DEVELOPMENTAL DYNAMICS 206.12-23 (1996)

Developmental Expression of Laminin-5 and HDI in the Intestine: Epithelial to Mesenchymal Shift for the Laminin y2 Chain Subunit Deposition V8RONIQUE ORIAN-ROUSSEAU, DANIEL ABERDAM, LIONEL FONTAO, LUC CHEVALIER, GUERRINO MENEGUZZI, MICHELE KEDINGER, AND PATRICIA SIMON-ASSMANN INSERM U.381 (V.0.-R. , L.F., M.K., P.S.-A,), and Department of Maternity, Centre Hospitalier Universitaire de Hautepierre (L.C.), 67200 Strasbourg, and INSERM U.385, Facultt! de Mbdecine, 06107 Nice Ct!dex 2 (DA. , G.M.) , France

ABSTRACT We report the expression pat- tern of hemidesmosome-associated proteins lami- nin-5, composed of a3p3y2 chains, and HD1 in the developing mouse and human intestine, an organ in which variations in structure and function par- allel morphogenesis and differentiation. Immuno- cytochemistry analysis revealed the coexpression of laminin-5 and HD1 at the basal pole of differ- entiating epithelial cells. Distinct noticeable vari- ations occurring in the location of laminin a 3 chain in development of mouse gut were stressed by the reverse transcriptase-polymerase chain re- action data. A peculiar finding was also the loca- tion of laminin y2 chain in the intestinal muscle coat. The cellular origin of laminin y2 chain was examined by immunocytochemistry on interspe- cies hybrid intestines with specific antibodies rec- ognizing mouse antigens. Complementary and se- quential production of laminin y2 chain was observed, by epithelial cells as establishment of the basement membrane occurs and by mesenchy- ma1 cells in the more differentiated organ. These results support the concept of mesenchymal in- volvement in deposition of basement membrane molecules, a crucial process for intestinal differ- entiation. Taken together these data provide the first evidence for the coexpression of hemidesmo- some-associated proteins in the gut, a non-strati- fied tissue. o 1996 Wiley-Liss, Inc.

Key words: Laminin, HD1, Intestine, Interactions

INTRODUCTION The basement membrane, a thin sheet of extracellu-

lar matrix, is a major regulator of cell behaviour in various organs. Within the intestine, the basement membrane separates the epithelial cells from the un- derlying connective tissue called the lamina propria. The mature epithelium derives from the embryonic in- testinal endoderm and the cellular elements of the lam- ina propria from the mesenchyme. It has been clearly demonstrated that dynamic epithelial/mesenchymal cell interactions are involved in the processes of mor- phogenesis and differentiation of adjacent cells during development and persists in the mature organ (Haffen

0 1996 WILEY-LISS, INC.

et al., 1989). Because of its strategic position, the base- ment membrane is believed to play a key role in these events through yet unresolved mechanisms. Among its constituents, special emphasis has been given to the major hetero-trimeric glycoprotein laminin-1, the first discovered molecule belonging to the large family of laminins (for reviews see Engel, 1992; Engvall, 1993; Timpl and Brown, 1994). Molecular biology techniques and availability of specific antibodies allowed to dem- onstrate the existence of collagen IV and laminin iso- forms that revealed an unexpected heterogeneity of basement membranes not only in different organs but even within the same organ. Concerning laminins, 7 variants have been discovered up to now (Burgeson et al., 1994).

Laminin-1 ( a l p l y l ) , laminin-2 (merosin: a2plyl), and laminin-3 (S-laminin: alp2yl) are expressed in the intestine (for a review see Simon-Assmann et al., 1995). In human and mouse mature intestine, the lami- nin a1 chain displays a species-specific distribution. In the adult mouse intestine, the polypeptide is restricted to the basement membrane underlying the crypt re- gion, while in humans it displays a decreasing gradient distribution from villi to crypts. In both species, the laminin a2 chain is confined to the crypt proliferation zone (Simon-Assmann et al., 1994). Expression of lami- nin p2 chain is confined to the smooth muscle coat (Glukhova et al., 1993).

It has been shown that a subset of epithelial base- ment membranes contains the laminin variant, lami- nin-5. Laminin-5, formerly termed kalinin, nicein, or epiligrin, consists of three disulfide linked polypeptide subunits-the a3, p3, and y2 chains-that constitute a mature 400 kDa protein. In the skin, laminin-5 was found to be prominently associated to the anchoring filaments of the lamina lucida (Verrando et al., 1987; Rousselle et al., 1991) underneath the hemidesmo- somes. Hemidesmosomes are stable adhesion devices at the basal pole of basal epithelial cells of specialized squamous, transitional, and pseudostratified epithelia that contribute to the interaction between the cells and

Received September 5, 1995; accepted November 13, 1995. Address reprint requestslcorrespondence to Dr. P. Simon-Assmann,

INSERM U.381, 3, avenue MoliBre, 67200 Strasbourg, France.

