Developmentally regulated expression and functional role of 7 integrin in the chick embryo

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Develop. Growth Differ. (2004)46, 299–307

Developmentally regulated expression and functional role of 7 integrin in the chick embryo

Nikolas Zagris,* Maria Christopoulos and Anastasia Giakoumaki

Division of Genetics and Cell and Developmental Biology, Department of Biology, University of Patras, Patras, Greece

Integrin 71 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of 7 in the neural tube was transient suggesting that 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of 7 by blocking antibodies showed that 7 integrin–laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that 7 integrin–laminin signaling may have played a role in segment re-specification in the mesoderm.

Key words: 7 integrin, heart tube morphogenesis, mesoderm re-specification, neural plate bending, somite epithelialization.

Introduction

The integrins, a family of heterodimeric () trans- membrane proteins, are unique cell surface receptors in that they mediate interactions of cells with extracellular matrix proteins and also interactions with other cells (Hynes 1992). The integrin extracellular domain forms a ligand-binding site recognizing one or more extracellular ligands or counter-receptors on other cells, and the cytoplasmic domain interacts with cytoskeletal proteins (Hynes 1992; Giancotti 1997).

These interactions may transmit or initiate signals between and within cells, promote cell movement or cell adhesion, cytoskeleton organization, cell prolifer-

ation, apoptosis and cell differentiation. The specificity of integrin binding and other functions are due to the variety of combinations of associations of the 17 known chains and 8 chains (Hynes 1992); in addition, an array of variant forms of integrins with alternatively spliced extracellular and cytoplasmic domains have been identified (Collo et al. 1993; Song et al. 1993; Ziober et al. 1993; Martin et al. 1996;

Ziober et al. 1997; von der Mark et al. 2002).

The integrin 71 was identified as a developmentally regulated cell surface antigen on cultured skeletal muscle and cardiac muscle cells and as a laminin-binding integrin of melanoma cells and myoblasts (Kaufman et al. 1985; Kramer et al. 1991). More recently the 7 subunit was localized to various types of smooth muscle cell lines, liver, stomach, spleen, vascular endothelia and restricted sites in the nervous system in addition to skeletal and cardiac muscle (Collo et al. 1993; Velling et al. 1996; Yao et al. 1997).

*Author to whom all correspondence should be addressed.

Email: [email protected]

Received 21 January 2004; revised 19 March 2004; accepted 22 March 2004.

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The 7 subunit has significant homology with the 3 and 6 subunits (Song et al. 1992), and, like 3 and

6, it associates with the 1 subunit, forming a sub- group of integrins which bind the E8 fragment of laminin (Kramer et al. 1991).

There is a great amount of information regarding the structural and biochemical characteristics of the 7 integrin and much has been learned from in vitro studies about its effects on cells as well as its interactions with other matrix components (Song et al. 1992; Ziober et al. 1993; Wang et al. 1995; Ziober &

Kramer 1996; Zolkiewska & Moss 1997). However, relatively little is known about the 7 integrin tissue specific distribution or function during development (Sutherland et al. 1993; Kil & Bronner-Fraser 1996).

Work on the chick embryo was limited to describing the distribution of 7 using immunofluorescence at a late stage HH17 (HH17–18, 36 somites, Hamburger &

Hamilton 1951) and stage HH21 (43–44 somites) and described immunoreactivity on the somites and the nervous system (Kil & Bronner-Fraser 1996). In our present work, we performed a systematic study aimed at detecting accurately the first appearance and subsequent distribution of the 7 integrin using immunofluorescence and immunoprecipitation in the chick embryo from stage X (morula) up to the onset of stage HH17 (29 somites), earlier than the stages described in Kil & Bronner-Fraser (1996). We also studied the role of 7 by blocking antibodies in a set of functional studies.

Materials and Methods Embryos

Chick embryos at stages X (morula), XIII (blastula), HH2–4 (primitive streak/gastrula), HH5–7 (neurula), HH8 (4 somites) and HH17 (29 somites) were removed from their eggs and adhering yolk was cleaned off with fine dissecting needles. Attention was paid to the exact developmental stage of the embryos. The embryos were flattened either upper (epiblast)- or lower (hypoblast)-side against the surface of vitelline membrane rafts (New 1955) to establish a firm support during fixation.

