ORIGINAL ARTICLE

Gabin Sihn . Katia Savary . Annie Michaud .

Marie-Claude Fournie-Zaluski . Bernard P. Roques .

Pierre Corvol . Jean-Marie Gasc

Aminopeptidase N during the ontogeny of the chick

Received July 12, 2005; accepted in revised form October 11, 2005

Abstract Little is known about the production andfunction of metallopeptidases in embryonic develop-ment. One such enzyme, aminopeptidase N (APN), ispresent in several epithelia, the brain and angiogenicvessels in adults. APN promotes vascular growth andendothelial cell proliferation in physiological and path-ological models of angiogenesis. However, its possiblerole in embryonic angiogenesis or other developmentalprocesses is unknown. Its expression profile in the earlyphase of embryonic development has not been reported.We report here the expression of this enzyme during theearly development of the chick embryo, using comple-mentary techniques for monitoring APN mRNA, pro-tein, and enzymatic activity. We detected APN in theembryo as early as gastrulation. In addition to theknown sites of APN production identified in bothadults and rat fetuses toward the end of gestation, APNwas found in unexpected sites, such as the primitivestreak, the dorsal folds of the neural tube, the somites,and the primordia of several organs. APN was presentmostly in the cardiovascular compartment during the

first 13 days of incubation, and in the hematopoieticcompartment (yolk sac and aorta–gonad–mesonephrosregion) early in development. This study provides cluesas to the possible role of APN in embryonic develop-ment.

Key words angiogenesis � aminopeptidase N � aorta–gonad–mesonephros (AGM) region � blood island �chorioallantoic membrane � chick embryo �hematopoiesis � intestine � liver � mesonephros �ontogeny

Introduction

Little is known about the production and function ofproteases during embryonic development, althoughsome proteases have been reported to be involved inprocesses such as hematopoiesis (Savary et al., 2005),kidney (Lelongt et al., 2001; Lelongt and Ronco, 2002),pancreas (Perez et al., 2005), and mammary (Vu andWerb, 2000) development or skeleton growth (Vu andWerb, 2000; Inada et al., 2004). Aminopeptidase N(APN, EC 3.4.11.2.) has been particularly studied inrecent years, following the discovery of its potential in-volvement in blood vessel formation (Pasqualini et al.,2000; Bhagwat et al., 2001).

APN is a membrane-bound zinc metallopeptidase(Hooper, 1994) that has been identified primarily inmammals (human, pig, rat) (Barrett et al., 1998). How-ever, an ortholog has also been purified from chick(Midorikawa et al., 1998; Gal-Garber and Uni, 2000).APN is found in almost all adult mammal tissues, but ispreferentially found in the re-absortive epithelia of theintestine and the kidney, the bile canaliculi of the liver,the pancreas (Bordessoule et al., 1993; Sjostrom et al.,2000), the blood-brain barrier-associated pericytes(Kunz et al., 1994), the central nervous system (Nobleet al., 2001), and the synaptic membranes (Matsas et al.,

1Fellowship from the Ministere de la Recherche et de laTechnologie, Paris, France

Gabin Sihn1 ( .*) � Katia Savary � Annie Michaud �Pierre Corvol � Jean-Marie GascLaboratoire de Pathologie Vasculaire et Endocinologie RenaleInserm U36, College de France11, place Marcelin Berthelot 75005 ParisFranceTel: 133 1 44 27 16 49Fax: 133 1 44 27 16E-mail: gabin.sihn@college-de-france.fr

Marie-Claude Fournie-Zaluski � Bernard P. RoquesDepartement de Pharmacochimie Moleculaire & StructuraleInserm U266, CNRS FRE 2463UFR des Sciences Pharmaceutiques & biologiques4, avenue de l’Observatoire, 75270 Paris CedexFrance

U.S. Copyright Clearance Center Code Statement: 0301–4681/2006/7402–119 $ 15.00/0

Differentiation (2006) 74:119–128 DOI:10.1111/j.1432-0436.2006.00058.x r 2006, International Society of Differentiation

1985). APN is also produced specifically in the hemato-poietic system, and has been shown to be identical tothe CD13 on blood cells of the myelomonocytic lineage(Look et al., 1989).

APN degrades short extracellular peptides. Its subst-rates include a number of bioactive peptides, such asangiotensin III (Zini et al., 1996), enkephalins (Waks-man et al., 1985), and nociceptin (Montiel et al., 1997).APN inhibitors have been used to investigate the role ofAPN in the regulation of blood pressure (Reaux et al.,1999), analgesia (Fournie-Zaluski et al., 1984), and in-flammation (Riemann et al., 1999). APN has been im-plicated in pathological and experimental angiogenesis(Pasqualini et al., 2000; Bhagwat et al., 2001), makingthis enzyme a potential target for anti-cancer therapies,and raising questions as to whether APN plays a role inembryogenesis, a critical period for development of thenormal vascular structures of the adult.

The APN gene inactivation has not been reported inmouse, and descriptions of APN onset and expressionpattern provide clues as to the potential role of APN inembryonic development. APN has been detected in hu-man placenta (Imai et al., 1994) and in rat fetuses late ingestation (E15–E20) (Jardinaud et al., 2004). However,no data are currently available for the early stages ofembryonic development, corresponding to the majormorphogenetic processes responsible for shaping theembryo and initiating tissue differentiation.

We report here the expression of APN throughoutdevelopment of the chick embryo, a well-establishedexperimental model for developmental studies. Thisstudy provides the first description of APN productionin the earliest stages of embryonic development, fromthe start of gastrulation (day 2 of incubation) up to day6 of incubation. Using complementary techniques, wedetected APN in unexpected sites (primitive streak,dorsal folds of the neural tube, somites, intermediatemesoderm, blood islands, and endoderm of the yolksac) and in sites predicted from work on adults but notdemonstrated previously in embryos (vascular tree,heart, primordia of the mesonephros, the duodenum,and the hepatic divericuli).

