the mammalian skin and its appendages are derived from ectoderm and mesoderm during embryogenesis ( fuchs , 2007 ; hardy , 1992 ) . the embryo surface emerges as a single layer of epithelial cells which give rise to the epidermis . subsequently , the epidermal - dermal interaction results in hair follicle formation ( fuchs , 2007 ; hardy , 1992 ) . morphologically , the development of hair follicles begins with a local thickening of the epidermis , known as hair placodes . upon successful initiation of the epithelial placode , a condensate of dermal cells is formed in the underlying mesenchyme ( driskell et al . , 2011 ; fuchs , 2007 ) . the signals sent between this mesenchymal condensate and the overlying epithelial placode dictate the behavior of both cell populations , ultimately orchestrating formation of the hair follicle and dermal papilla ( fuchs , 2007 ; hardy , 1992 ; millar , 2002 ; schmidt - ullrich and paus , 2005 ) . classical cross - species experiments indicate that mouse dermal tissue is capable of inducing the hair placode , feather bud or scale placode upon epithelial - mesenchymal recombination ( dhouailly , 1973 ; hardy , 1992 ; olivera - martinez et al . , 2004 ) . however , the first dermal signal initiating the developmental programming of the epidermis remains elusive . several families of secreted signaling molecules have been implicated in the communication between epidermis and dermis during hair follicle development . among them , the wnt family appears to be the earliest and most critical regulator for early development of the epidermis ( fuchs , 2007 ; millar , 2002 ; noramly et al . , 1999 ; this in turn elevates bmp , leading to fate determination of the epidermis ( fuchs , 2007 ; stern , 2005 ) . during formation of the hair follicles , activation of wnt/-catenin signaling is evident in both the epithelium and the underlying mesenchyme ( andl et al . , 2002 ; dasgupta and fuchs , 1999 ; zhang et al . , 2008 ) . deletion of -catenin in the epidermis impairs the formation of hair placodes ( huelsken et al . , 2001 ) . -catenin signaling is necessary and sufficient to initiate hair follicle development ( andl et al . , 2002 ; gat et al . , although the importance of wnt signaling in hair follicle development is well established , it is not clear whether the wnt signal arises intradermally or intraepidermally . whether wnt acts as the first dermal signal to initiate hair follicle development also remains to be determined . disruption of wnt production in the signal - producing cells can yield important insights into the mechanism underlying hair follicle development . however , due to overlapping expression of wnts in the developing skin , gene specific inactivation may not be practical , and is likely to encounter issues related to functional redundancy . we have recently identified gpr177 as the mouse orthologue of drosophila wls / evi / srt essential for proper sorting and secretion of wnt ( banziger et al . , 2006 ; bartscherer et al . , 2006 ; fu et al . , 2011 ; fu et al . , 2009 wls / evi / srt regulates the secretion of all wnts , except for wntd , due to its exclusion from lipid modifications ( ching et al . , 2008 ; herr and basler , 2012 ) . in mice , genetic studies have suggested that the gpr177-mediated regulation of canonical and noncanonical wnts is required for different cell types and tissues ( fu et al . , 2011 ; fu et al . , 2009 ; stefater et al . , the abrogation of wnt secretion caused by gpr177 deficiency may provide an excellent strategy to determine the source of wnt during organogenesis . to decipher wnt signaling regulation in hair follicle development , we created mouse models with cell - type specific disruption of gpr177 . this study not only defines the cell type responsible for wnt production , but also reveals a highly dynamic regulation of wnt signaling , at various steps of hair follicle development . our findings suggest a model for the role of wnt signaling in hair follicle morphogenesis . we examined the expression of gpr177 to determine the cell type potentially responsible for wnt production during hair follicle induction . using a well - characterized antibody specifically recognizing gpr177 ( fu et al . , 2009 ) , immunostaining analysis revealed that gpr177 is expressed in the epithelium and the underlying mesenchyme at e11.5 and e13.5 ( fig . gpr177 expression was restricted to the developing