Prominent Beta-5 Gene Expression in the

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Prominent Beta-5 Gene Expression in the Cardiovascular System and in the Cartilaginous Primordiae of the Skeleton During Mouse Development LAURENCE LE GAT, SEBASTIEN BONNEL, KARiN GOGAT, MARA BRIZARD, LOIC VAN DEN BERGHE, ALEXANDRA KOBETZ, STEPHANIE GADIN, PASCAL DUREAU, JEAN-LOUIS DUFIER, MARC ABITBOL, and MAURICE MENASCHE Centre d~ R L . ~ ~ K I ~ThJnrptwtiqnes IK.V en Ophtliulniologie. E q u i p d'ucciid t / u Minist& t/e lo Recherche ct tlr I 'Enseigiicrirmt Siipkieitr. Uiiivrr.siti.Re& DK.YCU~IKS (Purs v). Fucirltk iIe M&cine Neckrr. Puris. Fiiriiic~

Tlic alpha v bcta (uvp5) heterodimer has been implicated in many biological functions, including angiogenesis. We report the ,45 gene exprcssion pattern in cmbryonic and foetal mouse tissues as determined by Northcrn blotting and in situ hybridization. During the earliest stages, BS mRNA is widespread in the mesoderm. During later developmental stages, it remains mostly confined to tissues of mesodermal origin, although probable inductive effects trigger shifts of B5 gene expression from some mesenchymatous to epithelial structures. This was observed in the teeth, skin, kidneys, and gut. Of physiological importance is the b5 labeling in the developing cardiovascular and respiratory systems and cartilages. Furthermore, early b5 gene expression was observed within the intra- and extraembryonic sites of hematopoiesis. This suggests a major role f o r b 5 in the hernatopoietic and angiogenic stem cells and thus in the development of the vascular system. Later, the b 5 gene was expressed in endothelial cells of the vessels developing both by angiogenesis and vasculogenesis in the lung, heart, and kidneys. Moreover, the /J5 hybridization signal was detected in developing cartilages but not in ossified or ossifying bones. B5-lntegrin is a key integrin involved in angiogenesis, vasculogenesis, hematopoiesis, and bone formation.

Keywords Angiogenesis, chondrogenesis, p5-integrin. vasculogenesis

properties of the cells, and for tissue organization. The main family of cell surface receptors that mediate cell-matrix interactions is the integrins. These receptors are central to many normal

From the very first stages of embryogenesis, cells establish interactions with the extracellular matrix (ECM).These interactions are required for the full expression of the determination-differentiation

This work was supported by Association Retina France. We particuarly thank the Dean of Faculte de Medecine Necker, Patrick Berche, and Professor Philippe Even for generously helping our team. We thank the Ministere de L'Enseignement Supirieur de la Recherche et de la Technologie, INSERM, CNRS, Universitk Pans V, Association Franqaise de lutte contre les Myopathies, Fondation pour la Recherche Medicale, Fondation de I'Avenir, and Fondation de France. Laurence Le Gat and Karin Gogat are recipients of PhD grants from CERTO. Loic Van Den Berghe is the recipient of a postdoctoral grant from CERTO. Sibastien Bonnel is the recipient of a PhD grant from Association Franqaise des Amblyopes Unilateraux. Address correspondence to Maurice Menasche, Centre de Recherches Therapeutiques en Ophthalmologie Equipe d'accueil du Ministere de la Recherche et de I'Enseignement Sugrieur no. 2502, Universiti Rene Descartes (Paris V), Faculte de Medecine Necker, 156 rue de Vaugirard, 750 15 Pans, France. E-mail: [email protected] 99


