Selective expression of angiopoietin 1 and 2 in

Abstract. The expression of angiopoietin1 and 2 (ang1 and 2) and their receptor tie-2 was studied in avian embryos using in situ hybridization. Ang1.
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Mechanisms of Development 106 (2001) 133±136

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Gene expression pattern

Selective expression of angiopoietin 1 and 2 in mesenchymal cells surrounding veins and arteries of the avian embryo Delphine Moyon, Luc Pardanaud, Li Yuan, Christiane BreÂant, Anne Eichmann* Institut d'Embryologie Cellulaire et MoleÂculaire CNRS FRE 2160, 49bis, Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France Received 9 March 2001; received in revised form 20 April 2001; accepted 23 April 2001

Abstract The expression of angiopoietin1 and 2 (ang1 and 2) and their receptor tie-2 was studied in avian embryos using in situ hybridization. Ang1 was transcribed in the mesenchymal cells surrounding venous endothelium expressing tie-2. By contrast, ang2 was transcribed around arteries in which the endothelium down-regulated tie-2 mRNA. The aorta and large arteries of the heart out¯ow tract were never surrounded by ang2 positive cells and maintained tie-2 expression. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angiogenesis; Angiopoietin; Artery; Vein; Vessel wall; Embryo

1. Results The angiopoietin (ang) growth factor family consists of four members, ang1±4, all of which bind with high af®nities to the receptor tyrosine kinase tie-2 (see Yancopoulos et al., 2000 for review). Tie-2 and its ligands ang1 and 2 were recently cloned from chick (Jones et al., 1999). Using in situ hybridization, we examined their expression during the avian embryonic development in comparison to VEGFR2 and 3 (Eichmann et al., 1996), neuropilin-1 (NP1), which labels arterial endothelial cells (EC) in avian embryos (Moyon et al., submitted for publication), and ephrinB2, which labels arterial EC in mouse embryos (Wang et al., 1998; Adams et al., 1999). Quail and chick embryos were examined from presomitic stages to embryonic day (E)10. Tie-2 expression was ®rst detected at early somitic stages in the EC and some hematopoietic cells of forming yolk sac blood islands. Ang2 was expressed in Hensen's node, while no ang1 expression was observed (not shown). Ang1 was ®rst detected around the 8-ss in the myocardium, surrounding tie-2 1 endocardial cells (Fig. 1A,B). Ang2 showed a complementary expression in the foregut endoderm of the heart region (Fig. 1C). From E2.5 onward, ang1 and 2 expression appeared in the mesenchymal cells surrounding blood vessels (Fig. 2). Ang1 was strongly expressed around the cephalic segment of the * Corresponding author. Tel.: 133-1-45141506; fax: 133-1-48734377. E-mail address: [email protected] (A. Eichmann).

Fig. 1. Expression of tie-2, ang1, and ang2 in the cardiac region. Transverse sections through the heart of chick embryos at the indicated somitic stages (S) hybridized with antisense riboprobes as labeled. (A) Tie-2 labels EC of the aorta (Ao), perineural vascular plexus, and endocardium (arrowhead). (B) Ang1 is expressed exclusively in the myocardium (arrow). (C) Ang2 is expressed in the pharyngeal endoderm (asterisk). NT, neural tube; N, notochord. Bar: 140 mm.

0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00425-7

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D. Moyon et al. / Mechanisms of Development 106 (2001) 133±136

