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CONCISE REVIEW |
a Department of Surgery and
b Departments of Dermatology and Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, USA
Key Words. Epithelial stem cells • Regulation • Stem cell niche • Intestine • Adult stem cells
Correspondence: Melissa H. Wong, Ph.D., Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, Oregon, 97239 USA. Telephone: 503-494-8749; Fax: 503-418-4266; e-mail: wongme{at}ohsu.edu
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ABSTRACT |
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INTRODUCTION |
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The Epithelial Stem Cell Niche
The epithelial stem cell niche provides the protective environment for the stem cell to promote its overall survival and protect its genetic code. Although reliable markers for epithelial stem cells have not been established in most of the epithelial-based organs, we generally know where they reside through labeling experiments with bromodeoxyurindine (BrdU) or 3H-thymidine. In rapidly renewing epithelia, the stem cell resides in a physically defined region, whereas in epithelia that does not frequently turn over (persistent epithelia), the niche is less well defined.
Regardless of the rate of turnover, by definition, the epithelial stem cell remains anchored within its niche and rarely divides. When it does divide, the stem cell does so in an asymmetric fashion, renewing itself and giving rise to a daughter cell (Fig. 1
). The daughter cells are often referred to as the transient amplifying (TA) population. This population expands cell numbers, proliferating more frequently than the anchored stem cell. Although these daughter cells give rise to descendents that travel down the path to terminal differentiation, some investigators hypothesize that they can also dedifferentiate to replace the stem cell if it is damaged. In some epithelial-based organs, all of these cells populate the stem cell niche. This presents the challenge of imparting the functional diversity of these cells within a confined physical region.
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Epithelia can broadly be classified into two general categories based on its turnover time: rapidly self-renewing and persistent. Rapidly self-renewing epithelium is found in organs in which the epithelium is exposed to the external environment. In these tissues (e.g., skin, intestine, eye), the primary function of this epithelium is to act as a protective barrier. Therefore, the epithelium is rapidly replaced and the TA population is actively proliferating. In most of these organs, the epithelium is organized in discrete, easily replaceable units that are populated by a small number of active stem cells. Interestingly, these tissues have physically well-defined stem cell niches that promote an ordered stem cell hierarchy and support protection of the stem cell.
In persistent epithelium (e.g., kidney, liver, and mammary gland), the epithelium does not undergo constant turnover. In these organs, the organization of the stem cell niche and hierarchy of cellular structures are less understood. Recently, a putative kidney epithelial stem cell was identified by BrdU labeling [1]. The label-retaining cells were scattered among the tubular epithelial cells and did not seem to be confined to an organized protective niche. Likewise, in the mammary gland, BrdU labeling recently identified an asymmetrically dividing cell as a candidate for the mammary stem cell [2–4]. Although the mammary gland also does not undergo rapid renewal, it is unique in that it undergoes proliferation at puberty, during the monthly estrus cycle, and during pregnancy. It is likely that regulation of this epithelial stem cell is much more complex than in either rapidly renewed or persistent epithelium and therefore may fit into a category unto itself. Although our understanding of the persistent epithelial stem cell has grown in the last few years, much of the work defining the epithelial stem cell niche has been performed in rapidly self-renewing epithelium. Therefore, this review will focus on comparing the insights gained from studies in the intestine and the skin.
Rapidly Renewing Epithelium
Intestinal Epithelium
The adult intestinal epithelium of the gastrointestinal tract has a well-defined organizational structure (Fig. 2). The epithelium can be divided into two regions, a functional region that houses differentiated cells (villi) and a proliferative region (crypts of Lieberkühn) that represents the epithelial stem cell niche. In the small intestine, finger-like projections protrude from the floor of the intestine, forming villi. Multiple proliferative crypts surround each villus base. All epithelial cells that populate the villus originate from these crypts. Multipotent epithelial stem cells reside in the crypts and give rise to four principal epithelial lineages: absorptive enterocytes, mucin secreting-goblet cells, peptide hormone secreting enteroendocrine cells, and Paneth cells. Three of these cell lineages differentiate as they migrate up and out of the crypts onto the adjacent villus. It takes cells 3–5 days to migrate up the villus. As they reach the villus tip, they undergo apoptosis or are exfoliated into the lumen. The fourth lineage, Paneth cells, differentiates with a downward migration to reside at the base of the crypt. These cells are involved in mucosal immunity and secrete various proteins, including tumor necrosis factor, lysozyme, and cryptins [5]. Paneth cells are longer lived than cells that populate the villus, surviving 18–23 days before they are phagocytosed by surrounding cells [6].
