Single-cell RNA sequencing reveals novel markers of pituitary stem cells and hormone-producing cell-types

Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. Yet, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of non-endocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We  performed scRNAseq on approximately 13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell-types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms which suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. At the same time, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3’ transcript end sequences, and use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address longstanding questions regarding the development and function of the different populations of the pituitary gland throughout life.

调控垂体中干细胞与特化激素分泌细胞的转录因子及信号通路，一直是垂体研究领域的热点方向，相关研究已为垂体器官发生与疾病的分子机制提供了系统性阐释。然而，干细胞增殖与分化的调控机制、特化激素分泌细胞间的异质性，以及垂体内非内分泌细胞的功能，仍是尚未解决的重要科学问题。
近年来，单细胞RNA测序（single-cell RNA sequencing，scRNAseq）技术的进步为解答上述问题开辟了全新路径。本研究对7周龄C57BL/6雄性小鼠的6个完整垂体腺中混合收集的约13663个细胞开展了scRNAseq检测。我们通过硅基分析（in silico）鉴定出了垂体内分泌细胞与干细胞，同时还识别出内皮细胞、结缔组织、红细胞及白细胞等多种支持细胞类群。通过差异基因表达分析，我们筛选出了垂体内分泌细胞群与干细胞群的已知及新型标志物。我们针对新型促性腺激素细胞富集标志物Foxp2开展体内验证实验，证实了scRNAseq技术的应用价值。
本研究还提供了体内垂体组织的新型scRNAseq数据集，其中包含基于无偏聚类算法得到的分析结果，该结果提示存在一类富集固醇/胆固醇合成基因的生长激素细胞亚群。与此同时，我们发现部分仅能生成转录本3'端序列的scRNAseq测序平台，会因转录组注释不完整而产生假阴性结果，并通过体内实验数据成功找回了垂体转录因子Prop1的测序读段。
总而言之，scRNAseq技术为解答长期以来关于垂体不同细胞群在生命周期中的发育与功能相关的科学问题提供了重要机遇。