Methods

Chick embryos

Fertilized eggs from White Leghorn chickens were obtained from acommercial breeder (Haas, Kalten House, France) and incubated at381C, 70% humidity. Developmental stages were defined as de-scribed by Hamburger and Hamilton (1951) and on the basis ofincubation time.

Enzyme inhibitors and substrates

The APN inhibitor RB3014 and its radioactive equivalent, [125I]-RB129, were synthesized as described previously (Chen et al., 1999,

2000). The APN substrate, alanine b-naphthylamide (Ala b-NA),was obtained from Bachem, Weil am Rhein, Germany.

Semiquantitative reverse transcription-polymerase chain reaction(RT-PCR) analysis

Total RNA was extracted from single embryos and semi-quanti-tative RT-PCR carried out, as described previously (Larger et al.,2004). Primers were designed based on the published chickenAPN sequence as published in Midorikawa et al. (1998): 50-GACA-ACGCCTACTCCTCCATTGGC-3 0 (forward) and 50-CACAGA-AGGTCTCTCCACCGTGGA-3 0 (reverse). DNA was amplified bymeans of 24 standard PCR cycles, with an annealing temperature(Tm) of 631C.

APN enzymatic activity

Single embryos were homogenized in 50mmol/l HEPES buffer (pH7.4) containing 8mmol/l CHAPS (6:1 v/w), using a Potter–Elvej-hem tissue homogenizer, and were then solubilized overnight at41C. Homogenates were centrifuged for 10min at 5000� g, andAPN enzymatic activity was measured in the supernatants by mon-itoring Ala b-NA hydrolysis as follows: samples were incubated(27min, 371C) with 50mmol/l Ala b-NA in a final volume of 100 mlTris-HCl buffer (50mmol/l, pH 7.4). Fluorescence (emission l,330 nm; excitation l, 460 nm) was measured at the end of this in-cubation period, using a Fusiont fluorimeter (Packard Bioscience,Rungis, France). We checked that this hydrolysis resulted fromAPN activity by measuring fluorescence in the presence of 1 mmol/lRB3014. The total protein content of the samples was determinedwith the Bradford assay (BIO-RAD Protein Assay, Biorad,Marnes-la Coquette, France).

Partial re-cloning and riboprobe transcription of chicken APN

The chicken APN gene was subcloned by extracting total RNAfrom 10-day-old chick embryo mesonephroi, using the RNeasymini kit (Qiagen, Valencia, CA). A complementary DNA corres-ponding to the entire APN gene was amplified by RT-PCR (30cycles, Tm 5 561C), using primers designed from the published inchicken APN sequence Midorikawa et al. (1998): 50-ATGGCAG-CCGGCTTCTTCAT-3 0 (forward) and 50-GGCTAGCTGGAGG-CGGTCTC-30 (reverse). The product was inserted into the pCRIIvector, using the TOPO-TA cloning kit (Invitrogen, Cergy-Po-ntoise, France). The resulting plasmid was digested with XhoI andKpnI, generating a fragment that was inserted into pCRII (Invitro-gen). This subclone was linearized by XhoI digestion for the an-tisense probe and KpnI digestion for the sense probe and riboprobetranscription was performed as described previously (Sibony et al.,1995). The chicken Myb subclone was a gift from Luc Pardannaud.

Histological procedures

We used a radioactive in situ hybridization technique described indetail elsewhere (Sibony et al., 1995). Sections were counterstainedwith toluidin blue. In addition to microscopic pictures, we alsoanalyzed X-ray films. Whole-mount in situ hybridization wasperformed with digoxygenin-labeled riboprobes, as described byHenrique et al. (1995). The endodermal layer was removed byincubating HH8–HH18 embryos in Dulbecco’s modified Eagle’smedium supplemented with 15% chicken serum and 10% fetal calfserum (inactivated at 551C for 30min) at 41C for at least 1 hr, withgentle shaking. For both in situ hybridization and whole-mount insitu hybridization, controls were performed with sense riboprobes.Autoradiography with the APN inhibitor [125I]-RB129 (Chen et al.,

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2000) was performed as described previously (Noble et al., 2001),on frozen sections (10 mm thick).

Results

APN mRNA production and enzymatic activity duringearly development

We begun this study at stage HH6 (24 hr of develop-ment, neural fold stage; Hamburger and Hamilton,1951) of chick embryo development, corresponding tothe start of gastrulation and neurulation. RT-PCRanalysis and enzymatic activity measurements duringthe first 5 days of incubation showed a clear relationshipbetween relative APN transcript levels and APN-spe-cific enzymatic activity, measured as the rate of Alab-NA hydrolysis (Figs. 1A, 1B). We checked thespecificity of APN activity, using the selective APN in-hibitor RB3014 (Chen et al., 1999), which has an IC50 of30 nM for chicken APN (data not shown). Both mRNAand enzymatic activity were detected in whole-embryohomogenates as early as HH6, and their levels increased

from HH18 (67 hr of development, 29 pairs of somites)onwards. Levels of APN mRNA and activity then sta-bilized until at least HH26 (day 6 of development), inthe embryo and in the extra-embryonic area. The dis-tribution of APN in the embryo from HH7 to HH35 issummarized in Table 1. Only the most interesting sitesof expression are discussed in detail below.

HH7–HH10

At HH7 (24–25 hr of development, one to three pairs ofsomites), in situ hybridization (ISH) showed mediumlevels of APN mRNA in the epiblastic cells ingressingthrough the primitive streak. Moderate amounts ofmRNA were also detected in the neurectoderm of theneural groove (Fig. 2A). APN mRNA was also detectedin the mesodermal layer, in relatively large amounts inthe cephalic mesoderm (Figs. 2A–2C), in moderateamounts in the paraxial mesoderm (data not shown)and the early extra-embryonic region, and in largeamounts in the three pairs of somites (S1–S3) derivedfrom the paraxial mesoderm (Fig. 2A). Strong APNgene expression was also observed in the hemangioblas-tic condensations of the mesoderm in the area opaca.The horseshoe-shaped area caudal to the primitivestreak (Fig. 2A), derived from Koller’s sickle andknown to produce large amounts of blood islands andhemoglobin (Wilt, 1974), was also very strongly labeled(Fig. 2E). Finally, moderate to strong APN expressionwas observed in the thickened endoderm of the areaopaca (the extra-embryonic area) (Figs. 2E, 2G), butnot in the endoderm of the area pellucida (the embry-onic area), which was poorly labeled (not shown).