LAMININ-5 AND HD1 IN INTESTINAL DEVELOPMENT 13

12 dayfasl (iJ Isolation of pure tissue compartments m e i M

@ Recombinants between Chick / Mouse intestinal anlagen

c h i nmnd*n

Fig. 1. Schematic representation illustrating the main experimental steps leading to the development of intestinal hybrid intestines. Mm/Ce: mouse mesenchyme/chick endoderm, and Cm/Me: chick mesenchyme/ mouse endoderm recombinants.

the connective tissue. Their cellular components so far identified include bullous pemphigoid antigens (BP 230 and BP lSO), the protein HD1 and integrin a6p4 (for review see Jones et al., 1994). The expression of laminin-5 is specifically impaired in lethal junctional epidermolysis bullosa, an autosomal recessive blister- ing disease characterized by a dermal-epidermal disad- hesion at the level of the lamina lucida (Verrando et al., 1991; Meneguzzi et al., 1992). Recently, mutations in the genes encoding laminin-5 have been identified (Aberdam et al., 1994b; Pulkinnen et al., 1994a,b; Vidal et al., 1995). This suggests that laminin-5 plays a major role in cell adhesion and integrity of the base- ment membranes. In a comprehensive study, Aberdam and collaborators (1994a) reported that laminin-5 is expressed in different tissues with prominent secretory andlor protective function including the intestinal tis- sue, where despite the presence of the integrin a6p4 in the ventral plasma membrane of epithelial cells, bona fide hemidesmosomes have not been detected (Simon- Assmann et al., 1994).

The purpose of the present investigation was to define the existence and determine the precise expres- sion pattern of laminin-5 and HD1 in the developing intestine by immunocytochemistry and reverse tran- scriptase-polymerase chain reaction (RT-PCR) analy- sis. This should provide information on the potential role of these molecules in this tissue known to display important variations in structure and function paral- leling morphogenesis and crypt to villus positioning. Since epithelial-mesenchymal interactions have been

shown to be crucial for differentiation of the gut, we examined the cellular origin of laminin a 3 and y2 chains deposited at the epithelialimesenchymal inter- face using interspecies hybrid intestines and species- specific antibodies (see Fig. 1). We demonstrate the complementary production of the laminin y2 chain by epithelial and mesenchymal cells as formation of the basement membranes proceeds.

RESULTS Developmental Expression of Laminin-5 and of HD1 in the Human Intestine

Immunodetection of laminin-5 was performed using antibodies raised against the trimeric molecule (mAb GB3), the a3 (pAb SE85), p3 (mAb 6F12), and y2 (pAb SE144) chains described in Table 1. During human in- testinal development, the staining patterns of the three constituent chains of laminin-5 were identical whatever the antibody considered. At all developmen- tal stages studied, the labelling was exclusively re- stricted to the subepithelial basement membrane and was totally absent from the mesenchyme and the epi- thelial cells. At 8 weeks of gestation, the fetal human intestinal anlage is a simple tube of stratified endoder- ma1 cells surrounded by mesenchymal cells. Immuno- fluorescence analysis of 1x3, p3, y2 chain expression at this stage showed a continuous linear labelling at the epithelial/mesenchymal junction (Fig. 2A,B). At 10/12 weeks, expression of laminin-5 was revealed as a faint staining at the base of the protruding villi that inten- sified progressively to the top of the villi (Fig. 2 0 . At

14 ORIAN-ROUSSEAU ET AL.

15 weeks, when crypts develop by invagination of the epithelium in the stromal tissue, the increasing gradi- ent of intensity of the staining along the crypt-villus axis was still present (Fig. 2D). The weaker immuno- reactivity in the crypts and the prominent labelling along the villi persisted until adulthood (Figs. 2E-H, 3A-F). The colon (distal portion of the intestine) was characterized by a regular staining all along the deep glands, and a stronger labelling underneath the flat surface epithelium (Fig. 3G).

