Hair follicles develop via extensive interactions between cells of the surface ectoderm and underlying dermal cells (Hardy, 1992;Millar, 2002). The first morphological sign of hair follicle formation is the development of a regular array of placodes, or local thickenings, in the surface ectoderm, which are formed in response to signals from the dermis (Hardy, 1992). Signaling from each placode to the dermis causes the formation of a cluster of mesenchymal cells, known as a dermal condensate (DC) (Hardy, 1992). Reciprocal signaling from the DC to the epithelium results in the proliferation and downgrowth of epithelial cells into the dermis, forming a hair germ. The hair germ continues to elongate, to form a hair peg, and at this stage epithelial cells begin to surround the DC, which develops into the dermal papilla (DP) of the hair follicle (the "bulbous peg" stage). The follicular epithelium subsequently differentiates to form several concentric cell layers, which include the medulla, cortex, and cuticle of the hair shaft, and the cortex, Huxley's layer, and Henle's layer of the inner root sheath (IRS) that molds the developing hair shaft as it emerges from the follicle (Sperling, 1991). The IRS is surrounded by an outer root sheath (ORS) that is contiguous with the epidermis, and the follicle is bound by a connective tissue sheath. Production of the hair shaft and IRS is dependent on signaling between the DP and the follicular epithelium (Oliver and Jahoda, 1988), and is also likely to require communication between cells in different epithelial layers of the follicle (Millar et al, 1999;Lin et al, 2000).
After birth, hair follicles undergo successive cycles of growth (anagen), regression (catagen), and rest (telogen) (Dry, 1926;Muller-Rover et al, 2001). At the onset of a new growth cycle, stem cells in the permanent, bulge, region of the follicle are stimulated to divide, possibly by signals from the DP (Oliver and Jahoda, 1988;Cotsarelis et al, 1990). Stem cell progeny move to the lower part of the follicle and proliferate rapidly, forming the hair follicle matrix (Taylor et al, 2000;Oshima et al, 2001). Progeny of the matrix cells cease dividing and differentiate along specific pathways to form a new hair shaft and IRS, and the old hair is eventually shed. The movement of progenitor cells from the bulge to the matrix has been shown to continue throughout anagen in vibrissa follicles (Oshima et al, 2001); however, the mechanisms controlling these movements are unknown.
Among the intercellular signals active in developing and postnatal follicles, secreted WNT proteins appear to play particularly crucial roles (Millar, 2002). WNTs form a large family with 19 members in humans and mice (see http://www.stanford.edu/~rnusse/wntwindow.html). These bind to their receptors, the Frizzled (FZ) proteins, that also form a large family with ten members in humans and mice, to activate several different pathways that control cellular proliferation, fate, shape, and movements (Moon et al, 2002;Veeman et al, 2003). In the canonical WNT/
-catenin pathway (Moon et al, 2002), WNT ligands bind to low-density lipoprotein (LDL) receptor-related protein (LRP) 5/6 co-receptors as well as to an FZ receptor, causing activation of the intracellular protein Dishevelled (DSH), and inhibiting the function of a complex of proteins that normally targets cytoplasmic -catenin for degradation. As a consequence, -catenin accumulates in the cytoplasm, translocates to the nucleus, and forms transcriptional complexes with members of the lymphocyte enhancer factor (LEF)/T cell factor (TCF) family of DNA binding factors, resulting in the activation of target genes (Moon et al, 2002).
An alternate, non-canonical, WNT signaling pathway controls planar polarity in Drosophila embryos, and is required for planar polarity of cochlear hair cells in the mouse inner ear, and for the morphogenetic movements that occur during gastrulation in vertebrate embryos (Veeman et al, 2003). In non-canonical WNT signaling, binding of WNT to FZ receptors is enhanced by Knypek, a member of the glypican family of heparan sulfate proteoglycans (Topczewski et al, 2001), but does not involve LRP5/6. Non-canonical WNT signaling activates DSH, but utilizes different domains of the DSH protein than those involved in WNT/-catenin signaling (Axelrod et al, 1998). In addition, non-canonical WNT signaling does not require -catenin or LEF/TCF factors; instead, DSH activates Rho1 via a bridging molecule Daam1 (Habas et al, 2001). Activated DSH can cause calcium flux and activation of the calcium-sensitive kinases protein kinase C and CamKII (Slusarski et al, 1997;Kuhl et al, 2000). Whether Rho1 activation and activation of calcium flux occur in the same pathway, or represent different branches of the non-canonical FZ–DSH pathway, however, is currently unclear (Veeman et al, 2003). In some contexts, non-canonical WNT signaling can act to antagonize signaling through the WNT/-catenin pathway (Weidinger and Moon, 2003).
Certain WNT ligands appear to activate preferentially either the WNT/-catenin pathway or non-canonical WNT signaling; for instance, WNT5A is generally described as an activator of non-canonical signaling (e.g.,Weidinger and Moon, 2003), whereas WNT1 activates canonical signaling (Korinek et al, 1998). This distinction is context-dependent rather than absolute; for instance, WNT5A can activate the canonical pathway in Xenopus embryos when FZ5 is expressed (He et al, 1997), and WNT1 activates RhoA in 293T cells (Habas et al, 2001). Similarly, over-expression of FZ1 in Xenopus embryo animal cap explants activates the canonical pathway (Yang-Snyder et al, 1996), whereas over-expression in human 293T cells activates RhoA (Habas et al, 2001), and FZ7 has been implicated in both canonical and non-canonical pathways in different systems (Sumanas et al, 2000;Habas et al, 2001). Thus, activation of canonical versus non-canonical signaling is likely to depend on the precise pairing of WNT and FZ ligands, as well as on the presence of particular co-receptors, endogenous secreted inhibitors (Zorn, 2001;Pandur et al, 2002;Kawano and Kypta, 2003), and intracellular factors.