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The stem cells of the small intestine have developed mechanisms to ensure their viability when exposed to damaging agents. To preserve the original DNA content, it is proposed that sorting of DNA during stem cell division constitutes one protective mechanism. In this scenario, replication-induced errors are segregated to the daughter cells, thus protecting the stem cell from retaining genetic damage [11]. In addition, DNA damage that occurs to the stem cell might trigger a p53-dependent apoptosis, allowing a stem cell to sacrifice itself to prevent any retention of genetic errors that could be passed on to daughter cells [12, 13]. Potten and colleagues [14] propose a three-tiered hierarchical system of the intestinal epithelial stem cell niche. In this proposal, proliferation within the niche is sufficient for accommodating the upward migration and death of the differentiated epithelium. During crypt development, an ancestral stem cell is responsible for establishing the adult crypt niche and represents the first tier of cells. This cell is dormant in the adult crypt, after it has given rise to a subset of four to six active stem cells. These active stem cells represent the second tier of cells. They undergo asymmetric cell division, producing one daughter stem cell that will remain anchored in the niche and a second daughter cell that will undergo further division to populate the epithelium with terminally differentiated cells. The immediate daughter cells represent the third tier located just above the active stem cells. These daughter cells are thought to comprise a portion of the TA population. To ensure crypt survival, these 20 to 30 TA cells possess the ability to be committed to a differentiated cell fate and dedifferentiate into second-tier stem cells if an active stem cell is damaged or lost. Differences in susceptibility to gamma irradiation provide evidence that cells within these different cell tiers possess unique functional purposes within this hierarchy. Studies with low-dose gamma irradiation suggest that the second tier of cells is significantly more radio-resistant than the ancestral stem cells. The data suggest that these cells retain stem cell properties (able to self-renew) and can replenish the crypt in situations of crypt damage [15, 16]. In these studies, the third tier of cells is even more radio-resistant and also retains clonogenic capabilities. It is proposed that second-tier cells have the ability to replace first tier stem cells upon their death (Fig. 1
) [15]. In this fashion, cell–cell interactions between these epithelial cell layers influence stem cell behavior.
The stem cell niche also encompasses a mesenchymal component. The epithelium in the crypt directly contacts the underlying mesenchymal layer. This pericryptal mesenchyme is comprised of extracellular matrix, enteric neurons, blood vessels, intraepithelial lymphocytes, and pericryptal fibroblasts. Although it is clear that the cells in the mesenchyme are responsible for communicating with the overlying epithelium, the exact cells that secrete all of the important signaling factors have not been identified. The pericryptal fibroblasts have been shown to secrete various factors such as Hepatocyte growth factor, Tissue growth factor-ß (Tgf-ß), and Keratinocyte growth factor [17]. Corresponding receptors for these factors are located on the epithelial cells, supporting the importance of epithelial–mesenchymal cross-talk.
In addition, the pericryptal fibroblasts are thought to intimately influence the epithelium. Experiments using 3H-thymidine labeling indicate that the pericryptal fibroblasts migrate up along the crypt-villus axis at a similar rate as the differentiating epithelium [18].
In addition to factors secreted by the pericryptal fibroblasts, other cells within the pericryptal mesenchyme secrete factors that influence the overlying epithelium. Platelet-derived growth factor (Pdgf) is one such factor, because mice deficient for intestinal expression of Pdgf-
or its receptor Pdgfr- developed abnormal intestinal epithelium and depletion of the pericryptal mesenchyme [19]. Sonic hedgehog (Shh), Bone morphogenic protein (Bmp), Forkhead-6 (Fkh), Wnt, Notch, and the nuclear transcripton factor Nkx3-3 are among other factors that have been shown to influence the intestinal epithelium [20–24]. Although many of the factors originate in the mesenchymal compartment of the intestine, the epithelium also participates in this instructive cross-talk. Enteroendocrine cells secrete glucagon-like peptide, which stimulates mesenchymal enteric neurons to secrete an unknown factor that is thought to stimulate enterocyte production [25]. These observations all strongly support the existence of an intimate relationship between these two cell populations.
Epidermal Epithelium Like the intestinal epithelial lining, the mammalian skin represents a physical barrier between the body and the external environment. The epidermis receives the brunt of the damage caused by physical trauma and mutagenic ultraviolet radiation. Therefore, similar to the intestinal epithelium in protecting against accumulation of mutations, the epidermis is also characterized by rapid cellular turnover. The human epidermis sloughs off and is replaced approximately every 14–21 days, whereas mouse skin is renewed every 10–14 days [26, 27].
The mammalian epidermis is a stratified tissue comprised of four layers: a basal layer containing epidermal stem cells and a TA population, a spinous layer comprised of differentiating cells, a granular layer comprised of differentiated cells, and a stratum corneum populated by dead cells. Like the intestine, the epidermis is organized into discrete units. In the mouse, 10-cell-wide columns of maturing cell layers are organized into hexagonal units referred to as epidermal proliferative units (EPUs) [28–31]. Genetic tagging of keratinocytes revealed that keratinocyte stem cells populate each EPU and reside in the middle of the basal cell cluster. Although an exact number of these keratinocyte stem cells has not been determined, it is thought that as many as 10%–12% of murine basal layer cells might possess stem cell properties, allowing them to be capable of generating a single maturing column of cells [32, 33].
