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

Prostate organogenesis involves epithelial growth controlled by inductive signalling from specialised mesenchymal subsets. To identify pathways active in mesenchyme we used tissue and single cell transcriptomics to define mesenchymal subsets and subset-specific transcript expression. 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 female VMP were identified with Tag-seq of microdissected tissue, RNA-seq of cell populations, and single cells. We identified 400 transcripts as enriched in the VMP using bioinformatic comparisons of Tag-seq and RNA-seq data, and 44 were confirmed by single cell RNA-seq. Cell subset analysis showed that VMP and adjacent mesenchyme were composed of distinct cell types and that each tissue contained 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 female VMP and prostate mesenchyme. We conclude that prostate inductive mesenchyme shows limited cellular heterogeneity and that transcriptomic analysis identified new mesenchymal subset transcripts associated with prostate organogenesis. reproductive organogenesis. Pathways such as Fgf, Wnt, TGFbeta, Shh, Notch, and others have been identified as participating in prostate development, though it is uncertain whether our knowledge of regulatory pathways is comprehensive (reviewed in 1 ). Transcriptional profiling has been applied to whole prostate organs, both in development and adulthood 2–6 . These studies have identified dynamic expression of many pathways. However, the cellular complexity and proportions of different cell types within organs has led to difficulty in attribution of individual transcripts to defined cell subsets, as well as being confounded by changes in cell proportions over time or following hormonal manipulation. Within these datasets, it is difficult to deconvolute pathways expressed in either stromal or epithelial tissue compartments, although some studies have focussed upon mesenchymal and stromal tissue 7,8 . Since mesenchyme is known to regulate organogenesis as well as mediate the effects of hormones upon development, it is important to identify mesenchymally expressed pathways. During prostate development, several morphogens are expressed in a subset of mesenchyme termed the Ventral Mesenchymal Pad (VMP) and the peri-urethral mesenchyme. The VMP is most apparent on the ventral aspect of the urethra but it encircles the urethra. Its formation precedes the formation of the ventral, lateral and dorsal prostate lobes. It has been defined as a source of inductive mesenchyme using tissue recombination studies 9 , show the stroma subset-specific and and peri-urethral stroma. A anatomic descrip- of stromal subsets using specific markers 11–13 . The VMP forms in both males 9,14 constitutively expresses morphogens Fgf10 morphogen expression, experimental It that androgens the formation of a sexually dimorphic layer of smooth muscle that separates VMP mesenchyme from nascent prostatic buds 17,18 . This layer may regulate inductive signalling from the VMP, and constitutes part of the hormonal mechanism controlling prostate organogenesis. The smooth muscle hypoth- esis accounts for the non-dimorphic expression of Fgf10 and other morphogens 16 . A prediction of this hypothesis is that morphogens are constitutively expressed in both males and females but are regulated indirectly by andro- gens and AR acting in the smooth muscle compartment. We have used VMP isolated from females on the day of birth as our model of prostate mesenchyme, since this is when the tissue is largest and also because female VMP lacks prostatic epithelia and is of low cellular complexity. At the same age in males, the VMP has become the Ventral Prostate, and contains a high proportion of branching epithelia, while the mesenchyme is differentiating into smooth muscle and other fibroblast types. Thus, female VMP is a model system with low cellular complexity that is optimal for identification of molecules involved in prostate development. We have previously used to identify transcripts specifically expressed in the VMP 8 , and noted that mesenchymal pathways may be dys-regulated in cancer-associated fibroblasts, associated with EMT, or neuro-endocrine differentiation of tumours. identified Scube1, EfnB1/EphB3, and Dcn in prostate development One of the limitations of SAGE is its low sensitivity in transcript detection, and next generation RNA sequencing based methods such as Tag-sequencing (Tag-seq) and RNA-sequencing (RNA-seq) have considerably higher sensitivity and superior transcript quantitation, as well as high resolution techniques such as single cell RNAseq. The rationale for our study was to conduct a high resolution transcriptomic analysis of mesenchymal subsets and to examine homo/heterogeneity in regard to cellular composition, as well as to catalogue transcript expression. Cellular heterogeneity is a significant problem in whole organ and tissue transcriptional profiling. Comparison of transcript profiling from microdissected tissue and single cell RNA-seq (scRNA-seq) was used to identify transcripts with tissue and cell specific expression. The markers and pathways identified by such an approach can be deconvolved in whole organ datasets and prioritised for functional studies. We val- idated expression of VMP-specific transcripts by qPCR and also confirmed expression in neonatal prostate. model 34 . This demonstrates the significance of the VMP as a source of stromal-specific molecules with potent capacity to regulate epithelial growth and differentiation in both development and disease. We suggest that mesenchymal subsets are enriched for regulators and morphogens, as well as factors associated with paracrine signalling between mesenchyme and epithelium or juxtacrine signalling between mesenchymal subsets. Our studies are among the first to catalogue gene expression in inductive mesenchyme, and address cellular heterogeneity within the mesenchymal compartment. In this study, our goal was to identify molecules specific to the VMP and to examine cell and tissue heterogeneity within the mesenchyme. We performed both Tag-seq and RNA-seq on VMP as well as an adjacent mes- enchyme comprised of urethral epithelium and peri-urethral mesenchyme (termed SU). Comparison of VMP Tag/RNA-seq libraries to SU libraries identified 400 transcripts that were differentially expressed between the two compartments. Among these were several transcripts identified as VMP-specific in a previous SAGE study 8 , for the identification of mesenchyme specific molecules since it lacks branching epithelia. Inclusion of these in whole tissue transcriptomics yields more complex data in which it is difficult to deconvolve mesenchyme specific molecules. Comparison between male and female mesenchyme may be used to identify sexually dimor- phic gene expression and regulation by androgens and the androgen receptor, and we chose to focus upon mesenchyme-specific expression rather than sexually dimorphic expression. We note that some of our mark- ers exhibit differences between male and female mesenchyme which could be validated in future studies. This is important, since androgen action within the mesenchymal compartment regulates both prostate and genital tubercle growth, and we propose that identification of mesenchyme specific molecules is a first step in the discov-ery of such factors. We identified Meis homeobox 2 (Meis2) and early B-cell factor 3 (Ebf3) as specific to the VMP compared to SU, and also expressed in prostatic mesenchyme. Meis2 belongs to the TALE homeobox protein family and is a regulator of transcription 36 . Meis2 has been identified as essential for the development of cardiac, orofacial, gastro-esophageal and neural tissues 37–39 . Ebf3 is a DNA-binding transcription factor which is involved in the development of bone and neural tissues 40,41 . Here we have established specific expression and nuclear localisation of Meis2 and Ebf3 in developing prostate mesenchyme and are the first to associate these transcription factors with prostate development and expression within mesenchymal subsets. We propose that these molecules can be used as specific markers of mesenchyme or stroma and could be used to estimate the abundance of stroma vs epithelium in tissues of mixed cellular composition. In conclusion, we present a high-resolution transcriptomic analysis of inductive prostate mesenchyme that has documented limited cellular heterogeneity within subsets and identified markers and pathways expressed in mesenchyme during early prostate organogenesis.