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biological processes such as mesoderm development, epithelial morphogenesis, neural tube closure, anchorage to the extracellular matrix-basement membrane (ECM-BM), and central nervous system development. Their involvement in phenomena associated with pathology, such as neural wound healing, tumor metastasis, and inflammation, has also been described (Hogan 1999). The integrins are noncovalent alpha/beta (alp) heterodimers in which each subunit consists of a large N-terminal extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic tail. The extracellular domains of the a and /? subunits bind to the ligand, and the cytoplasmic tail is anchored to the cytoskeleton and cytoplasmic proteins. There are more than 14 a and 8 @ subunits, which together form more than 20 different heterodimers. Heterodimers sharing a common a subunit but with different B chains bind different ligands, suggesting that the B subunit is important in determining binding selectivity. Different integrin combinations may recognize the same ECM ligand, whereas others bind several different ECM proteins. Integrin-mediated adhesion leads to intracellular signaling events that regulate cell survival, growth, proliferation, and migration. The sites where cells contact the ECM via integrins, called focal adhesion sites, are multimolecular complexes that link integrins to the cytoskeletal network and to intracellular pathways. Thus, integrins act as bidirectional signaling machinery in the membrane, regulating numerous cellular processes (Giancotti and Ruoslahti 1999). The integrin B5 subunit predominantly associates with subunit alpha v (av) and mediates cell adhesion to vitronectin (Cheresh et al. 1989). The mouse /35 integrin cDNA has been cloned in human cells (GeneBank Accession 505633). Two different B5 integrin transcripts have been identified in the mouse; the cytoplasmic domains of the encoded proteins are dissimilar as a result of alternative splicing (Zhang et al. 1998). The promoter of the murine /?5 integrin has also been cloned recently, and a 19 bp ciselement has been identified that mediates the granulocyte-macrophage colony-stimulating factor

(GM-CSF)-induced downregulation of p5-integrin production (Feng et al. 1999). The avB5 heterodimer has been implicated in many biological functions including angiogenesis. Another integrin, called av s 3 , binds to vitronectin and is also involved in tumor angiogenesis, but does not trigger the same signaling pathway as avB5. Indeed, there appear to be two angiogenic pathways: avg5 has been implicated in vascularendothelial growth factor (VEGF)-induced angiogenesis, whereas avB3 has been implicated in basic fibroblast growth factor (b-FGF)-induced angiogenesis (Friedlander et al. 1995).Adenovirus interaction with avB5 induces virus internalization and penetration of the endosomal membrane (Wickham et al. 1994). The internalizationof the adenovirusdepends on a C-terminal motif of the B5 tail (Wang et al. 2000). avp5 is located on the retinal pigment epithelium (WE) membrane and also functions as a binding receptor for the phagocytosis of rod outer segment (ROS) (Finnemann et al. 1997). This activity is controlled by the protein kinase C (PKC) signaling pathway, which regulates the interactions between the integrin and cytoskeletal elements. Human B5 integrin has been detected in many cell types, but its mRNA is not found in lymphoblastoid cells or platelets (McLean et al. 1990; Ramaswamy and Hemler 1990; Suzuki et al. 1990). In a limited study, B5-integrin gene expression was detected in mouse, at a few stages of development, in a small number of embryonic organs (Ymada et al. 1995). However, no /35 gene expression has been previously reported in the mouse embryonic vasculature, despite the involvement of avB5 in angiogenesis (Friedlander et al. 1995). Here we report the B5 gene expression pattern in embryonic and foetal mouse tissues as determined by northern blotting and in situ hybridization. The /35 gene expression was strong as early as 9.5 days postconception (dpc) in endothelial cells during both vasculogenesis and angiogenesis and may be involved in both extraembryonic and intraembryonic hematopoiesis. We also demonstrate that B5-integrin is strongly expressed during the development in the cartilaginous models of the fbture bones.

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METHODS Tissue Preparation


Morphological normal mouse embryos and fetuses (n=20) 9.5 to 18.5 days postcoitum (dpc) were collected from NMRI mice. The embryos were microdissected under a microscope, frozen in dry ice, and stored at -80°C. As described in Sahly et al. (1998), cryostat serial sections (15 pm) were mounted on slides. Sections were fixed, dehydrated, air-dried, and stored at -80°C. DNA Probes for I n Situ Hybridization The 60-mer oligonucleotide probes were synthesized and purified by Eurogentec (Belgium). They were 3’ end labeled with a 35s dATP (NEN) using terminal deoxyribonucleotidyl transferase (Gibco BRL) and purified before use as described elsewhere (Sahly et al. 1998). Two sense and two antisense oligonucleotides were chosen according to the mouse B5-integrin cDNA (GeneBank Accession AF022110). All sequences were compared with the GeneBank and EMBL nucleotide sequence database procedures, to ensure that they were specific for the integrin g5 gene. The sequences of the oligomers were: Probe 5 sense: gcg tac agt agc atc cgg gct aaa gtg gag ctg tca gtg tgg gat cag cca gaa gac ctt (positions 1321-1380). Probe 5 antisense: aag gtc ttc tgg ctg atc cca cac tga cag Ctc cac ttt agc ccg gat gct act gta cgc. Probe 6 sense: tgg tta caa gtt att ccc caa ctg cgt ccc ctc ctt cgg gtt ccg gca tct gct gcc tct (positions 8 10-869). Probe 6 antisense: aga ggc agc aga tgc cgg aac ccg aag gag ggg acg cag ttg ggg aat aac ttg taa cca. Probe 3 sense: gcg tcc tat gct cag gcc atg gag agt gtc act gtg gag aat gca aat gcc acg cag gtt (positions 1880-1939). Probe 3 antisense: aac ctg cgt ggc att tgc att ctc cac agt gac act ctc cat ggc ctg agc ata gga cgc.

