PATTERNS & PHENOTYPES

Expression of Protocadherin-1 (Pcdh1) DuringMouse DevelopmentChristoph Redies,1* Jessica Heyder,1 Tomas Kohoutek,1† Katrien Staes,2,3 and Frans Van Roy2,3

Protocadherin-1 (Pcdh1) is a member of the �-protocadherin subgroup of non-clustered protocadherins. Westudied the expression of Pcdh1 from the early embryonic to the adult stage of mouse development bysemi-quantitative RT-PCR and in situ hybridization. Pcdh1 can be detected as early as embryonic day 9.5.In early embryogenesis, expression is especially prominent in blood vessels. During later development andin the adult mouse, organs derived from the embryonic gut, such as the esophagus, intestines, liver,lung, and submandibular gland, contain epithelia and other types of tissues that are Pcdh1-positive.Other positive organs include the brain, spinal cord, retina, peripheral ganglia, the inner ear, hairfollicles, kidney, vagina, uterus, placenta, testis, prostate, and the seminal gland. The tight spatial andtemporal regulation of Pcdh1 expression suggests that this protocadherin plays multiple roles not onlyduring development but also in mature tissues and organs in the mouse. Developmental Dynamics 237:2496–2505, 2008. © 2008 Wiley-Liss, Inc.

Key words: cell adhesion; placenta; blood vessel formation; organ development; brain; intestines; lung; liver; kidney;glomerulus; hair follicle

Accepted 29 May 2008

INTRODUCTION

Cadherins constitute a large family ofcell adhesion receptors that can beclassified into several subfamilies, in-cluding classic cadherins, protocad-herins, desmosomal cadherins, andflamingo cadherins (for reviews, seeNollet et al., 2000; Frank and Kemler,2002). Almost all cadherins show aspecific expression profile during thedevelopment of various tissues and or-gans. The protocadherin family (Sanoet al., 1993) comprises multiple sub-families, such as the �-, �-, and �-pro-

tocadherins, and the large (fat- anddachsous-related) protocadherins (forreviews, see Frank and Kemler, 2002;Hirano et al., 2003). Recently, a novelsubgroup of protocadherins, termed�-protocadherins, was identified by aphylogenetic comparison of mouse andhuman protocadherins (Vanhalst etal., 2005). On the basis of overall ho-mology, number of extracellular cad-herin repeats (seven versus six), andconservation of specific amino acidmotifs in the cytoplasmic domains,two subgroups were identified. The �1

subgroup comprises protocadherin-1,-7, -9, and -11(X/Y). The �2 subgroupcomprises protocadherin-8, -10, -17,-18, and -19 (for a review, see Redieset al., 2005).

The expression and function of onlysome �-protocadherins have been in-vestigated in detail during early Xeno-pus and zebrafish development (for areview, see Redies et al., 2005). Rela-tively little is known about the expres-sion of �-protocadherins in other ver-tebrate species and at later stages ofdevelopment, with the exception of

1Institute of Anatomy I, Friedrich Schiller University, Jena, Germany2Department for Molecular Biomedical Research, Ghent, Belgium3Department of Molecular Biology, Ghent University, Ghent, BelgiumGrant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Re 616/4-4; Grant sponsor: Interdisciplinary Center for Cancer ResearchJena; Grant number: TP1.16; Grant sponsor: Fund for Scientific Research (FWO), Flanders; Grant sponsor: Ghent University (GOA grant);Grant sponsor: The Foundation Against Cancer, Belgium.†Tomas Kohoutek’s present address is Institute of Histology and Embryology, Charles University Prague, Faculty of Medicine in Plzen,Karlovarska 48, CZ-30166 Plzen, Czech Republic.*Correspondence to: Christoph Redies, MD, PhD, Institute of Anatomy I, Friedrich Schiller University Jena, School ofMedicine, D-07740 Jena, Germany. E-mail: redies@mti.uni-jena.de

DOI 10.1002/dvdy.21650Published online 26 August 2008 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 237:2496–2505, 2008

© 2008 Wiley-Liss, Inc.

Pcdh19 in the developing mouse em-bryo (Gaitan and Bouchard, 2006) andseveral �-protocadherins that havebeen mapped in the developing andadult brain of mouse, rat, or chicken(Hirano et al., 1999; Yamagata et al.,1999; Muller et al., 2004; Vanhalst etal., 2005; Kim et al., 2007). Here wereport on the expression of protocad-herin-1 (Pcdh1) during mouse devel-opment from the early embryonicstage to the adult stage.

