ABSTRACT
The expression of laminin, a major glycoprotein constituent of basement membranes, was investigated in the rat developing intestine. The biosynthesis of laminin was studied after metabolic labeling of intestinal segments taken at various stages of development; the neosynthesized laminin was purified by affinity chromatography on heparin–Sepharose. Immunoblotting and immunoprecipitation experiments allowed us to analyze its constitutive chains. The data show that laminin is synthesized in very large amounts at 16–18 days of gestation concomitant with the onset of intestinal morphogenetic movements, i.e. villus emergence. Evaluation of the relative proportion of individual laminin polypeptides shows that laminin B1/B2 chains are produced in excess of A chains whatever the developmental stage considered. Interestingly at 17 days of gestation, levels of laminin A subunits are maximal. A second rise in the A/B chain ratio starts around birth and continues until adulthood. These quantitative data are corroborated by the immunocytochemical detection of laminin A and B chains, which revealed a specific spatiotemporal pattern. The finding that laminin A chains are located in the basement membrane of growing villi and of adult crypts raises the possibility that they may be involved in the process of cell growth and/or in the establishment of cell polarity by creating a specialized extracellular microenvironment.
Introduction
Laminin, an extracellular matrix glycoprotein, is known to play an important role in growth, adhesion and differentiation of cells (for reviews see Kleinman et al. 1984, 1985; Lissitzky et al. 1986; Martin and Timpl, 1987; Timpl, 1989). Furthermore, the fact that this molecule is expressed early during development (Leivo et al. 1980; Ekblom et al. 1980; Dziadek and Timpl, 1985) emphasizes its involvement in tissue interactions which are known to be of prime importance in morphogenesis. Laminin, which is a major component of basement membranes, was initially isolated from the mouse Engelbreth–Holm–Swarm (EHS) tumor (Timpl et al. 1979); it consists of three polypeptide chains, one A chain (with a Mr of about 440×103) and two B chains (Bl: 210×103 and B2 : 200×103) which form a crossshaped molecule (Timpl, 1989).
Many adult tissues (kidney, lung, heart and skin) have been shown to contain disproportionately low levels of mRNA for the A chain relative to the B chains of laminin, as well as variable levels of Bl and B2 mRNA (Kleinman et al. 1987; Martin and Timpl, 1987; Laurie et al. 1989; Olsen et al. 1989). Similarly, a noncoordinated expression of the three chains of laminin has been described during embryogenesis and development (Ekblom et al. 1990; Kücherer-Ehret et al. 1990). Indeed, in mouse blastulas and early embryos, laminin polypeptides are differentially synthesized, A chains being expressed later than the B chains (Cooper and MacQueen, 1983). In kidney, conversion of mesenchyme to epithelium is accompanied firstly by an increase of laminin Bl and B2 mRNA (Senior et al. 1988; Ekblom et al. 1990) followed by an upsurge of A chain expression (Ekblom et al. 1990). The importance of laminin A chains in the establishment of epithelial cell polarity has been emphasized by the inhibition of epithelial cell development with fragment-specific laminin antibodies (Klein et al. 1988). However, in a recent paper, Ekblom et al. (1990) point out that the relationship between laminin A chain synthesis and basement membrane development cannot be generalized to all epithelia since some basement membranes in developing or adult organs lack A chains or may be composed of A chain variants.
In the gut, laminin has been detected immunocytochemically at 12 days of gestation in the rat (the earliest stage studied) at the epithelial×mesenchymal junction and around a few cells scattered within the mesenchyme. At a later developmental stage, just before villus formation, the staining observed in the mesenchyme intensifies and becomes confined to two distinct areas: the zone immediately beneath the epithelial×mesenchyme interface, and in the most peripheral zone of the mesenchyme, which will differentiate into muscle layers (Simon-Assmann et al. 1986). These data emphasize that laminin is synthesized by specialized cells that are segregated during development.
In the present study, we have quantified the biosynthesis of laminin and analyzed its constitutive chains to assess whether expression of laminin can be correlated with morphogenetic events and epithelial differentiation features in the developing gut. In parallel, immunocytochemical detection of laminin A and B chains was carried out to investigate possible modifications in their localization or distribution.
