Prevention of cardiomyopathy in mouse models lacking the smooth muscle sarcoglycan-sarcospan complex.

Cardiomyopathy is a multifactorial disease, and the dystrophin-glycoprotein complex has been implicated in the pathogenesis of both hereditary and acquired forms of the disease. Using mouse models of cardiomyopathy made by ablating genes for components of the sarcoglycan complex, we show that long-term treatment with verapamil, a calcium channel blocker with vasodilator properties, can alleviate the severe cardiomyopathic phenotype, restoring normal serum levels for cardiac troponin I and normal cardiac muscle morphology. Interruption of verapamil treatment leads again to vascular dysfunction and acute myocardial necrosis, indicating that predilection for cardiomyopathy is a continuing process. In contrast, verapamil did not prevent cardiac muscle pathology in dystrophin-deficient mdx mice, which neither show a disruption of the sarcoglycan complex in vascular smooth muscle nor vascular dysfunction. Hence, our data strongly suggest that pharmacological intervention with verapamil merits investigation as a potential therapeutic option not only for patients with sarcoglycan mutations, but also for patients with idiopathic cardiomyopathy associated with myocardial ischemia not related to atherosclerotic coronary artery disease.

Dystroglycan binding to laminin alpha1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro.

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.

Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.

Limb-girdle muscular dystrophy type 2E (LGMD 2E) is caused by mutations in the beta-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscle. beta-sarcoglycan-deficient (Sgcb-null) mice developed severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. The sarcoglycan-sarcospan and dystroglycan complexes were disrupted in skeletal, cardiac, and smooth muscle membranes. epsilon-sarcoglycan was also reduced in membrane preparations of striated and smooth muscle. Loss of the sarcoglycan-sarcospan complex in vascular smooth muscle resulted in vascular irregularities in heart, diaphragm, and kidneys. Further biochemical characterization suggested the presence of a distinct epsilon-sarcoglycan complex in skeletal muscle that was disrupted in Sgcb-null mice. Thus, perturbation of vascular function together with disruption of the epsilon-sarcoglycan-containing complex represents a novel mechanism in the pathogenesis of LGMD 2E.

epsilon-sarcoglycan replaces alpha-sarcoglycan in smooth muscle to form a unique dystrophin-glycoprotein complex.

The sarcoglycan complex has been well characterized in striated muscle, and defects in its components are associated with muscular dystrophy and cardiomyopathy. Here, we have characterized the smooth muscle sarcoglycan complex. By examination of embryonic muscle lineages and biochemical fractionation studies, we demonstrated that epsilon-sarcoglycan is an integral component of the smooth muscle sarcoglycan complex along with beta- and delta-sarcoglycan. Analysis of genetically defined animal models for muscular dystrophy supported this conclusion. The delta-sarcoglycan-deficient cardiomyopathic hamster and mice deficient in both dystrophin and utrophin showed loss of the smooth muscle sarcoglycan complex, whereas the complex was unaffected in alpha-sarcoglycan null mice in agreement with the finding that alpha-sarcoglycan is not expressed in smooth muscle cells. In the cardiomyopathic hamster, the smooth muscle sarcoglycan complex, containing epsilon-sarcoglycan, was fully restored following intramuscular injection of recombinant delta-sarcoglycan adenovirus. Together, these results demonstrate a tissue-dependent variation in the sarcoglycan complex and show that epsilon-sarcoglycan replaces alpha-sarcoglycan as an integral component of the smooth muscle dystrophin-glycoprotein complex. Our results also suggest a molecular basis for possible differential smooth muscle dysfunction in sarcoglycan-deficient patients.

Biochemical characterization of the epithelial dystroglycan complex.

Dystroglycan is a widely expressed extracellular matrix receptor that plays a critical role in basement membrane formation, epithelial development, and synaptogenesis. Dystroglycan was originally characterized in skeletal muscle as an integral component of the dystrophin glycoprotein complex, which is critical for muscle cell viability. Although the dystroglycan complex has been well characterized in skeletal muscle, there is little information on the structural composition of the dystroglycan complex outside skeletal muscle. Here we have biochemically characterized the dystroglycan complex in lung and kidney. We demonstrate that the presence of sarcoglycans and sarcospan in lung reflects association with dystroglycan in the smooth muscle. The smooth muscle dystroglycan complex in lung, composed of dystroglycan, dystrophin/utrophin, beta-, delta-, epsilon-sarcoglycan, and sarcospan, can be biochemically separated from epithelial dystroglycan, which is not associated with any of the known sarcoglycans or sarcospan. Similarly, dystroglycan in kidney epithelial cells is not associated with any of the sarcoglycans or sarcospan. Thus, our data demonstrate that there are distinct dystroglycan complexes in non-skeletal muscle organs as follows: one from smooth muscle, which is associated with sarcoglycans forming a similar complex as in skeletal muscle, and one from epithelial cells.

