Maksim V. Plikus

Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration

In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic β-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/β-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought ‘chalone’ inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug delivery and stem cell engineering studies, because they highlight the acute need to differentiate supportive versus inhibitory regions in the host skin.

An Integrated Gene Regulatory Network Controls Stem Cell Proliferation in Teeth

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.

Morphoregulation of teeth: modulating the number, size, shape and differentiation by tuning Bmp activity

Summary During development and evolution, the morphology of ectodermal organs can be modulated so that an organism can adapt to different environments. We have proposed that morphoregulation can be achieved by simply tilting the balance of molecular activity. We test the principles by analyzing the effects of partial downregulation of Bmp signaling in oral and dental epithelia of the keratin 14‐Noggin transgenic mouse. We observed a wide spectrum of tooth phenotypes. The dental formula changed from 1.0.0.3/1.0.0.3 to 1.0.0.2(1)/1.0.0.0. All mandibular and M3 maxillary molars were selectively lost because of the developmental block at the early bud stage. First and second maxillary molars were reduced in size, exhibited altered crown patterns, and failed to form multiple roots. In these mice, incisors were not transformed into molars. Histogenesis and differentiation of ameloblasts and odontoblasts in molars and incisors were abnormal. Lack of enamel caused misocclusion of incisors, leading to deformation and enlargement in size. Therefore, subtle differences in the level, distribution, and timing of signaling molecules can have major morphoregulatory consequences. Modulation of Bmp signaling exemplifies morphoregulation hypothesis: simple alteration of key signaling pathways can be used to transform a prototypical conical‐shaped tooth into one with complex morphology. The involvement of related pathways and the implication of morphoregulation in tooth evolution are discussed.

‘Cyclic alopecia’ in Msx2 mutants: defects in hair cycling and hair shaft differentiation

Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously — although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype.

Self-Organizing and Stochastic Behaviors During the Regeneration of Hair Stem Cells

Cycling of active and quiescent states of the hair follicle integrates activator and inhibitor signals for patterning. Stem cells cycle through active and quiescent states. Large populations of stem cells in an organ may cycle randomly or in a coordinated manner. Although stem cell cycling within single hair follicles has been studied, less is known about regenerative behavior in a hair follicle population. By combining predictive mathematical modeling with in vivo studies in mice and rabbits, we show that a follicle progresses through cycling stages by continuous integration of inputs from intrinsic follicular and extrinsic environmental signals based on universal patterning principles. Signaling from the WNT/bone morphogenetic protein activator/inhibitor pair is coopted to mediate interactions among follicles in the population. This regenerative strategy is robust and versatile because relative activator/inhibitor strengths can be modulated easily, adapting the organism to different physiological and evolutionary needs.

Dermal adipocytes protect against invasive Staphylococcus aureus skin infection

Skin infection triggers fat responses Obesity is associated with chronic inflammation, but does fat tissue offer protection during infection? Zhang et al. noticed that the fat layers in the skin of mice thickened after inoculation with the pathogenic bacterium Staphylococcus aureus (see the Perspective by Alcorn and Kolls). Mutant mice incapable of forming new fat cells were more susceptible to infection. The differentiating fat cells secreted a small-molecule peptide called cathelicidin, specifically in response to the infection. By contrast, mature fat cells produce less cathelicidin, and are thus less protective. Science, this issue p. 67; see also p. 26 The subcutaneous fat layer thickens during infection and stimulates adipocytes to secrete a protective peptide. [Also see Perspective by Alcorn and Kolls] Adipocytes have been suggested to be immunologically active, but their role in host defense is unclear. We observed rapid proliferation of preadipocytes and expansion of the dermal fat layer after infection of the skin by Staphylococcus aureus. Impaired adipogenesis resulted in increased infection as seen in Zfp423nur12 mice or in mice given inhibitors of peroxisome proliferator–activated receptor γ. This host defense function was mediated through the production of cathelicidin antimicrobial peptide from adipocytes because cathelicidin expression was decreased by inhibition of adipogenesis, and adipocytes from Camp−/− mice lost the capacity to inhibit bacterial growth. Together, these findings show that the production of an antimicrobial peptide by adipocytes is an important element for protection against S. aureus infection of the skin.