Materials and methods
Reagents
Synthetic media, Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12, and fetal calf serum were purchased from Gibco Laboratories and the SerXtend serum substitute from NEN products. Methionine-free DMEM was obtained from J. Boy (France). D-[6-3H]glucosamine hydrochloride (specific activity 20×40 Ci mmole-1) and L-[35S]methionine (specific activity 800×1200 Ci mmole-1) were purchased from Amersham and NEN, respectively. 14C-labeled protein standards from Amersham were used. All the protease inhibitors and Tween 20 were obtained from Sigma. Sepharose 4B and C1-4B, heparin-Sepharose 6B were products of Pharmacia. Pansorbin (Staphylococcus aureus suspension) was purchased from Calbiochem. Goat antirabbit immunoglobulin G second antibody conjugated to peroxidase was obtained from Institut Pasteur and fluorescein (FITC)-conjugated Affinipure goat anti-rat IgG (H+L) from Jackson Immunoresearch Laboratories. Affinity-purified goat anti-rabbit immunoglobulin G second antibody conjugated to alkaline phosphatase and the BCIP/NBT color development solution from Bio-Rad were used for revelation of the immunoblots.
Laminin and anti-laminin antibodies
Laminin was selectively extracted from the murine Englebreth–Holm–Swarm (EHS) tumor using NaCl-contain-ing buffers and purified by anion-exchange chromatography as described by Timpl et al. (1979). Partial removal of nidogen was performed by dissociation of the laminin-nidogen complex after exposure to 2 ? guanidine–HCl and separation by molecular sieve chromatography (Sepharose C1-4B column) as reported previously (Paulsson et al. 1987). The purity of laminin was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions.
Rabbit polyclonal anti-laminin serum was produced by three repeated intradermal injections of 0.4 mg of laminin into rabbits. The anti-laminin antibodies were purified by chromatography on laminin–Sepharose 4B column. These antibodies recognized specifically laminin, and showed no reaction with type I collagen, type III collagen or fibronectin in dot-blot assays (Fig. 1A); on western blot (Fig. 1B), bands corresponding to A and B1/B2 chains of laminin as well as nidogen extracted from EHS tumor were labeled (lane 1); further specificity of the anti-laminin IgG was assessed by western blotting experiments of a crude preparation of intestinal proteins extracted by urea in which these bands were detected (lane 2). Using immunocytochemistry, anti-laminin serum was shown to bind to basement membranes in the intestine (Fig. 1C), as did antibodies provided by Dr Timpl (Simon-Assmann et al. 1986).
Validation of the rabbit polyclonal affinity-purified anti-laminin antibody (A) Dot assay of the binding of 1 μg of laminin (al), fibronectin (a2), type 111 collagen (bl) and type I collagen (b2) to anti-laminin antibodies as explained in Materials and methods, showing that the antibodies recognized specifically laminin. (B) SDS–gel electrophoresis on 4–15 % polyacrylamide gradient gels under reducing conditions and immunoreaction with anti-laminin antibody after electroblotting of EHS-tumor laminin (lane 1) and of a crude preparation of proteins extracted by urea from 17-day fetal rat intestine (lane 2). Note the presence of the laminin bands (A: ≈400 and B1/B2: ≈200–220×103) and of nidogen (=150×103). The position of marker proteins is indicated. (C) Immunofluorescence staining for laminin. Photomicrographs of 15-day fetal (1) and of adult (2) rat intestines stained with the anti-laminin antibody using standard immunofluorescence methods (see in Simon-Assmann et al. 1986). m, mesenchyme; e, epithelial cells; ml, muscular layers; Ip, lamina propria. Arrows point to the basement membrane at the epithelial–fibroblastic interface. Bar: 30μm.
Validation of the rabbit polyclonal affinity-purified anti-laminin antibody (A) Dot assay of the binding of 1 μg of laminin (al), fibronectin (a2), type 111 collagen (bl) and type I collagen (b2) to anti-laminin antibodies as explained in Materials and methods, showing that the antibodies recognized specifically laminin. (B) SDS–gel electrophoresis on 4–15 % polyacrylamide gradient gels under reducing conditions and immunoreaction with anti-laminin antibody after electroblotting of EHS-tumor laminin (lane 1) and of a crude preparation of proteins extracted by urea from 17-day fetal rat intestine (lane 2). Note the presence of the laminin bands (A: ≈400 and B1/B2: ≈200–220×103) and of nidogen (=150×103). The position of marker proteins is indicated. (C) Immunofluorescence staining for laminin. Photomicrographs of 15-day fetal (1) and of adult (2) rat intestines stained with the anti-laminin antibody using standard immunofluorescence methods (see in Simon-Assmann et al. 1986). m, mesenchyme; e, epithelial cells; ml, muscular layers; Ip, lamina propria. Arrows point to the basement membrane at the epithelial–fibroblastic interface. Bar: 30μm.
