Endopeptidases During Mammalian Craniofacial Development

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 4ûC. This homogenate was cen- systems (Kenny et al., 1989; LeBien and McCormack, 1989), trifuged for 5 minutes (1000 g) at 4ûC, 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 4ûC 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 −70ûC. 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 4ûC. 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 4ûC 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 −70ûC. The kidneys from the mothers were removed and demonstrable effects on embryogenesis and report the results of processed as above, as a positive control tissue. Cryostat sections (8 perturbation experiments using a whole embryo culture µm) cut from embryos and kidneys were thaw-mounted onto glass technique (Cockcroft, 1990) with two selective endopeptidase- slides precoated with 3-aminopropyltriethoxysilane (Sigma, UK) and 24.11 inhibitors, phosphoramidon and thiorphan. stored at −70ûC. For all stages used, representative sections from indi- Given that endopeptidase-24.11 and endopeptidase-24.18 vidual embryos from four separate litters were independently examined. have the ability to hydrolyse many simple biologically active peptides (and possibly some cytokines), our results suggest Immunohistochemistry possible roles for these enzymes in the control of cell growth, (i) Endopeptidase-24.11 differentiation and movement. This, we believe could consti- The presence of endopeptidase-24.11 on frozen sections was localized tute a previously unrecognised level of developmental control by an immunoperoxidase staining procedure using RAHE and PHM- during craniofacial morphogenesis. 6. Sections were incubated in 1% hydrogen peroxide in methanol to quench endogenous peroxidase activity. Non-specific protein binding was blocked by incubating sections with 20% non-immune goat serum for 15 minutes. The sections were subsequently incubated for MATERIALS AND METHODS 1 hour with 1¡ antibody (RAHE diluted 1/1000, or PHM-6 at 1/250 in PBS + 0.1% BSA), and then with a goat anti-rabbit (goat anti- Antibodies mouse for PHM-6) peroxidase conjugate for 30 minutes. This was RAHE, a polyclonal rabbit anti-human enkephalinase, (Genentech, diluted to 1/300 in PBS plus 2% normal rat serum and preabsorbed USA); PHM-6, a monoclonal mouse anti-human CALLA, (Monash with ‘rat powder’ (Barnes et al., 1989). Sections were finally Medical Centre, Australia); RRt151, a polyclonal rabbit anti-rat incubated in 2.5 mg/ml of diaminobenzidine (DAB) in PBS contain- endopeptidase-24.18 produced by one of the authors (A. J. K.). Both ing 0.01% hydrogen peroxide. This step was amplified with nickel RAHE and PHM-6 were found to cross-react with rat NEP. chloride when necessary. All reagents used were from Sigma, UK.

