Lydia Sorokin

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

The nature of the laminin α 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 α1, α2, and α5 chains and in situ hybridization for the α2, α3B, α4, and α5 mRNAs. Novel α1 chain‐specific monoclonal antibodies against E8 fragment revealed a restricted distribution of α1 chain in a subset of epithelial basement membranes in the embryo, in agreement with previous mRNA data. The α2 mRNA was produced by mesenchyme, although the protein was deposited in epithelial basement membranes. The α3B mRNA was found only in a small subset of endothelial cells. The α4 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 α5 mRNA was strongly expressed by ureter epithelium but not expressed at early stages of tubulogenesis. Immunofluorescence verified low levels of the α5 chain in the early stages of tubulogenesis. However, during the capillary loop stage, the α5 chain became strongly expressed in the developing glomerular basement membrane, which matches the in situ hybridization results. During subsequent maturation of the kidney, the α5 chain became ubiquitously expressed in basement membranes. Overall, the α5 chain exhibited the broadest pattern of expression, followed by the α1 chain, particularly in the adult stage. These chains were the only ones produced by epithelial cells. Although some basement membranes contained several α chains, we failed to detect any of the five studied chains in some basement membranes. Thus, the identity of the α chains of many embryonic kidney blood vessels and several basement membranes in the inner medulla in the developing and adult kidney remain unclear. Dev. Dyn. 1997;210:446–462.

Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development

Rat monoclonal antibodies were raised against fragment E3 of the mouse Engelbreth-Holm-Swarm (EHS) tumor laminin and selected according to their exclusive reaction with laminin A chain by immunoblotting and staining pattern in embryonic kidneys by immunofluorescence. Immunochemical studies of nine purified antibodies showed a comparable reaction with unfragmented laminin and fragment E3 but no cross-reaction with several other, unrelated laminin fragments including the major cell-binding fragment E8. Reduction or pepsin digestion of fragment E3 reduced or abolished antibody binding indicating that most of the epitopes involved are conformation dependent and do not include carbohydrates. They are, however, not identical as shown by different reactivities after proteolytic or chemical cleavage of E3. Four of the antibodies were highly active in inhibiting cell adhesion of the teratocarcinoma cell line F9 and the Schwannoma cell line RN22 on fragment E3 (IC50 approximately 1 microgram/ml), while the others were distinctly less active. No inhibition was observed for cell adhesion on unfragmented laminin, consistent with previous findings that this is largely mediated by binding of fragment E8 to alpha 6 beta 1 integrin. A distinct correlation was observed between cell adhesion inhibition and the inhibition of heparansulfate proteoglycan and heparin binding to fragment E3. Since heparin is not very efficient in inhibiting cell adhesion, it indicates that heparin- and cell-binding sites on fragment E3 are in close proximity but not identical. Two of the antibodies also showed partial inhibition of kidney tubule formation in organ culture of embryonic kidney mesenchyme while the other antibodies were inactive. It suggests some but probably minor involvement of the fragment E3 structure of laminin in this developmental process.

Expression of laminin α1, α5 and β2 chains during embryogenesis of the kidney and vasculature

Abstract Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of α, β and γ chain subunits. To resolve conflicting data in the literature concerning coexpression of α1 and β2 chains, expression of α1 chain was studied with two different antisera against the E3 fragment of laminin α1 chain. Expression of the α1 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, β2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, β2 chains were detected somewhat earlier, but in situ hybridization revealed that β chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of α1 and β2 chains was mutually exclusive. To explore whether the newly described α5 chain is expressed in locations lacking α1 chain, expression of α5 chain was studied by Northern blots and in situ hybridization. The α5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little α1 chain. There also appeared to be a developmental trend, with α1 chain appearing early and α5 later, in maturing epithelial sheets. The α5 chain could be a major α chain of the adult glomerular basement membrane.