Monoclonal antibody, mAb 193, was raised in rat against the E3 fragment of mouse laminin. It reacts with the A chain but not with the B chains of laminin as shown in western blot analysis of EHS-tumor extract (Fig. 2). Further characterization of this antibody is described in detail in Sorokin et al. (in preparation).
Western blot analysis of EHS-tumor laminin with mAb 193 antibodies. Lane 1 (intentionally overloaded) shows specific reaction of the monoclonal antibody with the A chain and no reaction with B chains. Lane 2 contains the relative molecular mass standard with the position of 200×103 marked.
Monoclonal antibody, mAb 4C12, raised in rat against mouse EHS laminin was kindly provided by Dr J. C. Lissitzky (UA CNRS 1175, Marseille, France). As described previously (Charpin et al. 1986; Lissitzky et al. 1988), this antibody recognizes a conformational epitope localized on the laminin B1/B2 heterodimer and in the Pl fragment of laminin.
Preparation of intestinal samples
Fetuses from pregnant Wistar rats, whose gestation had been accurately timed, were removed by cesarean section at various stages between the 15th day of gestation and birth. The day on which a vaginal plug was found was designated as day 0, and the developmental stages of the fetal rats were determined according to the number of days of gestation. Newborn rats (at delivery and on day 3 after birth) as well as adult rats were also used. Adult rats were subjected to pancreatico-biliary duct ligature 24 h before they were killed to diminish proteolytic activities. The intestines were cut into small fragments of about 0.5mm. Each experimental series was carried out with 1 or 2 litters of 15-to 17-day fetal rats, with 3 to 5 intestines of 18/19-day fetal rats, with 2 intestines for 20-day fetal and newborn rats and with the proximal portion of one adult rat jejunum.
Immunolocalization of A and B chains during gut development was performed on mouse species to avoid nonspecific reaction of the monoclonal and/or second antibodies with rat tissue since the monoclonals were raised in rat. Due to the fact that morphogenetic events in murine intestine occur 1–2 days earlier than in the rat, fetuses were taken from day 13 until birth; newborns at various developmental stages as well as adults were used.
Biosynthetic labeling and purification of laminin
The newly synthesized laminin was quantified by incubating the intestinal fragments for 24 h at 37°C in 4 ml of culture medium composed of a mixture (1:1) DMEM and Ham’s F12, supplemented with 7.5% fetal bovine serum (containing 2.5% SerXtend serum substitute) in which 4μCiml-1 of [3H]glucosarhine was added. The organ samples were separated from the labeled medium by centrifugation (1000g for 5 min) in presence of protease inhibitors (PMSF, 174μgml-1; pepstatin, lμgml-1; antipain, 1μgml-1; benzamidine, 15μgml-1; aprotinin, 10μgml, leupeptin, 10μgml1). The tissue pellets were washed with PBS, lysed by a brief sonication and proteins were extracted 4 times consecutively in 4? urea (60°C for 20min). The lysates of each extraction step were then clarified by centrifugation at 10 000 g for 15 min and pooled. Aliquots of 2 ml were stored at —80°C until use. The neosynthesized laminin molecules were purified from the lysate by affinity chromatography on a heparin-Scpharose 6B column previously tested with purified laminin from EHS tumor as described by Sakashita et al. (1980). After washing of the column with PBS, the absorbed material was eluted with 1 M NaCl in PBS containing protease inhibitors; fractions of 2.5 ml were collected and aliquots (0.2 ml) were counted using Biofluor scintillation fluid. The fractions eluted with 1 M NaCl, corresponding to a single radioactive peak were pooled, dialyzed and tested by SDS–PAGE.
Nitrocellulose dot-immunobinding
Aliquots of fractions obtained after affinity-chromatography as well as various extracellular matrix molecules were dotted onto nitrocellulose filter (Schleicher and Schuell, 0.45 μm) by suction, and filters were air dried. Filters were blocked with 3 % (w/v) bovine serum albumin (BSA) in Tris-HCl 0.01 M pH 7.4, NaCl 0.155 M for lh at 40°C and incubated with antilaminin antibodies (8μgml-1) for 2h at room temperature. After washings, the nitrocellulose was incubated 1 h in presence of peroxidase-conjugated secondary antibody in dilution of 1:2000 and revealed with 4-chloro-l-naphtol in methanol containing hydrogen peroxide (Ngai and Walsh, 1985).