Fgf9 from dermal γδ T cells induces hair follicle neogenesis after wounding

Understanding molecular mechanisms for regeneration of hair follicles provides new opportunities for developing treatments for hair loss and other skin disorders. Here we show that fibroblast growth factor 9 (Fgf9), initially secreted by γδ T cells, modulates hair follicle regeneration after wounding the skin of adult mice. Reducing Fgf9 expression decreases this wound-induced hair neogenesis (WIHN). Conversely, overexpression of Fgf9 results in a two- to threefold increase in the number of neogenic hair follicles. We found that Fgf9 from γδ T cells triggers Wnt expression and subsequent Wnt activation in wound fibroblasts. Through a unique feedback mechanism, activated fibroblasts then express Fgf9, thus amplifying Wnt activity throughout the wound dermis during a crucial phase of skin regeneration. Notably, humans lack a robust population of resident dermal γδ T cells, potentially explaining their inability to regenerate hair after wounding. These findings highlight the essential relationship between the immune system and tissue regeneration. The importance of Fgf9 in hair follicle regeneration suggests that it could be used therapeutically in humans.

Morpho-Regulation of Ectodermal Organs : Integument Pathology and Phenotypic Variations in K14-Noggin Engineered Mice through Modulation of Bone Morphogenic Protein Pathway

Ectodermal organs are composed of keratinocytes organized in different ways during induction, morphogenesis, differentiation, and regenerative stages. We hypothesize that an imbalance of fundamental signaling pathways should affect multiple ectodermal organs in a spatio-temporal-dependent manner. We produced a K14-Noggin transgenic mouse to modulate bone morphogenic protein (BMP) activity and test the extent of this hypothesis. We observed thickened skin epidermis, increased hair density, altered hair types, faster anagen re-entry, and formation of compound vibrissa follicles. The eyelid opening was smaller and ectopic cilia formed at the expense of Meibomian glands. In the distal limb, there were agenesis and hyperpigmentation of claws, interdigital webbing, reduced footpads, and trans-differentiation of sweat glands into hairs. The size of external genitalia increased in both sexes, but they remained fertile. We conclude that modulation of BMP activity can affect the number of ectodermal organs by acting during induction stages, influence the size and shape by acting during morphogenesis stages, change phenotypes by acting during differentiation stages, and facilitate new growth by acting during regeneration stages. Therefore during organogenesis, BMP antagonists can produce a spectrum of phenotypes in a stage-dependent manner by adjusting the level of BMP activity. The distinction between phenotypic variations and pathological changes is discussed.

Epithelial stem cells and implications for wound repair.

Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis has a mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new interfollicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover the cellular and signaling basis of this remarkable adult wound regeneration phenomenon.

Regeneration of fat cells from myofibroblasts during wound healing

Hair follicles: Secret to prevent scars? Although some animals easily regenerate limbs and heal broken flesh, mammals are generally not so gifted. Wounding can leave scars, which are characterized by a lack of hair follicles and cutaneous fat. Plikus et al. now show that hair follicles in both mice and humans can convert myofibroblasts, the predominant dermal cell in a wound, into adipocytes (see the Perspective by Chan and Longaker). The hair follicles activated the bone morphogenetic protein (BMP) signaling pathway and adipocyte transcription factors in the myofibroblast. Thus, it may be possible to reduce scar formation after wounding by adding BMP. Science, this issue p. 748; see also p. 693 Hair follicles convert wound myofibroblasts to adipocytes through the bone morphogenetic protein signaling pathway, revealing a target to decrease scarring. Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro. Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans.