Restricted distribution of laminin α1 chain in normal adult mouse tissues

Matrix Biology 18 Ž1999. 557]568

Restricted distribution of laminin a1 chain in normal adult mouse tissues Mats Falk a,1, Maria Ferlettaa,b,1, Erik Forsberg a , Peter Ekbloma,b,U a

Department of Cell and Molecular Biology, Uppsala Uni¨ ersity, Uppsala, Sweden b Department of Cell and Molecular Biology, Lund Uni¨ ersity, Lund, Sweden

Received 5 May 1999; received in revised form 9 September 1999; accepted 15 September 1999

Abstract The distribution of laminin a1 chain in adult mouse tissue was determined by immunofluorescence using monoclonal antibody 200, reacting with the globular carboxyterminus E3 fragment of a1 chain. Strong reactivity was noted only in a few tissues. Reactivity was restricted to epithelial basement membranes. Expression was noted in several epithelial basement membranes of the urinary tract, and male and female reproductive organs. In addition, expression was seen in some parts of the nervous system. Expression was seen in pia mater which surrounds the brain, and in the extracellular matrices covering the vitreous chamber and the lens of the eye. Staining was seen in the adrenal gland cortex, with strongest staining in the zona glomerulosa. Staining was negative in all other studied epithelial basement membranes, such as the lung Žtrachea or lung epithelium., epidermis, and all parts of the gastrointestinal tract Žliver, gut. except for weak staining in the ventricle and Brunner’s glands. No expression was seen in basement membranes of fat, Schwann, or endothelial cells in any studied parts of the body. Both small- and large-size vessel walls were negative both in endothelial basement membranes and blood vessel walls, with the exception of some larger brain blood vessels in locations where epithelial cells have invaginated. Neither smooth muscle, myocardium or striated muscle expressed a1 chain. We conclude that a1-containing heterotrimers including laminin-1 Ž a1b1g1. have a very restricted tissue distribution. Q 1999 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Laminin; Immunofluorescence; RT-PCR; Epithelium; Endothelium; Pia mater; Vitreous chamber; Lens; Adrenal gland; Eye; Tooth

1. Introduction Laminins are large glycoproteins of basement membranes, the thin extracellular sheets found on the basal side of epithelial and endothelial cells, and U

Corresponding author. Department of Cell and Molecular Biology, Lund University BOX 94, Solvegatan 39, Vaning 0, SE-22 ¨ ˚ 100 Lund, Sweden. Tel.: q46-46-222-0903; fax: q46-46-222-0855. E-mail address: [email protected] ŽP. Ekblom. 1 These two authors contributed equally.

surround muscle, Schwann and fat cells. All laminins so far found are heterotrimers of a , b and g chains, and in several species up to 5a , 3b and 2 g chains have been demonstrated ŽEngel, 1992; Yurchenco and O’Rear, 1994; Timpl and Brown, 1996. and several additional chains have been predicted. The number of chains is thus larger than anticipated when the first laminin was discovered 1979, and novel chains may still be discovered. Shared functions of the different laminin chains include participation in cell adhesion. Different

0945-053Xr99r$ - see front matter Q 1999 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 9 9 . 0 0 0 4 7 - 5