Characterization of bone marrow laminins and identification of alpha5-containing laminins as adhesive proteins for multipotent hematopoietic FDCP-Mix cells.

Laminins are extracellular matrix glycoproteins that influence the phenotype and functions of many types of cells. Laminins are heterotrimers composed of alpha, beta, and gamma polypeptides. So far five alpha, three beta, and two gamma polypeptide chains, and 11 variants of laminins have been proposed. Laminins interact in vitro with mature blood cells and malignant hematopoietic cells. Most studies have been performed with laminin-1 (alpha1beta1gamma1), and its expression in bone marrow is unclear. Employing an antiserum reacting with most laminin isoforms, we found laminins widely expressed in mouse bone marrow. However, no laminin alpha1 chain but rather laminin alpha2, alpha4, and alpha5 polypeptides were found in bone marrow. Our data suggest presence of laminin-2 (alpha2beta1gamma1), laminin-8 (alpha4beta1gamma1), and laminin-10 (alpha5beta1gamma1) in bone marrow. Northern blot analysis showed expression of laminin alpha1, alpha2, alpha4, and alpha5 chains in long-term bone marrow cultures, indicating upregulation of laminin alpha1 chain expression in vitro. Laminins containing alpha5 chain, in contrast to laminin-1, were strongly adhesive for multipotent hematopoietic FDCP-mix cells. Integrin alpha6 and beta1 chains mediated this adhesion, as shown by antibody perturbation experiments. Our findings indicate that laminins other than laminin-1 are functional in adhesive interactions in bone marrow.

Dystroglycan in development and disease.

Our understanding of the structure and function of dystroglycan, a cell surface laminin/agrin receptor, has increased dramatically over the past two years. Structural studies, analysis of its binding partners, and targeted gene disruption have all contributed to the elucidation of the biological role of dystroglycan in development and disease. It is now apparent that dystroglycan plays a critical role in the pathogenesis of several muscular dystrophies and serves as a receptor for a human pathogen as well as being involved in early development, organ morphogenesis, and synaptogenesis.

Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice.

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is an autosomal recessive disorder caused by mutations in the alpha-sarcoglycan gene. To determine how alpha-sarcoglycan deficiency leads to muscle fiber degeneration, we generated and analyzed alpha-sarcoglycan- deficient mice. Sgca-null mice developed progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, showed ongoing muscle necrosis with age, a hallmark of the human disease. Sgca-null mice also revealed loss of sarcolemmal integrity, elevated serum levels of muscle enzymes, increased muscle masses, and changes in the generation of absolute force. Molecular analysis of Sgca-null mice demonstrated that the absence of alpha-sarcoglycan resulted in the complete loss of the sarcoglycan complex, sarcospan, and a disruption of alpha-dystroglycan association with membranes. In contrast, no change in the expression of epsilon-sarcoglycan (alpha-sarcoglycan homologue) was observed. Recombinant alpha-sarcoglycan adenovirus injection into Sgca-deficient muscles restored the sarcoglycan complex and sarcospan to the membrane. We propose that the sarcoglycan-sarcospan complex is requisite for stable association of alpha-dystroglycan with the sarcolemma. The Sgca-deficient mice will be a valuable model for elucidating the pathogenesis of sarcoglycan deficient limb-girdle muscular dystrophies and for the development of therapeutic strategies for this disease.

Distribution of dystroglycan in normal adult mouse tissues.

Dystroglycan is a cell surface protein which, in muscle, links the extracellular matrix protein laminin-2 to the intracellular cytoskeleton. Dystroglycan also binds laminin-1 and the binding occurs via the E3 fragment of laminin-1. Recently, it was found that dystroglycan is expressed in developing epithelial cells of the kidney. Moreover, antibodies against dystroglycan can perturb epithelial development in kidney organ culture. Therefore, dystroglycan may be an important receptor for cell-matrix interactions in non-muscle tissues. However, information about the tissue distribution of dystroglycan is limited, especially in adult tissues. Here we show that dystroglycan is present in epithelial cells in several non-muscle organs of adult mice. Dystroglycan is enriched towards the basal side of the epithelial cells that are in close contact with basement membranes. We suggest that dystroglycan is involved in linking basement membranes to epithelial and muscle cells. Dystroglycan may be important for the maintenance of tissue integrity.

