Our central objective is to explore the mechanisms governing skin SCs and their remarkable ability to both self-renew long term and to commit to proliferate and differentiate along a particular lineage. Skin is one of the few systems where adult SCs can be maintained and propagated in the laboratory, a feature that greatly facilitates our studies. We also use technologies for targeting foreign genes to epidermis and hairs of transgenic animals and for removing single epidermal or hair genes from the chromosomes of either mouse embryos (knockout) or skin (conditional inducible knockout). By taking a molecular approach to skin biology and SC research, we aim to apply this knowledge to human genetics, cancers, and medicine.
During development, skin begins as a single layer of multipotent embryonic progenitors that are able to produce either hair follicles (HFs) or epidermis. If they receive a signal from underlying mesenchymal cells, skin SCs respond by developing into HFs, which then produce sebaceous glands; in the absence of this cue, the cells stratify to make epidermis. While embryonic progenitors must form tissues, fully grown adult skin requires SCs to replace tissue cells lost in wear and tear (homeostasis) and to repair wounds on injury. In addition, hairs must regrow periodically throughout life.
The upper portion of every adult HF maintains a reservoir (niche) of infrequently dividing, multipotent SCs, which are used for these purposes. What activates the niche to mobilize its precious residents? The initiation of new hair growth and the characteristics of the hair and its cycle are determined in part by interactions between the epithelial SCs and specialized mesenchymal cells, referred to as dermal papillae (DP). Similar to the epithelial-mesenchymal interactions that occur in embryonic skin, signals from the DP to the epithelial SC niche are important to initiate a new hair cycle. By contrast, upon injury, SCs respond to growth factors released by damaged tissue.
To monitor the divisions and movements of adult skin SCs, we devised methods to specifically label epithelial skin SCs and DP with fluorescently tagged proteins. Using these methods, we learned that within their niche, most SCs divide infrequently and only during the growing phase of the hair cycle. Following stimulation by the DP, SCs at the base of their niche become activated to re-form the new HF. During the hair growth phase, SCs continue to exit the niche and generate progeny that proliferate rapidly, fueling the production of hair. SCs remaining in the niche cycle slowly to replenish those SCs exhausted during this active hair growth period. Following this growth phase, the SC niche returns to quiescence until the next hair cycle or injury. In this way, most of the skin SCs are safely sequestered in their niche and are subjected less frequently to chromosomal replication, which might introduce genetic errors.
We have thus far identified five different transcription factors and also chromatin (epigenetic) and post-transcriptional (microRNA) modifications in skin SCs. Our loss-of-function mouse models suggest that these factors govern essential features of SC maintenance and integrity. Intriguingly, some of these factors are also expressed by intestinal and breast SCs.
We also have used our adult skin SCs to clone mice. Referred to as nuclear transfer, this technology can also be used to generate embryonic stem (ES) cells. If this technology can be successfully adapted to humans, ES cells might be able to be generated from a patient's skin without genetically altering the skin cell's DNA. Additionally, the recent demonstration that skin epidermal cells can be genetically reprogrammed to make induced pluripotent stem (iPS) cells is particularly intriguing, and the enormous tissue-regenerative capacity of HF SCs makes them an especially attractive host for iPS technology. In the future, it may be possible to turn an adult skin SC into an ES cell by epigenetically reprogramming a small number of genes.
While multipotent SCs are carefully guarded within their specialized niche in the HF, the proliferative innermost (basal) layer of epidermal cells also harbors SCs that produce the stratified, terminally differentiated layers that are continually shed from the skin surface. We discovered that in embryonic skin, stratification involves a reorientation of the mitotic spindle, causing basal cells to undergo asymmetric cell divisions. In this process, the epidermis appears to use a set of evolutionarily conserved genes, which are also used in asymmetric divisions in fruit flies and worms. As an asymmetrically dividing cell enters mitosis, an apical crescent of proteins assembles and anchors one of the two spindle poles. On the basal side of the cell, integrins and growth factor receptors maintain contact with the underlying basement membrane. By orienting the spindle in this way, the innermost daughter retains its ability to divide while the detached, uppermost daughter differentiates and continues its journey outward.