The embryos were subsequently fixed in Carnoy fixative (formula B), were dehydrated through graded ethanol solutions, embedded in paraffin and serially sectioned at 7 µm.

Antisera

A monoclonal antibody that recognizes an epitope on the core protein of the chick 7 integrin subunit (clone

H1B4, Bao et al. 1993) was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA). Fluorescein-conjugated goat antimouse IgG secondary antibody was obtained commercially (Santa Cruz Biotech, Santa Cruz, CA, USA)

7 integrin immunolabeling

Serial sections were deparaffinized and rehydrated sequentially in graded alcohols down to H₂O. Tissue sections were washed three times (5 min each) in phosphate-buffered saline (PBS) containing 0.1%

Triton X-100, and blocked for 30 min in 1% bovine serum albumin in PBS and 0.1% Triton X-100. The integrin 7 antibody was used at a final concentration of 10 µg/mL in PBS and the sections were incubated for 2 h at 37C in a moist chamber. After primary antibody incubation, the sections were rinsed three times (5 min each) in PBS containing 1% Triton X-100, 40 min in PBS (three changes), and incubated with fluorescein goat antimouse IgG secondary antibody at a final concentration of 5 µg/mL in PBS for 1 h at 37C in a moist chamber. The sections were rinsed three times (20 min each) in PBS and mounted in glycerol- Mowiol. Sections were observed and photographed with epifluorescent illumination, using filters selective for fluorescein. For control slides, primary antibody was omitted and sections were incubated with mouse IgG and with fluoresceinated secondary antibody alone.

Blocking 7 antibodies and culture of embryos Embryos between stages HH2 and HH3 were removed from their eggs, cleaned of adhering yolk, placed in 400 µl chick Ringer solution containing antibody to chick 7 integrin (1.14–2.28 µg/mL final concentration) in a microwell (G20 culture slide, Arthur Thomas Philadelphia, Philadelphia, PA, USA) and incubated for 1.5 h at 37C. At the end of incubation, embryos were washed in Ringer solution, were flattened with their epiblast sides against the surface of vitelline membrane support rafts (New 1955) and cultured on plain egg albumen at 37C. Embryos incubated in plain Ringer solution or in Ringer solution containing mouse IgG, then cultured on plain egg albumen in a parallel culture to serve as controls.

Embryos were photographed after 40 h in culture, fixed in Carnoy fixative, dehydrated, embedded in paraffin and serially sectioned at 7 µm. Sixty embryos (30 control, 30 experimental) were used in the course of this investigation. Embryos, culture media and glassware were handled under sterile conditions.

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Expression and role of 7 integrin in chick embryos 301

7 integrin immunoprecipitation

7 integrin subunit was identified from chick embryos between stages HH7 and HH8 (neural folds, 1–4 somites) by immunoprecipitation of material with a specific monoclonal antibody (Bao et al. 1993) and analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Embryos at between stages HH7 and HH8 (26 embryos) were solubilized in 300 µl lysing buffer (1% [v/v] Triton X-100, 0.02 M NaCl, 0.002 M ethylene- diamine tetra-acetic acid [EDTA], 0.004 M ethylene glycol bis [2-aminoethylether]-N,N-tetraacetic acid [EGTA], 0.04 M tris [hydroxymethyl] aminomethane hydrochloride [Tris-HCl], pH 7.5 containing 0.7 µg/mL pepstatin and 40 µg/mL phenylmethanesulfonyl fluoride [PMSF]). The supernatant was mixed with immunoprecipitation buffer (0.05% [v/v] Nonidet P-40, 0.05 M NaCl, 0.003 M EGTA, 0.01 M Tris-HCl, pH 7.4) (1:6 dilution) and was split into two equal aliquots, each containing the equivalent homo- genate from 13 embryos at stage HH7–8. One aliquot was reacted with the preformed antibody-protein A-Sepharose complex (experimental) and the other was reacted with the preformed mouse IgG protein A-Sepharose complex (control) by mild agitation.

