mouse embryos form five pairs of mammary rudiments ( mrs ) . their formation is preceded by the formation of three wnt10b - expressing mammary streaks in the axilla , inguen , and in between forelimb and hindlimb these streaks fuse into one continuous mammary line ( ml ) on each flank by e11.5 , on which mrs develop in asynchronous fashion and not in numerical sequence , with mr1 in the axilla ; mr2 , mr3 and mr4 on the flank ; and mr5 in the inguen . the hedgehog ( hh ) pathway was among the first signaling cascades tested for a role in murine mammogenesis , but is not required for it [ 2 , 3 ] . nonetheless , extratoes ( xt ) mice , which harbor a spontaneous , functional null - mutation for the transcription factor gli3 , exhibit multiple defects in prenatal mammary development [ 47 ] . given the role of gli3 in hh signaling , we provide a brief overview of hh signaling and gli transcription factors , followed by their functions in mammogenesis in mouse embryos . mammals possess three orthologues of the drosophila segment polarity gene hedgehog ; sonic hedgehog ( shh ) , indian hedgehog ( ihh ) , and desert hedgehog ( dhh ) [ 8 , 9 ] . while embryonic expression and function of dhh is restricted to spermatogenesis and schwann cells , shh and ihh are widely expressed and encode secreted morphogens with well - known roles for shh in neural tube , limb and somite development and for ihh in chondrogenesis [ 10 , 11 ] . in canonical hh signaling , hh molecules bind their twelve - transmembrane receptor patched1 ( ptch1 ) or ptch2 . ptch then derepresses the seven - transmembrane protein smoothened ( smo ) , which becomes activated . through intracellular signaling complexes -whose components have not been studied in embryonic mammary development and are thus not described here- smo activation converges on the gli family of krppel - type zinc - finger transcription factors , comprising gli1 , gli2 and gli3 . gli1 is expressed when hh signaling is active , and encodes the transcriptional activator gli1 . gli2 and gli3 can be expressed in the absence of hh signaling , in which case their protein products are cleaved to render transcriptional repressors ( gli2 , gli3 ) . gli2 requires less active hh signaling than gli3 to remain uncleaved and function as a transcriptional activator ( gli2 , gli3 ) . among gli transcriptional targets are gli1 and ptch1 , which provide regulatory feedback loops for hh signaling [ 12 , 13 ] . they need further exploration , but certainly point to three additional ,  non - canonical  signaling scenarios : 1 ) signaling involving hh pathway components independently of gli - mediated transcription , such as shh - mediated erk activation in mammary epithelial cells without smo - activation ; 2 ) direct interaction of hh signalling components with other molecular pathways ; potentially relevant for mammogenesis is the role of wnt signaling in expression and function of gli proteins , and 3 )  non - contiguous  or  atypical  interaction of core hh pathway components with each other . studies of the role of hh signaling components in mammogenesis began in the late 1960 s , when d.r . johnson identified and characterized a mouse strain which he named extra - toes ( xt ) , after the prominent polydactyly in all four paws of heterozygotes . in e13 embryos homozygous for this spontaneous , semi - dominant mutation , he also found a reduced number of mrs between the forelimb and hindlimb . because his wild type control had a supernumerary mr near mr3 the number of nipples visible in xt / xt mice at this stage is reduced from four to two pairs , the most anterior and posterior ones persisting  . note that johnson was mistaken in three aspects : 1 ) what he referred to as nipples were in fact mammary buds , which precede nipple formation ; 2 ) had he taken a proper control , he would have concluded that only one mr ( mr3 ) was missing on the flank ; 3 ) had he known that wild - types also form mrs behind the forelimb ( mr1 ) and hindlimb ( mr5 ) , he would have noticed an absence of mr5 in xt / xt embryos [ 57 , 18 ] . arose a spontaneous mouse mutant , named xt because it phenocopies johnson s xt mutant . xt mice carry a 51.5  kb deletion downstream of nucleotide 1670 of gli3 , including the dna - binding zinc fingers and thus abolishing both gli3 and gli3 functions [ 1921 ] , without loss of other known functional sequences . these renamed gli3 mice represent a good model for the human greig cephalopolysyndactyly syndrome ( gcps ) , which can be a sporadic or inherited trait , caused by heterozygous loss - of - function mutations of the gli3 gene . the syndrome is characterized by polysyndactyly of hands and feet , hypertelorism , and skull abnormalities including craniosynostosis at variable penetrance [ 19 , 22][omim 165240 , 175700 ] , the latter seen in mice only when homozygous mutant . while no mammary phenotypes have been reported for gcps patients and gli3 mouse mutants , gli3 embryos fail to induce mr3 and mr5 [ 57 , 18 ] . moreover , .  1 t with 1l ) , resembling mrs of wild - type male embryos , and also exhibits impaired branching morphogenesis ; while mr1 is hardly affected . the xt mutation thus exemplifies that all mammary glands are different and can develop inter - independently . the question then arises via which molecular mechanisms and tissue - interactions gli3 regulates formation and development of mr2-5.fig . 