BMP signaling: at the gate between activated melanocyte stem cells and differentiation [RNA-Seq]

Adult tissue stem cells protect their long-term potential by exerting precise control over their transitions between quiescence, activation, and differentiation. Through cyclic bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate to produce and transfer pigment to hair cells.  The signaling factors orchestrating this process are still poorly understood. Here, we use single cell RNA-sequencing with pseudotime analysis to elucidate the transcriptional trajectory of McSCs through quiescence, activation, and differentiation into mature melanocytes. Unearthing signs of increased WNT and BMP signaling along this progression, we lineage-ablate either pathway and see hair graying.  We show that BMP signaling functions downstream of McSCs but upstream of WNT signaling through LEF1. After stem cell activation, the two pathways trigger committed, proliferative progeny to fuel MITF-dependent differentiation. Analyses of the promoters required for melanosome maturation suggest a specific reliance upon MITF and LEF1 transcription factors, which  we show are dampened without BMP signaling. Our findings shed light upon the signaling interplay that orchestrates the melanocyte lineage. Moreover, the block in differentiation and enhanced proliferation observed in the absence of BMP signaling raises the disconcerting possibility that BMP may harbor not only tumor promoting but also tumor suppressing activity in melanoma. RNA-sequencing was performed on FACS-sorted second telogen (~P60) quiescent melanocyte stem cells (2 biological replicates) and full anagen (P10) melanocytes (3 biological replicates). Cells were FACS purified into TRI Reagent LS, and total RNA was purified using the Direct-zol RNA MicroPrep kit (Zymo Research) according to the kit protocol. RNA quality was determined by Agilent 2100 Bioanalyzer, and cDNA libraries were prepared using the Illumina TrueSeq mRNA sample preparation kit (non-stranded, poly-A selection) and sequenced on an Illumina HiSeq 4000 instrument.