SSAO/VAP-1 protein expression during mouse embryonic development

Authors

  • Tony Valente,

    Corresponding author
    1. Departament de Bioquimica i Biologia Molecular, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain
    • Departament de Bioquimica i Biologia Molecular, Institut de Neurociències, Facultat de Medicina, Torre M2, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
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  • Montse Solé,

    1. Departament de Bioquimica i Biologia Molecular, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain
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  • Mercedes Unzeta

    1. Departament de Bioquimica i Biologia Molecular, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain
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Abstract

SSAO/VAP-1 is a multifunctional enzyme depending on in which tissue it is expressed. SSAO/VAP-1 is present in almost all adult mammalian tissues, especially in highly vascularised ones and in adipocytes. SSAO/VAP-1 is an amine oxidase able to metabolise various endogenous or exogenous primary amines. Its catalytic activity can lead to cellular oxidative stress, which has been implicated in several pathologies (atherosclerosis, diabetes, and Alzheimer's disease). The aim of this work is to achieve a study of SSAO/VAP-1 protein expression during mouse embryogenesis. Our results show that SSAO/VAP-1 appears early in the development of the vascular system, adipose tissue, and smooth muscle cells. Moreover, its expression is strong in several epithelia of the sensory organs, as well as in the development of cartilage sites. Altogether, this suggests that SSAO/VAP-1 enzyme could be involved in the differentiation processes that take place during embryonic development, concretely in tissue vascularisation. Developmental Dynamics 237:2585–2593, 2008. © 2008 Wiley-Liss, Inc.

INTRODUCTION

Semicarbazide Sensitive Amine Oxidase [E.C.1.4.3.6, oxidoreductase (deaminating) (copper-containing), SSAO], also known as vascular adhesion protein-1 (VAP-1) (Salmi and Jalkanen,1992), constitutes a family of enzymes present in almost all mammalian species (Lewinshon,1984). SSAO/VAP-1 is encoded by the amine oxidase cooper-containing gene, AOC3 (Jalkanen and Salmi,2001). SSAO/VAP-1 is a homodimeric 180-kDa glycoprotein, mainly located in fat and highly vascularised tissues (endothelial and smooth muscle cells). SSAO/VAP-1 metabolizes primary amines, generating hydrogen peroxide (H2O2), ammonia (NH3), and the corresponding aldehyde (Lyles,1996). Aminoacetone and methylamine are considered physiological SSAO/VAP-1 substrates (Precious et al.,1988). SSAO/VAP-1 is considered a multifunctional enzyme whose function varies depending on the tissue where it is expressed (O'Sullivan et al.,2004). Moreover, plasma SSAO/VAP-1 activity is altered in several pathological conditions: diabetes type I and II (Boomsma et al.,1995), congestive heart failure (Boomsma et al.,1997), non-diabetic obesity (Mészáros et al.,1999), atherosclerosis (Grönvall-Nordquist et al.,2001; Karádi et al.,2002), inflammatory liver diseases (Kurkijärvi et al.,2000; Garpenstrand et al.,1999; Salmi et al.,1993), and Alzheimer's disease (del Mar et al.,2005). Moreover, SSAO/VAP-1 is found up-expressed in cerebrovascular amyloid angiopathy (AD-CAA) (Ferrer et al.,2002). Recently, Salmi and Jalkanen (2006) described the presence of SSAO/VAP-1 protein in some foetal human tissues. However, little is known about the expression of this protein during the development of mammalian embryos. The aim of this study is to characterize the expression pattern of SSAO/VAP-1 protein during the development of the mouse embryo.