Investigations of the possible functions of non-canonical WNT signaling in skin and hair follicles have not been reported to date. In contrast, multiple lines of evidence indicate that WNT signaling through the -catenin pathway is necessary for the formation and maintenance of hair follicle placodes (van Genderen et al, 1994;Huelsken and Birchmeier, 2001;Andl et al, 2002). Analysis of the in vivo expression of a TOPGAL WNT reporter transgene, in which the lacZ gene is placed under the control of multiple LEF/TCF binding sites and a minimal promoter, reveals activity of the canonical WNT pathway in both the epithelium and the mesenchyme of developing hair follicles (DasGupta and Fuchs, 1999), consistent with the involvement of WNT signals in the cross-talk between follicular epithelial and mesenchymal cells.
Roles for canonical WNT signaling in postnatal hair follicles are suggested by the finding that the TOPGAL WNT/-catenin reporter transgene is expressed in hair shaft precursor cells (DasGupta and Fuchs, 1999). A second type of WNT/-catenin reporter transgene, BATgal, containing the promoter regions of the Xenopus WNT target gene Siamois fused to lacZ, is additionally expressed in the hair follicle matrix and DP of some hair follicles, suggesting that these are also sites of WNT/-catenin signaling activity (Maretto et al, 2003). TOPGAL is reportedly expressed in hair follicle stem cells at anagen onset (DasGupta and Fuchs, 1999), suggesting that activation of WNT signaling might be one factor controlling the telogen–anagen transition. This hypothesis is consistent with the observation that progressive loss of epithelial -catenin causes failure of onset of the first postnatal anagen (Huelsken et al, 2001).
Multiple Wnt genes are expressed in developing and mature hair follicles, including Wnts known to be capable of activating the WNT/-catenin pathway and several Wnts that initiate non-canonical signaling in most developmental contexts (Reddy et al, 2001). As discussed above, the specificity of function of the different WNT proteins is likely to be determined in part by the availability of the various FZ receptors. However, with the exception of Fz3, which is expressed at several stages of hair follicle development (Hung et al, 2001), the Fz genes expressed in hair follicles are unknown. To address this question, we have carried out RT-PCR and in situ hybridization experiments to examine the expression profile of the known Fz genes in developing and postnatal mouse skin. We find that several Fz genes are expressed at sites of canonical WNT activity in developing and postnatal hair follicles, allowing us to identify candidate receptors for canonical WNT signals. In addition, certain Fz genes are expressed at locations and developmental stages that correlate with the expression of non-canonical Wnt genes and do not display WNT/-catenin reporter activity in transgenic mouse assays. These results suggest that WNT signaling through non-canonical pathways may contribute to the regulation of hair follicle development and hair growth.
Results and Discussion
Several Fz genes are specifically expressed in embryonic skin at the time of onset of hair follicle induction
To identify Fz genes expressed in embryonic skin at the time of onset of hair follicle induction, we carried out RT-PCR experiments using specific primers for mouse Fz1, Fz2, Fz3, Fz4, Fz5, Fz6, Fz7, Fz8 and Fz9, and RNA isolated from skin dissected from embryos at embryonic day 14.5 (E14.5). RNA preparations were treated with DNAse I to remove any contaminating genomic DNA, and control experiments in which the reverse transcriptase enzyme was omitted were performed for each primer pair and RNA preparation to ensure that PCR products did not arise from amplification of genomic DNA. Before experiments were carried out on embryonic skin RNA, each primer pair was tested on RNA isolated from whole embryos to check that a product of the appropriate size could be amplified. The expression of all of these genes was detected in E14.5 skin by this assay (Figure 1).
Figure 1.
RT-PCR analysis indicates that multiple members of the Fz gene family are expressed in embryonic mouse skin. RNA was extracted from the dorsal skin of single mouse embryos at E14.5 and cDNA was synthesized by RT. Fz cDNA fragments were amplified using specific primers. PCR products of the expected sizes were amplified using primers for Fz1 (336 bp), Fz2 (271 bp), Fz3 (407 bp), Fz4 (474 bp), Fz5 (270 bp), Fz6 (408 bp), Fz7 (403 bp), Fz8 (276 bp), and Fz9 (437 bp). No PCR products were amplified in control reactions lacking reverse transcriptase (RT-), indicating that the bands in the RT+ lanes do not result from amplification of genomic DNA.
Full figure and legend (19K)To confirm expression of Fz1, Fz2, Fz4, Fz5, Fz6, Fz7, Fz8, and Fz9, and determine the cellular localization of expression within developing skin, probes for these genes, and an additional, more recently cloned Fz, Fz10, were used for in situ hybridization experiments with sectioned embryos at E14.5. Fz3 expression in developing skin has previously been described in detail (Hung et al, 2001), and was not examined further here. Probes were designed to hybridize to regions of Fz cDNAs that did not contain regions of high homology to other Fz genes, and would not cross-hybridize with secreted Frizzled-related protein (sFrp) mRNAs that are similar to 5', but not 3', regions of Fz mRNAs (Rattner et al, 1997). In most cases, probes were complementary to non-conserved 3' coding regions or 3' non-coding regions of Fz genes. Hybridization to internal embryonic organs provided a positive control for each probe. Sense probes were used as negative controls. All of these Fz genes were expressed at low levels throughout the embryo. Markedly elevated expression in embryonic skin at E14.5 was only observed, however, for Fz1, Fz6, and Fz10. Of these genes, Fz6 was predominantly expressed in the epidermis, but was not expressed within the epithelium of developing placode and germ stage hair follicles (Figure 2b). Fz1 was expressed at low levels in the dermis, and was specifically elevated in hair follicle placodes (Figure 2a). Elevated expression of Fz10 was confined to developing hair follicles, where it was expressed both in the placode and in the DC (Figure 2c). In addition, Fz2 showed very slightly elevated expression in the placodes and DC of developing hair follicles (data not shown).