In parallel, the distribution of HD1 was analysed as a function of human intestinal morphogenesis. At 9 weeks of gestation, a positive staining visualized as a discontinuous faint linear band appeared at the base- ment membrane zone (Fig. 4A). At 12 weeks, this staining pattern was strongly enhanced at the top of the protruding villi (Fig. 4B). At 24 weeks of gestation and in the adult, HD1 staining of intestinal segments

TABLE 1. Antibodies Utilized for Detection of Laminin-5 and HD1 EDitoDes

Antibodies" Specificity Species recognized

mAb GB3 Lamin in -5 H u m a n mAb 6F12 p3 chain H u m a n pAb SE85 a3 chain H u m a n pAb SE513 a3 chain Mouse pAb SE144 y2 chain Human and mouse mAb HD-121 HD1 Human, bovine, r a t

amAb or pAb monoclonal or polyclonal antibodies.

was characterized by linear dot arrays along the base- ment membrane with a clear increasing base to tip gradient (Fig. 4C). Tangential sections of villi showed that HD1 staining in the mature organ appeared as

Fig. 2. lmmunostaining of laminin-5 and its individual chains during human fetal intestinal development with polyclonal Ab SE 144 recogniz- ing the y2 chain (A), mAb 6F 12 recognizing the p3 chain (B,H), poly- clonal Ab SE 85 recognizing the a3 chain (C,D,F,G) and with mAb GB3 reacting with the trimeric laminin-5 molecule (E). Intestinal segments were analyzed at 8 (A), 9 (B), 12 (C), 15 (D), 20 (E,F), and 24 (G,H) weeks of gestation. The staining pattern was identical whatever the an-

tibody used. At early stages of development (A,B), antibodies decorated linearly the endodermal/mesenchymal interface. From the 12th week on- wards (CH), an increasing gradient of intensity of the staining was de- tected from base to villus tip. (e) endoderm or epithelium; (m) mesen- chyme; (Lp) lamina propria; arrows point to the subepithelial basement membrane; (c) crypt area. Bars = 50 pm.

LAMININ-5 AND HD1 IN INTESTINAL DEVELOPMENT 15

Fig. 3. lmmunostaining of laminin-5 and its individual chains in the human intestine at term (A-C) and in the adult (D-G) with polyclonal Ab SE 85 (a3 chain; A,E,G), polyclonal Ab SE 144 (y2 chain; B), mAb 6F 12 (p3 chain; C,F), and with mAb GB3 reacting with the trimeric laminin-5 molecule (D). The staining pattern was identical whatever the antibody

used. Small intestinal (A-F) and colon (G) segments. Note the strong immunoreactivity along the small intestinal villi or underneath the flat surface of the colon contrasting with the weak or absent staining in the crypt region (c). (e) epithelium; (Lp) lamina propria; (mm) muscularis mucosae. Bars = 50 pm.

numerous patches localised at the base of the epithelial cells (Fig. 4D). In the colon, HD1 staining was exclu- sively localized at the flat surface epithelium (data not shown). Whatever the intestinal segment considered, HD1 staining was never observed in the crypt region.

Developmental Expression of Laminin-5 in the Mouse Intestine

Since mouseichicken hybrid intestines were used for xenoplastic combination experiments, it was first nec- essary to define the normal distribution pattern of laminin-5 in adult and developing mouse intestine. In rodents, the formation of villi occurs in the last days of gestation, and crypts start to develop during the first week after birth. As only the polyclonal SE144 anti- body raised against the human y2 chain crossreacting with the murine counterpart and the polyclonal anti- mouse a 3 chain SE513 antibody are available, the ex- pression of these 2 polypeptides was examined during the different developmental stages and in the mature intestine.

Expression of laminin y2 chain was detected in the

murine intestine as early as the 12th day of gestation. The basement membrane was strongly stained but a weak immunoreactivity could also be detected in the intercellular spaces within the mesenchyme (Fig. 5A). The staining pattern showing a regular labelling along the villi did not change from this stage onwards up to postnatal day 19 (Fig. 5B-E). At this later stage, as well as in the adult intestine, an increasing gradient of intensity settled from crypt to villus tip (Fig. 5E and F) that became identical to that observed in human ma- ture intestine (Fig. 3). An interesting feature of the y2 chain is its expression in the murine muscle coat. In- deed, at 18 days of gestation, the polyclonal SE144 an- tibody decorated a peripheral circular zone of cells within the mesenchyme corresponding to the future muscle layer (Fig. 5 0 . Later on, the fully differenti- ated muscular layers were strongly labelled (visible on Fig. 5D and F).