hair follicles and the basal layer of epidermis at e15.5 and e17.5 ( fig . co - labeling of gpr177 with ae3 , a marker for the entire epidermis , or k14 , a marker for the epidermal basal layer , further indicated that the epidermal basal cells and the hair follicular cells express high levels of gpr177 ( fig . to determine the requirement of gpr177 in the epidermis , we generated gpr177 mutant mice in which gpr177 was inactivated by k5-cre ( ramirez et al . , 2004 ) . using the r26r reporter allele , -gal staining of the e13.5 and e14.5 skins showed the effectiveness of cre recombination in the epidermis and developing hair follicles ( fig . immunostaining for gpr177 further indicated its successful ablation in the epidermis , but not dermis , of gpr177 ( fig . 2c , primary epidermal cells of control and gpr177 were used as the signal - producing cells , which were co - cultured with the signal - receiving cells harboring a topflash reporter for -catenin and lef / tcf - dependent transcription . while the control epidermal cells were capable of activating the topflash reporter , this activation was significantly reduced and close to the background level in the gpr177 dermal cells ( fig . s2a ) . furthermore , activation of several wnt signaling mediators was affected in the epidermis of gpr177 ( fig . no hair placodes and follicles were detected in the gpr177 mutants at e14.5 and e17.5 , respectively , indicating an essential role of gpr177 in the epidermis for hair follicle induction ( fig . next , we examined the expression of placode / follicle - specific genes affected by the loss of gpr177 ( botchkarev and sharov , 2004 ; laurikkala et al . , 2002 ; zhang et al . , 2009 ) no expression of ectodysplasin receptor ( edar ) , bmp2 , bmp4 and shh was found , indicating no formation of the placodes in the gpr177 mutants ( fig . the results imply that epidermal gpr177 is involved in the regulation of wnt , the earliest developmental signal known to turn on the programming for hair follicle induction ( fuchs , 2007 ; millar , 2002 ; noramly et al . multiple members of the wnt family were expressed in distinct patterns in the developing skin with a few candidates , e.g. wnt3 , wnt10a and wnt10b , implicated in hair follicle morphogenesis ( andl et al . , 2002 ; millar et al . , 1999 ; reddy et al . , 2001 ) . we first examined the expression of all 19 mouse wnt genes in the e14.5 skin by rt - pcr analysis , and detected transcripts of wnts2 , 3 , 4 , 5a , 6 , 7a , 7b , 10a , 10b , 11 and 16 , including those reported ( reddy et al . , 2001 ) , whose expression patterns were further characterized ( fig . s3 ) . in situ hybridization revealed that wnts2 , 3 , 7a , 7b , 10a and 16 are expressed in the epidermis ( fig . 3a , b , f , g , h ) ; wnts5a and 11 in the dermis ( fig . 3d , j ) ; wnts4 , 6 and 10b in both layers ( fig . furthermore , the wnts3 , 4 and 6 transcripts were evenly distributed throughout the epidermis , including the hair placode and interfollicular epithelium ( fig . 3b - c , e ) . while wnts2 , 7b , 10a and 10b showed elevated expression in the hair placode ( fig . 3a , g - i ) , wnts7a and 16 were expressed mainly in the interfollicular epithelium ( fig . the inactivation of gpr177 abolished the epidermal expression of wnts2 , 7a , 7b , 10a and 10b ( fig . in contrast , wnts3 , 4 , 6 and 16 genes remained active in the gpr177 mutants ( fig . furthermore , the dermal expression of wnt5a and wnt11 genes was affected by the epidermal ablation of gpr177 ( fig . 3d and j ) . their expression at low uniform levels was maintained in the dermis but absent in the dermal condensate due to the lack of hair placodes in the mutants . based on these data , we could categorize the hair follicle - expressing wnt genes into two groups : the first one , consisting of wnts3 , 4 and 6 , was evenly expressed in the hair placode and interfollicular epithelium and the second one , consisting of wnts2 , 7b , 10a and 10b , exhibited elevated expression in the hair placode . the second , but not the first , group was affected by the epidermal deletion of gpr177 . the results imply a potential hierarchy of wnt activation during hair follicle development . because -catenin signaling is required for early onset of hair follicle morphogenesis ( huelsken et al . , 2001 ; zhang et al . , 2009 ) , we examined if the canonical wnt pathway was affected by the gpr177 deletion . immunostaining of the control and gpr177 skins revealed a drastic reduction of an activated form of -catenin ( abc ) in the hair placode and the underlying mesenchyme at e14.5 ( fig . 