RESULTS AND DISCUSSION

Specificity of the hSSAO/VAP-1 and bSSAO/VAP-1 Antibodies

First, we compared the specificity of two different types of SSAO/VAP1 antibodies. bSSAO/VAP-1 is a rabbit polyclonal antibody raised against bovine SSAO/VAP-1 from lung microsomal membranes produced in our laboratory (Lizcano et al.,1998), whereas hSSAO/VAP-1 is a commercial antibody raised against the C-terminal extracellular domain of human SSAO/VAP-1 (H-43, Santa Cruz Biotechnology). When bSSAO/VAP-1 antibody was used, two molecular forms were detected in the lysates of the rat and mouse embryos: the trimeric glycoprotein (250–265 kd band) and the monomeric protein (90–100 kd band) (Fig. 1A, lane 1, rat embryo lysate, and lane 2, mouse embryo lysate). The same results were obtained when the bSSAO/VAP-1 antibody was used (Fig. 1A, lane 4, rat embryo lysate, and lane 5, mouse embryo lysate). The stable rat A7r5 cell line that overexpressed human SSAO/VAP-1 was used as positive control (Sole et al.,2007). In this case, both antibodies strongly detected the 90–100-kd band (Fig. 1A, lanes 3 and 6). All bands observed were correlated with those previously reported by Jaakkola et al. (1999) using human lysates from muscle samples and the human SSAO/VAP-1 antibody produced by themselves. Under immunohistochemical analysis, both antibodies showed the same expression pattern during mouse embryonic development (Fig. 1B). However, the intensity of the immunostaining was higher with the bovine antibody (Fig. 1B, b1, b3, and b5), than the human SSAO/VAP-1 antibody (Fig. 1B, b2, b4, and b6). In addition, in rat embryo sections similar SSAO/VAP-1 expression pattern was observed (data not shown). Moreover, no staining was detected when SSAO/VAP-1 antibodies were preabsorbed with the SSAO/VAP-1 (see Fig. 4B) peptide or when SSAO/VAP-1 was replaced by foetal bovine serum (Fig. 2J). These data allow us to conclude that in mammalians both antibodies b-SSAO/VAP-1 and h-SSAO/VAP-1 detected specifically the same protein: SSAO/VAP-1. Moreover, in embryonic mouse sections, the expression pattern of SSAO/VAP-1 observed is the same with both antibodies. The expression of SSAO protein during mouse embryonic development was described in detail in the Table 1.

Figure 1.

Immunospecificity of SSAO/VAP-1 antibodies by immunoblot and immunohistochemistry techniques. A: Immunoblot of SSAO/VAP-1 antibodies in human, rat, and mouse lysates. bSSAO/VAP-1 antibody (Lizcano et al.,1998) strongly detected two forms of SSAO/VAP-1 protein, the trimeric (250 kd) and monomeric (100 kd) in the lysates from rat and mouse whole embryos (lanes 1 and 2, respectively). In adult mouse spinal cord lysate, only the monomeric form was weakly detected by bSSAO/VAP-1 antibody (lane 3). The monomeric form was the main form shown (lane 4) when using bSSAO antibody in the human A7r5 stable cell line that overexpresses the human SSAO/VAP-1. The hSSAO/VAP-1 antibody (H-43, Santa Cruz Biotechnology), strongly detected the monomeric form of SSAO/VAP-1 protein, whereas in rat and mouse whole embryo lysates, only a weakly trimeric form was detected (lanes 5 and 6). Similar bands were observed in adult mouse spinal cord lysate (lane 7). In the A7r5 SSAO/VAP-1 cell line lysate, the trimeric and monomeric forms were strongly detected by hSSAO/VAP-1 antibody (lane 8). B: Immunohistochemistry of SSAO/VAP-1 antibodies in mouse embryo sections. Both bSSAO/VAP-1 and hSSAO/VAP-1 antibodies are strongly expressed in the choroids plexus of the fourth ventricle (B1 and B2, respectively), in the inner ear (B3 and B4, respectively), and in the heart (B5 and B6, respectively) of mouse embryos after 14 days of gestation. C: bSSAO/VAP-1 Western blotting in heart, liver, lung, and gut from embryonic and adult mice lysates. Low levels of SSAO/VAP-1 protein are found in embryo tissues whereas high levels are observed in adult tissues. These levels of SSAO/VAP-1 protein are correlated with their enzymatic activity (see bottom panel). A: Lanes: 1 and 5, rat whole embryo lysates; 2 and 6, mouse whole embryo lysates; 3 and 7, adult mouse spinal cord lysates; 4 and 8, A7r5 SSAO/VAP-1 cell line lysates. B: Scale bar = 50 μm. Cch, cochlea; cTem, cartilage of temporal bone; wrv, wall of right ventricle of the heart.

Figure 4.