Peptidases in craniofacial development 3215

All steps were separated by 2× 10 minute washes of PBS (or 0.1 M homology with the active site of endopeptidase-24.11, which is itself acetate buffer for the nickel enhancement) and all incubations took unique amongst mammalian metalloproteases (Jiang and Bond, place at room temperature in a humidity chamber. Sections were 1992), and therefore phosphoramidon efficiently inihibits endopepti- counterstained with Mayer’s Haematoxylin or Neutral Red (both from dase-24.11 (KI=2 nM). Phosphoramidon was dissolved in distilled BDH, UK), dehydrated, cleared and mounted in DPX (BDH, UK). water at 1 mg/ml and diluted to final inhibitor concentrations of 100 To control for non-specific binding, sections were routinely µM, 10 µM, 1 µM, 100 nM and 10 nM in diluted rat serum. Control incubated in the absence of 1û or 2û antibodies. Controls using RAHE, embryos were cultured in diluted rat serum only. which had been incubated overnight at 4ûC with 0.7 mg/ml of the Thiorphan (3-mercapto-2-benzylpropanoylglycine, Sigma, UK) kidney microvillar membrane preparation to preabsorb out endopep- was the first, potent synthetic inhibitor of endopeptidase-24.11 tidase-24.11, were also included. (KI=2.5 nM) (Roques et al., 1980). This was dissolved in 3% ethanol in water in the same concentration range as phosphoramidon. Control (ii) Endopeptidase-24.18 embryos were cultured in diluted serum ± 3% ethanol. Endopeptidase-24.18 was localized on rat tissue using the same indirect immunoperoxidase technique. Sections were incubated for 1 Histology hour with RRt151 diluted 1/100 in 0.1% BSA/PBS. Embryos were dissected from their yolk sac and amnion and fixed in In control experiments, RRt151 or the peroxidase conjugate were formalin for 24 hours, dehydrated through an ascending ethanol series omitted and replaced with PBS. Further controls to validate specific and embedded in filtered histology-grade wax. Sections were cut at 5 endopeptidase-24.18-positive staining involved replacing RRt151 µm and mounted onto APES-coated slides. Sections were then taken with preimmune rabbit serum, or with RRt151 that had been incubated through a routine Haematoxylin and Eosin staining procedure, and overnight at 4˚C with the purified enzyme (Kenny and Ingram, 1987) photographed using TMAX 100 film. or 0.7 mg/ml of the kidney microvillar membrane preparation to preabsorb out its immunoreactivity. Scanning electron microscopy All embryo sections were photographed under bright field on an Prior to fixation, extraembryonic membranes were removed and the Olympus BH2 photomicroscope using Kodak Ektachrome 64 T film. embryos washed in sterile Hanks’ saline. Embryos were fixed in 25% In situ hybridization glutaraldehyde, 0.2 M cacodylate buffer, pH 7.2 for 48 hours, rinsed This was carried out according to the protocol described by Wilkinson in cacodylate buffer then dehydrated. Embryos were dried in a and Green (1990). The antisense probe, labelled with [35S]UTP, was critical-point drier (Balzer, UK), and then sputter coated (Polaron, transcribed from complementary DNA which had been subcloned into UK) with a 60:40 gold-palladium alloy. Specimens were viewed on Bluescript (Strategene, UK). This cDNA corresponded to the full- a Cambridge 90 Stereoscan scanning electron microscope (Cambridge length sequence of rat endopeptidase-24.11 (Malfroy et al., 1987). A Instruments, UK). sense probe was also transcribed and used as a negative control. Each section received 10 µl of the appropriate probe, equivalent to 105 cts/minute/µl, overnight at 55ûC. Slides were dipped in Ilford K5 RESULTS emulsion (Ilford, UK), developed after 4 days, counterstained with haematoxylin, dehydrated and mounted. Western blots Whole embryo culture Protein bands with molecular weights corresponding to the pre- Pregnant Wistar rats (Charles Rivers, UK) were killed by cervical dis- viously shown values for both endopeptidase-24.11 and location whilst under CO2 anaesthesia. The E9.5 and E10.5 concep- endopeptidase-24.18, a single 94 kDa band and two bands at tuses were dissected free from maternal decidua and Reichert’s 80 kDa and 74 kDa respectively, were detected in both the rat membrane leaving the yolk sac, amnion and ectoplacental cone intact. kidney and E14 embryo membrane preparations (Fig. 1). They were then transferred into sterile 30 ml universals (Sterilin, UK), containing 1 ml/embryo of immediately centrifuged rat serum (prepared according to Cockcroft (1990) and supplied by Harlan Olac, UK.) diluted 3:1 with sterile Hank’s saline. 11 18 The embryos were cultured at 37ûC in a temperature-controlled rotator apparatus (Cockcroft, 1990). Embryos at E9.5 when culture commenced, received an initial gas mixture comprising 5% oxygen, 5% CO2 and the balance nitrogen. After 25 hours the oxygen concentration was increased to 20%, and after 44 hours to 40%. Embryos at E10.5 received an initial gas mixture containing 20% oxygen. After 21 hours this was increased to 40%, and to 95% after 29 hours. E9.5 Embryos were cultured for 48 hours and E10.5 for 45 hours. All gases were supplied by BOC, UK. Inhibitor treatment The two inhibitors of endopeptidase-24.11 used in this study are phosphoramidon and thiorphan, which differ in the functional moiety that co-ordinates the zinc ion at the active site of the enzyme. Both inhibitors are highly selective for this enzyme, for reviews see Wilkins Fig. 1. Western blot analysis of adult kidney microvillar membrane et al. (1993) and Roques et al. (1993). preparation (7 µg total protein/lane), lanes 1 and 3, and an E14 Phosphoramidon (N-(-L-rhamnopyranosyloxyhydroxyphosphinyl)- embryo membrane preparation (35 µg total protein/lane), lanes 2 and L-leucyl-L-tryptophan, Sigma, UK) is a natural metabolite produced 4. The endopeptidase-24.11 and endopeptidase-24.18 present in the by Streptomyces tanashiensis which is a specific, competitive adult rat kidney are identical to those found in the embryo. Bands are inhibitor of the bacterial enzyme thermolysin (Umezawa, 1972). The visible at 94 kDa for endopeptidase-24.11 and at 80 kDa and 74 kDa structure of the active site of thermolysin exibits a high degree of for endopeptidase-24.18. 3216 B. Spencer-Dene and others

