767 Discovering the signaling pathways underlying mouse Merkel cell development using FACS-based single cell RNA-seq

Abstract

Merkel cells (MC) are innervated light touch sensors that comprise less than 0.3% of the mouse epidermis. Terminally differentiated MC are derived from epithelial progenitors and possess both epidermal and neuronal features. Study of MC development provides a model of lineage-specific epithelial differentiation and gives insight into the tumorigenesis of Merkel cell carcinoma (MCC). Using fluorescence-activated cell sorting (FACS)-based single-cell RNA sequencing (scRNAseq) and MC-specific GFP reporter mice, we captured the transcriptome of single MC from embryonic, neonatal, and postnatal skin. Single GFP+ cells were isolated, lysed, and cDNA library for individual cells were generated using Smart-seq2 method. We determined the transcriptome of 1152 single cells including GFP+ MC at all states of differentiation and GFP- control cells. Sequencing yielded ∼3 million filtered reads per cell. Full-length mRNAs were mapped, and splice variants were identified with an average of 6000 genes per cell. Cell transcriptomes clustering identified MC at different differentiation stages, from epithelial precursor to terminally differentiated MC. Trajectory analysis traced the transcriptional changes occurring during MC differentiation. MC-specific genes and transcriptional regulators that define MC differentiation were identified. Transcriptional signatures associated with active cell signaling pathways were identified for each stage of MC differentiation. Comparison of MC progenitors to keratinocytes identified Shh, Egf, Bmp4, and Fgf signaling as being active in early MC specification. Taken together, our FACS-seq approach captured high quality scRNA-seq data to analyze the differentiation of MC from developing mouse skin. This analysis identified novel markers for the MC lineage, characterized the stages of MC differentiation, and defined transcriptional networks and signaling pathways underlying MC differentiation.