Mechanisms of Development 102 (2001) 259±262
Gene expression pattern
Metallocarboxypeptidase Z is dynamically expressed in mouse development Elena Novikova a, Lloyd D. Fricker a, Sandra E. Reznik b,* a
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forchheimer, Second Floor, Bronx, NY 10461-2373, USA Received 27 November 2000; received in revised form 23 January 2001; accepted 29 January 2001
Abstract Metallocarboxypeptidase Z (CPZ), a new member of the regulatory metallocarboxypeptidases, contains a 120-residue cysteine-rich region that has 20±35% amino acid sequence identity to Drosophila and mammalian frizzled proteins. In order to gain insights into the function of CPZ, we have examined the distribution of the protein by immunohistochemistry throughout mouse development. The expression of CPZ peaks at E9±E12, decreases in late gestation and falls further in adult tissues. CPZ expression in amnion cells, cochlear epithelial cells and surrounding mesenchyme, ventricular lining cells in the brain and cartilagenous condensations and surrounding connective tissue in ribs remains at high levels throughout mouse gestation. The expression pattern of CPZ overlaps with the expression pattern of several Wnt genes, consistent with the putative role of CPZ in Wnt signaling. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Carboxypeptidase Z; Frizzled; Wnt; Mouse embryo
Metallocarboxypeptidase Z (CPZ) was discovered during a search for enzymes that could compensate for the lack of carboxypeptidase E in Cpe fat/Cpe fat mice (Song and Fricker, 1997). CPZ most likely does not function in the intracellular processing of peptides, however, but probably has an extracellular function, based on its biochemical properties (Novikova and Fricker, 1999) and localization in the extracellular matrix (Novikova et al., 2000). In addition to the carboxypeptidase-homology domain, CPZ also contains a 120-residue cysteine-rich region that has 20±35% amino acid sequence identity to Drosophila and mammalian frizzled proteins (Xin et al., 1998). These proteins are receptors for members of the wingless/Wnt family, which are important signaling molecules in early development (Nusse and Varmus, 1992; Bhanot et al., 1996). Several extracellular proteins also contain this frizzled-like cysteine-rich region, including a form of collagen, proteins designated frzb and sizzled, and a family of frizzled-related proteins (Leyns et al., 1997; Salic et al., 1997; Mayr et al., 1997; Leimeister et al., 1998). These proteins are believed to bind to Wnt proteins extracellularly, thus inhibiting Wnts from interacting with frizzled receptor proteins and in¯uencing gene expression. In order to gain insights into the function of
* Corresponding author. Tel.: 11-718-430-2916; fax: 11-718-430-8954. E-mail address: [email protected]
Fig. 1. Expression of CPZ in early mouse embryos. CPZ immunoreactivity is present throughout the mouse decidualized endometrium (A,C). CPZ expression in the embryo itself increases between E5 and E7, with intense immunostaining present in the embryonic bilayer (E). Parallel sections reacted with preimmune serum show no immunostaining (B,D,F). (C,D) Higher magni®cations of the areas indicated in (A,B). DE, decidualized endometrium; Emb, embryo; PI, preimmune serum.
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00306-9
E. Novikova et al. / Mechanisms of Development 102 (2001) 259±262
CPZ, we have examined its expression pattern throughout mouse development. 1. Results Identical patterns of immunoreactivity were obtained using both the anti-C-terminal and the anti-N-terminalCPZ antiserum. Preimmune sera produced no signi®cant immunostaining. Both anti-CPZ antisera, which were raised against Schistosoma japonicum glutathione S-transferase (GST) fusion proteins, did not react with mammalian GST in Western blots, while they produced strong immunoreactive bands in lanes loaded with puri®ed CPZ (data not shown). Furthermore, immunostaining was blocked by preabsorbing the anti-CPZ antisera with the CPZ-GST fusion protein. On E5 both the decidualized endometrium and the embryo itself show strong immunoreactivity for CPZ (Fig.
1A). Parallel sections incubated with preimmune serum show no immunostaining (Fig. 1B) and the uterine cavity and embryo are shown at higher magni®cation in Fig. 1C,D. CPZ immunoreactivity intensi®es in the embryonic bilayer seen at E7 (Fig. 1E). No staining occurs when parallel sections of the embryonic bilayer are incubated with preimmune serum (Fig. 1F). CPZ is broadly expressed in the embryo at E9 and E12 (Fig. 2A,B), when immunostaining is most intense. Strong CPZ immunoreactivity in the amnion is noted at E9 (Fig. 2A). At E15 there is a decrease in CPZ expression in some tissues. In particular, the brain shows a dramatic fall in CPZ immunoreactivity with the exception of ventricular lining cells (Fig. 2C). Intense immunoreactivity persists at E15 in the lungs, liver, kidneys and intestine (Fig. 2C). CPZ immunoreactivity is decreased in most organs at E17 (Fig. 2D), but persists in cochlear epithelial cells and surrounding mesenchyme, ventricular lining cells in the brain and cartilagenous condensations and surrounding connective tissue
Fig. 2. Overview of CPZ expression in sagital sections of mouse embryos. Peak CPZ expression is present at E9 and E12 (A,B). CPZ immunostaining intensity decreases in some tissues at E15 (C) and falls further at E17 (D). Parallel sections reacted with preimmune serum (E,F,H) or with anti-CPZ antiserum preabsorbed with puri®ed CPZ-GST fusion protein (G) show no immunoreactivity. MB, midbrain; NT, neural tube; Am, amnion; FV, fourth ventricle; IE, inner ear; Ht, heart; Liv, liver; Sto, stomach; Lu, Lung; LV, lateral ventricle; Kid, kidney; NC, nasal cavity; PA, preabsorbed.
