Peggy Myung

Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.

Melanocyte stem cells (McSCs) intimately interact with epithelial stem cells (EpSCs) in the hair follicle bulge and secondary hair germ (sHG). Together, they undergo activation and differentiation to regenerate pigmented hair. However, the mechanisms behind this coordinated stem cell behavior have not been elucidated. Here, we identified Wnt signaling as a key pathway that couples the behavior of the two stem cells. EpSCs and McSCs coordinately activate Wnt signaling at the onset of hair follicle regeneration within the sHG. Using genetic mouse models that specifically target either EpSCs or McSCs, we show that Wnt activation in McSCs drives their differentiation into pigment-producing melanocytes, while EpSC Wnt signaling not only dictates hair follicle formation but also regulates McSC proliferation during hair regeneration. Our data define a role for Wnt signaling in the regulation of McSCs and also illustrate a mechanism for regeneration of complex organs through collaboration between heterotypic stem cell populations.

Differential Requirement for LAT and SLP-76 in GPVI versus T Cell Receptor Signaling

Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells. Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage. Both SLP-76 and LAT are important for optimal platelet activation downstream of the collagen receptor, GPVI. In addition, SLP-76 is involved in signaling mediated by integrin αIIbβ3. Because SLP-76 and LAT function coordinately in T cell signal transduction, yet their roles appear to differ in hemostasis, we investigated in detail the functional consequences of SLP-76 and LAT deficiencies in platelets. Previously we have shown that LAT−/− platelets exhibit defective responses to the GPVI-specific agonist, collagen-related peptide (CRP). Consistent with this, we find that surface expression of P-selectin in response to high concentrations of GPVI ligands is reduced in both LAT- and SLP-76–deficient platelets. However, platelets from LAT−/− mice, but not SLP-76−/− mice, aggregate normally in response to high concentrations of collagen and convulxin. Additionally, unlike SLP-76, LAT is not tyrosine phosphorylated after fibrinogen binding to integrin αIIbβ3, and collagen-stimulated platelets deficient in LAT spread normally on fibrinogen-coated surfaces. Together, these findings indicate that while LAT and SLP-76 are equally required for signaling via the T cell antigen receptor (TCR) and pre-TCR, platelet activation downstream of GPVI and αIIbβ3 shows a much greater dependency on SLP-76 than LAT.

Epithelial Wnt Ligand Secretion Is Required for Adult Hair Follicle Growth and Regeneration

β-catenin, a key transducer molecule of Wnt signaling, is required for adult hair follicle growth and regeneration. However, the cellular source of Wnt ligands required for Wnt/β-catenin activation during anagen induction is unknown. In this study, we genetically deleted Wntless, a gene required for Wnt ligand secretion by Wnt-producing cells, specifically in the hair follicle epithelium during telogen phase. We show that epithelial Wnt ligands are required for anagen, as loss of Wntless in the follicular epithelium resulted in a profound hair cycle arrest. Both the follicular epithelium and dermal papilla showed markedly decreased Wnt/β-catenin signaling during anagen induction compared to control hair follicles. Surprisingly, hair follicle stem cells that are responsible for hair regeneration maintained expression of stem cell markers but exhibited significantly reduced proliferation. Finally, we demonstrate that epidermal Wnt ligands are critical for adult wound-induced de novo hair formation. Collectively, these data show that Wnt ligands secreted by the hair follicle epithelium are required for adult hair follicle regeneration and provide new insight into potential cellular targets for the treatment of hair disorders such as alopecia.

β-Catenin Activation Regulates Tissue Growth Non–Cell Autonomously in the Hair Stem Cell Niche

Coordinated Hair Growth Wnt/β-catenin signaling is a key pathway that plays a conserved role in regulating stem cell function during adult tissue regeneration. Using time-lapse imaging of live mice, Deschene et al. (p. 1353) show that genetic activation of β-catenin within hair follicle stem cells generates axes of hair growth by coordinated cell divisions and cell movements, even when the normal niches—the dermal papillae—are laser-ablated. Activated β-catenin enhances Wnt ligand secretion, and these ligands can then activate Wnt signaling in adjacent cells that do not have activated β-catenin, indicating how activated stem cells could influence neighboring cells during normal growth and in cancer. Signals generated by mouse hair follicle stem cells generate new hair growth. Wnt/β-catenin signaling is critical for tissue regeneration. However, it is unclear how β-catenin controls stem cell behaviors to coordinate organized growth. Using live imaging, we show that activation of β-catenin specifically within mouse hair follicle stem cells generates new hair growth through oriented cell divisions and cellular displacement. β-Catenin activation is sufficient to induce hair growth independently of mesenchymal dermal papilla niche signals normally required for hair regeneration. Wild-type cells are co-opted into new hair growths by β-catenin mutant cells, which non–cell autonomously activate Wnt signaling within the neighboring wild-type cells via Wnt ligands. This study demonstrates a mechanism by which Wnt/β-catenin signaling controls stem cell–dependent tissue growth non–cell autonomously and advances our understanding of the mechanisms that drive coordinated regeneration.

Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool

Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression). In contrast to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration. Here we show by intravital microscopy in live mice that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbours. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through transforming growth factor (TGF)-β activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool, as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviours and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis.

Dissecting the bulge in hair regeneration

The adult hair follicle houses stem cells that govern the cyclical growth and differentiation of multiple cell types that collectively produce a pigmented hair. Recent studies have revealed that hair follicle stem cells are heterogeneous and dynamic throughout the hair cycle. Moreover, interactions between heterologous stem cells, including both epithelial and melanocyte stem cells, within the hair follicle are just now being explored. This review will describe how recent findings have expanded our understanding of the development, organization, and regeneration of hair follicle stem cells. At a basic level, this review is intended to help construct a reference point to integrate the surge of studies on the molecular mechanisms that regulate these cells.