SSAO/VAP-1 protein expression in the smooth muscle cells, chondrocytes and adipocytes. At embryonic day 17.5, ED17.5, the cutaneous muscle of the skin and many cells of the outer layer of skin expressed SSAO/VAP-1 (A). No SSAO/VAP-1 expression is found in the skin when the SSAO/VAP-1 antibody is preadsorbed with SSAO/VAP-1 peptide (B). Strong expression of SSAO/VAP-1 protein is detected in the adipose tissue of the dorsal part of the embryo at 17.5 days of gestation (C). SSAO/VAP-1-positive chondrocytes are observed at ED12 (arrowheads in D) only in the cartilage of vertebrae and ribs, whereas in the subsequent developmental stages, this immunolabelling progressively decreased in the differentiating chondrocytes (D, E) and at ED17.5 only some resting chondrocytes expresses SSAO/VAP-1 protein (arrowheads in F). Strong SSAO/VAP-1 expression is detected in the perichondrium cells of these cartilage sites at early embryonic stages (white solid arrows in D and E). At ED17.5, many smooth muscle cells of the gut are strongly immunolabelled for SSAO/VAP-1 protein (arrows in G, H). Strong expression of SSAO/VAP-1 protein is also observed in the umbilical vein (solid arrow in G), in the vibrissae follicles (arrows in I), and in the face muscle cells that surround the vibrissae follicles (solid arrows in I). Sagital sections. Scale bar = 50 μm. Aor, aorta; at, adipose tissue; cm, cutaneous muscle cells; cpr, cartilage primordium of ribs; g, gut; li, liver; ols, outer layer of skin; or, ossification of ribs.

Figure 2.

Expression pattern of SSAO/VAP-1 protein in the vascular system of mouse embryo. SSAO/VAP-1 is expressed in the heart of immunohistological sections of embryonic day 9, ED9 (A), 12 (B), and 14 (C). In the wall of ventricles (D, E, G, I), SSAO protein is detected weakly at embryonic day 9, ED9 (D), strongly between ED12 and 14 (E, G), and moderately at ED17.5 (I). At ED12 and ED14, strong expression of SSAO/VAP-1 protein is detected in the wall of aorta (F and H, respectively). No immunostaining is found when the primary antibody is replaced by foetal bovine serum (J). SSAO/VAP-1 is found in liver vessels of embryos of 17.5 days of gestation (K,L). Immunostaining for MECA-32 in liver vessels at ED17.5 (M). In the eye, many immunostained SSAO/VAP-1 cells were observed in the vascular hyaloid layer of the retina (arrows in N,O). Sagital sections. Scale bar = 50 μm in A–O. Aor, aorta; bvc, bulbo-ventricular canal; bvj, bulbo-ventricular junction; emc, eye muscle cells; hv, hyaloid vascular cells; le, lens; llv, lumen of left ventricle; lrv, lumen of right ventricle; lv, lung vessels; mz, marginal zone of spinal cord; nlr, neural layer of retina; otrv, outflow tract of right ventricle (pulmonary trunk leading to ductus arteriosus); rhs, right horn of sinus venosus; smg, submandibular gland; ta, truncus arteriosus; tst, tissue of septum transversal; whb, wall of heart at bulbo-ventricular junction; wlv, wall of left ventricle; wrv, wall of right ventricle.

Table 1. SSAO/VAP-1 Expression Pattern in Abryonic Tissues
Embryonic areaEmbryonic day
ED9ED12ED14ED17.5
  • a

    Sensory organs.

Vascular system    
 Heart    
  Wall of ventricular chamber of heart++++++++
  Lumen of ventricular chamber of heart+/−+/−
 Umbilical vein ++++++
 Umbilical artery +/−++
 Aorta ++++
 Lung    
  Vessels +/−
  Artery +
 Brain    
  Hypothalamus  +/−+
  Optic chiasma  +/−+
  Choroid plexus differentiating from roof of fouth ventricle ++++
 Pituitary gland    
  Infundibulum of pituitry (future pars nervosa) ++++ 
  Vascular differentiation in anterior wall of Rathke's pouch ++++ 
 Eyea    
  Hyaloid artery  +/−+
Cartilage    
 Cartilage primordium of head 
 Cartilage primordium of limbs 
 Cartilage primordium of ribs and vertebrae ++++/−
 Ossification sites of ribs and vertebrae +/−
Epithelia of sensory organs    
 Cochlear sensory epithelium ++++++
 Olfactory epithelium +++++++
 Tongue epithelium  +/−++
Digestive system    
 Epithelium of lips  +/−++
 Epithelium of oropharynx  +/−++
 Stomach  
 Liver  
 Gut    
  Lumen  +/−
  Smooth muscle  +/−++
Adipose tissue  +++
 Skina    
  Cutaneous muscle  ++
  Outer layer  +++

