Development 120, 3213-3226 (1994) 3213 Printed in Great Britain © The Company of Biologists Limited 1994 Distribution of, and a putative role for, the cell-surface neutral metallo- endopeptidases during mammalian craniofacial development Bradley Spencer-Dene1,*, Peter Thorogood2, Sean Nair1, A. John Kenny3, Malcolm Harris1 and Brian Henderson1 1Maxillofacial Surgery Research Unit, Eastman Dental Institute and University College Hospital London, Eastman Dental Hospital, 256 Grays Inn Road, London, WC1X 8LD, UK 2Developmental Biology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK 3Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 8BS, UK *Author for correspondence SUMMARY Endopeptidase-24.11 (neutral endopeptidase, neprilysin, been detectable in the craniofacial vasculature at E12 and ‘enkephalinase’, EC 3.4.24.11) and endopeptidase-24.18 E14, this was no longer apparent at E16. Significantly, the (endopeptidase-2, meprin, EC 3.4.24.18) are cell-surface distribution of endopeptidase-24.11 mRNA closely zinc-dependent metallo-endopeptidases able to cleave a matched the immunolocalization of the protein at all stages variety of bioactive peptides including growth factors. We investigated. report the first study of the cellular and tissue distribution In order to explore the functional role of these enzymes, of both enzymes and of the mRNA for NEP during inhibition studies were carried out using two selective embryonic development in the rat. Endopeptidase-24.11 inhibitors of endopeptidase-24.11, phosphoramidon and protein was first detected at E10 in the lining of the gut and, thiorphan. E9.5 and E10.5 embryos exposed to either at E12, the enzyme was present on the notochord, medial inhibitor displayed a characteristic, asymmetric abnor- and lateral nasal processes, otocyst, mesonephros, heart mality consisting of a spherical swelling, possibly associated and neuroepithelium. In contrast, at this time endopepti- with a haematoma, predominantly on the left side of the dase-24.18 was present only on the apical surface of the prosencephalon, and the severity of this defect appeared to neuroepithelial cells. By E14 and E16, NEP was also be a dose-dependent phenomenon. This study suggests that detected in a wide range of craniofacial structures, notably these enzymes play previously unrecognized roles during the palatal mesenchyme, the choroid plexus, tongue and mammalian embryonic development. perichondrium. The distribution of endopeptidase-24.18 at these stages was restricted to the inner ear, the nasal Key words: membrane metallo-endopeptidases, neutral conchae, and ependymal layer of the brain ventricles and endopeptidase-24.11, endopeptidase-24.18, craniofacial the choroid plexus. Although endopeptidase-24.11 had morphogenesis, mammal INTRODUCTION while endopeptidase-24.18 hydrolyses bonds adjacent to aromatic residues, but the attack may be on either side of such Mammalian cells synthesize two main classes of metallo- residues (Stephenson and Kenny, 1987; Wolz et al., 1991). In endopeptidases, those that are secreted and participate in recent years, these enzymes have been shown to be capable of remodelling of the extracellular matrix, such as collagenase hydrolysing a variety of neuropeptides and peptide hormones and stromelysin (Henderson and Blake, 1994) and the cell- (Erdös and Skidgel, 1989; Price et al., 1991; Choudry and surface metallo-endopeptidases which play a role in the inac- Kenny, 1991), including growth factors and cytokines (Kenny tivation of biologically active peptides (Kenny et al., 1989; and Ingram, 1987; Katayama et al., 1991). Additionally, two Erdös and Skidgel, 1989). Two well-studied examples of the NEP substrates, the tachykinin, substance P, and the major latter are endopeptidase-24.11 and endopeptidase-24.18. Both bacterial chemotactic peptide formyl-Met-Leu-Phe provoke enzymes are transmembrane, zinc-containing metallo- rapid changes in the migration, morphology and adhesion endopeptidases found on the outer aspect of the plasma molecule expression of human neutrophils. These changes are membrane of a variety of cells. Both are abundant on the brush potentiated when endopeptidase-24.11 is inactivated by the borders of the epithelial cells lining the adult kidney proximal selective inhibitor, phosphoramidon (Shipp et al., 1991). tubule and intestine (Ronco et al., 1988; Barnes et al., 1989; Molecular cloning and expression studies have demon- Corbeil et al., 1992). Endopeptidase-24.11 cleaves peptide strated that endopeptidase-24.11 is identical to CD10 bonds involving the amino function of hydrophobic residues, (CALLA, common acute lymphoblastic leukaemia antigen) 3214 B. Spencer-Dene and others (Letarte et al., 1988; Chen et al., 1992). This finding, and