Immunoblots
The material purified by affinity chromatography has been analyzed by immunoblotting. Samples were boiled for 5 min in Laemmli sample buffer with 5% (v/v) β-mercaptoethanol and proteins separated by SDS–PAGE on gels (4–15 % gradient gels). The gels were calibrated with a standard of laminin–nidogen complex extracted from EHS tumor. Proteins were subsequently electrotransferred overnight onto nitrocellulose in 0.025 mM Tris–HCl, 0.192? glycine, pH 8.2, 20% (V/V) methanol. After transfer, the nitrocellulose was blocked for lh at 40°C with 0.3% BSA in buffer A (0.01 M Tris–HCl, pH 7.4, 0.155 M NaCl containing 0.3 % V/V Tween 20). For immunological revelation, the nitrocellulose was incubated for 2h at room temperature with polyclonal antilaminin antibodies (8μgml-1 in buffer A). After several washes, the nitrocellulose was incubated with affinity-purified goat anti-rabbit IgG conjugated to alkaline phosphatase [1:3000 (V/V) in buffer A], Finally, the blot was rinsed and developed with BCIP/NBT-color reagents; the staining intensity of the bands was quantified by linear scanning with a densitometer (Shimadzu, Roucaire).
Immunoprecipitation
Intestinal fragments were incubated for 24 h in 4 ml of methionine-free DMEM with 7.5% of fetal bovine serum (containing 2.5% SerXtend serum substitute), supplemented with 100μCi of [35S]methionine. Tissue samples were recovered and rinsed as described above. Aliquots of tissue extracts (500 μl) were brought to 700 μl with immunoprecipitation buffer (buffer B: 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% Triton X-100; 2m? iV-ethylmaleimide, 2m? EDTA, 2m? PMSF). 100μl of 10% Pansorbin suspension (made up in the immunoprecipitation buffer) was then added. After preadsorption for 45 min at 4°C, the Pansorbin was removed by centrifugation (5000 revs min-1 for 2 min, Bio-fuge A), and 26μg of antiserum (1.62mgml-1) added to the supernatant. The mixture was shaken for 2h at 4°C; 100 μ l of the Pansorbin suspension was added and adsorption was carried out for 1 h. The Pansorbin pellets were then washed three times with buffer B and two times with buffer C (50 mM Tris-HCl, pH 7.8, containing l? NaCl, 1% Nonidet P-40, and protease inhibitors). Pellets were then boiled for 5 min in Laemmli sample buffer containing 4% SDS with 5% (V/V) β-mercaptoethanol, and subjected to SDS-PAGE on 4–15 % gradient gels. The immunoprecipitated 35S-labeled molecules were detected by fluorography at —70°C using Amersham Hyperfilm -βMAX. The gels were calibrated with a standard of laminin and l4C-labeled protein standards.
Immunolocalization of the laminin A and B chains
Small fragments of murine intestines were embedded in Tissue-Tek, frozen in Freon, cooled in liquid nitrogen and stored at —70°C. Frozen sections (5–6μm thick) were treated for lh with the specific monoclonal antibodies (mAb 193: 12μgml-1, mAb 4C12: 10μgml-1). Sections were then incubated with fluorescein (FITC)-conjugated affinipure goat anti-rat IgG (1/50 in PBS). Slides were washed, mounted in glycerol/PBS/phenylenediamine under coverslips and observed with an Axiophot Microscope (Zeiss).
Results
Pattern of laminin synthesis in the developing rat intestine
The amount of laminin synthesized by intestinal segments at various stages of development was evaluated after isolation of the metabolically [3H]glucosa-mine-labeled molecules on affinity chromatography (Fig. 3A). Validity of the purification method of laminin on heparin-Sepharose column was assessed as follows. First, when laminin extracted from EHS tumor was submitted to chromatography, the protein was recovered in the 1 M NaCl peak as shown by dot-immunobinding (Fig. 3B); this is also the case with laminin extracted from intestinal tissue (Fig. 3C). Second, dot immunoassay showed no specific binding of the 1 M NaCl fraction with anti-fibronectin antibody (not illustrated). Finally, electrophoresis under reducing conditions of the 1M NaCl fraction of laminin extracted from EHS tumor (Fig. 3D) demonstrated the presence of bands with relative molecular masses in the regions expected for laminin, ≈210×103 (B1/B2 chains) and ≈440×103 (A chains); these bands were recognized after immunoblotting by anti-laminin antibody. In addition a 150×103 band was detected, although in minute amounts.