The distribution of the laminin a3 chain is strikingly different from that of the laminin y2 chain. Indeed, the a 3 subunit was only detected in the adult small intes- tine and even then was totally restricted to the top of

16 ORIAN-ROUSSEAU ET AL

Fig. 4. lmmunofluorescence staining of HD1 in the developing human intestine with mAb HD-121 at 9 (A) and 12 (B) weeks of gestation, and in the adult (C,D). HD1 is found restricted to the basal pole of the epithelial cells facing the basement membrane region whatever the developmental stage. In the adult (C) an increasing gradient of intensity occurs from crypt to villus tip. On a tangential section of an intestinal villus, HDI staining appeared as rosette-like patterns located at the base of epithelial cells (D); similar pictures have already been obtained in the tongue using bullous pemphigoid autoantibodies recognizing the 230 kDa hemidesmo- soma1 plaque component or using a monoclonal antibody against integrin p4 (Jones et al., 1991). (e) endoderm or epithelium; (m) mesenchyme; (Lp) lamina propria; arrows point to the subepithelial region. Unspecific staining of mucus cells (M) was observed. Bars = 50 pm.

the villi where the most differentiated cells can be found (Fig. 5G,I). Interestingly, sections through the colon, distal part of the intestine showed detectable immunoreactivity with the antimouse a3 antibody. The staining was located at the interface between en- doderm and mesenchyme at 16 days of gestation (Fig. 5H). This subepithelial location was maintained until adulthood, the stage at which immunoreactivity was mainly confined to the basement membrane underly- ing the surface epithelium (not shown).

In the next step, we addressed the question whether the absence of a3 chain product in the small intestine was reflected at the mRNA level. Therefore, RNA were isolated from embryonic, neonatal, and adult mouse small intestine and analyzed by RT-PCR using specific oligoprimers to a3, 72, and p3 chains as described in Experimental Procedures. The results showed that the three genes are transcribed in every stage since PCR products of the expected length were found (Fig. 6).

Cellular Origin of Laminin-5 Using Mouse/Chicken Hybrid Intestines

The cellular origin of laminin-5 was studied by cre- ating hybrid intestines between mouse and chick tissue components (see Fig. 1). In this model, the basement membrane molecules located at the epithelial/mesen- chymal interface were screened with species-specific antibodies.

The SE144 and SE513 antibodies raised against the mouse laminin y2 and a3 chains that do not react with chick antigens (not shown) were used. In the chick mes- enchyme/mouse endoderm recombinant (Cm/Me) de- veloped for 4 days, SE144 polyclonal antibodies gave a continuous linear staining visible at the subepithelial basement membrane which is indicative of the pres- ence of epithelial-derived molecules (Fig. 7A). At 6 to 10 days of implantation, when primitive villi have arised, this staining pattern persisted (Fig. 7C). Later on, during villus elongation, the labelling became re- stricted to the villus base (illustrated at 11 days in Fig. 7E). At 17 days of implantation, the latest stage stud- ied, the overall intestinal structures were negatively stained; only occasionally a faint irregular staining was still visible in some crypts (Fig. 7G).

In the inverse hybrid associations corresponding to a mouse mesenchyme recombined to a chick endoderm (MdCe), SE144 polyclonal antibodies did not stain the mesenchyme/endoderm interface (Fig. 7B) until day 6 of grafting. At this stage (Fig. 7D) the staining of rare protruding villi was detected showing the contribution of the mesenchyme. After 11 and 17 days of implanta- tion (Fig. 7F,H), the staining pattern was indistin- guishable from that observed in mature mouse intes- tine with a gradient distribution increasing from the base to the top of the villi.

Results obtained from the analysis of mousekhick hybrid intestines show that y2 chain was deposited at the basement membrane zone successively by the en- doderm and the mesenchyme, according to a specific chronology. Neither C d M e nor M d C e grafted recom- binants were stained with the polyclonal Abs SE513 (not illustrated) in accordance with the fact that the laminin a3 chain is expressed only in adult mouse in- testine.

DISCUSSION Three major and novel findings can be drawn from

the present studies. First, laminin-5 and HDI are co- expressed in human intestine. The two proteins are,

Fig. 5. lmmunostaining of laminin-5 individual chains during mouse intestinal development with polyclonal Ab SE 144 (y2 chain; A-F) and polyclonal Ab SE 513 (a3 chain; GI). Intestinal segments were analyzed at 12 (A,G), 14 (B), 16 (H), and 18 (C) days of gestation, at birth (D), at postnatal day 19 (E), and in the adult (F,I). At the fetal stages (A-C), the y2 chain staining underlined prominently the endodermalhesenchymal interface as well as the serosal layer around the mesenchyme; some imrnunoreactivity to the y2 chain appeared in the newly formed muscular layers at 18 days of gestation (C). After birth, in addition to its location in

the subepithelial basement membrane, the y2 chain was clearly found located in well-developed muscle coat (Df). a3 chain staining is absent from the fetal small intestine at 12 days of gestation (G) but present at 16 days of gestation in the colon (H); immunolabelling in the small intestine was only obvious in the adult exhibiting a restricted pattern of expression at the villus tip (I). (e) endoderm or epithelium; (m) mesenchyme; (Lp) lamina propria; (mL) muscular layers; arrows point to the subepithelial basement membrane. Bars = 50 pm.

