RNA-seq Analysis of Pancreatic Epithelium and Mesenchyme from Wild Type and Pbx-mutant Embryos

The surrounding microenvironment plays a crucial role in pancreas formation and differentiation. Nevertheless, its cellular composition has not been analyzed in depth and whether different cell populations exist within the mesenchyme of the developing pancreas is an open question. Similarly, the molecular mechanisms by which the mesenchyme coordinates and integrates various signaling pathways to stimulate epithelial pancreatic progenitor proliferation and differentiation remain elusive. Here, we investigate the cell composition and molecular network in the pancreatic mesenchyme, which promote pancreas differentiation. Whole transcriptome analysis and functional experiments showed that PBX in the mesenchyme promotes endocrine differentiation non-cell autonomously, acting at two different levels in the epithelial-mesenchymal crosstalk: 1) the maintenance of BM integrity and ECM and 2) regulation of soluble molecules, including guidance cues. In conclusion, this study has identified distinct pancreatic mesenchymal niches and dissected their different roles during pancreatic development. This knowledge will be useful to improve current differentiation strategies based on ECM and soluble factors to generate beta-cell equivalents from pluripotent cells for future cell replacement therapies of diabetes. We performed sequencing-based expression profiling (RNA-Seq) of pancreatic epithelial and mesenchymal cells isolated from wild type (WT) and Pbx mutant mice at embryonic day (E) 12.5 and E14.5周围微环境在胰腺形成与分化过程中发挥关键作用。然而，发育中胰腺的间充质（mesenchyme）细胞组成尚未得到深入解析，而该间充质内是否存在异质性细胞群仍是一个悬而未决的科学问题。类似地，间充质通过协调整合多条信号通路以刺激上皮源性胰腺祖细胞增殖与分化的分子机制，目前仍未阐明。本研究聚焦于调控胰腺分化的胰腺间充质（mesenchyme）细胞组成与分子网络。全转录组分析与功能实验结果表明，间充质中的PBX以非细胞自主性方式促进内分泌分化，其在上皮-间充质串扰（epithelial-mesenchymal crosstalk）中通过两个层面发挥调控作用：1）维持基底膜（basement membrane, BM）完整性与细胞外基质（extracellular matrix, ECM）；2）调控包括导向信号分子在内的可溶性因子。综上，本研究鉴定出多种不同的胰腺间充质微环境龛（niche），并解析了其在胰腺发育进程中的差异化功能。该研究成果可用于优化当前基于细胞外基质与可溶性因子的分化策略，从而从多能干细胞（pluripotent cells）中诱导生成β细胞（beta-cell）类似物，为未来糖尿病的细胞替代疗法提供理论支撑。本研究对胚胎发育第12.5天（E12.5）与第14.5天（E14.5）的野生型（wild type, WT）及Pbx突变小鼠的胰腺上皮细胞与间充质细胞开展了基于测序的表达谱分析（RNA-Seq）。