At HH10 (35 hr of development, 10 pairs of somites),relatively strong APN expression persisted in the re-gressing primitive streak (Fig. 2D). In the neuralgroove, APN expression was detected primarily in thedorsal neural folds, especially in the region about toclose (Figs. 2D, 2I, 2J). In somites 1 and 2 (Figs. 2D, 2I–2J), the labeling was moderate and homogenous, where-as in somites 3–10 (Figs. 2D, 2K–2L), it depended onthe position along the anterioposterior axis of each so-mite—with stronger levels in the caudal halves—asshown by the unequal labeling on slightly oblique sec-tions (Figs. 2K–2L). High levels of APN mRNA werealso detected in the blood islands derived from conden-sations of the lateral plate mesoderm (Figs. 2D, 2F). Inthese blood islands, labeling was detected in both theperipherally located angioblasts and the central hem-atopoietic precursors following commitment to these 2cell lineages (Fig. 2F). Strong labeling was also ob-served in the primary capillary plexus, which started toform at this stage by the fusion of blood islands in boththe area opaca and the area pellucida (i.e., the extra-embryonic and intra-embryonic areas) (Fig. 2D),particularly in the endothelium. Expression in the end-

Fig. 1 Detection of aminopeptidase N (APN) mRNA and enzyma-tic activity between HH6 and HH26. (A) APN mRNA levels.Measurements were made by RT-PCR and normalized using 18SRNA. Values are the means � SEM of four samples for each stage.(B) APN enzymatic activity, expressed in nanomoles of b-nap-hthylamine released/min/mg protein (mean � SEM). The residualactivity, measured in the presence of 1 mM of the APN-specificinhibitor RB3014, has been subtracted from the whole-Ala b-NAhydrolysis. The number of samples is indicated at the top of thebars. RT-PCR and enzyme activity measurements were carried outon whole blastodiscs (striped bars) at HH6 and HH12, and onembryos strictly (white bars) and extra-embryonic areas (blackbars) separately between HH18 and HH26.

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oderm increased markedly overtime in the area opaca,and was very strong from HH10 onwards (Fig. 2D).

Also at HH10, new sites of APN expression not de-tected at HH7, were observed: (i) the two dorsal aortae,which displayed heterogeneous labeling, mainly restrict-ed to their dorsal sides (Figs. 2K, 2L), (ii) the interme-diate mesoderm, which was strongly labeled (Figs. 2I–2L), (iii) the two endocardial tubes, the epimyocardiumand endocardium of which were strongly and moder-ately labeled, respectively (Fig. 2H), and (iv) the ventro-lateral borders of the pharynx (not shown) and theanterior intestinal portal in the region of closure of thegut (Figs. 2I, 2J), which was strongly labeled. In con-trast, the rest of the area pellucida displayed little or noendodermal labeling.

HH17–HH18 (67 hr of development, 29 pairs ofsomites)

By HH17, APN expression had stopped in the neuraltube and persisted only in the neural roof plate (Figs.3A–3C). Expression in the somites also appeared het-erogeneous—strong in the caudal halves of the 23–29

pairs of somites, but only moderate in their rostralhalves (Fig. 3A). A gradient in expression was observed,decreasing from the caudal to the more anterior somites(Fig. 3A), the sclerotome being more strongly labeledthan the dermo-myotome (Figs. 3B, 3C). Particularlystrong APN expression was also observed (i) in the in-termediate mesoderm (Fig. 3A) and its derivatives, thenephrogenous mesenchyme and the nephric ducts (Figs.3D, 3E), (ii) in the peritoneal epithelium, which is de-rived from the splanchnic mesoderm covering the ne-phrogenic ridges (not shown), and (iii) in the duodenum(Figs. 3D, 3E) and in the hepatic diverticuli (Figs. 3B,3C). In contrast, no labeling was detected in the eso-phagus (Figs. 3B, 3C).

APN expression in the heart was strong in the end-ocardium and moderate in the myocardium (Figs. 3B,3C). Moderate to strong vascular expression was ob-served in the dorsal aortae, the aortic arches, the vitel-line arteries, the anterior and posterior cardinal veinsand the pericephalic vascular plexus (Figs. 3B–3G).Within the dorsal aorta, the endothelial layer was mod-erately labeled and the aorta-gonad-mesonephros re-gion (AGM) was strongly labeled (Figs. 3B, 3C, 3H). Inthis region, APN labeling colocalized with the hemato-

Table 1 Aminopeptidase N expression between HH7 and HH35 of chick embryo development

HH 7 HH10 HH19 HH27-35

Ectoderm + Ectoderm + Ectoderm + Epiderm NDPrimitive streak 1/11 Primitive streak 1/11Neural grooveand plate

11 Neural groove 1 Neural tube 1 Central nervous system NDNeural folds 11 Neural roof plate 11Cephalic vesicle + Cephalic vesicle +

Optic and otic vesicles 1 Eye and ear NDMesoderm

Somites 111 Somites 111 Sclerotome 11 Vertebrae/perichondrium 1/11Dermo-myotome 1/11 Muscles 1

Derm NDLateral(intraembryonic)

11 Somatopleura 11 Limb pouches 11 Long bonecartilages/perichondrium 1/11Sternum/perichondrium 1/11Intervertebral discs 11

Splanchnopleura 11 Germinal ridge 111 Gonad 1Heart1 Heart1 Heart1

Dorsal aorta1 Dorsal aorta andblood vessels1

Blood vessels1

Intermediatemesoderm

111 Mesonephros1 111 Mesonephros1 111

Spleen 11Lateral(extraembryonic)

11 Lateral(extraembryonic)

11 Yolk sac mesenchyme 11

Blood islands1 Blood islands1 Blood vessels andHematopoietic cells1

EndodermArea pellucida 1 Gut1 Gut1 Gut1

Hepatic diverticuli1 Liver1

Allantois and CAM1

Lung +Epimyocardium1 Heart1 Heart1

Area opaca 111 Area opaca 111 Area opaca 11

+, no or minimal expression; 1, 11 and 111, little, moderate and strong expression; ND, not determined.1Detailed report is given in the text.

