Expression of the β4 integrin subunit in the mouse heart during embryonic development: Retinoic...

DEVELOPMENTAL DYNAMICS 201:8%103 (1896)

Expression of the p4 Integrin Subunit in the Mouse Heart During Embryonic Development: Retinoic Acid Advances p4 Expression BEEREND P. HIERCK, ADRIANA C. GITTENBERGER-DE GROOT, LIESBETH VAN IPEREN, ANTJE BROUWER, AND ROBERT E. POELMANN Department of Anatomy and Embryology, University of Leiden, 2300 RC L e i a h (BP.H., A.C.G.-D.G., L.vJ., R X P J , and Hubrecht Laboratory, Uppsalalaun 8,3584 CT Utrecht (AS.), The Netherlands

ABSTRACT Using immunohistochemical techniques as well as in situ hybridization we were able to elicit the expression pattern of the $4 integrin subunit in the murine heart during development. We show that $4 is not expressed in the heart before El3 and is afterwards restricted to the endocardium of the atrioventricular canal, the outflow tract, and the venous valves in the right atrium. As these are all sites of high shear stress in the heart, we propose a role for a6$4 in the tight adhesion of the endocardial cells to their basement membranes in these segments. Moreover, mouse embryos were treated with all-trans retinoic acid, which was previously shown to induce congenital malformations, among which malformations of the heart. We show an advanced expression without ectopic localization of cardiac $4 after the administration of retinoic acid. This advanced appearance of $4 was also shown in ext- racardiac tissue like migrating neural crest cells. Several hypotheses on the mechanism of p4 upregulation and a possible role for a6$4 in the development of heart malformations after the administration of retinoic acid are discussed. 0 1996 Wiley-Liss, Inc.

Key words: p4 Subunit, a6$4 Integrin, Heart De- velopment, Mouse, Retinoic Acid

INTRODUCTION The development of the heart during mammalian

embryogenesis is a complex process. A tightly sched- uled and regulated sequence of events is necessary to develop from two heart forming regions in the lateral mesoderm to a fully septated, four chambered heart with functional valves providing unidirectional flow, and a conduction system to direct regulated contrac- tion. A complicating factor in this process is the fact that the heart is the first organ to be functional during embryogenesis and that most of the looping, septation, migration, and remodelling must occur in a beating environment. Therefore, adhesion of cells to their surrounding extracellular matrix and to neighbouring cells is essential.

Intercellular and cell-matrix interactions in the body

0 1996 WILEY-LISS, INC.

often involve integrin receptors. Integrins are heterodimer transmembrane molecules that consist of an a and p subunit that are non-covalently associated. To date 20 a, and 8 p subunits have been described and various combinations of an a and 0 subunit can give rise to receptors with their own ligand specificity and distinct functions in processes like migration, differentiation, and signal transduction (reviewed in Hynes, 1992). Recently, we described the differential expression of the a6 integrin subunit in the developing murine heart (Hierck et al., 1996). The p4 integrin subunit invariably associates to 016, regardless of its splice variants, e.g., A, B, X1, or X1X2 (Niessen et al., 1994b; Delwel et al., 19951, giving rise to the a6p4 receptor molecule that is predominantly involved in the adhesion of epithelial cells to their basement membrane. In stratified epithelia a6p4 is localized in specialized adhesion structures, termed hemidesmosomes, that mediate firm adhesion to extracellular matrix molecules in the basement membrane (Sonnenberg et al., 1991; Jones et al., 1994 and references therein). Hemidesmo- somes provide linking between the anchoring filaments in the basement membrane and the intermediate filament network in epithelial cells, with the a6p4 cell surface receptor as the most likely linking candi- date (Gmez et al., 1992). This integrin is also expressed in cell types that do not form these adhesion structures like Schwann cells and peripheral nerve perineural fibroblasts (Sonnenberg et al., 1990; Feltri et al., 1994; Niessen et al., 1994a), immature thymo- cytes (Phillips et al., 19911, vascular smooth muscle cells (Cremona et al., 19941, cytotrophoblast cells (Ap- lin, 19931, and subsets of endothelial cells (Kennel et al., 1992; Natali et al., 1992). Moreover, carcinomas of various origin express a6p4 (Kajiji et al., 1987; Kennel et al., 1989).