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laminins bind to distinct cell surface receptors and extracellular matrix components ŽHenry and Campbell, 1996; Kuhn, 1996; Aumailley and Smyth, 1998., ¨ raising the possibility that different laminins influence cells in a specific fashion. Genetic studies provide evidence for distinct roles of the various laminin chains. Absence or reduction of normal a 2 chain leads to muscular dystrophy in mice ŽSunada et al., 1994; Xu et al., 1994; Miyagoe et al., 1997. and mice lacking a 5 chain die during embryogenesis and show multiple morphological abnormalities in several tissues ŽMiner et al., 1998.. In mice lacking b2 chain, morphological development is normal but the mice die after birth, probably due to abnormal glomerular basement membranes or neuromuscular junctions ŽNoakes et al., 1995a,b.. Furthermore, mutations in the individual chains of laminin-5 Ž a 3b2 g2. lead to lethal blisters of the skin ŽBurgeson and Christiano, 1997.. Mice lacking the ubiquitous g1 chain show the most serious phenotype, and die shortly after implantation ŽSmyth et al., 1999.. The so far described specific abnormalities can to a large extent be explained by the different expression patterns of the genes, but it is likely that the proteins also have different functional properties. In order to clarify the role of individual laminin chains, it is valuable to know the distribution of the chains. The first described laminin isoform, laminin-1, is composed of a1, b1 and g1 chains. In many distribution studies polyclonal antibodies against laminin-1 have been used. Since such antibodies also detect the ubiquitous b1 and g1 chains, the observed distribution obviously does not reflect expression of laminin-1, although this is sometimes assumed. The issue can be clarified with probes specific for the a-chains. A few monoclonal and polyclonal antibodies have been suggested to be specific for the a1 chain, but two different types of results have been reported with these reagents. Our studies with polyclonal and monoclonal antibodies against the E3-fragment of laminin-1 suggested a restricted distribution of a1 polypeptide in some epithelial basement membranes in mouse embryos ŽEkblom et al., 1990; Klein et al., 1990; Sorokin et al., 1992, 1997a.. These findings were in agreement with RNA analyses which demonstrated low levels of the a1 chain mRNA by Northern blots in many tissues and cells ŽKleinman et al., 1987; Ekblom et al., 1990; Nissinen et al., 1991; Van den Heuvel and Abrahamson, 1993. and in situ hybridization studies which showed selective expression of the mRNA in embryonic epithelia ŽEkblom et al., 1990; Vuolteenaho et al., 1994. or in mesenchyme in close association with embryonic epithelium ŽThomas and Dziadek, 1993, 1994; Kadoya et al., 1995., with a1 protein deposition protein exclusively in the epithelial basement membrane ŽKlein et al., 1990; Kadoya et al.,

1995.. In contrast, a broad distribution of the a1 polypeptide in virtually all adult basement membranes was suggested by many investigators using the monoclonal 4C7 antibody ŽEngvall et al., 1986. reported to detect the a1 chain ŽEngvall et al., 1990; Sanes et al., 1990; Mundegar et al., 1995; Virtanen et al., 1995.. The issue recently became clarified in three independent studies, which all demonstrated that 4C7 reacts with laminin a 5 chain and not with the a1 chain ŽTiger et al., 1997; Church and Aplin, 1998; Kikkawa et al., 1998.. There is thus now agreement by most investigators in the field that the a1 chain has a ‘restricted’ expression pattern, whereas the a 5 chain has a ‘broad’ expression pattern both in mice and men ŽEkblom et al., 1996, 1998; Miner et al., 1997.. However, such general rules do not clarify the distribution pattern for most tissues and their different anatomical compartments. Most of the previous studies which employed well-characterized probes against the a1 chain have focused on mouse embryonic tissues, whereas the studies on adult tissues focused on human adult tissues, employing the 4C7 antibody. As a consequence of these biases, the distribution of the a1 chain in the adult stage is well known only for the kidney of mouse ŽEkblom et al., 1990; Sorokin et al., 1997a., rat ŽDurbeej et al., 1996. and human tissue sections ŽTiger et al., 1997., and a few other mouse tissues ŽFrojdman et al., 1995; Durbeej et al., 1998; ¨ Gu et al., 1999.. We have therefore here conducted a broad survey of the distribution of mouse laminin a1 chain in the adult mouse. We used a well-characterized rat monoclonal antibody 200 against laminin a1 chain ŽSorokin et al., 1992. in immunofluorescence of frozen sections. Results in heart, brain and lung were confirmed with RT-PCR.

2. Materials and methods 2.1. Immunofluorescence Frozen sections were stained with hybridoma supernatant of rat monoclonal antibody 200 against the E3 fragment of mouse laminin a1 chain ŽSorokin et al., 1992.. Double immunofluorescence was carried out with polyclonal antibody L9392 against mouse laminin-1 ŽSigma Chemical Co., St. Louis, MO, USA. and a rabbit polyclonal antibody against mouse von Willebrand factor ŽBehringwerke.. Secondary antibodies were coupled to the Cy 3 or to fluorescein isothiocyanate ŽFITC. labeled fluorochromes. Sections were analyzed and photographed by a Zeiss Axiophot microscope equipped for epifluorescence and appropriate filters as described ŽDurbeej et al., 1998., and analyzed with OpenLab software ŽImprovi-