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Probe 4 sense: ggg tag aca cca tcg tcc aag att gcc cag cag act tag gtt caa gct gaa ggt gcg cca (positions 2204-2226). Probe 4 antisense: tgg cgc acc ttc agc ttg aac cta agt ctg ctg ggc aat ctt gga cga tgg tgt cta ccc. All g5 integrin antisense probes gave the same hybridization pattern on several adjacent embryonic and fetal tissue sections. The integrin /35 sense probes did not give any hybridization signal.

In Situ Hybridization Procedure As described in Sahly et al. ( 1998), the slides were hybridized and incubated in a humidified chamber at 43°C for 20 h. The sections were then used to expose Amersham Hyperfilm betamax x-ray films for 4 d and then Kodak NTB2 photographic emulsion for 2 mo at + 4 T . Northern Blot Hybridization Procedure Tissue specificity of B5-integrin gene expression was assessed by hybridization of the same antisense probes, labeled with T4 polynucleotide kinase (Life Technologies), to Northern blot filters containing murine poly A+RNAs (2 pg for each lane) from mouse embryos and fetuses (7763-1) and various adult tissues (7762- 1) (Clontech, Palo Alto, CA). Hybridization and washing were performed as described elsewhere (Sahly et al. 1998).

RESULTS Northern Blot Analysis of p5 Gene Expression During Embryogenesis To determine whether the /35 gene is expressed during embryogenesis, we performed a Northern blot analysis of poly(A) RNA isolated from mouse embryos at different stages of development (Figure 1A). The p5 mRNA was first detected at 7 dpc, then less abundantly at I 1 and 15 dpc and slightly more abundantly at 17 dpc. One single

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FIG. 1. Northern blot analysis ofg5 gene expression (A) during mouse embryogenesis and (B) in adult tissues. (See Color Plate I.)


species of RNA, a 3.4-kb transcript, was recognized by each of the probes used. Northern Blot Analysis of PS Gene Expression in Adult Tissues The tissue distribution of the /35 mRNA was examined by Northern blot analysis of poly(A) RNA extracted from various adult organs (Figure 1B). No /35 mRNA was detected in spleen or skeletal muscles. All probes hybridized to the same species of transcript (3.4 kb) and showed similar pattern. These transcripts were abundant in heart, liver, kidney, and testis and less abundant in brain and lung.

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Expression of the P5 Gene During Mouse Embryogenesis as Detected by In Situ Hybridization PS Gene Expression in Mesenchymatous Derivatives Throughout the Earliest Stage of the Mouse Embryo Development

As early as 9.5 and 10.5 dpc, the g 5 gene was ubiquitously expressed throughout the mesenchyme (Figure 2, A and B), especially in the developing somites and the mandibular component of the first branchial arch. No significant /35 mRNA signal was detected in the developing central nervous system, endoderm, or ectoderm at these early developmental stages. PS Gene Expression Pattern in the Developing Cardiovascular System

The /35 integrin gene was significantly expressed as early as 11.5j12.5 dpc throughout the yolk sac. The hybridizatim signals were not aggregated within

structures resembling blood islands or developing vessels but displayed a diffise gene expression pattern. This yolk sac /I5 gene expression persisted at 14.5 dpc (Figure 2, C and D). At this developmental stage, g 5 gene expression contrasts with the near absence of /?5 transcripts in the central nervous system and the complete absence of g 5 transcripts in the liver. The g 5 hybridization signals were detected as early as 12.5 dpc in the developing heart, especially at its periphery. At the cellular level, #?5mRNA was first and abundalty detected at 13.5 dpc in the epicardium and in the outer myocardial layer, at which time deeper myocardial layers displayed moderate /?5 gene expression levels in myocardium and endocardium. There was an apparent transmural myocardial gradient of /I5 gene transcription during the mid and late developmental stages of the mouse heart, with the highest levels of g 5 transcripts in the compact region of myocardium, which is the closest to epicardium. During the latest mouse developmental stages (1 7.5 to 19.5 dpc), this gradient gradually decreased. At subsequent developmental stages, atrial /?5 hybridization signals were always stronger than the myocardial signals (Figure 2, E and F). This gene expression pattern in the heart continued until birth. Our observationsare consistent with, and extend, the partial data previously reported (Yamada et al. 1995). The /I5 hybridization signals were readily detected in developing vessels from 10.5 dpc. At this stage, developing aortas, splanchnopleural vessels, and cardinal veins contained #?5transcripts within their endothelial cells and in the mesenchyme surrounding them (Figure 3, A and B). The /35 gene transcription was also observed in developing branches of aortas, particularly in coronary arteries, and in veins of medium and large diameter. Additionally, the j?5 gene was strongly expressed in the choroidal vessels, including the developing capillaries of the choriocapillary layer, as early as 14.5 dpc (Figure 3, C to F). PS Gene Expression Pattern in the Respiratory System

