mir-212 and mir-132 are required for epithelial stromal interactions necessary for mouse mammary gland development

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1 mir-212 nd mir-132 re required for epithelil stroml interctions necessry for mouse mmmry glnd development Ahmet Ucr 1,5, Vid Vfizdeh 2, Huertus Jrry 3, Jn Fiedler 4, Petr A B Klemmt 2, Thoms Thum 4, Bernd Groner 2 & Kml Chowdhury 1 MicroRNAs re smll noncoding RNAs tht crry out post-trnscriptionl regultion of the expression of their trget genes. However, their roles in mmmlin orgnogenesis re only eginning to e understood. Here we show tht the microrna- 212/132 fmily (which comprises mir-212 nd mir-132) is indispensle during the development of the mmmry glnds in mice, prticulry for the regultion of the outgrowth of the epithelil ducts. Mmmry trnsplnttion experiments reveled tht the function of the mir-212/132 fmily is required in the strom ut not in the epitheli. Both mir-212 nd mir-132 re expressed exclusively in mmmry strom nd directly trget the mtrix metlloproteinse MMP-9. In glnds tht lck mir-212 nd mir-132, MMP-9 expression increses nd ccumultes round the ducts. This my interfere with collgen deposition nd led to hyperctivtion of the tumor growth fctor-β signling pthwy, therey impiring ductl outgrowth. Our results identify the mir-212/132 fmily s one of the min regultors of the epithelil-stroml interctions tht re required for proper puertl development of the mmmry glnd. MicroRNAs re endogenous regultory smll RNAs tht ssocite with the 3 untrnslted regions (UTRs) of their trget mrnas nd cuse their degrdtion or trnsltionl inhiition 1,2. MicroRNA-medited gene regultion is crucil for mny iologicl processes such s cellulr growth, differentition, moility nd deth 3. However, in contrst to fruit flies nd nemtodes, the functionl importnce of micrornas during verterte development is only eginning to e nlyzed 4. The mir-212/132 fmily is highly conserved in vertertes. MiR- 132 regultes neuronl morphogenesis nd the dendritic plsticity of cultured neurons 5 8. Moreover, ntgomir-medited knockdown studies in dult mice showed tht mir-132 regultes some spects of the circdin clock 9 nd inflmmtory pthwys 10. We used deletion mutgenesis to investigte the developmentl role(s) of mir-212 nd mir-132 in the mouse nd found tht their sence leds to severe impirment of mmmry glnd development. The mmmry glnd hs unique fetures, s most of its development tkes plce during postntl life In mice, n epithelil plcode is formed during emryogenesis. Upon irth, rudimentry ductl structure is present in the nipple re. During puertl development, these ducts extend throughout the ft pd until they rech the peripheries of the glnd. This invsive growth of the ducts occurs t their distl tips, clled terminl end uds (TEBs). Additionl ductl side rnches re lso formed during repeted estrus cycles. During pregnncy, these ductl structures differentite into loulolveolr structures, which produce nd secrete milk during the lcttion period. After wening, the mmmry glnd involutes nd looks similr to n dult virgin mmmry glnd. The mmmry glnd of virgin mouse consists of two seprte comprtments: the epithelil ducts nd the surrounding strom 15,16. Although epithelil ductl outgrowth is the most ovious mcroscopiclly visile structurl chnge during puertl mmmry glnd development, the strom hs crucil functions in the induction nd regultion of this ductl outgrowth y providing essentil growth fctors nd extrcellulr mtrix (ECM) proteins Here we show tht the function of mir-212 nd mir-132 in the mmmry strom is indispensle for the regultion of epithelil-stroml interctions tht re required for ductl outgrowth during puertl development of the mmmry glnds. RESULTS Genertion of mice lcking mir-212 nd mir-132 mir-212 nd mir-132 hve een reported to e generted from stle intron of non protein coding gene tht is expressed in primry neuronl cultures 5,19. However, we identified new trnscript vrint with different exon-intron structures tht encoded oth micrornas in its second exon (Supplementry Fig. 1). These two trnscript vrints oviously represent different lterntively spliced forms of the gene (mir-212/132) tht encodes mir-212 nd mir Deprtment of Moleculr Cell Biology, Mx Plnck Institute of Biophysicl Chemistry, Goettingen, Germny. 2 Georg-Speyer-Hus, Institute for Biomedicl Reserch, Frnkfurt m Min, Germny. 3 Deprtment of Endocrinology, University of Goettingen, Goettingen, Germny. 4 Institute for Moleculr nd Trnsltionl Therpeutic Strtegies, Hnnover Medicl School, Germny. 5 Present ddress: Cellulr Senescence Group, Germn Cncer Reserch Center (DKFZ), Heidelerg, Germny. Correspondence should e ddressed to K.C. (kchowdh@gwdg.de). Received 12 April; ccepted 13 Octoer; pulished online 7 Novemer 2010; doi: /ng.709 Nture Genetics VOLUME 42 NUMBER 12 DECEMBER

2 Figure 1 Genetic deletion of mir-212/132 in mice results in mmmry glnd defects ssocited with impired nourishment of pups. () Pup growth curve nlysis. Pups of homozygous mutnt femles hd retrded growth. The differences in ody weights of mir-212/132 / mothers pups were significnt t postntl dy 2 (P < 0.01) nd t ll susequent dys (P < 0.001) compred with pups of wild-type mothers. No significnt difference ws oserved etween the growth curves for pups of wild-type or heterozygous mothers. Three litters per genotype were used for this nlysis. Dt re expressed s men ± s.d.; n = 15 for ech group. () Neutrl-red Body weight (g) Pups of homozygous mother Pups of heterozygous mother Pups of wild-type mother Postntl dys stining of sections from lctting mmmry glnd smples. The dense epithelil stining (red) ws seen throughout the whole section of the glnds from wild-type nd heterozygous mice. Epitheli within homozygous mutnt glnds were oserved only in the proximl region close to the nipple. The oundries of the section for the homozygous mutnt smple re shown with dshed lines, s the non-epithelil ft pd does not show ny stining. +/ Isoform 1 is expressed only in the testes nd rin, wheres isoform 2 is expressed in the rin, testes, hert nd mmmry glnds (Supplementry Fig. 1). To investigte the physiologicl nd developmentl roles of mir-212 nd mir-132 in mice, we used homologous recomintion to delete the genomic region tht encodes pre mir-212 nd pre mir-132 (Supplementry Fig. 2). DNA lotting (Supplementry Fig. 2) nd quntittive RT-PCR nlyses (dt not