Low levels of SSAO/VAP-1 Protein Are Found Early in Embryonic Development

SSAO begins to express weakly in early embryonic development, at ED9 (Fig. 2A and Table 1). Its expression in embryos reaches moderate levels between ED12 and ED14 (Fig. 2B,C and Table 1). However, although its expression during embryonic development is weak to moderate, in adult its expression is very high. This is confirmed by Western blot analysis using heart, gut, and lung lysates from both ED14 and adult samples (Fig. 1C). Moreover, we used the same lysates for measurement of SSAO/VAP-1 enzyme activity and this activity in embryonic development was very low (Fig. 1C). The low levels of SSAO/VAP-1 protein during embryogenesis compared with the high levels detected in adult mice are correlated with the corresponding activities. The SSAO/VAP-1 activities from different embryonic and adult mouse tissues reported here are similar to those described by Salmi and Jalkenen (2006) in human foetal tissues. SSAO/VAP-1 is expressed early during embryogenesis and its expression is moderate in embryos with 14 days of gestation as observed by immunohistochemistry. Therefore, the early expression of the SSAO/VAP-1 in embryos doesn't seem to be only related to their enzymatic activity, suggesting that the SSAO/VAP-1 could have another role in development not yet described. Further studies will be necessary to reveal the role of SSAO/VAP-1 enzyme in early development.

Heart and Vascular Associated System

In general, the expression of SSAO/VAP-1 in embryonic development is very low on embryonic day 9, ED9, and moderate on embryonic day 12 and 14, ED12 and ED14 (Fig. 2A–C). In lower magnifications, SSAO/VAP-1 protein was detected at ED12 (Fig. 2B); however, at higher magnifications its expression was found at ED9 (Fig. 2D). Thus, low levels of SSAO/VAP-1 protein were detected in the myocardial progenitor cells of the ventricular wall of the heart at embryonic day nine, ED9 (Fig. 2D). At this embryonic stage, the heart is in a differentiated process. The outflow tract of the heart is differentiating and at this stage it is possible to distinguish the aortic-pulmonary septum formation. At ED12, the expression of SSAO/VAP-1 protein increases in the heart (Fig. 2E), when the differentiation of the vascular system begins to define the asymmetry of the branchial (pharyngeal and aortic) arch arterial system, and in this case it is possible to see the aorta (Fig. 2F). During subsequent embryonic stages, the vessels of the ascending aorta and the pulmonary trunk are very well defined and the pulmonary circulation is established. Thus, at ED14, the immunolocalization of SSAO/VAP-1 protein was found in the heart (Fig. 2G), aorta, and pulmonary vessels (Fig. 2H), as well as in the vessels and arteries of the lung (Fig. 2K,L). Moreover, this expression of SSAO/VAP-1 in the vessels and arteries co-localize with MECA-32, a common endothelial cell marker, as it was observed in the lung (Fig. 2L,M). At ED17.5, the expression of SSAO/VAP-1 decreases in the heart (Fig. 2I) and it is located in the endothelial layer and in the vascular smooth muscle cells. No staining was observed in the heart when the SSAO/VAP-1 antibody was replaced by foetal bovine serum (Fig. 2J). On the other hand, many vascular smooth muscle cells exhibit a wide range of different phenotypes at different stages of development and these cells are not completely differentiated in adult organisms (Owens et al.,1996; Owens and Wise,1997). During embryonic development, the SSAO/VAP-1 protein is expressed parallel to the myogenesis processes of the vascular smooth muscle cells, and its expression persists in adult mammalian tissue, where SSAO/VAP-1 plays an important role in the vascular system (Ochiai et al.,2005; Jaakkola et al.,1999; Castillo et al.,1998; Salmi et al.,1993). Under inflammatory conditions in endothelial cells, SSAO/VAP-1 behaves as an inducible vascular adhesion protein involved in lymphocyte trafficking (Smith et al,1998). Moreover, SSAO/VAP-1 has been described as being most active in the vascular smooth muscle cells and endothelial cells of the mammalian adult aortic wall (Langford et al.,1999). Functional study of arteries from adult SSAO/VAP-1 KO mice suggests that SSAO/VAP-1 might contribute to arterial wall remodelling (Mercier et al.,2006). Therefore, the expression of SSAO/VAP-1 protein in embryos seems to follow vasculogenesis of the heart and the associated vascular system, which suggests that this protein could play a role during the cardiogenesis. Further experimental work is necessary in order to confirm the role of SSAO/VAP-1 in heart and vascular system development.