3l , l ) , and in the hair follicular epithelial and dermal cells at e17.5 ( fig . the reduction of active -catenin was also accompanied by the loss of expression of its transcriptional targets , axin2 ( fig . 3q , q ) , all of which are critically involved in hair follicle development ( bazzi et al . an early activation of wnt/-catenin signaling in the upper dermis immediately adjacent to the epidermis occurs prior to the appearance of the placode at e13.5 ( fig . 3n ) , suggesting that wnt secretion from the epidermis drives the early response of -catenin signaling in the dermis . subsequently , the patterned signaling activity of wnt/-catenin necessary for hair follicle morphogenesis is impaired ( huelsken et al . in addition to cell fate specification and differentiation , wnt is also a key signal for cell proliferation and survival during skin organogenesis ( widelitz , 2008 ) . brdu incorporation analysis revealed that the number of proliferating cells was significantly reduced in the epidermis , but not dermis , of gpr177 , compared to the littermate control ( fig . s4a - c , mutant : 23.910.01% and control : 34.030.03% ; p value < 0.01 , n=3 ) . these results indicated a detrimental effect of the gpr177 deletion on the canonical wnt pathway , suggesting a mechanism underlying the induction of hair follicles mediated through gpr177-dependent -catenin signaling . to definitively assess that the impairment of -catenin signaling is responsible for the hair follicle defects , we introduced the -catex3fx allele into the gpr177 mice to generate the gpr177 ; scat model . in these mutants , a stabilized -catenin mutant was expressed in the developing epidermis due to the deletion of exon 3 of -catenin ( maruyama et al . , 2010 ; , we examined if the block in hair follicle induction caused by the gpr177 deletion could be alleviated . hair placode - like structures were apparent in the gpr177 ; scat skins , suggesting that induction has occurred at e15.5 ( fig . immunostaining analysis further showed that epidermal activation of -catenin takes place in the absence of gpr177 ( fig . 4d - i ) . nonetheless , dermal activation of -catenin remained absent in the gpr177 ; scat skins ( fig . , we first examined the expression of edar and shh , which are evident in the control but missing in the gpr177 skins ( fig . however , expression of the -catenin mutant was able to activate edar and shh in the placode - like structures of gpr177 ; scat , suggesting a restoration of placode signals ( fig . , -catenin activation was able to overcome the block in hair follicle induction caused by the epidermal deletion of gpr177 . the formation of hair placodes then triggers a clustering of underlying mesenchymal cells to form dermal condensates , leading to the development of dermal papilla expressing cd133 ( hardy , 1992 ; ito et al . the cd133 positive dermal papilla cells were present in the control , but absent in the gpr177 ; scat mutants ( fig . although epidermal activation of -catenin alleviated the defects associated with initial induction of hair follicles , it failed to induce the neighboring mesenchymal cells to form dermal condensates . these data indicate that wnt/-catenin signaling induced by the epidermal wnt is essential for initial induction of hair follicles within the epidermis . they also imply that the subsequent development of dermal papilla requires wnt secretion from the epithelial cells to induce wnt signaling in the mesenchymal cells . to investigate the requirement of wnt production contributing to the first dermal signal essential for hair follicle induction , we generated the gpr177 model where gpr177 was inactivated in the mesenchymal cells of the developing dermis by dermo1-cre ( tran et al . using r26r , -gal staining of the e14.5 skins showed the efficiency of cre recombination in the mesenchymal cells of developing dermis ( fig . immunostaining of gpr177 further indicated its removal in the dermis , but not epidermis , of gpr177 ( fig . the dermal deletion of gpr177 had no effect on the formation of hair placodes at e15.5 , suggesting that gpr177-mediated wnt production may be dispensable in the dermis for hair follicle initiation ( fig . immunostaining of cadherin proteins showed that the morphology of follicular epithelial cells in the mutants is comparable to that of control ( fig . although edar and shh were expressed in the hair placodes , their expression was affected by the mutation ( fig . 