Protein A-Sepharose CL-4B (3 mg, Sigma Chemi- cal, St Louis, MO, USA) was suspended in Tris-NaCl buffer (0.15 M NaCl, 0.05 M Tris-HCl, pH 8.5) by agitation for 10 min and washed five times in the same buffer. The monoclonal antibody (clone H1B4, Bao et al. 1993) (30 µl) or the mouse IgG that served as the negative control were diluted with Tris-NaCl buffer (1:3 dilution) and were added to the protein A-Sepharose pellet. Sepharose was suspended by mild agitation and the antibody-protein A-Sepharose complex was allowed to form for at least 1.5 h at room temperature.

After addition of the one supernatant aliquot to the preformed antibody-protein A-Sepharose complex and the other supernatant aliquot to the preformed mouse IgG-protein A-Sepharose complex, the immune complex was allowed to form at 4C for 3 h with end-over-end mixing. Immunoprecipitates were analyzed on 7.5% and 10% slab SDS-PAGE (Laemmli 1970) and the gels were stained with Coomassie blue and photographed.

Results

Restricted patterns of 7 integrin distribution We studied the expression patterns of 7 integrin in early chick embryos from stage X (morula) up to the onset of stage HH17 (29 somites) by immunofluorescence. In our results, the chick blastoderm at stages X, XIII (blastula) and HH3–4 (primitive streak/

gastrula) did not show 7 immunoreactivity but 7 expression was regulated beginning at the neurula stage.

In embryos at stage HH6–7 (early neurula), 7 integrin fluorescence was distinct in cells in the neural furrow in the midline of the neural plate (‘notoplate’) where the neural plate bends, and in the initial folds at the lateral margins on each side of the neural plate (Fig. 1A); a higher magnification view of the same region is shown in Fig. 1(B). In a more posterior section of the same embryo, 7 fluorescence was detected lining the apical surface of neuroepithelial cells in the developing midline neural furrow (i.e. the area underlain by notochord) of the neural plate (Fig. 1C). The inchoate neural plate (i.e. the flat neural plate) at stage HH4 did not show 7 fluorescence. In embryos at stage HH8 (4 somites) where the neural folds had converged dorsally, 7 fluorescence was strong in the neural tube and in the surface ectoderm

Fig. 1. Transverse sections of chick embryos at stage HH6–7 (Hamburger & Hamilton 1951) during primary neurulation treated with a monoclonal antibody against the 7 integrin subunit to reveal areas of specific immunoreactivity which are shown by fluorescence.

(A), 7 immunoreactivity first appeared in the neural furrow and in elevated neural folds during bending of the neural plate at the future midbrain level. (B), higher magnification

view of neural plate from section neighboring the section in (A). (C), 7 integrin is expressed in the neural plate during median hinge point formation (i.e. neural plate anchoring to mesoderm and furrowing) at the future trunk level of the embryo shown in (A). e, ectoderm;

en, endoderm; f, neural furrow; m, mesoderm; n, notochord; nf, neural fold; np, neural plate. Bars, 50 µm (A,C), 25 µm (B).

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302 N. Zagris et al.

overlying the neural tube, but lateral ectoderm did not express the integrin further (Fig. 2A). In the same embryo, the paired cardiac primordia of condensed mesoderm and endoderm apposing the cardiac primordia showed intense 7 fluorescence (Fig. 2A);

a part of the same region was enlarged in Figure 2(B).

7 labeling was not tightly restricted to the heart trough (primordia); 7 labeling occurred for a short distance beyond the heart trough boundaries and the overall degree of labeling reduced abruptly and the more lateral mesoderm showed virtually no 7 immunoreactivity (Fig. 2A,B). Intense 7 fluorescence was detected in blood clusters in the vitelline vascular plexus (Figs 2A,C).

By the onset of stage HH17 (the time when the first neurons begin to differentiate), 7 immunoreactivity was weak on neuroepithelial cells spanning the width of the neural tube except for the neuroepithelial cells in the germinal region of the neural tube toward its lumen which showed particularly high levels of 7 immunoreactivity (Fig. 3A); a higher magnification view of the same region is shown in Figure 3(B). The strong 7 immunoreactivity shown on the lateral margin and on the floor plate margin underlain by notochord of the neural tube may indicate the expression of this integrin on axonal bundles (Fig. 3A,B,C).