Purification of the laminin-nidogen complex by affinity chromatography. (A) Representative profile of labeled material, extracted from 18 day fetal rat intestine by urea, after chromatography on heparin-Scpharosc. Elution of neosynthesized laminin was achieved by the passage of l? NaCl. (B) Dot assay of the binding of EHS laminin with antilaminin antibodies before (al), and after its passage on heparin-Sepharose: fractions PBS (bl) and NaCl 1 M (a2); NaCl buffer served as control (b2). (C) Dot assay of the binding with anti-laminin antibodies of neosynthesized material extracted from rat intestine before (al), and in the fractions PBS (bl), 1 M NaCl (b2) after heparin-Sepharose column; NaCl buffer served as control (a2). (D) Laminin extracted from EHS tumor after its passage on affinity chromatography. Coomassie blue staining of the l? NaCl fraction (lane 1). Ferritine (220×103; lane 2), and myosin (205×103; lane 3) used as molecular weight markers.
Purification of the laminin-nidogen complex by affinity chromatography. (A) Representative profile of labeled material, extracted from 18 day fetal rat intestine by urea, after chromatography on heparin-Scpharosc. Elution of neosynthesized laminin was achieved by the passage of l? NaCl. (B) Dot assay of the binding of EHS laminin with antilaminin antibodies before (al), and after its passage on heparin-Sepharose: fractions PBS (bl) and NaCl 1 M (a2); NaCl buffer served as control (b2). (C) Dot assay of the binding with anti-laminin antibodies of neosynthesized material extracted from rat intestine before (al), and in the fractions PBS (bl), 1 M NaCl (b2) after heparin-Sepharose column; NaCl buffer served as control (a2). (D) Laminin extracted from EHS tumor after its passage on affinity chromatography. Coomassie blue staining of the l? NaCl fraction (lane 1). Ferritine (220×103; lane 2), and myosin (205×103; lane 3) used as molecular weight markers.
The quantitative study of the neosynthesized laminin molecules eluted with 1 M NaCl led to the following developmental profile in the rat intestine from 15 days of gestation to the adult (Fig. 4). Synthesis of laminin was already important at 15 days of gestation, the first stage studied; it approximately doubled to reach a maximum at day 17 of gestation; the amount of laminin molecules synthesized per mg of intestinal tissue protein then decreased drastically up to 19 days and from this stage onwards remained almost stable with values that were far lower than those found in the gestational period. Quantification of immunoprecipitated laminin led to a similar developmental profile; indeed the level of radiolabeled laminin was the highest in the 17-day-old fetal intestine (≈90 000 cts min-1 mg-1 protein) and was 4-fold decreased at 19 days (≈21000cts min-1 mg-1 protein); furthermore, values recovered in this latter stage were of similar range to those found in the mature adult organ (18 000 cts min-1 mg-1 protein)
Developmental profile of laminin biosynthesis (ctsmin-1 [3H]glucosamine mg-1 intestinal tissue protein; mean;s.E.M.) in rat intestines from 15 days of gestation until adulthood. Numbers in parenthesis represent the numbers of experiments.
The molecular form of laminin purified on heparin–Sepharose was analyzed at various developmental stages after separation on SDS-PAGE and immunoblotting. Fig. 5 illustrates representative immunoblots at various stages of development. Although bands were obvious in the 400 and 220×103 regions at almost all stages studied, variations in the ratio of laminin chains seemed to occur as a function of development. Scanning of the gels allowed us to determine the intensity of the bands corresponding to A and B1/B2 chains and their relative proportion (Fig. 6). At 15–16 days of gestation, A chains were undetectable in half of the experiments; when present, the relative number of A chains per 100 B chains was low. At 17 days of gestation, a stage coinciding with the upsurge of laminin biosynthesis (Fig. 4), this proportion increased (16 A/per 100 B). After a short time period (during late gestation) during which A chains were present in low amounts, their proportion again increased until adulthood reaching 12 A chains per 100 B1/B2 chains. These data, obtained by the analysis of immunoblots, concern the whole pool of laminin present at a given stage. Autoradiograms following immunoprecipitation of laminin metabolically labeled with [35S]methionine during a 24h-incubation period (depicted for the 17-day fetal intestine in Fig. 7) revealed that B1/B2 chains are synthesized at all developmental stages considered; in contrast, the A chains could never be detected. This result may be related to the fact that samples from which laminin was immunoprecipitated were not affinity purified as were the samples used in western blots. In addition, A chains are produced in smaller amounts than the B chains of laminin and may consequently be more difficult to visualize using immunoprecipitation and autoradiograms. Anti-lam-inin antibodies precipitated nidogen in all-cases.
