a PATTERNS & PHENOTYPES

Expression of Gpr177, a Wnt TraffickingRegulator, in Mouse EmbryogenesisHsiao-Man Ivy Yu, Ying Jin, Jiang Fu, and Wei Hsu*

Wls/Evi/Srt encoding a multipass transmembrane protein has been identified as a regulator for propersorting and secretion of Wnt in flies. We have previously demonstrated that Gpr177 is the mouse orthologrequired for axis determination. Gpr177 is a transcriptional target of Wnt that is activated to assist itssubcellular distribution in a feedback regulatory loop. We, therefore, proposed that reciprocal regulationof Wnt and Gpr177 is essential for the Wnt-dependent developmental and pathogenic processes. Here, weexamine the expression pattern of Gpr177 in mouse development. Gpr177 is expressed in a variety of tis-sues and cell types during organogenesis. Furthermore, Gpr177 is a glycoprotein primarily accumulatingin the Golgi apparatus in signal-producing cells. The glycosylation of Gpr177 is necessary for propertransportation in the secretory pathway. Our findings suggest that the Gpr177-mediated regulationof Wnt is crucial for organogenesis in health and disease. Developmental Dynamics 239:2102–2109, 2010.VC 2010 Wiley-Liss, Inc.

Key words: Gpr177; Wntless; Evi; Sprinter; b-catenin; Wnt production; Wnt signaling; organogenesis

Accepted 3 May 2010

INTRODUCTION

Members of theWnt family trigger cel-lular signals essential for proper de-velopment of organisms (Logan andNusse, 2004; Clevers, 2006). Aberrantregulation of an evolutionary con-served Wnt signal transduction path-way has been implicated in a varietyof cancers and congenital diseases(van Amerongen and Berns, 2006; Gri-goryan et al., 2008). Wnt signaling hasbeen repetitively proven to be criticalfor human health and disease. Com-pared with the enormous wealth ofknowledge on the events in signal-receiving cells, we know relatively lit-tle about the processes associated with

Wnt maturation, sorting, and secre-tion in signal-producing cells (Willertet al., 2003; Takada et al., 2006; Cou-dreuse and Korswagen, 2007; Haus-mann et al., 2007). At the primarysequence level, Wnt proteins share anearly invariant pattern of 23 Cys res-idues, an N-terminal signal sequenceand several potential N-glycosylationsites (Miller, 2002). Although the roleof glycosylation in Wnt secretion andfunction is not fully understood(Tanaka et al., 2002; Vincent andDubois, 2002; Eaton, 2006), two lipidmodifications present on the matureWnt seem to be required for correct in-tracellular targeting of Wnt and sig-naling activity of the secreted protein

(Willert et al., 2003; Zhai et al., 2004;Takada et al., 2006). Based on recentstudies in the fly, Wnt proteins mightbe secreted bound to lipoprotein par-ticles (Panakova et al., 2005; Eaton,2006). Alternatively, Wnt proteinsmight form high order complexes,thereby overcoming their hydrophobicproperty and potentiating their signal-ing capacity (Miller, 2002).The mechanism underlying the sort-

ing and secretion of Wnt remainslargely elusive. Studies in the fly sug-gest that Wnt accumulates in cellswhere nonconventional secretory routesmay be used (Strigini and Cohen, 2000;Marois et al., 2006). A significant frac-tion of Wnt is present in endocytic

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Department of Biomedical Genetics, Center for Oral Biology, James Wilmot Cancer Center, University of Rochester Medical Center,Rochester, New YorkGrant sponsor: National Institutes of Health; Grant numbers: CA106308, DE015654.*Correspondence to: Wei Hsu, Department of Biomedical Genetics, Center for Oral Biology, James Wilmot Cancer Center,University of Rochester Medical Center, 601 Elmwood Avenue, Box 611, Rochester, NY 14642.E-mail: wei_hsu@urmc.rochester.edu

DOI 10.1002/dvdy.22336Published online 14 June 2010 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 239:2102–2109, 2010

VC 2010 Wiley-Liss, Inc.

