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A large pool of actively cycling progenitors orchestrates self-renewal and injury repair of an ectodermal appendage

ABSTRACT: The classical model posits that a small number of quiescent stem cells (SCs) gives rise to proliferating transit-amplifying cells before terminal differentiation. However, recent evidence indicates that some tissues house multiple progenitor pools with distinct proliferative and differentiation potentials. Resolving the identity and spatial organization of these populations is therefore a fundamental requirement for understanding tissue renewal. Here, using a combination of single-cell RNA sequencing (scRNAseq), mouse genetics, and tissue injury approaches, we uncovered cellular hierarchies and mechanisms underlying the maintenance and repair of the ever-growing mouse incisor, an ectodermal appendage model system that requires high cell turnover for its homeostasis and quick healing capability. We found that during homeostasis, a group of actively cycling epithelial progenitors generates both the enamel-producing ameloblasts and the adjacent non-ameloblast cell layers, which were previously proposed to be quiescent SCs. Upon injury, transient adjustments to the proliferation kinetics of dental epithelial progenitor cells compensated for cell loss. Injury repair was also supported by direct conversion of supporting Notch1-expressing cells to ameloblasts. Our elucidation of epithelial SC identity, position, and function thus provides a mechanistic basis for the homeostasis and repair of a fast-turnover ectodermal appendage and illustrates how a cycling progenitor pool confers considerable epithelial plasticity during tissue renewal.

ORGANISM(S): Mus musculus

PROVIDER: GSE131204 | GEO | 2019/09/02

REPOSITORIES: GEO

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Project description:Background Quiescent (non-dividing) muscle satellite cells (SCs) transition into proliferating progenitor cells that differentiate to repair skeletal muscle after injury. Recently, the AP-1 family member, FOS, was found to be rapidly and transiently induced in SCs in response to muscle damage, and its activity is required for effective SC activation and muscle repair. However, why FOS is rapidly down-regulated before SCs enter the cell cycle as progenitor cells remains unclear. In addition, whether boosting FOS expression in primary cycling muscle progenitor cells can enhance their myogenic properties needs to be evaluated. Methods In this study, we established a lentiviral, doxycycline-inducible system to explore the molecular and functional consequences of continued FOS expression in SC-derived, cycling muscle progenitor cells ex vivo. Specifically, we performed cell growth and EdU incorporation assays to measure cellular proliferation; myotube formation assays to evaluate terminal differentiation potential; and RNA-Sequencing (RNA-Seq) in conjunction with high-throughput chromosome conformation capture (Hi-C) to uncover changes in gene expression and long-range chromatin interactions. Results Persistent FOS activity in cycling muscle progenitor cells modestly enhances progenitor cell proliferation, but severely antagonizes their ability to differentiate and form myotubes. Genome-wide RNA-Seq analysis revealed that ectopic FOS activity in cycling muscle progenitor cells suppresses a global pro-myogenic gene expression program, while activating a stress-induced Mitogen-Activated Protein Kinase (MAPK) transcriptional signature. Importantly, we found various chromosomal re-organization events in A/B compartments, TADs, and long-range genomic loops near FOS-regulated differentially expressed genes in cycling muscle progenitor cells ectopically expressing FOS. Conclusions Altogether, our results suggest that elevated FOS activity in proliferating muscle progenitor cells perturbs cellular differentiation by altering the 3-dimensional (3D) chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage, and raises the possibility that in states of chronic stress or disease, persistent FOS activity in muscle stem and/or progenitor cells may actually disrupt the muscle forming process.

Project description:A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function, the extent to which stem cells can regulate quiescence is unknown. Here, we show that the stem cell quiescent state is composed of two distinct functional phases: G0 and an “alert” phase we term GAlert, and that stem cells actively and reversibly transition between these phases in response to injury-induced, systemic signals. Using genetic models specific to muscle stem cells (or satellite cells (SCs)), we show that mTORC1 activity is necessary and sufficient for the transition of SCs from G0 into GAlert and that signaling through the HGF receptor, cMet is also necessary. We also identify G0-to-GAlert transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into GAlert possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into GAlert functions as an 'alerting' mechanism, a novel adaptive response that positions stem cells to respond rapidly under conditions of injury and stress without requiring cell cycle entry or a cell fate commitment. We performed microarray analysis on muscle stem cells, harvested immediately after isolation, to investigate the transcriptional response these cells have to injury and the role of mTORC1 and cMet have in this response At least 2 weeks after tamoxifen treatment, to induce recombination and expression of the Rosa26rYFP lineage tracer in Pax7 expressing muscle stem cells using a Pax7-CreER driver, YFP+ cells were FACS purified from hindlimb muscles of animals that were noninjured (quiescent satellite cells (QSCs)) or from hindlimb muscle contralateral to muscles where we induced a BaCl2 injury (contralateral satellite cells (CSCs)). We compared the response of wild-type (WT) muscle stem cells to the response elicited by muscle stem cells with conditional ablation of TSC1, Rptor, or cMet genes. Immediately after FACS purification of cells, total RNA was extracted, processed, labeled, and hybridized to Affymetrix Mouse Gene 1.0 ST arrays. Each biological replicate is a pooled sample of muscle stem cells isolated from 3-4 mice.

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A large pool of actively cycling progenitors orchestrates self-renewal and injury repair of an ectodermal appendage

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