shown) confirmed successful genomic deletion nd the complete loss of oth micrornas in homozygous mutnts. mir-212/132 / mice were orn with the expected Mendelin rtio nd hd norml life spn. However, lthough the femle mir-212/132 / mice showed norml nursing ehviors, their pups hd severe growth prolems (Fig. 1) nd usully died within 5 dys of irth. The survivl rte of these pups ws inversely proportionl to the litter size (dt not shown). Cross-fostering experiments showed tht wild-type femles pups fced the sme prolems under the cre of mir-212/132 / mothers, 3 weeks 5 weeks 10 weeks 40 weeks Lctting c e g i d f h j wheres the pups of mir-212/132 / femles grew nd survived normlly if they were fostered y wild-type mothers (dt not shown). Thus, mir-212/132 / femle mice hd difficulty in feeding their pups. To identify the reson for this difficulty, we exmined lctting mmmry glnds histologiclly (Fig. 1). In contrst to wild-type or heterozygous glnds, mir-212/132 / glnds hd n norml ppernce. Dense loulolveolr structures were restricted to the regions close to the nipple re, nd the rest of the ft pds hd no epithelil structures. Impired ductl outgrowth in mir-212/132 / glnds We chrcterized the oserved phenotype further y whole-mount stining of the mmmry glnds of puertl, pregnnt nd lctting mice (Fig. 2 nd Supplementry Fig. 3). The rudimentl ductl trees ppered norml in oth wild-type nd mir-212/132 / pre-puertl mice (Fig. 2,). At 5 weeks of ge, the ductl structures reched the region of the lymph nodes in wild type glnds nd TEBs were visile t their distl ends (Fig. 2c). However, the rudimentl ductl trees in Figure 2 mir-212/132 / mmmry glnds hve defect in puertl ductl outgrowth, ut not in ductl side rnching nd loulolveolr differentition. ( j) Whole-mount nlyses of inguinl (no. 4) mmmry glnds from wild-type nd homozygous mutnt littermtes t different stges of mmmry glnd development. Virgin stges re pre-puertl (3 weeks;,), erly puertl (5 weeks; c,d), lte puertl (10 weeks; e,f), nd 40-week-old dult (g,h). Lctting smples (i,j) were otined from mice killed 1 dy fter giving irth. Proximl-to-distl orienttions of ll smples re shown s left-to-right. The gryscle insets re highmgnifiction pictures of corresponding smples for detiled visuliztion. In wild-type glnds, TEBs were formed (rrow in c) nd ductl outgrowth took plce during puerty (c,e). No TEBs were oserved t the distl ends of the ducts in mutnt glnds (rrowhed in d) nd ductl outgrowth did not tke plce (d,f). Ductl side rnching ws oserved in mutnt glnds (rrows in h) similr to wild-type glnds (g). Loulolveolr structures were formed in mutnt glnds during pregnncy nd milk-producing lveoli were seen during lcttion (j) similr to wildtype glnds (i), ut restricted to the proximl region where the rudimentry ductl tree ws locted. At lest five mice were nlyzed for ech genotype t ech stge. Scle rs represent 1 cm; for gryscle insets, scle rs represent 1 mm VOLUME 42 NUMBER 12 DECEMBER 2010 Nture Genetics

3 Figure 3 mir-212/132 / mmmry epitheli hve norml ductl rchitecture. ( f) Mmmry glnd sections from 6-week-old wild-type (,c,e) nd homozygous mutnt (,d,f) littermtes were nlyzed y hemtoxylin-eosin (H&E) stining (,), nd immunohistochemistry for E-cdherin (E-cdh; c,d) nd cytokertin 14 (CK-14; e,f). E-cdherin is mrker for luminl epithelil cells, wheres cytokertin-14 is expressed only y myoepithelil cells within the ductl structures. Both wild-type nd homozygous mutnt mmmry ducts hve n inner single lyer of luminl epithelil cells nd outer lyer of myoepithelil cells, s shown y E-cdherin nd cytokertin-14 stining, respectively. Firolsts re distinguishle within the periductl strom for oth genotypes. Scle rs represent 100 μm. H&E c d mir-212/132 / glnds filed to initite ductl outgrowth nd produced no TEBs (Fig. 2d). At 10 weeks of ge, the ductl tree in wild type littermtes lmost reched the end of the ft pd (Fig. 2e), ut the ductl structures in mutnt glnds retined their pre-puertl ppernce (Fig. 2f). Wild-type glnds contined elorte epithelil structures t 40 weeks of ge owing to extensive ductl side-rnching tht hd occurred during repeted estrus cycles (Fig. 2g). mir-212/132 / glnds contined similr structures, lthough the ductl tree did not extend much further thn in the pre-puertl stge (Fig. 2h). The loulolveolr structures of mmmry glnds develop grdully during pregnncy nd rech their mture forms y prturition (Fig. 2i). Similr loulolveolr structures lso develop in mir-212/132 / glnds, ut they extend through much smller portion (only up to 20%) of the ft pd (Fig. 2j nd Supplementry Fig. 3). Thorcic mmmry glnds showed similr phenotypes (dt not shown). It seems resonle tht this difference ccounts for the decrese in milk production nd could explin the growth nd survivl prolems of the pups of homozygous mutnt femles. Mutnt ducts lck TEB ut retin the cpcity for outgrowth To determine whether there is structurl defect in mir-212/132 / epithelil ducts, we nlyzed the puertl mmmry glnds histologiclly. We found severl typicl TEB structures in wild-type glnds ut not in mir-212/132 / glnds (dt not shown). On the other hnd, the ductl structures of oth genotypes looked norml with n inner lyer of luminl epithelium nd n outer lyer of myoepithelium (Fig. 3,). Immunostining for E-cdherin nd cytokertin-14 confirmed the identity of luminl epithelil (Fig. 3c,d) nd myoepithelil (Fig. 3e,f) cells, respectively. Both genotypes lso hd firolsts in the periductl strom (Fig. 3,). Ductl outgrowth depends on extensive cellulr prolifertion of the cells in TEBs In ddition, the lumen of growing ducts is generted through poptosis of inner lyers of ody cells s the TEBs push the ductl outgrowth further 20. As mir-212/132 / glnds seem to contin no TEBs, we evluted the numers of poptotic nd proliferting cells only in the ductl regions of wild-type nd mir-212/132 / glnds. We found no significnt difference in the numer of TUNEL- or romodeoxyuridine (BrdU)-positive cells (dt not shown). This suggests tht the oserved phenotype is not due to defect in prolifertion or poptosis in lredy formed ducts. In mir-212/132 / mmmry glnds, the isometric growth of ducts continues during puerty (Supplementry Fig. 3), lthough the llometric growth driven y TEBs does not tke plce. Therefore, the min defect in mir-212/132 / glnds should e relted to the genertion or stility of TEBs, or oth. To find out whether the