Two variants of human 04 (Suzuki and Naitoh, 1990) have been described, containing an insert of 53 or 70 amino acids, respectively (Hogervorst et al., 1990; Tamura et al., 1990), and one variant in which a 7 amino acids domain was deleted (Clarke et al., 1994).

Received December 28, 1995; accepted April 16, 1996. Address reprint requestidcorrespondence to Robert E. Poelmann,

Dept. of Anatomy and Embryology, PO Box 9602, 2300 RC Leiden, “he Netherlands.

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Murine p4 was recently characterized (Kennel et al., 1993). Only the mouse homologues of the human p4(- 53, - 70) and the p4( + 53) variant were reported whereas the p4( + 70) variant could not be detected in mice. Although differences in localization have been reported for the p4 variants, all appear to associate to 016.

The physiological extracellular ligand for a6p4 seems to be cell-type dependent. In human colon ade- nocarcinoma cells u6p4 appears to bind laminin 1 (Lee et al., 1992) and in cells transfected with p4 this integrin binds to laminins 1 and 5 (Niessen et al., 1994b). In addition, laminin 2 and 4 were recently described to be ligands for a6p4 in a similar expression model (Spinardi et al., 1995). However, it cannot be excluded that other, probably yet unidentified, extracellular matrix components will serve as physiological ligand for a6p4 in vivo. This could well be a (new) member of the laminin family of extracellular matrix molecules. Laminin is a large extracellular matrix molecule com- posed of three covalently linked polypeptide chains termed a, p, and y (Burgeson et al., 1994). To date 4 laminin a chains, 3 p chains, and 2 y chains have been described that can give rise to at least 7 laminin molecules (reviewed in Timpl and Brown, 1994; Iivanainen et al., 1995), all with their own characteristics with respect to their structure, binding sites, etc. This diversity in laminin isoforms comprises a large potential of a6p4 ligands and complicates determining the actual ligand for this integrin receptor in vivo.

Retinoic acid (RA), a vitamin-A (retinol) derivative, is a powerful mediator of gene expression. It is impor- tant in normal embryonic development and both lack and excess of retinol or its active metabolites can cause various embryonic malformations (Wilson et al., 1953; Morriss-Kay, 1992; Twal et al., 1999, including heart malformations (Taylor, 1979, 1981; Lammer et al., 1985; Hart et al., 1990; Pexieder et al., 1992, 1995; Broekhuizen et al., 1995; Bouman et al., 1995).

In 1975 the effect of RA on cells in the skin was already described. Excess of RA caused an increase in the number of hemidesmosomes and increased adher- ence of keratinocytes to their basement membrane (Christophers and Wolff, 1975; Kwasigroch and Koch- har, 1975). Later, keratins, the intermediate filaments of epithelial cells, were shown to be upregulated by RA (Kim, 1992) as is the case for laminin, the major component of all basement membranes (Vasios et al., 1989; Vuolteenaho et al., 1990; Hierck et al., 1993; Huang and Chakrabarty, 1994; Ross et al., 1994). While in vitro data show that expression of some integrins is under RA control as well (Dedhar et al., 1991; Defilippi et al., 1991; Gaetano et al., 1994; Ross et al., 19941, and a6p4 is a major component of hemidesmosomes, linked to the intermediate filament network, we decided to study the expression of the p4 integrin subunit during normal and FtA induced abnormal development, with special emphasis on the heart.

Here we show that p4 is differentially expressed in

the heart and that the timing of expression is altered after treatment with RA. Based on these results we propose a role for a6p4 during normal cardiogenesis and in the development of cardiac malformations after embryonic administration of RA.

RESULTS Normal mouse embryos of distinct embryonic stages

(E9, E l l , E13, E15, EM), and retinoic acid treated embryos (E9, E l l , E13), were examined for protein and &A expression. Figures 1 and 2 show normal expression of the p4 integrin subunit, while Figures 3-7 show normal and RA-induced abnormal expression patterns in an ascending series of staged embryos. As mRNA and protein expression clearly correlate, results from both in situ hybridization and immunohistochemistry techniques are used complementarily, unless indicated.