This newfound mechanism for controlling epidermal growth and differentiation provides insights into how the delicate balance of growth and differentiation may be influenced. We are learning that when this balance is tipped by gene mutations that affect epidermal polarity, invasive squamous cell carcinoma (SQCC) is observed. One such gene encodes α-catenin, a cell-cell junction and regulatory protein.
Resident progenitors also exist at the base of the sebaceous gland. These cells are marked by expression of Blimp1, a transcriptional repressor for the gene encoding the cell cycle regulator c-Myc. When c-Myc is overexpressed, either transgenically or through ablation of the Blimp1 gene, sebaceous gland tumors develop. In the absence of Blimp1, multipotent SCs in HFs are mobilized to fuel production of gland cells, suggesting a precursor-product relationship between multipotent and resident SCs in skin.
We are also learning more about the multipotent and resident skin SCs and their differentiating progeny by exploiting fluorescence tagging to isolate and transcriptionally profile these cells. By identifying genes that are preferentially expressed in each of these cell populations, we are beginning to decipher the unique properties of skin SCs and how they relate to other SCs of the body. We have also developed methods to isolate pure populations of DP cells to learn how these cells communicate with the epithelial SCs to stimulate rounds of new hair growth. While we are still in the midst of these explorations, the ability to culture adult skin SCs in the laboratory is a major advantage in evaluating how SCs maintain a growth- and differentiation-inhibited environment in vivo, and how they become mobilized outside their niche.
We have learned that SCs express TCF3 and TCF4, DNA-binding proteins that appear to inhibit differentiation. In SC activation to make a HF, TCF3/TCF4 are converted from repressors to activators. Although the exact details remain to be elucidated, this switch involves the TCF-interacting protein β-catenin, which is stabilized and becomes nuclear upon convergent receipt of activating Wnt signals and inhibiting BMP signals. Among the genes that are subsequently transactivated are those involved in cell proliferation and migration. When Wnt signaling is overactivated or when BMP signaling is irreversibly compromised, HF tumorigenesis arises. Overall, we are learning that the very pathways involved in the activation of the SCs of epidermis, sebaceous gland, and HFs may contribute to cancer when deregulated. In the future, we will pursue the relation between SCs and cancers in skin.
As we continue to tackle our understanding of the environmental signaling and transcriptional changes that take place when SCs become activated to produce epidermis or hair follicles, we are pursuing our long-standing interest in how the extraordinarily distinct architectures of these two structures are established. Without the proper contacts between neighboring cells and extracellular matrix, epidermis is unable to keep harmful microbes out and body fluids in, hairs are not produced, and wounds cannot be repaired. Genetic defects in cell adhesion genes are prevalent in a variety of disorders, including human cancers, where tissue architecture is reduced to distorted masses of cells.
Over nearly three decades, we have uncovered many molecules that epidermal cells use to make adhesive connections and their associated mechanical frameworks (cytoskeletons). These investigations guided us to the genetic bases of blistering skin diseases as well as hyperproliferative disorders that resemble precancerous lesions. We are now gaining insights into how skin cells control and change the organization of their actin and microtubule cytoskeletons to achieve dynamic tissue organization. In this regard, we again focus on β-catenin and a relative, p120-catenin, this time in the context of their other well-known interacting partners, E-cadherin and α-catenin. These four proteins form the central components of cell-cell junctions, known as adherens junctions.
By integrating cell junctions and cytoskeletal dynamics, cells within the tissue can coordinate contractions and movements. Actin-microtubule-cell junction connections also provide the potential to polarize microtubule-based cargo transport and orient the mitotic spindle. Our studies are pointing to the view that such connections are important in orchestrating the orientation of cell divisions in skin and in controlling cellular movements upon SC activation and during wound repair.
We have begun to unravel the mechanisms governing these cellular dynamics and have uncovered new insights and new proteins involved. Of particular interest is that the integral components of adherens junctions each appear to have alternate roles. β-Catenin can function as a bipartite transcription factor in Wnt signaling, and p120-catenin and α-catenin regulate actin dynamics and proinflammatory and proliferative responses. We have developed new technologies that will facilitate our continued efforts in unfolding the molecular mechanisms underlying these complex communication networks in mice.
In closing, as we probe more deeply into an understanding of mammalian skin SCs and tissue morphogenesis, we are progressively learning more about how defects in this process can lead to various human genetic skin disorders, including skin cancers and inflammation.
