Discussion
During budding morphogenesis, intersecting signaling networks from the epithelium and mesenchyme govern transcriptional, adhesive, polarity, and motility programs in these select groups of cells. The dynamic nuclear and cytosolic changes that take place during this time form the cornerstone for organ morphogenesis. Two major challenges in understanding the mechanisms underlying a particular budding process are to order the temporal sequence of external cues involved and then to dissect how the cells of the developing bud translate these signals into the downstream events of cellular remodeling, proliferation, and differentiation. Our studies here provide some insights into how these events are orchestrated during hair bud formation in developing skin.

Signaling during Early Hair Follicle Morphogenesis
Recent studies on hair bud morphogenesis suggest that Wnt signals likely from the epithelium and BMP inhibitory signals from the underlying mesenchyme converge to produce an active transcription factor complex involving β-catenin and LEF-1, which in turn plays a key role in specifying the hair follicle fate [4,29,30,36,37]. Sonic hedgehog (Shh) and TGF-β2 signaling also play essential roles in follicle morphogenesis, but in contrast to β-catenin null skin, in which follicle invaginations are absent [30], some hair buds still form in the absence of LEF-1, Shh, or TGF-β2 [32,38]. These likely reflect the first wave of follicle (i.e., guard hair) morphogenesis, which accounts for a small number (fewer than 5%) of hairs and is under distinct regulatory control. Guard hairs form in the absence of LEF-1 and TGF-β2, and we have found that they also fail to express Snail at the budding stage of development (unpublished data). How E-cadherin is regulated in guard hairs remains to be determined. Several candidates include other Snail family members such as Slug or twist, or alternatively, transcription factors involving β-catenin and a different member of the LEF-1/TCF/Sry-type HMG box (commonly known as SOX) family [39,40]. Further investigation will be required to determine whether the signaling pathway we have elucidated here is a theme with multiple variations.
TGF-βs are known to promote withdrawal of keratinocytes from the cell cycle [41]. Hence, when TGF-β2 protein was detected at the transition between the growing and destructive phases of the adult hair cycle, research initially and naturally focused on a role for this family member in cessation of growth and/or triggering apoptosis ([42] and references therein). However, in contrast to TGF-β1-null skin, which exhibits an extended growing phase of postnatal hair follicles, TGF-β2-null skin displays an embryonic block in follicle bud progression [32]. Although this phenotype is consistent with TGF-β2's embryonic expression patterns [33], about 50% of TGF-β2 null buds appear unable to progress to the down-growth phase, a feature that cannot be explained readily on the basis of previously established effects of TGF-βs.
Our finding that TGF-β2 is upstream from Ki67 expression and MAPK activation lends further support to the notion that hair follicle keratinocytes at this early stage of development react to TGF-β2 signaling in a fashion opposite to that typically expected for TGF-β factors. This said, based upon pSMAD2 immunohistochemistry, the immediate steps of downstream signaling appeared to be intact. Thus, we surmise that the proliferative outcome is likely to be rooted in differences in the repertoire of activated SMAD target genes. In this regard, the positive effects of TGF-β2 on proliferation within the hair bud may be more analogous to what has been seen in progression of squamous cell carcinoma to metastatic carcinoma [43] rather than that typically observed for keratinocytes [44,45,46].
The prior identification of the Snail gene as a potential target of TGF-β signaling [15] was intriguing, given the temporal wave of Snail gene expression that occurs in the developing hair bud. The additional correlation between epithelial hyperproliferation and Snail transgene expression further fostered our interest in a possible link between TGF-β2 and Snail. Our functional studies demonstrate that without TGF-β2, Snail expression is abolished in the mutant hair buds, and conversely, in K14-Smad2 skin, Snail is ectopically activated. Moreover, our in vitro studies indicate that even sustained TGF-β2 exposure may cause only a transient induction of Snail, offering a possible explanation as to why Snail is so briefly expressed during hair follicle morphogenesis. An additional point worth mentioning is that prolonged expression of Tg Snail in postnatal skin resulted in morphological and biochemical signs of epithelial to mesenchymal transitions (unpublished data), underscoring why transient Snail expression may be so important during normal hair follicle morphogenesis [18].