Figure 2.
Expression of Fz genes in embryonic skin and developing hair follicles at the time of hair follicle initiation. Sagittal paraffin sections of E14.5 mouse embryos were subjected to in situ hybridization with probes for Fz1 (A), Fz6 (B), and Fz10 (C). Hybridization appears as red grains and is indicated by arrows. Nuclei are counterstained with Hoechst 33258 dye and appear blue. Developing hair follicles are indicated by brackets. The dermal–epithelial junction is indicated by a dashed white line in each panel. Fz1 is expressed at low levels in the dermis and is slightly elevated in the follicle epithelium (A) (yellow arrow). Fz6 is expressed in the interfollicular epithelium (yellow arrows) (B) but is downregulated in hair follicle placodes. Fz10 expression is elevated in the epithelium (yellow arrows) and DC (white arrows) of developing hair follicles (C). Sense control probes for these Fz genes gave only background hybridization. The photographs in all panels were taken at the same magnification. Scale bar=25 
m (panel C).
These data are summarized and compared with the previously described expression of Wnt genes in developing hair follicles at the placode stage (Reddy et al, 2001) in Figure 4 (left diagram). Expression of Fz1 and Fz10 in the placode and Fz10 in the developing DC correlates with expression of Wnt10a and Wnt10b in the placode. WNT10B, FZ1, and FZ10 are all known to be capable of activating canonical WNT signaling (Yang-Snyder et al, 1996;Kawakami et al, 2000b;Ross et al, 2000;Malbon et al, 2001;Terasaki et al, 2002). The signaling properties of Wnt10a have not been described, but its high degree of sequence similarity to, and overlapping expression with, Wnt10b suggests that it may have similar properties (Wang and Shackleford, 1996;Reddy et al, 2001). These data suggest that canonical WNT signaling is likely activated in the placodes and DC of developing hair follicles by WNTs 10A and 10B, expressed in and secreted from epithelial cells, binding to FZ1 and FZ10, expressed in the placode epithelium and DC.
Figure 4.
Summary of Fz expression in hair follicle morphogenesis and comparison with Wnt gene expression. Expression of Wnt10a and Wnt10b is represented by solid dark blue coloration. Fz1 expression is represented by violet circles, Fz10 by red circles, Fz6 by gray circles, Wnt5a by green circles, Fz5 by orange circles, and Fz7 by light blue circles. Wnt gene expression data are taken fromReddy et al (2001).
Full figure and legend (12K)Interestingly, Fz6 is expressed in the embryonic interfollicular epidermis, overlapping with the previously described expression of Fz3 (Hung et al, 2001). This expression persists in postnatal epidermis (see below) (Hung et al, 2001). In vivo assays indicate that the canonical WNT/-catenin signaling pathway is not active in embryonic or postnatal interfollicular epidermis (DasGupta and Fuchs, 1999;Maretto et al, 2003). Fz3 and Fz6, however, are able to stimulate non-canonical WNT signaling (Sheldahl et al, 1999), and the interfollicular epidermis displays expression of several Wnts, including Wnt4 and Wnt6, that can activate non-canonical signaling (Wong et al, 1994;Reddy et al, 2001;Westfall et al, 2003). In addition, two non-canonical Wnt genes, Wnt5a and Wnt11, are expressed in the underlying dermis (Reddy et al, 2001). These data raise the possibility that non-canonical WNT signaling is active in interfollicular epidermis.
Additional Fz genes are expressed as hair follicles undergo morphogenesis
To examine the profile of Fz gene expression at later stages of embryonic hair follicle development, sagittal sections of embryos at E15.5, E16.5, and E18.5 were subjected to in situ hybridization with all of the Fz probes. We found that expression of Fz4, Fz8, and Fz9 is not specifically elevated in developing skin or hair follicles at these stages; however, Fz4 transcripts localize to the muscle layer underlying the dermis. Fz2 expression is very slightly elevated within the epithelium and DC of germ and peg stage follicles, and is very weakly expressed in the epithelium above the bulb in bulbous peg stage follicles (data not shown). At the germ and peg stages, Fz1, and, more prominently, Fz10, are expressed in epithelial cells, and Fz10 is also expressed in DC (Figure 3a, e). Expression of Fz1 in follicular epithelium, and Fz10 in follicular epithelial and dermal cells, is maintained at the bulbous peg stage (Figure 3f, j), with expression of Fz1 concentrating particularly in cells of the developing ORS (Figure 3f). Fz10 is expressed at relatively high levels throughout the follicular epithelium and DP, and its expression is markedly elevated in hair follicles compared with interfollicular epidermis and dermis.
Figure 3.