Pcdh1, one of the first protocad-herins to be discovered (then calledprotocadherin-42), mediates cell adhe-sion upon ectopic expression in L cells(Sano et al., 1993). The ortholog ofPcdh1 in Xenopus, termed axial pro-tocadherin, plays a role in prenoto-chordal cell sorting in the gastrulatingembryo (Kuroda et al., 2002). At thetailbud stage of Xenopus, axial pro-tocadherin was shown to be expressedin the somites, pronephros, heart, oticvesicle, and brain (Kuroda et al.,2002). The results of the presentstudy, which is the first to describemouse Pcdh1 expression at the histo-logical level in detail, suggest thatPcdh1 expression is tightly regulatedin several other developing organsand tissues, shedding light on the pos-sible role of Pcdh1 in tumorigenesis orother pathogenetic processes in someof these tissues.

RESULTS

Overview of Expression

Results from semi-quantitative RT-PCR demonstrated that weak expres-sion of both short and long isoforms ofmouse Pcdh1 can be detected as earlyas embryonic day (E) 9.5 (Fig. 1). Ex-pression in whole embryos is strong onE11.5 and remains strong until atleast E16.5. In the adult, several or-gans, such as the liver, brain, kidney,heart, lung, and uterus, expressPcdh1 at different levels.

By in situ hybridization, which de-tected both short and long isoforms,Pcdh1 expression was demonstratednot only in mammalian brain (Sano etal., 1993; Kim et al., 2007; Hertel etal., 2008) and lung (Favre et al., 2003),but also in the placenta and in severalother tissues and organs derived fromall three germinal layers (Figs. 2, 3).Hybridization with a sense probeserved as a negative control.

Most striking is the expression ofPcdh1 by blood vessels during earlyembryogenesis of most organs (Fig.2A–F,K,L,T,U,W,W’). Prenatal ex-pression of Pcdh1 is seen in many ofthe epithelia derived from the embry-onic gut, such as the epithelial liningof the esophagus, the small and largeintestines, and organs such as theliver, the lung, and the submandibu-

lar gland (Figs. 2Q–W’, 3A–E). In ad-dition, Pcdh1 is expressed along theentire neuraxis, including the brain,spinal cord, sensory epithelia, andmost ganglia of the peripheral ner-vous system (Fig. 3L–P). These re-sults will be described in detail in thefollowing sections. Tissues and organsthat do not express Pcdh1 or do so onlyweakly (except in their blood vessels)include the epidermis, bone, cartilage,muscle, pancreas, and urinary blad-der.

Cardiovascular System

In situ hybridization showed thatblood vessels (bv) throughout the em-bryo express Pcdh1 as early as E10(compare staining for PECAM-1, amarker for endothelial cells, in Fig. 2Bwith 2A). At E10, the endothelial lin-ing of the heart expresses Pcdh1 (ar-rows in Fig. 2A,B). After E10, the en-dothelial lining of the heart and ofmajor blood vessels, such as the aorta,do not show a Pcdh1 signal. In con-trast, small blood vessels retain prom-inent mRNA expression during em-bryogenesis, as demonstrated, forexample, for blood vessels in a digit ofthe forelimb (Fig. 2C,C’). Neverthe-less, not all embryonic endothelia ex-press Pcdh1 throughout early devel-opment. In the heart, the blood vesselsof the compact (outer) part of the ven-tricular myocardium [com(o)] expressPcdh1 at E15, but the endothelial lin-ing of the trabeculated (inner) part[tr(i)] does not (Fig. 2D,D’). Smallblood vessels in both parts of the ven-tricle, however, show Pcdh1 expres-sion two days later (at E17; data notshown). The endothelial lining of theheart remains negative. Postnatally,Pcdh1 expression by blood vessels de-creases to low levels (Fig. 2E,F).

Chorioallantoic Placenta andYolk Sac

In the E10 maternal compartment,Pcdh1 is expressed strongly by the en-dothelial lining (end) of the sinusoids(ds) in the decidua (Fig. 2G). ThePcdh1 signal is also found in some tro-phoblast cells within the spongiotro-phoblast (sp) and labyrinthine layer(lb) of the chorioallantoic placenta(Fig. 2H) and in the yolk sac (data notshown). At E12, the yolk epithelium

Fig. 1. Results from semi-quantitative RT-PCR analysis showing the expression of the longisoform and the short isoform of Pcdh1 during embryogenesis at different stages of mousedevelopment (left) and in adult organs (right). Experiments were carried out with (�) and without (�)reverse transcriptase. GAPDH served as an internal control.