Immunoblot analysis of laminin isolated by affinity chromatography and electrophoresed on SDS– polyacrylamide gel of rat intestinal tissue at various developmental stages: 15-day (a), 17-day (b), 19-day (c) fetal, 2-day postnatal (d) and adult (e) rat intestines; EHS laminin standard (f). The amount of protein deposited on each track was not the same. Positions of A and B1/B2 chains of laminin and of nidogen are indicated by arrows; in addition, some other immunoreactive bands were seen that are probably degradation products.
Immunoblot analysis of laminin isolated by affinity chromatography and electrophoresed on SDS– polyacrylamide gel of rat intestinal tissue at various developmental stages: 15-day (a), 17-day (b), 19-day (c) fetal, 2-day postnatal (d) and adult (e) rat intestines; EHS laminin standard (f). The amount of protein deposited on each track was not the same. Positions of A and B1/B2 chains of laminin and of nidogen are indicated by arrows; in addition, some other immunoreactive bands were seen that are probably degradation products.
Relative proportion of A chains versus 100 B chains of laminin extracted at various developmental stages. Gel tracks of immunoblots were scanned using a dualwavelength scanner densitomer. Each point corresponds to the mean value (±S.E.M.) of at least 4 experiments.
Immunoprecipitation with anti-laminin antibody of [35S]methionine-labelcd proteins from 17-day fetal rat intestine under reducing conditions (lane 1); the corresponding densitometer scan shows B1/B2 chains as a doublet and nidogen. Laminin standard (lane 2) was detected by staining the gel with Coomassie Blue. The position of. l4C-labeled proteins is indicated.
Immunoprecipitation with anti-laminin antibody of [35S]methionine-labelcd proteins from 17-day fetal rat intestine under reducing conditions (lane 1); the corresponding densitometer scan shows B1/B2 chains as a doublet and nidogen. Laminin standard (lane 2) was detected by staining the gel with Coomassie Blue. The position of. l4C-labeled proteins is indicated.
Immunocytochemical localization of A and B chains during gut development
This study has been conducted in the developing murine intestine, since antibodies used against laminin A and B chains were raised in the rat. Fig. 8 illustrates the pattern of laminin A and B chains deposition at various stages, which have been chosen according to the fact that developmental events in the mouse species occur 1–2 days earlier than in the rat.
Representative indirect immunofluorescence micrographs of A (A,C,E–G) and B (B,D,H) chains of laminin, using respectively mAb 193 and mAb 4C12 antibodies raised in rat, in transverse sections of mouse intestines at various developmental stages: 13 (A,B), 16 (C,D), 18 (E) days of gestation; 3 days after birth (F, enlargment of the external muscular coat); adult (G,H). In G, some unspecific fluorescence is found within the lamina propria. The epithelial-mesenchymal or epithelial–lamina propria interface is indicated by arrows; e, endoderm or epithelium; m, mesenchyme; ml, muscular layers; Ip, lamina propria; *, enteric ganglia located between the two muscular layers. Bar: 30 µm.
Representative indirect immunofluorescence micrographs of A (A,C,E–G) and B (B,D,H) chains of laminin, using respectively mAb 193 and mAb 4C12 antibodies raised in rat, in transverse sections of mouse intestines at various developmental stages: 13 (A,B), 16 (C,D), 18 (E) days of gestation; 3 days after birth (F, enlargment of the external muscular coat); adult (G,H). In G, some unspecific fluorescence is found within the lamina propria. The epithelial-mesenchymal or epithelial–lamina propria interface is indicated by arrows; e, endoderm or epithelium; m, mesenchyme; ml, muscular layers; Ip, lamina propria; *, enteric ganglia located between the two muscular layers. Bar: 30 µm.
