Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

Prostate organogenesis involves epithelial growth in response to inductive signalling from specialised subsets of mesenchyme. To identify regulators and morphogens active in mesenchyme, we performed transcriptomic analysis using Tag-seq, RNA-seq, and single cell RNA-seq and defined new mesenchymal subsets and markers. We documented transcript expression using Tag-seq and RNA-seq in female rat Ventral Mesenchymal Pad (VMP) as well as adjacent urethra comprised of smooth muscle and peri-urethral mesenchyme. Transcripts enriched in VMP were identified in Tag-seq data from microdissected tissue, and RNA-seq data derived from cell populations and single cells. We identified 400 transcripts as enriched or specific to the VMP using bio-informatic comparisons of Tag-seq and RNA-seq data. Comparison with single cell RNA-seq identified transcripts yielded 45 transcriptscommon to both approaches. Cell subset analysis showed that VMP and adjacent mesenchyme were composed of distinct cell types and that each tissue was comprised of two subgroups. Markers for these subgroups were highly subset specific. Thirteen transcripts were validated by qPCR to confirm cell specific expression in microdissected tissues, as well as expression in neonatal prostate. Immunohistochemical staining demonstrated that Ebf3 and Meis2 showed a restricted expression pattern in VMP condensed mesenchyme. Taken together, we demonstrate that the VMP shows limited cellular heterogeneity and that our high-resolution transcriptomic analysis identified new mesenchymal subset markers associated with prostate organogenesis.前列腺器官发生过程中，上皮细胞的生长会响应特化间充质亚群所传递的诱导信号。为鉴定间充质中发挥活性的调控因子与形态发生原，我们采用Tag-seq、RNA-seq及单细胞RNA-seq开展转录组学分析，并明确了全新的间充质亚群及其标记物。我们借助Tag-seq与RNA-seq，记录了雌性大鼠腹侧间充质垫（Ventral Mesenchymal Pad, VMP）以及由平滑肌与尿道周围间充质构成的邻近尿道组织中的转录本表达情况。通过显微切割组织的Tag-seq数据，以及来源于细胞群体与单细胞的RNA-seq数据，我们鉴定出了在VMP中富集的转录本。通过对Tag-seq与RNA-seq数据进行生物信息学比对，我们共鉴定出400个在VMP中富集或特异性表达的转录本。结合单细胞RNA-seq鉴定的转录本进行比对后，两种方法共筛选得到45个重合的转录本。细胞亚群分析结果显示，VMP与邻近间充质由不同的细胞类型构成，且每种组织均包含两个亚群。这些亚群的标记物具有极强的亚群特异性。我们通过qPCR验证了13个转录本，证实其在显微切割组织中存在细胞特异性表达，且在新生大鼠前列腺中亦有表达。免疫组化染色结果证实，Ebf3与Meis2在VMP致密间充质中呈现限制性表达模式。综上，我们证实VMP的细胞异质性较低，且本研究通过高分辨率转录组学分析，鉴定出了与前列腺器官发生相关的全新间充质亚群标记物。