PROTOCADHERIN-1 EXPRESSION IN MOUSE 2497

(yo) is strongly Pcdh1-positive whereasthe Reichert’s membrane (R) exhibits aweak to moderate signal (Fig. 2I). At

E13, Pcdh1 expression becomes re-stricted to the glycogen cells (gly) withinthe spongiotrophoblast layer (sp in Fig.

2J) and the labyrinth is negative. Theexpression of Pcdh1 in the placentareaches low levels at E17.

Fig. 2.

).

2498 REDIES ET AL.

Kidney and Urinary Bladder

During kidney development, expressionof Pcdh1 in vascular structures predom-inates during the early embryonicstages. At E13, some Pcdh1-positive cel-lular aggregates are seen in the meta-nephros (data not shown). Epithelialstructures are negative. At E15, a hy-bridization signal is seen in the devel-oping blood vessels of the renal cortex.In the glomeruli (gl in Fig. 2K,L), thesignal has a distribution similar to PE-CAM-1. From E17 to P12, the glomeruliare strongly positive and the epithelialcomponents of the renal cortex are mod-erately positive (Fig. 2M; compare tothe sense control shown in Fig. 2M’).The tubular system in the medulla ismoderately positive at P5 and P12.Blood vessels show no signal in the me-dulla during the postnatal and adultstages, but prominent staining persistsin the glomeruli. In the cortex of theadult (cx in Fig. 2N,O), the glomeruliand the proximal tubules (pt) that arecharacterized by bluish Azan stainingof their ciliated borders (Fig. 2N’,P) ex-press Pcdh1, whereas the rest of thecortex and the medulla (med) are nega-tive (Fig. 2N). The urothelium of theurinary bladder is negative at allstages.

Gastrointestinal System andOther Derivatives of theEmbryonic Gut

In the embryonic gut, Pcdh1 expres-sion is first seen at E10 in PECAM-1-positive blood vessels (bv in Fig. 2Q-T). In addition, starting at E12, Pcdh1

is expressed also by the epithelial lin-ing of the midgut intestinal mucosa(mu) and by scattered cells in the sub-mucosa (smu in Fig. 2Q,R,T). The ep-ithelial lining of the esophagus (oe) isPcdh1-positive but that of the stom-ach (st) does not express Pcdh1 at E15(Fig. 2S) and E17. From P5 until theadult stage, there is a prominent ex-pression gradient in the epithelial lin-ing of the mucosal lamina in the intes-tines; the crypts (cr) express Pcdh1more strongly than the villi (vi in Fig.2V; compare to the sense controlshown in Fig. 2V’).

Pcdh1 is expressed in the embryoniclung by the parenchyma (p), wherePECAM-1-positive blood vessels (bv)predominate, but not by the bronchi(br in Fig. 2W,W’). This expressionprofile persists until the adult stage(Fig. 3A). The parenchyma of the liver(li) also expresses Pcdh1 from E12 tothe adult stage (Figs. 2W, 3B). Thepattern of Pcdh1 expression in theliver roughly resembles that ofPECAM-1 at E15 (Fig. 2W,W’).

In the submandibular gland, Pcdh1expression is rather ubiquitous in ep-ithelial cells and blood vessels at E17(Fig. 3C–E).

Reproductive System

The reproductive organs were studiedat the postnatal and adult stages. Infemale P5 pups, the endometrium (enin Fig. 3F) and the vaginal epithelium(ve) express Pcdh1 (Fig. 3G). In adultfemales, the tubular glands of themyometrium (arrows in Fig. 3H) arePcdh1-positive, but the endometrial

lining of the uterine cavity is negative;the ovaries do not express Pcdh1.

In postnatal males (P5, P10, andP12), the epithelia of the prostategland (Fig. 3I) and of the seminal ves-icles (Fig. 3J) express Pcdh1. The sem-iniferous tubules of the testis are alsopositive (Fig. 3K). In the adult, thesignal in the male reproductive organswas not above the sense control.

Nervous System

In the spinal cord, the neuroepithe-lium of the basal plate expressesPcdh1 at E10 (data not shown). AtE13, the entire marginal layer of thebasal and alar plates shows a promi-nent signal. At E15, expression is es-pecially strong in the superficial lay-ers of the dorsal column (dc in Fig.3L). The motor column (mc) of thebasal plate is also Pcdh1-positive atsome spinal cord levels (insert in Fig.3L). This general staining pattern per-sists in the spinal cord at least untilP12.