1expression patterns of gli1 , gli2 and gli3 , and models of tissue- and molecular interactions involving gli3 in mammary development . a q cross - sections of wild - type embryos and ( r  w ) gli3 embryos at e11.5-e13.5 hybridized with s - labeled rna - probes for gli1 , gli2 and gli3 . insets in t and u present wild - type mr2 from l respectively m at the same magnification . x cartoon of a lateral view of a mouse embryo . between e10.5 and e11.5 gli3 is expressed in the limb buds and all somites ( outlined in gray and numbered ) . gray shades represent overlapping in expression with fgf10 , the intensity positively correlating with level of fgf10 expression . note the proximity of the overlapping somitic expression to mr3 and mr2 , and overlapping limb mesenchymal expression to mr1 , mr2 , mr4 and mr5 . mr1 and mr5 are hidden behind the limbs . z model for gli3-mediated repression of gli1 in the me ( bud shape ) and contiguous mm ( darker shade of gray surrounding mammary bud ) , based on expression data in panels a  w and in [ 5 , 6 ] . in the absence of gli3 , gli1 is misexpressed in the mm of mr1 , mr2 and mr4 , indicating gli3 normally represses gli1 expression . whether this occurs tissue - autonomously ( intact block arrow ) or via tissue interactions ( broken block - arrow ) remains unclear . abbreviations : li : liver , r : rib primordium , st : stomach , wt : wild - type expression patterns of gli1 , gli2 and gli3 , and models of tissue- and molecular interactions involving gli3 in mammary development . a q cross - sections of wild - type embryos and ( r  w ) gli3 embryos at e11.5-e13.5 hybridized with s - labeled rna - probes for gli1 , gli2 and gli3 . insets in t and u present wild - type mr2 from l respectively m at the same magnification . x cartoon of a lateral view of a mouse embryo . between e10.5 and e11.5 gli3 is expressed in the limb buds and all somites ( outlined in gray and numbered ) . gray shades represent overlapping in expression with fgf10 , the intensity positively correlating with level of fgf10 expression . note the proximity of the overlapping somitic expression to mr3 and mr2 , and overlapping limb mesenchymal expression to mr1 , mr2 , mr4 and mr5 . mr1 and mr5 are hidden behind the limbs . z model for gli3-mediated repression of gli1 in the me ( bud shape ) and contiguous mm ( darker shade of gray surrounding mammary bud ) , based on expression data in panels a  w and in [ 5 , 6 ] . in the absence of gli3 , gli1 is misexpressed in the mm of mr1 , mr2 and mr4 , indicating gli3 normally represses gli1 expression . whether this occurs tissue - autonomously ( intact block arrow ) or via abbreviations : li : liver , r : rib primordium , st : stomach , wt : wild - type prior to and at the time of ml and mr formation , gli3 is not expressed in the surface ectoderm , from which the mrs derive , but is expressed in the limb mesenchyme , and in the somites in the region between fore- and hindlimbs   ( fig . 1c ) . given the earlier implications of somitic involvement in the establishment of the mammary streak along the flank and in mr induction [ 1 , 7 ] , the somitic gli3 expression was of interest . indeed , the ventral elongation of the somites including expression of gli3 in all thoracic and lumbar somites , and fibroblast growth factor ( fgf)10 in thoracic somites # 12 to # 18 , determines the position of the ml on the dorso - ventral body axis . the absence of mr3 in gli3 embryos   is due to reduction of somitic fgf10 expression , in particular the relatively high fgf10 expression in somite # 15 underlying mr3 . as mr2 is located above somite # 12 , reduced somitic fgf10 expression may be involved in impaired development of mr2 as well . it is most likely that somitic gli3 indirectly activates somitic fgf10 independent of shh signaling ( and references therein ) . somitic fgf10 may reach and activate the ectodermal fgfr2b via diffusion and/or transportation by dermal precursor cells that delaminate from the somites . this cascade leads to ectodermal cell elongation , wnt10b marker expression , and canonical wnt signaling as required for ml and mr formation , but only at the level of mr3 , and possibly mr2 . these insights finally gave some molecular explanation for the capacity of the dermal mesenchyme to induce mr formation as recognized four decades earlier [ 2527 ] . however , at the level of mr4 and mr5 , fgf10 is not expressed in the somites . given that null mutants for either gli3 or fgf10 fail to induce mr5 and both genes are expressed in the adjacent limb mesenchyme   ( fig . 1x ) , it is tempting to speculate that an epistatic interaction between these molecules may also exist in the limb mesenchyme and be required for induction of mr5 . the compromised induction of mr2 and mr4 in the absence of gli3 can perhaps be attributed to gli3-independent residual expression of fgf10 and other genes in the thoracic somites and limb mesenchyme . the cellular functions of gli3 during early mammary growth and morphogenesis are fourfold , and the same in all mrs : downregulation of 1 ) cell proliferation and 2 ) cell apoptosis in epithelial cells of the ml and mrs ; 3 ) mediation of ectodermal cell recruitment into mrs , which contributes to mr growth until at least e14.5 ; 4 ) hypertrophy of the basal / peripheral cells of the me , which provides a large proportion of the growth between e12.5 and e13.5 . while similar regulatory functions for gli3 have been identified in other cell types ( references in ) , it remains to be investigated how gli3 mediates so many distinct effects in the same cell population ; and whether these epithelial effects rely on gli3 expression prior to mammary induction , on cell - autonomous gli3 function in the me , or via tissue - interactions with and gli3 expression in the mm ( fig . at e11.5 , the somites also express gli2 , but not gli1 ( fig . expression of gli genes in the surface ectoderm or mammary tissues is arguable at e12.5 , except for notable gli3 expression in mr5 ( fig . 