Fz genes are expressed throughout hair follicle morphogenesis. Sagittal paraffin sections of mouse embryos at E16.5 (C) or E18.5 (A, B, D–J) were subjected to in situ hybridization with probes for Fz1 (A, F); Fz5 (B, G); Fz6 (C, H); Fz7 (D, I); and Fz10 (E, J). Secondary hair follicles at the germ and peg stages are shown in (A, B, D, E); a primary hair follicle at the early peg stage is shown in (C); primary hair follicles at the bulbous peg stage are shown in (F–J). Nuclei are counterstained with Hoechst 33258 dye and appear blue. The dermal–epithelial junction is indicated by a dashed white line in each panel. At the late germ–early peg stages, Fz1 is expressed at very low levels in the follicle epithelium (yellow arrow) (A); Fz5 is not specifically expressed in the follicle (B); Fz6 is expressed in the epidermis (yellow arrows) and is downregulated in follicle epithelium (C); Fz7 is expressed in proximal follicle epithelium (arrows) (D); and Fz10 is specifically expressed in the follicle epithelium (yellow arrows) and DC (white arrows) (E). At the bulbous peg stage, expression of Fz1 and Fz5 localizes to the developing ORS (arrows) (F, G); Fz6 is expressed in central hair follicle epithelial cells (arrow) as well as in the epidermis (H); Fz7 expression intensifies in central, proximal follicular epithelial cells (arrow) (I); and Fz10 expression is maintained in the DP (white arrow) and follicle epithelium (yellow arrow) (J). The smaller follicles in (J) (right side) are secondary follicles at the early peg stage. Sense control probes for these Fz genes gave only background hybridization. The photographs in all panels were taken at the same magnification. Scale bar=25 m (panel C). DC, dermal condensate; DP, dermal papilla.
By the late peg and bulbous peg stages, specific expression of several additional Fz genes is initiated. Expression of Fz5 appears in the developing ORS, particularly in the follicle bulb, at the bulbous peg stage (Figure 3g). Expression of Fz6 is maintained in the epidermis throughout later embryonic development, and appears in central cells of the hair follicle epithelium at the bulbous peg stage (Figure 3h). Expression of Fz7 is not elevated in the interfollicular epidermis, but specifically appears in central epithelial cells in the upper half of developing hair follicles at the late peg stage (Figure 3d), intensifying in these cells at the bulbous peg stage (Figure 3i).
These patterns of Fz expression are compared with Wnt gene expression (Reddy et al, 2001) at the germ and bulbous peg stages in Figure 4 (diagrams in the middle and right of the figure). Wnt10b and Wnt10a are expressed continuously in follicular epithelium during these later stages of morphogenesis. Canonical WNT signaling, however, assayed by TOPGAL expression, is downregulated in hair follicles at the germ to peg stages (DasGupta and Fuchs, 1999). It is therefore interesting to note that expression of Wnt5a, which is capable of antagonizing the WNT/-catenin pathway (Weidinger and Moon, 2003), is initiated in the DC from the early germ stage. How WNT5A signaling might interact with WNT10A/WNT10B-FZ1/FZ10 signaling to regulate follicle development remains to be investigated; however, it is tempting to speculate that non-canonical signaling driven by WNT5A may predominate at these time points.
At the bulbous peg stage, Wnt10a and Wnt10b are strongly expressed in a cone of epithelial cells surrounding the DP (Reddy et al, 2001), and activation of canonical WNT signaling also appears to localize to these cells (DasGupta and Fuchs, 1999). Expression of Fz6 and Fz10 in cells adjacent to the DP is therefore consistent with activation of canonical WNT signaling by WNT10A/10B binding to FZ6 and FZ10 receptors. Further experiments will be required to determine whether Wnt-expressing and Fz-expressing WNT activated cells are intermingled at this stage, or whether the same cells express Wnt and Fz genes and activate the WNT pathway. The latter situation would be consistent with an autocrine as well as a paracrine mode of signaling. Wnt5a is strongly expressed, and Wnt10a is weakly expressed, in the DP at the bulbous peg stage (Reddy et al, 2001), consistent with the absence of canonical WNT reporter gene expression from the DP itself (DasGupta and Fuchs, 1999).
The expression patterns of Wnt and Fz genes at the various stages of follicle development were essentially identical between primary and secondary hair follicles and vibrissa follicles, indicating that differences in the size and growth properties of different types of hair follicle are not mediated by differential expression of these genes. In contrast, comparison of the expression patterns of Wnt genes in embryonic mouse skin and hair follicles with expression of the equivalent genes in embryonic chick skin and feather follicles reveals several striking differences, suggesting that divergence in the morphogenesis of hair and feather follicles might be achieved in part through differential expression of WNT family members (Reddy et al, 2001;Chodankar et al, 2003;Millar, 2003). Interestingly, Fz1, Fz7, and Fz10 are expressed in developing feather follicles in chick embryos (Kawakami et al, 2000a;Chodankar et al, 2003) as well as in developing mouse hair follicles. More detailed analysis of Fz expression in chick skin and comparison with the results reported here may reveal additional mechanisms by which the diversity of appendage development is achieved.
Fz genes are expressed in specific subsets of cells in postnatal anagen follicles
To identify Fz genes expressed in mature anagen follicles, we used in situ hybridization of dorsal skin sections from mice at postnatal day 7 with probes for Fz1, Fz2, Fz4, Fz5, Fz6, Fz7, Fz8, Fz9, and Fz10. Of these, specific expression in hair follicles was not detected for Fz2, Fz4, Fz8, or Fz9. We found that Fz1 and Fz10 are expressed in the ORS, and in the DP (Figure 5a, e). Fz5 is expressed predominantly in the ORS (Figure 5b), and Fz6 and Fz7 are expressed in precursor cells of the hair shaft cortex, with Fz7 being expressed in mature precortical cells higher up in the follicle (Figure 5d), and Fz6 in precortical cells that are exiting the proliferative matrix compartment and beginning the process of differentiation, as well as in adjacent IRS precursor cells (Figure 5c).
Figure 5.