vesicles which retrieve back to thesurface (Pfeiffer et al., 2002). The func-tion of this retrieval is not clear, butit might allow Wnt to gain access to cel-lular compartments from which stand-ard secretory molecules are excluded.Alternatively, Wnt might directly enterthese vesicles during the secretion pro-cess as the fusion of secretory vesiclesfrom the Golgi with endosomes hasbeen reported (Futter et al., 1995; Anget al., 2004). The identification ofWntless (Wls/Evi/Srt; Banziger et al.,2006; Bartscherer et al., 2006; Good-man et al., 2006) has shown that thismultipass transmembrane protein is atransporting regulator for Wnt produc-tion in Drosophila. Given the extensiveWnt family in higher organisms, theessential role of Wls in production of allWnt proteins remains to be determined.

We have previously demonstratedthat Gpr177 is the mouse ortholog ofDrosophila Wls, required for Wnt-mediated embryonic axis determina-tion (Fu et al., 2009). In the Gpr177-deficient mutant, Wnt production isimpaired, leading to disruption ofWnt signaling in the establishment ofthe anterior–posterior axis. As a Wntdirect target, Gpr177 is activated byb-catenin and LEF/Tcf-dependenttranscription. This activation thenmodulates Wnt production in a posi-tive feedback loop. Our study has ledto a hypothesis in which reciprocalregulation of Wnt and Gpr177 isessential for normal developmentalprocesses. Alterations of this regula-tory circuit are causally linked topathogenesis in human diseases.

In this study, we have examined theexpression pattern of mouse Gpr177during development of various organs.This comprehensive survey revealsthat Gpr177 may modulate the Wntpathway in a variety of tissues and celltypes. Furthermore, Gpr177 is a glyco-protein predominantly localized in theGolgi apparatus. Disturbance of N-linked glycosylation prevents the Golgiaccumulation of Gpr177. Given theestablished role of Wnt signalingin health and disease (http://www.stanford.edu/�rnusse/wntwindow.html),our findings suggest that the Gpr177-mediated Wnt regulation is criticalfor organogenesis, including neuraldevelopment, craniofacial morpho-genesis, and the other developmentaland pathogenic processes, especially

related to epithelial–mesenchymalinteraction.

RESULTS AND DISCUSSION

Expression Pattern of Gpr177

mRNA and Protein in Mouse

Embryogenesis

To study the potential involvement ofGpr177 during mouse embryogenesis,we analyzed its expression by in situhybridization. A majority of organsbegan to form in the developing mouseembryo after 13.5 days post coitum(dpc). We detected the Gpr177 tran-script in various neural tissues, cranio-facial prominences, developing skele-ton, and several internal organs (Fig.1A,B). Strong expression of Gpr177was shown in certain regions of thebrain, which include forebrain, mid-brain, hindbrain, and Rathke’s pouch(future pituitary gland). Craniofacialtissues, such as tooth primordium,tongue, olfactory epithelium, Meckel’scartilage, inner ear, esophagus, andlip, were also positive for the staining.In the trunk region, the stained signalswere present in dorsal root ganglia,spinal cord, and cartilage primordium.The Gpr177 transcript could also befound in lung, kidney, intestine, thymicprimordium, and urethra.

We next investigated the presence ofGpr177 protein during embryogenesisusing an antibody, which we developedpreviously against the carboxyl termi-nus (Fu et al., 2009). In our previousreport, this antibody recognized a�60-Kd protein, which is absent in theGpr177 mutants, suggesting the speci-ficity of this antibody (Fu et al., 2009).In addition, immunostaining was ableto detect a strong presence of Gpr177in the control mesoderm at embryonicday (E) 7.5, while the Gpr177-positivemesoderm was absent in the mutant(Fu et al., 2009). The protein expres-sion analysis was in agreement withthe in situ hybridization study (Fig.1C,D). Strongest levels of Gpr177were identified in the brain regionswith a very restricted expression pat-tern around hippocampus, thalamus,hypothalamus, and ventricles. Similarto the in situ expression study,Rathke’s pouch, dorsal root ganglia,spinal cord, cartilage primordium,lung, kidney, intestine, thymic primor-dium, ovary, and urethra were positive

for the staining. Immunostained sig-nals were also observed in the cranio-facial regions, including tooth primor-dium, tongue, olfactory epithelium,Meckel’s cartilage, inner ear, esopha-gus, and lip. The protein expressionpattern coincides with that of theGpr177 transcript, indicating the spec-ificity of our Gpr177 antibody.