A key observation of our study is the detection of strong g 5 gene transcription at 14.5 dpc


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FIG. 2. The g5 labeling can be seen in the mesoderm of a 10.5dpc mouse embryo (A, B). The g5 mRNA is localized in the cardiovascular system: in the yolk sac at 14.5 dpc (C, D), and the heart at 15.5 dpc (E, F). (B, D, F) Bright-field aspect of the embryonic tissue sections (A, C, E). Scale bar (0.7 cm), 100 pm. YS,yolk sac; AC, auricular cavity. (See Color Plate 11.)

in the mesenchyme immediately surrounding the bronchioli. This contrasts with the absence of labeling in the respiratory epithelium of the bronchioli (Figure 3, G and H). After 16.5 dpc, g5 expression in the mesenchyme surrounding the bronchioli was almost undetectable. However, no significant #3 5 labeling was observed in the mesenchyme surrounding the walls of the medium and large vessels of the developing lung, whereas the endothelial cells

continued to contain j35 mRNA until later developmental stages. In the developing trachea, a p5 gene expression pattern similar to that detected in the bronchioli was observed between 14.5 dpc and 16.5 dpc. The mesenchyme surrounding the tracheal epithelium was labeled, as were the smooth muscle cells of the tracheal wall (Figure 4, E and F). A characteristic of the tracheal j35 gene expression pattern was early labeling of the developing tracheal


CELL COMMUNICATION & ADHESION VOLUME 8, NUMBER 3. COLOR PLATE I. See L. LE GAT et al., Figure 1.

CELL COMMUNICATION & ADHESION VOLUME 8, NUMBER 3. COLOR PLATE II. See L. LE GAT et al., Figure 2.

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FIG. 3. The j35 gene expression pattern in the developing vessels and respiratory system. The j35 labeling is detected in the aortic endothelial cells and their surrounding mesenchyme (A, B) at 17.5 dpc. The j35 hybridization signal detected in the developing vessels of the choroidal plexus (C, D) and the choriocapillary layer (E, F) at 14.5 and 15.5 dpc, respectively. In the lung, j35 gene expression is observed in the mesenchyme of the bronchiolae (G, H) at 14.5 dpc (B, D,F, H). Bright-field aspect of the same embryonic tissue sections (A, C, E, G). Scale bar (0.7 cm), 100 p m for (AHF); 25 p m for (B). Ao Endoth, aortic endothelium; Mes, mesenchyme; AoL, aortic lumen; AoW, aortic wall; Ch Plex. choroidal plexues; Ch layer, choriocapillary layer; NR. neural retina; Peribr Mes, peribronchiolar mesenchyme. (See Color Plate 111.)


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p5 GENE EXPRESSION IN THE MOUSE EMBRYO

FIG. 4. The g5 gene expression pattern in the developing skeleton. The gS labeling is observed in the cartilages and the surrounding mesenchyme of the vertebra at 17.5 dpc (A, B), of the ribs at 14.5 dpc (C, D), and in the mesenchyme surrounding the tracheal epithelium and the developing tracheal cartilages at 16.5 dpc (E, F). A p 5 labeling is also observed in olfactive structures at 12.5 dpc (G, H). (B. D, F, H) Bright-field aspect of the embryonic tissue stained with toluidine blue (A, C. E, G). Scale bar (0.7 cm). 100 pni for (AHH). Vert. vertebra labeling; periNP. pen nucleus pulpeosus; NP,nucleus pulpeosus; Rib Pc, rib perichondre; Trach Mes, tracheal mesenchyme; Tr Epith. tracheal epithelium; Tr L, tracheal lumen; Tr Cart, tracheal cartilage; Olf epith. olfactory epithelium: Mes, menchyme: Olf str, olfactory structure. (See Color Plate 1V.)