Expression of Fz genes during anagen. Sections of dorsal skin at postnatal day 7 were hybridized with probes for Fz1 (A), Fz5 (B), Fz6 (C), Fz7 (D), and Fz10 (E). (A) Fz1 is expressed in a subset of ORS cells above the follicle bulb (yellow arrow) and, at lower levels, in the DP (white arrow). (B) Fz5 is expressed in the ORS (yellow arrow). (C) Fz6 is expressed in precursor cells of the hair shaft cortex (arrow), as they exit the proliferative matrix compartment in the follicle bulb, and in adjacent IRS precursor cells. (D) Fz7 is expressed in more differentiated precursors of the hair shaft cortex, higher up in the follicle (arrow). (E) Fz10 is expressed in the ORS (yellow arrow), in matrix cells, and in the DP (white arrow). (F) Summary diagrams of Fz expression in anagen hair follicles and comparison with the expression of Wnt genes (Reddy et al, 2001). Major sites of Fz gene expression (the ORS; precursor cells of the hair shaft cortex; and the matrix and DP) are indicated above each diagram in which the domains of relevant Fz gene expression, and adjacent Wnt expression are indicated. Fz gene expression is represented by the following colors: Fz1, Fz10, red; Fz5, orange; Fz6, gray; Fz7, pale blue. Wnt gene expression is indicated by the following colors: Wnt11, brown; Wnt5a, green; Wnt3, dark pink; Wnt3a, pale pink; Wnt10a and Wnt10b, dark blue; Wnt4, yellow. The photographs in panels (A–E) were taken at the same magnification. Scale bar=25 m (panel E). ORS, outer root sheath; DP, dermal papilla; IRS, inner root sheath.
Expression of Fz6 and Fz7 in precursor cells of the hair shaft cortex is consistent with previous reports of expression of the intracellular WNT pathway component Dishevelled 2 (DVL2) (Millar et al, 1999) and activation of WNT/-catenin signaling (DasGupta and Fuchs, 1999) in these cells. Two canonical Wnts are expressed in cell layers immediately adjacent to the precortex: Wnt3 is expressed in differentiating hair shaft medulla cells, whereas the related gene Wnt3a is expressed in differentiating IRS cells (Millar et al, 1999;Reddy et al, 2001) (Figure 5f, middle diagram). These expression patterns suggest that activation of the WNT/-catenin pathway in precortical cells is mediated by interactions of WNT3 and WNT3A, secreted from the premedulla and IRS, with FZ6 expressed on early precortex cells, and FZ7 expressed in more differentiated precortical cells.
Expression of Fz1, Fz5, and Fz10 in ORS cells is intriguing, as canonical WNT signaling has not been reported in this cell layer. However, DVL2, which is a component of both canonical and non-canonical WNT signaling pathways, is specifically expressed in a subset of ORS cells above the hair follicle bulb (Millar et al, 1999), and two Wnts that generally activate non-canonical WNT signaling, Wnt5a and Wnt11, are expressed in, or immediately adjacent to, the ORS, specifically in this region (Reddy et al, 2001) (Figure 5f, left diagram). Thus, WNT5A and WNT11 may stimulate non-canonical WNT signaling via interactions with FZ1, FZ5, and/or FZ10. To date, non-canonical signaling activity has been demonstrated for FZ1 (Habas et al, 2001;Roman-Roman et al, 2004), but not FZ5 or FZ10, making FZ1 the strongest candidate for such a role. Possible functions for non-canonical WNT signaling in the ORS of anagen hair follicles include regulation of the movements of stem cell progeny from the bulge to the matrix (Oshima et al, 2001).
Expression of Fz1 and Fz10 is detected in the DP and matrix of anagen follicles, and several Wnts are expressed in the follicle bulb (Figure 5f, right diagram), and could potentially interact with DP and matrix cells, in particular Wnt10a, Wnt10b, and Wnt4 (Reddy et al, 2001). In addition, Wnt5a is weakly expressed in the DP itself (Reddy et al, 2001). Consistent with these data, expression of the BATgal WNT/-catenin reporter has been demonstrated in the matrix and DP of hair follicles examined at postnatal day 4 (Maretto et al, 2003). Reporter gene activity was not detected in the DP of all follicles, suggesting that canonical WNT signaling in the DP may show temporal variability, depending on the precise stage of anagen examined. Such variability might be regulated in part by the balance of canonical and non-canonical WNTs expressed in the follicle bulb.
Fz genes are expressed in the secondary germ at anagen onset
To determine the patterns of Fz gene expression at telogen and anagen onset, we carried out in situ hybridization experiments with the probes listed above, using cephalo-caudal strips of dorsal skin from mice at postnatal day 23. At this stage, follicles in posterior regions of the skin are in telogen and more anterior follicles are just entering the first postnatal anagen phase. Fz2, Fz4, Fz5, Fz8, and Fz9 were not specifically expressed in the skin or hair follicles of P23 skin. Fz1, Fz6, Fz7, and Fz10 showed low levels of expression in the epithelium of telogen hair follicles (Figure 6a, c, e, g). At anagen onset, all of these genes were expressed in the proliferating epithelium of the regenerating hair follicle (Figure 6b, d, f, h), with Fz10 giving the weakest signal among these Fz genes (Figure 6h). We were not able to identify any Fz genes that were specifically expressed at anagen onset, making it unlikely that this process is driven by regulated expression of Fz. Instead, expression of Fz genes at low levels during telogen and association of Fz expression with the proliferating secondary hair germ in very early anagen indicates that hair follicle cells are capable of receiving WNT signals at these stages and could potentially respond to Wnts 10a and 10b that are upregulated at anagen onset (Reddy et al, 2001).
Figure 6.
