-Catenin–Lef SignalsAs genomic sequences are identified, the next frontier toward effective genetic medicine will be to identify how gene expression is regulated, so ectopic genes can be expressed in appropriate locations, at appropriate times and to appropriate therapeutic levels. This is especially true for molecules that regulate the downstream expression of a cascade of genes. Like traffic control regulators, these molecules effectively act as molecular switches, which can favor one pathway or another with dramatic biological consequences. For years scientists have tried to map the transcriptional regulatory regions of structural effector molecules and found that gene promoters are composed of multiple small, positive, and negative gene response elements. But the study of the regulatory regions of signaling molecules has lagged. Gene regulation is based on a "committee level decision" of these transcriptional factors and co-activators. In recent years, several key signaling pathways have been shown to play major roles in regulation of skin/skin appendage morphogenesis (e.g., Wnt, 
-catenin, Shh, FGF, BMP). The regulation of these regulators has tremendous consequences. Here we will comment on Wnt signaling based upon new progress made on the lymphocyte enhancer factor (Lef)-1 promoter by Liu et al (2004).
Recent Progress in Wnt Signaling
Wnt signaling through the canonical pathway stabilizes -catenin and allows it to translocate to the nucleus, associate with Lef/T cell factor (Tcf) and induce transcriptional events. The Wnt--catenin signaling cascade has been shown to be important for developmental morphogenesis and cancer. It plays an essential role in the morphogenesis of several organs including skin appendages. Over expression of exogenous, constitutively active -catenin in otherwise normal mice led to the formation of ectopic hairs and the formation of hair tumors (Gat et al, 1998). Cre/loxP knockout mice demonstrated that -catenin plays a critical role in directing epidermal cell fate toward becoming hair follicle or interplacode epidermis (Huelsken et al, 1991). Similarly, inhibition of Wnt signaling with Dkk-1 blocked the initiation of hair follicle development and caused other developmental anomalies (Andl et al, 2002). Although -catenin is an important component in these events, it does not act alone to induce hair formation. It must interact with co-activators. Lef-1 mRNA and protein have been found in the developing hair. Lef-1 protein was detected by immunostaining the mesenchyme (E11) and epithelium (by E12) of wild-type mouse vibrissae. Constant expression in the mesenchyme but transient expression of Lef-1 in the epithelium is required for normal whisker formation. Lef-1 knockout mice lack hairs, teeth, mammary glands, and other epithelial-derived skin appendages (van Genderen et al, 1994). Mice expressing an NH2-terminal truncation of Lef-1 in the skin epidermis form interfollicular skin rather than hairs or form sebaceous tumors (Niemann et al, 2002). -Catenin and Lef-1 form a complex to suppress E-cadherin expression presumably while inducing the expression of other genes within the hair follicle (Jamora et al, 2003). These studies clearly show the importance of the canonical Wnt signaling pathway and of Lef-1 to skin appendage formation.
-Catenin can interact with a number of downstream genes to activate different pathways. The different Lef/Tcf family members can form a complex with -catenin to induce a different set of downstream genes, perhaps by attracting different transcriptional co-activators. For example, Lef-1 can induce Siamois, and Tcf-4E can induce the caudal-related homeobox gene, Cdx1 (Hecht and Stemmler, 2003). Tcf-1 is essential for thymocyte differentiation (Verbeek et al, 1995), Tcf-3 is more involved in axis duplication (Merrill et al, 2001), Tcf-4 regulates the formation of intestinal epithelium (Korinek et al, 1998), and Lef-1 affects epithelial-mesenchymal interactions in many organ systems (van Genderen et al, 1994). In the skin, -catenin interacting with Lef-1 leads to hair formation, but interactions with TCF-3 lead to sebaceous gland formation (Merrill et al, 2001).
Additionally, -catenin has been shown to directly interact with a number of other nuclear transcriptional repressor and co-activator proteins including XSox17, Xsox3, Smad proteins, the retinoic acid receptor, and androgen receptor leading to the transcriptional activation of other genes. -Catenin association with either of the steroid receptors reduces its association with Lef/Tcf and thereby inhibits Lef/Tcf mediated transcription. Thus, there is competition for binding to -catenin, which may further regulate the facilitative recruitment of other co-activators to stimulate or repress the transcription of different cohorts of downstream genes. In this way, the interaction of -catenin with its varied partners can have dramatic consequences on the activation of downstream effector pathways. A cell's predilection toward one pathway versus another is dependent to a certain extent on the differential expression of these regulatory molecules within the cellular milieu (Figure 1).
