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Keywords:

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

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES

Protocadherin-1 (Pcdh1) is a member of the δ-protocadherin subgroup of non-clustered protocadherins. We studied the expression of Pcdh1 from the early embryonic to the adult stage of mouse development by semi-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 and in 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, hair follicles, kidney, vagina, uterus, placenta, testis, prostate, and the seminal gland. The tight spatial and temporal regulation of Pcdh1 expression suggests that this protocadherin plays multiple roles not only during development but also in mature tissues and organs in the mouse. Developmental Dynamics 237:2496–2505, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES

Cadherins constitute a large family of cell adhesion receptors that can be classified into several subfamilies, including classic cadherins, protocadherins, desmosomal cadherins, and flamingo cadherins (for reviews, see Nollet et al.,2000; Frank and Kemler,2002). Almost all cadherins show a specific expression profile during the development of various tissues and organs. The protocadherin family (Sano et al.,1993) comprises multiple subfamilies, such as the α-, β-, and γ-protocadherins, and the large (fat- and dachsous-related) protocadherins (for reviews, see Frank and Kemler,2002; Hirano et al.,2003). Recently, a novel subgroup of protocadherins, termed δ-protocadherins, was identified by a phylogenetic comparison of mouse and human protocadherins (Vanhalst et al.,2005). On the basis of overall homology, number of extracellular cadherin repeats (seven versus six), and conservation of specific amino acid motifs in the cytoplasmic domains, two subgroups were identified. The δ1 subgroup comprises protocadherin-1, -7, -9, and -11(X/Y). The δ2 subgroup comprises protocadherin-8, -10, -17, -18, and -19 (for a review, see Redies et al.,2005).

The expression and function of only some δ-protocadherins have been investigated in detail during early Xenopus and zebrafish development (for a review, see Redies et al.,2005). Relatively little is known about the expression of δ-protocadherins in other vertebrate species and at later stages of development, with the exception of Pcdh19 in the developing mouse embryo (Gaitan and Bouchard,2006) and several δ-protocadherins that have been mapped in the developing and adult brain of mouse, rat, or chicken (Hirano et al.,1999; Yamagata et al.,1999; Müller et al.,2004; Vanhalst et al.,2005; Kim et al.,2007). Here we report on the expression of protocadherin-1 (Pcdh1) during mouse development from the early embryonic stage to the adult stage.

Pcdh1, one of the first protocadherins to be discovered (then called protocadherin-42), mediates cell adhesion upon ectopic expression in L cells (Sano et al.,1993). The ortholog of Pcdh1 in Xenopus, termed axial protocadherin, plays a role in prenotochordal cell sorting in the gastrulating embryo (Kuroda et al.,2002). At the tailbud stage of Xenopus, axial protocadherin was shown to be expressed in the somites, pronephros, heart, otic vesicle, and brain (Kuroda et al.,2002). The results of the present study, which is the first to describe mouse Pcdh1 expression at the histological level in detail, suggest that Pcdh1 expression is tightly regulated in several other developing organs and tissues, shedding light on the possible role of Pcdh1 in tumorigenesis or other pathogenetic processes in some of these tissues.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES

Overview of Expression

Results from semi-quantitative RT-PCR demonstrated that weak expression of both short and long isoforms of mouse Pcdh1 can be detected as early as embryonic day (E) 9.5 (Fig. 1). Expression in whole embryos is strong on E11.5 and remains strong until at least E16.5. In the adult, several organs, such as the liver, brain, kidney, heart, lung, and uterus, express Pcdh1 at different levels.

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

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By in situ hybridization, which detected both short and long isoforms, Pcdh1 expression was demonstrated not only in mammalian brain (Sano et al.,1993; Kim et al.,2007; Hertel et al.,2008) and lung (Favre et al.,2003), but also in the placenta and in several other tissues and organs derived from all three germinal layers (Figs. 2, 3). Hybridization with a sense probe served as a negative control.