Table 1. Summary of immunostaining of rat embryos at E12, E14 and E16 for Endopeptidase-24.11 and Endopeptidase- 24.18 in the craniofacial region Tissue Endopeptidase-24.11 immunoreactivity Endopeptidase-24.18 immunoreactivity Ependymal cells Positive at all stages, strong at E14 Positive at all stages Choroid plexus: mesenchyme Positive at E14 and E16 Strongly positive at E14, positive at E16 epithelium Positive at E14 and E16 Negative at E14 and E16 Otocyst/Ear Positive at E12-E16 Positive at all stages Eye (lens and neural retina) Positive at E14 and E16 Weakly positive at E14 and E16 2û Palate mesenchyme Positive at E14, strong at E16 Negative at all stages Tongue Positive at E14 and E16 Negative at all stages Craniofacial vasculature Positive at E12 and E14, negative at E16 Negative at all stages Perichondrium Positive at E14 and E16 Negative at all stages Nasal epithelium Weakly positive at E14 and E16 Positive at all stages Nasal mesenchyme Strongly positive at E12 and E14, negative at E16 Negative at all stages Notochord Positive at all stages Negative at all stages

Overnight incubation of both antibodies with the kidney luminal surface of both the gut (Fig. 2D) and mesonephric microvillar membrane preparation blocked staining of both epithelium. In addition, the pericardium and endocardial kidney and embryo membranes. Similarly, incubation of cushions in the heart were strongly positive, whilst the myocar- RRt151 with purified rat endopeptidase-24.18 completely dial cells exhibited weaker positive staining. By E14, positive abolished binding of the antibody to the endopeptidase-24.18 staining was observed in the cells enveloping the vertebrae, bands from kidney and embryo preparations (data not shown). epithelial lining of the gut, mesonephros and bronchi, These western blot experiments therefore demonstrated that diaphragm, notochord, the dura mater surrounding the spinal the antibodies used in this study recognised antigens with cord and the heart. We have no data regarding endopeptidase- identical molecular weights as observed in the adult rat kidney 24.11 localization in postcranial sites in the E16 rat embryo membranes. since only the craniofacial region was investigated at this later stage. Immunohistochemical distribution of endopeptidase-24.11 in the craniofacial region In situ hybridization of endopeptidase-24.11 The tissue localization of endopeptidase-24.11 was studied by Gene expression of endopeptidase-24.11 closely matched the an indirect immunoperoxidase method with nickel chloride distribution of the endopeptidase-24.11 antigen both in enhancement where necessary; the results are summarised in location and signal intensity at E12 and E14. Of particular note Table 1. All positive staining was abolished by preabsorption was the expression in the medial and lateral nasal process mes- of RAHE with the rat kidney membrane preparation. Binding enchyme, and to a lesser extent on the otocyst epithelium and in the adult rat kidney was confined to the brush border of the on the first branchial arches. The expression throughout the proximal convoluted tubules and Bowman’s capsule (Fig. 2A). notochord was extremely intense at this stage (Fig. 4B). At E10 the luminal surface of the gut ectoderm exhibited At E14, the strongest expression in the craniofacial region strong staining. At E12, endopeptidase-24.11 was detectable was in the oral and nasal tissues (Fig. 4C), and the choroid mainly in mesenchymal component of the medial and lateral plexus was also positive (Fig. 4D). Many other postcranial sites nasal processes, the notochord and on the luminal surface of were strongly positive (Fig. 4A) including the lungs, the otocyst epithelium and the branchial arteries. The rest of mesonephros, intestine, notochord and surrounding the the craniofacial vasculature exhibited moderately strong vertebrae. The craniofacial vasculature expression at both positive staining. In E14 embryos, strong staining in the stroma and on the luminal/ventricular surface of the choroid plexus (Fig. 2E), the ependymal lining of the brain ventricles, the Fig. 2. (A) Immunolocalization of endopeptidase-24.11 in the adult rat kidney. Positive staining is confined to the brush border of the basilar and carotid arteries and the basilar sulcus in the pons. epithelia lining the proximal convoluted tubules and on the At E16, the palate, root of the tongue, choroid plexus, Bowman’s capsule (arrow); Distal convoluted tubules are negative. ependymal lining and several discrete sites in the eye and inner (B) Immunolocalization of endopeptidase-24.18 in the adult rat ear all exhibited positive staining of various degrees of kidney. Positive staining is confined to the brush border of the intensity (see Fig. 3A,B). All the facial vasculature, cartilage epithelia lining the proximal convoluted tubules. (C) The and bone including the nasal septum and conchae and Meckel’s perichondrial layer (arrowheads) surrounding the developing hyoid cartilage were negative. However, there was a distinct popula- cartilage, like all perichondria, exhibit strong endopeptidase-24.11 tion of positively staining cells in a perichondrial layer immunoreactivity at E16; differentiated chondrocytes are negative. enveloping Meckel’s cartilage and other craniofacial skeletal (D) At E12, endopeptidase-24.11 is strongly immunolocalized on the elements (Fig. 2C). luminal surface of the hind gut (G) endoderm and on the notochord (N). The immunoperoxidase staining has been amplified using a Immunolocalization of endopeptidase-24.11 in other DAB-enhancement technique. (E) Positive endopeptidase staining is evident in the mesenchymal stroma and on the apical surface of the tissues epithelia (arrows) covering the choroid plexus at E14. (F) At E14, Endopeptidase-24.11 was localized in several other areas the luminal surface of the choroid plexus, exhibits strongly positive across the developmental period studied. At E12 there was endopeptidase-24.18 staining, and in contrast to endopeptidase-24.11 intense positive staining within the notochord and on the immunoreactivity, the stroma is completely negative. Bars, 50 µm. Peptidases in craniofacial development 3217 3218 B. Spencer-Dene and others Peptidases in craniofacial development 3219