mir-212/132 / epitheli hve n priori defect in forming stle TEBs, we performed reciprocl mmmry epithelil trnsplnttion experiments. We trnsplnted mmmry epithelil ducts from pre-puertl mir-212/132 / mice into clered ft pds of pre-puertl wild-type E-Cdh. CK-14 e mice nd vice vers (Fig. 4). When we trnsplnted pre-puertl mir-212/132 / mmmry epitheli into the clered wild-type ft pd, they formed norml TEBs (rrows in Fig. 4) nd invded the whole ft pd (Fig. 4c,d). Moreover, they lso developed proper loulolveolr structures during pregnncy (Fig. 4e). By contrst, pre-puertl wild-type mmmry epitheli tht were trnsplnted into clered ft pds from mir-212/132 / mice did not form TEBs (Fig. 4f) nd consequently did not invde the mutnt ft pds 12 weeks (Fig. 4g,h) or 25 weeks fter trnsplnttion (dt not shown). However, loulolveolr differentition took plce during pregnncy in the limited ductl regions round the trnsplnttion site (Fig. 4i) t similr level s in the non-trnsplnted contrlterl glnd of mir-212/132 / mice (dt not shown). These dt suggest tht the mir-212/132 / epitheli hve no pprent histologicl defects or inherent functionl deficiencies nd cn form TEBs to chieve norml ductl outgrowth. Our oservtion tht wild-type epitheli show impired ductl outgrowth in clered mir-212/132 / ft pds lso indictes tht either the mmmry strom or the hormonl environment of mir-212/132 / mice is the primry cuse of the impired ductl outgrowth phenotype. Mutnt strom is non-permissive for ductl outgrowth From puerty, ll susequent stges of mmmry glnd development re under the control of endocrine systems Estrogen nd growth hormones re the min endocrine regultors of the puertl stge of mmmry glnd development. Therefore, we mesured the serum levels of oth hormones in mir-212/132 / mice nd found no significnt differences when compred with femle littermtes of ll genotypes (Supplementry Fig. 4). As mir-212/132 / epitheli mintin the functionl cpcity to undergo ductl outgrowth, nd estrogen nd growth hormone levels re norml in the mutnt mice, the impired ductl outgrowth in mutnt mmmry glnds is proly due to stroml deficiency. To verify this hypothesis, we performed whole mmmry glnd trnsplnttions. We trnsplnted whole mmmry glnds from 5-week-old donor mice into 5-week-old wild-type nd mir-212/132 / recipient mice f Nture Genetics VOLUME 42 NUMBER 12 DECEMBER

4 3 weeks p.o. c E & S E & S f g Figure 4 mir-212/132 / mmmry epitheli hve norml cpcity for ductl outgrowth. () The strtegy of the mmmry epithelil trnsplnttion. The inguinl (no. 4) glnds re shown together with rudimentry epithelil tree in the proximl region close to the nipple re. The loctions of the cuts for clering the ft pds re shown s dshed lines. The rrows show the origin of trnsplnts nd the loction to which they were trnsplnted. ( i) Whole-mount stining of the trnsplnted mmmry glnds nlyzed 3 nd 12 weeks fter the opertion (p.o.) nd in very erly lcttion. Asterisks show the region where the trnsplnts were plced during the opertion. Homozygous mutnt epitheli ( / E) formed TEBs (rrow in ) nd underwent proper ductl outgrowth within the wild-type ft pd strom (S; d). During pregnncy these ductl structures differentited into loulolveolr structures (e). By contrst, wild-type epitheli (E) did not generte TEBs to invde the homozygous mutnt ft pd strom ( / S; f h). However, these underdeveloped ductl structures, which were restricted to the region of trnsplnttion, underwent loulolveolr differentition during pregnncy (i). c nd g re low-mgnifiction pictures of d nd h, respectively. Scle rs represent 1 cm (c,e,g,i) nd 2 mm (,d,f,h). 12 weeks p.o. Lctting d e (Fig. 5) nd nlyzed them 5 or 10 weeks lter for ductl outgrowths. Five weeks fter eing trnsplnted into either mir-212/132 / (Fig. 5) or wild-type recipient mice (Fig. 5), wild type or heterozygous glnds contined proper TEBs nd ductl outgrowth, which eventully covered the whole glnd with epithelil ducts (Fig. 5c,d). By contrst, mir-212/132 / glnds showed no signs of ductl outgrowth when they were trnsplnted into either mir-212/132 / (Fig. 5e) or wild-type mice (Fig. 5f). To investigte the possiility tht the mutnt phenotype ws cused y n erlier endocrine defect which might hve n effect only during the onset of puerty, we lso trnsplnted whole mmmry glnds from pre-puertl mice into 5-week-old recipients nd nlyzed them 5 weeks lter for the initition of ductl outgrowth. Wild-type glnds from pre-puertl donors formed proper TEBs nd showed norml ductl outgrowth in mir-212/132 / mice (Fig. 5g). By contrst, we found no ductl outgrowths in glnds tht were trnsplnted from pre-puertl mir-212/132 / donors into wild-type mice (Fig. 5h). These findings clerly show tht the cuse of the mutnt phenotype is not n endocrine defect, which could prevent the genertion of TEBs during the onset of puerty. Together, these results suggest tht the cuse of the impired ductl outgrowth phenotype in mir-212/132 / mmmry glnds is tht the mutnt strom is not permissive for puertl outgrowth of epithelil ducts. h i mir-212 nd mir-132 re restricted to mmmry glnd strom We used quntittive RT-PCR to determine the expression kinetics of mir-212 nd mir-132 during different stges of mmmry glnd development (Supplementry Fig. 5). Their expression is low t the pre-puertl stge nd grdully increses throughout puerty, reching mximum t round 10 weeks of ge, which coincides with the completion of ductl outgrowth. Although the expression of mir-212 nd mir-132 sustntilly decreses nd reches its minimum y the end of pregnncy, it strts to increse gin immeditely fter prturition. High vriility in expression during puerty nd dulthood might indicte tht the expression is controlled y the estrus cycle. Indeed, this vriility is much lower efore puerty nd during pregnncy, when the estrus cycle is not ctivted or hs een discontinued, respectively. The expression of mir-212 nd mir-132 in periovultory grnulos cells is highly upregulted fter the induction of luteinizing hormone or humn chorionic gondotrophin 21. To determine which cell types in the mmmry glnd express mir-212 nd mir-132, we isolted mmmry epithelil orgnoids nd stroml cells seprtely nd checked the expression of their primry trnscript (Supplementry Fig. 1) nd mture forms (Fig. 6). The purity of isolted frctions ws confirmed y quntittive RT-PCR results for vimentin nd E-cdherin, which re cell mrkers for firolsts nd luminl