In the same embryo, the myotome showed strong strand-like fluorescence, but dermatome and sclero- tome were not labeled (Fig. 3A,C). In the heart of the same embryo (Fig. 3A), 7 fluorescence was intense in the myocardium and milder in the endocardial cushions of the ventricle, intense in the sinus venosus, but the dorsal mesocardium showed weak fluorescence; a higher magnification view of a similar region in a neighboring section is shown in

Figure 3(D). 7 was not detectable on premigratory neural crest cells but a subpopulation of neural crest cells on each lateral edge of the dorsal aorta showed intense 7 fluorescence (Figs 3A,C). Expression of 7 was strong in the dorsal aorta, common cardinal vein, and in blood cells (Fig. 3A,C,D). 7 immunofluorescence was distinct in individual cells of the foregut, especially at the border of the thick gut wall and the secretory lining, and at the border of the gut lumen (Figs 3A,E).

All of the control labeling experiments using mouse IgG in place of the antibodies gave rise to negative results (not shown).

Functional role of 7 integrin

The 7 antibodies perturbed specific morphogenetic processes consistent with the 7 expression pattern.

Embryos at stages HH2 and HH3 were cultured in the presence of a monoclonal antibody of the 7 subunit for 1.5 h and then cultured further on plain egg albumen (New 1955) for about 40 h. One characteristic anti-7-treated embryo, which started culture at stage HH2–3, formed an apparently normal looking anterior–posterior axis, shown after 40 h of culture on plain albumen in Figure 4b. This anti-7-treated embryo showed distinct somites and it was of particular interest to note that median somites seemed to have replaced the notochord at its cranial region (Fig. 4b, pointer). A HH2–3 stage embryo that had been incubated in Ringer solution containing non-immune mouse IgG for 1.5 h, and then cultured on plain egg albumen in a parallel culture, was photographed at the same time point for comparison (Fig. 4a); this embryo was similar to

Fig. 2. Localization of ^{7 integ-} rin in chick embryo at stage HH8 shown on transverse sections.

(A), 7 immunoreactivity is most intense in the heart primordia, the endoderm apposing the heart primordia, the neural tube and the surface ectoderm overlying the neural tube, and in blood cells in the vitelline vascular plexus. (B), heart primordium and (C) blood cells in vitelline vessel of embryo that are shown in (A) shown at higher magnification. b, blood cells; e, ectoderm; en, endoderm;

h, heart primordium; i, anterior intestinal portal; m, mesoderm;

n, notochord; nt, neural tube;

sp., splachnopleure; st, somato- pleure; v, vitelline membrane.

Bars, 100 µm (A), 25 µm (B,C).

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other control embryos cultured in the absence of serum.

Transverse sections through the heart (Fig. 4A) and somite (Fig. 4B) regions of the control embryo

Fig. 3. Localization of ^{7 integ-} rin in the developing cardio- vascular, muscular and nervous systems in chick embryo at the onset of stage HH17 shown on transverse sections. (A), ⁷ immunoreactivity is intense in the sinus venosus and ventricle in the heart, and is distinct in the myotome in the somite, in the ventrolateral margins and at the lumenal surface of the neural tube. (B,C,E) Higher magnification view of the neural tube, somite, gut and dorsal mesocardium, respectively, as shown in (A); (D) higher magnification view of the ventricle walls in a heart from a transverse section neighboring the section shown in (A). b, blood cells; cv, common cardinal vein; d, dorsal aorta;

dm, dorsal mesocardium; ec, endocardium; g, foregut; mc, myocardium; mt, myotome; n, notochord; nt, neural tube; sp., splachnopleure; st, somato- pleure; sv, sinus venosus; ve, ventricle; vv, vitelline vessel. Bars, 100 µm (A), 25 µm (B–E).

Fig. 4. A monoclonal antibody against the 7 integrin subunit perturbed specific morphogenetic processes in chick development.