As in other organs, Pcdh1 is ex-pressed by blood vessels in andaround the brain at E13 (Fig. 3M).The first regionalized staining in theneuroepithelium of the brain is ob-served at E12. The expression ofPcdh1 in gray matter structures of de-veloping and adult brain has been de-scribed previously (Sano et al., 1993;Kuroda et al., 2002; Kim et al., 2007;Hertel et al., 2008). The present find-ings confirm the results of Kim et al.(2007), who showed that Pcdh1 is ex-pressed in a subset of gray matterstructures of the postnatal forebrain.As an example, in Figure 3N, whichshows a transverse section throughthe P5 forebrain, expression is espe-cially strong in the hippocampus (h)and in the indusium griseum (ig),whereas neurons of the dentate gyrus(dg) show a weak signal. In neocortex,specific layers show signal. For exam-ple, layer V of the somatosensory cor-tex (V) is more strongly stained thanthe other layers. Other Pcdh1-positiveforebrain structures include nuclei ofthe amygdalar complex (amy), thedorsal and ventral lateral geniculatenuclei in the thalamus (dlg, vlg), andthe substantia nigra (sn). For a morecomplete mapping of Pcdh1 expres-sion in mouse brain development, seethe study done by Kim et al. (2007).

Fig. 2. Pcdh1 expression by the endothelial lining of the heart and blood vessels (bv) of themandibular arch (ba1; A,B) and of a toe (C,C’), in the heart ventricle (D,D’,E,F), in the placenta (G–J),in the kidney (K–P), in the midgut (Q,R,T,U), at the transition between esophagus and stomach (S),in the colon (V,V’), and in the embryonic liver and lung (W,W’) at different stages of development,as indicated at the top and right side of each panel. Ad, adult; E, embryonic day; P, postnatal day.Sections were hybridized in situ with an anti-sense mRNA probe (A,C,D,E,G–K,M,N,O,Q–T,V,W) ora sense (control) mRNA probe (M’,V’) for Pcdh1. B, C’, D’, F, L, U, and W’ show sections that areadjacent to A, C, D, E, K, T, and W, respectively, and were stained with antibodies againstPECAM-1 (PECAM). N’ and P show sections that are adjacent to N and O, respectively, and werestained with azan dye (Azan). at, atrium of heart; ba1, first branchial arch; br, bronchi; bv, bloodvessel; com(o), compact (outer) part of the ventricle; cr, intestinal crypts; cx, renal cortex; dia,diaphragm; dph, distal phalanx; ds, sinusoids of the decidua; dt, distal tubulus; end, endotheliallining; gl, glomerulus; gly, glycogen cells; iph, intermediate phalanx; lb, labyrinthine layer; li, liver;med, renal medulla; mu, mucosa of intestine; oe, esophagus; p, parenchyma of lung; pph, proximalphalanx; pl, placenta; pt, proximal tubulus; R, Reichert’s membrane; smu, submucosa of intestine;sp, spongiotrophoblast; st, stomach; tr(i), trabeculated (inner) part of the ventricle; ve, ventricle ofheart; vi, intestinal villi; yo, yolk sac. Scale bars � 200 �m in C (for C,C’) and N (for N,N’); 100 �min A (for A,B), J, R, S, T (for T,U), V (for V,V’), and W (for W,W’); 50 �m in D (for D,D’), G, H, I, K (forK,L), M (for M,M’), O (for O,P) and Q; and 20 �m in E (for E,F).

PROTOCADHERIN-1 EXPRESSION IN MOUSE 2499

Pcdh1 is also expressed in the pro-spective ganglion cell layer (gcl) andinner nuclear layer (inl) of the retina

during embryonic development (fromabout E15; E19 in Fig. 3W) and atpostnatal stages (Fig. 3O). The glo-

merular and granular layers of the ol-factory bulb (gl, gr in Fig. 3P), theaccessory olfactory bulb (data not

Fig. 3

2500 REDIES ET AL.

shown), and the anterior olfactory nu-cleus (ao in Fig. 3P) also expressPcdh1.

In the peripheral nervous system,most neurons in the spinal ganglia(sg), cranial sensory ganglia, and vis-ceral ganglia (sy) express Pcdh1 (Fig.3L) from the early stages of embryo-genesis (E10-E12) to postnatal stages.Schwann cells in the peripheralnerves are negative. The vomeronasalorgan (vno in Fig. 3Q) is also positivefor Pcdh1, at least from E15 to P12. Inthe developing P5 cochlea, Pcdh1mRNA is detected in the stria vascu-laris (sv) of the cochlear duct and insuperficial cells of the organ of Corti,possibly including prospective haircells and Hensen cells (arrowheads inFig. 3R); expression in other struc-tures of the inner ear is weak or ab-sent.

