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

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. RNA-Seq of E17.5 wildtype and Sufu;Spop double knockout intestine in biological duplicates间充质-上皮相互作用（mesenchymal-epithelial interactions）在器官发育、干细胞生物学及疾病发生中发挥关键作用。在肠道发育过程中，假复层上皮会经历被称为绒毛发生（villification）的显著形态发生过程，形成指状突起；此过程中，间充质细胞聚集与肌层发挥核心调控功能。成年个体的肠道间充质被认为是关键的肠道干细胞巢（intestinal stem cell niche），可提供Wnt配体（Wnt ligands）等必需的巢信号；而转化生长因子β（TGF-β）信号介导的肠道基质程序对癌症进展至关重要。然而，目前尚不清楚这些信号的具体产生机制。 在肠道中，Hedgehog（Hh）信号通路严格以旁分泌方式发挥作用：Hh配体（Hh ligands）在上皮细胞中表达，且仅在间充质细胞中激活信号通路。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，还参与肠道肿瘤发生过程。为探究Hh信号介导的调控机制，本研究对肠道间充质中关键Hh负调控因子Sufu和/或Spop敲除的小鼠进行分析，证实了二者在Hh信号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及肌层功能缺陷，但绒毛发生过程仍可通过正常的间充质细胞聚集完成，这提示Hh信号通路仅发挥许可性作用。通过降低Gli2的表达可部分挽救这些间充质缺陷，证实了其转录调控的重要性。令人意外的是，与此前已知的Hh信号通路对上皮增殖的抑制作用相反，肠道间充质中异常的Hh激活会促进上皮细胞增殖。相关实验数据验证表明，降低Sufu的表达足以促进肠道肿瘤发生，而Gli2杂合缺失则可抑制该进程。 为明确GLI2介导的下游调控机制，本研究对其结合位点进行了定位，并开展了全基因组基因表达分析，最终获得了迄今报道的最可靠的Hh直接靶标组（targetome）数据集之一。本研究揭示了GLI2通过转录调控Wnt与TGF-β通路，参与干细胞增殖及肌细胞分化的过程，为理解肠道发育与肿瘤发生中的肠道干细胞巢调控机制提供了全新的机制性见解。本研究附带了E17.5（胚胎第17.5天）野生型与Sufu;Spop双敲除小鼠肠道组织的生物学重复RNA测序（RNA-Seq）数据集。