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Figure 2. Pcdh1 expression by the endothelial lining of the heart and blood vessels (bv) of the mandibular 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) or a sense (control) mRNA probe (M',V') for Pcdh1. B, C', D', F, L, U, and W' show sections that are adjacent to A, C, D, E, K, T, and W, respectively, and were stained with antibodies against PECAM-1 (PECAM). N' and P show sections that are adjacent to N and O, respectively, and were stained with azan dye (Azan). at, atrium of heart; ba1, first branchial arch; br, bronchi; bv, blood vessel; 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, endothelial lining; 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, proximal phalanx; 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 of heart; vi, intestinal villi; yo, yolk sac. Scale bars = 200 μm in C (for C,C') and N (for N,N'); 100 μm in A (for A,B), J, R, S, T (for T,U), V (for V,V'), and W (for W,W'); 50 μm in D (for D,D'), G, H, I, K (for K,L), M (for M,M'), O (for O,P) and Q; and 20 μm in E (for E,F).

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Figure 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 and peripheral ganglia (L), blood vessels entering the hindbrain (M), forebrain (N), retina (O), olfactory bulb (P), vomeronasal organ (Q), cochlea (R), hair follicles (S,T), thymus (U,V), epithelial lining of the nasal cavity (W,Y), and nasal glands (X), 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 mRNA probe for Pcdh1 (A–C,F–U,W–Y). D shows a section that is adjacent to C and was stained with antibodies against PECAM-1 (PECAM). E and V show sections that are adjacent to C and U, respectively, and were stained with hematoxylin/eosin dye (HE). The arrows 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 show magnifications of the areas boxed in W. V, layer V of cortex; amy, amygdala; ao, anterior olfactory nucleus; bh, hair bulb; br, terminal bronchus; bv, blood vessel; cd, cochlear duct; cp, caudoputamen; 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 root sheath; pir, piriform cortex; po, posterior thalamic nuclear complex; r, retina; rm, Reissner's membrane; sc, supporting cells; sg, spinal ganglion; sn, substantia nigra; sv, stria vascularis; sy, sympathetic ganglion; ve, vaginal epithelium; vlg, ventral lateral geniculate nucleus; vno, vomeronasal (Jacobson's) organ. Scale bars = 1 mm in N and W; 500 μm in P; 200 μm in L, Q, and T; 100 μm in H, L (insert), R, and X (for X,Y); and 50 μm in A, B, C (for C–E), F (for F,G), I, J (for J,K), M, O, S, and U (for U,V).

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Most striking is the expression of Pcdh1 by blood vessels during early embryogenesis of most organs (Fig. 2A–F,K,L,T,U,W,W'). Prenatal expression of Pcdh1 is seen in many of the epithelia derived from the embryonic gut, such as the epithelial lining of the esophagus, the small and large intestines, and organs such as the liver, the lung, and the submandibular gland (Figs. 2Q–W', 3A–E). In addition, Pcdh1 is expressed along the entire neuraxis, including the brain, spinal cord, sensory epithelia, and most ganglia of the peripheral nervous system (Fig. 3L–P). These results will be described in detail in the following sections. Tissues and organs that do not express Pcdh1 or do so only weakly (except in their blood vessels) include the epidermis, bone, cartilage, muscle, pancreas, and urinary bladder.

Cardiovascular System

In situ hybridization showed that blood vessels (bv) throughout the embryo express Pcdh1 as early as E10 (compare staining for PECAM-1, a marker for endothelial cells, in Fig. 2B with 2A). At E10, the endothelial lining of the heart expresses Pcdh1 (arrows in Fig. 2A,B). After E10, the endothelial lining of the heart and of major blood vessels, such as the aorta, do not show a Pcdh1 signal. In contrast, small blood vessels retain prominent mRNA expression during embryogenesis, as demonstrated, for example, for blood vessels in a digit of the forelimb (Fig. 2C,C′). Nevertheless, not all embryonic endothelia express Pcdh1 throughout early development. In the heart, the blood vessels of the compact (outer) part of the ventricular myocardium [com(o)] express Pcdh1 at E15, but the endothelial lining of the trabeculated (inner) part [tr(i)] does not (Fig. 2D,D′). Small blood vessels in both parts of the ventricle, however, show Pcdh1 expression two days later (at E17; data not shown). The endothelial lining of the heart remains negative. Postnatally, Pcdh1 expression by blood vessels decreases to low levels (Fig. 2E,F).