Other Organs

The hair follicles contain Pcdh1-posi-tive cells from the beginning of theirformation until at least P12 (Fig.3S,T). Except for the dermal papilla(dp), which expresses Pcdh1 weakly,all layers of the hair bulb (bh) expressPcdh1, including those giving rise tothe medulla and cortex of the hairshaft. The inner root sheath (irs)shows a strong Pcdh1 signal, but theouter root sheet (ors) is negative. Inthe embryonic thymus, the endothe-

lial lining of the sinuses expressesPcdh1 at E15 (Fig. 3U,V). Thymicblood vessels are strongly positive atE17 but the signal decreases to lowlevels at P5.

The staining pattern in the nasalcavity (nc) is especially striking (Fig.3W) from E15 to at least P12, withregional variations in the Pcdh1 ex-pression level. The serous glands (ng)in the lateral wall of the middle me-atus express moderate levels of Pcdh1in the glandular epithelium (Fig. 3X).In the epithelial lining (el) of the nasalcavity, the most superficial layer isPcdh1-negative whereas the deeperlayer shows a very strong signal (Fig.3Y).

DISCUSSION

The results demonstrate that Pcdh1 isexpressed under tight spatial andtemporal control in a subset of tissuesand organs during embryogenesis andin the adult mouse. Kuroda et al.(2002) showed that the ortholog ofPcdh1 in Xenopus, axial protocad-herin, is expressed during early devel-opment by the somites, pronephros,heart, otic vesicle, and brain. Our re-sults extend these earlier findings inseveral respects. First, we demon-strate that, in the mouse, other organsalso express Pcdh1. Second, we alsodescribe expression at later stages ofdevelopment and in the adult. Third,

by performing a histological analysisin addition to semi-quantitative RT-PCR experiments, we identify thetypes of tissue that express Pcdh1 inthe various organs. For example, inthe heart, Pcdh1 is expressed by bloodvessels but not by cardiomyocytes;skeletal muscle cells are also negative.

Our in situ hybridization approachcannot discriminate between tran-scripts encoding either short or longisoforms of Pcdh1. It is a recurrentfinding that �-protocadherins, includ-ing Pcdh1, are expressed as a mixtureof short and long isoforms (Vanhalstet al., 2005; Redies et al., 2005). Thedifference between the isoforms isbased on alternative splicing and re-sults in a shorter versus extendedlength of the cytoplasmic domain. Thefunctional implications of differentialexpression may be significant but arepresently unclear. Indeed, the ex-tended cytoplasmic domain comprisesseveral conserved motifs (CM1, CM2,and CM3 in the case of Pcdh1). Pro-tein phosphatase-1� is known to bindto the CM3 motif (Vanhalst et al.,2005), but molecular binding partnersof the CM1 and CM2 motifs remain tobe identified.

Endothelial Cells ExpressPcdh1

In general, Pcdh1 is expressed promi-nently by developing blood vesselsduring angiogenesis (e.g., in brain ves-sels) and vasculogenesis (e.g., in vis-ceral and somitic vessels) (for a re-view, see Risau, 1995). However, notall endothelia are Pcdh1-positive. Forexample, in the heart at E15, no ex-pression is observed in the endotheliallining of the inner trabeculated part ofthe myocardium, while blood vesselsin the outer compact part show a hy-bridization signal. Interestingly, thetwo parts of the myocardium repre-sent different stages of cardiomyocytedifferentiation (Sedmera et al., 2000).

Other cadherins expressed by bloodvessels are cadherin-5 (VE-cadherin;Lampugnani et al., 1992), cadherin-2(N-cadherin; Salomon et al., 1992;Redies et al., 1993), cadherin-13 (T-cad-herin), cadherin-4 (R-cadherin), andprotocadherin-12 (VE-cadherin-2; for areview, see Cavallaro et al., 2006). Inthe mouse and human brains, bloodvessels that contribute to the blood-

