Endopeptidase-24.11 (neutral endopeptidase, neprilysin, ‘enkephalinase’, EC 3.4.24.11) and endopeptidase-24.18 (endopeptidase-2, meprin, EC 3.4.24.18) are cell-surface zinc-dependent metallo-endopeptidases able to cleave a variety of bioactive peptides including growth factors. We report the first study of the cellular and tissue distribution of both enzymes and of the mRNA for NEP during embryonic development in the rat. Endopeptidase-24.11 protein was first detected at E10 in the lining of the gut and, at E12, the enzyme was present on the notochord, medial and lateral nasal processes, otocyst, mesonephros, heart and neuroepithelium. In contrast, at this time endopeptidase-24.18 was present only on the apical surface of the neuroepithelial cells. By E14 and E16, NEP was also detected in a wide range of craniofacial structures, notably the palatal mesenchyme, the choroid plexus, tongue and perichondrium. The distribution of endopeptidase-24.18 at these stages was restricted to the inner ear, the nasal conchae, and ependymal layer of the brain ventricles and the choroid plexus. Although endopeptidase-24.11 had been detectable in the craniofacial vasculature at E12 and E14, this was no longer apparent at E16. Significantly, the distribution of endopeptidase-24.11 mRNA closely matched the immunolocalization of the protein at all stages investigated.

In order to explore the functional role of these enzymes, inhibition studies were carried out using two selective inhibitors of endopeptidase-24.11, phosphoramidon and thiorphan. E9.5 and E10.5 embryos exposed to either inhibitor displayed a characteristic, asymmetric abnormality consisting of a spherical swelling, possibly associated with a haematoma, predominantly on the left side of the prosencephalon, and the severity of this defect appeared to be a dose-dependent phenomenon. This study suggests that these enzymes play previously unrecognized roles during mammalian embryonic development.

Mammalian cells synthesize two main classes of metallo-endopeptidases, those that are secreted and participate in remodelling of the extracellular matrix, such as collagenase and stromelysin (Henderson and Blake, 1994) and the cell-surface metallo-endopeptidases which play a role in the inactivation of biologically active peptides (Kenny et al., 1989; Erdös and Skidgel, 1989). Two well-studied examples of the latter are endopeptidase-24.11 and endopeptidase-24.18. Both enzymes are transmembrane, zinc-containing metallo-endopeptidases found on the outer aspect of the plasma membrane of a variety of cells. Both are abundant on the brush borders of the epithelial cells lining the adult kidney proximal tubule and intestine (Ronco et al., 1988; Barnes et al., 1989; Corbeil et al., 1992). Endopeptidase-24.11 cleaves peptide bonds involving the amino function of hydrophobic residues, while endopeptidase-24.18 hydrolyses bonds adjacent to aromatic residues, but the attack may be on either side of such residues (Stephenson and Kenny, 1987; Wolz et al., 1991). In recent years, these enzymes have been shown to be capable of hydrolysing a variety of neuropeptides and peptide hormones (Erdös and Skidgel, 1989; Price et al., 1991; Choudry and Kenny, 1991), including growth factors and cytokines (Kenny and Ingram, 1987; Katayama et al., 1991). Additionally, two NEP substrates, the tachykinin, substance P, and the major bacterial chemotactic peptide formyl-Met-Leu-Phe provoke rapid changes in the migration, morphology and adhesion molecule expression of human neutrophils. These changes are potentiated when endopeptidase-24.11 is inactivated by the selective inhibitor, phosphoramidon (Shipp et al., 1991).

Molecular cloning and expression studies have demonstrated that endopeptidase-24.11 is identical to CD10 (CALLA, common acute lymphoblastic leukaemia antigen) (Letarte et al., 1988; Chen et al., 1992). This finding, and the observation that CD10 is expressed by foetal haematopoietic cells (Hokland et al., 1983), has led to speculation that endopeptidase-24.11 may play a role in the control of growth and differentiation in both haematopoietic and epithelial cell systems (Kenny et al., 1989; LeBien and McCormack, 1989), possibly regulating local concentrations of active peptides, such as growth factors, at the cell surface.

It is apparent from the limited information available that the expression of various peptide growth factors and their receptors is under developmental regulation during craniofacial morphogenesis (reviewed by Slavkin, 1990; Lee and Han, 1991; Vainio et al., 1993). Recent evidence strongly suggests that some of these factors, such as transforming growth factors α and β, are likely to be critically important in normal growth and development of the facial primordia (Wilcox and Derynck, 1988; Mahmood et al., 1992; Frenz et al., 1992). Thus, although their precise and respective contributions remain to be fully defined, it is clear that regulatory growth factors have pivotal roles in craniofacial morphogenesis and, presumably, dysmorphogenesis.

We postulate that, given their known role in postembryonic tissues, endopeptidase-24.11, endopeptidase-24.18 and related enzymes may have a significant, but as yet unrecognised, morphogenetic role in the growth and development of embryonic craniofacial tissues. Here we report, for the first time, the successful immunolocalization of both endopeptidase-24.11 and endopeptidase-24.18, and the in situ hybridization of endopeptidase-24.11 mRNA in postimplantation rat embryos at various critical stages of craniofacial development. We describe patterns of distribution of both message and gene product in the craniofacial tissues and consider possible roles of these enzymes during craniofacial morphogenesis.