Laminin isoforms and epithelial development.

Several different approaches suggest that basement-membrane assembly is important for epithelial development. Basement membranes contain isoforms of collagen IV, proteoglycans, and noncollagenous glycoproteins such as the laminins and nidogens. The expression and role of laminins for epithelial morphogenesis is reviewed. Laminins are large heterotrimeric proteins composed of alpha, beta, and gamma chains. Many major epithelial laminins and their receptors have been identified recently, and the extracellular protein-protein interactions that drive basement-membrane assembly are beginning to be understood. Three laminin alpha-chains are typically made by epithelial, alpha 1, alpha 3, and alpha 5. Three major epithelial heterotrimers can at present be distinguished--laminin-1 (alpha 1 beta 1 gamma 1), laminin-5 (alpha 3 beta 3 gamma 2), and laminin-10 (alpha 5 beta 1 gamma 1)--but other heterotrimers may exist in epithelia. Laminins containing either alpha 1 or alpha 3 chains are largely limited to epithelia, whereas the alpha 5 is also found in endothelial and muscle basement membranes, particularly in the adult. Some epithelial cell types express several laminin alpha-chains, so it is relevant to test how the different laminins affect epithelial cells. Laminins interact with integrin type of receptors on the cell surface, but binding to other proteins has also recently been demonstrated. Two important recent discoveries are the identification of dystroglycan as a major laminin receptor in muscle and epithelia, and nidogen as a high-affinity laminin-binding protein important for basement-membrane assembly. Antibody perturbation experiments suggest that these protein-protein interactions are important for epithelial morphogenesis.

Dystroglycan and laminins: glycoconjugates involved in branching epithelial morphogenesis.

Branching epithelial morphogenesis is crucial for the development of several organs, such as lung, submandibular gland, mammary gland, tooth, pancreas, and kidney. During early embryogenesis, these organs are composed of a small epithelial rudiment surrounded by mesenchymal cells. Interactions between the two tissue compartments induce growth and branching of the epithelium into the mesenchyme. In each tissue, the epithelial branching has tissue-specific features, but there are many similarities both at the morphological and molecular level. Basement membrane components such as laminin have been implicated in the regulation of epithelial morphogenesis. Here data are reviewed that suggest that interactions between laminin-1 and other basement membrane components and the cell surface are important for epithelial morphogenesis in the kidney, lung, and salivary gland.

Transient expression of Dp140, a product of the Duchenne muscular dystrophy locus, during kidney tubulogenesis.

Dystroglycan is a cell surface complex which in muscle links the extracellular matrix protein laminin-2 to the membrane associated cytoskeletal protein dystrophin. Recently it was found that dystroglycan is also expressed in developing epithelial cells. Moreover, antibodies against dystroglycan can perturb epithelial cell development in kidney organ culture. Dystroglycan could provide a link between the basement membrane and the intracellular space also in epithelial cells. However, there is no dystrophin in epithelial cells. By in situ hybridization here we show prominent expression of a shorter isoform of dystrophin, Dp140, in embryonic kidney tubules. In addition, another isoform, Dp71, is expressed by all studied embryonic epithelial cells. Both isoforms share the dystroglycan-binding region of dystrophin but lack the region known to bind to actin. Here we also characterized monoclonal antibodies against different domains of dystrophin and used these to study the distribution of Dp140 protein. In embryonic kidney tubules the dystrophin antibody VIA4(2)A3 stained an intracellular antigen close to the basal cells. In contrast, no staining was observed in adult kidney. We suggest that Dp140 is a structural component during kidney tubulogenesis but it may also be involved in signal transduction.

Differential expression of five laminin alpha (1-5) chains in developing and adult mouse kidney.

The nature of the laminin alpha chains in the embryonic and adult kidney is still being debated. The present study attempted to clarify this issue by immunofluorescence study using monoclonal antibodies against mouse alpha1, alpha2, and alpha5 chains and in situ hybridization for the alpha2, alpha3B, alpha4, and alpha5 mRNAs. Novel alpha1 chain-specific monoclonal antibodies against E8 fragment revealed a restricted distribution of alpha1 chain in a subset of epithelial basement membranes in the embryo, in agreement with previous mRNA data. The alpha2 mRNA was produced by mesenchyme, although the protein was deposited in epithelial basement membranes. The alpha3B mRNA was found only in a small subset of endothelial cells. The alpha4 mRNA was found transiently in embryonic mesenchyme, with particularly high levels in condensed mesenchyme, close to the tips of the ureteric tree where tubulogenesis is initiated. The alpha5 mRNA was strongly expressed by ureter epithelium but not expressed at early stages of tubulogenesis. Immunofluorescence verified low levels of the alpha5 chain in the early stages of tubulogenesis. However, during the capillary loop stage, the alpha5 chain became strongly expressed in the developing glomerular basement membrane, which matches the in situ hybridization results. During subsequent maturation of the kidney, the alpha5 chain became ubiquitously expressed in basement membranes. Overall, the alpha5 chain exhibited the broadest pattern of expression, followed by the alpha1 chain, particularly in the adult stage. These chains were the only ones produced by epithelial cells. Although some basement membranes contained several alpha chains, we failed to detect any of the five studied chains in some basement membranes. Thus, the identity of the alpha chains of many embryonic kidney blood vessels and several basement membranes in the inner medulla in the developing and adult kidney remain unclear.

