Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs

The pancreas of vertebrates is separately derived from both the dorsal and ventral endodermal domains. However, the difference between these two programs has been unclear. Here, using a pancreatic determination gene, Pdx1, driven GFP transgenic mouse strain, we identified Pdx1-GFP highly expressing cells (Pdx1high) and Pdx1-GFP lowly expressing cells (Pdx1low) in both embryonic dorsal Pdx1-expressing region (DPR) and ventral Pdx1-expressing region (VPR). We analyzed the transcriptomes of single Pdx1low and Pdx1high cells from the DPR and VPR. In the VPR, Pdx1low cells have an intermediate progenitor identity and can generate hepatoblasts, extrahepatobiliary cells, and Pdx1high pancreatic progenitor cells. In the DPR, Pdx1high cells are directly specified as pancreatic progenitors, whereas Pdx1low cells are precocious endocrine cells. Therefore, our study defines distinct road maps for dorsal and ventral pancreatic progenitor specification. The findings provide guidance for optimization of current ß-cell induction protocols by following the in vivo dorsal pancreatic specification program. Overall design: The overall goal of this study was understanding the programs of ventral and dorsal pancreatic progenitor development. Specifically, to get an overall gene expression profiles during ventral and dorsal pancreas development, we performed bulk cell RNA-seq in E10.5 ventral and dorsal pancreatic progenitor cells purified from Pdx1-GFP transgene embryos; E14.5 ventral and dorsal endocrine progenitor cells from Ngn3-GFP transgene embryos; and E17.5 ventral and dorsal beta cells from Insulin-RFP transgene embryos. To determine the progress and cell type components during the specification of pancreatic progenitor in ventral and dorsal domains, we performed single-cell transcriptomic analysis in sorted Pdx1-GFP+ cells from ventral and dorsal pancreatic buds.

脊椎动物的胰腺分别源自背侧与腹侧内胚层区域，但这两套发育程序间的差异此前尚未明确。本研究利用受胰腺决定基因Pdx1驱动的绿色荧光蛋白（Green Fluorescent Protein, GFP）转基因小鼠品系，在胚胎期背侧Pdx1表达区域（Dorsal Pdx1-expressing Region, DPR）与腹侧Pdx1表达区域（Ventral Pdx1-expressing Region, VPR）中分别鉴定出Pdx1-GFP高表达细胞（Pdx1high）与低表达细胞（Pdx1low）。我们对来自DPR与VPR的Pdx1low及Pdx1high单细胞进行了转录组分析。在VPR中，Pdx1low细胞具有中间祖细胞身份，可分化为肝母细胞、肝胆管外细胞以及Pdx1high胰腺祖细胞；在DPR中，Pdx1high细胞直接特化为胰腺祖细胞，而Pdx1low细胞则为早熟内分泌细胞。因此，本研究明确了背侧与腹侧胰腺祖细胞特化的差异化发育路径，相关发现可为遵循体内背侧胰腺特化程序优化现有β细胞诱导方案提供重要指导。本研究的总体目标为阐明腹侧与背侧胰腺祖细胞的发育程序。具体而言，为获取腹侧与背侧胰腺发育过程中的全景式基因表达谱，我们对三类样本开展了批量RNA测序：从Pdx1-GFP转基因胚胎中纯化的胚胎发育第10.5天（E10.5）腹侧与背侧胰腺祖细胞、从Ngn3（Neurogenin 3）-GFP转基因胚胎中分离的胚胎发育第14.5天（E14.5）腹侧与背侧内分泌祖细胞，以及从Insulin-RFP（Insulin-Red Fluorescent Protein, RFP）转基因胚胎中获取的胚胎发育第17.5天（E17.5）腹侧与背侧β细胞。为解析胰腺祖细胞在腹侧与背侧区域特化过程中的进程与细胞类型组成，我们对分选得到的腹侧与背侧胰腺芽中的Pdx1-GFP阳性细胞进行了单细胞转录组分析。