TrxG complex catalytic and non-catalytic activity play distinct roles in pancreas progenitor specification and differentiation (E13.5 Dpy30deltaP pancreas scRNA-Seq)

Purpose: To investigate whether pancreas progenitor specification may be altered, we performed single-cell RNA-sequencing (scRNA-seq) using E13.5 control and Dpy30deltaP pancreas. To determine whether Dpy30deltaP cells have a different developmental trajectory than control cells, we performed pseudo-temporal analysis (Trapnell et al., 2014). Methods: Floxed Dpy30 mice were crossed to Pdx1-Cre driver mice to obtain conditional deletion of Dpy30 exon 4 in the pancreas. In all studies, knockout mice (Dpy30deltaP, Pdx1-Cre; Dpy30flox/flox) were compared to littermate controls (Dpy30flox/flox or Dpy30flox/wt). Results: In total, we sequenced 3,900 control and 3,044 Dpy30deltaP cells from which 786 control and 477 Dpy30deltaP epithelial cells were identified for further analysis. As expected, control progenitor cells gave rise to cells in two distinct branches corresponding to the acinar and endocrine lineages, with terminal markers upregulated in cells at the ends of these branches. Dpy30deltaP progenitors differentiated along these same trajectories; however, relatively few Dpy30deltaP cells were found at the ends of these branches, and acinar and endocrine terminal marker expression was reduced in Dpy30deltaP cells. Interestingly, a higher fraction of Dpy30deltaP cells were found within the MPC/BPC and acinar clusters, predominantly at the expense of endocrine cells. Analyzing the fraction of cells in G1 phase across pseudo-time showed that a higher fraction of Dpy30deltaP acinar lineage cells were in G1 phase. Conclusions: Overall, this suggests that MPC/BPCs and acinar cells may have a prolonged G1 phase due to a failure to appropriately induce genes essential for progression through G2M or S phase. Single-cell RNA-sequencing was performed using one E13.5 control and one E13.5 Dpy30deltaP pancreas研究目的：为探究胰腺祖细胞特化是否可被改变，我们使用E13.5期对照组与Dpy30deltaP胰腺样本开展了单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）。为明确Dpy30deltaP细胞与对照组细胞的发育轨迹是否存在差异，我们进行了拟时序分析（pseudo-temporal analysis，Trapnell等，2014）。 实验方法：将携带Dpy30 flox等位基因的小鼠与Pdx1-Cre驱动小鼠杂交，以实现胰腺中Dpy30第4外显子的条件性敲除。本研究中，所有实验均以敲除小鼠（Dpy30deltaP，即Pdx1-Cre; Dpy30flox/flox）与同窝对照小鼠（Dpy30flox/flox或Dpy30flox/wt）作为对比组别。 实验结果：本研究共测序得到3900个对照组细胞与3044个Dpy30deltaP细胞，其中分别鉴定出786个对照组上皮细胞与477个Dpy30deltaP上皮细胞用于后续分析。正如预期，对照组祖细胞可分化为两个独立分支的细胞，分别对应腺泡细胞系与内分泌细胞系，且这些分支末端的细胞均上调了终末分化标志物。Dpy30deltaP祖细胞虽可沿上述相同轨迹分化，但在这些分支末端发现的Dpy30deltaP细胞数量相对较少，且其腺泡与内分泌终末标志物的表达水平均出现下调。值得注意的是，在MPC/BPC簇与腺泡细胞簇中，Dpy30deltaP细胞的占比更高，这主要是以内分泌细胞的占比减少为代价的。通过分析拟时序过程中处于G1期的细胞占比，我们发现Dpy30deltaP腺泡系细胞中处于G1期的比例更高。 研究结论：综合来看，这一结果提示MPC/BPC与腺泡细胞可能存在G1期延长的现象，原因是其无法正常诱导G2M或S期进程所必需的基因表达。本研究的单细胞RNA测序分别针对1个E13.5期对照组胰腺样本与1个E13.5期Dpy30deltaP胰腺样本开展。