GLI2-mediated mesenchymal Hh signaling induces stem cell niche signals in intestinal development and tumorigenesis [ChIPSeq]

Mesenchymal-epithelial interactions play a critical role in organ development, stem cells and disease. During intestinal development, pseudostratified epithelia undergo dramatic morphogenesis called villification, to form finger-like projections, in which mesenchymal cell clustering and muscle layers play a key role. In the adult, the gut mesenchyme is proposed as a key intestinal stem cell niche providing essential niche signals such as Wnt ligands, while the TGF beta signaling mediated gut stromal program is critical for cancer progression. However, how these signals are produced is currently unknown. In the gut, Hedgehog (Hh) signaling acts strictly in a paracrine manner: Hh ligands are expressed in the epithelium and activate signaling exclusively in the mesenchyme. Notably, Hh signaling is not only essential for mesenchymal clustering and muscle differentiation, it is also involved in intestinal tumorigenesis. To investigate Hh mediated mechanisms, we analyzed mice deleted for key Hh negative regulators, Sufu and/or Spop in the gut mesenchyme, and demonstrated their dosage dependent role in the negative regulation of Hh signaling. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction, demonstrating the significance of its transcriptional regulation. Surprisingly, in contrast to its known inhibitory role in epithelial proliferation, abnormal Hh activation in the gut mesenchyme leads to increased epithelial proliferation. Corroborating this data, Sufu reduction is sufficient to promote intestinal tumorigenesis, while Gli2 heterozygosity suppresses it. To define GLI2-mediated downstream mechanisms, we mapped its binding sites and analyzed gene expression genome-wide, identifying one of the most robust Hh direct targetome data sets ever reported. This work reveals the GLI2 transcriptional regulation of Wnt and TGF beta pathways in stem cell proliferation and muscle differentiation, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis.

间充质-上皮相互作用（Mesenchymal-epithelial interactions）在器官发生、干细胞生物学及疾病进程中发挥关键调控作用。在肠道发育阶段，假复层上皮会经历被称为绒毛形成（villification）的剧烈形态发生过程，最终形成指状肠绒毛；其间充质细胞聚集与肌层结构在此过程中扮演核心角色。在成体肠道中，肠道间充质被认为是肠道干细胞巢（intestinal stem cell niche）的关键组成部分，可提供Wnt配体等必需的巢信号；而转化生长因子β（TGF beta）信号介导的肠道间质程序则对癌症进展至关重要。然而目前学界尚未明确这些信号的具体产生机制。在肠道组织中，刺猬（Hedgehog, Hh）信号通路严格以旁分泌方式发挥功能：Hh配体在上皮细胞中表达，并仅在间充质细胞中激活下游信号通路。值得注意的是，Hh信号通路不仅对间充质细胞聚集与肌细胞分化不可或缺，还直接参与肠道肿瘤发生过程。为探究Hh介导的分子调控机制，我们对肠道间充质中关键Hh负调控因子Sufu和/或Spop敲除的小鼠模型进行了系统分析，证实了二者在Hh信号通路负调控中的剂量依赖性作用。尽管这些突变小鼠表现出间充质细胞生长异常与肌层功能缺陷，但绒毛形成过程仍可通过正常的间充质聚集完成，这提示Hh信号通路仅发挥许可性调控作用。通过降低Gli2的表达水平可部分挽救上述间充质缺陷，这证明了其转录调控的核心重要性。令人意外的是，与已知的Hh信号抑制上皮增殖的功能相悖，肠道间充质中异常激活的Hh信号会导致上皮增殖水平显著升高。为验证这一实验结果，我们进一步发现：降低Sufu的表达足以促进肠道肿瘤发生，而Gli2杂合缺失则可有效抑制该过程。为明确GLI2介导的下游调控机制，我们在全基因组范围内绘制了其结合位点并分析了基因表达谱，鉴定出了迄今为止报道的最具稳定性的Hh直接靶标组（direct targetome）数据集之一。本研究揭示了GLI2通过转录调控Wnt与TGF beta通路，参与干细胞增殖与肌细胞分化的具体过程，为理解发育及肿瘤发生过程中的肠道干细胞巢提供了关键的机制层面新见解。