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poietic marker Myb (Vandenbunder et al., 1989), but wasnot restricted to Myb-positive cells, being observed inmost cells of the disorganized aortic endothelial layer andthe underlying somatopleural mesenchyme (Fig. 3H).

Moderate to strong vascular expression was alsoobserved in the vitelline sac, depending on the vesselconsidered: strong labeling was detected in the vitellinearteries and veins and in the area vasculosa, and mod-erate labeling was observed in the sinus terminalis (Fig.3J). Finally, the endodermal layer of the area opaca wasvery strongly labeled (Fig. 3J).

HH26–HH35

From HH26 onwards, the APN gene was strongly ex-pressed in the liver, mesonephros and duodenum. Liver

expression remained strong and homogeneous through-out the rest of embryonic development (Figs. 4A). Incontrast, APN expression in the mesonephros rapidlybecame heterogeneous, disappearing altogether in thenephric duct, the distal tubules and the glomeruli, andremaining strong only in the proximal tubules, as ob-served at HH29 (day 6–6.5 of development) (Figs. 4A–C). High levels of APN expression were observed in thegut, in the brush border of the duodenum and the ileum,from HH29 onwards (Figs. 4A, 4D, 4E). In contrast,little labeling was observed in the esophagus. At laterstages, the crop gland and gizzard also displayed littleor no APN expression (Figs. 4M, 4N).

In the heart, moderate levels of APN expression wereobserved in the myocardium in the compact andtrabecular zones from HH29 onwards, with lower lev-els of expression in the atria than in the ventricles.

Fig. 2 Aminopeptidase N (APN) mRNA expression at HH7–HH10 by whole-mount in situ hybridization (ISH) (A and D) andradioactive ISH (B–C and E–L). (A) at HH7, expression in theneural groove (NG), the cephalic mesoderm (CMe), the first tothird somite pairs (S1–S3), the primitive streak (PS), the horseshoe-shaped area (HSA) and the area opaca (AO). (B–C) transversalanterior section of a HH7 embryo, showing expression in the NGand the CMe. (D) at HH10, expression in the neural folds in theregion of closure of the neural tube (NF), the somites S1–S10, theremnants of the primitive streak, the inflow tract (IFT) and thevascular plexus of the area pellucida (%); in the area opaca, theextra-embryonic endoderm (En), strongly positive, was removedfrom the right side of the embryo, revealing expression in the bloodislands (BI), and the vascular plexus (�); inset: higher magnificationof the boxed area, showing strong and moderate labeling in thecaudal (c) and the rostral (r) halves, respectively, of somites S6–S9.E: section through the posterior horseshoe-shaped area at HH7,showing strong expression in the hemangioblastic condensations of

the mesoderm (HC) and in the extra-embryonic endoderm, but notin the ectoderm layer (Ec). (F) section through an extra-embryonicblood island at HH10, showing expression in the peripheral an-gioblasts (An) and in the central hematopoietic precursors (HP).(G) controls of the figures E (left panel) and F (right panel) withAPN sense probe, showing background labeling. (H–L) are rostralto caudal sections through HH10 embryos, showing expression inthe endocardium (Endc) and epimyocardium (EM) of the heart, theendoderm of the anterior intestinal portal (arrows), the splanchnicmesoderm (SpMe), the NF and the roof plate (NRP) of the neuraltube and the intermediate mesoderm (IMe); strong expression wasalso observed in the somites (S), with the precise level of expressiondepending on the position along the anterioposterior axis of eachsomite (see D); finally, expression was observed in the dorsal aortae(DAo), particularly in the dorsal parts of these vessels (arrow-heads). Scale bars: 1mm for (A and D); 20 mm for (B, C and E–G);100 mm for (H–L).

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Moderate expression was also observed in the end-ocardium (Figs. 4A, 4F, 4G).

Large intra-embryonic vessels, such as the dorsalaorta (Figs. 4H–4I), the pulmonary arteries and thecardinal veins (not shown) were weakly labeled, withthe exception of the outflow tract, where relatively

strong endothelial and moderate medial labeling wasobserved, as at HH31 (Figs. 4H–4I). APN was also ex-pressed to different levels in the small interstitial vesselsof the various tissues tested (data not shown).

In contrast to what was observed in the intra-em-bryonic vessels, large amounts of APN mRNA were

Fig. 3 Aminopeptidase N (APN) mRNA expression at HH18 bywhole-mount ISH (A and J) and radioactive ISH (B–I). (A) ex-pression in the neural roof plate (NRP) and the intermediate mes-oderm (IMe); in the somites, the most posterior (S15–S29) exhibitstronger labeling in their caudal (c) compared to their rostral (r)halves. (B–C) dark and bright field view of a section, showing ex-pression in the neural roof plate (NRP), the sclerotome (Sc), thedermomyotome (DM) and the hepatic diverticuli (HD) but not inthe esophagus (Es); expression was also visible in the dorsal aorta(DAo), in the AGM (boxed area), in the anterior cardinal veins(ACV) and in the endocardium (arrowheads) of the sinus venosus(SV), the left atrium (LA) and the bulbus cordis (BC) of the heart;moderate labeling was also visible in the epimyocardium (EM). (D–

E) Dark and bright field view of a more posterior section, showingexpression in the mesonephros (Ms) and the duodenum (Du); DAo:dorsal aorta; the inset is a higher magnification of the mesonephros,

showing expression in the nephrogenous mesenchyme (NM) andthe nephric duct (ND). (F–G) Dark and bright field view of a sec-tion, showing vascular expression in the anterior cardinal veins(ACV), the internal carotid arteries (ICA), the pericephalic vascularplexus (PCVP) and the aortic arches (AA); moderate expressionwas also visible in the mesenchyme of the first (BA1) and second(BA2) branchial arches. H: higher magnification of the AGM,showing widely distributed expression in the disorganized ventralendothelial layer (Endth) and somatopleural mesenchymal cells(SoMC). (I) Comparative expression of the hematopoietic markercMyb in the AGM, indicated by the arrowheads. (J) Embryo withextra-embryonic endoderm (En) pealed off on its left side, showingvascular expression in the left vitelline artery (LVA) and the areavasculosa (AV); weak to moderate expression was observed inthe sinus terminalis (ST). Scale bars: 150 mm for (A–G); 10mm for(H–I); 1mm for (J).