the Detergent solubilization of a membrane preparation from observation that CD10 is expressed by foetal haematopoietic embryos cells (Hokland et al., 1983), has led to speculation that E14 rat embryos were removed from the uterine horns and homoge- endopeptidase-24.11 may play a role in the control of growth nized in 50 mM Tris/HCl buffer (pH 6.5) containing 0.1 mM phenyl- and differentiation in both haematopoietic and epithelial cell methylsulfonylfluoride (PMSF) at 4˚C. This homogenate was cen- systems (Kenny et al., 1989; LeBien and McCormack, 1989), trifuged for 5 minutes (1000 g) at 4˚C, and the supernatant was possibly regulating local concentrations of active peptides, retained and centrifuged at 100,000 g for 1 hour, using a Kontron such as growth factors, at the cell surface. ultracentrifuge, to collect a membrane fraction. Membranes were resuspended in 10 mM Tris-HCl (pH 7.5) containing 0.1 mM PMSF, It is apparent from the limited information available that the 0.1 mM pepstatin A, 0.1 mM 1,10 phenanthroline and 0.5% Triton expression of various peptide growth factors and their X-100 and left at 4˚C overnight to solubilize the membrane enzymes. receptors is under developmental regulation during craniofa- Insoluble material was removed by centrifugation at 100,000 g for 1 cial morphogenesis (reviewed by Slavkin, 1990; Lee and Han, hour and the supernatant retained and stored at −70˚C. 1991; Vainio et al., 1993). Recent evidence strongly suggests that some of these factors, such as transforming growth factors Microvillar membranes from rat kidneys α and β, are likely to be critically important in normal growth These were prepared as described previously (Booth and Kenny, and development of the facial primordia (Wilcox and Derynck, 1974). The final pellet was resuspended in the same Tris-HCl/protease 1988; Mahmood et al., 1992; Frenz et al., 1992). Thus, inhibitor buffer as used for the embryo membranes. although their precise and respective contributions remain to Western blotting be fully defined, it is clear that regulatory growth factors have Kidney microvilli and embryo membranes were separated by SDS- pivotal roles in craniofacial morphogenesis and, presumably, PAGE according to Laemmli (1970) and were transferred to nitro- dysmorphogenesis. cellulose membrane according to Towbin et al. (1979). Blots were We postulate that, given their known role in postembryonic incubated with either RAHE or RRt151 (1:1000) for 1 hour, rinsed tissues, endopeptidase-24.11, endopeptidase-24.18 and related with distilled water and Tris-buffered saline-Tween-20 (TBST), then enzymes may have a significant, but as yet unrecognised, mor- incubated with goat anti-rabbit peroxidase-conjugate (DAKO, UK; phogenetic role in the growth and development of embryonic diluted 1:500 with 5% skimmed milk in TBST+5% rat serum) for 1 craniofacial tissues. Here we report, for the first time, the suc- hour, rinsed and washed as above and developed with 4-chloro-1- cessful immunolocalization of both endopeptidase-24.11 and naphthol. endopeptidase-24.18, and the in situ hybridization of endopep- To establish the specificity of antibody binding both to the embryo tidase-24.11 mRNA in postimplantation rat embryos at various and kidney membrane preparations, the antibodies were preincubated with either purified endopeptidase-24.18 or purified kidney membrane critical stages of craniofacial development. We describe preparation prior to blotting. patterns of distribution of both message and gene product in the craniofacial tissues and consider possible roles of these Preparation of embryos for immunohistochemistry enzymes during craniofacial morphogenesis. Wistar rats (Charles Rivers, UK) were mated and the date of vaginal The increasing understanding of the role played by endopep- plug detection designated day 0 (E0). Rats were killed by cervical dis- tidase-24.11 in hydrolysing and inactivating enkephalins and location following CO2 anaesthesia at E10, 12, 14 or 16 of gestation. natriuretic peptides has led to many synthetic inhibitors of Embryos were dissected out in PBS and fixed in 4% paraformaldehyde endopeptidase-24.11 being clinically evaluated as analgesics and in PBS overnight at 4˚C. In the case of the E16 embryos, only the heads as therapeutic agents in cardiac failure (reviewed by Wilkins et were used. Embryos were transferred into cryoprotectant (20% (w/v) al., 1993). We have taken advantage of these inhibitors to test sucrose/PBS solution) for 5-10 hours at 4˚C and then each embryo was mounted and orientated in OCT (Miles Inc., USA) rapidly frozen and whether inhibition of endopeptidase-24.11 may have any stored at −70˚C. The kidneys from the mothers were removed and demonstrable effects on embryogenesis and report