The keratinocyte stem cell possesses a low rate of mitotic activity. Upon division, the keratinocyte stem cell not only self-renews but also gives rise to a rapidly dividing TA population of cells. As the TA population detaches from the basal layer and migrates into the spinous layer, it begins differentiation. Cells that reside above the basal layer are connected by small bridge-like structures called desmosomes and synthesize a host of granules and proteins, including keratohyaline granulaes, profilagrin, loricrin, incolucrin, and cornifin. These molecules are important for formation of the stratum corneum. As the terminally differentiated keratinocytes migrate to the body’s surface, they begin a self-destruction program, becoming enucleated and sloughed off [34, 35].
The keratinocyte stem cell is derived from a multipotent stem cell that resides in the hair follicle bulge. This multipotent stem cell also gives rise to cells of the dermal papillae and sebaceous gland (Fig. 2). 3H-thymidine labeling studies indicated that most label-retaining cells (LRCs) in the skin reside in the bulge region of the hair follicle, with only a small fraction found in the basal layer [36, 37]. Transplant and tracking experiments of LacZ-marked bulge cells into wild-type animals elegantly illustrate that stem cells originating from the bulge give rise to basal and dermal papilla cells [38]. The bulge region provides an attractive niche for the epidermal stem cell, because it constitutes a protective region at the base of the epithelial portion of the hair follicle. Many of the classic developmental signaling pathways have been implicated in epidermal development and also in adult epidermal homeostasis. The factors that are required to stimulate the bulge stem cell to asymmetrically divide, migrate, and reanchor in the epidermis or epidermal appendages have recently been identified in a series of elegant microarray studies [39, 40]. Using a doxycycline-inducible Green Fluorescent Protein (GFP) construct, Tumbar et al. labeled stem cells in the hair follicle of mice. Then they turned GFP off for several days to identify and isolate long-lived, label-retaining cells by fluorescence-activated cell sorter. Microarray analysis comparing three populations of cells (high GFP-expressing, low GFP-expressing, and all skin cells) revealed molecules that could be classified into categories, including cell signaling, cell-cycle inhibitors, basal lamina, and extracellular matrix molecules. Interestingly, 68% of the mRNAs identified were also identified in similar studies examining hematopoietic stem cells, embryonic stem cells, and neural stem cells [39, 40]. Similar studies performed by Morris et al. [41] showed similar results. These elegant studies highlight the importance of cell signaling pathways in influencing the stem cell.
Factors Regulating the Stem Cell
Identifying how signaling pathways regulate epithelial stem cells either indirectly by influencing the niche or directly by regulating proliferative status is a critical step toward gaining the ability to manipulate these adult cells for therapeutic approaches. This challenge is not an easy one. Currently, major developmental signaling pathways have been implicated in regulation of epithelial stem cells [39–42]. We will examine how four of the major developmental signaling pathways influence stem cells by comparing what is known in the skin with what is known in the gut.
Wnt Signaling
The canonical Wnt signaling pathway plays a key role in development, cellular homeostasis, and disease. In the context of stem cells, the Wnt signaling pathway and its downstream transcription factors Lymphoid enhancer factor-1/T-cell factor (Lef/Tcf) are intimately involved in the maintenance of the niche (Fig. 3). The secreted Wnt proteins bind to two receptors, Frizzled and Low density lipoprotein-related receptor protein (Lrp), to transduce the signal. Activation of the signaling cascade results in inhibition of Glycogen-synthase kinase-3ß (Gsk-3ß), which under non-Wnt signaling conditions phosphorylates ß-catenin when bound to the Adenomatous polyposis coli (Apc)/Axin/Gsk-3ß scaffold to promote ß-catenin degradation. In the presence of the Wnt signal, the cytoplasmic levels of unphosphorylated ß-catenin increase, allowing ß-catenin to enter the nucleus and interact with Lef/Tcf high motility group (HMG) box transcription factors. This interaction promotes transcription of Wnt target genes. Many of the target genes are important for cell proliferation, cell polarity, and cell fate decisions [43].