epithelium, respectively (Fig. 6). We lso nlyzed the expression of oth micrornas in the clered ft pd, from which the epithelium ws surgiclly removed t the pre-puertl stge nd nlyzed 5 weeks fter the opertion. Our results show tht clered ft pd smples contined threefold more firolsts nd out threefold higher expression of oth mir-212 nd mir-132 thn did 10-week-old mmmry glnd smples (Fig. 6). Moreover, the expression of oth micrornas ws detected in the mmmry stroml cell frctions ut not in the mmmry epithelil orgnoids. Therefore, we conclude tht mir-212 nd mir-132 re expressed exclusively y the stroml cells in the mmmry glnd. Structurl defects in mutnt periductl strom The mmmry strom comprises the ft pd nd the periductl strom. The ft pd contins minly firolsts, predipocytes, rown dipose tissue nd white dipose tissue. The periductl strom tht surrounds the epithelil ducts is minly composed of firolsts nd collgen-rich ECM nd is essentil for the regultion of mmmry glnd development 16, VOLUME 42 NUMBER 12 DECEMBER 2010 Nture Genetics

5 Figure 5 The mir-212/132 / strom is not permissive for puertl outgrowth of the epithelil ducts. ( h) Whole-mount stining of trnsplnted whole mmmry glnds nlyzed 5 nd 10 weeks fter the opertion (p.o.). Outlines in, nd e h show loctions of the gryscle insets imges. Asterisks show the prts of nipple tissues tht were lso trnsplnted. Mmmry glnds from 5-week-old wild-type or heterozygous donor mice hd the proper TEBs nd ductl outgrowth fter eing trnsplnted into homozygous (,c) or wild-type (,d) recipient mice. By contrst, mmmry glnds of 5-week-old mir-212/132 / donor mice showed no ductl outgrowth or TEB structures when they were trnsplnted into mir-212/132 / (e) or wildtype (f) mice. Mmmry glnds of 3-week-old wildtype mice were lso le to initite ductl outgrowth in mir-212/132 / recipient mice (g), wheres, the glnds of 3-week-old mutnt mice filed to form proper TEBs in wild-type mice (h). (i) The strtegy of the whole mmmry glnd trnsplnttion. The inguinl (no. 4) glnds re shown on oth sides of mice in the lower domen. Both right nd left glnds of donor mice were used for trnsplnting, ech into different recipient mouse (rrows). The loction where the trnsplnts were plced is shown in the upper domen in perpendiculr orienttion to the endogenous inguinl glnds. Arrows in, nd g show TEBs on the distl tips of primry ducts. Scle rs represent 1 cm. 5-week-old donors As mir-212 nd mir-132 were exclusively expressed in the mmmry strom nd stroml defect cused the impired ductl outgrowth phenotype in mir-212/132 / glnds, we investigted the integrity of the periductl strom in mutnt glnds y Msson s trichrome stining. The periductl strom is rich in firolsts round the neck region of TEBs nd in the distlmost ducts (Supplementry Fig. 6,). In these regions, there re low levels of collgen. However, more proximl primry ducts re generlly surrounded with collgen-rich periductl strom ut re ssocited with fewer firolsts (Supplementry Fig. 6c). We did not detect such collgen-rich periductl strom round the proximl or reltively distl ducts of mir-212/132 / glnds, lthough firolsts were still seen close to them (Supplementry Fig. 6d f). Mutnt glnds contined lue-stining mucus-like structures in lmost every ductl lumen. In reltively distl ducts, it ws mostly seen djcent to the peripheries of the lumen, wheres it covered the lumen entirely in the more proximl ducts (Supplementry Fig. 6d f). We lso nlyzed collgen deposition in trnsplnted glnds composed of wild-type epitheli nd mir-212/132 / strom or vice vers (Supplementry Fig. 7). Trnsplnted mir-212/132 / epitheli cn form TEBs nd undergo ductl outgrowth in the wild-type clered ft pds (Fig. 4). Mmmry ducts formed in such trnsplnts lso hd similr levels of collgen deposition to the non-trnsplnted contrlterl glnds (Supplementry Fig. 7 c). On the other hnd, trnsplnted wild-type epitheli in mutnt clered ft pds showed impired ductl outgrowth nd lso showed the sme collgen deposition defect s oserved in the non-trnsplnted contrlterl glnds (Supplementry Fig. 7d f). These results indicte tht the collgen deposition defect in mir-212/132 / mmmry glnds is due to defect not in the mutnt epithelium, ut in the mutnt strom. mir-212 nd mir-132 negtively regulte MMP-9 expression The collgen in the periductl strom of mmmry glnds is secreted mostly y the epitheli under the control of the tumour growth fctor-β +/ 3-week-old donors c e g h Recipients d f Recipients 5 weeks p.o. (TGF-β) pthwy 22. Collgen deposition is lso regulted within the ECM y severl mtrix metlloproteinses (MMPs) nd tissue inhiitor of metlloproteinses (TIMPs). MMPs tht hve collgense function degrde the collgen in the ECM, wheres TIMPs specificlly inhiit the destructive functions of MMPs 23,24. In the mmmry glnd, these MMPs nd TIMPs re produced y epitheli, firolsts, mst cells nd dipocytes. MMP-9, lso known s collgense type IV-B, is predicted trget of mir-212 nd mir-132 (Mirnd dtse). We tested this ioinformtic prediction using luciferse ssy pproch. We cloned the 3 UTR of MMP-9 downstrem of the firefly luciferse gene nd found tht the normlized luciferse ctivity ws sustntilly reduced upon cotrnsfection with either mir-212 or mir-132 or with oth micrornas together ( MMP-9 in Supplementry Fig. 8). However, this decrese in luciferse ctivity ws olished when the inding sites of mir-212 nd mir-132 on the 3 UTR of MMP-9 were mutted in the luciferse construct ( MMP-9-mut. in Supplementry Fig. 8). These results indicte tht MMP-9 is direct trget of oth mir-212 nd mir-132. Previous in situ hyridiztion dt showed tht MMP-9 is expressed t low levels throughout the mmmry glnd in the epitheli nd strom, ut high levels of MMP-9 mrna were lso detected in some cells scttered within the ft pd 25. We used immunohistochemistry to nlyze MMP-9 expression in oth wild-type nd mir-212/132 / mmmry glnds. In wild-type glnds, we detected low levels of MMP-9 throughout the whole ft pd, with slightly higher levels in distl regions thn in proximl regions. Around the TEBs, there were severl firolsts in which MMP-9 ws highly expressed (rrows in Fig. 7). We found low, diffuse stining in the periductl strom of distl ducts; however, proximl ducts hd lmost no MMP-9 in their periductl strom (Fig. 7,c). Some scttered cells in the ft pd hd high MMP-9 levels (lue rrows in Fig. 7c), consistent with the results of the in situ hyridiztion experiments 25. By contrst, MMP-9 ws much more i Donor 5 weeks p.o. 10 weeks p.o. 5 weeks p.o. Nture Genetics VOLUME 42 NUMBER 12 DECEMBER