Distinct alpha 7A beta 1 and alpha 7B beta 1 integrin expression patterns during mouse development: alpha 7A is restricted to skeletal muscle but alpha 7B is expressed in striated muscle, vasculature, and nervous system.

The laminin binding alpha 7 beta 1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of alpha 7 integrin undergoes alternative splicing to generate two major forms, denoted alpha 7A and alpha 7B. In the current paper, we have examined the developmental expression patterns of the alpha 7A and alpha 7B splice variants in the mouse. The alpha 7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin-alpha 1 and laminin-alpha 2 chains. Alpha 7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for alpha 7B integrin and in situ hybridization revealed the presence of alpha 7 mRNA and alpha 7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, alpha 7B integrin was not detectable in the endocardium or myocardium during embryonic and fetal heart development. Northern blot analysis and immunohistochemistry revealed a postnatal induction of alpha 7B in the myocardium. In addition to striated muscle, alpha 7B integrin was localized to previously unreported nonmuscle locations such as a subset of vascular endothelia and restricted sites in the nervous system. Comparison of the alpha 7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, alpha 7 integrin, and laminin-alpha 2, but not laminin-alpha 1, in the forming skeletal muscle. We conclude that the alpha 7A and alpha 7B integrin variants are expressed in a developmentally regulated, tissue-specific pattern suggesting different functions for the two splice forms.

Expression of laminin alpha 1, alpha 5 and beta 2 chains during embryogenesis of the kidney and vasculature.

Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of alpha, beta and gamma chain subunits. To resolve conflicting data in the literature concerning coexpression of alpha 1 and beta 2 chains, expression of alpha 1 chain was studied with two different antisera against the E3 fragment of laminin alpha 1 chain. Expression of the alpha 1 chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, beta 2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, beta 2 chains were detected somewhat earlier, but in situ hybridization revealed that beta 2 chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of alpha 1 and beta 2 chains was mutually exclusive. To explore whether the newly described alpha 5 chain is expressed in locations lacking alpha 1 chain, expression of alpha 5 chain was studied by Northern blots and in situ hybridization. The alpha 5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little alpha 1 chain. There also appeared to be a developmental trend, with alpha 1 chain appearing early and alpha 5 later, in maturing epithelial sheets. The alpha 5 chain could be a major alpha chain of the adult glomerular basement membrane.

Integrin alpha 6B beta 1 is involved in kidney tubulogenesis in vitro.

Laminin-1 has previously been shown to be of major importance for the development of kidney tubules. Antibodies against fragments E8 and E3 of laminin-1 perturb kidney development in vitro. We here studied expression of integrins alpha 6 beta 1 and alpha 6 beta 4, two known laminin receptors, during kidney development. Integrin beta 1 subunit could be detected by immunofluorescence on all cell types of embryonic mouse kidney, but we could not detect integrin beta 4 subunit in embryonic kidney by immunofluorescence or by in situ hybridization. The presence of integrin alpha 6 subunit in all epithelia of embryonic kidney was demonstrated by immunofluorescence and by in situ hybridization. RT-PCR showed that alpha 6B is the major splice variant in embryonic kidney. During in vitro conversion of nephrogenic mesenchyme to epithelial tubules, a strong increase in the expression of the 6 kb mRNA for alpha 6 integrin subunit was seen by northern blotting at the onset of epithelial morphogenesis, on day two of culture. Immunoprecipitation of extracts from embryonic kidney with antibodies against alpha 6 subunit yielded bands corresponding to the expected size of beta 1 integrin subunit but not of beta 4 subunit. Monoclonal antibodies against either alpha 6 or beta 1 subunit but not against E-cadherin blocked kidney tubulogenesis in vitro. This suggests that integrin alpha 6B beta 1 is involved in kidney tubulogenesis in vitro. Another possibility is that the antibodies against integrin alpha 6 and beta 1 subunit cause abnormal signalling by the integrin.