The increasing understanding of the role played by endopeptidase-24.11 in hydrolysing and inactivating enkephalins and natriuretic peptides has led to many synthetic inhibitors of endopeptidase-24.11 being clinically evaluated as analgesics and as therapeutic agents in cardiac failure (reviewed by Wilkins et al., 1993). We have taken advantage of these inhibitors to test whether inhibition of endopeptidase-24.11 may have any demonstrable effects on embryogenesis and report the results of perturbation experiments using a whole embryo culture technique (Cockcroft, 1990) with two selective endopeptidase -24.11 inhibitors, phosphoramidon and thiorphan.

Given that endopeptidase-24.11 and endopeptidase-24.18 have the ability to hydrolyse many simple biologically active peptides (and possibly some cytokines), our results suggest possible roles for these enzymes in the control of cell growth, differentiation and movement. This, we believe could constitute a previously unrecognised level of developmental control during craniofacial morphogenesis.

Antibodies

RAHE, a polyclonal rabbit anti-human enkephalinase, (Genentech, USA); PHM-6, a monoclonal mouse anti-human CALLA, (Monash Medical Centre, Australia); RRt151, a polyclonal rabbit anti-rat endopeptidase-24.18 produced by one of the authors (A. J. K.). Both RAHE and PHM-6 were found to cross-react with rat NEP.

Detergent solubilization of a membrane preparation from embryos

E14 rat embryos were removed from the uterine horns and homogenized in 50 mM Tris/HCl buffer (pH 6.5) containing 0.1 mM phenyl-methylsulfonylfluoride (PMSF) at 4°C. This homogenate was centrifuged for 5 minutes (1000 g) at 4°C, and the supernatant was retained and centrifuged at 100,000 g for 1 hour, using a Kontron ultracentrifuge, to collect a membrane fraction. Membranes were resuspended in 10 mM Tris-HCl (pH 7.5) containing 0.1 mM PMSF, 0.1 mM pepstatin A, 0.1 mM 1,10 phenanthroline and 0.5% Triton X-100 and left at 4°C overnight to solubilize the membrane enzymes. Insoluble material was removed by centrifugation at 100,000 g for 1 hour and the supernatant retained and stored at −70°C.

Microvillar membranes from rat kidneys

These were prepared as described previously (Booth and Kenny, 1974). The final pellet was resuspended in the same Tris-HCl/protease inhibitor buffer as used for the embryo membranes.

Western blotting

Kidney microvilli and embryo membranes were separated by SDS-PAGE according to Laemmli (1970) and were transferred to nitro-cellulose membrane according to Towbin et al. (1979). Blots were incubated with either RAHE or RRt151 (1:1000) for 1 hour, rinsed with distilled water and Tris-buffered saline-Tween-20 (TBST), then incubated with goat anti-rabbit peroxidase-conjugate (DAKO, UK; diluted 1:500 with 5% skimmed milk in TBST+5% rat serum) for 1 hour, rinsed and washed as above and developed with 4-chloro-1-naphthol.

To establish the specificity of antibody binding both to the embryo and kidney membrane preparations, the antibodies were preincubated with either purified endopeptidase-24.18 or purified kidney membrane preparation prior to blotting.

Preparation of embryos for immunohistochemistry

Wistar rats (Charles Rivers, UK) were mated and the date of vaginal plug detection designated day 0 (E0). Rats were killed by cervical dislocation following CO2 anaesthesia at E10, 12, 14 or 16 of gestation. Embryos were dissected out in PBS and fixed in 4% paraformaldehyde in PBS overnight at 4°C. In the case of the E16 embryos, only the heads were used. Embryos were transferred into cryoprotectant (20% (w/v) 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 stored at −70°C. The kidneys from the mothers were removed and processed as above, as a positive control tissue. Cryostat sections (8 μm) cut from embryos and kidneys were thaw-mounted onto glass slides precoated with 3-aminopropyltriethoxysilane (Sigma, UK) and stored at −70°C. For all stages used, representative sections from individual embryos from four separate litters were independently examined.

Immunohistochemistry

(i)Endopeptidase-24.11

The presence of endopeptidase-24.11 on frozen sections was localized by an immunoperoxidase staining procedure using RAHE and PHM-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 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 antimouse for PHM-6) peroxidase conjugate for 30 minutes. This was diluted to 1/300 in PBS plus 2% normal rat serum and preabsorbed with ‘rat powder’ (Barnes et al., 1989). Sections were finally incubated in 2.5 mg/ml of diaminobenzidine (DAB) in PBS containing 0.01% hydrogen peroxide. This step was amplified with nickel chloride when necessary. All reagents used were from Sigma, UK. All steps were separated by 2× 10 minute washes of PBS (or 0.1 M acetate buffer for the nickel enhancement) and all incubations took place at room temperature in a humidity chamber. Sections were counterstained with Mayer’s Haematoxylin or Neutral Red (both from BDH, UK), dehydrated, cleared and mounted in DPX (BDH, UK).