18 ORIAN-ROUSSEAU ET AL.

P3 Y2 a3 --- M 1 2 3 1 2 3 1 2 3 M

603 -

31 0, 281 - 271-

Fig. 6. Analysis of laminin-5 gene expression in mouse intestine by RT-PCR. PCR using primers specific for p3, y2, and a3 was carried out on 1 pl of random-primed first strand cDNA synthesis from total RNA derived from 15 days fetal (lane l), 6 day postnatal (lane 2), and adult (lane 3) mouse small intestine. PCR reaction products were resolved by 1.5% agarose gel electrophoresis. The positions of the marker bands are indicated at the left.

respectively, located in the basement membrane region and at the basal pole of differentiating epithelial cells. Second, the intestinal muscle cells synthesize laminin y2 chain whose expression increases with muscle dif- ferentiation. Third, using the strategy of interspecies (mousekhick) chimaeric intestines combined to mouse- specific antibodies, we show that deposition of the lami- nin y2 chain is developmentally regulated; y2 chain is from successively epithelial and mesenchymal origin, according to a temporal chronology linked to the mor- phogeneticldifferentiation stage of the intestine and to the activation of the epithelial and mesenchymal cells.

In stratified epithelia such as epidermis, laminin-5 is a putative component of the anchoring filaments of hemidesmosomes. The hemidesmosomes are special- ized cell-matrix junctions promoting the attachment of the epithelium to the underlying connective tissue and more precisely to type VII collagen of the anchoring fibrils. The cellular core of this complex entity is basi- cally composed of bullous pemphigoid antigens (BP230 and BP180), HD1 protein and integrin a6P4, the puta- tive receptor of laminin-5 (Jones et al., 1994). It was very tempting to speculate that similar anchoring com- plexes could exist in the gut, in which the epithelium is stratified during early organogenesis and in which, at adulthood, stable stem cell populations reside within the crypt in the mature intestine (for review see Ked- inger, 1994). In support of this possibility, we could show that both laminin-5 and HD1 are indeed ex- pressed in the human intestine. Moreover, their distri- bution pattern was superimposable to that of integrin a6P4 and reactivity with all these proteins presented an increasing gradient of intensity from the base to the villus tip (Simon-Assmann et al., 1994). However, type VII collagen is absent from the human intestine (Leigh et al., 1987; Visser et al., 1993) and the hemidesmo- some components BP230 and BP180 could not be de-

tected in human intestine either by immunocytochem- istry or by Western blot (data not shown). In accordance are the data obtained by Owaribe et al. (1990) who demonstrated the lack of bullous pemphig- oid antigens in all simple epithelia tested including the intestinal mucosa. In a descriptive study of rat intesti- nal development, Mathan et al. (1972) showed conden- sations of electron-opaque material occasionally seen along apposing surfaces of epithelial and mesenchymal cell plasma membranes during fetal life. Our own ob- servations of human and mouse intestines by transmis- sion electron microscopy did not allow us to verify the existence of canonical hemidesmosome structures char- acterized by intracytoplasmic dense plaques. These ob- servations together with the fact that only some of the hemidesmosomal components are found in the intes- tine indicate the possible existence in this tissue of early hemidesmosomes or type I1 hemidesmosomes. Contrariwise to true hemidesmosomes, which play a role in the epidermis attachment to the underlying der- mis, the “hemidesmosome-like structures” found in the intestine do not seem to function as stable anchorage devices. Indeed, laminin-5 and HD1 are mostly located at the basal pole of epithelial cells that migrate up the villi where these molecules display a gradient of inten- sity from the crypt mouth to the tip of the villi paral- leling cell differentiation. These data, and the fact that laminin-5 and HD1 are present early in development, suggest a role in intestinal epithelial cell differentia- tionfmigration beside cell anchorage. Although the role of laminin-5 in intestine is not yet defined, the fact that patients affected by junctional epidermolysis bullosa present erosions of the gastrointestinal mucosa argues that it plays a major role in intestinal physiology (Lund et al., 1995). Other manifestations like nutrient mal- absorption, constipation, painful bowel movements, and perianal erosions are described (Ergun et al., 1992). The possible involvement of laminin-5 in epithe- lial cell migration is supported by enhanced expression of laminin-5 in human cancers including colon cancer, where the y2 chain is preferentially located in inva- sively malignant cells (Pyke et al., 1994).