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In the intestine, Wnt is an important developmental and adult signaling factor. During development, Wnt signaling is critical for maintaining the proliferative pressure in the stem cellniche. The intestines from mice deficient for the HMG box transcription factor, Tcf-4, developed normally until embryonic day (E) 16.5, when crypt structures began to form [47]. In these intestines, the proliferative compartment was devoid of proliferating cells based on lack of Ki-67 and BrdU staining. Interestingly, cells that were normally proliferating underwent inappropriate differentiation. This suggests that Wnt signaling is critical for either maintenance of proliferation within the stem cell niche or inhibition of differentiation of the TA cells. In homozygous Dickoff-1 (Dkk-1) transgenic intestines, suppression of Wnt signaling also resulted in suppression of proliferation in the stem cell niche [24]. Although Wnt signaling was suppressed in the intestine at E14.5, similarly to the Tcf-4–/– intestines, these mice survived to adulthood. In addition, temporal induction of Dkk-1 expression in the adult mouse intestine via adenoviral infection resulted in suppression of proliferation without an effect on survival of the animal [48]. The discrepancy in phenotypes elicited from Wnt signaling suppression by Dkk-1 expression compared with Tcf-4 ablation reflects additional function for Tcf-4 in maintaining the stem cell, perhaps, or an incomplete suppression of the Wnt signal by Dkk-1. Regardless, these experiments suggest that Wnt is a potent growth factor in the intestine.
Although Wnt signaling is critical for sustaining proliferation in the intestinal stem cell niche, different levels of Wnt signaling may result in different effects on stem cell behavior. We and our colleagues demonstrated that expression of a Wnt signaling molecule, a fusion between ß-catenin and Lef-1, in the chimeric mouse small intestine stimulated apoptosis in stem cells expressing the fusion transgene [49]. Intestines from these chimeric mice were devoid of transgene-expressing cells, suggesting that Wnt signaling is a critical factor in designating which stem cells will be anchored in each adult stem crypt during crypt morphogenesis. These experiments suggest that although Wnt signaling is critical for maintaining proliferation in the stem cell niche, the gradient of Wnt (or levels of Wnt) may be received and interpreted differently by these cells.
The Wnt pathway may also play a role in directing cell differentiation. Using Caco-2 cells in culture, Mariadason et al. [50] showed that downregulation of Wnt/ß-catenin signaling resulted in an increase in promoter activities of alkaline phosphatase and fatty acid binding protein, two markers of epithelial cell differentiation [50]. In addition, Clevers and colleagues [51] used DNA microarrays and a colon carcinoma cell line to identify genes that respond to Wnt signaling. Most of the genes they identified were localized to proliferative crypts. Identification of c-MYC (myelocytomatosis oncogene) supported the previous report that this gene is a Wnt target [52]. Furthermore, they went on to show that expression of c-MYC disrupted expression of the cell cycle inhibitor p21CIP1/WAF1. Because p21CIP1/WAF1 has previously been shown to be expressed in differentiated colon epithelium [53], van der Wetering et al. [51] propose that Wnt signaling results in increased c-MYC expression to allow cell proliferation and concomitantly inhibits p21CIP1/WAF1 to suppress epithelial differentiation. Additional support for a role for Wnt signaling in epithelial differentiation comes from the transgenic mice expressing Dkk-1. Intestines from these mice were devoid of secretory lineages, suggesting that Wnt expression actively promotes secretory lineage differentiation [24].
Wnt signaling also plays a role in maintaining the intestinal stem cell by directing migration of cells within the niche. The Wnt target genes EphB2 and EphB3 [51] play a role in cellular migration within the stem cell niche. Eph receptors are part of the tyrosine kinase receptor family and are involved in vascular development, tissue border formation, regulation of cell shape, and migration [54]. EphB2 and EphB3 are expressed in the crypts of wild-type adult mice. EphB3 expression is restricted to the Paneth cell population. EphB3+/– and EphB3–/– mice developed normal-appearing villi but display abnormal distribution of Paneth cells. This suggests that Ephrins are involved in restricting intermingling of proliferative and differentiated cell populations. Although it appears that Wnt signaling within the stem cell niche touches all aspects of stem cell maintenance, some of the influences may be through indirect means. The effects of Wnt signaling on the epidermal stem cell niche may provide us with insight into this.
In the skin, the canonical Wnt signaling pathway clearly impacts the behavior of the epidermal stem cell during development and in the adult tissue. A Wnt reporter mouse designed in Elaine Fuchs’ laboratory designates robust Wnt signaling via LacZ expression during both mouse development and adulthood [55]. LacZ activation in the embryonic epithelial progenitor cell of the hair follicle suggests that multipotent skin stem cells may communicate with their environment at least in part through Wnt signals. In addition, components of the Wnt signaling pathway, ß-catenin, Lef-1, and Tcf-3, are expressed in the dermal papillae and the matrix cells, indicating that these epidermal structures can also respond to the Wnt signal [56–58]. Members of the Wnt signaling pathway are expressed during epidermal development and adulthood. Lef-1, the downstream transcription factor of the Wnt signaling pathway, was shown to regulate developmental expression of keratin genes [57]. Lef-1 is expressed in matrix cells and in the nucleus of hair precursor cells just before keratins are expressed [55, 59, 60].