Table 2. Results of phosphoramidon exposure on E9.5 rat embryos in vitro Yolk sac Crown-rump % Cranio- Inhibitor No. of diameter length % Somite % Heart % Yolk sac facial *Severity conc. embryos mm±s.d. mm±s.d. Turned no. ±s.d. beating circulation defect of defect 0 (Control) 25 3.19±0.24 3.06±0.52 85.7 24.8±1.79 100 100 0 − 10 nM 16 2.63±0.25 2.35±0.18 35 21.75±0.5 100 100 25 + 100 nM 19 3.25±0.2 2.88±0.63 100 24.5±0.58 100 100 25 ++ 1 µM 22 2.88±0.14 3.06±0.31 75 22.25±2.22 75 75 42.5 ++ 10 µM 16 3.13±0.25 2.94±0.43 75 22.75±0.96 100 100 25 +++ 100 µM 12 3.17±0.29 3.08±0.14 100 23.67±1.15 100 100 33 +++

Table 3. Results of phosphoramidon exposure on E10.5 rat embryos in vitro Yolk sac Crown-rump Inhibitor No. of diameter length % Somite % Heart % Yolk sac % Cranio- *Severity conc. embryos mm±s.d. mm±s.d. Turned no. ±s.d. beating circulation facial defect of defect 0 (Control) 11 4.43±2.51 4.3±2.0 100 31.16±1.52 100 100 0 − 10 nM 27 4.1±3.6 4.03±3.78 100 27.33±3.05 100 100 46 + 100 nM 23 4.2±3.6 4.05±3.01 100 28.33±3.21 100 100 25 + 1 µM 9 3.78±4.91 3.36±7.95 100 28.4±4.39 72 54 72 ++ 10 µM 9 4.26±0.57 4.3±1.0 100 29.33±1.15 100 100 33 ++ 100 µM 9 3.56±8.14 3.4±3.6 100 28.67±1.15 100 67 33 ++