Hardwiring Stem Cell Communication through Tissue Structure

Adult stem cells across diverse organs self-renew and differentiate to maintain tissue homeostasis. How stem cells receive input to preserve tissue structure and function largely relies on their communication with surrounding cellular and non-cellular elements. As such, how tissues are organized and patterned not only reflects organ function, but also inherently hardwires networks of communication between stem cells and their environment to direct tissue homeostasis and injury repair. This review highlights how different methods of stem cell communication reflect the unique organization and function of diverse tissues.

Defining the hair follicle stem cell (Part I).

Stem cells possess the salient features of multipotency and self-renewal, enabling them with the ability to differentiate into the multiple cell types required to generate new tissue throughout the development and the lifetime of the organism. Their maintenance and activation during homeostasis and in response to injury provide resiliency to the organism, while their dysregulation can lead to the tissue compromise and dysfunction that underlie aging and tumorigenesis. Large strides have been made to understand how stem cells are regulated in an effort to develop novel treatments in the fields of cancer, aging and regenerative medicine. To this end, several animal and organ models have been vital to the synthesis of principles explaining adult stem cell maintenance and differentiation. The hair follicle (HF) has emerged into the forefront as one of the most illustrative and accessible models to examine adult stem cell regulation in mammals, attributed to its ability to predictably cycle through well-characterized phases of degeneration (catagen), rest (telogen) and regeneration (anagen) throughout life. The ability of the follicle to maintain its regenerative capacity rests upon resident stem cells that lie in a region called the bulge, located at the base of the upper permanent portion of the follicular outer root sheath (ORS).1 Similar to many other somatic stem cells, bulge cells are slow cycling in nature. This feature has permitted their initial identification and isolation as label-retaining cells (LRCs) that can retain a pulse of nucleotide label following a long chase period and is frequently regarded as a defining characteristic of the HF stem cell.1,2 In addition, the availability of several immunohistochemical markers, including keratin-15 (K15) and CD34, that specifically label murine follicular stem cells has given researchers the ability to carefully examine the signals required for adult stem cell activation and renewal.3,4 Subsequently, different groups have utilized these tools to label and track the fate of bulge cells and their progeny during homeostasis and following epithelial injury. These studies demonstrate that the bulge serves as a repository of long-lived multipotent stem cells, imparted with the capacity to differentiate into all cell types that constitute the lower cyclic portion of the HF, as well as the interfollicular epidermis during wound repair.5– 9 Since then, the explosion of genetic and molecular tools available to target and profile specific HF cell populations in mice has fueled the rapid growth of information regarding the molecular mechanisms governing follicular morphogenesis and the hair cycle. Additionally, the identification of other stem cell markers has provided further resolution of the bulge and has revealed that the bulge is composed of distinct subpopulations.4,10– 14 Despite these advancements, our understanding of how HF stem cells are initially generated and what cells constitute the HF stem cell niche is still incomplete. In particular, it is unclear if immunohistochemically distinct bulge populations represent stem cells with different lineage commitment and self-renewing potential. Furthermore, even less is known about how other cell types, including dermal fibroblasts, regulate their identity and behavior during development and the hair cycle. This two-part review will focus on some of the recent studies that have examined these questions and have resurrected earlier seminal efforts to define the germinative origin of the anagen follicle.

Dissecting Wnt Signaling for Melanocyte Regulation during Wound Healing

Abnormal pigmentation is commonly seen in the wound scar. Despite advancements in the research of wound healing, little is known about the repopulation of melanocytes in the healed skin. Previous studies have shown the capacity of melanocyte stem cells in the hair follicle to contribute skin epidermal melanocytes after injury in mice and humans. Here, we focused on the Wnt pathway, known to be a vital regulator of melanocyte stem cells in efforts to better understand the regulation of follicle-derived epidermal melanocytes during wound healing. We showed that transgenic expression of Wnt inhibitor Dkk1 in melanocytes reduced epidermal melanocytes in the wound scar. Conversely, forced activation of Wnt signaling by genetically stabilizing β-catenin in melanocytes increases epidermal melanocytes. Furthermore, we show that deletion of Wntless (Wls), a gene required for Wnt ligand secretion, within epithelial cells results in failure in activating Wnt signaling in adjacent epidermal melanocytes. These results show the essential function of extrinsic Wnt ligands in initiating Wnt signaling in follicle-derived epidermal melanocytes during wound healing. Collectively, our results suggest the potential for Wnt signal regulation to promote melanocyte regeneration and provide a potential molecular window to promote proper melanocyte regeneration after wounding and in conditions such as vitiligo.

Cutaneous immunohistochemical staining pattern of p53β isoforms

p53 is considered the guardian of the genome and as such has numerous functions. The TP53 gene is the most commonly mutated gene in cancer, and yet the exact biological significance of such mutations remains unclear. There are at least 12 different isoforms of p53, and the complexity of the p53 pathway may be in part related to these isoforms. Prior research has often not teased out what isoforms of p53 are being studied, and there is evidence in the literature that p53 isoforms are expressed differently. In this paper, we document the staining pattern of p53β isoforms in the skin and correlate it with mutational status in a subgroup of squamous proliferations of the skin. p53β isoforms are present in the cytoplasm of the differentiated layer of the epidermis and hair follicles (granular layer, infundibular and isthmus–catagen). p53β isoforms are diffusely expressed within the cytoplasm of well-differentiated squamous tumours with tetramerisation (C-terminal) domain mutations in TP53. Our results lend support to p53β isoforms being a marker of differentiation in keratinocytes.