Chorioallantoic Placenta and Yolk Sac

In the E10 maternal compartment, Pcdh1 is expressed strongly by the endothelial lining (end) of the sinusoids (ds) in the decidua (Fig. 2G). The Pcdh1 signal is also found in some trophoblast cells within the spongiotrophoblast (sp) and labyrinthine layer (lb) of the chorioallantoic placenta (Fig. 2H) and in the yolk sac (data not shown). At E12, the yolk epithelium (yo) is strongly Pcdh1-positive whereas the Reichert's membrane (R) exhibits a weak to moderate signal (Fig. 2I). At E13, Pcdh1 expression becomes restricted to the glycogen cells (gly) within the spongiotrophoblast layer (sp in Fig. 2J) and the labyrinth is negative. The expression of Pcdh1 in the placenta reaches low levels at E17.

Kidney and Urinary Bladder

During kidney development, expression of Pcdh1 in vascular structures predominates during the early embryonic stages. At E13, some Pcdh1-positive cellular aggregates are seen in the metanephros (data not shown). Epithelial structures are negative. At E15, a hybridization signal is seen in the developing blood vessels of the renal cortex. In the glomeruli (gl in Fig. 2K,L), the signal has a distribution similar to PECAM-1. From E17 to P12, the glomeruli are strongly positive and the epithelial components of the renal cortex are moderately positive (Fig. 2M; compare to the sense control shown in Fig. 2M′). The tubular system in the medulla is moderately positive at P5 and P12. Blood vessels show no signal in the medulla during the postnatal and adult stages, but prominent staining persists in the glomeruli. In the cortex of the adult (cx in Fig. 2N,O), the glomeruli and the proximal tubules (pt) that are characterized by bluish Azan staining of their ciliated borders (Fig. 2N′,P) express Pcdh1, whereas the rest of the cortex and the medulla (med) are negative (Fig. 2N). The urothelium of the urinary bladder is negative at all stages.

Gastrointestinal System and Other Derivatives of the Embryonic Gut

In the embryonic gut, Pcdh1 expression 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 lining of the midgut intestinal mucosa (mu) and by scattered cells in the submucosa (smu in Fig. 2Q,R,T). The epithelial lining of the esophagus (oe) is Pcdh1-positive but that of the stomach (st) does not express Pcdh1 at E15 (Fig. 2S) and E17. From P5 until the adult stage, there is a prominent expression gradient in the epithelial lining of the mucosal lamina in the intestines; the crypts (cr) express Pcdh1 more strongly than the villi (vi in Fig. 2V; compare to the sense control shown in Fig. 2V′).

Pcdh1 is expressed in the embryonic lung by the parenchyma (p), where PECAM-1-positive blood vessels (bv) predominate, but not by the bronchi (br in Fig. 2W,W′). This expression profile persists until the adult stage (Fig. 3A). The parenchyma of the liver (li) also expresses Pcdh1 from E12 to the adult stage (Figs. 2W, 3B). The pattern of Pcdh1 expression in the liver roughly resembles that of PECAM-1 at E15 (Fig. 2W,W′).

In the submandibular gland, Pcdh1 expression is rather ubiquitous in epithelial cells and blood vessels at E17 (Fig. 3C–E).

Reproductive System

The reproductive organs were studied at the postnatal and adult stages. In female P5 pups, the endometrium (en in Fig. 3F) and the vaginal epithelium (ve) express Pcdh1 (Fig. 3G). In adult females, the tubular glands of the myometrium (arrows in Fig. 3H) are Pcdh1-positive, but the endometrial lining of the uterine cavity is negative; the ovaries do not express Pcdh1.

In postnatal males (P5, P10, and P12), the epithelia of the prostate gland (Fig. 3I) and of the seminal vesicles (Fig. 3J) express Pcdh1. The seminiferous tubules of the testis are also positive (Fig. 3K). In the adult, the signal in the male reproductive organs was not above the sense control.