First cardiac expression of the p4 integrin subunit was observed at E l 3 in a subset of endocardial cells. At this stage the murine heart is fully looped, but septation has yet to be completed. The endocardium lining the endocardial cushions in the atrioventricular (AV) canal and outflow tract was highly positive (Fig. lA,C). In these segments p4 clearly colocalized with the a6 integrin subunit (compare Fig. 1A,C with B,D). More- over, endocardium lining the developing venous valves in the atrium, i.e., remnants of the walls of the right superior cardinal vein after incorporation in the right atrium, was positive for p4 (Fig. 1C). Epicardial cells, especially a small subset of cells in the AV-sulcus, also showed p4 staining (not shown).

This endocardial and epicardial expression pattern was very consistent during development. At El5 and El8 essentially the same pattern was observed (Fig. 21, although epicardial staining had progressed over the heart a t this stage, probably due to completed epicardial coverage of the myocardium.

Extracardially, endothelial staining for 04 was evident from El3 onward. Venous endothelium, especially that of the cardinal veins entering the heart, was highly positive (Fig. 2 0 . Likewise, the pulmonary veins that arise in the dorsal mesocardium at this stage showed a high p4 expression. In contrast with the high venous expression, p4 staining in the arterial endothelium was much less. Only the most proximal part of the pulmonary trunk (Fig. 1A) and that of the aorta (not shown), close to the developing semilunar valves, is slightly positive for p4, whereas other arteries as well as the microvascular endothelium are negative.

In epithelia 84 expression was already observed at E l l . At this stage skin epithelium as well as the epithelium of the oesophageal-tracheal diverticulum showed a diffuse p4 staining throughout the cells (Fig. 4C). From E l 3 onward p4 was distinctly localized at the basal side of the cells, although in the oesophagus also the lateral epithelial membranes were stained for p4 (Fig. 2F).

From E9 to E l l a small cell population just beneath

p4 INTEGRIN IN THE DEVELOPING HEART 91

Fig. 1. lmmunohistochemical localization of p4 (A,C) and a6 (B,D) in an El3 mouse embryo. A: p4 staining is localized in the endocardium lining the developing semilunar valves (s) and pulmonary trunk (p). C: Moreover, expression is evident on the endocardium lining the atrio-

ventricular endocardial cushions (ec), the atrial valves (arrows), and in the pulmonary veins (arrowheads). Note that at all sites where 84 is expressed a6 is colocalized (B,D) although a6 staining exceeds p4 (Hierck et al., 1996). A0 = aorta. Bar = 100 Fm.

Fig. 2.


Fig. 3. Staining for p4 protein in an untreated embryo at E9 (A$) and an E9 embryo treated with RA at E7 (B,D). B: Note the particular high staining intensity at the sites of dispersing somites, suggestive for migrating neural crest cell expression (arrows), whereas the untreated em-

bryo (A) is negative in this area. Positive endocardial cells (arrowheads) appear in the AV-canal after RA treatment (D) whereas these cells are negative in an untreated embryo (C) at the same stage. cj = cardiac jelly in the AV-canal, v = ventricle, nt = neural tube. Bar 5 100 pm.

Fig. 2. Antibody staining for the p4 integrin subunit in an El5 (A,C,E) and an El 8 (B,D,F) embryo. Expression in the endocardial cells lining the developing av (C,D) and semilunar valves (5) (A,B) is still evident. En- dothelial staining in the cardinal vein (vc) exceeds arterial staining in the aorta (Ao) and pulmonary trunk (p). C: Epicardial staining is indicated by the large arrows. E: High p4 expression can be observed in the ventral horn (vh) and peripheral nerves (pn), whereas the spinal ganglia (sg) contain positive cells (small arrows). F: The vagal nerve (v) and emerging recurrent nerve (r), as well as presumptive neural connections to the cardiac ganglia (asterisks), are highly positive at E18. In addition, oesophageal (0) and tracheal (t) epithelia are highly positive, although p4 staining in the trachea appears to be restricted to the basal cell surface. st = sympathetic trunk. Bar = 100 Fm.

the skin ectoderm in the pharyngeal arches and dorso- lateral thoracal wall was diffusely positive for p4 (Fig. 4A). This staining could not be detected after E13. At El3 neuronal j34 expression became evident. Spinal ganglia and their efferent nerves contained cells that were positive for this integrin subunit whereas the sympathetic trunk and its ganglia were negative. Dor- sal and ventral roots, emerging from the neural tube, were highly positive. These patterns were consistent until E l 5 (see Fig. 2E). Thereafter these structures were not included in this study. The vagal nerve, responsible for the parasympathetic innervation of the heart, and its derivatives showed a minor p4 expres-

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Fig. 4.


sion at El3 and were highly positive from El5 onward (Fig. 2F).