Smooth Muscle

In the smooth muscle tissue, the expression of SSAO/VAP-1 appeared weakly at ED12 in the skin and moderately at ED14 in the gut and tail. However, the high levels of SSAO/VAP-1 were observed in the smooth cells of the digestive tract and oesophagus at ED16 (Fig. 3E), and in lower levels at ED17.5. Moreover, at this embryonic stage moderate levels of SSAO/VAP-1 protein were detected in the smooth muscle cells of the lips (Fig. 3H,I), skin (Fig. 4A and I), and gut (Fig. 4G,H), as well as in the smooth muscle cells of the tail (Fig. 3A), abdominal cavity, and reproductive organs (data not shown). No staining was found in the smooth muscle cells when SSAO/VAP-1 antibody is preabsorbed previously with SSAO/VAP-1 peptide (Fig. 4B). Moreover, smooth muscle cells of the tail co-expressed SSAO/VAP-1 and nestin (Fig. 3A–C) at ED12, while the smooth muscle cells of the lips co-expressed SSAO/VAP-1 and vimentin (Fig. 3C,D) at ED14. Nestin is a marker of multi-lineage progenitor cells (neural progenitors, myogenic cells, epithelial cells, and so on) that include smooth muscle progenitor cells (Wiese et al.,2004), and vimentin is an intermediate filament highly expressed during the differentiation process of smooth muscle cells (Van Muijen et al.,1987; Pixley et al.,1984). Smooth muscle precursor cells mostly derive from the mesoderm (gut), but also from the neuroectoderm (skin and tail) and endoderm (lips). The growth and differentiation of smooth muscle cells start at different times in different organs and many growth factors and neurohumoral agents regulate smooth muscle growth and differentiation. SSAO/VAP-1 expression during the growth and differentiation of smooth muscle from different tissues could be related to the myogenesis of smooth muscle.

Figure 3.

Expression of SSAO/VAP-1 protein in the epithelia of nasal and oral cavities in mouse embryos. At ED12, the smooth cells of the tail co-expressed SSAO/VAP-1 and nestin (A and B, respectively). At ED14, the smooth cells of lips co-expressed SSAO/VAP-1 and vimentin (arrowheads in C and D). At ED16, the expression of SSAO/VAP-1 is very strong in the smooth cells of the digestive tract, oesophagus (E), and its expression decrease at ED17.5 (F). At ED17.5, SSAO/VAP-1 is detected in the epithelium of the nasal cavity (G) and in the epithelial layer of lips and tongue (white and black solid arrows, respectively, in H). Similar expression is observed in the smooth muscle cells of the most internal part of lips (arrows in I). Sagital sections. Scale bar = 50 μm. el, epithelium of lip; ne, nasal epithelium; orp, orporopharynx.

Adipose Tissue

The expression of the SSAO/VAP-1 protein is observed weakly in adipose tissue of embryos with 14 days of gestation. At ED17.5, the SSAO/VAP-1 protein is strongly expressed in the embryonic adipose tissue (Fig. 4C), at a developmental stage where high adipocyte differentiation occurs. In adults, high levels of SSAO/VAP-1 protein were observed at the caveolae of differentiating and mature adipocytes (Souto et al.,2003) and could be involved in triggering terminal adipocyte differentiation (Fontana et al.,2001). In adults, SSAO/VAP-1 activity stimulates glucose transport by mimicking the insulin effect (Enrique-Tarancón et al,1998), Therefore, the SSAO/VAP-1 could play a role in differentiating adipocytes during development just as it does in adults.