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present in the vessels of the chorioallantoic membrane(CAM) from day 5 until at least day 13 of development.APN expression was moderate at HH26, and increasedthereafter. Strong labeling was observed in the vessels ofthe ectomesodermal layer and in the arteries and veinsof the mesodermal layer, especially in the endothelium.Moderate expression was also observed in a subend-othelial layer in these arteries. Finally, strong expressionwas observed in the lymphatic vessels (Figs. 4J–4L).

From HH35 (day 9 of development) onwards, westudied the distribution of APN by autoradiography(Figs. 4M, 4N), using the radioactive APN-specificinhibitor [125I]-RB129 (Chen et al., 2000; Jardinaudet al., 2004), which had an IC50 of 4 nmol/l for chicken

APN (data not shown). This technique allowed us tostudy the presence, in situ, of an active protein, able tobind the specific inhibitor. The pattern observed withAPN protein was almost identical to that for APNmRNA, in terms of both the location and the strengthof expression.

Discussion

APN is a peptidase involved in various physiologicalprocesses, and has been reported to be produced inpathological disorders like acute myeloblastic leukemia

Fig. 4 Detection of APN mRNA and protein at HH29–HH35 byradioactive ISH (A–M) and [125I]-RB129 binding/autoradiography(N). (B, D, F, H and J) are dark-field images and (C, E, G, I and K),and (L) are the corresponding bright-field images. (A) X-ray filmISH picture of a sagittal section at HH29, showing strong expres-sion in the liver (Li), duodenum (Du), mesonephros (Ms) and inter-vertebral discs (ID), and moderate expression in the heart (He) andthe gonad (Go); no significant expression is observed in the lung(Lu). (B and C) in the mesonephros, strong expression in the prox-imal tubules (PT) but not in the distal tubules (DT), the Wolffianduct (WD) and the Mullerian duct (MD). (D and E) in the duo-denum, strong expression at brush border (DBB) and moderate tostrong expression in the epithelium of the ileum (IEp). (F and G) inthe outflow tract of the heart (upper panel), the ascending aorta(Ao) displays a strong APN expression in the endothelium (arrow-head) and the expression is lower in the tunica media (%); in bothtissues, the labeling decreases as one goes distally (d) to the heart;within the heart (upper and lower panels), a moderate expression isobserved in the endocardium (arrow) and the myocardium (�) ofthe left and right ventricles (LV and RV); in contrast, weak labeling

is observed in the left atrium (LA); very strong expression is ob-served in the liver (Li). H and I: within the outflow tract (left panel),relatively strong and moderate expression is observed in the en-dothelium (arrowheads) and the tunica media (%), respectively, inboth the ascending aorta (Ao) and the pulmonary artery (PA); littleexpression is observed in the descending part of the dorsal aorta(Ao) (right panel). (J and K) sections of a HH35 chorioallantoicmembrane, showing expression in the ectomesoderm (EcMe), in thelarge arteries and veins (Ar and Ve), in smaller vessels (arrowheads)and in lymphatic vessels (Ly). (L) Higher magnification of theboxed area in (K), showing expression in the capillaries (arrow-heads) of the ectomesoderm. (M and N) X-ray film pictures ofHH35 embryos, showing similar that the distribution of the tran-scripts (M) is similar to that of the protein (N), with strong ex-pression in the liver (Li), the mesonephros (Ms), the intestine (In)and the perichondria (Pe), and moderate expression in the heart(He) and the gizzard (Gi); in contrast no expression was observedin the esophagus (Es) and the crop (Cr). Scale bars: 2mm for (A, Mand N); 150 mm for (B–K); 20mm for (L).

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(Griffin et al., 1981; Drexler, 1987) and tumors of var-ious origins (Mori et al., 2001; Tokuhara et al., 2001;Hashida et al., 2002; Ikeda et al., 2003; Kehlen et al.,2003). It has recently been reported to have anangiogenic activity (Pasqualini et al., 2000; Bhagwatet al., 2001), and to be produced toward the end of fetaldevelopment in rat (E15–E20) (Jardinaud et al., 2004),raising questions about a possible role in earlier stagesof development, particularly in vascular differentiation.We describe here the pattern of APN expressionthroughout chick embryo development, with a parti-cular emphasis on the earliest stages of development.In situ hybridization and [125I]-RB129 binding/auto-radiography, 2 technologically different approaches,showed that APN mRNA and protein were expressedin various tissues, among which only some were in-volved in differentiation of the vascular system. Thisstudy provides the first global description of APN ex-pression in chicks, as APN expression has been reportedpreviously only in the egg yolk (Midorikawa et al.,1998) and the intestine of adult chickens (Gal-Garberand Uni, 2000).

Table 1 recapitulates our results and we will discussonly those, which open new lines of research onembryonic development. A first interesting observationis the selective, transient, and polarized pattern ofexpression of APN found in the immature somitesat HH10 and HH18. Such expression is indicativeof a possible involvement of APN in the patterningof the paraxial mesoderm leading to its segmentationand/or the establishment of rostral/caudal somiticpolarity.