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cartilaginous primordia, followed, once the tracheal cartilageshad been formed, by perichondral labeling of these cartilages.


p5 Gene Expression, Pattern in the Developing Skeleton At the earliest developmental stages studied (from 9.5 to 10.5 dpc), the #?5 gene was transcribed throughout the mesoderm. By 11.5 dpc, #?5 gene expression was restricted essentially to the somites and especially to their sclerodermal component. On 12.5 dpc the aggregations of mesenchymal cells constituting developing ribs were significantly labeled. This costal g 5 gene transcription continued throughout development. Some of the mesenchyme surrounding the developing ribs that will eventually become the intercostal ligaments displayed significant #?5 hybridization signals (Figure 4, C and D). The core of the vertebral bodies did not seem to exhibit any significant #?5 gene expression. However, the perichondral mesenchyme surrounding the developing vertebrae was consistently labeled from 13.5 dpc to 18.5 dpc. Interestingly, at 17.5 and 18.5 dpc, a strong j?5 hybridization signal was detected along the surface of the developing vertebrae (Figure 4, A and B; see also Figure 6, B and C) and in the mesenchyme surrounding the nucleus pulpeosus but not within the ossified and the ossifying parts of the vertebrae (Figure 4, A and B). As can be seen at 15.5 dpc, it is not only the segmented axial skeletal that gave rise to #?5hybridization signals but also the limb cartilage primordia. During the first stages of cartilage formation, a b5 mRNA signal was detected in the most active region of cartilage growth. Once the cartilagesreached the preossification stage, only the perichondral mesenchymes remained intensely labeled. From 14.5 dpc, in addition to the endocranial cartilages undergoing endochondral ossification,the superficial developingcartilages of the face and sM1, undergoingan intramembranousbone formation, displayed intense g5 hybridization signals prior to any ossification (see Figure 6, A X ) . The cartilagesthat will form the bony walls of the olfactive structures displayed intense g5 hybridization

signalsboth within their intracartilagenousstructure and in their surrounding mesenchyme. There was also significant @5 gene expression early during the development of teeth (see Figure 6, A and C). This particular finding is consistent with previous observations (Yamada et al. 1995). p5 Gene Expression Pattern During Kidney Development

Extremely localized, high levels of g5 rnRNAs were first detected in glomerular epithelial cells at 13.5 dpc. In contrast, other cell types of the kidney, including ureter epithelium, endothelial cells, and stromal mesenchyme, displayed extremely faint and diffuse #?5gene transcription at this developmental stage, which corresponds to the early differentiation of the glomerular epithelial cells. The g5 gene expression continued in the glornerular epithelium until birth and later on in the adult kidney as demonstrated by Northern blot analysis. At 14.5 dpc and subsequently, exceptionally high levels of #?5mRNA were produced in the podocytes of the nephrogenic zone (Figure 5 , A and B). These observations are consistent with a previous report (Yamadaet al. 1995). The #?5transcripts were first detected in adrenal medulla at the same developmental stage (13.5 dpc) as in the kidney. The homogeneous and d i ffi e labeling of the adrenal medulla increased in intensity as development proceeded (Figure 5 , C and D).

pS Gene Expression Pattern in the Digestive System At 10.5 dpc, the #?5 gene was significantly expressed in the mesenchyme of the mesenterium, where blood islands are forming. The #?5 gene expression shifted from the mesenchymal ceIls of the mesenterium, which were the first digestive sites of #?5 gene transcription, to the epithelial cells of the walls of the digestive tube, where g5 transcripts were detected during the later mouse developmental stages (Figure 5, E and F). No g5 gene expression was detected at any stage of development anywhere in liver, pancreas, spleen, thyroid, parathyroid

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$ 5 GENE EXPRESSION IN THE MOUSE EMBRYO

FIG. 5. The g.5 gene expression pattern in the developing kidney and gut. High levels of P5 mRNA are produced in the podocytes of the nephrogenic zone at 14.5 dpc (A, B). The ,B5 transcripts are also produced in the adrenal medulla at 15.5 dpc (C, D). The intestinal epithelium is labeled at 18.5 dpc (E, F). The g5 gene is also expressed in neurectodermic structures. At 13.5 and 15.5 dpc, g5 transcripts are detected throughout the ependymal layer lining the cerebrospinal fluid (G, H). (B,D, F, H) Bright-field aspect of the embryonic tissue section stained with toluidine blue (A, C, E, G). Scale bar (0.7 crn), 100 p m for ( C H H ) ; scale bar (0.7 crn). 50 prn for (A) and 25 p m for (B). Nephr. Nephron; Adr M, adrenal medulla; lep, intestinal epithelium. (See Color Plate V )

CELL COMMUNICATION & ADHESION VOLUME 8, NUMBER 3. COLOR PLATE V: See L. LE GAT et al., Figure 5 .