An experimental group of animals was treated with a single low dose of Retinoic Acid of 20 mg/kg at E7. Pilot experiments showed that this treatment resulted in minor to microscopically undetectable cardiac malformations, a t least up to E13. Animals treated this way show a distinct altered p4 expression as compared to the controls. No ectopic localization was observed, but p4 expression was clearly advanced, i.e., earlier in time. Cardiac mRNA and protein expression wag already evident at E l l in all treated animals and even at E9 in one embryo, as shown in Figures 3-5. Strong and basally localized p4 staining was observed in the outflow tract endocardium and in the endocardium of the AV-canal a t these stages (Figs. 3,4). In addition, in situ hybridization showed a high p4 mRNA expression in these cells (Fig. 5). The a6 integrin subunit showed no change in expression pattern after RA treatment in the heart, but still clearly colocalized with p4 in these embryos. Figure 6 shows that at El3 no differences in cardiac p4 protein staining could be observed any more between treated animals and controls, even though fusion of endocardial cushion appears delayed (Fig. 6D). p4 mRNA expression, however, was still clearly increased in these embryos (Fig. 7).

At E l l , thoracal epithelia, like the skin and laryn- geal epithelium, showed a more prominent p4 expression upon RA treatment than controls. Moreover, cells were not diffusely positive, but a distinct basal localization of this integrin subunit was observed (Fig. 4C,D).

Interestingly, a t E9 early migrating neural crest cells in the region of dispersing somites were highly positive for p4 (Fig. 3B). The subectodermal cell population in the pharyncheal arches and dorso-lateral thoracal wall, presumptively late migrating neural crest cells, could not be stained for p4 at E l l after RA treatment (compare Fig. 4A with B).

DISCUSSION Both laminins and integrins play major roles during

development. Laminin is the first extracellular matrix molecule to appear during embryogenesis (Cooper and MacQueen 1983; Wu et al., 1983; Dziadek and Timpl, 1985) and can mediate various processes like cellular

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Fig. 4. lmmunolocalization of the p4 integrin subunit in an RA treated embryo at E l 1 (B,D,F) and one untreated at the same stage of development (A,C,E). E and F are magnifications of the outflow tract region shown in C and D, respectively. A: p4 is normally expressed in the area directly underneath the pharyngeal ectoderm (arrowheads). an area in which extensive neural crest migration occurs (Smits-VanProoije et al., 1988), and in the ectoderm (ect) itself, although diffusely. No expression could be observed in the heart (C,E). After RA treatment the endocardium in the outflow tract and AV-canal (arrows) is positive (D,F), ectcdermalp4 is sharply localized to the basal cell surface (B,D), and subectodermal p4 staining is absent (B). ot = outflow tract, av = atrioventricular canal, otd = oesophageal-tracheal diverticulum. A,B: bar = 200 pm. C F : bar = 100 pm.

outgrowth, migration and differentiation (Timpl, 1989; Poelmann et al., 1990). Moreover, various integrins are expressed very early and show differential expression throughout development (Hierck et al., 1993; Suther- land et al., 1993; Tarone et al., 1993). Recently, we and others have shown differential expression of the a6 integrin subunit during development of the murine heart and proposed a role in tissue stability and valve-formation (Buck et al., 1993; Baldwin and Buck, 1994; Collo et al., 1995; Hierck et al., 1996). a6 can be associated to pl or p4 of which the latter combination is far more restricted in its expression pattern.

p4 in Normal Development In the heart, first expression of p4 was observed at

El3 on the endocardium lining the endocardial cushions that will contribute to the mesenchymal part of the valves. In the AV-canal and outflow tract, expression was restricted to those endocardial cells that are in close apposition to each other. In these segments the orifices are narrow and the blood-flow is very high, resulting in a high level of shear stress on the endocardial cells (Icardo, 1989). This also applies to the endocardium lining the venous valves in the atrium. The expression of p4 specifically a t these sites in the heart indicates a role for this cell-matrix receptor in the firm adhesion of these cells to their basement membrane. Although endothelial cells do not form hemidesmosomes this integrin receptor appears to function in the heart in a way comparable to that in epithelial cells, p4 invariably colocalized with a6 while a basement membrane containing laminin, as assessed by staining with a polyclonal antiserum, always underlies P4-positive areas (data not shown). Together with the distinct basal localization of p4 in these endocardial cells it suggests a functional role for a6P4 in these areas.