Nervous System

In the spinal cord, the neuroepithelium of the basal plate expresses Pcdh1 at E10 (data not shown). At E13, the entire marginal layer of the basal and alar plates shows a prominent signal. At E15, expression is especially strong in the superficial layers of the dorsal column (dc in Fig. 3L). The motor column (mc) of the basal plate is also Pcdh1-positive at some spinal cord levels (insert in Fig. 3L). This general staining pattern persists in the spinal cord at least until P12.

As in other organs, Pcdh1 is expressed by blood vessels in and around the brain at E13 (Fig. 3M). The first regionalized staining in the neuroepithelium of the brain is observed at E12. The expression of Pcdh1 in gray matter structures of developing and adult brain has been described previously (Sano et al.,1993; Kuroda et al.,2002; Kim et al.,2007; Hertel et al.,2008). The present findings confirm the results of Kim et al. (2007), who showed that Pcdh1 is expressed in a subset of gray matter structures of the postnatal forebrain. As an example, in Figure 3N, which shows a transverse section through the P5 forebrain, expression is especially 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 example, layer V of the somatosensory cortex (V) is more strongly stained than the other layers. Other Pcdh1-positive forebrain structures include nuclei of the amygdalar complex (amy), the dorsal and ventral lateral geniculate nuclei in the thalamus (dlg, vlg), and the substantia nigra (sn). For a more complete mapping of Pcdh1 expression in mouse brain development, see the study done by Kim et al. (2007).

Pcdh1 is also expressed in the prospective ganglion cell layer (gcl) and inner nuclear layer (inl) of the retina during embryonic development (from about E15; E19 in Fig. 3W) and at postnatal stages (Fig. 3O). The glomerular and granular layers of the olfactory bulb (gl, gr in Fig. 3P), the accessory olfactory bulb (data not shown), and the anterior olfactory nucleus (ao in Fig. 3P) also express Pcdh1.

In the peripheral nervous system, most neurons in the spinal ganglia (sg), cranial sensory ganglia, and visceral ganglia (sy) express Pcdh1 (Fig. 3L) from the early stages of embryogenesis (E10-E12) to postnatal stages. Schwann cells in the peripheral nerves are negative. The vomeronasal organ (vno in Fig. 3Q) is also positive for Pcdh1, at least from E15 to P12. In the developing P5 cochlea, Pcdh1 mRNA is detected in the stria vascularis (sv) of the cochlear duct and in superficial cells of the organ of Corti, possibly including prospective hair cells and Hensen cells (arrowheads in Fig. 3R); expression in other structures of the inner ear is weak or absent.

Other Organs

The hair follicles contain Pcdh1-positive cells from the beginning of their formation until at least P12 (Fig. 3S,T). Except for the dermal papilla (dp), which expresses Pcdh1 weakly, all layers of the hair bulb (bh) express Pcdh1, including those giving rise to the medulla and cortex of the hair shaft. The inner root sheath (irs) shows a strong Pcdh1 signal, but the outer root sheet (ors) is negative. In the embryonic thymus, the endothelial lining of the sinuses expresses Pcdh1 at E15 (Fig. 3U,V). Thymic blood vessels are strongly positive at E17 but the signal decreases to low levels at P5.

The staining pattern in the nasal cavity (nc) is especially striking (Fig. 3W) from E15 to at least P12, with regional variations in the Pcdh1 expression level. The serous glands (ng) in the lateral wall of the middle meatus express moderate levels of Pcdh1 in the glandular epithelium (Fig. 3X). In the epithelial lining (el) of the nasal cavity, the most superficial layer is Pcdh1-negative whereas the deeper layer shows a very strong signal (Fig. 3Y).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES

The results demonstrate that Pcdh1 is expressed under tight spatial and temporal control in a subset of tissues and organs during embryogenesis and in the adult mouse. Kuroda et al. (2002) showed that the ortholog of Pcdh1 in Xenopus, axial protocadherin, is expressed during early development by the somites, pronephros, heart, otic vesicle, and brain. Our results extend these earlier findings in several respects. First, we demonstrate that, in the mouse, other organs also express Pcdh1. Second, we also describe expression at later stages of development and in the adult. Third, by performing a histological analysis in addition to semi-quantitative RT-PCR experiments, we identify the types of tissue that express Pcdh1 in the various organs. For example, in the heart, Pcdh1 is expressed by blood vessels but not by cardiomyocytes; skeletal muscle cells are also negative.