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labeled. During development, there was a shift of the g5 gene expression in the skin, with the cutaneous labeling becoming mostly confined to the epithelium (data not shown). At the earliest developmental stages examined, fiom 9.5 to 11.5 dpc, no labeling was detected in the marginal and the mantle layers of the developing central nervous system (CNS) (Figure 6A). From 12.5 to 16.5 dpc, /35 transcripts were detected throughout the ependymal layer lining the cerebrospinalfluid (CSF) containing cavities of the whole CNS. However, the expression of the gS gene in the CNS was also detected from 12.5 to 17.5 dpc in neurons surrounding the ependymal layer in the floor of mesencephalon (Figure 6, G and H).No expression of g 5 was detected in the neural retina or in the retinal pigment epithelial layer.

DISCUSSION

FIG. 6. The g5 gene expression pattern in the whole mouse embryo at various developmental stages: 14.5 dpc (A), 17.5 dpc (23, C),

18.5dpc(D).Scale bar(0.7cm), I.Mmmfor(A);scalebar(0.7cm). 1.8 mm for (BHD). CNS, central nervous system; 0s Cart, ossifying cartilages; bo Cart, basiaccipital cartilages; Vert, vertebra; Lep, ieptomeninge;Ch, choroid; Mand,mandibular structure.

glands, or thymus. Nevertheless, at 13.5 and 14.5 dpc ,65 mRNA was produced in the Ratke pouch and the pituitary stalk (data not shown). This mesenchyma1 labeling was not detected at later developmental stages. Although no significant labelingwas detected during development in the liver, strong /I5 gene expression was detected from 14.5 dpc to 18.5 dpc in Glisson’s capsule surro&ding the liver (data not shown). This labeling is probably related to g5 mRNA production in developing mesenterium, epicardium, and pleura. PS Labeling of Neuroectodermic Structures

At 10.5 dpc, the mesenchyme underlying the ectodermal epithelium was clearly labeled and the developing cutaneousepithelium itself was moderately

During the earliest stages ofmouse embryonic development, g 5 gene expression is widespread in the mesoderm. During later developmental stages, @5 gene transcription remains mostly confined to tissues of mesodermal origin, although probable inductive effects and tissue interactions trigger shifts of g 5 gene expression, during particular phases, from some mesenchymatous tissues to epithelial structures. Examples of this were observed in the teeth, skin, kidneys, and gut. Of great physiological importance is the g 5 labeling in the developing cardiovascular and respiratory systems and the cartilages. A limited study of g 5 gene expression in the mouse has been reported previously (Yimada et al. 1995). The g5 labeling in the heart, kidney, ependymal layer of the CNS, and developing teeth has been already described. However, no systematic analysis of the mouse embryonic g 5 gene expression pattern has been previously reported. Our data indicate strong #?5 gene transcription in developing vessels, lung, cartilaginous models, and perichondral mesenchymes. The cardiovascular system is the first functional system of the vertebrate embryo. Very early during ontogeny, a primitive vascular plexus is laid down


PS G E N E EXPRESSION IN T H E MOUSE EMBRYO

when the embryo is still being nourished by diffusion. The precursors of endothelial and blood cells are determined very early during gastrulation. These precursors migrate and differentiate, within the wall of the yolk sac, into solid clumps of epithelioid cells, which are called blood islands. Once the vascular network is perfused by the beating heart, blood cells derived from blood islands enter the circulation and are gradually replaced by definitive blood cells that are independently derived from intraembryonic sites ofhematopoiesis, like the paraaortic splanchnopleurdaorta, gonads (Psp/AG), and mesonephros region (AGM) (Dieterlen-Lievre et al. 1988; 1993; Olah et al. 1988). At 1 1.5 and 12.5 dpc, the /35 gene is expressed at moderate levels in mesenchymal cells of the yolk sac and the AGM region. The g5 gene expression in mesenchymal cells may participate either in the determination of the blood stem cell phenotype or in the migration of these cells. The /35 gene expression pattern in these regions is somewhat similar to the VEGF gene expression pattern in the same areas at the same developmental stages. Two cytokine-dependent pathways of angiogenesis exist and are defined by their dependency on distinct vascular cell integrins. Monoclonal antibody of a v g 5 selectively blocks angiogenesis stimulated by VEGF, whereas it has minimal effects on that induced by bFGF. Conversely, antibody to a v s 3 blocks selectively angiogenesis in response to bFGF, but has a slight effect on angiogenesis induced by VEGF. These pathways are further distinguished by their sensitivity to calphostatin C, an inhibitor of PKC that blocks angiogenesis potentiated by crvg5 but not by a v g 3 (Friedlander et al. 1995). The colocalization of VEGF and /?5-integrin mRNAs in several areas during mouse development further supports the existence of two angiogenic pathways linked to different integrins and suggests that these pathways may be involved in distinct developmental angiogenic processes occurring in different organs or at different developmental stages in the same organs. These two possibilities are probably not mutually exclusive and may represent only a limited sample of the versatility characterizing developmental angiogenesis. The concomitant g5 and VEGF gene