M. Falk et al. r Matrix Biology 18 (1999) 557]568

sion, Coventry, England.. Unaltered computer images were prepared with OpenLab and Adobe Photoshop ŽAdobe Systems Incorporation, San Jose, CA, USA. software. 2.2. Re¨ erse transcriptase polymerase chain reaction (RT-PCR) Total RNA from several adult mouse organs was isolated from deep-frozen samples using guanidinium cesium chloride extraction and ultracentrifugation. Reverse transcription was carried out using Superscript II ŽGibco BRL., poly dŽT. primers and 2 mg RNA. Primers detecting laminin a chain sequences were: v

v

v

v

a1 sense 59-ACTTGGTGCTCCCTCTGAATC-39 and antisense 59-ATGCGGTGCTTGCTTTTGTGG-39 ŽSasaki et al., 1988. expecting a 626-bp product; a 2 sense 59-GGGTCGGATCAATCATGCTG-39 and antisense 59-AAAGGTCAGCTGGTGGGTGG-39 ŽBernier et al., 1994. expecting a 589-bp product; a 3 sense 59-AAAGGTCACCTGGGTGGTGG-39 and antisense 59-TTCGGTGGGAAGGAAAGCT-39 ŽGalliano et al., 1995. expecting a 587-bp product; and a4 sense 59-CATGGGATCCTATTGGCCTG-39 and antisense 59-CACATAGCCGCCTTCTGTGG-39 ŽLiu and Mayne, 1996. expecting a 664-bp product.

PCR conditions were: initial denaturation was for 3 min at 948C, then 948C for 30 s, 608C for 60 s and 728C for 60 s for 35 cycles. Products were then analyzed on a 1.2% agarose gel containing ethidium bromide.

3. Results

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pia mater with antibody against von Willebrand factor and mAb 200 showed that the laminin a1 chain staining was confined to the vessels walls ŽFig. 1E. and pia mater ŽFig. 1F.. No expression of a1 chain was seen in other parts of the brain. In sensory organs, selective staining for a1 chain was seen in the extracellular matrices, the vitreous chamber and the lens of the eye ŽFig. 2A.. In contrast, no staining was seen in the olfactory epithelium ŽTable 1.. 3.2. Gastrointestinal tract In the unerupted tooth, a1 chain was detected in the basal side of the odontoblast dentine and also as long strands within the dentine ŽFig. 2B.. No other basement membranes or extracellular matrices stained for a1 chain in the unerupted tooth, as shown with double immunofluorescence for laminin a1, b1 and g1 chains ŽFig. 2C. and a1 chain ŽFig. 2D.. Interestingly, the polyclonal antibody against laminin a1, b1 and g1 chains did not show any staining in the basal side of the dentine ŽFig. 2C. although mAb 200 against a1 chain did. In salivary glands, and the epithelium lining the esophagus ŽFig. 3A., ventricle, duodenum, small intestine and colon, no staining for a1 chain was detected. The only other tissue in the gastrointestinal tract, except for the odontoblast extracellular matrix, which stained for a1 chain was one area with glands in the ventricle ŽFig. 3B., probably representing Brunner’s glands ŽTable 1.. 3.3. Endocrine organs With the polyclonal antibody against laminin a1, b1 and g1 chains, staining was seen in basement membranes of the zona glomerulosa of the adrenal gland cortex, while more faint expression was seen in the zona fasciculata and reticularis ŽFig. 3D.. A similar gradient of expression of a1 chain was noted in the cortex with antibody 200 ŽFig. 3C.. In other studied endocrine organs, no staining for a1 chain was seen ŽTable 1..

