Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling [RNA-seq]

In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. To characterise the basis for region-specific mesenchymal-epithelial crosstalks during late small intestinal morphogenesis, we combined transcriptional profiling of epithelial and mesenchymal cell populations isolated from different intestinal regions with ChIP-seq, 3D imaging and functional ex vivo and organoid studies. We find that a mesenchymal PDGFRA-high population located in close proximity to the developing epithelium is enriched for genes involved in signalling and support the epithelium in a region-specific manner via the establishment of a proximal to distal gradient of epithelial Wnt activity. Furthermore, we provide evidence that Wnt signalling in the distal SI directly regulates epithelial expression of Sonic Hedgehog (SHH), which in turn acts on mesenchymal cells driving villi formation. Our results therefore uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments central for anterior-posterior regionalisation and correct organ formation. RNA-seq of epithelial - EpCAM+ - and mesenchymal - PDGFRA-HIGH (PDGFRA-high/CD29-med), PDGFRA-MED (PDGFRA-med/CD29-med) and CD29-HIGH (CD29-high/PDGFRA-med) - cell populations isolated from proximal, mid and distal parts of the mouse intestine at embryonic day 16.5 (E16.5). RNA-seq of epithelial cells isolated from different regions of the mouse E16.5 intestine upon treatment with PORCN inhibitor from E12.5 to E16.5. RNA-seq data of intestinal epithelial cells isolated from proximal and distal parts of the mouse intestine at embryonic day 13.5 (E13.5).在小肠（Small Intestine, SI）中，区域化特性可实现食物的循序加工与营养吸收。为阐明小肠晚期形态发生过程中区域特异性间充质-上皮细胞串扰（mesenchymal-epithelial crosstalk）的分子基础，我们将从不同肠段分离的上皮细胞与间充质细胞群体的转录组谱分析（transcriptional profiling），与染色质免疫共沉淀测序（ChIP-seq）、三维成像（3D imaging）、体外功能实验（ex vivo）及类器官（organoid）研究相结合。 我们发现，紧邻发育中上皮细胞的高表达PDGFRA的间充质群体，其信号通路相关基因显著富集，并通过建立上皮Wnt信号活性的近-远端梯度，以区域特异性方式支持上皮细胞发育。此外，本研究证实，远端小肠中的Wnt信号可直接调控音猬因子（Sonic Hedgehog, SHH）的上皮表达，而后者又可作用于间充质细胞，促进绒毛形成。因此，本研究揭示了跨不同细胞区室的Wnt与Hedgehog信号通路之间的机制性联系，该联系对于小肠的前后轴区域化与器官正常形成至关重要。 转录组测序（RNA-seq）样本：从胚胎第16.5天（embryonic day 16.5, E16.5）小鼠小肠的近端、中段及远端分离的EpCAM阳性（EpCAM+）上皮细胞，以及三类间充质细胞群体——高表达PDGFRA（PDGFRA-high/CD29-med）、中表达PDGFRA（PDGFRA-med/CD29-med）与高表达CD29（CD29-high/PDGFRA-med）的细胞群。 转录组测序数据：从E12.5至E16.5期间经PORCN抑制剂处理的小鼠E16.5小肠不同区域分离的上皮细胞。 转录组测序数据：从胚胎第13.5天（embryonic day 13.5, E13.5）小鼠小肠近端与远端分离的肠上皮细胞。