Embryos at stage HH2–3 (initial primitive streak/early gastrula) were placed in Ringer solution containing either antibodies to 7 integrin subunit (experimental) or non-immune mouse IgG (control) for 1.5 h, and then placed on rafts and cultured on plain egg albumen for 40 h. The embryo presented in (a) is the control. The experimental embryo (b) formed median somites which seemed to have replaced the notochord at its cranial end (pointer). Transverse sections through the heart (C) and the somite (D) regions of the embryo presented in (b) showed open an neural tube, abnormal heart and absence of epithelial somites. Transverse sections through the heart (A) and somite (B) regions of the embryo in (a) served as the control. Sections

(7 µm) stained with eosin (A,B) and with Alcian blue-eosin (C,D). Bars, 100 µm (A,B), 50 µm (C,D), 0.94 mm (a,b). e, ectoderm; ec, endocardium; en, endoderm; g, foregut; h, heart; m, mesoderm; mc, myocardium; n, notochord; np, neural plate; nt, neural tube; s, somite; v, vitelline membrane.

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presented in Figure 4(a) showed characteristic brain, heart, notochord, somite and gut morphology.

Figure 4(C,D) shows transverse sections at the level of heart and somites, respectively, of the embryo presented in Figure 4(b). Figure 4(C) shows how the neural plate wall was disorganized and had failed to form a neural tube. The heart primordia had fused to form an abnormal heart tube; the myocardial tissue was thickened and appeared disorganized, and the primordial endothelial cells that exhibit the characteristics of migratory cells were congregated and encased by cells corresponding to the early myocardium but they were not organized into the single hollow channel that corresponds to the definitive endocardium of the heart tube.

Figure 4(D) shows opened neural plate and mesodermal clusters that had not lost contact to the neural plate. A striking feature to note was the rosette of epithelializing mesodermal tissue underneath the neural plate in place of a notochord which corresponded to the median somites that had replaced the notochord in the embryo presented in Figure 4(b) (pointer).

The neural tube, neural crest due to incomplete closure of neural tube and somite abnormalities of embryos presented in Fig. 4(C,D), were practically consistently reproduced in 10 of the sectioned embryos that had been treated in a similar manner and resembled in gross morphology the embryo presented in Fig. 4(b). The cardiac rudiments had either migrated toward the anterior intestinal portal

and had formed an abnormal heart tube at the ventral midline (five embryos including the embryo in Fig. 4b) or had migrated and were closely apposed at the ventral midline but were not united into a single tube (four embryos) or were absent (one embryo) in the sectioned embryos.

Identification of 7 integrin by immunoprecipitation In our present work, embryos at stage HH7–8 were extracted with EDTA-containing buffer and were sub- jected to an antibody affinity column specific for its core protein. Homogenates immunoprecipitated with a monoclonal antibody against chick 7 integrin subunit recognized a band running slightly slower than the marker at M_r approximately 120 kDa (Fig. 5, lane c in panel A, arrow) although there were two other bands at M_r approximately 50–60 kDa, which could correspond to the IgG heavy chains, that were also recognized by the antibody and by the non-immune mouse IgG as well (Fig. 5, lane b in panel B); an additional band at M_r 20 kDa was recognized by the non-immune mouse IgG only (Fig. 5, lane b in panel B). The band at M_r approximately 120 kDa (Fig. 5, lane c in panel A, arrow) was absent in precipitations with non-immune mouse IgG (Fig. 5, lane b in panel B) and it is therefore likely to correspond to the chick

7 integrin subunit (Bao et al. 1993). Molecular mass of identified polypeptides was determined according to electrophoretic migration of standard markers in lanes a (BlueRanger, Pierce, Rockford, IL, USA;