Fig. 3. Pcdh1 expression in lung (A), liver (B), submandibular gland (C–E), uterus (F,H), vagina (G),prostate gland (I), seminal vesicles (J), seminiferous tubules of the testis (K), spinal cord andperipheral ganglia (L), blood vessels entering the hindbrain (M), forebrain (N), retina (O), olfactorybulb (P), vomeronasal organ (Q), cochlea (R), hair follicles (S,T), thymus (U,V), epithelial lining of thenasal cavity (W,Y), and nasal glands (X), at different stages of development, as indicated at the topand right side of each panel. Ad, adult; E, embryonic day; P, postnatal day. Sections werehybridized in situ with an mRNA probe for Pcdh1 (A–C,F–U,W–Y). D shows a section that isadjacent to C and was stained with antibodies against PECAM-1 (PECAM). E and V show sectionsthat are adjacent to C and U, respectively, and were stained with hematoxylin/eosin dye (HE). Thearrows in H point to tubular glands in the myometrium. The inset in L shows the motor column (mc)of the spinal cord at another level of sectioning. The asterisks in N indicate an artifact (tissue fold).The arrowheads in R indicate superficial Pcdh1-expressing cells in the organ of Corti. X and Y showmagnifications of the areas boxed in W. V, layer V of cortex; amy, amygdala; ao, anterior olfactorynucleus; bh, hair bulb; br, terminal bronchus; bv, blood vessel; cd, cochlear duct; cp, caudoputa-men; cv, central vein; dc, dorsal column; dg, dentate gyrus; dlg, dorsal lateral geniculate nucleus;dp, dermal papilla; el, epithelial lining of the nasal cavity; en, endometrium; gcl, ganglion cell layer;gl, glomerular layer; gr, granular layer; h, hippocampus; hb, hindbrain; hy, hypothalamus; ig,indusium griseum; inl, inner nuclear layer; ipl, inner plexiform layer; irs, inner root sheath; le, lens;mc, motor column; nc, nasal cavity; ng, nasal glandular tissue; ns, nasal septum; ors, outer rootsheath; pir, piriform cortex; po, posterior thalamic nuclear complex; r, retina; rm, Reissner’smembrane; sc, supporting cells; sg, spinal ganglion; sn, substantia nigra; sv, stria vascularis; sy,sympathetic ganglion; ve, vaginal epithelium; vlg, ventral lateral geniculate nucleus; vno, vomero-nasal (Jacobson’s) organ. Scale bars � 1 mm in N and W; 500 �m in P; 200 �m in L, Q, and T; 100�m in H, L (insert), R, and X (for X,Y); and 50 �m in A, B, C (for C–E), F (for F,G), I, J (for J,K), M,O, S, and U (for U,V).

PROTOCADHERIN-1 EXPRESSION IN MOUSE 2501

brain barrier express cadherin-10 (Wil-liams et al., 2005). It has been sug-gested that all these cadherins playdifferent roles in the formation of bloodvessels (for a review, see Cavallaro etal., 2006). Cadherin-5 mediates the in-tercellular adhesion between endothe-lial cells. Cadherin-5-deficient mice canstill form primitive vascular plexus andtubular endothelial structures, but vas-cular remodeling and endothelial adhe-sion is impaired (Carmeliet et al., 1999).Pcdh1 is a candidate mediator of thisresidual adhesive function of endothe-lial cells in the absence of cadherin-5.N-cadherin is involved in linking theendothelial cells to the surrounding tis-sues (Navarro et al., 1998; Gerhardt etal., 2000). Protocadherin-12 has beenrelated to vasculogenesis rather thanangiogenesis (Rampon et al., 2005).

Like VE-cadherin and protocad-herin-12, Pcdh1 is not expressed byendothelial cells in the adult in mosttissues. However, Favre et al. (2003)isolated Pcdh1-positive endothelialcells from adult lung, which is in linewith our visualization of Pcdh1 ex-pression in sections through the adultlung (Fig. 3A).

Pcdh1 Expression in OtherOrgans and Tissues

Kidney

In the kidney, Pcdh1 is expressed inthe glomeruli throughout develop-ment and in the proximal tubules ofthe adult mouse. Apart from Pcdh1,several other members of the cadherinsuperfamily are expressed differen-tially in the developing and adult kid-ney (for a review, see Dressler, 2002),including some classic cadherins, suchas E-cadherin (Vestweber et al.,1985), R-cadherin (Dahl et al., 2002),and cadherin-6 (Xiang et al., 1994;Cho et al., 1998), as well as the �-pro-tocadherins Pcdh7 (BH-protocad-herin; Rudnicki et al., 2007) andPcdh19 (Gaitan and Bouchard, 2006).Like these other (proto-)cadherins,the expression of Pcdh1 is restricted toparticular renal tissues. For example,Pcdh1 is expressed in proximal butnot in distal tubules of the adult kid-ney (Fig. 2O,P). The similarity be-tween the expression patterns ofPcdh1 and PECAM-1 in the glomeruliindicates that Pcdh1 is expressed byglomerular endothelial cells.

Placenta and reproductiveorgans.