3.1. Central ner¨ ous system and sensory organs 3.4. Urinary system With a polyclonal antibody detecting a1, b1 and g1 chains, staining was seen in pia mater covering the central nervous system, and smaller and larger blood vessel basement membranes ŽFig. 1A.. In the same area, mAb 200 against a1 chain only stained pia mater ŽFig. 1B.. In areas containing small vessels as well, the antibody against a1, b1 and g1 chains stained basement membranes of both larger and smaller vessels ŽFig. 1C., whereas antibody 200 only stained basement membranes of the larger vessels ŽFig. 1D.. Double immunofluorescence of the larger vessels and

As previously reported, expression of a1, b1 and g1 chains was confined to basement membranes of proximal tubules in the adult kidney ŽEkblom et al., 1990; Sorokin et al., 1992., but other parts of the urinary system were not studied. Although antibodies for a1, b1 and g1 chains stained both the epithelial and endothelial basement membranes in the urinary bladder ŽFig. 3E., very little or no staining for a1 chain was seen in the urinary bladder ŽFig. 3F; Table 1..

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M. Falk et al. r Matrix Biology 18 (1999) 557]568 Table 1 Ž Continued.

3.5. Male and female sexual organs Laminin a1 chain could be detected by immunofluorescence in the epithelial basement memTable 1 Detection of laminin a1 chain in adult mouse tissues by immunofluorescence Organs studied Ner¨ ous system and sensory organs Central nervous system Žnerves, glia. Brain blood vessels Arachnoidea Pia mater Dura mater Eye Sclera Inner and outer limiting membranes Choroid Lining of the vitreous chamber Lining of the lens Lens Olfactory epithelium Peripheral nervous system neurons Schwann cells

Staining with mAb 200 y q y q y y y y q q y y y y

Digesti¨ e system Tooth Dentine of unerupted tooth Other parts of unerupted tooth Submandibular gland Esophagus Ventricle Brunner’s glands Small intestine Large intestine Liver Pancreas, exocrine glands Mesothelium

q y y y y q y y y y y

Endocrine organs Thyroidea Parathyroid Adrenal cortex Adrenal medulla Pancreas, endocrine

y y q y y

Excretory system Kidney Proximal tubules Distal tubules Collecting ducts GBM Bowman’s capsule Ureter Urinary bladder

q y y y y y qry

Male reproducti¨ e system Penis, corpus cavernosum Testis Ductus epididymis Prostate

q q q q

Organs studied

Staining with mAb 200

Female reproducti¨ e system Ovary Uterine tube Uterus Placenta Mammary gland

q y y q q

Cardio¨ ascular system outside brain Heart atrium Heart ventricle Pulmonary artery, valves, aorta Medium-sized arteries and veins Capillaries

y y y y y

Hematopoietic and lymphatic organs Bone marrow Lymph node Thymus Spleen

y y y y

Respiratory system Trachea Lung

y y

Integument Epidermis

Not studied

Bone and cartilage

y

Muscle Smooth muscle Striated muscle Main basement membrane, muscle fibers Myotendinous junction Neuromuscular junction

y y y

Adipose tissue

y

Loose connecti¨ e tissue

y

y

branes of the testis, epididymis ŽFig. 4A. and prostate gland ŽFig. 4B.. In the penis, discontinuous staining was seen in basement membrane of corpus cavernosum ŽFig. 4C.. In the ovary, expression was seen in basement membranes of follicles ŽFig. 4D. at all stages, as reported ŽFrojdman et al., 1995.. In addi¨ tion, staining for a1 chain was seen in epithelial basement membranes of female breast glands ŽTable 1., as reported ŽDurbeej et al., 1998.. 3.6. Other tissues Staining for a1 chain was negative in all other studied tissues ŽTable 1., including cartilage were laminin-1 has been suggested to be present ŽDurr et al., 1996.. Due to difficulties to obtain suitable sections from skin of adult mice, it was not possible with certainty to study a1 chain expression in epidermis,

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Fig. 1. Laminin a1 chain in the adult central nervous system and supporting structures. Immunostaining was performed with polyclonal antibody detecting either a1, b1 or g1 chain ŽA, C., and monoclonal antibody 200 detecting a1 chain ŽB, D., and double immunofluorescence using antibody 200 and polyclonal antibody against von Willebrand factor ŽE, F.. Shown are areas containing parts of pia mater within the brain ŽA, B, F., larger and smaller vessels within the brain ŽC, D., and a single larger vessel within the brain ŽE..

sweat glands or hair follicles. In the studied skin sections, the histology was poor, but no specific staining was seen in any area Ždata not shown.. In suitable sections obtained from penis, no staining was seen in the epidermis.

a 3 and a4 was seen in all three studied tissues ŽFig. 5..