6 Figure 6 In the mmmry glnd, mir-212 nd mir-132 re exclusively expressed in the stroml cells, ut not in the epitheli. () Quntittive RT-PCR nlyses were performed to detect the expression of mir-212 nd mir-132 in 10-week-old whole mmmry glnds, clered ft pds nd stroml nd epithelil orgnoid frctions from digested glnds. Clered ft pd smples, which re epithelium-free, hve three-fold higher expression of mir-212 nd mir-132 thn whole glnds. Isolted stroml frctions from mmmry glnds hve the highest expression of oth mir-212 nd mir-132, wheres isolted epithelil orgnoids express neither mir-212 nor mir-132. All expression levels were clculted s fold-chnges compred to their levels in intct mmmry glnds from 10-week-old mice. () Quntittive RT-PCR nlyses were performed for E-cdherin nd vimentin to determine the presence of epithelil nd firolst cells, respectively, in the sme smple set s in. The sence of E-cdherin expression in clered ft pd nd isolted 2,400 2,200 2,000 1,800 1,600 1,400 1,200 1, mir-132 mir week old Clered ft pd Stroml frction Epithelil orgnoids 1,400 1,300 1,200 1,100 1, old 10 week Vimentin E-Cdherin Clered ft pd Stroml frction Epithelil orgnoids stroml frction smples proved tht these smples were epithelium-free. The sence of vimentin expression in isolted epithelil orgnoids lso showed tht there ws no firolst contmintion in their preprtion. Dt re expressed s men ± s.d.; n = 3 for ech smple set. Aritrry vlues Aritrry vlues highly expressed throughout the mir-212/132 / glnds. All epithelil ducts contined high levels of diffuse MMP-9 stining in their periductl strom (Fig. 7d f). In ddition, we found firolsts tht expressed MMP-9 in the periductl strom of mutnt ducts (lck rrows in Fig. 7f), similr to the firolsts oserved round the neck region of TEBs in wild-type glnds (Fig. 7). Also within the ftty strom, there were mny scttered cells, presumly firolsts, tht contined high concentrtions of MMP-9 (lue rrows in Fig. 7f). These results indicte tht in the sence of negtive regultion y mir-212 nd mir-132, the expression of MMP-9 in stroml cells is mrkedly incresed. The ccumultion of MMP-9 in the periductl strom might explin the collgen deposition defect in mir-212/132 / glnds. Hyperctivtion of TGF- pthwy in mir-212/132 / glnds TGF-β regultes ductl outgrowth nd side rnching during mmmry glnd development. 23,24 Overexpression of TGF-β in c d e f g h i j k l puertl mmmry glnds cuses impirment of ductl outgrowth nd results in simplified roriztion ptterns of ducts 23,26. All three isoforms of TGF-β re expressed in mmmry epitheli with different sptiotemporl distriutions 23,26. The TGF-β pthwy suppresses the prolifertion of epithelil cells nd regultes the synthesis of ECM proteins y these cells. All three isoforms of TGF-β ind to the sme TGF-β receptors nd ctivte downstrem pthwys through the phosphoryltion of Smd2 nd Smd3 (ref. 27). However, TGF-β is secreted in ltent form s covlently ssocited with the ltency-ssocited polypeptide (LAP) 23,27. Within the ECM, ltent TGF-β inding proteins (LTBPs) lso ind to the LAP TGF-β complexes to keep TGF-β in ltent stte 27. TGF-β ccumultes in the periductl strom within the mmmry glnds 23. It is elieved tht the collgenrich ECM is importnt for mintining reservoir of ltent TGF-β round the epithelil ducts nd lso for keeping TGF-β in ltent MMP-9 p-smd2/3 Figure 7 Loss of mir-212 nd mir-132 cuses high MMP-9 levels nd hyperctivity of TGF-β pthwy in mutnt mmmry glnds. Immunohistochemicl stining for MMP-9 ( f) nd phosphorylted Smd2 or 3 (p-smd2/3; g l) on mmmry glnd sections from 6-weekold wild-type mice ( c, g i) nd homozygous mutnt littermtes (d f, j l). In wild-type mmmry glnds, high MMP-9 ws seen only in the periductl strom of TEBs. Arrows show the MMP-9 expressing firolsts in. The periductl strom of growth-quiescent ducts in wild-type glnds hs low MMP-9 stining (rrowheds in nd c). In the ft pd etween ducts, there re few scttered firolsts expressing high levels of MMP-9 (lue rrows in c). The periductl strom in mutnt glnds shows high MMP-9 (rrowheds in d nd e), nd oth periductl strom nd ft pd contin mny MMP-9 expressing firolsts (lck nd lue rrows, respectively, in f). In wildtype mmmry glnds, p-smd2/3 is present in the epithelil cells of TEBs nd firolsts round TEBs (g). However, p-smd2/3 is not detectle in the epitheli or strom of growthquiescent ducts of wild-type glnds (h,i). In mutnt glnds, most epithelil cells of ll ducts re stined for p-smd2/3 (j l). There were lso severl p-smd2/3-positive firolsts in oth periductl strom nd ft pds of mutnt glnds (lue rrowheds in j l). Scle rs represent 50 μm for h,i,k,l nd 100 μm for other pnels VOLUME 42 NUMBER 12 DECEMBER 2010 Nture Genetics

7 form y mking it inccessile to the proteins or environmentl conditions tht re necessry for its ctivtion 23,24. MMP-9 cn ctivte ltent TGF-β y dissociting the ltency complex tht contins TGF-β, LAP nd LTBPs 28,29. Therefore, high levels of MMP-9 nd mrked reduction in collgen in the periductl strom might result in the hyperctivtion of ltent TGF-β round the mir-212/132 / mmmry ducts. To test this hypothesis, we performed immunohistochemicl nlyses with n ntiody tht recognizes only the phosphorylted forms of oth Smd2 nd Smd3. The presence of phosphorylted Smd2 or Smd3 in cell is strong indictor for the ctivtion of the TGF-β pthwy. In wild-type mmmry glnds, the mjority of epitheli nd firolsts in the prolifertive zones, such s TEBs (Fig. 7g) nd ductl side-uds (dt not shown), were positive for phospho- Smd2 or phospho-smd3. By contrst, in the growth-quiescent ductl regions of wild-type glnds, lmost no epitheli or firolsts showed detectle stining for phospho-smd2 or phospho-smd3 (Fig. 7h,i). However, in mir-212/132 / glnds, most of the epitheli in ll ductl regions contined high levels of phospho-smd2 or phospho-smd3, indicting tht the TGF-β pthwy ws highly ctive (Fig. 7j l). We lso found severl firolsts tht contined phospho-smd2 or phospho-smd3 in the mutnt strom round the ducts (lue rrowheds in Fig. 7j l). The min moleculr pthwy tht is known to induce epithelil prolifertion in puertl mmmry glnds involves the trnscriptionl ctivtion of the mphiregulin (Areg) gene y the estrogen receptor ERα in mnner tht depends on the