Early Neural Development

Closer examination of Gpr177 in neu-ral development found that it is highlyexpressed in the neural epithelial cellsof various tissues (Fig. 2). Most promi-nent expression was detected in thedentate gyrate epithelium of hippo-campus (Fig. 2A,E,I,M) and epitheliumof the thalamus surrounding the thirdventricle (Fig. 2I,M). The Gpr177expression was distinguished in theneural epithelial cells of midbrain (Fig.2B,F,J,N), cerebellum (Fig. 2C,G,K,O),pons (Fig. 2D,H), medulla (Fig.2D,K,O), and spinal cord (Fig. 2L,P)that line the aqueduct, fourth ventri-cle, and central canal, especially in theventral and dorsal regions. The choroidepithelial cells of the choroid plexus inthe lateral ventricle (Fig. 2I,M) andfourth ventricle (Fig. 2C,G,K,O) alsoexhibited the Gpr177 transcript andprotein at high levels. The expressionin the proliferating zones of these tis-sues suggests that Gpr177 may have arole in the expansion of neural stem/progenitor cells. Mouse genetic analy-ses have demonstrated that membersof the Wnt family are essential forearly neural development (McMahonand Bradley, 1990; Thomas and Capec-chi, 1990; Hall et al., 2000; Lee et al.,2000). In addition, several Wnt signal-ing molecules, e.g., GSK3 and Axin1,are essential for development of neuro-ectoderm (Zeng et al., 1997; Kim et al.,2009). The deletion of Axin1 has beenreported in human medulloblastoma(Dahmen et al., 2001). Therefore, it ishighly possibly that the Gpr177-medi-ated Wnt production is critically linkedto neural development in health anddisease.

Craniofacial Morphogenesis

During craniofacial morphogenesis,the expression of Gpr177 was detectedin many cell types and tissues (Fig. 3).In the developing incisors and molars,

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both epithelial cells and surroundingmesenchymal cells show expression ofGpr177 (Fig. 3A–D). In humans,odonto-onycho-dermal dysplasia, arare autosomal recessive inheritedform of ectodermal dysplasia, wasrecently shown to be caused by a non-sense WNT10A mutation (Adaimyet al., 2007). The mutation of AXIN2,a gene encoding a negative regulatorof Wnt essential for targeting b-cate-nin degradation, was linked to fami-lial tooth agenesis (Lammi et al.,2004). Many previous studies (Jarvi-nen et al., 2006; Wang et al., 2009;Cobourne and Sharpe, 2010; Rookeret al., 2010) also support a potentialrole of Gpr177 in mediating Wnt pro-duction during tooth development.

Nonsyndromic cleft lip with orwithout cleft palate is one of the mostcommon birth defects. Genetic varia-tions in several Wnt family memberswere shown to be associated with thisphenotype in humans (Chiquet et al.,2008). Mouse genetic analysis also

indicated that Wnt5a (He et al., 2008)and Wnt9b (Juriloff et al., 2006) areessential for palate development.Indeed, we were able to identify thepresence of Gpr177 mRNA and pro-tein during embryonic development ofthe lip (Fig. 1A,C) and palate (Fig.3E,F). Strong Gpr177 expression wasalso found in the tongue (Fig. 3E,F)that maybe related to the role of Wntsignaling in formation of taste papilla(Iwatsuki et al., 2007).

The Wnt pathway has a well-estab-lished role in development of the skinand hair follicles (Alonso and Fuchs,2003; Haegebarth and Clevers, 2009).There was no surprise to identify theGpr177 transcript and protein inthese regions (Fig. 3G,H). Other sen-sory organs, including inner ear (Fig.3I,J), olfactory epithelium (Fig. 3K,L),and eye (Fig. 3M–P), were also posi-tive for the expression of Gpr177.Both canonical and noncanonical Wntproteins were shown to control forma-tion of the inner ear (Riccomagnoet al., 2005; Qian et al., 2007). It hasbeen suggested that Wnt3 acts as anaxon guidance molecule to mediatemedial–lateral retinotectal topogra-phy (Schmitt et al., 2006). The local-ization of the Gpr177 mRNA and pro-