Non-muscle alpha-dystroglycan is involved in epithelial development.

The dystroglycan complex is a transmembrane linkage between the cytoskeleton and the basement membrane in muscle. One of the components of the complex, alpha-dystroglycan binds both laminin of muscle (laminin-2) and agrin of muscle basement membranes. Dystroglycan has been detected in nonmuscle tissues as well, but the physiological role in nonmuscle tissues has remained unknown. Here we show that dystroglycan during mouse development in nonmuscle tissues is expressed in epithelium. In situ hybridization revealed strong expression of dystroglycan mRNA in all studied epithelial sheets, but not in endothelium or mesenchyme. Conversion of mesenchyme to epithelium occurs during kidney development, and the embryonic kidney was used to study the role of alpha-dystroglycan for epithelial differentiation. During in vitro culture of the metanephric mesenchyme, the first morphological signs of epithelial differentiation can be seen on day two. Northern blots revealed a clear increase in dystroglycan mRNA on day two of in vitro development. A similar increase of expression on day two was previously shown for laminin alpha 1 chain. Immunofluorescence showed that dystroglycan is strictly located on the basal side of developing kidney epithelial cells. Monoclonal antibodies known to block binding of alpha-dystroglycan to laminin-1 perturbed development of epithelium in kidney organ culture, whereas control antibodies did not do so. We suggest that the dystroglycan complex acts as a receptor for basement membrane components during epithelial morphogenesis. It is likely that this involves binding of alpha-dystroglycan to E3 fragment of laminin-1.

Evidence for a role of protein phosphatases 1 and 2A during early nephrogenesis.

Although most transcriptional events appear to be modulated by reversible protein phosphorylation, little is known about the role of this regulatory system during the development of mammalian organs. Here we have studied the serine/threonine protein phosphatases (PP) 1 and 2A in the early embryonic rat kidney with regard to expression and effects on growth and differentiation. All isoforms of PP-1 and PP-2A were ubiquitously expressed in 15-day embryonic (E15) kidneys (in situ hybridization studies). In contrast, mRNA for inhibitor-1 (I-1), an endogenous inhibitor of PP-1, was detected only in undifferentiated stem cells in the outer cortical area. I-1 is a novel marker for these cells. The abundance of the PP-1 protein, confirmed with immunoblotting, was high in the embryonic kidney. In organ culture of E13 kidneys, okadaic acid (OA), an exogenous inhibitor of PP-1 and PP-2A, dose-dependently inhibited growth and nephron formation (apparent half-maximal effect at 6 nM). OA 10 nM had little effect on the growth of cultured E15 kidneys, whereas nephron formation was disturbed and morphological evidence of apoptosis was seen. In summary, this study points towards important roles for protein phosphatases 1 and/or 2A in regulation of mitogenic activity in the early embryonic kidney.

Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes.

Branching epithelial morphogenesis requires interactions between the surrounding mesenchyme and the epithelium, as well as interactions between basement membrane components and the epithelium. Embryonic submandibular gland was used to study the roles of two mesenchymal proteins, epimorphin and tenascin-C, as well as the epithelial protein laminin-1 and one of its integrin receptors on branching morphogenesis. Laminin-1 is a heterotrimer composed of an alpha 1 chain and two smaller chains (beta 1 and gamma 1). Immunofluorescence revealed a transient expression of laminin alpha 1 chain in the epithelial basement membrane during early stages of branching morphogenesis. Other laminin-1 chains and alpha 6, beta 1, and beta 4 integrin subunits seemed to be expressed constitutively. Expression of epimorphin, but not tenascin-C, was seen in the mesenchyme during early developmental stages, but a mAb against epimorphin did not perturb branching morphogenesis of this early epithelium. In contrast, inhibition of branching morphogenesis was seen with a mAb against the carboxy terminus of laminin alpha 1 chain, the E3 domain. An inhibition of branching was also seen with a mAb against the integrin alpha 6 subunit. The antibodies against laminin alpha 1 chain and integrin alpha 6 subunit perturbed development in distinct fashions. Whereas treatment with the anti-E3 resulted in discontinuities of the basement membrane at the tips of the branching epithelium, treatment with the mAb against alpha 6 integrin subunit seemed to leave the basement membrane intact. We suggest that the laminin E3 domain is involved in basement membrane formation, whereas alpha 6 beta 1 integrin binding to laminin-1 may elicit differentiation signals to the epithelial cells.