Figure 1.
Co-activator competition for 
-catenin can route cell fate.-Catenin can directly interact with several competing transcriptional co-activators which preferentially transcribe different downstream genes. Here this is depicted as a traffic circle with pathways diverging in different directions based on the co-activator associated with -catenin. In this scenario, the relative levels of expression of these important regulatory molecules can route cells towards alternative fates. Therefore it is important to study how these regulators are themselves regulated.
Signaling through the Wnt pathway has been implicated in tumor formation in a number of different tissues. In the skin over expression of -catenin led to the induction of hair tumors (Gat et al, 1998). Mutations of -catenin have also been found in naturally occurring hair tumors (Chan et al, 1999). One might envision that differential expression of the -catenin partner molecules would influence tumor formation? This again highlights the importance of understanding the regulation of -catenin and its associated proteins.
Regulation of Wnt Signaling Regulators
Much attention has been focused on the role of nuclear translocation of -catenin in activating the canonical pathway. Regulation, however, also takes place at the transcriptional level. We found in chicken skin that -catenin transcripts were dynamically regulated (Widelitz et al, 2000). In the rat, mouse, and human,Li et al (2004) have examined the -catenin promoter and found many regulatory elements by gel shift, reporter assays on serial deletion constructs in vitro and protein/DNA analyses. The function of some regions of the promoter region had been previously tested in vitro (Nollet et al, 1996). Similar studies have been performed on the Lef-1 promoter (Hovanes et al, 2000;Filali et al, 2002).Filali et al (2002) found that a 
2.5 kb region of the human promoter was inducible by Wnt-3a and -catenin. Some regions of the promoter led to increased expression while others suppressed expression. They then went on to identify a 110 bp Wnt responsive element (WRE) within the Lef-1 promoter. Wnt3a relieved inhibition from this site and promoted Lef-1 transcription.
In this issue, Liu et al have further examined regulation of the Lef-1 promoter in the context of chromatin structure which places more constraints on gene expression in vivo. Using transgenic mice with LacZ expression driven by different forms of the Lef-1 promoter, they report that the 2.5 kb human promoter region is sufficient to direct expression within hairs and vibrissae, although the timing of expression was delayed compared to earlier reports based on immunostaining. Expression from the transgene was only observed in the mesenchyme, not in the epithelium in contrast to these earlier reports. A longer construct, containing 3 kb downstream of the initiation codon (including intron 1 and a portion of intron 2) in addition to the 2.5 kb promoter, directed expression to the sebaceous glands. This expression, although not previously described, was confirmed in wild-type embryos. This demonstrates that intronic regulatory sequences further restrict expression. Transgenic expression of a 2.5 kb construct lacking the WRE failed to express in the mesenchyme, raising the likelihood that Wnt function directs mesenchymal expression of Lef-1. Furthermore, expression in one of the transgenic mice mimicked the normal epithelial pattern. Hence, activation or repression of the 110 bp WRE may direct expression to either the mesenchyme or epithelium. They then go on to show that Shh can induce -galactosidase expression from the 2.5 kb Lef-1 promoter as is normally seen in wild-type mice after Shh induces telogen phase hairs to enter anagen phase. Discrepancies between endogenous and transgene expression suggest that some elements of the Lef-1 promoter are missing in their tested constructs or may be configured differently than in their normal state.
Studies of this type will begin to elucidate positive and negative regulatory elements controlling the expression of signaling molecules. Cellular conditions can favor the activation of one pathway or another. If the expression of these Lef/Tcf factors is transcriptionally regulated, -catenin will preferentially bind to another partner protein, ultimately changing cell fate. Given the region-specific, hormone-dependent sexual dimorphism of skin appendages in humans and other species (i.e., beards grow on males but not females; male pattern baldness, etc.), this is an area that warrants further exploration. Once this information is ascertained, it can be utilized for modulating the course of disease. Therefore, it is essential that we understand how these regulatory molecules are themselves regulated at the transcriptional, translational, and post-translational levels.
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