At first glance, the sparsity in hair coat of K14-Snail Tg mice seemed indicative of a defect in follicle formation (see Figure 2A). Closer inspection, however, revealed that not all hairs penetrated the hyperthickened Tg epidermis. Several factors may contribute to the seemingly normal follicle development in these mice. One obvious factor is the K14 promoter, which is elevated in the basal layer of the epidermis and the outer root sheath (ORS) of the hair follicle, but is markedly down-regulated in developing embryonic hair buds as well as in the postnatal hair progenitor cells. The K14 promoter is also less active in the ORS than epidermis and hence this might also account for the lack of apparent response of the ORS to ectopic Snail. Additional contributing factors could be the multiplicity of Snail family members and their differential expression, the saturation, and/or diversity of regulatory mechanisms that govern AJ formation, migration, and proliferation in the follicle ORS. Distinguishing between these possibilities must await the generation of mice harboring skin-specific loss-of-function Snail mutations.

Links between Signaling, Transcriptional Cascades, Epithelial Remodeling, and Proliferation in the Hair Bud
Previously, we discovered that early during hair follicle morphogenesis, E-cadherin gene expression is down-regulated concomitantly with the invagination of developing bud cells into the skin [4]. Because the timing of this event correlated with the activation of a LEF-1/β-catenin transcription factor complex [20], we were intrigued by the presence of a putative LEF-1/TCF binding site in the E-cadherin promoter. This prompted an investigation that subsequently led to our discovery that LEF-1/β-catenin can contribute to repression of E-cadherin gene expression in skin keratinocytes [4]. In the course of these studies, we also noted that Snail can also contribute to this process in keratinocytes in vitro, and our present studies revealed that Snail is expressed at the right place and time to be physiologically relevant in the process.
In noggin-null embryonic skin, LEF-1 expression and subsequent activation of the LEF-1/β-catenin reporter gene is abrogated in the developing placodes. The corresponding failure of E-cadherin down-regulation underscores the importance of Wnt/noggin signaling in regulating this event in follicle morphogenesis [4]. Conditional gene targeting studies will be necessary to establish whether Snail family members also contribute to the down-regulation in E-cadherin gene expression that occurs during follicle formation. However, it is intriguing that K14-Snail Tg epidermis displayed a marked down-regulation in E-cadherin expression, thereby demonstrating its potential to do so in skin. Our prior findings showed that by elevating E-cadherin levels or by conditionally ablating α-catenin, hair follicle morphogenesis can be impaired [4,7]. The marked epidermal hyperproliferation seen in the K14-Snail Tg skin, coupled with the converse suppression of proliferation and Snail in TGF-β2-null hair buds led us to wonder whether the down-regulation of E-cadherin during follicle morphogenesis might have a direct impact on elevating the proliferative state of these cells.
Our Tg studies suggested that, at least in part through its regulation of E-cadherin, Snail is able to influence the subcellular localization of a variety of AJ-associated proteins. One of these appears to be Ajuba, which was previously shown to have the dual capacity to bind Grb-2 as well as α-catenin [9,10]. Our studies revealed that in skin keratinocytes that either harbor a conditional null mutation in α-catenin or that overexpress Snail, Ajuba develops an interaction with Grb-2 that is otherwise not observed in WT keratinocytes. The corresponding abilities of either Snail-transfected or Ajuba-transfected keratinocytes to exhibit elevated activation of the Ras-MAPK pathway suggest that the Grb-2 association of Ajuba under conditions of reduced levels of AJ proteins may be directly relevant to the parallel in hyperproliferation.
In stable epithelial (i.e., Madin-Darby canine kidney, or MDCK) cell lines, Snail has been shown to block cell cycle progression and promote motility and shape changes for invasion [47]. While our in vivo studies are consistent with a role for Snail in motility and epithelial remodeling, they differ with respect to Snail's apparent proliferative effects. A priori, this could be simply due to variations in the response of different cell types to Snail expression. Alternatively, these differences may be relevant to the benefit of using mouse models to reveal functions not always recapitulated in stable cell line models. Future studies should highlight the underlying reasons for these opposing results.
Irrespective of these differences, our in vivo studies do not stand alone, as there are many situations in which a down-regulation in AJ proteins correlate with enhanced proliferation. In fact, a myriad of diverse mechanisms have been implicated in activating epithelial proliferation upon down-regulation of AJ proteins [7,23,24,48]. Sifting through these converging pathways is likely to be a difficult and painstaking process. This said, by identifying the status of different players involved in specific cell types and at specific stages in development, our mechanistic understanding of how intercellular remodeling is linked to proliferation in epithelial morphogenesis should begin to surface in the future. Elucidating the molecular mechanisms through which these networks converge is also a prerequisite for understanding how these processes go awry during tumorigenesis.