tein supports the role of Wntsignaling in retinal and lens develop-ment (Van Raay and Vetter, 2004;Lovicu and McAvoy, 2005; Silver andRebay, 2005). However, the importantfunction of Gpr177- and Wnt-medi-ated development in the olfactory sys-tem remains largely to be explored. Inthe salivary gland, Wnt stimulationwas linked to pleomorphic adenoma,the most common type of salivarygland tumor in humans (Zhao et al.,2006; Declercq et al., 2008). Strongexpression of Gpr177 was detected inthe epithelial components of majorsalivary glands to a lesser extent inthe mesenchymal regions (Fig. 3Q–T).Nevertheless, the requirement of Wntsignaling in normal development ofthe salivary glands remains to bedetermined.

Internal Organs

We next examined if Gpr177 is alsoexpressed in development of othermajor organs. In the kidney, theGpr177 mRNA and protein weredetected predominantly in the ure-teric epithelium, but not the differen-tiated comma and S-shape bodies(Fig. 4A–D). A weak mesenchymal

Fig. 1. The Gpr177 transcript and protein aredetected in multiple tissues and organs duringmouse embryogenesis. A–D: In situ hybridization(A,B) and immunostaining (C,D) analyses showthe expression pattern of Gpr177 in embryonicday (E) 14.5 and E13.5 embryos, respectively.Cb, cerebellum; ChP, choroid plexus; CC, cere-bral cortex; CP, cartilage primordium; ccSC, cen-tral canal of spinal cord; DRG, dorsal rootganglia; Es, esophagus; Hc, hippocampus; Ht,hypothalamus; HB, hyoid bone; IE, inner ear; K,kidney; Lp, lip; Lu, lung; Mb, midbrain; MG, midgut; MC, Meckel’s cartilage; OE, olfactory epi-thelium; Ov, ovary; PP, pancreatic primordium;RP, Rathke’s pouch; Tg, tongue; Th, thalamus;TP, tooth primordium; TyP; thymic primordium;Ur, urethra. Scale bar¼ 1 mm.

Fig. 2. Gpr177 gene is active in neural development. A–P: The expression pattern of Gpr177 invarious neural tissues and cell types of the developing embryos is analyzed by in situ hybridiza-tion (A–D,I–L, embryonic day [E] 14.5) and immunostaining (E–H, E13.5; M–P, E14.5). Cb, cere-bellum; ChP, choroid plexus; CC, cerebral cortex; Hc, hippocampus; Ht, hypothalamus; Mb,midbrain; Me, medulla; SC, spinal cord; Th, thalamus. Scale bar ¼ 200 mm.

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expression was also observed, sug-gesting that Gpr177 may regulate thesorting and secretion of Wnt4, Wnt7b,Wnt9b, and Wnt11, which have beenimplicated in kidney organogenesis(Stark et al., 1994; Majumdar et al.,2003; Park et al., 2007; Karner et al.,2009; Yu et al., 2009). In agreementwith mouse genetic studies of the Wntfamily proteins (Li et al., 2002; Shuet al., 2002; Rajagopal et al., 2008;Goss et al., 2009), our data showed

that Gpr177 is expressed in both epi-thelial and mesenchymal cells, poten-tially involved in lung development(Fig. 4E,F). A selective expression ofGpr177 was found in the thymic pri-mordium (Fig. 4G,H). Given the well-established role of Wnt in hematopoie-sis (Staal and Clevers, 2005; Gri-goryan et al., 2008; Malhotra and Kin-cade, 2009), Gpr177 is likely to play arole in development of the hematopoi-etic lineages. In the developing gut,

the expression was found in both epi-thelium and mesenchyme of theesophagus (Fig. 4I,J), mid gut (Fig.1B), and duodenum (Fig. 4K,L), sug-gesting that the Gpr177-dependentregulation of Wnt may be critical fordevelopment of the digestive system.

