Arterial Identity of Endothelial Cells Is Controlled by Local Cues

Developmental Biology 237, 398–409 (2001) doi:10.1006/dbio.2001.0383, available online at http://www.idealibrary.com on Arterial Identity of Endothelial Cells Is Controlled by Local Cues Karim Othman-Hassan,* Ketan Patel,† Maria Papoutsi,* Marc Rodriguez-Niedenfu¨hr,* Bodo Christ,* and Jo¨rg Wilting*,1 *Anatomisches Institut II der Albert-Ludwigs-Universita¨t Freiburg, Albertstrasse 17, 79104 Freiburg, Germany; and †Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Mu¨llerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process. © 2001 Academic Press Key Words: angiogenesis; HTKL; ephrin-B2; arterial endothelial cell; quail–chick chimera. INTRODUCTION venules, and veins. The mechanisms that determine vascular heterogeneity have not been studied in detail, The cardiovascular system is a vital requirement in but recently it has been shown that some of the members large organisms for the continuous transport of nutrients of the ephrin-family of ligands and their Eph receptors are and metabolites to and from every single part of the body. expressed in specific parts of the vasculature. The family It is the first functional organ system of the embryo, and of Eph receptors consists of 14 members, and 8 ephrin numerous mutations affecting the cardiovascular system ligands are known (for review, see Yancopoulos et al., cause embryonic lethality (for review, see Auerbach and 1998). The ephrins can be divided into two groups, A and Auerbach, 1997). The heart is a derivative of the splanch- B. The ephrin-B ligands are transmembrane proteins, nic mesoderm induced by the endoderm, and is formed capable of bidirectional signalling, and preferentially from a promyocardium and a proendocardium (for review, bind Eph-B receptors (Gale and Yancopoulos, 1999; see Lough and Sugi, 2000). The first blood vessels are Holder and Klein, 1999). Ephrin-B1 and Eph-B3 are ex- made up exclusively of endothelial cells, and are formed pressed in both arteries and veins (Adams et al., 1999), by a variety of angiogenic mechanisms (for reviews, see but the expression pattern of other family members is Benninghoff et al., 1930; Wilting et al., 1995a, 1998; Risau, restricted to specific parts of the vascular tree. Ephrin-B2 1997). In most parts of the body, a hierarchy of vessels is is expressed in the endothelium of arteries, whereas formed consisting of arteries, arterioles, capillaries, Eph-B4 is a marker of venous endothelial cells (Wang et al., 1998; Adams et al., 1999; Helbling et al., 1999; Gale 1 To whom correspondence should be addressed. Fax: 49 761 203 et al., 2001; Shin et al., 2001). Mutant mice that lack 5091. E-mail: [email protected]. either ephrin-B2 or Eph-B4 function die of cardiovascular 0012-1606/01 $35.00 Copyright © 2001 by Academic Press 398 All rights of reproduction in any form reserved. Arterial Identity of Endothelial Cells 399 FIG. 1. Grafting scheme: Arterial and venous endothelium was isolated from day-14 quail embryos and grafted into the proximal wing bud region of day-3 chick embryos, which were then reincubated until day 6. malformations. These mice exhibit abnormal vascular known about the mechanisms that control and maintain remodelling. The formation of arteries and veins is dis- the arterial and venous identity of endothelial cells in turbed and the vascular system seems to retain the differentiated vessels during later stages of development. character of a primitive vascular plexus (Wang et al., In order to investigate these mechanisms, we have cloned 1998; Gerety et al., 1999). The results show that the the avian homolog of ephrin-B2 (HTKL) and performed arterial and venous identity of embryonic endothelial descriptive and experimental studies. We show that cells is determined very early. However, nothing is ephrin-B2 is expressed in endothelial cells of arteries and FIG. 2. Comparison of the 3Ј untranslated region of chick ephrin-B2 (HTKL) cDNA sequence with the mouse ephrin-B2/HTK ligand (Bennett et al., 1995). (A) The sequence of the 506 bases of the 3Ј end of the probe is shown, and possesses 62% homology with the mouse sequence. (B) Comparison of two highly conserved regions of this sequence shows 90% homology between chick and mouse. Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 400 Othman-Hassan et al. FIG. 3. (A) Whole-mount in situ hybridization of a stage-10 (HH) chick embryo (11 somites) with the chick ephrin-B2 probe. Dorsal view. Note expression in the caudal neural tube, somites and segmental plate, and specific areas of the lateral plate. (B) Same embryo as in (A); ventral view. The aorta (arrows) and the umbilical arterial plexus (star) are positive. (C) Expression of ephrin-B2 in the vitelline arteries of a stage-17 (HH) chick embryo. (D) Expression in the mesenchyme of the head and the cardiac region (arrows) of a stage-7 (HH) chick embryo. n, neural tube; p, pericardiac cavity. arterioles during all stages examined. In addition, there is MATERIALS AND METHODS expression in the smooth muscle cells of the aorta, and in some nonvascular tissues such as the somites, neural Embryos tube, Mu¨llerian duct, and joint regions. Grafting of ephrin-B2-positive quail arterial endothelial cells into Fertilized chick and quail eggs were incubated in a humidified atmosphere at 37.8°C. Staging was performed according to Hamburger chick embryos shows that expression of ephrin-B2 in and Hamilton (1952). Embryos and organs were fixed at various stages these cells is dependent on the integration site, and only of development (from day 2 to day 16) for descriptive studies. Inter- cells that integrate into arteries retain the ephrin-B2 specific grafting experiments were performed as described below. signal. Ephrin-B2-negative venous endothelial cells that integrate into arteries become ephrin-B2-positive. The results show that the expression of the arterial marker Grafting Experiments ephrin-B2 is dependent on local cues which may be Quail–chick chimeras (Le Douarin, 1969) were produced as derived from the smooth muscle cells of the media. follows. The ephrin-B2-positive and ephrin-B2-negative vessels Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. Arterial Identity of Endothelial Cells 401 isolated from day-14 quail embryos and grafted into the coelomic cavity of day-3 chick embryos (3 out of 12 experiments were successful). The host embryos were reincubated until day 6 and studied as follows. Cloning of the Chick Ephrin-B2 (HTKL) Probe and in Situ Hybridization The full-length mouse ephrin-B2 (hepatoma transmembrane kinase ligand, HTKL) cDNA, kindly provided by Drs. A. Flenniken and D. Wilkinson (Mill Hill, London), was used to screen a stage-11 whole chick cDNA library. A total of 2.5 ϫ 105 clones were screened yielding eight positive clones. The clone with the longest sequence contained the 3Ј coding and untranslated sequence, and was used in this study. Comparison of the 3Ј end of the probe showed that the 3Ј untranslated region possessed on avarage 62% homology between chick and mouse, and also yielded 90% homology in highly conserved regions (Fig. 2). For in situ hybridization on sections, normal and experimental embryos were fixed overnight at 4°C in Serra’s solution (Serra, 1946). The samples were dehydrated, embedded in paraffin wax, and 8-␮m sections mounted onto silanized slides. The sections were postfixed in 4% paraformalde- hyde solution (PFA) and, in older specimens, treated with protein- ase K and refixed in 4% PFA. The chick ephrin-B2 (HTKL) was linearized with ClaI and NotI to produce sense and anti-sense riboprobes, respectively. The probes were labeled as described before (Wilting et al., 1997) with digoxigenin RNA-labeling kit as recommended (Boehringer, Mannheim, Germany). A hybridization mixture of 40% formamide, 25% 20ϫ SSC, 1% Denhardt‘s solution, 1% tRNA, 1% herring sperm DNA, 2% labeled sense or anti-sense probe, and 30% DEPC-treated water was prepared. Sixty microliters of hybridization mixture were placed on each slide and the slides were incubated overnight at 65°C. After standard wash- ing, the location of the digoxigenin was detected by using a 1:4000 solution of an alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer) in a blocking agent (1% bovine serum albu- min in malate buffer) at 4°C overnight.

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