*Severity of the defect is measured on a scale from − (normal appearance) to +++ (grossly abnormal appearance, including open cranial folds, clearly discernible haematoma and dysmorphic branchial arches). stages was not appreciably intense. The sense control gave no displayed no detectable staining for this enzyme. In older positive signal and extremely low background. embryos, the luminal surface of the neuroepithelium demonstrated a particularly striking pattern of distribution, and this Distribution of endopeptidase-24.18 in the was best exemplified by the choroid plexus and the ependymal craniofacial region lining of the developing brain ventricles (Fig. 2F). The The tissue distribution localization of this enzyme was studied intensity of staining on the epithelial cells lining the choroid by both indirect immunoperoxidase and immunofluorescence. plexus decreased between E14 and E16. The developing lens When sections of adult rat renal cortex were stained (as a and pigmented layer of the retina exhibited moderately positive positive control), staining was restricted to the brush border of staining at E16, but earlier embryos displayed only negligible the proximal convoluted tubules (Fig. 2B). staining at these locations. Immunolocalization of endopeptidase-24.18 in the develop- The most striking pattern of endopeptidase-24.18 distribution ing head and face was confined to discrete sites with a distri- was that displayed in the developing inner ear. At E12, there was bution which seems to be temporally regulated; this is sum- a diffuse and moderately intense positive stain on the apical marised in Table 1. All positive staining disappeared when surface of the cells lining the otic vesicle. By E14 the positive RRt151 was preabsorbed with either rat kidney microvilli or staining appeared considerably stronger, specifically localized to purified endopeptidase-24.18 (Fig. 2E,F). E10 embryos the stria vascularis and not detectable on the developing hair cells, Organ of Corti or the tectorial membrane. This distribution Fig. 3. (A) Central regions of the palatal shelf mesenchyme pattern is still apparent in the cochlea at E16 (Fig. 3C-F). In some (asterisks) display positive endopeptidase-24.11 immunoreactivity at E16 embryos, the epithelia lining the oral and nasal cavities E16; nasal septum (NS) is negative, (no counterstain). Bar, 50 µm. showed a variable distribution of endopeptidase-24.18. A few (B) At E16 endopeptidase-24.11 is localized on the genioglossus other postcranial sites in the E14 embryos stained positively for muscle (asterisks) in the root of the tongue; Meckel’s cartilage (M) is endopeptidase-24.18, notably in the bladder, ureter and gut. negative, (no counterstain). Bar, 50 µm. (C) In the E16 inner ear, endopeptidase-24.18 is confined to the luminal surface of the stria vascularis and Reissner’s membrane, within the cochlear duct Perturbation studies (arrows); otic capsule (OC) is negative. Bar, 100 µm. (D) Higher The results of the perturbation studies are summarized in magnification of part of C to show positive immunoreactivity on stria Tables 2, 3 and 4. Following culture with two inhibitors of vascularis (SV) and the adjacent part of Reissner’s membrane (RM); endopeptidase-24.11, SEM revealed that a proportion of the the region from where the sensory epithelium (asterisk) will differentiate remains negative. Bar, 50 µm. (E,F) Transverse section inhibitor-treated E9.5 embryos displayed an asymmetric, pre- through an E16 semicircular canal. (F) Endopeptidase-24.18 is only dominantly left-sided, facial deformity which appeared to be localized on the surface of the non-sensory cells. (E) An adjacent due to the presence of a haematoma-like swelling adjacent to section incubated with RRt151 which was preabsorbed overnight the prosencephalon. This often had the effect of distending the with purified endopeptidase-24.18 shows that all positive staining left side of the head which in turn disrupted the normal posi- has been extinguished. Bar, 50 µm. tioning of the first and second branchial arches. In the most 3220 B. Spencer-Dene and others severe cases, observed at the highest inhibitor concentrations, proportionately swollen, in a midline position and seemed not the swelling was so great that the anterior neural folds could to have undergone normal looping morphogenesis. not close. Cultured E9.5 embryos are shown in Fig. 5A-D. The E10.5 embryos also displayed an inhibitor dose- Control embryos, cultured in diluted serum only, developed dependent abnormal left-sided prosencephalic swelling (Fig. normally. The severity of the defect, but not the incidence, 6A-D). None of the E10.5 embryos had open anterior neural generally increased as inhibitor concentration increased. In folds but the branchial arches were often displaced asymmet- addition to these craniofacial effects, the heart appeared dis- rically and of abnormal appearance, whilst after exposure at

Fig. 4. (A) Bright-field image of a sagittal section through an E14 embryo probed for endopeptidase-24.11 mRNA using in situ hybridization. The mRNA is localized in several discrete locations including the lining of the lungs (asterisk), the cells enveloping the vertebrae (small arrowheads), the lining of the intestine (small arrow), within the choroid plexus (large arrow) and in the notochord, shown here caudally in oblique section (large arrow head). Bar, 100 µm. (B) Bright-field image of a transverse section through the notochord (arrow) of an E12 embryo probed for endopeptidase-24.11 mRNA. Bar, 50 µm. (C) Dark-field image showing the oral and nasal tissues at a higher magnification. The silver grains (white) are intensely localized throughout most of the nasal mesenchyme (N), throughout the lower jaw (M) and tongue (asterisk). Also in the roof of the oral cavity/ secondary palate (arrow) and in the oesophagus (arrow head). Bar, 100 µm. (D) Higher magnification bright-field image of the E14 choroid plexus. In addition to the mesenchymal distribution, there appears to be some degree of heterogeneity shown by the epithelial localization of the mRNA. Bar, 50 µm. Peptidases in craniofacial development 3221 this later stage, the heart morphogenesis appeared grossly developmentally regulated manner. We have therefore studied normal. Our assessment of relative normality was based upon the distribution of endopeptidase-24.11 mRNA using a radio- the following criteria; yolk sac diameter and circulation, labelled full-length complementary RNA probe, containing crown-rump length, turning and presence of a beating heart sequences common to all known transcripts, to assess the pos- compared with the control embryos. sibility of transcriptional control occurring during embryoge- Subsequently, histological analysis of embryos treated at nesis. In situ hybridisation reveals the regional presence of E9.5 revealed that the swelling was typically the result of both message as early as E12, and reverse transcriptase-PCR a localised overgrowth of the prosencephalic neurectoderm on analysis has enabled the detection of message as early as E8 the left side (Fig. 7A) and a gross distension of the internal and indicates the existence of a novel, embryo-specific spliced carotid artery on the same side (Fig. 7B). More caudally, this variant (work in progress). vascular disturbance extended to include the first branchial The present investigation substantiates the previous work of artery, which like the internal carotid arises from the dorsal others, in which the various locations of endopeptidase-24.11 aorta, and displayed distension. A presumed secondary effect in the adult rat central and peripheral nervous systems are of this was the lateralward displacement of the first branchial described employing both immunocytochemical (Ronco et al., arch (Fig. 7C) as seen in the SEM. 1988) and autoradiographic techniques (Waksman et al., 1986). Of the few related studies, endopeptidase-24.11 distribution in the foetal and postnatal rat has been localized indi- DISCUSSION