During further development endocardial p4 staining essentially remains restricted to the same areas as at E13. At El8 p4 was still only expressed on the endocardium of the AV-valves, semilunar valves, and venous valves. Kennel and colleagues (1993) mentioned essentially the same pattern in mice 12 weeks after birth, suggesting a similar expression pattern in young adults. As has been suggested for the a6 integrin subunit (Hierck et al., 1996), the localization of p4 a t areas of high mechanical force could be mediated by transforming growth factor-p that can be upregulated in areas of increased shear stress (Pelton et al., 1991; Ohno et al., 1992; Brand and Schneider, 1995). However, only the endocardial cells lining the cushions, and not the subendocardial cells inside the endocardial cushions, show p4 staining (Fig. 1). a6 is expressed in both cell types, suggesting a role for a6Pl in the cushion tissue.

In non-cardiac endothelia outside the heart, p4 protein expression is restricted to the large veins whereas arterial and microvascular endothelium are negative. An exception is the proximal endothelium lining the aorta and pulmonary trunk, close to the semilunar

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Fig. 5.


valves. The nature of the gradual decrease in p4 staining in these large vessels remains unexplained. Re- markably, p4 mRNA was not detected at E l 3 at sites of protein expression whereas mRNA expression was distinct at E l5 (not shown). A low level of mRNA with a high translation rate is likely to account for this dis- crepancy between detectable amounts of mRNA and protein.

Although the techniques we used do not allow assess- ment of ultrastructural localization of a6p4, it is ex- pected that the staining in the peripheral nervous system reflects expression in satellite cells rather than neuronal p4 expression. High p4 staining coincides with areas of myelinization of axons, like the dorsal and ventral roots emerging from the neural tube, areas in the spinal ganglia, and the peripheral nerves. On the other hand, areas in which no or few Schwann cells are present, like the sympathetic trunk and the neural tube itself, show less (34 staining. Therefore, in vitro data assigning p4 expression to Schwann cells and perineural fibroblasts (Niessen et al., 1994a) correlate with our in vivo data on expression of this integrin subunit in these satellite cells during mouse embryogenesis.

Retinoic Acid and Development The transcription and translation of integrin sub-

units and extracellular matrix components can be influenced by several agents. Transforming growth factor p (TGF-P) and retinoic acid (RA) are the most effective mediators of expression. Retinoic acid, a vitamin-A derivative, has various effects on cell lines in vitro. In F9 mouse teratocarcinoma cells, laminin and fibronectin expression was upregulated upon RA exposure (Hierck et al., 1993; Huang and Chakrabarty, 1994; Ross et al., 1994). Integrin subunits (al , a5,a6, av, p l , 03) are usually upregulated except that p4 is downregulated in lung carcinomas (Dedhar et al., 1991; Defilippi et al., 1991; Gaetano et al., 1994; Ross et al., 1994). In vivo, both lack and excess of retinol and its active metabolites can cause congenital malformations, although the mechanisms might differ. The heart and vascular malformations include ventricular septa1 defects (VSD), transposition of the great arteries (TGA), double outlet right ventricle (DORV), and various aortic arch malformations (Wilson et al., 1953; Taylor, 1979, 1981; Pexieder et al., 1992, 1995; Broekhuizen et al., 1995; Bouman et al., 1995). Fur- thermore, Morriss-Kay (1992) showed that a refined balance in retinoid metabolism is needed for normal morphogenesis and that the RA level has to be low. The embryo cannot synthesize RA by itself and is therefore

Fig. 5. In situ hybridization for the p4 integrin subunit in untreated (A,B) and RA treated (CF) E l 1 embryos. Bright (A,C,E) and dark-field (B,D,F) micrograph of the same sections, respectively. D,F: Note the high mRNA expression in the endocardium of the outflow tract (ot) and AV- canal after RA treatment. In addition, expression in the endocardium of the AV-canal (small arrows) and in the developing skin epithelium (large arrows) is elevated. Bar = 100 pm.

