Past and Current Research
Stem Cells of the Skin and Their Lineages.
Skin begins as a single layer of multipotent epithelial cells, which give rise to epidermis, hair follicles, sebaceous and sweat glands. In adult skin, epidermis and hair follicles constantly renew, and our recent studies show that each adult hair follicles contains a reservoir of stem cells, called the bulge, which in vitro can self-renew and generate all of these different lineages when isolated and grafted onto Nude mice (Blanpain et al., 2004). To understand how a stem cell chooses its differentiation pathway, we have taken several approaches. An ongoing approach of the lab is to express different fluorescent proteins under the control of various skin promoters, active at different stages in stem cells and their lineages. Through fluorescent activated cell sorting (FACS), we've purified cells at different time points along the lineages and generated a battery of lineage-specific profiles, enabling us to define at an mRNA and chromatin level how stem cells change as they transition from quiescence to activation to lineage determination. The long term goal is to use this information to understand how stem cells receive signals, change their program of gene expression and select a lineage, and to understand the functional significance of these changes. Thus far, our focus has been on the stem cell compartment, where we have identified >150 mRNAs upregulated selectively in this niche (Tumbar et al., 2004; Blanpain et al., 2004). Among them are transcription factors Tcf3/Tcf4, Lhx2, NFATc1 and Sox9, all of which by our conditional targeting studies, turn out to be important for stem cell behavior and/or maintenance (Nguyen et al., 2006; 2009; Rhee et al., 2006; Horsley et al., 2008; Nowak et al., 2008). Additionally, the polycomb chromatin repressor complex plays a critical role, serving as a double-edged sword to control the fate switch from a stem cell to a committed, transit-amplifying state (Ezhkova et al., 2009; 2011; Lien et al., 2011). We continue to systematically work our way through the functional significance and mechanism of action of epigenetic and transcriptional controls on stem cells as they transit from a quiescent to activated to committed state. When coupled with our recent ability to efficiently knockdown genes in a few days using lentiviral-mediated shRNA delivery (Beronja et al., 2010), this now becomes a powerful tool for exploiting bioinformatics analyses to gain biological insights.
Another approach of the lab is to explore how these lineages are controlled by external signaling pathways that lead to transcriptional changes during skin development. An example of this approach is our interest in Wnt signaling and the skin. The Wnt/Wingless pathway was first studied in fly development, where defects affect the patterning of denticles on the body surface of the fly. Studies from the Fuchs' laboratory and others now reveal that this pathway also affects hair follicle development in mammals. At the heart of the Wnt signaling pathway is β-catenin, which normally is used only for cell-cell adhesion (see the previous section). In the presence of a Wnt signal, however, extra β-catenin is stabilized, enabling it to interact with and activate members of the Lef/Tcf family of DNA binding proteins. The Grosschedl lab showed that mice lacking Lef1 can't make proper hair follicles, while our lab showed that excess Lef1 or excess stabilized β-catenin results in excess hair follicles in inappropriate places (Zhou et al., 1995; Gat et al., 1998; Lowry et al., 2005).
To identify where and when Wnt signals are received in the skin, we engineered transgenic mice harboring a gene which expresses β-galactosidase (scored by a blue dye assay) whenever a cell receives a Wnt signal and is also expressing a Lef/Tcf protein. From these analyses, we learned that Wnt signals play key roles in mesenchymal-epithelial cross-talk and in the stem cell lineage involved in hair production (DasGupta and Fuchs, 1999; Merrill et al.,2001; Greco et al., 2009).
Relatives of Lef1, Tcf3 and Tcf4 are expressed by follicle stem cells. Our biochemical studies have suggested that these proteins can act in the absence of Wnt signaling (Merrill et al., 2001; Nguyen et al., 2009). To test the function of these Tcfs more rigorously, we've engineered mice that express a skin-specific tetracycline transactivator protein, enabling us to switch on the expression of genes and monitor the consequences (Nguyen et al., 2006). We've learned that Tcf3/4 control a group of genes that are characteristic of embryonic and adult skin stem cells, and that at high levels, Tcf3/4 inhibit all three programs of epidermal differentiation.
We are also beginning to examine how other signal transduction pathways in the skin impact on stem cell differentiation, and we've learned that interfering with the bone morphogenetic protein (BMP) pathway is also critical for follicle stem cells to be activated (Jamora et al., 2003; Kobielak et al., 2003; 2007). Interestingly, BMPs appear to act in part to control the expression of NFATc1, a calcium-sensitive transcription factor which represses genes involved in the G1 to S transition (Horsley et al., 2008). Recently, we discovered that intriguingly, at the end of a bout of hair regeneration, some committed stem cell progeny home back to the niche where they transmit strong BMP6 and FGF18 signals to the stem cells that tells them to stop making hair (Hsu et al., 2011). It will be interesting in the future to see if other stem cells niches have such negative feedback loops, which could be important to understanding how stem cells know not only when to be activated but also when to quit making tissue. Such responses are critical in understanding stem cell action in response to injury.
Finally, Notch signaling acts to regulate the switch from the progenitor to commitment state (Blanpain et al., 2006). Based upon our most recent studies, it appears to be linked to asymmetric cell division, as it is in the fly neuroblast (Williams et al., 2011). In the future, we hope to uncouple the underlying mechanisms involved.
Our ultimate goal is to understand how external signals from surrounding cells impact on skin stem cells to induce the expression of specific genes that enables them to remodel their cytoskeleton and adhesive contacts and either form a stratified epidermis or an epithelial bud that can then develop into a hair follicle (Perez-Moreno et al., 2003; Blanpain and Fuchs, 2009). While our model is the skin, the problem is a general one of how a single epithelial stem cell gives rise to a spatially organized, functional tissue. It is also integrally linked to understanding the basis of cancer progression, and to this end, we've focused our attention on the tumor-initiating, cancer stem cells of SCCs as delineated above.