Expression of Fz genes in telogen and at anagen onset. Sections of dorsal skin at postnatal day 23 were probed with Fz1 (A, B), Fz6 (C, D), Fz7 (E, F), and Fz10 (G, H). Telogen stage follicles are shown in panels (A, C, E, G); follicles entering anagen are shown in panels (B, D, F, H). Hybridization is indicated by arrows. All four Fz genes are expressed at low levels in the epithelium of telogen follicles (A, C, E, G). Fz6 is also expressed in the epidermis (C, D). All four Fz genes are expressed in the secondary germ at anagen onset (B, D, F, H) (arrows). Scale bar=25 m (panel H). DP, dermal papilla; B, bulge.
In summary, here we show that Fz genes are expressed in complex patterns during hair follicle morphogenesis and postnatal hair growth. Fz gene expression patterns exhibit extensive overlap, with two or more Fz genes often being expressed at the same site. In addition to expression of the Fz genes described here, Fz3 shows transient expression in hair follicle placodes at E15 (Hung et al, 2001), overlapping with the expression of Fz1 and Fz10. In postnatal hair follicles in full anagen, Fz3 expression in the ORS (Hung et al, 2001) overlaps with that of Fz1, Fz10 and Fz5. Taken together, these observations suggest that Fz genes may perform redundant or partially redundant functions in the skin, and that analysis of the effects of loss of Fz function is likely to require the generation of mice bearing multiple mutations.
At each developmental stage, the pattern of Fz expression in developing and postnatal hair follicles can be clearly correlated with the expression of genes encoding WNT ligands, allowing us to identify candidate receptors for each of the WNTs. Specific Fz genes are expressed at each site where activity of the canonical WNT signaling pathway has been described. In addition, Fz genes are expressed at sites in the skin where the canonical pathway is not active, but that overlap with, or are adjacent to, cell populations displaying expression of non-canonical WNT ligands. These sites include the interfollicular epidermis, and specific sub-populations of cells in hair follicles. It will be interesting in the future to determine whether the distribution of WNT co-receptors associated with canonical signaling (LRP5/6 (Moon et al, 2002)) or non-canonical signaling (Knypek (Topczewski et al, 2001)) reflects the expression of canonical and non-canonical WNT ligands. The dynamic nature of hair follicles, the known roles of non-canonical WNT signaling in the regulation of cell movements in vertebrates (Habas et al, 2003;Takeuchi et al, 2003;Ulrich et al, 2003), and recent data indicating antagonism between canonical and non-canonical WNT pathways (Weidinger and Moon, 2003) suggest that the functions of non-canonical WNT signaling in the skin will be a fruitful area for further investigation.
Materials and Methods
Mice and tissues
All studies involving animals were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania School of Medicine. BALB/c postnatal mice and FVB/N embryos from timed natural matings were used as the sources of tissue.
RT-PCR
For RT-PCR experiments, we dissected dorsal skin from FVB/N mouse embryos at embryonic day 14.5 (E14.5) and extracted RNA using Trizol (Gibco BRL, Rockville, Maryland). RNA preparations were treated with DNAse I (Roche-Boehringer-Mannheim, Indianapolis, Indiana) to remove any contaminating genomic DNA, and were purified using Rneasy columns (Qiagen, Valencia, California). cDNA was synthesized using 5 g of RNA in a volume of 40 L. Two microliters of each reverse transcription (RT) reaction was subjected to 30 rounds of PCR using specific primers for mouse Fz genes. Each experiment was repeated on skin from at least three embryos. Primers were designed to amplify non-conserved regions of the cDNAs. Eighteen base pair primers were used to amplify the following sequences whose Genbank accession numbers are shown in brackets: Fz1, 1522–1857 (af054623); Fz2, 41–311 (af139183); Fz3, 1967–2373 (u43205); Fz4, 1491–1964 (u43317); Fz5, 1502–1771 (xm_192878); Fz6, 1864–2271 (u43319); Fz7, 1791–2193 (u43320); Fz8, 1791–2066 (U43321); and Fz9, 1323–1759 (AF088850).
In situ hybridization
Anterior-posterior strips of skin were dissected from the mid-dorsum of postnatal mice prior to fixation. A single slit was made in the mid-ventrum of all embryos, and embryos aged E15.5 and older were also decapitated to allow easier penetration of fixative. Fixation was carried out overnight in 4% paraformaldehyde at 4°C prior to dehydration and embedding. Embryos were sectioned sagittaly, and dorsal skin strips were sectioned parallel to the anterior–posterior axis. 35S-labeled antisense riboprobes for Fz2 and Fz4 were prepared from linearized pBluescript plasmid subclones using either T3 or T7 RNA polymerase as described previously (Wang et al, 1996). Probe templates for Fz1, Fz5, Fz6, Fz7, Fz8, and Fz9 were synthesized by PCR of mouse embryo cDNA using the primers described above. Probe template for Fz10 was synthesized by PCR of mouse embryo cDNA using 18 base pair primers to amplify nucleotides 1501–2030 of Fz cDNA (accession number af206321). Sequences for the binding of T7 RNA polymerase were added to the 3' primers to create templates for the synthesis of antisense probes and to the 5' primers for creating sense probe templates. In situ hybridization was carried out as described previously (Reddy et al, 2001). Sections were counterstained with 2 g/mL Hoechst 33258 dye (Sigma, St Louis, Missouri), mounted in 50% Canada balsam in methyl salicylate (Sigma), and were photographed using an Olympus Bx60 microscope with an MVI Darklite stage adaptor and Photometrics Coolsnap digital camera.