Pcdh1 is expressed by specific tissueelements of the placenta and yolk sac.The expression of Pcdh1 by the endo-thelial cell lining of the decidual sinu-soids and by the glycogen-rich cells issimilar to that described for protocad-herin-12 (VE-cadherin-2; Rampon etal., 2005; Bouillot et al., 2006). Classiccadherins expressed differentially inplacental tissues include cadherin-3(P-cadherin; Nose et al., 1987), cad-herin-6, cadherin-11 (for a review, seeMacCalman et al., 1998), VE-cadherin(Bulla et al., 2005), and cadherin-1 (E-cadherin). Several of the epithelia ofthe reproductive organs expressPcdh1. It is unknown whether Pcdh1expression underlies hormonal regu-lation, as has been shown for some ofthe classic cadherins expressed in theuterus (for a review, see Horne et al.,2002).

In the testis, Pcdh1 signal wasfound in the seminiferous tubules.Cadherin-mediated cell–cell adhesionmediates the close contact betweendifferentiating germ cells, the seminif-erous epithelium, and the Sertoli cells(for a review, see Goossens and vanRoy, 2005). Many cadherins and pro-tocadherins are expressed in testis,amongst them several classic cad-herins (Munro and Blaschuk, 1996)and clustered protocadherins (John-son et al., 2000). The present study isthe first to identify a �-protocadherinin the testis.

Nervous system.

In the developing brain, Pcdh1 is ex-pressed in a subset of neuroanatomi-cal structures in all major brain re-gions. Our results are in agreementwith findings in the rat brain (Kim etal., 2007) and in the mouse basal gan-glia (Hertel et al., 2008). Extendingthese results, we demonstrate thatPcdh1 is expressed also in restrictedregions and layers of the spinal cordand retina, and in several sensory andvisceral ganglia of the peripheral ner-vous system (Fig. 3L,O). The expres-sion pattern of Pcdh1 in these struc-tures is distinct from that of manyother classic cadherins and �-pro-tocadherins, which are also expressedin highly regionalized patterns in thevertebrate brain (for reviews, see

Redies, 2000; Hirano et al., 2003;Redies et al., 2005). Thus, it is likelythat Pcdh1 contributes to the cad-herin-based adhesive code that regu-lates diverse morphoregulatory pro-cesses during brain development (forreviews, see Redies, 2000; Takeichi,2007).

Pcdh1 in PathogeneticProcesses

The expression of Pcdh1 is alteredduring tumorigenesis in several typesof tissues and organs (Castilla et al.,2004; Neben et al., 2004; Rush et al.,2005; Nonnenmacher et al., 2006) thatwere found to express Pcdh1 in thepresent study. For example, Pcdh1 isdown-regulated in a carcinogenesismodel of renal tumors in rat (Stemmeret al., 2007). Another �-protocadherin,Pcdh10, was found to be methylated incarcinomas and hematologic malig-nancies (Ying et al., 2006, 2007). Inhair follicles, Pcdh1 can act as a recep-tor for cottontail rabbit papillomavi-rus (Nonnenmacher et al., 2006). Thispathogen targets keratinocytes andcan cause lesions that have a high riskof progression to carcinoma (Schmittet al., 1996; Nonnenmacher et al.,2006). Whether Pcdh1 plays a similarrole in the epithelial components ofthe female genital tract, for examplein the development of papilloma virus-induced cervical cancer, remains to bestudied. An interaction of Pcdh1 withSmad3, which is involved in TGF betasignaling, has been demonstrated(Colland et al., 2004; Rual et al.,2005).

Besides its possible role in tumori-genesis, Pcdh1 is upregulated in re-sponse to skin irritation induced bysodium lauryl sulfate in the skin(Fletcher et al., 2001). In the lung,Pcdh1 was identified as a candidatesusceptibility gene for bronchial hy-perresponsiveness in the pathogene-sis of asthma (Whittaker, 2003; Hol-gate et al., 2007).

General Conclusion

The results of this study demonstratethat Pcdh1 is expressed not only dur-ing development but also in maturetissues and organs in vertebrates, ashas been reported for other membersof the cadherin superfamily (for a re-

2502 REDIES ET AL.

view, see Halbleib and Nelson, 2006).Consequently, Pcdh1 may play moreroles in tissue development and orga-nogenesis than previously assumed onthe basis of studies on Pcdh1 duringearly Xenopus development (Kurodaet al., 2002).

