作用和调节器官特异性转录网络的机制仍有待建立。最近来自Richie和Manley实验室的合作实验表明，转录因子TBX1是决定胸腺和甲状旁腺细胞命运的关键调节因子。我们发现Tbx1在第三pp内胚层的甲状旁腺结构域表达，而不在胸腺腹侧宿命结构域表达，并且Tbx1异位足以抑制Foxn1的表达。基于我们的初步数据，我们建立了一个可测试的胸腺命运规范模型，该模型表明FGF8和BMP4是阳性调节因子，而SHH和Tbx1则是胸腺发育的拮抗因子。本项目结合胚胎学、遗传学、分子生物学、生物信息学和表观遗传学，采用一种新的方法来破译指定胸腺命运的内胚层祖细胞的分子网络。结果将为编程早期祖细胞以产生支持胸腺发育的功能性胸腺上皮细胞网络提供理论基础。我们将在以下具体目标中测试我们的模型。目的1。验证多种机制抑制Tbx1在第3pp内胚层腹侧结构域的表达，并且这种抑制是胸腺命运建立所必需的假设。目标2。测试TBX1抑制胸腺分化的能力是否受到暂时限制，并确定异位TBX1和/或SHH信号阻断胸腺命运的机制。目标3。验证器官特异性表观遗传特征的逐步建立决定胸腺命运的假设，并取决于BMP和FGF信号通路之间的合作

DESCRIPTION (provided by applicant): Despite the critical role of the thymus in T cell development, the mechanisms required to specify initial thymus fate during fetal development are not known. Understanding these mechanisms will facilitate the development of strategies to generate functional thymus organs in culture, and to rejuvenate the aged, involuted thymus. Thymus fate is specified in the ventral 3rd pharyngeal pouch (pp) endoderm between E9.5 and E11 during mouse fetal development; the dorsal domain of this pouch gives rise to the parathyroid glands. As the 3rd pp must be patterned into two distinct cell fates, mechanisms must be in place to ensure that both organs are properly specified. Previous work from our labs and others have implicated BMP2/4 and FGF8/10 signals in establishing thymus fate, while SHH signaling is required for parathyroid fate. These two signaling pathways appear to act in opposition; however, the mechanisms by which they interact with each other and regulate organ-specific transcriptional networks remain to be established. Recent collaborative experiments from the Richie and Manley labs suggest that the transcription factor TBX1 is a key regulator of the decision between thymus and parathyroid cell fate. We have shown that Tbx1 is expressed in the parathyroid domain of the 3rd pp endoderm, but not in the ventral thymus fated domain, and that ectopic TBX1 is sufficient to suppress Foxn1 expression. Based on our preliminary data we generated a testable model of thymus fate specification in which suggest that FGF8 and BMP4 are positive regulators, whereas SHH and Tbx1 antagonize thymus development. This project uses a novel approach to decipher the molecular network that specifies endodermal progenitors to a thymus fate, combining embryology, genetics, molecular biology, bioinformatics, and epigenetics. The results will provide a rationale basis for programming early progenitors to generate a functional thymic epithelial cell network that supports thymopoiesis. We will test our model in the following Specific Aims. Aim 1. Test the hypothesis that multiple mechanisms suppress Tbx1 expression in the ventral domain of 3rd pp endoderm, and that this suppression is required to establish thymus fate. Aim 2. Test whether the ability of TBX1 to inhibit thymus differentiation is temporally restricted, and identify the mechanism by which ectopic TBX1 and/or SHH signaling blocks thymus fate. Aim 3. Test the hypothesis that progressive establishment of organ-specific epigenetic signatures determines thymus fate, and depends on cooperation between the BMP and FGF signaling pathways