5m - p ) . quantitative measurement confirmed their expression domains reduced in the mutants ( fig . 5y ) . nonetheless , there were sufficient placode signals to promote the subsequent development in the underlying mesenchyme as the dermal condensate formation was not affected in the gpr177 mutants ( fig . although the dermal deletion of gpr177 had minimal effects on hair follicle induction , the dermal layer of gpr177 exhibited a loose structure with significant abnormalities ( fig . the number of proliferating cells was significantly reduced in the dermis , but not the epidermis , of gpr177 , compared to the littermate controls ( fig . s5a - c , mutant : 19.010.03% and control : 32.210.03% ; p value < 0.01 , n=3 ) . the dermal deletion affected cell - type specification of mesenchymal cells into fibroblasts , smooth muscle cells and adipocytes modulated by wnt ( wei et al . , 2011 ) . we detected an apparent shift of dermal cell identity to favor adipocytes in the gpr177 mutants ( fig . this is consistent with prior reports indicating the differential effects of wnt on development of the mesenchymal derived cell types ( cristancho and lazar , 2011 ; wei et al . , 2011 ) . analysis of abc and axin2 expression further indicated no significant difference in the pattern of wnt/-catenin signaling ( fig . the early activation of wnt/-catenin signaling remained detectable prior to the appearance of hair placodes at e13.5 ( fig . 5u - v ) and was unaffected in the placode and dermal condensate at e14.5 ( fig . 5w - x ) . to ensure that gpr177 is required for wnt production in the dermis while the control dermal cells were capable of activating the topflash reporter , this activation was reduced by the gpr177 deletion , suggesting an impairment of wnt secretion ( fig . activation of several wnt signaling effectors was not affected in the dermis and epidermis of gpr177 most likely due to the presence of epidermal wnt ( fig . however , using the ex vivo culture of primary cells , we found that wnt signaling activation is impaired in the gpr177 dermal cells ( fig . although gpr177 regulates wnt secretion in the dermis , dermal wnt is not required for hair follicle induction . the findings thus do not support the idea that wnt is secreted from the dermis to induce -catenin signaling in the epidermis essential for hair follicle initiation . this study reveals that epidermal gpr177 is essential for the wnt - mediated induction of hair follicles . epidermal , but not dermal , deletion of gpr177 disrupts the early activation of wnt/-catenin signaling in the upper dermis prior to initiation of the hair follicle . subsequently , the expression of edar , bmp2/4 and shh , critical for hair follicle morphogenesis is disrupted ( botchkarev and sharov , 2004 ; laurikkala et al . , 2002 ; st - jacques et al . , 1998 ) , suggesting that wnt signaling acts upstream of these pathways . these results are consistent with recent findings but provide additional insights into hair follicle induction mediated by wnt signaling crosstalk between the epidermis and dermis . our data also support the requirement of gpr177 for secretion of all canonical wnts , and imply a hierarchy of wnt actions during hair follicle formation . furthermore , the lack of hair placode formation upon deletion of gpr177 in the epidermis can be alleviated by -catenin stimulation , suggesting that epidermal autocrine signaling of wnt promotes the induction process . however , epidermal restoration of -catenin signaling fails to correct the subsequent formation of dermal condensates . due to the lack of epidermal wnt epidermal wnt is therefore a part of the placode signals which induce the dermis through an extra - epidermal signaling mechanism . our dermo1-mediated deletion of gpr177 causes defects in dermal cell proliferation and differentiation , as well as in full induction of the placode signal during hair follicle morphogenesis , not detected in the deletion mediated by en1-cre ( chen et al . although wnt secretion is impaired , -catenin signaling remains intact in both epidermis and dermis of the mutants . we propose a mechanism underlying hair follicle development mediated by the wnt