dependent on the mother’s supply. Within a cell RA can be metabolized or transferred into the nucleus to serve as ligand for retinoic acid receptors (RAR-a, -p, -y) or heterodimer complexes of RAR and retinoid X receptors (RXR-a, p, -7) (for reviews see De Luca, 1991; Leid et al., 1992). These activated receptors can act as transcription factors via retinoic acid responsive elements (RAREs). Knock-out experiments in which null-mutants for RARs and RXRs show cardiac malformations suggest a key role for these receptors in the development of RA-induced heart malformations (Kastner et al., 1994; Mendelsohn et al., 1994; Dyson et al., 1995). Various genes are under such an RA control, among which RAR-p itself and the laminin p l chain and in vitro RA can influence expression of these genes. How- ever, only a limited number of reports exist regarding the in vivo effects of RA on the expression of extracellular matrix molecules and their receptors.

RA and p4 Expression Taking in mind the cardiac malformations after RA

treatment and the expression of extracellular matrix molecules and their ligands, it was hypothesized that altered expression of (one of) these components could (in part) be responsible for abnormal development. In this study, we have chosen to use a low dose of RA to circumvent development of major morphological malformations. With this approach we show that the cardiac expression of the p4 integrin subunit, but not a6, is influenced by excess of RA. Although no ectopic expression was observed, p4 expression was clearly advanced. Instead of cardiac expression arising between E l l and E13, p4 was already detected in the heart as early as E9. Whether altered p4 expression is a direct effect of RA on the transcription of this integrin subunit remains to be investigated. To date, no data are available on possible RAREs in the promoter of murine p4. An indirect effect through an upregulation of laminin expression seems likely because a RARE has been described for the LAMB1 gene (Vasios et al., 1989; Vuolteenaho et al., 1990), in vitro laminin is upregulated after treatment with RA (Hierck et al., 19331, and dermal endothelial cells increase extracellular matrix production after exposure to RA in vitro (Vora and Karasek, 1994). However, our own semiquantitative in situ hybridization and immunohistochemical data do not show such an upregulated expression of laminins in these areas, while p4 mRNA expression is affected by RA. Therefore, it seems likely that RA has a more gen- eral differentiation inducing effect on RA responsive cells, resulting in a more differentiated phenotype too early in development.

p4 in Malformed Cardiogenesis Several hypotheses can be proposed for the role of p4

in the development of cardiac malformations. One problem caused by RA during development is delay or absence of fusion of endocardia1 cushions (Pexieder et al., 1992; Broekhuizen et al., 1995; Bouman et al.,

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Fig. 6. lmmunohistochemical localization of p4 in untreated (A,C) and RA treated (B,D) El3 mouse embryos. Staining is evident in the endocardium lining the endocardial cushions in the outflow tract (s) (A,B) and AV-canal (ec) (C,D). 0: Although in the RA treated embryo a ventricular

septum defect appears to develop (arrow), expression of p4 is unaltered. A0 = aorta, p = pulmonary trunk, rv = right ventricle, Iv = left ventricle. Bar = 100 pm.


Fig. 7. mRNA expression of p4 in €13 embryos treated with RA (B,D) and untreated (A$). Dark-field micrographs (C,D) show Increased p4 expression after RA treatment in the endothelium of the pulmonary trunk (p) and Aorta (Ao) as compared to the control (C). Moreover, endocar-

dium in the outflow tract and endothelium in the aorta and pulmonary trunk (small arrows) as well as skin epithelium (large arrows) show increased p4 expression. Bar = 100 pm.