3.7. RT-PCR

The current study defines the location of a1 chain in the adult mouse body and thus possible sites for laminin-1, but the chain associations have to be established in each case. There are other b and g chains and it cannot without experimentation be con-

By RT-PCR of total RNA, no expression of a1 chain mRNA was seen in adult heart or lung, whereas some expression was seen in brain. Expression of a 2,

4. Discussion

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Fig. 2. Laminin a1 chain in the adult eye and unerupted tooth. Immunostaining performed with antibody 200 ŽA, B, D. and with polyclonal antibody detecting either a1, b1 or g1 chain ŽC. shows selective bright staining of a1 chain in extracellular matrices of the vitreous body and lens of the eye ŽA., and in dentine of the unerupted tooth ŽB]D..

cluded that the a1 invariably associates only with b1 and g1. Expression was largely restricted to epithelium, and thus no a1 chain was seen in muscle, fat cells or Schwann cells. Moreover, only a limited set of epithelial basement membranes stained for laminin a1 chain. Prominent expression was noted in proximal tubules of the kidney and in epithelial basement membranes of the ovary and testis, as described previously ŽEkblom et al., 1990; Frojdman et al., 1995.. In ¨ the current study, we found that several other epithelial basement membranes of the reproductory organs both in male and female adult mice expressed the a1 chain. Sites with equally prominent expression, also not described previously, include the lining of the vitreous chamber and lens of the eye, pia mater covering the brain, one basement membrane of the unerupted tooth, larger vessels of the brain, and the cortex of adrenal glands. Interestingly, polyclonal antibodies against a1, b1 and g1 chains did not stain the dentine, suggesting a novel laminin isoform or technical problems due to the unusual composition of this immature dentine. In contrast, several other major epithelial sheets were negative for a1 chain. Thus no staining was seen in lung, epidermis, or peri-

toneum. In the gut and liver, expression was noted only in one type of gland in the ventricle. Although our current description is in complete agreement with our previous suggestions that the a1 chain is found almost exclusively in epithelial basement membranes ŽEkblom et al., 1990, 1998., the extremely limited distribution was nevertheless slightly unexpected. The a1 chain is largely limited to epithelial basement membranes also in the embryo, but the number of different types of epithelial sheets and other tissues expressing is much larger in the embryo ŽKlein et al., 1990; Kucherer-Ehret et al., 1990; Tho¨ mas and Dziadek, 1993, 1994; Schuger et al., 1997. than in the adult body. Lack of a1 chain in adult lung has been noted before with the same antibody used in the present study ŽMiner et al., 1997. and by RNA analyses of human lung tissues and cells ŽPierce et al., 1998. and RT-PCR of adult mouse heart ŽHirohata et al., 1998.. Both immunofluorescence and RT-PCR are extremely sensitive assays to demonstrate expression. Finally, the findings are also in agreement with previous Northern blot studies of adult tissues, which failed to detect a1 chain mRNA from total RNA of several adult mouse tissues ŽKleinman et al., 1987., and we have also failed to detect truncated forms of a1 chain

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Fig. 3. Staining with antibodies detecting laminin a1 chain in adult esophagus ŽA., ventricle ŽB., adrenal gland ŽD. and urinary bladder ŽF., and with antibodies detecting laminin a1, b1 or g1 chains of adrenal gland ŽC. and of urinary bladder ŽE..

from polyAŽq. mRNA from several embryonic tissues using probes covering both the 39 and 59 ends as well as the middle part of the mRNA ŽKlein et al., 1990.. We conclude that maturation of epithelial sheets, with only a few exceptions, is accompanied by a gradual loss of laminin a1 chain from basement membranes, as first noted for collecting ducts of the kidney ŽEkblom et al., 1990.. The apparent complete lack of a1 chain in basement membranes of adult endothelium, except for larger brain vessels, is in agreement with our previous studies of several embryonic tissues, and adult kidney. We have noted expression in embryonic mouse heart

atrium ŽKlein et al., 1990., but not in the adult heart. Lack of a1 chain in most adult endothelial basement membranes is notable since this chain repeatedly has been proposed to be important for angiogenesis and endothelial cell physiology ŽGrant et al., 1990; Malinda et al., 1999.. We therefore carefully analyzed most adult endothelial sheets in the present study. Rapid transitions in the expression pattern of the different laminin a chains occur during embryogenesis in many organs at different developmental stages ŽMiner et al., 1997; Salmivirta et al., 1997; Sorokin et al., 1997a., but possible gradual changes occurring during postnatal maturation stages have not been