pathway based on previous evidence and current findings ( fig . -catenin signaling is first uniformly activated in the upper dermis prior to its activation in the epithelium of hair placodes or feather buds ( noramly et al . this is followed by activation in the placode epithelium , and then in the underlying mesenchyme and dermal condensate ( dasgupta and fuchs , 1999 ; fuchs , 2007 ) . epidermal but not dermal production of wnt is responsible for activating -catenin signaling in the epithelium and mesenchyme during hair follicle and dermal papilla induction . epidermal wnt is required for activation of -catenin signaling not only within the epidermis ( epidermal autocrine ) but also across the epidermis to the dermis ( epidermal paracrine ) . before the receipt of the initial dermal signal , -catenin signaling is blocked by an inhibitor (  ) in the epidermis ( stage : pre - placode ) . however , the epidermal wnt signal permits a uniform activation of -catenin signaling in the upper dermis via epidermal paracrine regulation . when the initial dermal signal arises , it prohibits the inhibitory effect on -catenin to induce the placode - specific signaling in the epidermis , followed by induction of the hair placode via epidermal autocrine regulation ( stage : placode ) . a reciprocal induction occurs subsequently between the placode and underlying mesenchyme ( stage : dermal condensate ) . the placode signals including epidermal wnt are sent back to the underlying mesenchyme to pattern the signaling activity of -catenin for the formation of dermal condensate and papilla . the concept of the  first dermal message  as the initial signal for hair follicle induction was described decades ago ( hardy , 1992 ) . because of the essential role of -catenin and its spatiotemporal expression pattern during hair follicle development , wnt has been suggested to be the first signal in the dermis ( fuchs , 2007 ; millar , 2002 ; noramly et al . our genetic studies imply that wnt probably is not the  first dermal message  . instead of activation , we hypothesize that the initial dermal signal is required for de - repression of -catenin signaling in the epidermis . smad7-mediated regulation of smurf2 has been shown to modulate -catenin in hair follicle formation ( han et al . , 2006 ) , suggesting a potential mechanism underlying the inhibition effect of wnt . how the first signal arising in the dermis to regulate the wnt pathway in the epidermis remains an important question to be addressed . the dermal abnormalities detected in the gpr177 mutants suggest that the gpr177-mediated secretion of wnt play a critical role in the dermis . these abnormalities are most likely caused by alteration in noncanonical , but not canonical , wnt because the -catenin signaling activity is comparable in the dermis of control and gpr177 . furthermore , dermal wnt is not required for hair follicle initiation , consistent with wnt5a dispensable for this process ( hu et al . , 2010 ) . another question remaining to be addressed is which of the wnt family members regulates the development of hair follicles . our findings suggest that the epidermal deletion of gpr177 interferes with the expression of wnts2 , 7b , 10a and 10b , which exhibit elevated expression in the placode . however , the expression of wnts3 , 4 and 6 uniformly in the interfollicular epithelium and placode is not affected in the mutants . as a trafficking regulator for wnt proteins , the removal of gpr177 the first group of wnt genes , including wnts3 , 4 and 6 , is unaffected by the gpr177 deletion in the epidermis . thus they represent candidates for the  primary wnt  which mediates hair follicle initiation . the second group of wnt genes , including wnts2 , 7b , 10a and 10b , whose expression is disrupted by the gpr177 deletion , depends on epidermal activation of -catenin signaling . this is supported by the observation that ectopic expression of the wnt inhibitor dkk1 and ectodermal deletion of -catenin impair hair follicle development through disruption of patterning signaling mediated by wnts 10a and 10b ( andl et al . , 2002 ; zhang et al . therefore , wnts 2 , 7b , 10a and 10b likely act as the  secondary wnt  , which is a part of the placode signal , essential for hair follicle development . defining the specific role of these wnts in hair follicle induction the gpr177fx , k5-cre , dermo1-cre , r26r , -catex3fx and axin2 mouse