Fig. 5. Immunodetection of 7 integrin subunit isolated from chick embryo. Embryos at stage HH7–8 (neural folds/1–4 somites) were lysed, precipitated either with a monoclonal antibody against an 7 integrin subunit (lane c in panel A) or with a non- immune mouse IgG (lane b in panel B). Precipitates were analyzed in 7.5% (panel A) or in 10% (panel B) slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and polypeptides were visualized by Coomassie blue stain (lane c in panel A and b in panel B). The antiserum against 7 recognized a band at molecular mass (Mr) approximately 120 kDa (lane c in panel A, arrow) not recognized by the non-immune mouse IgG and it is likely to correspond to the chick 7 integrin subunit. There were two other bands at M^r approximately 50–60 kDa, which could correspond to the IgG heavy chains, that were also recognized by the antibody (lane c in panel A) and by the non-immune mouse IgG (lane b in panel B); an additional band at Mr 20 kDa was recognized by the non-immune mouse IgG only (lane b in panel B). Molecular mass of identified polypeptides was determined according to electrophoretic migration of standard markers BlueRanger/Pierce (in lanes a) and Rainbow/Amersham (in lane b in panel A). Numbers to the left indicate position of migration of Mr protein markers in kilodaltons.

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panels A and B) and b (Rainbow, Amersham Biosciences, Piscataway, NJ, USA; panel A).

Discussion

Integrin 71 is a specific cellular receptor for laminin-1 as well as for the laminin isoforms-2 and -4 but not laminin isoform-5 (Kramer et al. 1991; Yao et al. 1996; von der Mark et al. 2002) and it appears likely that integrin 71 is expressed in numerous sites in the vicinity of its natural ligand. Laminins are major components of the extracellular matrix, including the basement membrane, and of embryonic tissues and promote adhesion, cell attachment, directional migration, proliferation, differentiation, neurite outgrowth, axon guidance and survival (Zagris 2000; Zagris et al. 2000).

The immunolabeling results of the present work showed that 7 was first detectable in the neural furrow and in the neural folds in embryos at stage HH6–7 and showed a dynamic expression pattern in the developing neural tube. The 7 expression domain spanned the entire neural tube by stage HH8, and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in the embryos by stage HH17. The expression of the

7 integrin in the neural plate and in the adjacent ectoderm (Fig. 1A–C) during neural plate anchoring to the mesoderm and furrowing to form the neural tube could implicate the 7 integrin in this major morphogenetic event of primary neurulation. Primary neurulation involves induction and thickening of the neural plate and formation of the neural tube by bending of the neural plate (Schoenwolf & Smith 1990; Tiede- mann et al. 1998). The detailed cellular mechanisms underlying neural plate bending to form the neural tube are not yet established, but it has been dis- cussed in the literature that neural plate and surface ectoderm are both taking an active, cooperative part in the bending movement in which the extracellular matrix may be playing an active part. The strong 7 immunoreactivity shown on the lateral and floor plate margins of the neural tube in embryos at the onset of stage HH17 may point to expression of this integrin on axonal bundles (Fig. 3A,B,C). Laminin is present surrounding the neural tube and notochord (Duband

& Thiery 1987; Zagris 2000). It is possible that laminin serves as a permissive substrate for com- missural nerve cells which may extend their axons along the laminin-rich basement membrane on the lateral and ventral margins of the neural tube (Tessier- Lavigne et al. 1987). Expression of 7 in the neural tube was transient suggesting that 7 functions

during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. The

7 expression on axonal bundles was stronger in embryos by late stage HH17–18 as has been shown in previous work (Kil & Bronner-Fraser 1996). Pertinent to our work are findings that have shown the 71 integrin present on axons and their growth cones and that it played an important role on the axonal outgrowth during peripheral nerve regeneration in the adult nervous system in the mouse (Werner et al.

2000).

The 7 integrin immunoreactivity was intense in the heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8 (Fig. 2A,B). By the onset of stage HH17, 7 expression was intense in the myocardium, milder in the endocardial cushions of the ventricle, intense in the sinus venosus but the dorsal mesocardium showed weak fluorescence (Fig. 3A,D).

Molecules known to exist in the extracellular matrix between the heart primordia and the apposing endoderm include fibronectin, cytotactin, collagens I and IV and laminin which could influence heart formation in a variety of ways including mediating cell shape changes, migration, proliferation and differentiation (Little & Rongish 1995; Sugi & Markwald 1996).