To control for non-specific binding, sections were routinely incubated in the absence of 1° or 2° antibodies. Controls using RAHE, which had been incubated overnight at 4°C with 0.7 mg/ml of the kidney microvillar membrane preparation to preabsorb out endopeptidase-24.11, were also included.

(ii)Endopeptidase-24.18

Endopeptidase-24.18 was localized on rat tissue using the same indirect immunoperoxidase technique. Sections were incubated for 1 hour with RRt151 diluted 1/100 in 0.1% BSA/PBS.

In control experiments, RRt151 or the peroxidase conjugate were omitted and replaced with PBS. Further controls to validate specific endopeptidase-24.18-positive staining involved replacing RRt151 with preimmune rabbit serum, or with RRt151 that had been incubated overnight at 4°C with the purified enzyme (Kenny and Ingram, 1987) or 0.7 mg/ml of the kidney microvillar membrane preparation to preabsorb out its immunoreactivity.

All embryo sections were photographed under bright field on an Olympus BH2 photomicroscope using Kodak Ektachrome 64 T film.

In situ hybridization

This was carried out according to the protocol described by Wilkinson and Green (1990). The antisense probe, labelled with [35S]UTP, was transcribed from complementary DNA which had been subcloned into Bluescript (Strategene, UK). This cDNA corresponded to the fulllength sequence of rat endopeptidase-24.11 (Malfroy et al., 1987). A 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 55°C. Slides were dipped in Ilford K5 emulsion (Ilford, UK), developed after 4 days, counterstained with haematoxylin, dehydrated and mounted.

Whole embryo culture

Pregnant Wistar rats (Charles Rivers, UK) were killed by cervical dislocation whilst under CO2 anaesthesia. The E9.5 and E10.5 concep-tuses were dissected free from maternal decidua and Reichert’s membrane leaving the yolk sac, amnion and ectoplacental cone intact. 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.

The embryos were cultured at 37°C 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 phos-phoramidon 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 et al. (1993) and Roques et al. (1993).

Phosphoramidon (N-(-L-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptophan, Sigma, UK) is a natural metabolite produced by Streptomyces tanashiensis which is a specific, competitive inhibitor of the bacterial enzyme thermolysin (Umezawa, 1972). The structure of the active site of thermolysin exibits a high degree of homology with the active site of endopeptidase-24.11, which is itself unique amongst mammalian metalloproteases (Jiang and Bond, 1992), and therefore phosphoramidon efficiently inihibits endopepti-dase-24.11 (KI=2 nM). Phosphoramidon was dissolved in distilled water at 1 mg/ml and diluted to final inhibitor concentrations of 100 μM, 10 μM, 1 μM, 100 nM and 10 nM in diluted rat serum. Control embryos were cultured in diluted rat serum only.

Thiorphan (3-mercapto-2-benzylpropanoylglycine, Sigma, UK) was the first, potent synthetic inhibitor of endopeptidase-24.11 (KI=2.5 nM) (Roques et al., 1980). This was dissolved in 3% ethanol in water in the same concentration range as phosphoramidon. Control embryos were cultured in diluted serum ± 3% ethanol.

Histology

Embryos were dissected from their yolk sac and amnion and fixed in formalin for 24 hours, dehydrated through an ascending ethanol series and embedded in filtered histology-grade wax. Sections were cut at 5 μm and mounted onto APES-coated slides. Sections were then taken through a routine Haematoxylin and Eosin staining procedure, and photographed using TMAX 100 film.

Scanning electron microscopy

Prior to fixation, extraembryonic membranes were removed and the embryos washed in sterile Hanks’ saline. Embryos were fixed in 25% glutaraldehyde, 0.2 M cacodylate buffer, pH 7.2 for 48 hours, rinsed in cacodylate buffer then dehydrated. Embryos were dried in a critical-point drier (Balzer, UK), and then sputter coated (Polaron, UK) with a 60:40 gold-palladium alloy. Specimens were viewed on a Cambridge 90 Stereoscan scanning electron microscope (Cambridge Instruments, UK).

Western blots

Protein bands with molecular weights corresponding to the previously shown values for both endopeptidase-24.11 and endopeptidase-24.18, a single 94 kDa band and two bands at 80 kDa and 74 kDa respectively, were detected in both the rat kidney and E14 embryo membrane preparations (Fig. 1).

Fig. 1.

Western blot analysis of adult kidney microvillar membrane preparation (7 μg total protein/lane), lanes 1 and 3, and an E14 embryo membrane preparation (35 μg total protein/lane), lanes 2 and 4. The endopeptidase-24.11 and endopeptidase-24.18 present in the adult rat kidney are identical to those found in the embryo. Bands are visible at 94 kDa for endopeptidase-24.11 and at 80 kDa and 74 kDa for endopeptidase-24.18.