References
| 1. | Andl T, Reddy ST, Gaddapara T & Millar SE. Wnt signals are required for the initiation of hair follicle development. Dev Cell (2002) 2: 643–653. | Article | PubMed | ISI | ChemPort | |
| 2. | Axelrod JD, Miller JR, Shulman JM, Moon RT & Perrimon N. Differential recruitment of dishevelled provides signaling specificity in the planar cell polarity and wingless signaling pathways. Genes Dev (1998) 12: 2610–2622. | PubMed | ISI | ChemPort | |
| 3. | Chodankar R, Chang CH & Yue Z et al. Shift of localized growth zones contributes to skin appendage morphogenesis: Role of the Wnt/beta-catenin pathway. J Invest Dermatol (2003) 120: 20–26. | Article | PubMed | ISI | ChemPort | |
| 4. | Cotsarelis G, Sun TT & Lavker RM. Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell (1990) 61: 1329–1337. | Article | PubMed | ISI | ChemPort | |
| 5. | DasGupta R & Fuchs E. Multiple roles for activated Lef/Tcf transcription complexes during hair follicle development and differentiation. Development (1999) 126: 4557–4568. | PubMed | ISI | ChemPort | |
| 6. | Dry FW. The coat of the mouse (Mus musculus). J Genet (1926) 16: 287–340. |
| 7. | Habas R, Dawid IB & He X. Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev (2003) 17: 295–309. | Article | PubMed | ISI | ChemPort | |
| 8. | Habas R, Kato Y & He X. Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel formin homology protein Daam1. Cell (2001) 107: 843–854. | Article | PubMed | ISI | ChemPort | |
| 9. | Hardy MH. The secret life of the hair follicle. Trends Genet (1992) 8: 55–60. | Article | PubMed | ISI | ChemPort | |
| 10. | He X, Saint JJ, Wang Y, Nathans J, Dawid I & Varmus H. A member of the Frizzled protein family mediating axis induction by wnt-5a. Science (1997) 275: 1652–1654. | Article | PubMed | ISI | ChemPort | |
| 11. | Huelsken J & Birchmeier W. New aspects of Wnt signaling pathways in higher vertebrates. Curr Opin Genet Dev (2001) 11: 547–553. | Article | PubMed | ISI | ChemPort | |
| 12. | Huelsken J, Vogel R, Erdmann B, Cotsarelis G & Birchmeier W. Beta-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell (2001) 105: 533–545. | Article | PubMed | ISI | ChemPort | |
| 13. | Hung BS, Wang XQ, Cam GR & Rothnagel JA. Characterization of mouse Frizzled-3 expression in hair follicle development and identification of the human homolog in keratinocytes. J Invest Dermatol (2001) 116: 940–946. | Article | PubMed | ISI | ChemPort | |
| 14. | Kawakami Y, Wada N, Nishimatsu S, Komaguchi C, Noji S & Nohno T. Identification of chick Frizzled-10 expressed in the developing limb and the central nervous system. Mech Dev (2000a) 91: 375–378. | Article | ISI | ChemPort | |
| 15. | Kawakami Y, Wada N, Nishimatsu S & Nohno T. Involvement of Frizzled-10 in Wnt-7a signaling during chick limb development. Dev Growth Differ (2000b) 42: 561–569. | Article | PubMed | ISI | ChemPort | |
| 16. | Kawano Y & Kypta R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci (2003) 116: 2627–2634. | Article | PubMed | ISI | ChemPort | |
| 17. | Korinek V, Barker N & Willert K et al. Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse. Mol Cell Biol (1998) 18: 1248–1256. | PubMed | ISI | ChemPort | |
| 18. | Kuhl M, Sheldahl LC, Malbon CC & Moon RT. Ca(2+)/calmodulin-dependent protein kinase Ii is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J Biol Chem (2000) 275: 12701–12711. | Article | PubMed | ISI | ChemPort | |
| 19. | Lin MH, Leimeister C, Gessler M & Kopan R. Activation of the notch pathway in the hair cortex leads to aberrant differentiation of the adjacent hair-shaft layers. Development (2000) 127: 2421–2432. | PubMed | ISI | ChemPort | |
| 20. | Malbon CC, Wang H & Moon RT. Wnt signaling and heterotrimeric G-proteins: Strange bedfellows or a classic romance? Biochem Biophys Res Commun (2001) 287: 589–593. | Article | PubMed | ISI | ChemPort | |
| 21. | Maretto S, Cordenonsi M & Dupont S et al. Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci USA (2003) 100: 3299–3304. | Article | PubMed | ChemPort | |
| 22. | Millar SE. Molecular mechanisms regulating hair follicle development. J Invest Dermatol (2002) 118: 216–225. | Article | PubMed | ISI | ChemPort | |
| 23. | Millar SE. Wnts: Multiple genes, multiple functions. J Invest Dermatol (2003) 120: 7–8. | Article | PubMed | ISI | ChemPort | |
| 24. | Millar SE, Willert K, Salinas PC, Roelink H, Nusse R, Sussman DJ & Barsh GS. Wnt signaling in the control of hair growth and structure. Dev Biol (1999) 207: 133–149. | Article | PubMed | ISI | ChemPort | |
| 25. | Moon RT, Bowerman B, Boutros M & Perrimon N. The promise and perils of Wnt signaling through beta-catenin. Science (2002) 296: 1644–1646. | Article | PubMed | ISI | ChemPort | |
| 26. | Muller-Rover S, Handjiski B & van der Veen C et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol (2001) 117: 3–15. | Article | PubMed | ISI | ChemPort | |
| 27. | Oliver RF & Jahoda CA. Dermal–epidermal interactions. Clin Dermatol (1988) 6: 74–82. | PubMed | ISI | ChemPort | |