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Fig. 4. Laminin a1 chain in reproductive organs of adult mice. Immunostaining was performed with monoclonal antibody 200 of sections containing testis and epididymis ŽA., prostate ŽB., penis ŽC. and the ovary ŽD..

much studied. In one study, it was convincingly shown that the a 5 chain gradually becomes detectable in mouse endothelial basement membranes after birth ŽSorokin et al., 1997a.. Except for liver, embryonic vessels lack the a 5 chain ŽDurbeej et al., 1996., but in the adult stage virtually all endothelial basement membrane were brightly positive when stained with antibodies to a 5 chain ŽSorokin et al., 1997b.. A similar slow appearance of laminin a1 in maturing endothelial basement membranes was thus a distinct

possibility. This is shown here not to be the case. A peculiar expression pattern in the wall of adult mouse brain blood vessel walls was recently reported ŽJucker et al., 1996; Hagg et al., 1997.. We confirm this but we found that expression was limited to some larger blood vessels. This could be conclusively demonstrated by double immunofluorescence comparing expression of b1 and g1 chains of laminin. This does not necessarily mean that endothelial cells produce these chains, since glial cells and invaginated epithe-

Fig. 5. RT-PCR of total mRNA of adult heart, brain and lung with probes detecting mRNA for a1, a 2, a 3 and a4 laminin. When RT was omitted, no signals were detected.

M. Falk et al. r Matrix Biology 18 (1999) 557]568

lial cells are located here also, and the epithelial cells are the likely sources. Lack of a1 chain in adult mouse muscle basement membranes, and their myotendinous and neuromuscular junctions, was demonstrated in the present study, in agreement with data from human adult muscle ŽTiger et al., 1997. and several recent reports of mouse adult muscle ŽPatton et al., 1997; Tiger et al., 1997; Ringelmann et al., 1999.. Lack of a1 chain in basement membranes of mouse embryonic muscle has also been demonstrated by affinity purified polyclonal a1-specific antibodies ŽKlein et al., 1990., and the only exception seems to be faint expression locally in the myotendinous junction during a short period of embryogenesis ŽPatton et al., 1997; Ringelmann et al., 1999.. Our findings are in sharp contrast to some previous views of a broad distribution of a1 chain in several types of basement membranes. The view of widespread expression of a1 chain in endothelium, muscle, epithelia, and Schwann cells in human adult tissues was largely due to the use of monoclonal antibody 4C7 ŽEngvall et al., 1986., which for a long time was marketed as a specific antibody for a1 chain ŽEngvall et al., 1990; Virtanen et al., 1995; Beaulieu, 1997.. However, this antibody detects laminin a 5 chain ŽTiger et al., 1997.. Combined with current knowledge about the broad distribution of laminin a 5 chain in mouse tissues ŽSorokin et al., 1992, 1997a,b; Miner et al., 1997., it seems evident that a 5 chain rather than a1 chain has a widespread distribution in adult tissues in many species. Consequently, it is still unclear where a1 chain is expressed in human adult tissues, except for kidney ŽTiger et al., 1997.. This will be important to clarify. Preliminary studies ŽVirtanen et al., 1998. suggest that the distribution in mouse and human tissues is very similar. The specificity of the used antibody ‘200’ towards the a1 chain has been amply verified in a number of studies, and similar antibodies against the E3 fragment are available ŽSorokin et al., 1992.. Antibody 200 reacts exclusively with the G4 globular domain within the E3 fragment of a1 chain, based on immunoblotting, immunoprecipitation and ELISA assays ŽSorokin et al., 1992, 1997a; Andac et al., 1999.. Polyclonal antibodies against whole E3 fragment from different laboratories have been shown to react in an identical fashion in immunofluorescence both in mouse ŽKlein et al., 1988, 1990. and rat kidney sections ŽDurbeej et al., 1996.. Other similar antibodies against laminin a1 chain are available. Rat monoclonal antibody AL-4 ŽSkubitz et al., 1988; Schuger et al., 1997. also reacts with some region of the globular part of mouse a1 chain. Like polyclonal anti-E3 antibodies ŽKlein et al., 1988. and antibody 200 ŽSorokin et al., 1992; Kadoya et al., 1995., AL-4 has been