Wnt signaling plays a proliferative role in the epidermis. Lef-1 has been both knocked-out and overexpressed in mouse skin. In the absence of Lef-1, mice have sparse hair and loss of whiskers and secondary follicles [56]. When Lef-1 is overexpressed, mice form ectopic hair [57]. Likewise, when a stabilized ß-catenin molecule is expressed in the skin, de novo hair follicles are formed [59]. In addition, these mice also displayed unchecked proliferation in their epidermis. Although these important studies suggest that Wnt signaling plays an important role in hair follicle morphogenesis, they also implicate Wnt signaling in proliferation.
Wnt signaling within the skin plays a role in cellular differentiation. When Wnt signaling was suppressed by expressing a mutant Lef-1 molecule that does not bind to ß-catenin, a shift in epidermal fates toward interfollicular epidermis and sebocytes was observed [61]. These mice exhibited sebaceous tumors that express high levels of Indian Hedgehog (Ihh) and the receptor Patched (Ptch). Additionally, in mice lacking epidermal expression of ß-catenin, stem cells fail to differentiate into follicular keratinocytes [62]. Furthermore, c-Myc, a downstream target of Wnt signaling, can stimulate differentiation of epidermis in vitro [63]. In vivo studies overexpressing c-Myc in the basal layer of the epidermis resulted in an increase in sebaceous gland size, suggesting that Wnt signaling promotes sebocyte differentiation [64]. Tcf3 and Lef1 are also thought to control differentiation of epidermal lineages, although by different mechanisms. Epidermal expression of dominant-negative mutations in Tcf3 or Lef-1 revealed that Lef-1 acts to determine hair follicle fate, whereas Tcf-3 acts independently of Wnt to maintain the stem cell compartment [60].
Wnt signaling may also impact cellular adhesion and migration within the epidermal stem cell niche. Forced expression of the downstream Wnt target c-Myc resulted in depletion of the stem cells within the epidermal stem cell niche as the animal aged [65, 66]. Frye et al. [67] suggest that c-Myc acts to stimulate cells to exit the stem cell compartment by modulating the adhesiveness of the stem cell niche. They go on to suggest that a cell’s failure to differentiate may reflect its failure to migrate from the niche [67].
Wnt signaling impacts the proliferative status of the epithelial stem cell niche in both the intestine and the skin and seems to affect cell fate decisions. It is easy to see how Wnts might have these diverse roles in the skin, because reception of the Wnt signal may be context dependent; stem cells of the bulge may be influenced differently from stem cells in the outer root sheath (ORS) or basal cell layer. However, the intestinal stem cell niche is a relatively small, regionally defined area posing the conundrum of how a single factor could influence neighboring cells differently. One possibility is that Wnts may act as a morphogen within this region, allowing cells to respond differently to different levels of Wnt. Alternatively, differentiation of cell lineages may be indirectly regulated by Wnt, through regulation of Notch signaling (see below). Although this seems to be a possibility in the intestinal stem cell niche, it remains to be seen if this scenario might exist in the epidermis.
Notch Signaling
Notch and Wnt signaling are intimately regulated in the intestine. Intestines from mice expressing the Wnt inhibitor, Dkk-1, had a depletion of secretory cell lineages and suppressed expression of the Notch pathway molecule, Math-1 [24]. Notch proteins are involved in various aspects of vertebrate cell fate determination, including lateral inhibition of adjacent cells, such that they do not adopt the same cell fate [68, 69]. The Notch gene encodes a transmembrane receptor that interacts with neighboring cell surface ligands, Delta and Jagged (Fig. 3). Upon ligand binding, the intracellular domain of Notch undergoes cleavage and translocates to the nucleus to activate the transcription factor Suppressor of Hairless (SuH). This in turn results in upregulation of downstream target genes such as hairy/enhancer of split (HES) [69]. HES proteins inhibit the activity of various basic helix-loop-helix transcriptional activators, including Math-1 and neurogenin-3. Immunohistochemistry of the mouse intestine reveals expression of the four Notch receptors (Notch 1 through 4), five ligands (Delta 1, 3, and 4 and Jagged 1 and 2), and four Hes genes (Hes 1, 5, 6, 7) at both embryonic and adult time points [70].
Notch signaling actively designates the intestinal secretory cell lineage. Intestinal phenotypes described from a series of knockout mice support the role of Notch signaling in defining the intestinal stem cell hierarchy. Hes1–/– mice revealed precocious development of endocrine cells in the stomach and small intestine at embryonic time points as well as an increased number of goblet cells and fewer enterocytes [71]. These changes in differentiation were independent of proliferative effects. Because normal expression of Hes-1 is restricted to the nonproliferating villus in E17 mouse intestines, a role for Hes-1 in securing the secondary fate of differentiated cells is likely. In this scenario, Notch signaling would impact cellular differentiation rather than proliferation of the TA cell population.