It is worth noting that in contrast to the expression pattern depicted in the human intestine, the mouse a3 subunit is detectable in the small intestine only at the adult stage with a distribution restricted to the very top of the villi. At this level, the epithelial cells are shed into the lumen. Such a different expression be- tween human and mouse intestine was also noted in the case of the laminin a1 chain (Simon-Assmann et al., 1994). Yet, in the distal part of the intestine, the colon, the laminin a 3 chain protein is expressed al- ready at fetal stages in the mouse. The fact that in RT-PCR analysis using primers specific for a 3 chain, products could be obtained in the fetal and postnatal small intestine as well as in the mature organ suggests a region-specific posttranscriptional control in the tem- poral expression of the a3-polypeptide chain. The dif- ferential expression between the colon and the small

Fig. 7. lmmunodetection of laminin-5 72 chain with polyclonal Ab SE144 recognizing solely mouse antigens on Cm/Me (A,C,E,G) and on Mm/Ce (B,D,F,H) hybrid intestines developed in the coelomic cavity of chick embryos and recovered after 4 (A,B), 6 (C,D), 11 (E,F), and 17 (G,H) days of implantation. In Cm/Me hybrid intestines, a clear linear staining occurred at 4 and 6 days of implantation (A,C) becoming re- stricted to the villus base at 11 days (E); at 17 days, the overall intestinal structures were negative (G). In the inverse associations, MrniCe intes-

tines, the staining appeared first at the upper part of the protruding villi (D) and then underlined the whole villus basement membrane (F) depicting a gradient of intensity from crypt to villus tip. In H, the well-developed muscle coat derived from the murine mesenchyme was labelled with the anti 72 chain antibody. In 6, a positive staining was obvious in the ex- ternal serosa. (e) endoderm or epithelium; (m) mesenchyme; (Lp) lamina propria; (mL) muscular layer; (lu) lumen; v, villus; arrows point to the subepithelial basement membrane. Bars = 50 pm.

20 ORIAN-ROUSSEAU ET AL.

intestine is of potential interest, although no clear ex- planation could be advanced yet.

The fact that laminin-5 is found restricted in major- ity to basement membranes underlying epithelia, would argue for its epithelial origin. In accordance are the data of Kallunki et al. (19921, Aberdam et al. (1994a1, and Sugiyama et al. (1995) who demonstrated that the strongest mRNA signals for the y2 chain are restricted to specific epithelial cells in skin, lung, and kidney. The strategy of creating interspecies hybrid intestines combined to species-specific antibodies al- lowed us to approach the origin of laminin-5 in the intestine. No conclusion could be drawn for the constit- uent laminin-5 a 3 chain due to its lack of detection with the antibodies used during early phases of devel- opment. Concerning the y2 subunit, the fact that in the hybrid intestines composed of mouse endoderm and of chick mesenchyme, the basement membrane was la- belled with the antimouse antibody clearly demon- strates the epithelial origin of the chain at least during the first week following the re-establishment of the basement membrane. The expression analysis of the y2 chain in the inverse association-chick endoderm com- bined to mouse mesenchyme- demonstrated that the mesenchyme is also involved in the synthesis of lami- nin-5 although its participation is delayed; it is worthy to note that in the most developed grafts the gradient of staining obtained is that observed at the adult stage. This is the first indication in the literature showing a dual and developmentally regulated expression of laminin y2 chain, by undifferentiated epithelial cells at early phases of development and by the mesenchymal cells at later stages giving rise to the adult pattern.

Using cocultures of bovine keratinocytes and human dermal fibroblasts exposed to antibodies specific to hu- man a3 chain, Marinkovich et al. (1993) concluded that the fibroblasts do not deposit this chain into the base- ment membrane. Two hypotheses could explain the ap- parent discrepancy with our results. Firstly, laminin-5 production occurs in an organ specific manner. Sec- ondly, 1 week coculture might not be suficient to detect the intervention of the fibroblasts which might not be differentiated enough as compared to the grafting ex- periments. Related to this last point, it should be men- tioned that a similar delayed deposition of the laminin a1 chain by the mesenchyme has been noted (Simo et al., 1992). These observations show the necessity of in- ductive cell interaction process for synthesistsecretion of laminin cxl and y2 chains, possibly accompanied by the acquirement of a certain level of mesenchymal cell differentiation. Similarly, tenascin synthesis by the mesenchymal cells has been shown to result from epi- thelial-mesenchymal interaction (Aufderheide and Ek- blom, 1988). In this regard, the interspecies recombi- nation model has a major advantage as compared to the coculture experiments as it allows the attainment of a more advanced stage of differentiation.