Glycosylation of Gpr177

We previously showed that Gpr177 isdifferentially localized in the Wnt-

Fig. 3. Expression of Gpr177 during craniofacial morphogenesis. A–T: In situ hybridization (A,C,E,G,I,K,M,O,Q,S) and immunostaining(B,D,F,H,J,L,N,P,R,T) analyses reveal that a variety of craniofacial tissues and cell types express Gpr177 at embryonic day (E) 14.5. Co, cochlea;HF, hair follicle; Ln, lens; OE, olfactory epithelium; OM, ocular muscle; ON, optic nerve; OR, optic recess; P, palate; SC, semicircular canal; SG,salivary gland; SL, sublingual duct; SM, submandibular duct; Tg, tongue; TP, tooth primordium. Scale bars ¼ 100 mm in A–D,G,H,S,T, 50 mm inQ,R, 200 mm in E,F,I–P.

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producing and non–Wnt-producingcells (Fu et al., 2009). The differentialcompartmentalization is dependentupon the positive feedback regulationof Wnt to activate the Gpr177 expres-sion at the transcriptional level (Fuet al., 2009). The Golgi accumulationof Gpr177 can only be identified in thesignal-producing cells, such as neuralstem cells (Fu et al., 2009). Becausemembrane and secreted proteins aremodified by saccharides, we examinedwhether inhibition of glycosylationinterferes with the Golgi accumulationof Gpr177. In the culture of neuro-sphere cells, Gpr177 is colocalizedwith a Golgi marker GM130 (Fig. 5A).The addition of tunicamycin, whichinhibits the enzyme GlcNAc phospho-transferase involved in the first step ofglycoprotein synthesis, affects concen-trations of Gpr177 in the Golgi appa-ratus (Fig. 5A). Treatment of tunica-mycin prevented the colocalization ofGpr177 with GM130, suggesting thatglycosylation is required for the Golgiaccumulation. Immunoblot analysisindicated that a slow migrating banddisappears after the tunicamycintreatment (Fig. 5B). Furthermore, theaddition of Endo Hf diminished thedetection of Gpr177 with slow mobility(Fig. 5C). The results suggest that

Fig. 4. Expression of Gpr177 in development of multiple organs. A–L: The mRNA and protein expression of Gpr177 is examined by in situ hybridization(A,B,E,G,I,K) and immunostaining (C,D,F,H,J,L) of embryonic day (E) 14.5 embryos, respectively. Insets in A, C denote higher magnification in B, D, respec-tively. Du, duodenum; Es, esophagus; K, kidney; Lu, lung; TyP, thymic primordium. Scale bars ¼ 200 mm in A,C, 100 mm in B,E,F,I,K, 50 mm in D,G,H,J,L.

Fig. 5. Glycosylation is required for Golgi distributions of Gpr177. A: Neurosphere cells weretreated with Tunicamycin (TM) for different concentrations and time periods as indicated. Three-dimensional (3D) imaging of the immunostained cells shows the localization of endogenous Gpr177(green) and GM130 (red), counterstained with DAPI (40,6-diamidine-2-phenylidole-dihydrochloride;blue). Note that the TM treatment disrupts the colocalization of Gpr177 and GM130 in Golgi.B: Immunoblot analysis of Gpr177 reveals that TM abolishes the detection of Gpr177 with slowmobility (arrow) in neural stem cells. The number indicates the drug concentration (mg/ml) present inculture media. The expression level of Actin was analyzed as a loading control. C: Immunoblot anal-ysis shows that the slow migrating band (arrow) detected by the Gpr177 antibody disappears afterthe addition of Endo Hf. The number presents the amount of enzyme (ml: 1,000 units per ml) addedto the in vitro assays. The expression level of Actin was analyzed as a loading control.

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Gpr177 is modified by N-linked glyco-sylation required for proper sortingwithin the cells.