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1995). This can lead to abnormal septation and valves, development of VSD, and probably other malformations (van Mierop et al., 1962). p4 that is expressed on the endocardial cushions at a too early stage coincides with or could even be responsible for the delay of fusion. Although the process of fusion is not fully under- stood it is clear that two cushions, each with their own endocardial lining, meet to form a continuity. This im- plies that two opposed endocardial cell layers must dis- appear. Various mechanisms can be suggested in this process: apoptosis (programmed cell death), migration, or incorporation of the endocardial cells in the cushion tissue. Apoptosis has been shown not to appear under normal conditions at these sites (Los and van Eijnd- thoven, 1973; Icardo, 1984). This is in contrast to incorporation of cells in the underlying tissue. Markwald and coworkers (1975, 1977) first described the process of “epithelial to mesenchymal transformation” as a way to fill the cardiac jelly with cells recruited from the endocardium. The end of this process coincides in time with the normal appearance of p4 in the endocardium. It is comprehensive that endocardial cells that express this adhesion molecule at a too early stage in development adhere to the underlying basement membrane too firmly and that transformation is disturbed. In- creased adhesion could also inhibit or delay migration of these cells to other areas. The latter is interesting while endothelial cells actively downregulate a6P4 after they have been induced to migrate (Sepp et al., 1995), indicating that high amounts of a6p4 are not favourable for migration. Moreover, a computer model, in which parts of the development of the heart can be simulated, correlates increased adhesiveness of endocardial cells in the AV-canal to development of atrioventricular defects (Kurnit et al., 1985).

A further mechanism to be postulated is that an en- hanced adhesion of the endocardial cells lining the AV- canal and outflow tract to their basement membranes could influence the looping of the heart, by making the inner lining of these segments too stiff, In this way the normal apposition of cushions could be prohibited. Ab- normal looping after the administration of RA has been suggested as a mechanism leading to a spectrum of cardiac malformations (Bouman et al., 1995). Changes in volume and extracellular matrix composition of endocardial cushions after RA-treatment (Davis and Sa- dler, 1981; Nakajima et al., 1995) and changes in the expression of endocardial cell adhesion molecules (this study) indicate that the endocardial cushions are main targets for excess of RA.

The expression of p4 in the subectodermal region in the pharyngeal arches at E l l suggests expression in migrating neural crest cells. In RA treated embryos this expression is lost. The neural crest cells appear to advance their p4 expression after RA treatment, as early migrating cells in E9 embryos were clearly positive. The advanced expression of the 04 integrin subunit in these cells could be the cause of altered migration (Lee et al., 19951, and therefore account for the

similarity between phenotypes of neural crest ablated and RA-treated embryos (Yasuda et al., 1986; Nishiba- take et al., 1987; Hart et al., 1990; Kirby, 1990).

Conclusions We showed differential expression of the p4 integrin

subunit during mouse embryonic development in the heart and surrounding tissue. Cardiac expression appears correlated with regions of high mechanical force. Upon RA treatment p4 expression is advanced in the endocardial cells in the heart, but also in migrating neural crest cells. Coexpression with the a6 integrin subunit and localization to the basal cell surface suggest the functionality of the a6p4 receptor. The altered expression of this cell-matrix receptor molecule could influence both migration of cells and stiffness of cardiac segments and, therefore, contribute to the development of congenital malformations. Whether RA directly acts on p4 expression or through its extracellular ligand is a subject of further investigation.

EXPERIMENTAL PROCEDURES Tissue Preparation

Female Swiss mice were caged overnight with males. Mice showing a vaginal plug were selected on the next morning and housed separately under standard CAHPA (Comitb Ad Hoc pour la Protection des Ani- maux) conditions. On the day of interest the mice were killed by cervical dislocation and the embryos were dissected free (EO is designated as the day of the vaginal plug). For immunohistochemistry, embryonic tissue was collected and immersion-fixed overnight in acetone at 4°C. Acetone was removed by rinsing in several changes of PBS-sucrose (6.8% sucrose), followed by immersion in OCT Compound (Tissue-Tek, Miles Inc., Elkhart, IN). The embryos were embedded in fresh OCT Compound, frozen in isopentanol, chilled in liquid nitrogen, and stored at -20°C. Six micron serial sections were cut transversely through the thoracal region of the embryo on a 2800 Frigocut-E cryostat (Leica, NussLoch, Germany), collected on Starfrost slides (Knittel Glaser, Braunschweig, Germany), dehydrated in acetone (-20’0, and stored at -20°C. For in situ hybridization, embryos or isolated hearts were collected and immersion-fixed overnight in 4% parafor- maldehyde in PBS at 4°C. Tissue was routinely prepared for paraffin embedding, sectioned at 5 pm and mounted on aminopropyltriethoxysilane (Sigma, St. Louis, MO) treated glass slides.

Retinoic Acid Treatment On E7 20 mgkg all-trans Retinoic Acid (Sigma), dis-

solved in vegetable oil, was injected intraperitoneally in pregnant mice. On the day of interest embryos were dissected free and examined for macroscopic malformations. The embryos were routinely prepared for immunohistochemistry or in situ hybridization (see above).