Finally, an unexpected finding was the staining of the intestinal muscle coat by the antibodies recogniz-

ing the y2 chain. This was clearly obvious in the adult mouse intestine when the two smooth muscle layers, longitudinal and circular, were fully differentiated. The onset of y2 chain in the muscle coat occurs just before birth, paralleling the cytodifferentiation of smooth muscle cells as assessed by the expression of smooth muscle a and y actins (Kedinger et al., 1990). In the adult human intestine, a similar pattern of distri- bution of y2 chain was noted, the staining being how- ever discontinuous and weak (not shown). It is of in- terest to note that the HD1 protein was not detected in the smooth muscle cells, whatever the developmental stage examined. Immunoprecipitation experiments confirmed the presence of y2 chain in the murine mus- cle coat and allowed us to show that it is synthesized as a precursor form (155 kDa; not shown). Yet, the other coprecipitated bands that migrated at 180 and 120 kDa do not correspond to the classical molecular weights of a3 and p3 chains. This raises the possibility of the ex- istence of a new a?p?y2 isolaminin whose identification requires additional experiments since p l and p2 con- taining laminin variants have been identified in the intestinal muscle coat (Glukhova et al., 1993; Simon- Assmann et al., 1994).

In summary, our data show that laminin-5 and HD1 are expressed and deposited in the intestine with spe- cies and/or chains specific spatial and temporal pat- terns. The focalised location of laminin-5 in the intes- tine suggests a role distinct from that exerted in the skin.

EXPERIMENTAL PROCEDURES Antibodies

The monoclonal and polyclonal antibodies used for indirect immunofluorescence are listed in Table 1. Monoclonal antibody GB3 raised against amnion epi- thelium (Hsi and Yeh, 1986) reacts with human lami- nin-5 (Verrando et al., 1987). Monoclonal antibody 6F12 (generous gift from Prof. R.E. Burgeson, Massa- chussets General Hospital, Charlestown, MA) recog- nizes the human laminin p3 chain (Rousselle et al., 1991). Affinity purified polyclonal antibodies against the individual chains of laminin-5 were SE85 and SE513 specific for the human and mouse laminin a3 chain, respectively, SE144 specific for both human and mouse laminin y2 chain (Aberdam et al., 1994a). Mono- clonal antibodies against HD1 (mAb HD-121) were pre- pared with electrophoretically purified HD1 protein (Hieda et al., 1992).

Intestinal Samples Human intestines were obtained from foetuses be-

tween 9 and 12 weeks of gestation after legal abortion (collaboration with Dr. M. Dreyfus, Department of Gy- necology and Obstetrics, CHU Hautepierre, Stras- bourg, France). Other specimens were obtained from fetuses at 15,20, and 24 weeks of gestation and at term after therapeutic or spontaneous abortion (Department of Maternity, CHU, Strasbourg, and Dr. A. Zweibaum,

LAMININ-5 AND HD1 IN INTESTINAL DEVELOPMENT 21

U178 INSERM, Paris). The specimens were taken with the informed consent of the mothers within 2 hr follow- ing abortion. Early fetal ages were calculated from the last menstruation of the mother and verified by the developmental pattern of hand morphology (Lacroix et al., 1984). Adult ileal and colonic mucosa were taken from surgically resected material (CHU Hautepierre, Strasbourg); only healthy and undamaged intestinal segments were used.

Small intestinal and colonic segments were taken at various developmental stages from fetal, suckling, and adult mice bred in our institute. Fetuses were removed by cesarean sections at various stages between day 12 post-coitum and birth. The day on which the vaginal plug was found was designated as day zero, and the developmental stages of the fetal mice were deter- mined according to the number of days of gestation.

Interspecies Intestinal Recombinants Associations between chick and mouse intestinal tis-

sue components have been performed using an experi- mental procedure described previously (Kedinger et al., 1981). Experimental design is schematically repre- sented in Figure 1. In the chick embryo, the presump- tive small intestine was removed at 5?h days of incu- bation. In the fetal mouse, the segment corresponding to the small intestine was excised at 12 days of gesta- tion. In preliminary experiments, we have determined that these developmental stages were the most appro- priate. The mesenchyme was separated from the endo- derm after incubation of the intestinal segments in a 0.03% solution of collagenase (Boehringer, Mannheim, Germany, 0.54 U/mg) in culture medium for 1 hr a t 37°C according to Gumpel-Pinot et al. (1978). Interspe- cies recombinations of the isolated endodermal and mesenchymal components: chick mesenchyme/mouse endoderm (Cm/Me) and mouse mesenchyme/chick en- doderm (MdCe) were performed. The separation of the endoderm from the mesenchyme was val-idated in ear- lier studies by using chickfquail associations in which quail cells were recognized by their peculiar nuclear marker (see Haffen et al., 1981). In order to ensure their cohesion, the associations were maintained on agar-solidified medium for 24 hr. The recombinants were then grafted into the coelomic cavity of 3-day-old chick embryos. The grafts were recovered at various periods of time up to 17 days after their implantation. In this latter case, the theoretical stage reached by the grafts corresponds to 11 days of postnatal life for the mouse.