Sensory Organs

In the eye, the expression of SSAO/VAP-1 protein was found weakly in the vascular cells of the hyaloid cavity surrounding the internal part of the neural layer of the retina at ED14, and increased at ED17.5 (Fig. 2K,L). During development, the angiogenic mesenchyme forms the hyaloid artery and vein, which later become the central artery and vein of the retina (Lang,1997), and during later embryonic stages both were immunolabelled with SSAO/VAP-1. We previously described SSAO/VAP-1 activity in the optic nerve, choroid, iris, and retina of adult bovine eyes, and the role of this enzyme in the metabolism of dopamine in retinal tissue (Fernandez de Arriba et al.,1991). Therefore, SSAO/VAP-1 enzyme could be required for the vascularisation that takes place during eye development. In the nasal and oral cavities, the expression of SSAO/VAP-1 protein was detected in several epithelia of the embryo between 14 and 17.5 days of gestation (Fig. 3). SSAO/VAP-1 immunostaining was found in the epithelial cells of the marginal layer of the oral cavity, mainly in the lips (Fig. 3H,I), where during development the external surface of the lip passes through a transitional zone to merge with the oral mucosa on the inner surface of the lip (Kang and Svoboda,2005). Its expression is also found in the epithelium of the tongue (Fig. 3H), specifically at the specialized lingual papillae, which is strongly vascularised in adulthood. Moreover, SSAO/VAP-1 protein is expressed in the epithelium of the nasal cavity (Fig. 3G) and in the epithelia of the organ of Corti and the cochlea of the inner ear (Fig. 1B3,4). During inner ear development, the vestibulocochlear artery supplies the cochlea at the same time that the epithelia of the cochlear region and the organ of Corti begins to express SSAO/VAP-1 protein (ED14). Therefore, SSAO/VAP-1 expression appeared when the oral cavity began to mature during the later embryonic stages, just before birth, in parallel with both the vascular system and the smooth muscle cells in the nasal and oral cavities. All these findings suggest that SSAO/VAP-1 could play a role in vasculogenesis and/or myogenesis of the sensory organs.

Cartilage Development

At ED12 and ED14, SSAO/VAP-1 expression was found in the chondrocytes showing active differentiating processes, mainly in the vertebrae and ribs (Fig. 4D,E), as well as in the vascular cells that surround the cartilage primordium of these skeletal sites (Fig. 4D). However, no immunostaining for SSAO/VAP-1 protein was observed at the cartilage sites of the head, concretely in the nasal area (Fig. 3G) and limbs (data not shown). At ED17.5, the ossification process is predominant and many cartilages are replaced by bones. At the same time, SSAO/VAP-1 protein is expressed very weakly in the resting chondrocytes (Fig. 4F). Therefore, SSAO/VAP-1 expression in the skeletal elements of the mouse embryo shows a regional pattern during differentiation process. Previous studies showed the expression of SSAO/VAP-1 protein in adult rat chondrocytes from articular cartilage (Lyles and Bertie,1987). However, the role of SSAO/VAP-1 in chondrogenic sites is still unknown. Chondrocytes are a dynamic cell type that plays an important role in bone maintenance. In embryo, the vertebrate skeleton is formed from mesodermic cells for endoskeleton primarily in the trunk (ribs, vertebrae, and limbs), and from neural crest cells for exoskeleton and endoskeleton of the head and branchial region. The expression of SSAO/VAP-1 is found essentially in the vertebrae and ribs (endoskeleton from mesodermic cells) and concretely in endochondral bones, since its expression decreases with the osteogenesis process. Therefore, the regional expression of SSAO/VAP-1 and its basal expression after ossification in the resting chondrocytes suggests that this enzyme could be involved in the development and/or maintenance of cartilage sites.

Conclusion

SSAO/VAP-1 expression is spatially and temporally regulated in several mouse embryonic tissues, first in the vascular system and later in the sensory organs, smooth muscle tissue, and skeletal elements. In parallel, its enzymatic activity was low during mouse embryonic development. SSAO/VAP-1 could play a different role during mouse development beyond its role in amine metabolism and vascular adhesion described in adult mammalians. Further studies will be necessary in order to clarify the role that SSAO/VAP-1 protein expression plays during mouse embryogenesis.