Protein and mRNA distributions This study is the first report describ- ing the localization of both endopeptidase-24.11 and endopeptidase- 24.18 immunohistochemically during mammalian embryogenesis, and to demonstrate a functional role for one of these endopeptidases during craniofacial development. Using immunohistochemistry, we have established that both endopeptidase-24.11 and 24.18 are present in discrete locations in the rat embryo during a period of active craniofacial morphogenesis. Certain regions within the adult rat brain, for example the globus pallidus, have been shown to display different distributions of NEP protein compared to mRNA (Wilcox et al., 1989). In addition, at least five alternative splice variants of NEP have been identified (D’Adamio et al., 1989; Llorens- Cortes et al., 1990; Iijima et al., 1992). Given that the NEP gene is constitutively expressed in some tissues and is developmentally regulated in other cell types (i.e. lymphocytes and granulocytes), it is possible that the transcription of endopeptidase-24.11 is controlled by alternative promoter activation. Fig. 5. (A) E9.5 rat embryos cultured in control serum for 48 hours; frontal view. Bar, 200 µm. (B) E9.5 embryo cultured for 48 hours in serum containing 10 nM phosphoramidon; note the The substantial conservation of µ 5′ untranslated regions between failure of neural fold fusion over forebrain (arrow) Bar, 200 m. (C) E9.5 embryo cultured for 48 hours in serum containing 100 nM phosphoramidon. Note complete failure of cephalic neural folds different species and the existence µ ′ closure and lateral distension of the left side of the head (arrow). Bar, 100 m. (D) E9.5 embryo of 5 alternative splicing suggest cultured for 48 hours in serum containing 100 µM phosphoramidon; note open neural tube, a that endopeptidase-24.11 gene general asymmetry to the head and the swelling (arrow) of the telencephalic neuroepithelium into expression may be differentially the lumen, caused by a subadjacent haematoma. The normal looping morphogenesis of the heart controlled in a tissue-specific and/or (asterisk) has also clearly been disturbed. Bar, 100 µm. 3222 B. Spencer-Dene and others rectly by the binding of a selective tritiated inhibitor of murine foetal lung maturation (King et al., 1993). The palatal endopeptidase-24.11, [3H]HACBO-Gly, (Dutriez et al., 1992). localization of endopeptidase-24.11 might reflect the presence A limited range of selected and unrelated tissues have been of TGFα or epidermal growth factor in the palatal mesenchyme studied, namely microvilli from human placental syncytiotro- (Dixon et al., 1991), although cleavage of either of these growth phoblast; foetal rabbit, rat and human small intestinal brush factors by endopeptidase-24.11 has yet to be demonstrated. border membranes; and nuchal ligaments from late-stage fetal However, TGFα is a known substrate of endopeptidase-24.18 calves (Johnson et al., 1984, 1990; Lecavalier et al., 1989). The (Choudry and Kenny, 1991). A recent study demonstrated that distribution patterns described here extends significantly the TGFβ1 could down-regulate endopeptidase-24.11 activity via a onset of endopeptidase-24.11 expression back into much reduction in the levels of the transcribed gene or possibly by earlier development and demonstrates, for the first time, an decreasing endopeptidase-24.11 mRNA stability (Casey et al., embryonic presence of endopeptidase-24.18. 1993). Indeed, it has been proposed that the role of TGFβ1