trnscriptionl cofctor Cited1 (ref. 11). TGF-β negtively regultes this pthwy to suppress epithelil prolifertion 11. Therefore, we checked the levels of Cited1 nd Areg mrna in puertl mmmry glnds nd found tht they were oth significntly (P < 0.005) downregulted in mir-212/132 / glnds (Supplementry Fig. 9). This provides dditionl evidence tht the TGF-β pthwy is hyperctivted in mir-212/132 / mmmry glnds. To find out whether there is ny deficiency of ERα expression in mir-212/132 / mmmry glnds, which might lso cuse the downregultion of Areg expression, we checked its expression pttern y imunohistochemistry (Supplementry Fig. 10). The expression of ERα in oth epitheli nd strom of mir-212/132 / mmmry glnds ws similr to tht seen in wild-type glnds. These results indicte tht the downregultion of Areg mrna in mir-212/132 / glnds is not due to defect in ERα expression, ut might e due to the hyperctivtion of the TGF-β pthwy. Together, our findings indicte tht in the sence of mir-212 nd mir-132, MMP-9 levels increse in the mutnt mmmry strom, which proly cuses the defect in collgen deposition nd the hyperctivtion of the TGF-β pthwy. High TGF-β ctivity cn downregulte Cited1 mrna, therey preventing the trnscriptionl ctivtion of Areg in mmmry epitheli, which in turn leds to impired ductl outgrowth in mir-212/132 / mmmry glnds. DISCUSSION We hve shown tht the mir-212/132 fmily is required nd indispensle for proper ductl outgrowth during puertl development of the mmmry glnds in mice. Trnsplnttion experiments showed tht the impired ductl outgrowth in mir-212/132 / mmmry glnds ws cused y defects not in the epitheli ut in the strom. Levels of circulting estrogen nd growth hormone were norml in mir-212/132 / mice. mir-212 nd mir-132 were exclusively expressed y stroml cells in the mmmry glnd, nd they directly downregulted the expression of MMP-9. In the sence of regultion y mir-212 nd mir-132, MMP-9 incresed nd ccumulted in the periductl strom of mutnt glnds. The oserved collgen deposition defects in mir-212/132 / glnds were proly due to this increse in MMP-9. The periductl strom ccommodtes lrge reservoir of ltent TGF-β, which is necessrily kept in n inctive stte within the collgen-rich ECM 23. As MMP-9 cn ctivte ltent TGF-β 28,29, the sence of collgen nd high MMP-9 levels in mutnt periductl strom my eventully cuse hyperctivtion of ltent TGF-β. Consistent with this model, we hve shown tht the TGF-β pthwy ws hyperctivted in mir-212/132 / mmmry glnds. The TGF-β pthwy positively regultes oth the production nd secretion of MMP-9 in vrious cell types Such possile upregultion of MMP-9 expression y TGF-β might generte positive feedck loop in mir-212/132 / mmmry glnds, s MMP-9 cn lso ctivte the ltent TGF-β 28,29. Therefore, the essentil function of mir-212 nd mir-132 might e to suppress this positive feedck loop, which otherwise might e deleterious for collgen-rich ECM formtion round the mmmry ducts nd lso might completely suppress ductl outgrowth. Mice deficient in Cited1, Areg or Egfr (the receptor for mphiregulin) show impired mmmry ductl outgrowth phenotypes similr to tht of mir-212/132 / mice The downregultion of the expression of Cited1 nd mphiregulin in mir-212/132 / glnds might explin the similrity in the oserved phenotypes. Cited1 lso cts s cofctor for Smd4 in the TGF-β pthwy 39. Therefore, high TGF-β ctivity in mir-212/132 / epitheli might selectively regulte the expression of specific set of genes, whose regultion would not depend on the interction etween Cited1 nd Smd4. The signling proteins Wnt5 nd Fgfr2 re lso downregulted in mir-212/132 / mmmry glnds (our unpulished microrry dt). As Fgfr2 is essentil for TEB mintennce 40,41, its downregultion might lso explin the sence of TEBs in mir-212/132 / glnds. Wnt5 is the min downstrem meditor in the suppression of ductl outgrowth y TGF-β 42. Its downregultion in mir-212/132 / glnds might e explined y the possiility tht its trnscriptionl control depends on the interction etween Smd4 nd Cited1. However, in the sence of Wnt5 upregultion, ductl outgrowth is still impired in mir-212/132 / glnds, suggesting tht other meditor(s) of the TGF-B pthwy lso regulte ductl outgrowth. Previous studies showed the functionl importnce of other micro- RNAs in mmmry epithelil cell lines However, our results provide the first in vivo exmple of microrna function eing required for mmmry glnd development. Moreover, we hve shown tht micrornas not only regulte utonomous cell functions, ut lso re centrl to communiction etween stroml nd epithelil cells. URLs. Mirnd dtse, Methods Methods nd ny ssocited references re ville in the online version of the pper t Accession codes. The full-length sequence of primry trnscript encoding the trnscript vrint 2 of mir-212/132 gene hs een deposited in the GenBnk dtse with the following ccession numer: HM Note: Supplementry informtion is ville on the Nture Genetics wesite. Acknowledgments We thnk S. Geisendorf for technicl ssistnce, S. Hille for DNA sequencing, S. Mhsur nd U. Frnke for technicl help in genertion of knockout mouse Nture Genetics VOLUME 42 NUMBER 12 DECEMBER

8 line, A. Kurth, A. Driehorst, K. Kiel nd U. Teichmn for niml cre work, M. Smlley for discussion out mmmry epithelil trnsplnttion method, N. B Tiep for technicl help with collecting lood smples, A. Ylcin for cretive contriution to the initil phse of the work, nd P. Gruss nd G. Eichele for encourgement nd support. Funded y the Mx Plnck Society. Additionl funding ws provided y the Deutsche Forschungsgemeinschft (DFG, GR 536/9-2 nd GR 536/11-1 to B.G. nd TH 903/7-2 to T.T.). AUTHOR CONTRIBUTIONS A.U. nd K.C. developed the concept of this study. A.U. performed ll experiments except the mmmry epithelil trnsplnttion nlyses, which were performed y V.V. nd P.A.B.K. in the lortory of B.G., serum hormone level nlyses, which were done y H.J., nd luciferse ssys, which were done y J.F. in the lortory of T.T. The mnuscript ws written y A.U. nd K.C. Importnt suggestions were mde y B.G., V.V. nd T.T. which improved the qulity of the mnuscript. All uthors contriuted to the discussion of the dt nd commented on the finl version of the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Vlenci-Snchez, M.A., Liu, J., Hnnon, G.J. & Prker, R. Control of trnsltion nd mrna degrdtion y mirnas nd sirnas. Genes Dev. 20, (2006). 2. Brtel, D.P. MicroRNAs: Trget recognition nd regultory functions. Cell 136, (2009). 