Project description:We compared the transcriptomics of mouse melanocyte stem cells at the activation stage of the hair cycle (anagen onset) and mature melanocytes from the hair bulb by single cell RNAseq
Project description:Melanocytes are pigment-producing cells of neural crest origin responsible for protecting the skin against UV-irradiation. Melanocyte dysfunction leads to pigmentation defects including albinism, vitiligo, and piebaldism and is a key feature of systemic pathologies such as Hermansky-Pudlak (HP) and Chediak-Higashi (CH) Syndromes. Pluripotent stem cell technology offers a novel approach for studying human melanocyte development and disease. Here we report that timed exposure to activators of WNT, BMP and EDN3 signaling triggers the sequential induction of neural crest and melanocyte precursor fates under dual-SMAD inhibition conditions. Using a SOX10::GFP hESC reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human neural crest induction. Surprisingly, suppression of BMP signaling does reduce neural crest yield. Subsequent differentiation of hESC-derived melanocyte precursors under defined conditions yields pure populations of pigmented cells matching the molecular and functional properties of adult melanocytes. Melanocytes from patient-specific iPSCs faithfully reproduce the ultrastructural features of the HP- and CH-specific pigmentation defects with minimal variability across lines. Our data define a highly specific requirement for WNT signaling during neural crest induction and enable the generation of pure populations of hiPSC-derived melanocytes for faithful modeling of human pigmentation disorders. Total RNA obtained from embryonic stem cells (ESCs), ESC-derived melanocyte progenitors, ESC-derived mature melanocytes, primary melanocytes, and disease-specific induced pluripotent stem cell-derived melanocytes.
Project description:Melanocytes are pigment-producing cells of neural crest origin responsible for protecting the skin against UV-irradiation. Melanocyte dysfunction leads to pigmentation defects including albinism, vitiligo, and piebaldism and is a key feature of systemic pathologies such as Hermansky-Pudlak (HP) and Chediak-Higashi (CH) Syndromes. Pluripotent stem cell technology offers a novel approach for studying human melanocyte development and disease. Here we report that timed exposure to activators of WNT, BMP and EDN3 signaling triggers the sequential induction of neural crest and melanocyte precursor fates under dual-SMAD inhibition conditions. Using a SOX10::GFP hESC reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human neural crest induction. Surprisingly, suppression of BMP signaling does reduce neural crest yield. Subsequent differentiation of hESC-derived melanocyte precursors under defined conditions yields pure populations of pigmented cells matching the molecular and functional properties of adult melanocytes. Melanocytes from patient-specific iPSCs faithfully reproduce the ultrastructural features of the HP- and CH-specific pigmentation defects with minimal variability across lines. Our data define a highly specific requirement for WNT signaling during neural crest induction and enable the generation of pure populations of hiPSC-derived melanocytes for faithful modeling of human pigmentation disorders.
Project description:MIcrophthalmia-associated Transcription Factor (MITF) regulates melanocyte and melanoma physiology. ShRNA-mediated silencing of the NURF subunit BPTF revealed its essential role in several melanoma cell lines and in untransformed melanocytes in vitro. Comparative RNA-seq shows that MITF and BPTF co-regulate overlapping gene expression programs in cell lines in vitro. Somatic and specific inactivation of Bptf in developing murine melanoblasts in vivo shows that Bptf regulates their proliferation, migration and morphology. Once born, Bptf-mutant mice display premature greying where the second post-natal coat is white. This second coat is normally pigmented by differentiated melanocytes derived from the adult melanocyte stem cell (MSC) population that is stimulated to proliferate and differentiate at anagen. An MSC population is established and maintained throughout the life of the Bptf- mutant mice, but these MSCs are abnormal and at anagen, give rise to reduced numbers of transient amplifying cells (TACs) that do not express melanocyte markers and fail to differentiate into mature melanin producing melanocytes. MSCs display a transcriptionally repressed chromatin state and Bptf is essential for reactivation of the melanocyte gene expression program at anagen, the subsequent normal proliferation of TACs and their differentiation into mature melanocytes.
Project description: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.
Project description:DICER is a central regulator of microRNA maturation. However little is known about mechanisms regulating its expression in development or disease. While profiling miRNA expression in differentiating melanocytes, two populations were observed: some upregulated at the pre-miRNA stage, and others upregulated as “mature” miRNAs (with stable pre-miRNA levels). Conversion of pre-miRNAs to fully processed miRNAs appeared to be dependent upon stimulation of DICER expression—an event found to occur via direct transcriptional targeting of DICER by the melanocyte master transcriptional regulator MITF. MITF binds and activates a conserved regulatory element upstream of DICER’s transcriptional start site upon melanocyte differentiation. Targeted knockout of DICER is lethal to melanocytes, at least partly via DICER-dependent processing of the pre-miRNA-17~92 cluster thus targeting BIM, a known pro-apoptotic regulator of melanocyte survival. These observations highlight a central mechanism underlying miRNA regulation which could exist for other cell types during development. Primary melanocytes were obtained from 7 independent donors. Mature-miRNA levels were detected using Applied Biosystem's TaqMan PCR assay.
Project description:Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single cell RNA-sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated during differentiation. Employing conditional lineage-specific genetic ablation studies in mice, we find that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream of activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and promoter analyses, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1 and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation.
Project description:We report single cell transcriptomes from human skin samples that have been enriched for melanocytes using FACS. This work revealed anatomical site-specific melanocyte expression programs. In addition, by surveying melanocytes at different stages of development, we identified development-associated expression programs that are reflected in the dedifferentiation of melanomas.
Project description:Both benign nevi and melanomas exhibit high rates of activating mutations in BRAF kinase, suggesting that this event is important for melanocyte proliferation and melanoma initiation in vivo; however, the precise mechanism of BRAF kinase action in these lesions remains to be elucidated. We have used comprehensive gene expression profiling to evaluate the downstream effectors of activated BRAFV600E kinase in primary human melanocytes and identified a dominant proliferative target gene signature for BRAF kinase in these cells. We used microarrays to detail the global gene expression pattern change induced by the activation of RAS/BRAF/MEK/ERK signaling pathway and identified distinct classes of up-regulated genes during this process. Experiment Overall Design: Primary human melanocytes were isolated from neonatal foreskins. Melanocytes isolated from five individual donors were pooled together in order to reduce genetic variations and transduced by BRAFV600E expressing lentivirus and control virus with MOI 10 respectively. Cells were harvested 5 days following infection and total RNA was extracted and purified by RNeasy kit by following manufacturerâs protocol. RNA quality check, double strand complementry DNA synthesis, hybridization with Human Genome U133 Plus 2.0 Array Chips (Affymetrix Inc. Santa Clara, CA), initial data extraction, normalization using RMAExpress software.
Project description: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.