strains , and genotyping methods were reported previously ( fu et al . , 2011 ; , 1999 ; soriano , 1999 ; sosic et al . , 2003 ; tarutani et al . , 1997 ; yu et al . , 2005a ; yu et al . , 2003 ) . care and use of experimental animals described in this work comply with guidelines and policies of the university committee on animal resources at the university of rochester . epidermal and dermal cells isolated from the specific skin layer were cultured in the cnt - pct and dmem media , respectively , with 10% fbs . for wnt secretion assay , cells transfected with topflash were used as indicator for receiving of wnt signals determined by relative luciferase activity . for histology and immunostaining , samples were fixed and embedded for sections , stained with h&e and specific antibodies using the avidin : biotinlylated based method ( chiu et al . , 2008 ; fu et al . , 2011 ; fu et al . , 2009 ; maruyama et al . , 2010 ; yu et al . , 2005a ; yu et al . , 2010 ; yu et al . , 2005b ) . -gal staining was performed with standard protocols described previously ( fu et al . , 2009 ; to detect apoptotic cells , tunel staining was performed as described ( maruyama et al . in situ hybridization was performed to detect gene expression using the digoxygenin - labeled probes , followed by recognition with an alkaline phosphatase conjugated anti - digoxygenin antibody ( chiu et al . , 2008 ; david and wedlich , 2001 ; fu et al . , 2009 ; yu et al . , 2010 ) . the gpr177fx , k5-cre , dermo1-cre , r26r , -catex3fx and axin2 mouse strains , and genotyping methods were reported previously ( fu et al . , 2011 ; , 1999 ; soriano , 1999 ; sosic et al . , 2003 ; tarutani et al . , 1997 ; yu et al . , 2005a ; yu et al . , 2003 ) . care and use of experimental animals described in this work comply with guidelines and policies of the university committee on animal resources at the university of rochester . epidermal and dermal cells isolated from the specific skin layer were cultured in the cnt - pct and dmem media , respectively , with 10% fbs . for wnt secretion assay , cells transfected with topflash were used as indicator for receiving of wnt signals determined by relative luciferase activity . for histology and immunostaining , samples were fixed and embedded for sections , stained with h&e and specific antibodies using the avidin : biotinlylated based method ( chiu et al . , 2008 ; fu et al . , 2011 ; fu et al . , 2009 2005a ; yu et al . , 2010 ; yu et al . , 2005b ) . -gal staining was performed with standard protocols described previously ( fu et al . , 2009 ; to detect apoptotic cells , tunel staining was performed as described ( maruyama et al . , 2010 ; yu et al . , 2007 ) total rna isolated from e14.5 mouse skins was subject to rt - pcr analysis . in situ hybridization was performed to detect gene expression using the digoxygenin - labeled probes , followed by recognition with an alkaline phosphatase conjugated anti - digoxygenin antibody ( chiu et al . , 2008 ; david and wedlich , 2001 ; fu et al . , 2009 ; yu et al . , 2010 )
a signal first arising in the dermis to initiate the development of hair follicles has been described for many decades . 
 wnt is the earliest signal known to be intimately involved in hair follicle induction . 
 however , it is not clear whether the inductive signal of wnt arises intradermally or intraepidermally . 
 whether wnt acts as the first dermal signal to initiate hair follicle development also remains unclear . 
 here , we report that wnt production mediated by gpr177 , the mouse wls orthologue encoding a wnt trafficking regulator , is essential for hair follicle induction . cell - type specific abrogation of the signal reveals that only epidermal , but not dermal , production of wnt is required . 
 an intra - epidermal wnt signal is necessary and sufficient for hair follicle initiation . 
 but , the subsequent development depends on reciprocal signaling crosstalk of epidermal and dermal cells . 
 wnt signals within the epidermis and dermis , and crossing between the epidermis and dermis , have distinct roles and specific functions in skin development . 
 this study not only defines the cell type responsible for wnt production , but also reveals a highly dynamic regulation of wnt signaling at different steps of hair follicle morphogenesis . 
 our findings uncover a mechanism underlying hair follicle development orchestrated by the wnt pathway .