Candidate substrates for endopeptidase-24.11 and endopeptidase-24.18 The hypothesis that these enzymes are involved in early developmental processes is based on the proposition that expression implies function and that the main function of these enzymes in the embryo is to deactivate peptide signals. The present work does not attempt to identify specific peptide substrates within the various embryonic tissues in which the enzymes are found. However, some likely candidate substrates can be recognised. In the adult, the roles of endopeptidase-24.11 include pain modulation via enkephalin degradation at central and spinal levels, osmoregulation via atrial natriuretic peptide (ANP) and degradation in the kidney and other peripheral sites. We have found that both endopeptidase-24.11 and 24.18 are expressed during eye development, and this correlates with the presence of their putative substrates such as substance P, vasoac- tive intestinal peptide, enkephalins, calcitonin-gene-related peptide and Neu- ropeptide Y in the adult eye (Stone et al., 1987). In the choroid plexus (which is involved in the production of cerebrospinal fluid), ependymal lining of the brain ventricles and developing cardiovascular system, the substrate could possibly be ANP (Kenny and Stephen- son, 1988). High densities of ANP receptors have been localized autoradi- ographically at these sites (Tong and Pelletier, 1990), which closely matches Fig. 6. (A) E10.5 rat embryo cultured for 45 hr in serum containing 10 nM phosphoramidon. the distribution of both endopeptidase- Embryo appears morphologically normal; lateral view. Bar, 200 µm. (B) E10.5 rat embryo 24.11 and 24.18. cultured for 45 hours in serum containing 100 nM phosphoramidon. Note the swollen In the developing lung, the endopepti- position of the anterior part of the side of the head (asterisk) and lateral distension of the left side of the head (large arrow). This asymmetry is associated with a lateral/ventral dase-24.11 substrate may be substance P displacement of the left mandibular arch (arrowhead). Frontal view. Bar, 100 µm. (C) E10.5 (Shepherd et al., 1988) or, more likely, rat embryo cultured for 45 hours in serum containing 1 µM phosphoramidon; note the grossly the bombesin-like peptides (Shipp et al., abnormal left side to the head with a swollen dysmorphic first arch and a distorted second 1991). Indeed, it has recently been arch. Lateral view. Bar, 200 µm. (D) E10.5 rat embryo cultured for 45 hours in serum reported that the hydrolysis of the containing 10 µM phosphoramidon; Note the relatively distended left side of the bombesin-like peptides by endopepti- prosencephalon (asterisk) and the asymmetry of the first branchial arch, similar to B. Frontal dase-24.11 could control the rate of view. Bar, 100 µm. Peptidases in craniofacial development 3223

Table 4. Results of thiorphan exposure on E9.5 rat embryos in vitro Yolk sac Crown-rump Inhibitor No. of diameter length % Somite % Heart % Yolk sac % Cranio- *Severity conc. embryos mm±s.d. mm±s.d. Turned no. ±s.d. beating circulation facial defect of defect

Serum only control 4 3.75±0.44 3.3±0.54 100 29±1.15 100 100 0 − Serum + ethanol control 3 3.8±0.87 2.9±0.36 33 27±1.41 100 100 0 − 100 nM 6 1.85±0.33 1.53±0.38 33 17.83±1.33 100 100 0 + 1 µM 6 3.2±0.62 3.06±0.64 33 24±1.73 50 50 33 ++ 10 µM 6 1.8±0.44 1.63±0.20 0 17.3±1.15 50 50 33 +++

*Severity of the defect is measured on a scale from − (normal appearance) to +++ (grossly abnormal appearance, including open cranial folds, clearly discernible haematoma and dysmorphic branchial arches). during chondrocyte differentiation is to regulate the expression is an oligomeric tetramer of subunits linked by disulphide of the matrix proteins and metalloproteases (Ballock et al., bonds. Initial research on the cloning and sequencing of the 1993). Such a relationship might explain the distribution of amino terminus of endopeptidase-24.18 identified it as a endopeptidase-24.11 that we have observed surrounding the member of the ‘astacin family’ of metallo-endopeptidases developing skeletal elements in the older embryos. (Dumermuth et al., 1991). All the enzymes in this family have Many of the regions where we have immunolocalized a zinc-binding metalloprotease domain which shares a high endopeptidase-24.18 in the rat embryo, particularly on the degree of homolgy with the domain found in astacin (EC choroid plexus and in the inner ear, are sites of ion trans- 3.4.24.21), a protease from the crayfish Astacus fluviatilis portation. This enzyme may, therefore, have an important role (Shimell et al., 1991). Other enzymes attributed to this family in the production of cerebrospinal fluid and endolymph respec- are ‘PABA-peptide hydrolase’ (Sterchi et al., 1982) and BMP- tively during mammalian craniofacial development. In the 1, bone morphogenetic protein-1 (both in humans), UVS.2 (in adult rat, comparison of the distribution patterns of these two Xenopus), the tolloid gene product (in Drosophila) (Finelli et neutral metallo-endopeptidases reveals a considerable differ- al., 1994), and suBMP, blastula protein 10 and SpAN proteins ence, in terms of the number and variety of organs where these (in sea urchin) (Lepage et al., 1992; Reynolds et al., 1992; enzymes have been detected (Ronco et al., 1988; Barnes et al., Hwang et al., 1994). Since the members of the astacin gene 1989). This correlates with the comparative paucity of family are present in a wide variety of organisms and are endopeptidase-24.18 distribution in the embryo compared to conserved, they are likely to have similar functions. The con- the relatively greater distribution of endopeptidase-24.11. clusion that they are involved in controlling the activity of growth factors, and thus are vital for specifying cell determi- Endopeptidase-24.18 and the astacin family nation, developmental and differentiation events (Dumermuth Endopeptidase-24.18 is an unconventional peptidase in that it et al., 1991), therefore strengthens our contention that the cell-