In the 13-day embryo, when the intestinal anlage consists of a simple tube of radially arranged stratified endodermal cells surrounded by mesenchymal cells, both types of laminin chains were present in the basement membrane (Fig. 8A,B); at this stage, laminin B chains were also detected around a few scattered cells in the mesenchyme (Fig. 8B). At 16–17 days of gestation, during villus formation, laminin A chains were still restricted to the epithelial–lamina propria interface (Fig. 8C). As villus elongation proceeds, laminin A labeling became sporadically interrupted towards the top of the villi (illustrated at 18 days of gestation, Fig. 8E). The fluorescence for laminin B chains at this period of time was strikingly different (Fig. 8D); it was located at the epithelium-lamina propria region, within the mucosal connective tissue and in the newly formed muscular layers. Thereafter, the staining pattern of laminin B chains remained unchanged as shown in the adult organ (Fig. 8H). Laminin A chains progressively appeared around the enteric ganglia at 18 days of gestation (Fig. 8E), and in the muscle coat at birth, the intensity of fluorescence increasing during the postnatal period (Fig. 8F). In the adult organ, laminin A chain staining at the epithelium-lamina propria interface was restricted to the crypt zone (Fig. 8G), while laminin B chains were present all along the crypt–villus axis (Fig. 8H).
Discussion
The present study emphasizes that changes in the biosynthesis and the molecular forms of laminin, a major component of the basement membrane, occur as a function of intestinal development. Results of immunofluorescence staining of laminin A and B chains corroborate the quantitative data and point to spatiotemporal modifications related to key events of morphogenesis and differentiation.
We found that the maximal biosynthetic activity of laminin occurs in rat intestinal tissue during the gestational period (16–18 days) showing that the fetal intestine synthesizes very large amounts of laminin concomitantly with the commencement of intestinal differentiation, i.e. villus emergence and individualization of the smooth muscle layers. The involvement of this basement membrane molecule in morphogenetic and differentiation processes is supported by the following observations. First, segregation of some mesenchymal cells decorated with anti-laminin antibodies in the subepithelial region occur at the time of villus outgrowth (Simon-Assmann et al. 1986). Second, deposition of a true basement membrane requires the presence of both epithelial and fibroblastic cells (Simon-Assmann et al. 1988) and precedes the expression of differentiation markers (Kedinger et al. 1989) as demonstrated by an in vitro coculture model system simulating the in vivo epithelial-mesenchymal entity (Kedinger et al. 1987). Thus one can assume that epithelial-mesenchymal interactions that influence development of the gut (for reviews see Kedinger et al. 1986; Haffen et al. 1989) may be mediated in part by changes in the synthesis and accumulation of laminin.
Later on, around birth, another upsurge of laminin synthesis seems to occur at precisely the moment at which the formation of the crypts (zone of the stem cells) by invagination of the base of the villi into the mucosal connective tissue occurs. Related to this, it has been shown that the highest concentration of laminin mRNA was found in the crypt zone of the mature intestine (Weiser et al. 1990) and that the IEC-17 cell line, derived from postnatal intestinal crypt cells, is able to synthesize laminin (Scarpa et al. 1988). Furthermore, administration of glucocorticoid hormones to suckling rats, which causes a precocious induction of a mature pattern of enzymatic activities as well as of structural features, increases laminin amounts and their respective mRNA levels (Walsh el al. 1987). In the mature organ, laminin biosynthesis is about 4 times lower than that of 17-day fetal intestinal tissue suggesting that the turnover of laminin is very slow in the adult. This assumption is corroborated by the low level in the adult intestine of messenger of laminin (Senior el al. 1988) and of type IV collagen, another basement membrane molecule, (Simon-Assmann et al. 1990) assessed by in situ hybridization or/and by northern blot analysis. Moreover, Trier et al. (1990) demonstrated, by injections of anti-laminin IgG, that laminin turnover occurs focally in the basement membrane of adult mouse jejunum over a period of weeks.