3. Zho, Y. & Srivstv, D. A developmentl view of microrna function. Trends Biochem. Sci. 32, (2007). 4. Smiert, P. & Li, E.C. Lessons from microrna mutnts in worms, flies nd mice. Cell Cycle 7, (2008). 5. Vo, N. et l. A camp-response element inding protein-induced microrna regultes neuronl morphogenesis. Proc. Ntl. Acd. Sci. USA 102, (2005). 6. Wymn, G.A. et l. An ctivity-regulted microrna controls dendritic plsticity y down-regulting p250gap. Proc. Ntl. Acd. Sci. USA 105, (2008). 7. 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Biol. Reprod. 79, (2008). 22. Silerstein, G.B., Stricklnd, P., Colemn, S. & Dniel, C.W. Epithelium-dependent extrcellulr mtrix synthesis in trnsforming growth fctor-et 1-growth-inhiited mouse mmmry glnd. J. Cell Biol. 110, (1990). 23. Dniel, C.W., Roinson, S. & Silerstein, G.B. The role of TGF-et in ptterning nd growth of the mmmry ductl tree. J. Mmmry Glnd Biol. Neoplsi 1, (1996). 24. Silerstein, G.B. Postntl mmmry glnd morphogenesis. Microsc. Res. Tech. 52, (2001). 25. Wisemn, B.S. et l. Site-specific inductive nd inhiitory ctivities of MMP-2 nd MMP-3 orchestrte mmmry glnd rnching morphogenesis. J. Cell Biol. 162, (2003). 26. Roinson, S.D. et l. Regulted expression nd growth inhiitory effects of trnsforming growth fctor-et isoforms in mouse mmmry glnd development. Development 113, (1991). 27. Kng, J.S., Liu, C. & Derynck, R. New regultory mechnisms of TGF-et receptor function. Trends Cell Biol. 19, (2009). 28. Yu, Q. & Stmenkovic, I. Cell surfce-loclized mtrix metlloproteinse-9 proteolyticlly ctivtes TGF-et nd promotes tumor invsion nd ngiogenesis. Genes Dev. 14, (2000). 29. Wilson, T.J., Nnnuru, K.C. & Singh, R.K. Cthepsin G-medited ctivtion of pro-mtrix metlloproteinse 9 t the tumor-one interfce promotes trnsforming growth fctor-et signling nd one destruction. Mol. Cncer Res. 7, (2009). 30. Okmoto, T. et l. Trnsforming growth fctor-et 1 induces mtrix metlloproteinse-9 expression in humn meningel cells vi ERK nd Smd pthwys. Biochem. Biophys. Res. Commun. 383, (2009). 31. Sinpitkskul, S.N., Pimkhokhm, A., Snchvnkit, N. & Pvsnt, P. TGF-et 1 induced MMP-9 expression in HNSCC cell lines vi Smd/MLCK pthwy. Biochem. Biophys. Res. Commun. 371, (2008). 32. Seomun, Y., Kim, J.T. & Joo, C.K. MMP-14 medited MMP-9 expression is involved in TGF-et 1-induced kertinocyte migrtion. J. Cell. Biochem. 104, (2008). 33. Chou, Y.T., Wng, H., Chen, Y., Dnielpour, D. & Yng, Y.C. Cited2 modultes TGFet-medited upregultion of MMP9. Oncogene 25, (2006). 34. Konrd, L., Scheier, J.A., Schwrz, L., Schrder, A.J. & Hofmnn, R. TGF-et1 nd TGF-et2 strongly enhnce the secretion of plsminogen ctivtor inhiitor-1 nd mtrix metlloproteinse-9 of the humn prostte cncer cell line PC-3. Regul. Pept. 155, (2009). 35. Howlin, J. et l. CITED1 homozygous null mice disply errnt puertl mmmry ductl morphogenesis. Oncogene 25, (2006). 36. Luetteke, N.C. et l. Trgeted inctivtion of the EGF nd mphiregulin genes revels distinct roles for EGF receptor lignds in mouse mmmry glnd development. Development 126, (1999). 37. Cirloni, L., Mllepell, S. & Brisken, C. Amphiregulin is n essentil meditor of estrogen receptor lph function in mmmry glnd development. Proc. Ntl. Acd. Sci. USA 104, (2007). 38. Wiesen, J.F., Young, P., Wer, Z. & Cunh, G.R. Signling through the stroml epiderml growth fctor receptor is necessry for mmmry ductl development. Development 126, (1999). 39. Shiod, T. et l. Trnscriptionl ctivting ctivity of Smd4: roles of SMAD heterooligomeriztion nd enhncement y n ssociting trnsctivtor. Proc. Ntl. Acd. Sci. USA 95, (1998). 40. Prs, S. et l. Terminl end ud mintennce in mmmry glnd is dependent upon FGFR2 signling. Dev. Biol. 317, (2008). 41. Lu, P., Ewld, A.J., Mrtin, G.R. & Wer, Z. Genetic mosic nlysis revels FGF receptor 2 function in terminl end uds during mmmry glnd rnching morphogenesis. Dev. Biol. 321, (2008). 42. Rorty, K. & Serr, R. Wnt5 is required for proper mmmry glnd development nd TGF-β-medited inhiition of ductl outgrowth. Development 134, (2007). 43. Irr, I., Erlich, Y., Muthuswmy, S.K., Schidnndm, R. & Hnnon, G.J. A role for micrornas in mintennce of mouse mmmry epithelil progenitor cells. Genes Dev. 21, (2007). 44. Kong, W. et l. MicroRNA-155 is regulted y the trnsforming growth fctor et/ Smd pthwy nd contriutes to epithelil cell plsticity y trgeting RhoA. Mol. Cell. Biol. 28, (2008). 45. Tnk, T., Hned, S., Imkw, K., Ski, S. & Ngok, K. A microrna, mir- 101, controls mmmry glnd development y regulting cyclooxygense-2 expression. Differentition 77, (2009) VOLUME 42 NUMBER 12 DECEMBER 2010 Nture Genetics

9 ONLINE METHODS Genertion of mir-212/132 / mice. We generted mir-212/132 / mice y homologous recomintion trgeting the genomic region tht encodes oth pre-mir-212 nd pre-mir-132. Detils of the trgeting vector nd the strtegy for otining the mir-212/132 / mouse lines re descried in the Supplementry Note. All phenotypicl results were otined from mice of 129/Sv ckground. Similr results were lso otined from mir-212/132 / mice of C57BL6/N ckground for the whole-mount mmmry glnd nlyses in ll puertl, pregnncy nd lcttion stges. RT-PCR nlysis. Totl RNA smples from dissected tissues or mmmry cell frctions were isolted with TRI regent (Sigm). To detect mture microrna levels, we crried out reverse trnscription (RT) nd rel-time PCR nlyses using TqMn MicroRNA Reverse Trnscription kit (Applied Biosystems) nd TqMn Universl PCR mster mix (Applied Biosystems) ccording to the mnufcturer s instructions. Primers for RT nd Q-PCR were otined from TqMn MicroRNA ssy kits for hs-mir-132, mmu-mir-212, sno-142 nd sno-202 (Applied Biosystems). Fold chnges re clculted from the determined Ct vlues nd the expression levels of mir-132 nd mir-212 were determined y normlizing these vlues with the sum of fold chnge vlues for sno-142 nd sno-212. For the detection of CITED1 nd mphiregulin mrna levels, cdnas were synthesized with the ThermoScript RT-PCR System (Invitrogen) using rndom hexmers ccording to the mnufcturer s instructions. Rel-time PCR nlyses were performed using iq SYBR green supermix (Biord) nd QuntiTect Primer ssys (Qigen) for Cited1, Areg, Hprt1 nd Act. Fold chnges re clculted from the determined Ct vlues nd the expression levels of Cited1 nd Areg were determined y normlizing these vlues to the fold-chnge vlues for Hprt1 nd Act. For the