In summary, we have performed acomprehensive survey on the expres-sion of Gpr177 using in situ hybrid-ization and immunostaining analyses.The expression patterns of mRNAand protein are in agreement witheach other, supporting the notion thatWnt signaling is critical for develop-ment of multiple organs. Our priordiscovery that Gpr177 is essential formodulating canonical Wnt in earlyembryogenesis suggests that their re-ciprocal regulations are likely to beimportant for other developmentalprocesses. Gpr177 might also be asso-ciated with the Wnt-mediated malig-nant transformation of these organs.The expression of Gpr177 in develop-ment of several organs requiring theepithelial–mesenchymal interactionadds another layer of regulation forsignal-producing and signal-receivingcells. In addition, the Wnt-producingcells are able to initiate paracrine aswell as autocrine signaling effects.Gpr177 seems to be expressed in pla-ces where noncanonical Wnt proteinshave essential functions. However,whether noncanonical Wnt is regu-lated by Gpr177 remains an impor-tant question to be determined, espe-cially to be assessed functionally bygenetic analysis. Using large-scalemeta-analysis of genome-wide associ-ation, a recent study has identifiedGPR177 as 1 of the 20 bone-mineral-density loci in humans (Rivadeneiraet al., 2009). Among them, CTNNB1,encoding b-catenin, is another locusidentified at the genome-wide studylevel. The finding suggests thatGpr177 and b-catenin are master reg-ulators for Wnt production and sig-naling, respectively. One can expectfurther discovery revealing the signif-icance of the Gpr177-mediated regu-lation of Wnt in skeletal developmentand disease.

EXPERIMENTAL

PROCEDURES

Mouse embryos were first fixed, paraf-fin embedded, sectioned, and stainedwith hematoxylin/eosin for histologicalevaluation as described (Yu et al., 2007;Hsu et al., 2010). Sections were thensubject to in situ hybridization with

digoxigenin-labeled antisense RNAprobes or immunological staining withavidin:biotinlylated enzyme complex asdescribed (Yu et al., 2005a; Liu et al.,2007; Chiu et al., 2008). For in situhybridization, RNA probes for detectingthe Gpr177 transcript as well as thedetailed method were described previ-ously (Chiu et al., 2008; Fu et al., 2009).Rabbit polyclonal antibodies were gen-erated for the immunostaining analysisof Gpr177 as described (Fu et al., 2009).Briefly, sections were deparaffinizedand hydrated, followed by antigenunmasking (Vector Laboratories, Bur-lingame, CA). Endogenous peroxidaseactivity was blocked by incubating sec-tions with 3% hydrogen peroxide. Afteraddition of primary antibodies, sectionswere incubated with horseradish perox-idase-conjugated secondary antibodies.The staining was then visualized by en-zymatic color reaction according to themanufacture’s specification (VectorLaboratories). Images were analyzedusing Zeiss AXIO OBSERVER withAXIOCAM or Nikon SMZ-1500 withSPOT-RT microscope imaging systems(Yu et al., 2005b; Chiu et al., 2008; Liuet al., 2008).

Isolation and culture of primary neu-rospheres were performed as described(Fu et al., 2009). Briefly, telencepha-lons of E12.5 mouse embryos wererecovered andmechanically dissociatedin DMEM/F12 medium (1:1; Invitro-gen, Carlsbad, CA), followed by filter-ing through a 70-mm nylon mesh (BDBiosciences, Bedford, MA). Cells werethen cultured in DMEM/F12 medium,supplemented with Insulin-Transfer-rin-Selenium supplements (Invitro-gen), epithelial growth factor (20 ng/ml; Sigma-Aldrich, St. Louis, MO), andantibiotic solution (Sigma-Aldrich), at37�C in a humidified 5% CO2 incuba-tor. After 5 days, neurospheres wereharvested by centrifugation, dissoci-ated by Trypsin-ethylenediaminetetra-acetic acid (Sigma-Aldrich), and contin-ued to be cultured for next passages orused in experimental procedures.

Immunostaining of cultured neuro-sphere cells was performed using indi-rect fluorescent staining techniquesdescribed previously (Fu et al., 2009).Images were taken using AXIO OB-SERVER with AXIOCAM microscopeimaging system, followed by deconvo-lution, three-dimensional imaginganalysis. Immunoblot analysis was

performed as described (Liu et al.,2008; Fu et al., 2009). Bound primaryantibodies were detected with horse-radish peroxidase-conjugated second-ary antibodies, followed by enhancedchemical luminescence-mediated vis-ualization (GE Healthcare, Piscat-away, NJ) and autoradiography.

ACKNOWLEDGMENTSWe thank Anthony Mirando for prepa-ration of the manuscript. W.H. wasfunded by the National Institutes ofHealth (CA106308 andDE015654).

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