Fig 7. Haematoxylin and Eosin stained frontal section through an E9.5 rat embryo cultured for 48 hours in the presence of 1 µM phosphoramidon. Bar, 20 µm. (A) Section through the forebrain region. Note the greatly overgrown neurectoderm (asterisk) on the left side of the prosencephalon. (B) Slightly more caudal section. Note the grossly dilated left internal carotid artery (large arrow) compared to its contralateral partner (small arrow). The optic vesicle is also indicated (hollow arrow). (C) Section at the level of the first branchial arch. Note the abnormally displaced left arch (arrow) and the relatively dilated first branchial artery (asterisk). 3224 B. Spencer-Dene and others surface metallo-endopeptidases play a major role in embryo- CONCLUSION genesis. The disturbance in morphogenesis, which appears to be a con- Endopeptidase-24.11 and craniofacial development sequence of endopeptidase-24.11 inhibition during culture, Here we have successfully shown the localization of both leads us to believe that this enzyme plays a critical role during endopeptidase-24.11 and 24.18 protein in postimplantation rat normal craniofacial development. This raises the possibility embryos. Both enzymes appear to be developmentally that cell-surface metallo-endopeptidases, such as endopepti- regulated at several discrete loci and the patterns of distribu- dase-24.11, may constitute a hitherto unrecognised level of tion of these enzymes and their putative substrates strongly control through the cleavage and inactivation of biologically suggest that they play a significant role during craniofacial active small peptides, growth factors and cytokines in cranio- development in the rat. In order to explore a possible functional facial tissues. role, we have carried out inhibition studies. The use of two different selective inhibitors of endopeptidase-24.11 phospho- We would especially like to thank the Collaborations Dept. at ramidon and thiorphan, in whole embryo culture consistently Genentech Inc. California, USA for their generous gift of rabbit anti- human enkephalinase antibody and the rat endopeptidase-24.11 resulted in the formation of an asymmetric facial lesion in a cDNA, Professor R. C. Atkins, Director of Nephrology at the Monash proportion of embryos. Significantly, the specific anomalies Medical Centre for generously sending us the PHM-6 antibody, Nicky observed resemble those found in the human birth defect hemi- Mordan for her technical help and advice with the SEM, Dr Andrew facial microsomia or ‘oculo-auriculo-vertebral spectrum’ Copp for advice on embryo culture, Dr Nick Lench for advice on (Gorlin et al., 1990). Hemifacial microsomia is asymmetric, probe preparation and Ms Monique Doherty for technical assistance. 70% of cases exhibit unilateral deformities in the facial This work was supported by a grant from the Medical Research skeleton, presenting as hypoplasia and malformation, particu- Council to B. H. and P. T. larly in the mandibular, auditory, maxillary and zygomatic bones. Possible causes of this defect include haemorrhaging from the primordial stapedial artery following teratogen insult REFERENCES (Poswillo, 1973) and perturbation of auriculofacial chondro- genesis (Cousley and Wilson, 1992). Children born with hemi- Ballock, R. T., Heydemann, A., Wakefield, L. M., Flanders, K. C., Roberts facial microsomia are also highly likely to suffer from skeletal and A. B., Sporn, M. B. (1993). TGF-β1 prevents hypertrophy of epiphyseal abnormalities, various forms of heart disease, pulmonary and chondrocytes: Regulation of gene expression for cartilage matrix proteins and metalloproteases. 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