Recent studies reported a marked endothelial expres-sion and an involvement of APN during some situationsof pathological and physiological angiogenesis ofthe adult (Arap et al., 1998; Pasqualini et al., 2000;Bhagwat et al., 2001). However, it remains unclearwhether APN is involved in the development of normalvascular structures, which takes place mainly duringembryonic development. Our observations of a selectiveAPN expression appearing as early as the formation ofthe hemangioblastic condensations in the yolk sac, andpersisting in the intra- as well as extra-embryonic ves-sels, are in favor of such a role.

The expression of APN observed in the early sites ofembryonic hematopoiesis, the yolk sac blood islandsand the AGM (Dieterlen-Lievre and Martin, 1981;Peault et al., 1988; Cumano et al., 1996; Medvinskyand Dzierzak, 1996), is also indicative of its possibleinvolvement in this process. No significant expressionwas observed between E5.5 and E8.5 in the para-aorticfoci responsible for colonization of the definitive hem-atopoiesis sites (Dieterlen-Lievre and Martin, 1981),suggesting that APN may be selectively associatedwith early hematopoiesis in the yolk sac and AGM.The diffuse APN expression within the AGM, not re-stricted to the hematopoietic cells budding into the

aorta lumen, and the strong expression observed in theendoderm of the area opaca, suggest that APN may beinvolved in establishing a suitable environment forhematopoiesis. Such a putative role for APN is evoc-ative of the role described for angiotensin-convertingenzyme (ACE) in hematopoiesis of the yolk sac (Savaryet al., 2005).

APN expression in the kidney, intestine, liver, andheart had been previously shown during late rat fetaldevelopment (E15–E20) (Jardinaud et al., 2004) and inadults. We show here that APN expression in thesedifferentiated organs and structures is preceded by aprecocious and selective expression in the primordialanlage of these structures.

Some data on the regulation of APN expression arenow available in adults, and showed that several keydevelopmental factors can modulate APN expressionin vitro, like Ets-1 (Shapiro, 1995) or Myb (Wilt, 1965;Miura and Wilt, 1969; Bielinska et al., 1996). These 2factors, which are coexpressed with APN in a numberof developing organs and structures (Vandenbunderet al., 1989; Fafeur et al., 1997; Meyer et al., 1997;Tahtakran and Selleck, 2003), may participate to theregulation of APN expression during embryonicdevelopment. Several pro-angiogenic cytokines capableof stimulating APN endothelial expression in vitro, likeVascular Endothelial Growth Factor (VEGF) or basicFibroblast Growth Factor (FGF) (Bhagwat et al.,2001), may also account for the strong expression ofAPN in the developing vascular structures.

The actual role of APN in early embryonic develop-ment and its mode of action remain uncertain. APNmay modulate the concentration of bioactive peptides,as reported in adults. APN enzymatic activity in adultshas often been reported to be complementary to theactivity of other peptidases, such as neutral end-opeptidase (NEP) (Fournie-Zaluski et al., 1992) andACE (Reaux et al., 1999). Interestingly, APN and ACEshare common sites of production in the chick embryo(Savary et al., 2005). NEP has been implicated in ratembryonic development (Spencer-Dene et al., 1994),and is produced in several sites of APN production,notably the first and second branchial arches and thedeveloping skeletal structures. Several factors, such asTGF-b1, modulate the expression of both APN andNEP (Casey et al., 1993; Lendeckel et al., 1999; Kehlenet al., 2004). It would therefore be interesting to inves-tigate whether APN cooperates with other peptidasesduring embryonic development.

Acknowledgments We thank M. T. Morin and F. Mongiat forskillful assistance, and M. Brand, J. Favier, N. Lamande, E. Larg-er, F. Le Noble, L. Pardanaud, and R. Rosenfeld for helpful advice.This work was supported by INSERM, CNRS and the EuropeanVascular Genomics Network. G. S. holds a doctoral fellowshipfrom the French Ministry of Research and Technology.

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References

Arap, W., Pasqualini, R. and Ruoslahti, E. (1998) Cancer treat-ment by targeted drug delivery to tumor vasculature in a mousemodel. Science 279:377–380.

Barrett, A.J., Rawlings, N.D. and Woessner, J.F. (1998) Membranealanyl aminopeptidase. In: Barrett, A.J., Rawlings, N.D. andWoessner, J.F. (eds.) Handbook of proteolytic enzymes. Aca-demic Press, London, pp 996–1000.

Bhagwat, S.V., Lahdenranta, J., Giordano, R., Arap, W., Pas-qualini, R. and Shapiro, L.H. (2001) CD13/APN is activated byangiogenic signals and is essential for capillary tube formation.Blood 97:652–659.

Bielinska, M., Narita, N., Heikinheimo, M., Porter, S.B. andWilson, D.B. (1996) Erythropoiesis and vasculogenesis in em-bryoid bodies lacking visceral yolk sac endoderm. Blood88:3720–3730.

Bordessoule, D., Jones, M., Gatter, K.C. and Mason, D.Y. (1993)Immunohistological patterns of myeloid antigens: tissue distri-bution of CD13, CD14, CD16, CD31, CD36, CD65, CD66 andCD67. Br J Haematol 83:370–383.

Casey, M.L., Smith, J.W., Nagai, K. and MacDonald, P.C. (1993)Transforming growth factor-beta 1 inhibits enkephalinase (EC3.4.24.11) gene expression in human endometrial stromal cellsand sex skin fibroblasts in culture. JClin Endocrinol Metab77:144–150.

Chen, H., Bischoff, L., Fournie-Zaluski, M.C. and Roques, B.P.(2000) Synthesis of 2(S)-benzyl-3-[hydoxy(1’(R)-amino ethyl)phosphinyl]propanoyl-L-3-[125I]-iodotyrosine: a radiolabelled in-hibitor of aminopeptidase N. J Labelled Compd Radiopharm43:103–111.

Chen, H., Roques, B.P. and Fournie-Zaluski, M.C. (1999) Designof the first highly potent and selective aminopeptidase N (EC3.4.11.2) inhibitor. Bioorg Med Chem Lett 9:1511–1516.

Cumano, A., Dieterlen-Lievre, F. and Godin, I. (1996) Lymphoidpotential, probed before circulation in mouse, is restricted tocaudal intraembryonic splanchnopleura. Cell 86:907–916.