detection of specific isoforms of the mir-212/132 gene cdnas were synthesized s ove. PCR primers were designed from exon junctions to prevent ny mplifiction from contminting genomic DNA within the RNA smples (Supplementry Tle 1). RT ( ) controls were lso included in the experiments. Whole-mount nlysis of mmmry glnds. Whole-mount crmine-lum stining of mmmry glnds ws performed s descried 46. Briefly, inguinl (no. 4) mmmry glnds were dissected from mice, spred onto glss slides, fixed in 6:3:1 mixture of ethnol:chloroform:glcil cetic cid, hydrted, stined overnight in 0.2% crmine (Sigm) nd 0.5% AlK(SO 4 ) 2, dehydrted in grded solutions of ethnol, clered in Histocler nd mounted. Animl studies. All niml studies were performed in ccordnce with the relevnt guidelines nd regultions nd with the pprovl of the responsile locl nd ntionl uthorities. For the pup growth curve nlysis, femle littermtes were mted nd the numer of pups per litter ws reduced to five fter prturition. Litters contining fewer thn five pups were not used. The pup weights were recorded during the indicted postntl dys of life. For hormone nlysis in lood serum, mice were nesthetized nd the lood smples were collected y retro-oritl puncture. Mmmry epithelil trnsplnttion. Reciprocl mmmry epithelil trnsplnttions were done with 3-week-old femle littermtes (Fig. 4). Ft pd clering nd trnsplnttions of epithelil ducts were done s descried 47. Briefly, mice were nesthetized nd inguinl mmmry glnds were exposed through smll incisions in the skin of the lower domen nd long the right hind leg. For ft pd clering, the connection etween glnds no. 4 nd 5 ws disrupted y cuteriztion nd the endogenous epitheli were removed y removing the region etween the nipple nd the line ove the lymph node. Smll pieces of the removed glnds (~2 mm 3 nd close to the nipple region) were cut nd trnsplnted into the clered ft pds of recipient mice. The left inguinl (no. 4) mmmry glnds were used s controls. For nlyses of ductl outgrowth, trnsplnted mice were killed 3, 12 or 25 weeks fter the opertion. For the determintion of lveolr differentition, the trnsplnted mice were mted 2 months fter the opertion nd killed within the first 6 h post-prtum. From these mice, oth trnsplnted nd contrlterl endogenous control glnds were dissected nd either nlyzed y whole-mount crmine-lum stining or emedded into prffin for Msson s trichrome stining. In totl, 22 sets of reciprocl trnsplnttions were done nd reproducile results were otined from ll of them. Whole mmmry glnd trnsplnttion. These trnsplnttions were done s descried 48. Briefly, oth right nd left inguinl (no. 4) mmmry glnds were dissected from killed donor mice nd kept for short time in DMEM until trnsplnttion. Menwhile, recipient mice were nesthetized nd smll incisions were mde in the skin of the upper domen. The trnsplnts were plced on the peritonel wll just under the sternum s shown in Figure 5i. Trnsplnted nd endogenous inguinl glnds were dissected 5 or 10 weeks fter the opertion nd nlyzed y whole mount crmine-lum stining. At lest three sets of trnsplnttion were done for ech experimentl condition nd reproducile results were otined from ll of them. Histologicl stining. Dissected mmmry glnds were fixed in 4% prformldehyde nd then either frozen in cryomtrix or emedded in prffin. Cryosections (25 μm) were used for neutrl-red stining. Prffin sections (7 μm) were used for Msson s trichrome stining, hemtoxylin-eosin stining or immunohistochemistry. Trichrome stining ws performed using the Msson s trichrome stining kit (Dignostic Biosystems) ccording to the mnufcturer s instructions. For immunohistochemicl detection of proteins, tissue sections were treted with 3% H 2 O 2 in methnol for 10 min, followed y oiling in 0.1 mm citrte uffer (ph 6.0) for 15 min for ntigen retrievl. The sections were locked in got serum nd then primry ntiodies for E-cdherin (Snt Cruz Biotechnology, sc-7870, 1:50 dilution), cytokertin-14 (Acm, 53115, 1:100 dilution), MMP-9 (Acm, :500 dilution), phospho-smd2 (Cell Signling Technology, #3101, 1:200 dilution) or ERα (Acm, 37438, 1:50 dilution) were pplied. For detection of primry ntiodies, the ABC stining system (Snt Cruz Biotechnology, sc-2018) ws used ccording to the mnufcturer s instructions. Seprtion of mmmry stroml frctions nd epithelil orgnoids. Twenty wild-type femle mice (8 12-weeks-old) were used for the isoltion of epithelil orgnoids nd mmmry stroml frctions. Inguinl glnds (no. 4) were dissected y excluding the lymph nodes. Afterwrds, they were minced into smll pieces (~1 mm 3 ) nd lysed in 10 ml of digestion cocktil (1.5 mg/ml trypsin, 3 mg/ml collgense A, 10% FCS in Hm s F12 medium) for 2 h t 37 C with slow stirring. DNAse (finl concentrtion of 0.1 mg/ml) ws dded nd the smples incuted for 10 min t room temperture. Lystes were left to settle nd the floting ftty lyer ws discrded. Epithelil orgnoids ccumulted in the ottom of the tue. The superntnt ws crefully collected into new tue s the stroml frction nd centrifuged t 500g, nd the pellet ws frozen for RNA isoltion. The remining portion of the lyste, contining epithelil orgnoids, ws wshed five times with Hm s F12 medium y centrifugtion t 10g with superntnts discrded to remove remining firolsts from epithelil orgnoids. Purified epithelil orgnoids were collected nd frozen for RNA isoltion. Serum nlyses. Estrdiol in the lood serum ws mesured using the Ultr- Sensitive Estrdiol kit (DSL) ccording to the mnufcturer s guidelines. Growth hormone in the lood serum ws mesured y using the specific rdioimmunossy (RIA) nd the stndrd protocol supplied y the Ntionl Hormone nd Pituitry Progrm of the NIDDK. Briefly, 50 μl serum ws dded to 200 μl rit nti-gh ntiody (1:5,000) nd incuted overnight t 4 C. Susequently, 100 μl 125 I-leled trcer (20,000 c.p.m.) ws dded nd smples were incuted for 24 h t 4 C. After the ddition of the secondry ntiody (sheep nti-rit, 1:30), smples were incuted for 48 h t 4 C nd finlly centrifuged for 1 h t 2,400g. Rdioctivity in the pellets ws counted with Wllc-LKB 1270 counter nd nlyzed with the RIA-CALC progrm. Luciferse ssy. A 398-p frgment from the 3 UTR of MMP-9, contining predicted inding sites for mir-212 nd mir-132, ws mplified y PCR nd cloned s the MMP-9 construct into the 3 UTR of the luc gene in the pmir-report plsmid (Amion). For the MMP-9 mut construct, muttions were generted doi: /ng.709 Nture Genetics