Additional evidence implicating Notch signaling in cellular differentiation decision was observed in the Math-1–null mice [23]. The loss of Math-1 in embryonic mouse intestines resulted in complete depletion of Paneth, goblet, and enteroendocrine cells, the three secretory cell lineages. The proliferative regions of these mice also exhibited an increase in the number of cycling cells, which might reflect a compensatory mechanism to maintain villus cell census by increasing the number of absorptive enterocytes. Finally, neurogenin-3–null mice also failed to develop enteroendocrine cells within their intestines [72]. Interestingly, Paneth and goblet cells were detected, suggesting that perhaps additional factors are important in defining these three secretory cell lineages. For example, activation of Rac1, a member of the Rho GTPase family of GTP-binding proteins, in the mouse intestinal epithelium resulted in Paneth and goblet cell depletion, suggesting that Rac1 may be involved in differentiation of a Paneth/goblet precursor cell [73].
The Notch signaling pathway is clearly implicated in cellular differentiation in the intestinal epithelium and has a similar role in cellular epidermal differentiation [74]. Components of the Notch signaling pathway are expressed in the epidermis. Further, the Notch receptor, Delta, is expressed primarily in the basal layer of the epidermis, where the keratinocyte stem cells reside [75]. Inactivation of Notch1 in the epidermis resulted in depression of Wnt/ß-catenin signaling in cells that normally undergo differentiation. This suggests that Notch1 acts to suppress pathways that normally promote proliferation [76].
In vitro overexpression of Delta in keratinocytes resulted in failure of cells to respond to neighboring cells and blocked these cells from undergoing terminal differentiation [77]. This observation suggests that Notch signaling promotes epidermal differentiation by promoting migration of differentiating cells. In this fashion, stem cells that are more cohesive will cluster within the niche, whereas cells that are less cohesive respond to Notch signaling by migrating away from the stem cell cluster and differentiating. A computerized model of the basal cell layer nicely illustrates how Notch signaling could promote clustering of stem cells within the niche through lateral inhibition [78]. In vitro induction of Notch signaling induced expression of early epidermal differentiation markers and suppressed late markers [76, 79]. In addition, epidermal specific ablation of Notch1 resulted in aberrant keratinocyte differentiation, whereas activated Notch1 expression resulted in keratinocyte growth arrest [79, 80].
In both the epidermal and intestinal stem cell niche, Notch signaling is implicated in determining cellular differentiation. In the epidermis, it is clear that the overall levels of Delta are important in Notch responsiveness. Likewise, the effect of Notch signaling on actively defining the secretory lineages of the intestine may also be defined by Notch responsiveness, because neurogenin-3–null mice only have defects in one secretory cell lineage, whereas Math-1–null mice have defects in all three secretory cell lineages. Although it is unclear what supports the proliferation to differentiation gradient within the crypt, it may in part be influenced by Notch-induced lateral inhibition similar to what is seen during drosophila eye development [81]. However, one issue is clear: Notch does not act on its own to direct cellular differentiation. Interestingly, inducible Notch1 ablation in skin and in primary keratinocyte cultures stimulated expression Gli2 and led to formation of basal cell carcinoma–like tumors. This illustrates a close relationship between Notch and hedgehog (Hh) signaling pathways in directing cellular differentiation [76].
Hedgehog Signaling
The Hh gene is involved in various aspects of embryonic development such as left-right asymmetry, anterior-posterior patterning of the limb bud, and neural tube formation [82–84]. In vertebrates, there are three Hh genes that share similar homology; Shh, Ihh, and Desert hedgehog (Dhh). Shh is a protein secreted by endodermal epithelium and induces the expression of its receptor, Ptch, in the surrounding mesenchyme (Fig. 3). Hh proteins bind to a transmembrane receptor Ptch, which normally inhibits downstream signaling through a second transmembrane protein, Smoothened (Smo) [85]. Uninhibited Smo acts on downstream transcription factors Gli and HRK4 through unknown mechanisms to transduce the signal [86, 87].