Fig. 1.

Western blot analysis of adult kidney microvillar membrane preparation (7 μg total protein/lane), lanes 1 and 3, and an E14 embryo membrane preparation (35 μg total protein/lane), lanes 2 and 4. The endopeptidase-24.11 and endopeptidase-24.18 present in the adult rat kidney are identical to those found in the embryo. Bands are visible at 94 kDa for endopeptidase-24.11 and at 80 kDa and 74 kDa for endopeptidase-24.18.

Overnight incubation of both antibodies with the kidney microvillar membrane preparation blocked staining of both kidney and embryo membranes. Similarly, incubation of RRt151 with purified rat endopeptidase-24.18 completely abolished binding of the antibody to the endopeptidase-24.18 bands from kidney and embryo preparations (data not shown). These western blot experiments therefore demonstrated that the antibodies used in this study recognised antigens with identical molecular weights as observed in the adult rat kidney membranes.

Immunohistochemical distribution of endopeptidase-24.11 in the craniofacial region

The tissue localization of endopeptidase-24.11 was studied by an indirect immunoperoxidase method with nickel chloride enhancement where necessary; the results are summarised in Table 1. All positive staining was abolished by preabsorption of RAHE with the rat kidney membrane preparation. Binding in the adult rat kidney was confined to the brush border of the proximal convoluted tubules and Bowman’s capsule (Fig. 2A). At E10 the luminal surface of the gut ectoderm exhibited strong staining. At E12, endopeptidase-24.11 was detectable mainly in mesenchymal component of the medial and lateral nasal processes, the notochord and on the luminal surface of the otocyst epithelium and the branchial arteries. The rest of the craniofacial vasculature exhibited moderately strong 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 basilar and carotid arteries and the basilar sulcus in the pons. At E16, the palate, root of the tongue, choroid plexus, ependymal lining and several discrete sites in the eye and inner ear all exhibited positive staining of various degrees of intensity (see Fig. 3A,B). All the facial vasculature, cartilage and bone including the nasal septum and conchae and Meckel’s cartilage were negative. However, there was a distinct population of positively staining cells in a perichondrial layer enveloping Meckel’s cartilage and other craniofacial skeletal elements (Fig. 2C).

Table 1.

Summary of immunostaining of rat embryos at E12, E14 and E16 for Endopeptidase-24.11 and Endopeptidase24.18 in the craniofacial region

Summary of immunostaining of rat embryos at E12, E14 and E16 for Endopeptidase-24.11 and Endopeptidase24.18 in the craniofacial region
Summary of immunostaining of rat embryos at E12, E14 and E16 for Endopeptidase-24.11 and Endopeptidase24.18 in the craniofacial region
Fig. 2.

(A) Immunolocalization of endopeptidase-24.11 in the adult rat kidney. Positive staining is confined to the brush border of the epithelia lining the proximal convoluted tubules and on the Bowman’s capsule (arrow); Distal convoluted tubules are negative. (B) Immunolocalization of endopeptidase-24.18 in the adult rat kidney. Positive staining is confined to the brush border of the epithelia lining the proximal convoluted tubules. (C) The perichondrial layer (arrowheads) surrounding the developing hyoid cartilage, like all perichondria, exhibit strong endopeptidase-24.11 immunoreactivity at E16; differentiated chondrocytes are negative. (D) At E12, endopeptidase-24.11 is strongly immunolocalized on the luminal surface of the hind gut (G) endoderm and on the notochord (N). The immunoperoxidase staining has been amplified using a DAB-enhancement technique. (E) Positive endopeptidase staining is evident in the mesenchymal stroma and on the apical surface of the epithelia (arrows) covering the choroid plexus at E14. (F) At E14, the luminal surface of the choroid plexus, exhibits strongly positive endopeptidase-24.18 staining, and in contrast to endopeptidase-24.11 immunoreactivity, the stroma is completely negative. Bars, 50 μm.

Fig. 2.

(A) Immunolocalization of endopeptidase-24.11 in the adult rat kidney. Positive staining is confined to the brush border of the epithelia lining the proximal convoluted tubules and on the Bowman’s capsule (arrow); Distal convoluted tubules are negative. (B) Immunolocalization of endopeptidase-24.18 in the adult rat kidney. Positive staining is confined to the brush border of the epithelia lining the proximal convoluted tubules. (C) The perichondrial layer (arrowheads) surrounding the developing hyoid cartilage, like all perichondria, exhibit strong endopeptidase-24.11 immunoreactivity at E16; differentiated chondrocytes are negative. (D) At E12, endopeptidase-24.11 is strongly immunolocalized on the luminal surface of the hind gut (G) endoderm and on the notochord (N). The immunoperoxidase staining has been amplified using a DAB-enhancement technique. (E) Positive endopeptidase staining is evident in the mesenchymal stroma and on the apical surface of the epithelia (arrows) covering the choroid plexus at E14. (F) At E14, the luminal surface of the choroid plexus, exhibits strongly positive endopeptidase-24.18 staining, and in contrast to endopeptidase-24.11 immunoreactivity, the stroma is completely negative. Bars, 50 μm.