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expression in the yolk sac and the AGM region at the same developmental stages is consistent with previous observations that cell motility mediated by crvg5, but not by crvg3, depends on prior activation ofa PKC-dependent signaling pathway (Klemke et al. 1994) and suggests a potential role for VEGF and /35-integrin in the migration of hematopoietic stem cells rather than in their determination. Further experiments are required for testing this hypothesis. A large body of data suggests similarity or overlap of the functional responses of cells to engagement of the VEGFNEGFR and extracellular matrixhntegrin systems during a variety ofbiological responses. One of the general mechanisms for regulation of integrin functions involves their activation, which enhances their apparent affinity or avidity for extracellular ligands (Bazzoni and Hemler 1998). VEGF can induce activation of the key integrins involved in angiogenesis: avg3, crvg5, a5p1, and a 2 8 1 (Byzova et al. 2000). Thus, the VEGFNEGFR system can regulate cellular adhesive and migratory responses of diverse cell types, via expression of diverse integrins. The cells types concerned possibly include blood stem cells and developing migratory presumptive hemopoietic cells. The /35 gene is not expressed in the liver itself throughout mouse development; thus, g5 is probably not involved at all in the process of hemopoiesis occurring in fetal liver. As g5 is not expressed in the spleen during development, we can also infer that g5 does not participate in any step of the hemopoiesis process in the spleen. Once laid down by vasculogenesis, the vascular pelxus expands rapidly. In contrast to vasculogenesis-the in situ differentiation of the primitive vascular plexus from its precursors-the expansion of blood vessels from pre-existing vessels is termed angiogenesis (Flamme et al. 1997). VEGF may play a key role in the early formation of major blood vessels in the embryo. Our study demonstrates /35 gene expression during mouse embryonic development of major blood vessels such as the aortas and cardinal veins. The coronary vasculature begins to develop during primary cardiac morphogenesis. Both processes of vasculogenesis and angiogenesis are involved in this event (Tomanek 1996). The


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myocardial g5 gene expression pattern during mouse heart development, reported in this study, closely matches the pattern of development of coronary vascularization. The highest levels of VEGF mRNAs in rat embryonic heart, as assessed by in situ hybridization, are in the myocardium adjacent to the epicardium during the early stages of tube formation (Tomanek et al. 1999). The similarity of VEGF and g5 gene expression patterns suggests that both genes may be involved in vasculogenesis during mouse embryonic coronary vascular development. The g5 gene expression is also detected in developing sprouting vessels: choroidal plexuses, leptomeninges, and of the choriocapillaris layer surrounding the neural retina. Hence, #I5 gene transcription also plays a role in angiogenesis during the development of the vascular system. During angiogenesis, it is likely that a number of integrins expressed on the surface of activated endothelial cells regulate critical adhesive interactions with a variety of ECM proteins. Each of these adhesive interactions may regulate a distinct biological event such as cell migration, proliferation, and differentiation (Byzova et al. 2000; Eliceiri and Cheresh 1999). Development of the lung and its vascular bed begins as establishment of the systemic circulation is completed. Both vasculogenesis and angiogenesis are thought to contribute to the development of the pulmonary circulation (Akeson et al. 2000). The first step in vasculogenesis is the transformation of a subpopulation of mesenchymal cells into haemangioblast clusters or blood islands (de Mello et al. 1997). As lung development progresses, adjacent blood islands form lumenized connections, through budding and branching of cells from the blood islands, suggesting that angiogenesis also occurs in the lung periphery. From 14.5 to 16.5 dpc, there is a significant gS gene expression in mesenchymal cells adjacent to the bronchiolar and tracheal epithelium. These cellular sites of /?5 gene transcription might correspond to clusters of cells that will form blood islands andor to subpopulations of progenitor cells that will transform into smooth muscle cells or fibroblasts. Thus, the pattern of g5 gene expression in the mouse suggests that the /?5 integrin is involved