E. Novikova et al. / Mechanisms of Development 102 (2001) 259±262
seen in cross-sections of ribs. Negative control sections of embryos reacted with either preimmune serum or antiserum preabsorbed with CPZ GST fusion protein show no staining (Fig. 2E±H). Particularly intense immunostaining is present in the epithelial cells lining the ventricles in the brain at E12 (Fig. 3A). Parallel sections of brain reacted with preimmune serum show no staining (Fig. 3B). The dorsal root ganglia also show intense CPZ immunoreactivity at E12 (Fig. 3C),
Fig. 3. CPZ expression in speci®c mouse embryonic tissues. Intense CPZ immunoreactivity is present throughout gestation in the brain ventricular epithelial cells (cells lining the fourth ventricle at E12 shown in A). In other neural tissues, high CPZ expression persists in the dorsal root ganglia through the gestational time points studied (shown in C at E12). CPZ expression also persists throughout gestation in the cochlear lining cells of the inner ear and surrounding mesenchymal cells (E) and in cartilagenous condensations and surrounding mesenchyme seen in cross-sections of ribs (F). Placental tissues from E5 through E17 were tested for CPZ expression and shown to contain uniformly high CPZ immunoreactivity (shown at E17 in H). Parallel sections reacted with preimmune sera showed no immunoreactivity (B,D,G,I). DRG, dorsal root ganglia; Co, cochlea; FCV, fetal chorionic villi.
which is not seen in parallel sections reacted with preimmune serum (Fig. 3D). At E15 the cochlear epithelial cells and surrounding mesenchymal cells maintain CPZ immunoreactivity (Fig. 3E). Cartilagenous condensations and surrounding connective tissue seen in cross-sections of ribs show CPZ expression at E17 (Fig. 3F), which is not seen in parallel sections reacted with preimmune serum (Fig. 3G). Previous Northern blot analysis showed low levels of CPZ expression in a variety of rat adult tissues tested and high CPZ expression in rat placenta (Xin et al., 1998). Mouse placental tissues from E5 to E17 are found to have uniformly high CPZ immunoreactivity both in the amnion and in the fetal chorionic villi throughout gestation (Fig. 3H). Sections of mouse placenta reacted with preimmune serum show no staining (Fig. 3I). CPZ expression is found in adult mouse skin, but is restricted to surface epithelial cells and hair follicles (Fig. 4A). No staining is present in negative control sections of adult skin reacted with preimmune serum (Fig. 4B). In addition, adult mouse brain and liver show very low CPZ expression (liver shown in Fig. 4C; preimmune control in Fig. 4D), which is in contrast to the intense immunostaining seen in these tissues during embryogenesis. Adult mouse spleen shows CPZ expression enriched in the extracellular matrix (Fig. 4E), which is not
Fig. 4. Expression of CPZ in adult mouse tissues. Intense CPZ immunoreactivity is present in hair follicles in adult mouse skin (A). Minimal CPZ expression is seen in the liver (C). CPZ is enriched in the extracellular matrix in spleen (E). Parallel sections reacted with preimmune serum show no immunostaining (B,D,F). Fol, follicle.
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seen in parallel sections reacted with preimmune serum (Fig. 4F). The expression pattern of CPZ shown in these studies overlaps with the expression patterns of several Wnt genes. Many Wnts show maximum levels of expression during the ®rst half of mouse development (Jakobovits et al., 1986; Wilkinson et al., 1987; McMahon and McMahon, 1989; Roelink et al., 1990; Gavin et al., 1990). Wnt 5a in particular is expressed in decidualized endometrium at E6.5, in the midbrain at E9.5, and in cartilagenous condensations and surrounding mesenchyme at E11.5±E14.5 (Gavin et al., 1990). Wnt proteins have very restricted expression in adult tissues. Wnt 3, for example, is expressed in speci®c hair follicle cells adjacent to the hair shaft cortex and cuticle (Millar et al., 1999). The overlap in expression patterns of Wnt genes and CPZ is consistent with the putative role of CPZ in Wnt signaling. 2. Methods Gestational tissues from C57Bl/6 mice were ®xed in formaldehyde and embedded in paraf®n. Serial sections (4 mm) of both embryonic and adult tissues were mounted on glass slides. Representative sections were then deparaf®nized, rehydrated and washed. Sections were immersed in prewarmed (378C) 3% H2O2 to quench endogenous peroxidase activity and then microwaved in antigen retrieval solution (8.2 mM sodium citrate, 1.8 mM citric acid (pH 5.7), containing 0.01% Triton X-100). Sections were incubated with 1:2000 dilutions of two different primary polyclonal rabbit anti-CPZ antisera raised against bacterially expressed GST fusion proteins containing either the C-terminal 73 amino acids or residues 163±212 of rat CPZ (Novikova et al., 2000). After 1 h of incubation with primary antiserum, the sections were washed and incubated with biotinylated anti-rabbit Ig secondary antibody (Dako). Immunostaining was achieved using an avidin±biotin complex developer kit (Dako) and 3,3 0 -diaminobenzidine as substrate. Tissue sections from all gestational ages tested were processed in a single experiment. At least three embryonic mice per time point were studied. Acknowledgements This study was supported by National Institutes of Health
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