EXPERIMENTAL PROCEDURES

Animals

We used embryos of NMRI mice and Sprague-Dawley rats (Iffa Credo, Lyon, France).

The day on which a vaginal plug was detected was considered embryonic day 0 (E0). E1 began 24 hr later. The fetal animals (E8, E9, E10, E11, E12, E14, E15, E16, E17.5, and E18.5) were removed from the mother under anaesthesia by intraperitoneal injection of ketamine (100 mg/Kg) and Xylazine (10 mg/Kg). All animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PFA) and processed for paraffin-embedded and for frozen PFA-fixed samples. The paraffin-embedded samples were cut into 5-μm sagital sections and frozen samples were cut into 25-μm sagital free-floating sections. The animals were kept under controlled temperature, humidity, and light conditions and were treated according to European Community Council Directive 86/609/EEC.

Immunohistochemistry

Paraffin and free-floating sections were processed for immunohistochemistry in accordance with the Valente protocol (Valente et al.,2005). The sections were incubated with rabbit polyclonal antibodies against bovine SSAO/VAP-1 protein at 1:200 (Lizcano et al.,1998) or human SSAO/VAP-1 protein at 1:50 (H-43, Santa Cruz Biotechnologies). Thereafter, sections were sequentially incubated with biotinylated goat anti-rabbit antibody (1:200) and with the avidin-biotin-peroxidase complex (ABC, 1:200). Peroxidase was developed with 0.05% diaminobenzidine in 0.1 M PB and 0.01% H2O2, and immunoreacted sections were mounted onto gelatinised slides. Alternatively, some sections were counterstained with hematoxylin. SSAO/VAP-1 immunohistochemical controls were made. First, we performed immunohistochemistry replacing the primary antibody by bovine foetal serum; second, we performed immunohistochemistry using the SSAO-VAP-1 antibody previously preadsorbed 1 hr at room temperature with the SSAO/VAP-1 peptide used for antibody production (Lizcano et al.,1998). For double immunohistochemistry and immunofluorescence, the sections immunostained with SSAO/VAP-1 were incubated with a second primary antibody: mouse anti-Nestin (1:200, Hybridoma Bank); mouse anti-Vimentin (1:500, DAKO); and a rabbit anti-MECA-32 (1:100, Hybridoma Bank). After, several washes, the sections were incubated 1 hr at room temperature with the respective secondary antibody for immunofluorescence: goat anti-mouse Alexa 488 (1:1,000, Invitrogene). The sections were mounted in mowiol medium.

Sections were photographed in a NIKON Eclipse 901 microscope/Nikon digital sight camera, using a 10× and 20× objective lens. Embryonic anatomy was defined and termed according to The Atlas of Mouse Development by Kaufman (1999).

Preparation of Embryonic Extracts and Western Blotting

Whole embryos with ED16 from rat and mouse were homogenized in Ripa buffer at 4°C to obtain a total cellular fraction. Total extracts (20 μg/lane) were prepared in sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 30% Glycerol, and 2,5% β-mercaptoethanol) and were resolved on 7.5% SDS-PAGE gels (using the Bio-Rad Mini-PROTEAN 3 system) and transferred to nitrocellulose membranes for Western blot. The membranes were incubated with rabbit polyclonal against SSAO/VAP-1 protein (Lizcano et al1998) or with rabbit polyclonal anti-SSAO/VAP-1 (Santa Cruz Biotechnologies). The bands were visualized using the ECL chemiluminescence system (Amersham Pharmacia Biotech).

Measure of SSAO/VAP-1 Activity

A modification of the Otsuka and Kobayhashi (1964) method was used to determine the SSAO activity. SSAO activity was determined radiochemically using 100 μM (14°C)-benzylamine (2 mCi/mmol, Amersham, UK) as substrate. L-deprenyl (1 μM) was used to pre-inhibit MAO B. Cell lysate activities are expressed as pmol product/min · mg protein.

Acknowledgements

We thank John Bates and Christian Brassington for assistance with the English language.

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