| 28. | Oshima H, Rochat A, Kedzia C, Kobayashi K & Barrandon Y. Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell (2001) 104: 233–245. | Article | PubMed | ISI | ChemPort | |
| 29. | Pandur P, Lasche M, Eisenberg LM & Kuhl M. Wnt-11 Activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature (2002) 418: 636–641. | Article | PubMed | ISI | ChemPort | |
| 30. | Rattner A, Hsieh JC, Smallwood PM, Gilbert DJ, Copeland NG, Jenkins NA & Nathans J. A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of Frizzled receptors. Proc Natl Acad Sci USA (1997) 94: 2859–2863. | Article | PubMed | ChemPort | |
| 31. | Reddy S, Andl T, Bagasra A, Lu MM, Epstein DJ, Morrisey EE & Millar SE. Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of sonic hedgehog in hair follicle morphogenesis. Mech Dev (2001) 107: 69–82. | Article | PubMed | ISI | ChemPort | |
| 32. | Roman-Roman S, Shi DL & Stiot V et al. Murine Frizzled-1 behaves as an antagonist of the canonical Wnt/beta-catenin signaling. J Biol Chem (2004) 279: 5725–5733. | Article | PubMed | ISI | ChemPort | |
| 33. | Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL & MacDougald OA. Inhibition of adipogenesis by Wnt signaling. Science (2000) 289: 950–953. | Article | PubMed | ISI | ChemPort | |
| 34. | Sheldahl L, Park M, Malbon C & Moon R. Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr Biol (1999) 9: 695–698. | Article | PubMed | ISI | ChemPort | |
| 35. | Slusarski DC, Yang-Snyder J, Busa WB & Moon RT. Modulation of embryonic intracellular Ca2+ Signaling by Wnt-5a. Dev Biol (1997) 182: 114–120. | Article | PubMed | ISI | ChemPort | |
| 36. | Sperling LC. Hair anatomy for the clinician. J Am Acad Dermatol (1991) 25: 1–17. | PubMed | ISI | ChemPort | |
| 37. | Sumanas S, Strege P, Heasman J & Ekker SC. The putative Wnt receptor Xenopus Frizzled-7 functions upstream of beta-catenin in vertebrate dorsoventral mesoderm patterning. Development (2000) 127: 1981–1990. | PubMed | ISI | ChemPort | |
| 38. | Takeuchi M, Nakabayashi J, Sakaguchi T, Yamamoto TS, Takahashi H, Takeda H & Ueno N. The prickle-related gene in vertebrates is essential for gastrulation cell movements. Curr Biol (2003) 13: 674–679. | Article | PubMed | ISI | ChemPort | |
| 39. | Taylor G, Lehrer MS, Jensen PJ, Sun TT & Lavker RM. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell (2000) 102: 451–461. | Article | PubMed | ISI | ChemPort | |
| 40. | Terasaki H, Saitoh T, Shiokawa K & Katoh M. Frizzled-10, up-regulated in primary colorectal cancer, is a positive regulator of the Wnt–beta-catenin–Tcf signaling pathway. Int J Mol Med (2002) 9: 107–112. | PubMed | ISI | ChemPort | |
| 41. | Topczewski J, Sepich DS & Myers DC et al. The zebrafish Glypican knypek controls cell polarity during gastrulation movements of convergent extension. Dev Cell (2001) 1: 251–264. | Article | PubMed | ISI | ChemPort | |
| 42. | Ulrich F, Concha ML & Heid PJ et al. Slb/Wnt11 controls hypoblast cell migration and morphogenesis at the onset of zebrafish gastrulation. Development (2003) 130: 5375–5384. | Article | PubMed | ISI | ChemPort | |
| 43. | van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L & Grosschedl R. Development of several organs that require inductive epithelial–mesenchymal interactions is impaired in Lef-1-deficient mice. Genes Dev (1994) 8: 2691–2703. | PubMed | ISI | ChemPort | |
| 44. | Veeman MT, Axelrod JD & Moon RT. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Developmental Cell (2003) 5: 367–377. | Article | PubMed | ISI | ChemPort | |
| 45. | Wang J & Shackleford GM. Murine Wnt10a and Wnt10b: Cloning and expression in developing limbs, face and skin of embryos and in adults. Oncogene (1996) 13: 1537–1544. | PubMed | ISI | ChemPort | |
| 46. | Wang Y, Macke JP & Abella BS et al. A large family of putative transmembrane receptors homologous to the product of the drosophila tissue polarity gene Frizzled. J Biol Chem (1996) 271: 4468–4476. | Article | PubMed | ISI | ChemPort | |
| 47. | Weidinger G & Moon RT. When Wnts antagonize Wnts. J Cell Biol (2003) 162: 753–755. | Article | PubMed | ISI | ChemPort | |
| 48. | Westfall TA, Brimeyer R, Twedt J, Gladon J, Olberding A, Furutani-Seiki M & Slusarski DC. Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/beta-catenin activity. J Cell Biol (2003) 162: 889–898. | Article | PubMed | ISI | ChemPort | |
| 49. | Wong GT, Gavin BJ & McMahon AP. Differential transformation of mammary epithelial cells by Wnt genes. Mol Cell Biol (1994) 14: 6278–6286. | PubMed | ISI | ChemPort | |
| 50. | Yang-Snyder J, Miller JR, Brown JD, Lai CJ & Moon RT. A Frizzled homolog functions in a vertebrate Wnt signaling pathway. Curr Biol (1996) 6: 1302–1306. | Article | PubMed | ChemPort | |
| 51. | Zorn AM. Wnt signalling: Antagonistic dickkopfs. Curr Biol (2001) 11: R592–R595. | Article | PubMed | ISI | ChemPort | |
Acknowledgments
We thank Jeremy Nathans for Fz plasmid probe templates, and George Cotsarelis for comments on the manuscript.
