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

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. Overall design: Tag-sequencing, RNA-sequencing and single-cell RNA-sequencing on 2 female inductive mesenchymal tissues of the developing prostate/urogenital tract.