Immunofluorescence Staining of Laminin-5 and of HD1

Intestinal segments at different developmental stages and interspecies recombinants were processed similarly. They were embedded in Tissue-Tek com- pound, and frozen with isopentane cooled in liquid ni- trogen and stored at -70°C until use. Transverse sec- tions (5 pm thick) performed at -25°C were placed on

gelatin-coated slides. Cryostat sections were incubated for 1-2 hr at room temperature in a moist chamber with the specific primary monoclonal or polyclonal antibod- ies diluted in PBS (phosphate buffered saline). Slides were rinsed with PBS and washed in two changes of PBS for 5 min each. Sections were incubated either with fluorescein (F1TC)-conjugated sheep anti-mouse IgG (Institut Pasteur, Paris, France) or goat anti-rabbit IgG (Nordic, Tilburg, The Netherlands). In some cases, an avidin-biotin amplification system was used. First, the sections were incubated with a sheep anti-mouse or donkey anti-rabbit IgG biotinylated whole antibody (Amersham International, Buckinghamshire, UK) and secondly with f luorescein-avidin DCS (Vector Labora- tories, Burlingame, CA) with several washes with PBS in between. Slides were mounted in Vectashield under a coverslip, observed under an Axiophot microscope (Zeiss, Oberkochen, Germany), and photographed using HP5 films (ASA 400, Ilford Ltd, Cheshire, England). Control sections were processed as above, omitting the primary antibodies. These controls showed some unspe- cific yellowish fluorescence mainly found within the lamina propria of the mature intestinal organ, or lo- calized in the mucus cells.

RNA Preparation and Reverse Transcriptase Polymerase Chain Reaction

Isolation of total RNA from fetal and adult mouse intestine was performed with trizol reagent (total RNA Isolation reagent) from Gibco-BRL (Life Technologies, Inc., Gaithersburg, MD). Random-primed first strand cDNA synthesis was carried out on total RNA (2 1.1) using MMLV reverse transcriptase as devised by the manufacturer. An aliquot (1 pl) of the first strand cDNA reaction was added directly to the polymerase chain reaction (PCR) mix containing 0.2 mM dNTP, 2.5 U Taq DNA polymerase (Gibco-BRL), sense primers in 25 pl 1 x Taq DNA polymerase (20 mM Tris-HC1, pH 8.4, 50 mM KC1, 1.5 mM MgC12). To amplify the lami- nin a3 cDNAs, the PCR cycling conditions were 94°C for 5 min, followed by 30 cycles at 94°C for 45 sec, 42°C for 45 sec, 72°C for 45 sec, and then extension at 72°C for 5 min. The same protocol was used to amplify by RT-PCR the laminin p3 and y2 chain transcripts ex- cept that the annealing temperatures were 66" and 60"C, respectively. Aliquots of 10 p1 were run on 1.5% agarose gels. The primer pairs used in these experi- ments were: L 5'-GTCAGG!M'GAACGATACAGTG-3', R 5'-CAAACTGCAGGGCTCTGTGA-3' for a 3 tran- script; L 5'-GACCCTCGCAACTCCCTAAGC-3', R 5' - TCGTTGGCATTGGTCACAGCG-3' for p3 transcript, and L 5'-TGGTTGGAAGGCGGTTCAGAG-3', R 5'- TCACGTTATCAATGTACCCTG-3' for y2 transcript. The expression of laminin-5 genes was demonstrated by amplification of DNA fragments in the size of 275 bp, 446 bp, and 729 bp for p3, y2, and a3, respectively. Reaction products were separated by 1.5% agarose gel electrophoresis and visualized under UV light in the presence of ethidium bromide and photographed.

22 ORIAN-ROUS!: SEAU ET AL.

ACKNOWLEDGMENTS We thank C. Arnold and C. Leberquier for invalu-

able technical help for this work. We are grateful to Prof. R.E. Burgeson (Massachusetts General Hospital, Charlestown, MA) for generously providing us with the 6F12 mAb and to Dr. K. Owaribe (Nagoya University, Nagoya, Japan) for the HD-121 mAb. We are indebted to Dr. J.F. Launay (INSERM U381, Strasbourg) for helpful discussion and suggestions. We also thank I. Gillot, C . Haffen, B. Lafleuriel, and L. Mathern for preparation of the manuscript and illustrations. Finan- cial support was given by INSERM, CNRS, the Asso- ciation pour la Recherche sur le Cancer, the Ligue Na- tionale contre le Cancer, INSERM-CNAMTS, and Groupement de Recherche et $Etude sur le G6nome (GIP-GREG).

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