Probes for INS

In the adult mouse spinal cord, the ependymal cell population that surrounds the central canal is thought to be a promising source of quiescent stem cells to treat spinal cord injury. Relatively little is known about the cellular origin of ependymal cells during spinal cord development, or the molecular mechanisms that regulate ependymal cells during adult homeostasis. Using genetic lineage tracing based on the Wnt target gene Axin2, we have characterized Wnt-responsive cells during spinal cord development. Our results revealed that Wnt-responsive progenitor cells are restricted to the dorsal midline throughout spinal cord development, which gives rise to dorsal ependymal cells in a spatially restricted pattern. This is contrary to previous reports that suggested an exclusively ventral origin of ependymal cells, suggesting that ependymal cells may retain positional identities in relation to their neural progenitors. Our results further demonstrated that in the postnatal and adult spinal cord, all ependymal cells express the Wnt/β-catenin signaling target gene Axin2, as well as Wnt ligands. Genetic elimination of β-catenin or inhibition of Wnt secretion in Axin2-expressing ependymal cells in vivo both resulted in impaired proliferation, indicating that Wnt/β-catenin signaling promotes ependymal cell proliferation. These results demonstrate the continued importance of Wnt/β-catenin signaling for both ependymal cell formation and regulation. By uncovering the molecular signals underlying the formation and regulation of spinal cord ependymal cells, our findings thus enable further targeting and manipulation of this promising source of quiescent stem cells for therapeutic interventions.

Basal cell carcinoma (BCC) is the most frequent cancer in humans and results from constitutive activation of the Hedgehog pathway1. Several Smoothened inhibitors are used to treat Hedgehog-mediated malignancies, including BCC and medulloblastoma2. Vismodegib, a Smoothened inhibitor, leads to BCC shrinkage in the majority of patients with BCC3, but the mechanism by which it mediates BCC regression is unknown. Here we used two genetically engineered mouse models of BCC4 to investigate the mechanisms by which inhibition of Smoothened mediates tumour regression. We found that vismodegib mediates BCC regression by inhibiting a hair follicle-like fate and promoting the differentiation of tumour cells. However, a small population of tumour cells persists and is responsible for tumour relapse following treatment discontinuation, mimicking the situation found in humans5. In both mouse and human BCC, this persisting, slow-cycling tumour population expresses LGR5 and is characterized by active Wnt signalling. Combining Lgr5 lineage ablation or inhibition of Wnt signalling with vismodegib treatment leads to eradication of BCC. Our results show that vismodegib induces tumour regression by promoting tumour differentiation, and demonstrates that the synergy between Wnt and Smoothened inhibitors is a clinically relevant strategy for overcoming tumour relapse in BCC.

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.

Gut mesenchyme provides key stem cell niche signals such as Wnt ligands, but how these signals are regulated is unclear. Because Hedgehog (Hh) signaling is critical for gut mesenchymal development and tumorigenesis, we investigated Hh-mediated mechanisms by analyzing mice deleted for key negative regulators of Hh signaling, Sufu and/or Spop, in the gut mesenchyme, and demonstrated their dosage-dependent roles. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction. Consistent with increased epithelial proliferation caused by abnormal Hh activation in development, Sufu reduction promotes intestinal tumorigenesis, whereas Gli2 heterozygosity suppresses it. Our analyses of chromatin and GLI2 binding genomic regions reveal its transcriptional regulation of stem cell niche signals through enhancers, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis

Under injury conditions, dedicated stem cell populations govern tissue regeneration. However, the molecular mechanisms that induce stem cell regeneration and enable plasticity are poorly understood. Here, we investigate stem cell recovery in the context of the hair follicle to understand how two molecularly distinct stem cell populations are integrated. Utilizing diphtheria-toxin-mediated cell ablation of Lgr5+(leucine-rich repeat-containing G-protein-coupled receptor 5) stem cells, we show that killing of Lgr5+ cells in mice abrogates hair regeneration but this is reversible. During recovery, CD34+ (CD34 antigen) stem cells activate inflammatory response programs and start dividing. Pharmacological attenuation of inflammation inhibits CD34+ cell proliferation. Subsequently, the Wnt pathway controls the recovery of Lgr5+ cells and inhibition of Wnt signalling prevents Lgr5+ cell and hair germ recovery. Thus, our study uncovers a compensatory relationship between two stem cell populations and the underlying molecular mechanisms that enable hair follicle regeneration.

Transit-amplifying cells (TACs) are an early intermediate in tissue regeneration. Here, using hair follicles (HFs) as a paradigm, we show that emerging TACs constitute a signaling center that orchestrates tissue growth. Whereas primed stem cells (SCs) generate TACs, quiescent SCs only proliferate after TACs form and begin expressing Sonic Hedgehog (SHH). TAC generation is independent of autocrine SHH, but the TAC pool wanes if they can't produce SHH. We trace this paradox to two direct actions of SHH: promoting quiescent-SC proliferation and regulating dermal factors that stoke TAC expansion. Ingrained within quiescent SCs' special sensitivity to SHH signaling is their high expression of GAS1. Without sufficient input from quiescent SCs, replenishment of primed SCs for the next hair cycle is compromised, delaying regeneration and eventually leading to regeneration failure. Our findings unveil TACs as transient but indispensable integrators of SC niche components and reveal an intriguing interdependency of primed and quiescent SC populations on tissue regeneration.

The murine epidermis with its hair follicles represents an invaluable model system for tissue regeneration and stem cell research. Here we used single-cell RNA-sequencing to reveal how cellular heterogeneity of murine telogen epidermis is tuned at the transcriptional level. Unbiased clustering of 1,422 single-cell transcriptomes revealed 25 distinct populations of interfollicular and follicular epidermal cells. Our data allowed the reconstruction of gene expression programs during epidermal differentiation and along the proximal-distal axis of the hair follicle at unprecedented resolution. Moreover, transcriptional heterogeneity of the epidermis can essentially be explained along these two axes, and we show that heterogeneity in stem cell compartments generally reflects this model: stem cell populations are segregated by spatial signatures but share a common basal-epidermal gene module. This study provides an unbiased and systematic view of transcriptional organization of adult epidermis and highlights how cellular heterogeneity can be orchestrated in vivo to assure tissue homeostasis.