Dieterlen-Lievre, F. and Martin, C. (1981) Diffuse intraembryonichemopoiesis in normal and chimeric avian development. DevBiol 88:180–191.

Drexler, H.G. (1987) Classification of acute myeloid leukemias—acomparison of FAB and immunophenotyping. Leukemia 1:697–705.

Fafeur, V., Tulasne, D., Queva, C., Vercamer, C., Dimster, V.,Mattot, V., Stehelin, D., Desbiens, X. and Vandenbunder, B.(1997) The ETS1 transcription factor is expressed during epithe-lial-mesenchymal transitions in the chick embryo and is activatedin scatter factor-stimulated MDCK epithelial cells. Cell GrowthDiffer 8:655–665.

Fournie-Zaluski, M.C., Chaillet, P., Bouboutou, R., Coulaud, A.,Cherot, P., Waksman, G., Costentin, J. and Roques, B.P. (1984)Analgesic effects of kelatorphan, a new highly potent inhibitor ofmultiple enkephalin degrading enzymes. Eur J Pharmacol102:525–528.

Fournie-Zaluski, M.C., Coric, P., Turcaud, S., Lucas, E., Noble,F., Maldonado, R. and Roques, B.P. (1992) ‘‘Mixed inhibitor-prodrug’’ as a new approach toward systemically activeinhibitors of enkephalin-degrading enzymes. J Med Chem35:2473–2481.

Gal-Garber, O. and Uni, Z. (2000) Chicken intestinal amino-peptidase: partial sequence of the gene, expression and activity.Poultry Sci 79:41–45.

Griffin, J.D., Ritz, J., Nadler, L.M. and Schlossman, S.F. (1981)Expression of myeloid differentiation antigens on normal andmalignant myeloid cells. J Clin Invest 68:932–941.

Hamburger, V., Hamilton, H.L. (1951) A series of normal stages inthe development of the chick embryo. Journal of Morphology88:49–92.

Hashida, H., Takabayashi, A., Kanai, M., Adachi, M., Kondo, K.,Kohno, N., Yamaoka, Y. and Miyake, M. (2002) Amino-

peptidase N is involved in cell motility and angiogenesis: itsclinical significance in human colon cancer. Gastroenterology122:376–386.

Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. andIsh-Horowicz, D. (1995) Expression of a Delta homologue inprospective neurons in the chick. Nature 375:787–790.

Hooper, N.M. (1994) Families of zinc metalloproteases. FEBS Lett354:1–6.

Ikeda, N., Nakajima, Y., Tokuhara, T., Hattori, N., Sho, M.,Kanehiro, H. and Miyake, M. (2003) Clinical significance ofaminopeptidase N/CD13 expression in human pancreatic carci-noma. Clin Cancer Res 9:1503–1508.

Imai, K., Kanzaki, H., Fujiwara, H., Maeda, M., Ueda, M.,Suginami, H. and Mori, T. (1994) Expression and localization ofaminopeptidase N, neutral endopeptidase, and dipeptidyl pep-tidase IV in the human placenta and fetal membranes. Am JObstet Gynecol 170:1163–1168.

Inada, M., Wang, Y., Byrne, M.H., Rahman, M.U., Miyaura, C.,Lopez-Otin, C. and Krane, S.M. (2004) Critical roles for colla-genase-3 (Mmp13) in development of growth plate cartilageand in endochondral ossification. Proc Natl Acad Sci USA101:17192–17197.

Jardinaud, F., Banisadr, G., Noble, F., Melik-Parsadaniantz, S.,Chen, H., Dugave, C., Laplace, H., Rostene, W., Fournie-Zaluski, M.C., Roques, B.P. and Popovici, T. (2004) Ontogenicand adult whole body distribution of aminopeptidase N inrat investigated by in vitro autoradiography. Biochimie 86:105–113.

Kehlen, A., Geisler, M., Olsen, J., Sablotzki, A., Langner, J. andRiemann, D. (2004) IL-10 and TGF-beta differ in their regula-tion of aminopeptidase N/CD13 expression in monocytes. Int JMol Med 13:877–882.

Kehlen, A., Lendeckel, U., Dralle, H., Langner, J. and Hoang-Vu,C. (2003) Biological significance of aminopeptidase N/CD13 inthyroid carcinomas. Cancer Res 63:8500–8506.

Kunz, J., Krause, D., Kremer, M. and Dermietzel, R. (1994) The140-kDa protein of blood-brain barrier-associated pericytes isidentical to aminopeptidase N. J Neurochem 62:2375–2386.

Larger, E., Marre, M., Corvol, P. and Gasc, J.M. (2004) Hype-rglycemia-induced defects in angiogenesis in the chicken chor-ioallantoic membrane model. Diabetes 53:752–761.

Lelongt, B., Legallicier, B., Piedagnel, R. and Ronco, P.M. (2001)Do matrix metalloproteinases MMP-2 and MMP-9 (gelatinases)play a role in renal development, physiology and glomerulardiseases? Curr Opin Nephrol Hypertension 10:7–12.

Lelongt, B. and Ronco, P. (2002) Role of matrix metalloproteinasesin kidney development and glomerulopathy: lessons fromtransgenic mice. Nephrol Dial Transplant 17:28–31.

Lendeckel, U., Arndt, M., Frank, K., Wex, T. and Ansorge, S.(1999) Role of alanyl aminopeptidase in growth and function ofhuman T cells (review). Int J Mol Med 4:17–27.

Look, A.T., Ashmun, R.A., Shapiro, L.H. and Peiper, S.C. (1989)Human myeloid plasma membrane glycoprotein CD13 (gp150)is identical to aminopeptidase N. J Clin Investigation 83:1299–1307.

Matsas, R., Stephenson, S.L., Hryszko, J., Kenny, A.J. and Turner,A.J. (1985) The metabolism of neuropeptides. Phase separationof synaptic membrane preparations with Triton X-114 reveals thepresence of aminopeptidase N. Biochem J 231:445–449.