in both vasculogenesis and angiogenesisduring lung development. Neoangiogenesis and vasculogenesis are major hallmarks of endochondral bone formation (Descalzi Cancedda et al. 1995). In those bones undergoing endochondral ossification, the /?5 gene is actively transcribed in the most active region of cartilage growth. As soon as the cartilage pieces have reached a preossification stage, only the perichondral mesenchyme remains intensely labeled. Ultimately, this perichondral mesenchyme will transform into periosteum. In addition to the cartilages undergoing endochondral ossification, the supeficia1 developing cartilages of the face and skull, undergoing an intramembranous bone formation, display intense /I5 hybridization signals. Recognition of bone matrix proteins by osteoblasts and osteoclast precursors profoundly affects their differentiation and function. Such interactions are mediated principally by integrins. The osteoclast is a polycaryon derived by fusion of macrophages in a process apparently requiring attachment of mononuclear precursors to bone matrix. The integrin uvg3 is expressed predominantly in mature osteoclasts (McHugh et al. 2000), whereas the integrin arv,95 is expressed on ostoclast precursors (Feng et al. 2000). Furthermore, VEGF and VEGFR gene expression is crucial for cartilaginous primordia development and bone formation (Niida et al. 1999). Our data emphasize the importance of interactions between the VEGFNEGFR and integrinECM systems during vasculogenesis, angiogenesis, and chondrogenesis. However, it seems that the developing vasculature might be dependent on integrins other than uv/?5. In the kidneys of rodents, VEGF is expressed at the latest developmental stages and exclusively in glomerular epithelial and tubular cells. No colocalization of g5 gene expression and the expression of any of the VEGF receptor genes has been reported in developing rodent kidneys (Tufro et al. 1999). The g5 gene expression pattern during mouse development is suggestive of a special role for the /?5 integrin subunit in angiogenesis, vasculogenesis, cartilage and bone formation, and possibly


g5 GENE EXPRESSION IN THE MOUSE EMBRYO

the genesis of blood stem cells and/or angiopoietic and hemopoietic progenitor cells. Thus g5 knockout mice were expected to display vascular pathologies, possibly associated with bone abnormalities and/or blood and vascular cell anomalies. However, B5 knockout mice are normal (Huang et al. ZOOO), whereas 83 knockout null mice display osteosclerosis (McHugh et al. 2000). Most mice deficient in av integrins die in utero, although approximately 20% survive to term but die within hours of birth (Bader et al. 1998). This lethal mutation is associated with extensive brain and intestinal vessel abnormalities and haemorrhaging. Thus a v integrins are essential during blood vessel formation and/or maturation in these tissues. However, other organs show apparently normal vascularization, suggesting that animals lacking a v integrins can compensate for this deficiency. Several integrins have been implicated in vasculogenesis and angiogenesis. The functional redundancy among integrins in angiogenesis may reside in their capacity to be activated to enhance their recognition of available ligands within a particular microenvironment (Byzova et al. 2000). We can draw various conclusions from this study. The ,65 gene expression in the yolk sac, in the PspIAGM region, and in the lung and the myocardium suggests the involvement of the /35 integrin subunit in vasculogenesis, angiogenesis, and hematopoiesis. REFERENCES Akeson, A. L., B. Wetzel, F. Y. Thompson, S. K. Brooks, H. Paradis, R. L. Gendron, and J. M. Greenberg. 2000. Embryonic vasculogenesis by endothelial precursor cells derived from lung mesenchyme. Do.Dyn. 2 17: I 1-23. Bader, B. L., H. Raybum, D. Crowley, and R. 0. Hynes. 1998. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins. Cell 95507-5 19. Bazzoni, G., and M. E. Hemler. 1998. Are changes in integrin a&ity and conformation overemphasized? Trendr Biochem. Sci. 23: 30-34. Byzova, T. V., C. K. Goldman, N. Pampori, K. A. Thomas, A. Be& S. J. Shattil, and E. F. Plow. 2OOO. A mechanism for modulation of cellular responses to VEGF. Activation of the integrins. Mol.Cell. 6:851-860. Cheresh, D. A., J. W. Smith, H. M. Cooper, and V. Quaranta. 1989. A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells. Cell 57: 59-69.

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