Single-cell RNAseq analysis of murine salivary gland development

Development of branching organs involves a series of events regulated by proliferation, differentiation, and intricate cell-cell communication networks. Understanding the molecular drivers for these developmental stages is instrumental to device effective regenerative therapies. Here, we use single cell RNAseq of murine submandibular gland (SMG) from developmental stages corresponding to end bud initiation (E12), branching morphogenesis (E14), cytodifferentiation (E16) and postnatal development (P1, P30, and adult) to generate an atlas of SMG development. The generated resource highlights the heterogeneity in the salivary gland epithelium throughout development and allowed us to identify putative transcription factors involved in secretory acinar cell specification that occurs around E16, including Ybx1, Eno1, Atf3, and Atf4. Clustering and trajectory inference (pseudotime) analyses of this atlas illustrate and suggest that end bud and acinar populations represent the most differentiated state relative to other populations from the same developmental stage, which was associated with a global decrease in expression of numerous transcription factors. Lastly, we identified and characterized two subsets of intercalated duct cells, a Kit+ population defined by Gfra3, and a subset characterized by Gstt1 with sexually dimorphic Smgc co-expression in females and high Serpinb11 co-expression in males. Our analysis indicates that the Gstt1+ population originates from proacinar precursors suggesting plasticity in this population. This atlas can be combined with other available resources to learn about specific cell functions and to predict mechanisms involved in development of branching organs. Examination of epithelial and mesenchymal cells in mouse SMG at five developmental stages分支型器官的发育涉及一系列受增殖、分化以及复杂细胞间通讯网络调控的事件。解析这些发育阶段的分子驱动因素，对于开发有效的再生治疗策略至关重要。本研究利用来自小鼠颌下腺（murine submandibular gland, SMG）不同发育阶段的单细胞RNA测序（single cell RNAseq）数据，构建了小鼠颌下腺发育的细胞图谱，所涵盖的发育阶段分别为终末芽起始（end bud initiation, E12）、分支形态发生（branching morphogenesis, E14）、细胞分化（cytodifferentiation, E16）以及出生后发育（postnatal development, P1、P30及成年个体）。本研究所生成的资源揭示了唾液腺上皮细胞在整个发育过程中的异质性，并帮助我们鉴定出参与E16左右发生的分泌性腺泡细胞特化过程的潜在转录因子，包括Ybx1、Eno1、Atf3及Atf4。对该图谱进行聚类及轨迹推断（trajectory inference, pseudotime）分析后显示，相较于同一发育阶段的其他细胞群，终末芽与腺泡细胞群代表了分化程度最高的细胞状态，这一特征与大量转录因子的整体表达下调密切相关。最后，我们鉴定并表征了两类闰管细胞亚群：一类是以Gfra3为标记的Kit+细胞群，另一类则以Gstt1为特征，且该亚群在雌性个体中伴随Smgc的两性差异性共表达，在雄性个体中则伴随高表达的Serpinb11共表达。本研究分析表明，Gstt1+细胞群起源于前腺泡前体细胞，提示该细胞群具有可塑性。该细胞图谱可与其他现有研究资源结合，用于探究特定细胞的功能，并预测分支型器官发育的潜在机制。对小鼠颌下腺上皮细胞与间质细胞在五个发育阶段的检测