Shh and Ihh expression during gastrointestinal development mediates anterior-posterior patterning, radial patterning, and epithelial stem cell proliferation and differentiation. Mice null for Shh or Ihh died before birth but exhibited interesting intestinal phenotypes [22]. Additionally, these mice primarily displayed gross developmental defects that included intestinal transformation of the stomach, duodenal stenosis, aganglionic colon, and imperforate anus. Interestingly, only Ihh–/– mice displayed a reduction in villus size and a repression of cell proliferation within the stem cell niche. Expression of the Wnt signaling mediator Tcf-4 was normal in these intestines, suggesting that suppression of proliferation was not due to loss of Tcf-4 expression. Contrarily, experiments suppressing Hh signaling by systemic injection of an anti-Hh antibody resulted in disorganized intestines that contained vacuolated epithelium and defective lipid processing but no affect on proliferation within the stem cell compartment [88]. Perhaps this difference in phenotype is the result of a critical temporal requirement for Hh signaling during morphogenesis that can only be appreciated if the pathway is perturbed during embryogenesis. Alternatively, systemic suppression of Hh signaling may elicit a different phenotype by indirectly affecting the intestine.
Although Hh signaling impacts overall intestinal morphogenesis, it also plays a role in epithelial differentiation. Inhibition of Ihh signaling by injection of the Hh inhibitor, cyclopamine, in the colon epithelium resulted in abnormal villin expression and loss of carbonic anhydrase IV expression, two enterocyte differentiation products. This suggests that Hh signaling may directly impact enterocyte differentiation [89]. In addition, Ihh may coordinately regulate Wnt expression within the colonic stem cell niche, because downstream Wnt target genes engrailed-1, Cyclin D1, and Bmp-4 were upregulated and mislocalized. These studies suggest that Ihh may act to restrict Wnt-responsive cells to the stem cell compartment.
In the skin, mutations in the Shh signaling pathway are most notably associated with occurrence of basal cell carcinoma [90]. The phenotype of this cancer reflects the relatively undifferentiated phenotype of the hair follicle ORS, suggesting that Shh signaling plays a role in basal cell differentiation. In addition, skin grafts from Shh-null embryos onto severe combined immunodeficiency (SCID) mice resulted in formation of large abnormal follicles containing small dermal papilla and high rates of proliferation with little differentiation of hair matrix cells [91, 92]. Interestingly, Wnt10b and Bmp2/4 expression was normal in the skin of these grafts. This suggests that Shh is not required for hair placode formation or for the initial growth of the epidermis but it is critical for hair formation [91, 92].
Overexpression of Shh in basal cells resulted in a wide variety of epidermal phenotypes that depended on temporal expression [93]. Furthermore, overexpression of Ptch also resulted in a variation of epidermal tumor formation that was correlated with the hair follicle cycle [94]. These studies suggest that Shh requires additional factors to regulate its temporal expression during hair follicle morphogenesis.
Shh is implicated in epidermal differentiation. Activation of Shh resulted in increased hair follicle development [95]. Furthermore, when Shh was inhibited, sebaceous gland differentiation was blocked. Interestingly, when Shh was overexpressed from a keratin promoter, inappropriate development of sebaceous glands occurred in the mouse footpads [96].
Hh signaling pathways seem to influence cellular differentiation in both the intestine and the skin. Temporal regulation of Hh expression is important for patterning during intestinal morphogenesis. Although it does not seem to play a critical role in hair placode morphogenesis in the skin, precise temporal Hh expression is critical in defining epidermal cell fates in coordination with the cycling follicle. In addition, dysregulation of Shh within the skin is associated with a lineage shift to basal cells, whereas in the intestine, suppressing Hh signaling results in a shift to the enterocyte lineage. Interestingly, a recent report suggested that Ihh acts to restrict Wnt-responsive epithelium to the proliferative zone of the colon crypt, and these signaling pathways act to reciprocally inhibit one another [89]. This suggestion might also apply in the epidermal stem cell niche, in which ß-catenin–null epidermis resulted in depletion of Shh expression [62]. Furthermore, both suppression of Wnt signaling and stimulation of Shh signaling resulted in sebocyte differentiation, supporting this notion.
BMP Signaling
Bmps are members of the Tgf-ß superfamily of secreted signaling molecules. Bmps have important functions in many biological contexts, including those important during embryogenesis. Bmps bind to specific serine/theronine kinase receptors, which transduce the signal to the cell nucleus through Smad proteins (Fig. 3).
Although not much is known about Bmp signaling within the intestinal stem cell niche, a role for this pathway is clear, because mutations in BMP-4 are associated with the polyp-forming disease juvenile polyposis in humans [97]. Mice deficient in the Bmp molecule, Smad4, also form polyps in their intestinal epithelium but lacked some of the hallmarks of the human disease [98]. Most recently, intestinal expression of the Bmp inhibitor Noggin resulted in de novo, ectopic crypt formation perpendicular to the villus axis in the mouse small intestine [99]. Except for their inappropriate location, these crypts appear normal, expressing factors such as Wnt target genes c-Myc and EphB3 that are found in normal crypts. This provides evidence that Bmp-4 is involved in shaping the intestinal stem cell niche.