Evaluation of relative proportions of individual laminin polypeptides shows that the intestine produces laminin B subunit forms in excess of A subunit forms whatever the developmental stage considered. However, the relative proportion of A chains versus B chains varies showing that the subunits of laminin are not synthesized/deposited synchronously during morphogenesis. Recently, Klein et al. (1990) studied the expression of laminin A and B chain mRNA and polypeptides in the murine embryonic intestine. Trace amounts of A chain mRNA was found in 13-day-old embryonic intestine, a stage equivalent to 14–15 days in rat, but, with the assay used, no A chain mRNA could be seen in 15-day-old embryos or newborn animals. Low levels of A chain mRNA despite the presence of high levels of Bl and B2 chain mRNA is apparently common in embryonic cells (Ekblom et al. 1990; Klein et al. 1990; Kücherer-Ehret et al. 1990). We likewise find only low amounts of A chain, but the current study shows that changes in the ratio between the amounts of A chain and B chain polypeptides nevertheless occur during gut morphogenesis. These changes seem to correlate with important morphogenetic events. Such changes could be due to increased or decreased synthesis of the individual mRNAs, but post-translational control mechanisms may also be operative. The previous (Klein et al. 1990) and current observations do not allow a distinction between these alternatives for the embryonic intestine. Immunocytochemical data obtained with the anti-laminin A and anti-laminin B chain antibodies agree well with the biochemical findings, showing in particular that A chains are found in a more restricted pattern. Indeed, in the undifferentiated intestinal anlage where laminin A chains are restricted to the basement membrane region, levels of A chains are very low or even undetectable biochemically. Later on, during villus formation, when A chains are still found exclusively at the epithelial-mesenchymal interface, their levels are maximal suggesting that, during this short period of time, A chains are produced at a higher rate than B chains. The second rise in the relative amount of A chains to B chains, which progressively occurs from birth until adulthood, can be correlated to the appearance of laminin A chains within the muscular layers and to their subsequent thickening. Furthermore, once the crypts are formed, a phenomenon that starts at birth, A chains present at the epithelium-lamina propria interface become restricted to this crypt zone while B chains are found in the mature organ all over the crypt-villus axis. Interestingly, during the fetal period, intestinal epithelial cells are able to divide along the whole villus axis and subsequently to differentiate, while in the mature intestine the crypts compartmentalize the profiferating and predifferentiated cells, which undergo morphological and functional maturation from the crypt depth to the villus tip. The restricted presence of laminin A chains in the basement membrane underlying embryonic or crypt epithelial cells could indicate that these chains are involved either in the process of cell growth or in the establishment of cell polarity. These observations can be related to the interesting findings in the kidney that, while B chains are constitutively expressed, appearance of A chains correlates with the initiation of morphogenesis of kidney tubules (Klein et al. 1988). Furthermore addition of antisera against the carboxy-terminal end of laminin, which recognize A chain domains, inhibits epithelial cell polarization in vitro (Klein et al. 1988).
The asynchronous expression of the laminin polypeptide chains revealed in the present work by both biochemistry and immunocytochemistry, is difficult to interpret. However, from literature, it appears that synthesis and assembly of laminin chains is a complex event, which varies according to cell types or tissues (Edgar et al. 1988; Schittny et al. 1988; Leblond and Inoue, 1989; Paulsson and Saladin, 1989; Timpl, 1989). The conclusion emerging from various studies leads to the concept of a minor proportion of A chains versus B ones in tissues or cell cultures as compared to laminin extracted from EHS tumor (for reviews see Martin and Timpl, 1987; Ekblom, 1989; Timpl, 1989). Taken together, our present data underline a clear correlation between the rate of laminin synthesis and deposition and morphogenetic events in intestine. As far as the molecular form of laminin is concerned, an imbalance between the proportion of A versus B chains is obvious. Determination of the cell-binding site(s) of laminin, which is currently being investigated by many groups (Gehlsen et al. 1989; Grant et al. 1989; Nurcombe et al. 1989; Sorokin et al. 1990), will help in the understanding of the function of the qualitative variations of laminin.
ACKNOWLEDGEMENTS
We are indebted to Dr J. C. Lissitzky (UA CNRS 1175, Marseille, France) for providing monoclonal antibodies against B chains. We thank Drs M. Vigny (Unité 118, Paris, France) and M. Aumailley (Max-Planck Institut fur Bioche-mie, Munich, RFA) for helpful discussion and Dr K. Haffen for her encouragement and support throughout this project. We are very grateful to E. Alexandre and C. Arnold for excellent technical assistance, to C. Haffen for photographic processing, to L. Mathem for typing the manuscript and to B. Lafleuriel for assistance in the preparation of the illustrations. Financial support was given by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique and the Association pour la Recherche contre le Cancer.