Cardiogenesis can be separated into two phases, a pretubular phase of cell migrations that leads to the formation of the heart tube, and a tubular heart phase of complex morphogenetic changes that leads to the definitive looped, multichambered morphology of the mature beating heart (Jacobson & Sater 1988;

Fishman & Chien 1997; Mohun & Sparrow 1997;

McFadden & Olson 2002). In embryos treated with antibodies to 7 (Fig. 4b), the cardiac rudiments migrated toward the anterior intestinal portal and in some cases they formed an abnormal heart tube (Fig. 4C). Our results indicated that the 7 integrin–

laminin signaling may not be involved primarily in the directional migration of precardiac cells but may regulate the expansion of the population of precardiac cushion mesenchyme and, especially, it may play a critical role in tissue morphogenesis of the heart tube.

The major blood vessels, such as the dorsal aorta, were malformed or absent in the embryos treated with

7 antibodies (Fig. 4C). The dorsal aorta, common cardinal vein and blood cells expressed 7 strongly (Figs 2A,C and 3A,C,D). A subpopulation of migrating neural crest cells on each lateral edge of the dorsal aorta showed intense 7 fluorescence (Figs 3A,C) while 7 was not detectable on premigratory neural crest cells. These migrating neural crest cells belong to the sympathoadrenal sublineage which differentiate into catecholamine-containing cells of

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the sympathetic ganglia, chromaffin cells of the adrenal medulla and cells of the aortic plexuses.

Another point of particular interest was that even though in gross morphological observation of the embryo the paraxial mesoderm showed a meristic (segmental) pattern, albeit with some abnormalities (Fig. 4b), in transverse histological sections epithelial somites were absent but condensations of cells suggestive of incipient somites were observed (Fig. 4D). It was striking to note a rosette of epithelializing mesodermal tissue underneath the neural plate which corresponded to the median somites that had replaced the notochord in the embryo presented in Figure 4(b) (pointer). This was intriguing and it is tempting to think that 7-laminin signaling might have played a role in segment re-specification in the mesoderm. Pertinent to this may be work which showed that the 7 integrin gene is clustered with Hox genes on the human chromosome 12q13 (Wang et al. 1995).

The clustering of integrin and Hox genes implies parallel evolution and interaction of these gene families and places in concert the evolution of the information (the Hox genes) that specifies the patterning of the body plan with the realisator integrin genes that mechanistically underlie the cell migrations and interactions that fulfill this body plan. In most of the sectioned embryos, however, we observed multiple rosettes of epithelializing paraxial mesodermal tissue with retracted (rounded up) cell morphology lateral to the neural tube (not shown) where a somite pair would be forming in normal embryos. The presence of metameric units in the anti

7-treated embryos suggested that 7 integrin–

laminin signaling does not therefore seem to be crucial for segment formation in the presomitic mesoderm; rather, it seems to be required for somite epithelialization. 7 immunoreactivity was strong in the newly formed somites (not shown) although the expression of this molecule was restricted only to the myotome in the mature somites by the onset of stage HH17 (Fig. 3A,C). Expression of 7 was stronger in the myotome in older embryos at stage HH17–18 and stage HH21 as was seen in previous work (Kil &

Bronner-Fraser 1996). This may show that 7 expressed in the myotome at this stage is upregulated and developmentally regulated.

Our results showed that the 7 subunit of integrin has a restricted and developmentally regulated expression pattern in the early chick embryo. 7 was first detected in the neural furrow and neural folds in embryos at stage HH6–7 during primary neurulation and had spanned the entire neural tube by stage HH8;

it was downregulated in the neural tube but was high on axonal bundles such as the ventral root in embryos

by the onset of stage HH17. Intense 7 immunoreactivity was detected in the somites as they epithelialized but 7 expression was restricted only to the myotome in the mature somite. The most intense and most constant levels of 7 fluorescence were detected in the heart primordia and the developing heart. The distribution pattern of 7 suggested a distinct role for integrin 71 in specific morphogenetic processes. The fact that antibodies directed against the 7 subunit of integrin perturbed the neural tube closure, somite epithelialization and the heart tube and major blood vessel morphogenesis demon- strated the organizational role of 7 integrin–laminin signaling in these processes, consistent with 7 expression pattern in the early chick embryo.

Acknowledgements

This work was supported by grants from the General Secretariat for Research and Technology of Greece and the European Social Fund (99 ÅÄ 352), and from the University of Patras (‘K. Karatheodoris’ grant 2435).

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