Fig. 3.

(A) Central regions of the palatal shelf mesenchyme (asterisks) display positive endopeptidase-24.11 immunoreactivity at E16; nasal septum (NS) is negative, (no counterstain). Bar, 50 μm. (B) At E16 endopeptidase-24.11 is localized on the genioglossus muscle (asterisks) in the root of the tongue; Meckel’s cartilage (M) is 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 (arrows); otic capsule (OC) is negative. Bar, 100 μm. (D) Higher magnification of part of C to show positive immunoreactivity on stria vascularis (SV) and the adjacent part of Reissner’s membrane (RM); the region from where the sensory epithelium (asterisk) will differentiate remains negative. Bar, 50 μm. (E,F) Transverse section through an E16 semicircular canal. (F) Endopeptidase-24.18 is only localized on the surface of the non-sensory cells. (E) An adjacent section incubated with RRt151 which was preabsorbed overnight with purified endopeptidase-24.18 shows that all positive staining has been extinguished. Bar, 50 μm.

Fig. 3.

(A) Central regions of the palatal shelf mesenchyme (asterisks) display positive endopeptidase-24.11 immunoreactivity at E16; nasal septum (NS) is negative, (no counterstain). Bar, 50 μm. (B) At E16 endopeptidase-24.11 is localized on the genioglossus muscle (asterisks) in the root of the tongue; Meckel’s cartilage (M) is 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 (arrows); otic capsule (OC) is negative. Bar, 100 μm. (D) Higher magnification of part of C to show positive immunoreactivity on stria vascularis (SV) and the adjacent part of Reissner’s membrane (RM); the region from where the sensory epithelium (asterisk) will differentiate remains negative. Bar, 50 μm. (E,F) Transverse section through an E16 semicircular canal. (F) Endopeptidase-24.18 is only localized on the surface of the non-sensory cells. (E) An adjacent section incubated with RRt151 which was preabsorbed overnight with purified endopeptidase-24.18 shows that all positive staining has been extinguished. Bar, 50 μm.

Immunolocalization of endopeptidase-24.11 in other tissues

Endopeptidase-24.11 was localized in several other areas across the developmental period studied. At E12 there was intense positive staining within the notochord and on the luminal surface of both the gut (Fig. 2D) and mesonephric epithelium. In addition, the pericardium and endocardial cushions in the heart were strongly positive, whilst the myocardial cells exhibited weaker positive staining. By E14, positive staining was observed in the cells enveloping the vertebrae, epithelial lining of the gut, mesonephros and bronchi, diaphragm, notochord, the dura mater surrounding the spinal cord and the heart. We have no data regarding endopeptidase-24.11 localization in postcranial sites in the E16 rat embryo since only the craniofacial region was investigated at this later stage.

In situ hybridization of endopeptidase-24.11

Gene expression of endopeptidase-24.11 closely matched the distribution of the endopeptidase-24.11 antigen both in location and signal intensity at E12 and E14. Of particular note was the expression in the medial and lateral nasal process mesenchyme, and to a lesser extent on the otocyst epithelium and on the first branchial arches. The expression throughout the notochord was extremely intense at this stage (Fig. 4B).

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.

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.

At E14, the strongest expression in the craniofacial region was in the oral and nasal tissues (Fig. 4C), and the choroid plexus was also positive (Fig. 4D). Many other postcranial sites were strongly positive (Fig. 4A) including the lungs, mesonephros, intestine, notochord and surrounding the vertebrae. The craniofacial vasculature expression at both stages was not appreciably intense. The sense control gave no positive signal and extremely low background.

Distribution of endopeptidase-24.18 in the craniofacial region

The tissue distribution localization of this enzyme was studied by both indirect immunoperoxidase and immunofluorescence. When sections of adult rat renal cortex were stained (as a positive control), staining was restricted to the brush border of the proximal convoluted tubules (Fig. 2B).

Immunolocalization of endopeptidase-24.18 in the developing head and face was confined to discrete sites with a distribution which seems to be temporally regulated; this is summarised in Table 1. All positive staining disappeared when RRt151 was preabsorbed with either rat kidney microvilli or purified endopeptidase-24.18 (Fig. 2E,F). E10 embryos displayed no detectable staining for this enzyme. In older embryos, the luminal surface of the neuroepithelium demonstrated a particularly striking pattern of distribution, and this was best exemplified by the choroid plexus and the ependymal lining of the developing brain ventricles (Fig. 2F). The intensity of staining on the epithelial cells lining the choroid plexus decreased between E14 and E16. The developing lens and pigmented layer of the retina exhibited moderately positive staining at E16, but earlier embryos displayed only negligible staining at these locations.