cardinal vein (Fig. 2B,E). At high magni®cation, mesenchymal cells, but not EC, clearly expressed ang1 (Fig. 2, data not shown). Ang1 expression appeared in the ventral ventricular zone of the anterior neural primordium and in two stripes on both sides of the ¯oor plate (Fig. 2B). Ventral to the notochord, prospective cartilage expressed ang1 from E2.5 until about E5 (Fig. 2B). Ang2 labeled mesenchymal cells around the aortic arches and internal carotid artery (Fig. 2C,F). EC of these vessels were ang2 2. Ang2 was expressed in the ventral perineural vascular plexus (Fig. 2C) and later in some brain capillary EC (not shown). In the trunk, ang2 labeled the medial part of the somite (not shown) and subsequently the myotome (Fig. 2C). Tie-2 mRNA levels remained high in cardinal vein and aortic EC, but decreased in the regions of the aortic arches where ang2 was expressed (Fig. 2A,C). VEGFR2 labeled both arterial and venous EC (not shown), while NP-1 labeled arterial EC only (Fig. 2D,G). EC starting to invade the neural primordium transcribed tie-2 (Fig. 2A). In the trunk, ang1 was expressed around the posterior

cava vein while ang2 1 mesenchyme surrounded the omphalomesenteric artery (Fig. 2H,I). Venous EC highly expressed tie-2, while its levels declined in most arteries (Fig. 2G). An exception was the aorta, the only artery surrounded by ang1 expressing cells until E4 (not shown), which maintained the tie-2 expression until late stages (Fig. 3A). To distinguish arteries, veins, and lymphatic vessels up to E10, sections were counterstained with the EC-speci®c QH1 monoclonal antibody (mAb), and with a mAb against smooth muscle actin (aSMA). The dorsal aorta remained tie-2 1, while small arteries branching from it were only weakly tie-2 1 (Fig. 3A). Ang2 was precisely expressed in regions of arterial branching from the aorta, both at E6 and at E10 (Fig. 3D). Lymphatic EC were tie-2 2 (Fig. 3A,E,I), but expressed VEGFR3 (not shown). In virtually all internal organs, connective tissue, cardiac and skeletal muscle, tie-2 was expressed at high levels in veins, while arteries, surrounded by a thick aSMA-positive coat, were only weakly positive (Fig. 3E±G,I). Ang2 was highly expressed

Fig. 2. Ang1 and 2 are selectively expressed around veins and arteries, respectively. (A±C) Adjacent sections through the rhombencephalon (Rh) of an E4 chick embryo hybridized with the indicated probes. (A) Note strong tie-2 expression in the cardinal vein (CV), while aortic arches (arrows) show signi®cantly less expression. (B) Ang1 is expressed around the anterior segment of the cardinal vein as well as in prospective cartilage ventral to the notochord (N). (C) Ang2 labels mesenchyme surrounding the aortic arches (arrows) as well as the myotome (arrowhead). Asterisks in B and C indicate ang1 and 2 expressions in the ventricular zone (B) and the perineural vascular plexus (C), respectively. (D±F) Higher magni®cation of the anterior cardinal vein (V) and the internal carotid artery (A) of an E6 chick embryo. The rhombencephalon is to the right. (D) Double in situ hybridization with NP-1 (brown staining) and tie-2 (blue staining). NP-1 labels arterial EC, while tie-2 is only weakly expressed there; in contrast, tie-2 strongly labels the venous EC. (E) Ang1 expression is observed around the vein. (F) Ang2 is expressed around the artery. (G±I) Adjacent sections through the abdominal region of an E6 chick embryo. (G) Double in situ hybridization with NP-1 (brown staining) and tie-2 (blue staining). Note weak tie-2 expression in the omphalomesenteric artery (A) and strong staining in the inferior cava vein (V). (H) Ang1 labels mesenchymal cells around the cava vein. (I) Ang2 labels mesenchymal cells around the omphalomesenteric artery. Ao, aorta; CV, cardinal vein; M, mesonephros; G, gizzard; L, liver. Bars: 270 mm in (A±C), 130 mm in (D±I).