Our in situ hybridization approach cannot discriminate between transcripts encoding either short or long isoforms of Pcdh1. It is a recurrent finding that δ-protocadherins, including Pcdh1, are expressed as a mixture of short and long isoforms (Vanhalst et al.,2005; Redies et al.,2005). The difference between the isoforms is based on alternative splicing and results in a shorter versus extended length of the cytoplasmic domain. The functional implications of differential expression may be significant but are presently unclear. Indeed, the extended cytoplasmic domain comprises several conserved motifs (CM1, CM2, and CM3 in the case of Pcdh1). Protein phosphatase-1α is known to bind to the CM3 motif (Vanhalst et al.,2005), but molecular binding partners of the CM1 and CM2 motifs remain to be identified.

Endothelial Cells Express Pcdh1

In general, Pcdh1 is expressed prominently by developing blood vessels during angiogenesis (e.g., in brain vessels) and vasculogenesis (e.g., in visceral and somitic vessels) (for a review, see Risau,1995). However, not all endothelia are Pcdh1-positive. For example, in the heart at E15, no expression is observed in the endothelial lining of the inner trabeculated part of the myocardium, while blood vessels in the outer compact part show a hybridization signal. Interestingly, the two parts of the myocardium represent different stages of cardiomyocyte differentiation (Sedmera et al.,2000).

Other cadherins expressed by blood vessels are cadherin-5 (VE-cadherin; Lampugnani et al.,1992), cadherin-2 (N-cadherin; Salomon et al.,1992; Redies et al.,1993), cadherin-13 (T-cadherin), cadherin-4 (R-cadherin), and protocadherin-12 (VE-cadherin-2; for a review, see Cavallaro et al.,2006). In the mouse and human brains, blood vessels that contribute to the blood-brain barrier express cadherin-10 (Williams et al.,2005). It has been suggested that all these cadherins play different roles in the formation of blood vessels (for a review, see Cavallaro et al.,2006). Cadherin-5 mediates the intercellular adhesion between endothelial cells. Cadherin-5-deficient mice can still form primitive vascular plexus and tubular endothelial structures, but vascular remodeling and endothelial adhesion is impaired (Carmeliet et al.,1999). Pcdh1 is a candidate mediator of this residual adhesive function of endothelial cells in the absence of cadherin-5. N-cadherin is involved in linking the endothelial cells to the surrounding tissues (Navarro et al.,1998; Gerhardt et al.,2000). Protocadherin-12 has been related to vasculogenesis rather than angiogenesis (Rampon et al.,2005).

Like VE-cadherin and protocadherin-12, Pcdh1 is not expressed by endothelial cells in the adult in most tissues. However, Favre et al. (2003) isolated Pcdh1-positive endothelial cells from adult lung, which is in line with our visualization of Pcdh1 expression in sections through the adult lung (Fig. 3A).

Pcdh1 Expression in Other Organs and Tissues

Kidney

In the kidney, Pcdh1 is expressed in the glomeruli throughout development and in the proximal tubules of the adult mouse. Apart from Pcdh1, several other members of the cadherin superfamily are expressed differentially in the developing and adult kidney (for a review, see Dressler,2002), including some classic cadherins, such as 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 δ-protocadherins Pcdh7 (BH-protocadherin; Rudnicki et al.,2007) and Pcdh19 (Gaitan and Bouchard,2006). Like these other (proto-)cadherins, the expression of Pcdh1 is restricted to particular renal tissues. For example, Pcdh1 is expressed in proximal but not in distal tubules of the adult kidney (Fig. 2O,P). The similarity between the expression patterns of Pcdh1 and PECAM-1 in the glomeruli indicates that Pcdh1 is expressed by glomerular endothelial cells.

Placenta and reproductive organs.