Medvinsky, A. and Dzierzak, E. (1996) Definitive hematopoiesis isautonomously initiated by the AGM region. Cell 86:897–906.

Meyer, D., Durliat, M., Senan, F., Wolff, M., Andre, M., Hourdry,J. and Remy, P. (1997) Ets-1 and Ets-2 proto-oncogenesexhibit differential and restricted expression patterns duringXenopus laevis oogenesis and embryogenesis. Int J Dev Biol41:607–620.

Midorikawa, T., Abe, R., Yamagata, Y., Nakajima, T. and Ichi-shima, E. (1998) Isolation and characterization of cDNA encod-ing chicken egg yolk aminopeptidase Ey. Comp Biochem PhysiolBiochem Mol Biol Part B 119:513–520.

127

Miura, Y. and Wilt, F.H. (1969) Tissue interaction and the forma-tion of the first erythroblasts of the chick embryo. Dev Biol19:201–211.

Montiel, J.L., Cornille, F., Roques, B.P. and Noble, F. (1997)Nociceptin/orphanin FQ metabolism: role of aminopeptidaseand endopeptidase 24.15. J Neurochem 68:354–361.

Mori, A., Tagawa, T., Kamei, T., Murata, T., Inui, M. and Ohse, S.(2001) Characterization of four cell lines derived from a humanmalignant fibrous histiocytoma of the maxillary sinus. OralOncol 37:527–536.

Noble, F., Banisadr, G., Jardinaud, F., Popovici, T., Lai-Kuen, R.,Chen, H., Bischoff, L., Parsadaniantz, S.M., Fournie-Zaluski,M.C. and Roques, B.P. (2001) First discrete autoradiographicdistribution of aminopeptidase N in various structures of ratbrain and spinal cord using the selective iodinated inhibitor[125I]RB 129. Neuroscience 105:479–488.

Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Saka-moto, M., Stryhn, A., Ashmun, R.A., Shapiro, L.H., Arap, W.and Ruoslahti, E. (2000) Aminopeptidase N is a receptor fortumor-homing peptides and a target for inhibiting angiogenesis.Cancer Res 60:722–727.

Peault, B., Coltey, M. and Le Douarin, N.M. (1988) Ontogenicemergence of a quail leukocyte/endothelium cell surface antigen.Cell Diff 23:165–174.

Perez, S.E., Cano, D.A., Dao-Pick, T., Rougier, J.P., Werb, Z. andHebrok, M. (2005) Matrix metalloproteinases 2 and 9 aredispensable for pancreatic islet formation and function in vivo.Diabetes 54:694–701.

Reaux, A., Fournie-Zaluski, M.C., David, C., Zini, S., Roques,B.P., Corvol, P. and Llorens-Cortes, C. (1999) AminopeptidaseA inhibitors as potential central antihypertensive agents. ProcNatl Acad Sci USA 96:13415–13420.

Riemann, D., Kehlen, A. and Langner, J. (1999) CD13—not just amarker in leukemia typing. Immunol Today 20:83–88.

Savary, K., Michaud, A., Favier, J., Larger, E., Corvol, P. andGasc, J.M. (2005) Role of the renin-angiotensin system in prim-itive erythropoiesis in the chick embryo. Blood 105:103–110,Epub 2004 Sep 2014.

Shapiro, L.H. (1995) Myb and Ets proteins cooperate to transac-tivate an early myeloid gene. J Biol Chem 270:8763–8771.

Sibony, M., Commo, F., Callard, P. and Gasc, J.M. (1995) En-hancement of mRNA in situ hybridization signal by microwaveheating. Lab Invest 73:586–591.

Sjostrom, H., Noren, O. and Olsen, J. (2000) Structure and func-tion of aminopeptidase N. Adv Exp Med Biol 477:25–34.

Spencer-Dene, B., Thorogood, P., Nair, S., Kenny, A.J., Harris, M.and Henderson, B. (1994) Distribution of, and a putative rolefor, the cell-surface neutral metallo-endopeptidases duringmammalian craniofacial development. Development 120:3213–3226.

Tahtakran, S.A. and Selleck, M.A. (2003) Ets-1 expression isassociated with cranial neural crest migration and vasculogenesisin the chick embryo. Gene Expr Patterns 3:455–458.

Tokuhara, T., Adachi, M., Hashida, H., Ishida, H., Taki, T.,Higashiyama, M., Kodama, K., Tachibana, S., Sasaki, S. andMiyake, M. (2001) Neutral endopeptidase/CD10 and amino-peptidase N/CD13 gene expression as a prognostic factor in non-small cell lung cancer. Jpn J Thoracic Cardiovascular Surg 49:489–496.

Vandenbunder, B., Pardanaud, L., Jaffredo, T., Mirabel, M.A. andStehelin, D. (1989) Complementary patterns of expression ofc-ets 1, c-myb and c-myc in the blood-forming system of thechick embryo. Development 107:265–274.

Vu, T.H. and Werb, Z. (2000) Matrix metalloproteinases: effectorsof development and normal physiology. Genes Dev 14:2123–2133.

Waksman, G., Bouboutou, R., Devin, J., Bourgoin, S., Cesselin, F.,Hamon, M., Fournie-Zaluski, M.C. and Roques, B. (1985) Invitro and in vivo effects of kelatorphan on enkephalin metab-olism in rodent brain. Eur J Pharmacol 117:233–243.

Wilt, F.H. (1965) Erythropoiesis in the chick embryo: the role ofendoderm. Science 147:1588–1590.

Wilt, F.H. (1974) The beginnings of erythropoiesis in the yolk sacof the chick embryo. Ann NY Acad Sci 241:99–112.

Zini, S., Fournie-Zaluski, M.C., Chauvel, E., Roques, B.P., Corvol,P. and Llorens-Cortes, C. (1996) Identification of metabolicpathways of brain angiotensin II and III using specific amino-peptidase inhibitors: predominant role of angiotensin III in thecontrol of vasopressin release. Proc Natl Acad Sci USA93:11968–11973.

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