EXPERIMENTALPROCEDURES

Animals

Tissues for RNA extraction were dis-sected from mice with a mixed back-ground that were killed by cervicaldislocation. Timed pregnant NMRImice were killed between 10 days(E10) and 19 days (E19) after visual-ization of a vaginal plug by inhalationof chloroform. E10, E15, E17, and E19embryos as well as postnatal mice (P5,P10, and P12) and adult tissues wereflash frozen in 2-methyl-butanechilled to �40°C by adding dry ice. Allspecimens were stored at �80°C untilsectioning. Some E12 and E13 em-bryos were fixed in 4% formaldehydein HEPES-buffered salt solution (pH7.4; HBSS) on ice. For cryo-protection,fixed tissues were immersed in an as-cending sucrose solution (12, 15, and18% in HBSS) for 6 to 24 hr each,depending on the size of the embryo.Tissues were embedded in Tissue-TekO.C.T. Compounds (Sakura Finetek,Heppenheim, Germany) and frozen inliquid nitrogen. Sections of 20-�mthickness were cut in a refrigeratedmicrotome, mounted on coated glassslides, and dried.

RT-PCR

For RNA extraction, dissected tissueswere washed in phosphate-bufferedsaline (PBS) and frozen at �70°C. Tis-sues were crunched with liquid nitro-gen and RNA was prepared with theRNAeasy method (Qiagen, Hilden,Germany). cDNA was prepared us-ing a commercial kit (Superscript IIReverse Transcriptase, Invitrogen,Karlsruhe, Germany). For detectingthe long isoform, primers MCB3759(5-AAGGATCCAAGCCCTGGCAG-TACTAGTG-3) and MCB3760 (5–CGGGAATTCAGTCACAGGTAGAT-CTCACGCTTG-3) were used,yielding a product of 1,123 bp. Detec-tion of the short isoform was by com-

bination of primers MCB3759 withMCB3761 (5-AGAATTCGGTAAGA-CACACCTGCTCTATCA-3) result-ing in a 607-bp product. As an inter-nal control, a 452-bp fragment ofmouse GAPDH was amplified by useof primers MCB4078 (5-ACCA-CAGTCCATGCCATCAC-3) andMCB4079 (5-TCCACCACCCTGTT-GCTGTA-3).

CRNA PROBEPREPARATION

The mouse Pcdh1 probe plasmidpGEMte-mPcdh1-ISH comprises a1.6-kb PCR fragment encoding a largepart of the cytoplasmic domain plusthe transmembrane domain (positions1,195–2,781 of sequence GenBankAcc. No. NM_029357). This fragmentwas cloned by ligating an ApaI-SpeIrestriction fragment from the mouseEST clone BF236228 into a pGEM-Teasy backbone vector (Promega,Madison, WI). The sequence was ver-ified by DNA sequencing.

Using digoxygenin-labeled ribo-nucleotides and SP6 and T7 RNApolymerase, respectively, anti-senseand sense cRNA synthesis was per-formed with a riboprobe labeling kit(Promega), according to the manufac-turer’s specifications. Labeled probeswere precipitated with 3 M KAc (pH6.0) and ethanol and resuspended inRNAse-free water (Gibco, Paisley,Scotland). Digoxigenin incorporationwas tested by RNA gel electrophoresisfollowed by blotting and detectionwith anti-digoxygenin antibody.

In Situ Hybridization

In situ hybridization was performedon cryosections as described by Redieset al. (1993). In brief, sections werepostfixed with 4% formaldehyde dis-solved in phosphate-buffered saline.Following pretreatment with protein-ase K and acetic anhydride, sectionswere hybridized overnight with a senseor an anti-sense RNA probe at 70°C inhybridization solution (50% formamide,3 SSC, 1 Denhardt’s solution, 10mM EDTA, 42 �g/ml yeast transferRNA, and 42 �g/ml salmon spermDNA). The sections were washed andunbound cRNA was removed by RNAseA, followed by incubation with alkalinephosphatase-coupled anti-digoxigenin

Fab fragments (Roche, Mannheim, Ger-many) at 4°C overnight. For visualiza-tion of the labeled mRNA, sections wereincubated with a substrate solution of5-bromo-4-chloro-3-indoyl phosphate(BCIP) and nitroblue tetrazolium salt(NBT). The sections were viewed andphotographed under a microscope(Olympus BX40, Hamburg, Germany)equipped with a digital camera (Olym-pus DP70).

ACKNOWLEDGMENTSThe authors thank Elke Winterhagerfor comments on the placental expres-sion patterns, Monique Nuernbergerand Krishna-K. for cRNA probe prep-aration, and members of the labora-tory for a critical reading of the manu-script.

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