1d  k ) as confirmed by rna - profiling . by e13.5 , gli3 and gli2 are convincingly expressed in overlap in the ectoderm , mammary epithelium ( me ) and mammary mesenchyme ( mm ) ( fig .  1m , n , p , q ) , the latter confirmed for gli3 by rna - profiling   and immunohistochemistry . thus , the initial growth defects of gli3 mr2 and mr4 at e11.5 could subsequently be compounded by the lack of gli3 in any of the tissue compartments . although gli2 and gli3 overlap in expression , homozygous gli2-null mutants have no mammary induction defect [ 6 , 28 ] . neither have gli1-null , gli2 or gli2 mutants which express gli1 from the endogenous gli2 promoter however , removing one gli3 allele in gli2 embryos abolishes induction of mr3 and mr5 as in gli3 embryos . moreover , gli1 is misexpressed in the mm of mr2 in e13.5 gli3 embryos ( fig .  1 t ) . the high gli1 expression in mr1 and mr4 at e13.5 ( fig . 1r , v ) may also reflect misexpression in the mm , similar to such misexpression reported at e14.5 . together , these data indicate that gli3 acts as gli3 during mr formation   ( fig . 1y , z ) , and is the only gli family member with functional relevance for embryonic mammary development , by repressing gli1 transcription , and antagonizing gli2 function . moreover , these data indicate that canonical hh signaling needs to be in an off - state to allow mammogenesis , at least at the positions of mr3 and mr5 . the above - described repressor function of gli3 and absence of shh , ihh , gli1 and ptc1 expression in the surface ectoderm or dermal mesenchyme at the time of ml and mr induction   ( our unpublished data and fig . 1a ) , explain why shh and ihh mutant mice have no mammary induction defect [ 2 , 3 ] . all together , these data indicate hh signaling is not required for induction of any of the five mrs . while normally , shh , ihh and ptc1 are expressed in the me from e12.5 onward and in postnatal stages , the normal outgrowth of transplanted e12.5 shh and ihh me in cleared wild - type mammary fat pads or under the kidney capsule , indicates that epithelial hh signaling or mesenchymal shh is not either required during later embryonic and postnatal stages . interestingly , activating components of the hh signaling cascade become expressed in the surface ectoderm and me only after the formation of mr , but prior to hair follicle formation . we therefore propose that the off - state of hh signaling is a discriminatory factor for the choice of a skin appendage between mammary and either hair or non - mammary fate , and that therefore mrs must develop prior to hair follicle formation , i.e. prior to activation of hh signaling in the trunk ectoderm [ 29 , 30 ] . such a decisive role for hh signaling appendage fate requires further investigation , but seems to be confirmed by the glandular appearance of hair follicles when hh signaling in the skin is ablated in k14cre;smo embryos . gli3 function is critical for embryonic development of mr2 - 5 ; in particular gli - mediated repression of canonical hh signaling is critical for induction of mr3 and mr5 . its variable requirement for the five mrs in mouse gives perhaps some insight in mechanisms underlying the variation in number of mammary glands among mammalian species . for example , mr3 requires somitic gli3 as the earliest known effector in a cascade involving fgf10/fgfr2b and subsequent wnt signaling ( fig . 1y ) , but other tissue - interactions and perhaps molecular mechanisms act downstream of gli3 in early development of other mrs , and remain to be elucidated . examples of additional interesting avenues for investigation pertain to possible interactions with other important mesenchymal factors in mr3 formation ( e.g. nrg3 , tbx3 , or raldh2 that mediates retinoic acid signaling ) [ 3234 ] ( fig . 1y ) ; whether the sustained protrusion of mr2 in gli3 embryos bears any mechanistic similarity to the protrusion of mrs in wild - type males ; and whether gli3 is involved in non - canonical hh signaling in mammogenesis . given the many molecular similarities between embryonic mammary development and mammary / breast tumors [ 35 , 36 ] , it is of interest whether gli3 plays a role in breast cancer . it is noteworthy that gli(1 ) was discovered as an oncogene , and like gli2 and shh , is upregulated in various cancers . hh signaling is considered a therapeutic target for breast cancer , as gli(1 ) upregulation predicts a poor prognosis for estrogen receptor negative or triple negative breast cancers [ 3739 ] . yet , gli3 is seldom reported in association with cancer . in absolute number and relative gli1 , only few somatic mutations in gli3 have been found in breast cancer samples [ cosmic database , sanger institute ] . it would be of interest to determine whether these gli3 mutations relate to gli1 upregulation , and whether they have any diagnostic or prognostic value .