The winged helix transcription factor Fkh6 is a regulatory protein expressed only in the pericryptal mesenchyme. Fkh6–/– mice developed proliferation of cells in the intervillus region as well as along the villi [21]. The villi of the small intestine contained all four cell lineages; however, there was an increase in goblet cells, suggesting a role of Fkh6 in epithelial differentiation. Possible downstream mediators involved may include Bmp2 and Bpm4, because these were both reduced in the mutant mice. Interestingly, the morphological changes seen in the Fkh6–/– mice were restricted to the stomach and proximal small intestine, even though Fkh6 is expressed along the entire intestinal tract in a wild-type mouse.
Bmps play a critical role in skin development [100]. Bmps, their receptors, and their downstream mediators display differential expression patterns that are highly suggestive of a critical role in epidermal morphogenesis. In postnatal skin, Bmps stimulate keratinocyte differentiation by inhibiting anagen initiation of the hair follicle cycle and stimulating hair shaft–specific differentiation [101]. Likewise, Bmp-4 has been shown to induce epidermal differentiation during development in mice [102]. Because the adult epidermis is a rapidly renewing tissue, Bmp-4 may also be important for adult epidermal homeostasis. Other Bmps are implicated in stimulation of cell proliferation and differentiation based on mRNA expression in the basal epidermal layer during embryonic development [103]. Furthermore, Blanpain et al. [42] suggest that BMP6 is involved in slowing down keratinocyte cell growth without inducing terminal differentiation. This provides evidence that BMPs are involved in maintaining stem cells in a quiescent state.
In both the intestine and the skin, Bmp signaling appears to negatively modulate ß-catenin signaling during development. In the skin, Bmps and Wnts are interregulated [58]. When mouse skin null for the Bmp inhibitor Noggin was engrafted onto SCID mice, ß-catenin expression was downregulated and secondary follicles were not supported [103]. This result suggests that there is a regulatory crosstalk between Wnt and Bmp signaling during hair follicle development. Yet how these pathways intersect to maintain or influence the stem cell/niche is not yet clear.
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Although the skin and the intestine have specific structures and diverse functions, their epithelial surfaces are rapidly self-renewing. Both niches are populated by a multi-potent stem cell that gives rise to multiple lineages that populate the epithelium. Both niches undergo a rapid expansion of the TA population to accommodate replacement of the epithelium. Both niches are situated in a protected environment in which epithelial-mesenchymal crosstalk is promoted. Finally, both niches depend on cellular migration within the niche to promote differentiation of their lineages. Therefore, it is compelling to think that these two stem cell niches are regulated in a similar fashion.
Lessons from the skin provide greater insight into the complex interactions between signaling pathways that regulate the intestinal stem cell niche. Given the current experimental findings from both systems, we propose the following model for regulation of the intestinal stem cell niche (Fig. 4). Signaling factors are responsible for defining the physical stem cell niche. Bmp expression within the mesenchymal compartment of the small intestine is maintained by epithelial expression of Ihh. Bmp-4, in turn, has an inhibitory effect on Wnt expression and restricts localization of Wnt-expressing cells to the mesenchyme around the crypt base. Wnt-expressing cells also actively restrict their physical location by stimulating Bmp-4 expression. In addition, Ihh expression is restricted to the differentiated villus epithelium by a (yet-to-be-identified) Wnt-responsive factor within the crypt. Furthermore, Ihh may also modulate the Wnt expression domain indirectly through its regulation of Bmp4. These factors act together to define a border between the proliferative stem cell niche and the differentiated villus epithelium.
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It is clear that the signaling pathways that define the stem cell niche are intertwined. The maintenance and propagation of the stem cell critically depends on the tight regulatory relationships within this signaling network. A systematic approach to manipulating these signaling pathways during development and an inducible approach for manipulation in the adult is greatly needed. Additionally, comparison of epithelial stem cell niches from different organs will continue to broaden our understanding of how these regions are defined and regulated.
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-secretase activity of Presenilin. The ICN of Notch receptor translocates to the nucleus, displaces the CoR, and binds to the transcription factor CSL along with p300, CoA, and MAML. (C): Hh pathway. Upon binding of Ptch with Hh, Smo is unrepressed. Through an unknown mediator, Gli is released from the Sufu-Fused-Cos2 complex and translocates into the nucleus and binds to DNA to activate transcription. (D): BMP pathway. BMP antagonists normally regulate extracellular BMP ligand levels. Binding of BMP ligand to BMP receptor 2 results in phosphorylation of BMP receptor 1. This results in downstream phosphorylation of R-Smad and Co-Smad, with subsequent translocation of this complex to the nucleus to regulate gene transcription. Abbreviations: BMP, bone morphogenic protein; CoR, corepressor complex; Hh, hedgehog; ICN, intracellular domain; LRP, lipoprotein-related receptor protein; Ptch, Patched; Smo, Smoothened.