Pcdh1 is expressed by specific tissue elements of the placenta and yolk sac. The expression of Pcdh1 by the endothelial cell lining of the decidual sinusoids and by the glycogen-rich cells is similar to that described for protocadherin-12 (VE-cadherin-2; Rampon et al.,2005; Bouillot et al.,2006). Classic cadherins expressed differentially in placental tissues include cadherin-3 (P-cadherin; Nose et al.,1987), cadherin-6, cadherin-11 (for a review, see MacCalman et al.,1998), VE-cadherin (Bulla et al.,2005), and cadherin-1 (E-cadherin). Several of the epithelia of the reproductive organs express Pcdh1. It is unknown whether Pcdh1 expression underlies hormonal regulation, as has been shown for some of the classic cadherins expressed in the uterus (for a review, see Horne et al.,2002).

In the testis, Pcdh1 signal was found in the seminiferous tubules. Cadherin-mediated cell–cell adhesion mediates the close contact between differentiating germ cells, the seminiferous epithelium, and the Sertoli cells (for a review, see Goossens and van Roy,2005). Many cadherins and protocadherins are expressed in testis, amongst them several classic cadherins (Munro and Blaschuk,1996) and clustered protocadherins (Johnson et al.,2000). The present study is the first to identify a δ-protocadherin in the testis.

Nervous system.

In the developing brain, Pcdh1 is expressed in a subset of neuroanatomical structures in all major brain regions. Our results are in agreement with findings in the rat brain (Kim et al.,2007) and in the mouse basal ganglia (Hertel et al.,2008). Extending these results, we demonstrate that Pcdh1 is expressed also in restricted regions and layers of the spinal cord and retina, and in several sensory and visceral ganglia of the peripheral nervous system (Fig. 3L,O). The expression pattern of Pcdh1 in these structures is distinct from that of many other classic cadherins and δ-protocadherins, which are also expressed in highly regionalized patterns in the vertebrate brain (for reviews, see Redies,2000; Hirano et al.,2003; Redies et al.,2005). Thus, it is likely that Pcdh1 contributes to the cadherin-based adhesive code that regulates diverse morphoregulatory processes during brain development (for reviews, see Redies,2000; Takeichi,2007).

Pcdh1 in Pathogenetic Processes

The expression of Pcdh1 is altered during tumorigenesis in several types of tissues and organs (Castilla et al.,2004; Neben et al.,2004; Rush et al.,2005; Nonnenmacher et al.,2006) that were found to express Pcdh1 in the present study. For example, Pcdh1 is down-regulated in a carcinogenesis model of renal tumors in rat (Stemmer et al.,2007). Another δ-protocadherin, Pcdh10, was found to be methylated in carcinomas and hematologic malignancies (Ying et al.,2006,2007). In hair follicles, Pcdh1 can act as a receptor for cottontail rabbit papillomavirus (Nonnenmacher et al.,2006). This pathogen targets keratinocytes and can cause lesions that have a high risk of progression to carcinoma (Schmitt et al.,1996; Nonnenmacher et al.,2006). Whether Pcdh1 plays a similar role in the epithelial components of the female genital tract, for example in the development of papilloma virus-induced cervical cancer, remains to be studied. An interaction of Pcdh1 with Smad3, which is involved in TGF beta signaling, has been demonstrated (Colland et al.,2004; Rual et al.,2005).

Besides its possible role in tumorigenesis, Pcdh1 is upregulated in response to skin irritation induced by sodium lauryl sulfate in the skin (Fletcher et al.,2001). In the lung, Pcdh1 was identified as a candidate susceptibility gene for bronchial hyperresponsiveness in the pathogenesis of asthma (Whittaker,2003; Holgate et al.,2007).

General Conclusion

The results of this study demonstrate that Pcdh1 is expressed not only during development but also in mature tissues and organs in vertebrates, as has been reported for other members of the cadherin superfamily (for a review, see Halbleib and Nelson,2006). Consequently, Pcdh1 may play more roles in tissue development and organogenesis than previously assumed on the basis of studies on Pcdh1 during early Xenopus development (Kuroda et al.,2002).