The most striking pattern of endopeptidase-24.18 distribution was that displayed in the developing inner ear. At E12, there was a diffuse and moderately intense positive stain on the apical surface of the cells lining the otic vesicle. By E14 the positive staining appeared considerably stronger, specifically localized to the stria vascularis and not detectable on the developing hair cells, Organ of Corti or the tectorial membrane. This distribution pattern is still apparent in the cochlea at E16 (Fig. 3C-F). In some E16 embryos, the epithelia lining the oral and nasal cavities showed a variable distribution of endopeptidase-24.18. A few other postcranial sites in the E14 embryos stained positively for endopeptidase-24.18, notably in the bladder, ureter and gut.

Perturbation studies

The results of the perturbation studies are summarized in Tables 2, 3 and 4. Following culture with two inhibitors of endopeptidase-24.11, SEM revealed that a proportion of the inhibitor-treated E9.5 embryos displayed an asymmetric, predominantly left-sided, facial deformity which appeared to be due to the presence of a haematoma-like swelling adjacent to the prosencephalon. This often had the effect of distending the left side of the head which in turn disrupted the normal positioning of the first and second branchial arches. In the most severe cases, observed at the highest inhibitor concentrations, the swelling was so great that the anterior neural folds could not close. Cultured E9.5 embryos are shown in Fig. 5A-D. Control embryos, cultured in diluted serum only, developed normally. The severity of the defect, but not the incidence, generally increased as inhibitor concentration increased. In addition to these craniofacial effects, the heart appeared dis-proportionately swollen, in a midline position and seemed not to have undergone normal looping morphogenesis.

Table 2.

Results of phosphoramidon exposure on E9.5 rat embryos in vitro

Results of phosphoramidon exposure on E9.5 rat embryos in vitro
Results of phosphoramidon exposure on E9.5 rat embryos in vitro
Table 3.

Results of phosphoramidon exposure on E10.5 rat embryos in vitro

Results of phosphoramidon exposure on E10.5 rat embryos in vitro
Results of phosphoramidon exposure on E10.5 rat embryos in vitro
Table 4.

Results of thiorphan exposure on E9.5 rat embryos in vitro

Results of thiorphan exposure on E9.5 rat embryos in vitro
Results of thiorphan exposure on E9.5 rat embryos in vitro
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 failure of neural fold fusion over forebrain (arrow) Bar, 200 μm. (C) E9.5 embryo cultured for 48 hours in serum containing 100 nM phosphoramidon. Note complete failure of cephalic neural folds closure and lateral distension of the left side of the head (arrow). Bar, 100 μm. (D) E9.5 embryo cultured for 48 hours in serum containing 100 μM phosphoramidon; note open neural tube, a general asymmetry to the head and the swelling (arrow) of the telencephalic neuroepithelium into the lumen, caused by a subadjacent haematoma. The normal looping morphogenesis of the heart (asterisk) has also clearly been disturbed. Bar, 100 μm.

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 failure of neural fold fusion over forebrain (arrow) Bar, 200 μm. (C) E9.5 embryo cultured for 48 hours in serum containing 100 nM phosphoramidon. Note complete failure of cephalic neural folds closure and lateral distension of the left side of the head (arrow). Bar, 100 μm. (D) E9.5 embryo cultured for 48 hours in serum containing 100 μM phosphoramidon; note open neural tube, a general asymmetry to the head and the swelling (arrow) of the telencephalic neuroepithelium into the lumen, caused by a subadjacent haematoma. The normal looping morphogenesis of the heart (asterisk) has also clearly been disturbed. Bar, 100 μm.

The E10.5 embryos also displayed an inhibitor dosedependent abnormal left-sided prosencephalic swelling (Fig. 6A-D). None of the E10.5 embryos had open anterior neural folds but the branchial arches were often displaced asymmetrically and of abnormal appearance, whilst after exposure at this later stage, the heart morphogenesis appeared grossly normal. Our assessment of relative normality was based upon the following criteria; yolk sac diameter and circulation, crown-rump length, turning and presence of a beating heart compared with the control embryos.

Fig. 6.

(A) E10.5 rat embryo cultured for 45 hr in serum containing 10 nM phosphoramidon. Embryo appears morphologically normal; lateral view. Bar, 200 μm. (B) E10.5 rat embryo cultured for 45 hours in serum containing 100 nM phosphoramidon. Note the swollen 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 displacement of the left mandibular arch (arrowhead). Frontal view. Bar, 100 μm. (C) E10.5 rat embryo cultured for 45 hours in serum containing 1 μM phosphoramidon; note the grossly abnormal left side to the head with a swollen dysmorphic first arch and a distorted second arch. Lateral view. Bar, 200 μm. (D) E10.5 rat embryo cultured for 45 hours in serum containing 10 μM phosphoramidon; Note the relatively distended left side of the prosencephalon (asterisk) and the asymmetry of the first branchial arch, similar to B. Frontal view. Bar, 100 μm.

Fig. 6.