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Fig. 3. Expression during late embryonic stages. Transverse truncal sections of an E10 quail embryo. (A±C) Triple staining of a section through the dorsal aorta (Ao) with the indicated markers. (A) Note strong tie-2 labeling in EC of the dorsal aorta, but not small arteries branching from it (arrows). Lymphatic EC of the thoracic duct (Th) are tie-2 negative. (B) aSMA staining of the wall of the aorta and small tie-2 negative arteries (arrows). (C) QH1 staining of all vascular and lymphatic EC. (D) Ang2 is expressed in regions of arterial branching (arrow). (E±G) Triple labeling of a section through the wall of the gizzard. (E) Tie-2 is expressed at highest levels in venous EC (V, arrowhead). (F) aSMA staining of the arterial, but not the venous or lymphatic vessel wall. (G) QH1 staining of all vascular and lymphatic EC. (H) Adjacent section hybridized with ang2, which is transcribed around the artery. (I±L) Adjacent sections of connective tissue hybridized with the indicated probes. (I) Note weak tie-2 staining of arterial compared to venous EC and the absence of lymphatic EC staining. (J) Ang2 is expressed in smooth muscle cells surrounding arterial EC. (K) No signi®cant ang1 staining was observed. (L) Section through connective tissue hybridized with ephrinB2 shows staining of arterial, but not venous or lymphatic EC. Ao, aorta; Th, thoracic duct; A, artery; V, vein; L, lymphatic vessel. Bars: 90 mm in (A±D), 60 mm in (E±H), 90 mm in (I±L).

in a few smooth muscle cell layers immediately adjacent to arterial EC (Fig. 3H,J). Ang1 expression was weak or absent around blood vessels of E10 embryos (Fig. 3K) but high in the ventricular zone of the neural tube (not shown). To clearly identify arterial EC, we cloned a fragment of chick ephrinB2. In E8 chick and E10 quail embryos, arterial EC were ephrinB2 1, while veins were negative (Fig. 3L, data not shown). In summary, we detected differential expression of ang1 and 2 in the mesenchyme surrounding, respectively, developing veins and arteries. Tie-2 mRNA levels decreased in arterial EC, surrounded by ang2 1 mesenchyme, while veins, surrounded by ang1 1 mesenchyme, maintained high expression levels. The dorsal aorta and large arteries of the heart out¯ow tract were never surrounded by ang2 1 cells and maintained tie-2 expression. A similar expression pattern was not reported in mice (Schnurch and Risau, 1993; Sato et al., 1995; Suri et al., 1996; Maisonpierre et al., 1997), and may thus be speci®c for birds. Species divergence of angiopoietins was previously noted: the mouse and human orthologues ang3 and 4 represent, respectively, a widely expressed receptor antagonist and a receptor agonist expressed in lung (Valenzuela et al., 1999). Further studies will determine if ang1 and 2 function as receptor agonist and antagonist in avian EC. Artero-venous differentiation in birds and mice may thus be regulated differently, but involves at least some common molecules, since ephrinB2

is expressed selectively by the arterial EC in both species (Wang et al., 1998; Adams et al., 1999; this study). 2. Methods Chick tie-2, ang1, ang2, and NP-1 clones were described (Takagi et al., 1995; Jones et al., 1999). Chick ephrinB2 (GenBank Accession No.: AF180729.1) was recloned by RT-PCR using two primer pairs (forward (f)1: CGC CTG CAA GTA CTG CTG G, reverse (r)1: GTG GTA ACC CTG CAG AAT TG, (f)2: CAA TTC TGC AGG GTT ACC TC, (r)2: GTA CTA TGT ACA CTG GAT GTC) to amplify 504 and 446 bp of coding sequence from E9 chick embryo total RNA. Bands were subcloned into pCRIITOPO (Invitrogen). In situ hybridizations on paraf®n sections were described (Eichmann et al., 2000; Etchevers et al., 2001). 1A4 aSMA (Sigma) (IgG2a) was diluted 1/400 in QH1 hybridoma supernatant (IgM). Acknowledgements We thank Drs G. Yancopoulos and H. Fujisawa for probes, and F. Beaujean for illustrations. This work was supported by the Association pour la Recherche contre le Cancer and the MinisteÁre de la Recherche (ACI jeune) to A.E.

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