565

shown to perturb development of epithelial sheets in embryonic organ cultures ŽSchuger et al., 1997.. Another rat monoclonal antibody 8B3 shows a similar reactivity in kidney ŽAbrahamson et al., 1989. as antibody 200, and was recently used to analyze a1 chain distribution in mice embryos lacking laminin a 5 ŽMiner et al., 1998.. Although the specificity of 8B3 antibody has not yet been fully documented, available data suggest that it also is a suitable antibody for the a1 chain. Since it could be argued that E3 fragment expression does not reflect expression of the complete chain Ždue to possible proteolysis or masking., two other a1-specific monoclonal antibodies ŽE8-1, E8-4. have been raised against the E8 fragment, and they react in embryonic and adult mouse kidney in an identical fashion as monoclonal antibody 200 and polyclonal anti-E3 antibodies ŽSorokin et al., 1997a.. In order to clarify the distribution of a1 chain in human samples, new antibodies against human a1 chain have recently been developed by us. Preliminary studies show that these react in an identical fashion in adult human tissues as monoclonal antibody 200 in mouse ŽVirtanen et al., 1998.. It is well known that mouse laminin-1 strongly stimulates the attachment, metabolism and differentiation of a vast number of different cell types ŽKleinman et al., 1993; Dziadek, 1995; Simon-Assmann et al., 1995; Dunsmore and Rannels, 1996; Ekblom et al., 1996.. Most such studies used embryonic cells or organs, but there is also ample evidence that many adult cell types bind to laminin-1 ŽKleinman et al., 1993; Ferletta and Ekblom 1999.. Thus, adult epithelial, endothelial, nerve, Schwann, endocrine, muscle, and even circulating hematopoietic cells react in a seemingly positive manner to the binding of laminin-1. Several laminin-1 receptors have been identified from these cells ŽHenry and Campbell, 1996; Timpl and Brown, 1996; Talts et al., 1999.. Given the current results, some of the results with cultured adult cell types should be reinterpreted. One possibility is that a chains share similar receptors and that the different a chains have overlapping functions. Another possibility is that the influence of pure mouse laminin-1 on diverse adult cell types is in part mediated by the b1 and g1 chains. In this context, it is important to note that several studies have used blocking antibodies against these chains or their fragments to study function. We therefore emphasize that only those functional studies which used specific probes against the a1 chain can be used as evidence for a role of this chain. In order to suggest a physiological role in vivo, the chain should also be expressed in the suggested site of action. So far, there are no genetic studies of the function of a1 chain in mouse or human cell or tissues. In an attempt to suppress a1 chain mRNA expression in a

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human colon carcinoma cell line Caco-2, anti-sense constructs made from the mouse sequence were transfected ŽDeArcangelis et al., 1996.. The selected cell lines lost cell polarity, and decreased expression of the a1 chain polypeptide was claimed. Unfortunately, polypeptide expression was monitored with the 4C7 antibody, and thus the treatment had apparently indirectly suppressed synthesis of the a 5 chain. No information about expression of a1 chain mRNA or protein was in fact demonstrated ŽDeArcangelis et al., 1996.. Since the untreated Caco-2 cell line produces a truncated a1 chain message of 8 kb rather than a full-length 10 kb, and no a1 protein ŽVelling et al., 1999., there is at present no genetic evidence for a role of a1 chain in human cell lines or tissues. It will therefore be important to test whether lack of laminin a1 chain in mice leads to abnormal embryonic development, or dysfunction in the few organs which in the adult stage express this chain. Based on expression ŽEkblom et al., 1990, 1998. and functional studies of embryonic organs ŽKlein et al., 1988; Schuger et al., 1997. we predict that a main function of laminin a1 chain is to promote development of epithelial sheets in the embryo ŽEkblom, 1996.. Our current study suggests that it might be of some importance for a limited set of basement membranes in the adult stage.

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