Hedgehog signaling in early development of the gastrointestinal tract

描述(由申请人提供)：尽管许多胃肠道(GI)疾病与胚胎发育过程中建立的细胞关系密切相关，但目前对GI发育机制的了解还不完全。胃肠道器官发生需要来自肠道特异性内胚层的细胞与邻近的内脏中胚层细胞之间的信号交换；研究这种交换的性质和机制是很重要的。在调节几乎所有发育信号的少数几个途径中，Hedgehog(HH)蛋白在肠道中的作用尤其突出。两种HH配体Sonic(Shh)和Indian(IHH)在胎儿肠道内胚层早期高度表达，从那里它们向邻近的间充质发出信号。在小鼠中，任何一个基因的中断都会导致特定的但令人惊讶的有限的胃肠道发育异常；因为两个HH因子之间的冗余可能会限制缺陷的范围，我们研究了在肠道内胚层中缺乏这两个HH因子的小鼠。Shh/IHH-Double突变小鼠由于胃肠道间充质细胞的显著磨损而出现严重的消化道发育异常。在具体目标1中，我们建议定义HH信号在早期GI发育中的作用和机制。首先，我们将剖析HH信号在胃和肠间充质细胞的增殖、存活和分化中的相对重要性，特别是在平滑肌细胞系中。其次，我们在缺乏HH信号的小鼠胚胎中的观察结果与之前文献中提出的观点一致：HH信号诱导分泌因子骨形态发生蛋白(BMP)-4和转录因子FoxF1和FoxF2的表达。我们建议进行实验，以确定这些假定的HH信号下游靶点在多大程度上对GI器官发生的全球影响负责。Shh/IHH-双零胚胎中的胃异常与我们在转基因小鼠中发现的相同，在转基因小鼠中，Notch信号通路在胎儿胃间充质中异常激活。其他初步数据也支持根据这些发现得出的新假设：胎儿胃中的HH信号在一定程度上限制了间充质Notch的激活，从而阻止了Notch诱导的细胞死亡。特定目的2概述了一系列体内和体外研究，以严格检验这一假说，特别是利用几个小鼠遗传模型来确定HH和Notch信号在GI器官发生中相互作用的性质和程度。尽管现有的实验模型允许对胃发育进行深入的实验，但类似的原理可能适用于整个消化道。我们提出的研究解决了重要的悬而未决的问题，探索了发育信号和调控机制的分子细节，并将有助于改善对常见胃肠道疾病的理解和治疗。与公共卫生相关。在过去十年的发育生物学研究中，一个特别令人惊讶的是，不同组织中复杂的发育过程仅依赖于几个细胞信号通路。Hedgehog途径在消化道的发育和动态平衡的许多步骤中起着至关重要的作用，也与内皮源性组织的癌症有关。Hedgehog信号在肠道发育中的作用和机制还知之甚少，尽管它们在人类胃肠道疾病中有有用的含义和应用。这项建议试图应用遗传和细胞培养工具来阐明刺猬信号在小鼠胃和肠道发生中的确切作用和机制。

DESCRIPTION (provided by applicant): Although many gastrointestinal (GI) disorders are intimately related to cellular relationships established during embryogenesis, current understanding of GI developmental mechanisms is incomplete. GI tract organogenesis requires exchange of signals between cells derived from gut-specific endoderm and the adjacent splanchnic mesoderm; investigating the nature and mechanisms of this exchange is important. Among the handful of pathways that mediate virtually all developmental signaling, the role of Hedgehog (Hh) proteins is especially prominent in the gut. Two Hh ligands, Sonic (Shh) and Indian (Ihh) are highly expressed early in fetal gut endoderm, from where they signal to the adjacent mesenchyme. Disruption of either gene in mice results in specific but surprisingly limited GI developmental anomalies; because redundancy between the two Hh factors may restrict the scope of defects, we studied mice lacking both Hh factors in gut endoderm. Shh/Ihh-double mutant mice have profoundly abnormal digestive tract development as a result of marked attrition of GI mesenchymal cells. In Specific Aim 1 we propose to define the roles and mechanisms for Hh signaling in early GI development. First, we will dissect the relative importance of Hh signaling in proliferation, survival and differentiation of stomach and intestinal mesenchymal cells, especially with respect to smooth muscle lineages. Second, our observations in mouse embryos that lack all Hh signaling agree with ideas previously proposed in the literature: that Hh signals induce expression of the secreted factor Bone morphogenetic protein (BMP)-4 and transcription factors FoxF1 and FoxF2. We propose experiments to determine to what extent these putative downstream targets of Hh signaling are responsible for the global effects on GI organogenesis. The stomach anomalies in Shh/Ihh-double null embryos are identical to those we identified in transgenic mice where the Notch signaling pathway is aberrantly activated in fetal stomach mesenchyme. Other preliminary data also support the novel hypothesis to follow from these findings: that Hh signaling in fetal stomach functions in part to restrict mesenchymal Notch activation and hence prevents Notch- induced cell death. Specific Aim 2 outlines a series of in vivo and ex vivo studies to test this hypothesis critically, making particular use of several mouse genetic models to define the nature and extent of interactions between Hh and Notch signaling in GI organogenesis. Although the available experimental models permit incisive experiments in stomach development, similar principles are likely to apply throughout the digestive tract. The studies we propose address important outstanding questions, explore developmental signaling and regulatory mechanisms in molecular detail, and will contribute toward improved understanding and treatment of common GI disorders. PUBLIC HEALTH RELEVANCE. One particular surprise from the last decade of developmental biology research is that complex developmental processes in diverse tissues depend on just a few cell signaling pathways. The hedgehog pathway plays a vital role at many steps in development and homeostasis of the digestive tract and is also implicated in cancers of endodermally derived tissues. The functions and mechanisms of hedgehog signaling in gut development are poorly understood, in spite of their useful implications and applications for human gastrointestinal disorders. This proposal seeks to apply genetic and cell culture tools to elucidate the precise role and mechanisms of hedgehog signaling in genesis of the mouse stomach and intestine.