(A) E10.5 rat embryo cultured for 45 hr in serum containing 10 nM phosphoramidon. Embryo appears morphologically normal; lateral view. Bar, 200 μm. (B) E10.5 rat embryo cultured for 45 hours in serum containing 100 nM phosphoramidon. Note the swollen 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 displacement of the left mandibular arch (arrowhead). Frontal view. Bar, 100 μm. (C) E10.5 rat embryo cultured for 45 hours in serum containing 1 μM phosphoramidon; note the grossly abnormal left side to the head with a swollen dysmorphic first arch and a distorted second arch. Lateral view. Bar, 200 μm. (D) E10.5 rat embryo cultured for 45 hours in serum containing 10 μM phosphoramidon; Note the relatively distended left side of the prosencephalon (asterisk) and the asymmetry of the first branchial arch, similar to B. Frontal view. Bar, 100 μm.

Subsequently, histological analysis of embryos treated at E9.5 revealed that the swelling was typically the result of both a localised overgrowth of the prosencephalic neurectoderm on the left side (Fig. 7A) and a gross distension of the internal carotid artery on the same side (Fig. 7B). More caudally, this vascular disturbance extended to include the first branchial artery, which like the internal carotid arises from the dorsal aorta, and displayed distension. A presumed secondary effect of this was the lateralward displacement of the first branchial arch (Fig. 7C) as seen in the SEM.

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).

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).

Protein and mRNA distributions

This study is the first report describing 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; LlorensCortes 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. The substantial conservation of 5′ untranslated regions between different species and the existence of 5′ alternative splicing suggest that endopeptidase-24.11 gene expression may be differentially controlled in a tissue-specific and/or developmentally regulated manner. We have therefore studied the distribution of endopeptidase-24.11 mRNA using a radiolabelled full-length complementary RNA probe, containing sequences common to all known transcripts, to assess the possibility of transcriptional control occurring during embryogenesis. In situ hybridisation reveals the regional presence of message as early as E12, and reverse transcriptase-PCR analysis has enabled the detection of message as early as E8 and indicates the existence of a novel, embryo-specific spliced variant (work in progress).

The present investigation substantiates the previous work of others, in which the various locations of endopeptidase-24.11 in the adult rat central and peripheral nervous systems are described employing both immunocytochemical (Ronco et al., 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 indirectly by the binding of a selective tritiated inhibitor of endopeptidase-24.11, [3H]HACBO-Gly, (Dutriez et al., 1992). A limited range of selected and unrelated tissues have been studied, namely microvilli from human placental syncytiotrophoblast; foetal rabbit, rat and human small intestinal brush border membranes; and nuchal ligaments from late-stage fetal calves (Johnson et al., 1984, 1990; Lecavalier et al., 1989). The distribution patterns described here extends significantly the onset of endopeptidase-24.11 expression back into much earlier development and demonstrates, for the first time, an embryonic presence of endopeptidase-24.18.

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, vasoactive intestinal peptide, enkephalins, calcitonin-gene-related peptide and Neuropeptide 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 Stephenson, 1988). High densities of ANP receptors have been localized autoradi-ographically at these sites (Tong and Pelletier, 1990), which closely matches the distribution of both endopeptidase-24.11 and 24.18.

In the developing lung, the endopeptidase-24.11 substrate may be substance P (Shepherd et al., 1988) or, more likely, the bombesin-like peptides (Shipp et al., 1991). Indeed, it has recently been reported that the hydrolysis of the bombesin-like peptides by endopeptidase-24.11 could control the rate of murine foetal lung maturation (King et al., 1993). The palatal localization of endopeptidase-24.11 might reflect the presence of TGFα or epidermal growth factor in the palatal mesenchyme (Dixon et al., 1991), although cleavage of either of these growth factors by endopeptidase-24.11 has yet to be demonstrated. However, TGFα is a known substrate of endopeptidase-24.18 (Choudry and Kenny, 1991). A recent study demonstrated that TGFβ1 could down-regulate endopeptidase-24.11 activity via a reduction in the levels of the transcribed gene or possibly by decreasing endopeptidase-24.11 mRNA stability (Casey et al., 1993). Indeed, it has been proposed that the role of TGFβ1 during chondrocyte differentiation is to regulate the expression of the matrix proteins and metalloproteases (Ballock et al., 1993). Such a relationship might explain the distribution of endopeptidase-24.11 that we have observed surrounding the developing skeletal elements in the older embryos.

Many of the regions where we have immunolocalized endopeptidase-24.18 in the rat embryo, particularly on the choroid plexus and in the inner ear, are sites of ion transportation. This enzyme may, therefore, have an important role in the production of cerebrospinal fluid and endolymph respectively during mammalian craniofacial development. In the adult rat, comparison of the distribution patterns of these two neutral metallo-endopeptidases reveals a considerable difference, in terms of the number and variety of organs where these enzymes have been detected (Ronco et al., 1988; Barnes et al., 1989). This correlates with the comparative paucity of endopeptidase-24.18 distribution in the embryo compared to the relatively greater distribution of endopeptidase-24.11.