Immunohistochemistry

Slides were post-fixed in ice-cold acetone for 10 min at -20°C and air dried. Endogenous peroxidase was inactivated by incubation in 0.3% HzO, in PBS-azide (PBS containing 0.1% Na-azide) for 30 min at room temperature. The slides were washed in PBS, incubated for 15 min in a background reducing solution (150 mM NaC1,lO mM Tris-HC1,5 mM EDTA, 0.25% (w/v) gelatin, 0.05% (v/v) Tween-20, pH 8.0) at room temperature and washed again (3 x 5 min PBS). The sections were incubated overnight with the pri-

mary antibody, diluted in PBSB (PBS containing 1% bovine serum albumin), in a moist chamber. Rat monoclonal antibodies were visualized by a standard DAB reaction (0.04% diaminobenzidine tetrahydrochloride in 50 mM Tris-Maleic acid, pH 7.6, containing 0.06%0 H202) after subsequent incubation with Rabbit-anti- Rat antiserum (Dako, Carpinteria, CA; 1:1,000 in PBSB), Goat-anti-Rabbit antiserum (Nordic, Tilburg, The Netherlands, 1 5 0 in PBSB), and Rabbit-Peroxi- dase-anti-Peroxidase (Nordic, 1500 in PBSB). Sec- tions were counterstained in haematoxylin, dehydrated and mounted in Entellan (Merck, Darmstadt, Germany).

In Situ Hybridization

In situ hybridization was performed according to Wilkinson and Green (1990) with minor modifications. Briefly, tissue sections were deparaffnized in xylene, rehydrated in a descending series of ethanol to PBS and post-fixed for 20 min. After treatment with Pro- teinase K (Merck), post-fixation and acetylation, sections were dehydrated and used for hybridization.

Linearized cDNA was transcribed to anti-sense cRNA probes in the presence of 75 pCi [35SlUTP (Am- ersham, Arlington Heights, IL), 10 U placental RNAse inhibitor (Pharmacia, Uppsala, Sweden) and 10 U T3- or T7-RNA polymerase (Pharmacia). Sections were hy- bridized overnight to 1 X lo5 cpm/p1 of [35SlcRNA in hybridization solution (50% deionized formamide, 0.3 M NaC1, 20 mM Tris-HCl pH 8.0, 5 mM EDTA, 100 mM dithiothreitol [DTT], 1 x Denhardt's, 0.5 mg/ml calf t-RNA, 10% dextran sulphate) at 55°C. High stringency washing was performed in 100 mM Dl" in 50% formamide, 2 x SSC for 45 min at 65°C. After RNAse treatment (RNAse-A, Sigma) the sections were submit- ted again to high stringency washing. Sections were washed in 2 x SSC and 0.1 x SSC at 55"C, respectively, dehydrated through ascending series of etha- nols, and air dried.

Subsequently, sections were coated with Ilford G5 emulsion and exposed for 6 to 15 days at 4°C. Slides were developed in Kodak D19 developing solution, fixed in Polymax I (Kodak, Rochester, NY) and counterstained with haematoxylin. Examination was performed with a Leitz Dialux 20E8 microscope equipped with a dark field condenser.

Antibodies and Probes Antibodies used in this study were (1) Monoclonal

Ftat-anti-p4 (346-11A, Kennel et al., 1989; Sonnenberg et al., 19901, used 1:ZOO in PBSB; (2) Monoclonal Rat- anti-a6 (GoH3, Sonnenberg et al., 19861, used 1:lO in PBSB.

Probes used in this study were (1) a 370 bp (EcoRV- NotI) fragment of human p4 integrin subunit (Hoger- vorst et al., 1990); (2) a 410 bp (EcoRI-Pat11 fragment of murine a6 integrin subunit (Hierck et al., 1993).

High sequence homology between human and murine p4 cDNA resulted in a strong hybridization signal of mouse tissue with human probes. All probes used in this study were designed to recognize all splice variants described to date. All cDNAs were subcloned in pBluescript (Stratagene, La Jolla, CA) and linearized. One microgram was used for probe preparation.

ACKNOWLEDGMENTS The authors thank Dr. Arnoud Sonnenberg for gen-

erous gifts of antibodies.

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