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES

Animals

Tissues for RNA extraction were dissected from mice with a mixed background that were killed by cervical dislocation. Timed pregnant NMRI mice were killed between 10 days (E10) and 19 days (E19) after visualization of a vaginal plug by inhalation of chloroform. E10, E15, E17, and E19 embryos as well as postnatal mice (P5, P10, and P12) and adult tissues were flash frozen in 2-methyl-butane chilled to −40°C by adding dry ice. All specimens were stored at −80°C until sectioning. Some E12 and E13 embryos were fixed in 4% formaldehyde in HEPES-buffered salt solution (pH 7.4; HBSS) on ice. For cryo-protection, fixed tissues were immersed in an ascending sucrose solution (12, 15, and 18% in HBSS) for 6 to 24 hr each, depending on the size of the embryo. Tissues were embedded in Tissue-Tek O.C.T. Compounds (Sakura Finetek, Heppenheim, Germany) and frozen in liquid nitrogen. Sections of 20-μm thickness were cut in a refrigerated microtome, mounted on coated glass slides, and dried.

RT-PCR

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES

For RNA extraction, dissected tissues were washed in phosphate-buffered saline (PBS) and frozen at −70°C. Tissues were crunched with liquid nitrogen and RNA was prepared with the RNAeasy method (Qiagen, Hilden, Germany). cDNA was prepared using a commercial kit (Superscript II Reverse Transcriptase, Invitrogen, Karlsruhe, Germany). For detecting the long isoform, primers MCB3759 (5′-AAGGATCCAAGCCCTGGCAGTACTAGTG-3′) and MCB3760 (5′–CGGGAATTCAGTCACAGGTAGATCTCACGCTTG-3′) were used, yielding a product of 1,123 bp. Detection of the short isoform was by combination of primers MCB3759 with MCB3761 (5′-AGAATTCGGTAAGACACACCTGCTCTATCA-3′) resulting in a 607-bp product. As an internal control, a 452-bp fragment of mouse GAPDH was amplified by use of primers MCB4078 (5′-ACCACAGTCCATGCCATCAC-3′) and MCB4079 (5′-TCCACCACCCTGTTGCTGTA-3′).

cRNA Probe Preparation

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES

The mouse Pcdh1 probe plasmid pGEMte-mPcdh1-ISH comprises a 1.6-kb PCR fragment encoding a large part of the cytoplasmic domain plus the transmembrane domain (positions 1,195–2,781 of sequence GenBank Acc. No. NM_029357). This fragment was cloned by ligating an ApaI-SpeI restriction fragment from the mouse EST clone BF236228 into a pGEM-Teasy backbone vector (Promega, Madison, WI). The sequence was verified by DNA sequencing.

Using digoxygenin-labeled ribonucleotides and SP6 and T7 RNA polymerase, respectively, anti-sense and sense cRNA synthesis was performed with a riboprobe labeling kit (Promega), according to the manufacturer's specifications. Labeled probes were precipitated with 3 M KAc (pH 6.0) and ethanol and resuspended in RNAse-free water (Gibco, Paisley, Scotland). Digoxigenin incorporation was tested by RNA gel electrophoresis followed by blotting and detection with anti-digoxygenin antibody.

In Situ Hybridization

In situ hybridization was performed on cryosections as described by Redies et al. (1993). In brief, sections were postfixed with 4% formaldehyde dissolved in phosphate-buffered saline. Following pretreatment with proteinase K and acetic anhydride, sections were hybridized overnight with a sense or an anti-sense RNA probe at 70°C in hybridization solution (50% formamide, 3× SSC, 1× Denhardt's solution, 10 mM EDTA, 42 μg/ml yeast transfer RNA, and 42 μg/ml salmon sperm DNA). The sections were washed and unbound cRNA was removed by RNAse A, followed by incubation with alkaline phosphatase-coupled anti-digoxigenin Fab fragments (Roche, Mannheim, Germany) at 4°C overnight. For visualization of the labeled mRNA, sections were incubated with a substrate solution of 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT). The sections were viewed and photographed under a microscope (Olympus BX40, Hamburg, Germany) equipped with a digital camera (Olympus DP70).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES

The authors thank Elke Winterhager for comments on the placental expression patterns, Monique Nuernberger and Krishna-K. for cRNA probe preparation, and members of the laboratory for a critical reading of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. RT-PCR
  8. cRNA Probe Preparation
  9. Acknowledgements
  10. REFERENCES