Endopeptidase-24.18 and the astacin family

Endopeptidase-24.18 is an unconventional peptidase in that it is an oligomeric tetramer of subunits linked by disulphide bonds. Initial research on the cloning and sequencing of the amino terminus of endopeptidase-24.18 identified it as a member of the ‘astacin family’ of metallo-endopeptidases (Dumermuth et al., 1991). All the enzymes in this family have a zinc-binding metalloprotease domain which shares a high degree of homolgy with the domain found in astacin (EC 3.4.24.21), a protease from the crayfish Astacus fluviatilis (Shimell et al., 1991). Other enzymes attributed to this family are ‘PABA-peptide hydrolase’ (Sterchi et al., 1982) and BMP-1, bone morphogenetic protein-1 (both in humans), UVS.2 (in Xenopus), the tolloid gene product (in Drosophila) (Finelli et al., 1994), and suBMP, blastula protein 10 and SpAN proteins (in sea urchin) (Lepage et al., 1992; Reynolds et al., 1992; Hwang et al., 1994). Since the members of the astacin gene family are present in a wide variety of organisms and are conserved, they are likely to have similar functions. The conclusion that they are involved in controlling the activity of growth factors, and thus are vital for specifying cell determination, developmental and differentiation events (Dumermuth et al., 1991), therefore strengthens our contention that the cell-surface metallo-endopeptidases play a major role in embryogenesis.

Endopeptidase-24.11 and craniofacial development

Here we have successfully shown the localization of both endopeptidase-24.11 and 24.18 protein in postimplantation rat embryos. Both enzymes appear to be developmentally regulated at several discrete loci and the patterns of distribution of these enzymes and their putative substrates strongly suggest that they play a significant role during craniofacial development in the rat. In order to explore a possible functional role, we have carried out inhibition studies. The use of two different selective inhibitors of endopeptidase-24.11 phospho-ramidon and thiorphan, in whole embryo culture consistently resulted in the formation of an asymmetric facial lesion in a proportion of embryos. Significantly, the specific anomalies observed resemble those found in the human birth defect hemifacial microsomia or ‘oculo-auriculo-vertebral spectrum’ (Gorlin et al., 1990). Hemifacial microsomia is asymmetric, 70% of cases exhibit unilateral deformities in the facial skeleton, presenting as hypoplasia and malformation, particularly in the mandibular, auditory, maxillary and zygomatic bones. Possible causes of this defect include haemorrhaging from the primordial stapedial artery following teratogen insult (Poswillo, 1973) and perturbation of auriculofacial chondro-genesis (Cousley and Wilson, 1992). Children born with hemifacial microsomia are also highly likely to suffer from skeletal abnormalities, various forms of heart disease, pulmonary and renal anomalies. Significantly, all of these locations are sites of intense endopeptidase-24.11 immunolocalization in the embryonic and neonatal rat. Furthermore, the histological findings demonstrating a vascular disturbance involving the internal carotid and first branchial arch arteries argue strongly for a similar aetiology in this system. The involvement of a substrate which is a vaso-active peptide is an interesting possibility and the asymmetry of effect may well reflect an inherent asymmetry in the development of the head vasculature. Therefore, rat embryos cultured in the presence of endopeptidase-24.11 inhibitors may be a possible animal model not only for hemifacial microsomia, but also for analysis of branchial arch morphogenesis and arterial arch development in particular..

Endopeptidase-24.11 shares several substrates, notably angiotensin I and bradykinin, with a related enzyme ‘angiotensin-converting enzyme’, also known as peptidyl dipeptidase-A. Recently it has become apparent that the use of angiotensin-converting enzyme inhibitors to treat hypertension during pregnancy can result in significantly increased incidence of foetal abnormalities and mortality (Mehta and Modi, 1989; Hanssens et al., 1991; Brent and Beckman, 1991). Moreover, the fact that secretion of one endopeptidase-24.11 substrate, atrial natriuretic peptide, is stimulated by the endothelial vasoconstrictor endothelin-1 (Fukada et al., 1988), and that the endothelin-1 knock-out mouse displays craniofacial and cardiovascular abnormalities (Kurihara et al., 1994), furher implicates endopeptidase-24.11 function as a key component of normal development. Clearly, it is appropriate now to reevaluate the possible teratogenicity of pharmaceutical endopeptidase-24.11 inhibitors being developed as analgesics and anti-hypertensive agents.

The disturbance in morphogenesis, which appears to be a consequence of endopeptidase-24.11 inhibition during culture, leads us to believe that this enzyme plays a critical role during normal craniofacial development. This raises the possibility that cell-surface metallo-endopeptidases, such as endopeptidase-24.11, may constitute a hitherto unrecognised level of control through the cleavage and inactivation of biologically active small peptides, growth factors and cytokines in craniofacial tissues.

We would especially like to thank the Collaborations Dept. at Genentech Inc. California, USA for their generous gift of rabbit antihuman enkephalinase antibody and the rat endopeptidase-24.11 cDNA, Professor R. C. Atkins, Director of Nephrology at the Monash Medical Centre for generously sending us the PHM-6 antibody, Nicky Mordan for her technical help and advice with the SEM, Dr Andrew Copp for advice on embryo culture, Dr Nick Lench for advice on probe preparation and Ms Monique Doherty for technical assistance. This work was supported by a grant from the Medical Research Council to B. H. and P. T.

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