Variable clinical phenotype in merosin-deficient congenital muscular dystrophy associated with differential immunolabelling of two fragments of the laminin α2 chain

Approximately half the cases of classical congenital muscular dystrophy (CMD) have a pronounced deficiency or absence of the laminin alpha 2 chain of laminin-2 (merosin). This is caused by mutations in the LAMA2 gene that codes for laminin alpha 2, and all informative cases so far studied show linkage to the appropriate region on chromosome 6q. Most CMD patients with a deficiency of laminin alpha 2 have a severe phenotype that involves skeletal muscle, and the central and peripheral nervous system. We have identified four cases that have minimal reduction of laminin alpha 2 using a commercial antibody that only recognises a C-terminal 80 kDa fragment, but show a pronounced reduction using an antibody to the 300 kDa fragment. Haplotype analysis is compatible with linkage to the LAMA2 locus in three informative families, whilst the fourth family was not informative. Two of the affected children are ambulant and have a mild phenotype. The third case is unusual in having severe muscle weakness but does not show the white matter changes on magnetic resonance imaging of the brain that is usually seen in merosin-deficient cases of CMD; the fourth case has a severe phenotype, typical of merosin-deficient patients but shows good immunolabelling of the 80 kDa fragment of laminin alpha 2, corresponding to the C-terminal region. Our data show that there is a broad spectrum of phenotype and protein expression associated with a primary deficiency in laminin alpha 2, and that a wider range of clinical cases need to be screened for a deficiency of merosin. It is also important to study the expression of laminin alpha 2 with more than one antibody.

Secondary reduction of α7B integrin in laminin α2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The α7β1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the α7 integrin are laminin-1 (α1-β1-γ1), laminin-2 (α2-β1-γ1) and laminin-4 (α2-β2-γ1). Targeted deletion of the gene for the α7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the α7B and β1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the α7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the β1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of α7B integrin was significantly reduced at the sarcolemma in six patients with laminin α2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin α2 chain expressed. In contrast, the expression of the laminin α2 chain was not altered in the skeletal muscle of the α7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the α7 integrin subunit and that of the laminin α2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of α7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the β1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of β1D integrin was significantly reduced in the α7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of α7B in laminin α2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in α7 deficient mice, implies that the α7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Distinct α7Aβ1 and α7Bβ1 integrin expression patterns during mouse development: α7A is restricted to skeletal muscle but α7B is expressed in striated muscle, vasculature, and nervous system

The laminin binding α7β1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of α7 integrin undergoes alternative splicing to generate two major forms, denoted α7A and α7B. In the current paper, we have examined the developmental expression patterns of the α7A and α7B splice variants in the mouse. The α7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin‐α1 and laminin‐α2 chains. α7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for α7B integrin and in situ hybridization revealed the presence of α7 mRNA and α7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, α7B 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 α7B in the myocardium. In addition to striated muscle, α7B 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 α7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, α7 integrin, and laminin‐α2, but not laminin‐α1, in the forming skeletal muscle. We conclude that the α7A and α7B integrin variants are expressed in a developmentally regulated, tissue‐specific pattern suggesting different functions for the two splice forms.

Laminin α1 chain G domain peptide, RKRLQVQLSIRT, inhibits epithelial branching morphogenesis of cultured embryonic mouse submandibular gland

Active sequences from the laminin α1 and α2 chain carboxyl‐terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583–20590; Nomizu et al. [1996] FEBS Lett. 396:37–42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin α1 chain peptide, RKRLQVQLSIRT (residues 2719–2730 and designated AG‐73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin α1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG‐73, a homologue of AG‐73 from the laminin α2 chain, did not inhibit the branching. The α2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin α2 chain‐specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E‐cadherin and α6 integrin, as detected by immunofluorescence, were unchanged in both AG‐73 and control scramble peptide‐treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG‐73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG‐73 sequence of the laminin α1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG‐73‐treated explants may indicate an important role for this region in basement membrane assembly. Dev. Dyn. 1998;212:394–402.

Differential expression of laminin α chains during murine tooth development

Basement membranes of the developing tooth have been previously shown to contain laminins, but the nature of the laminins have not been described. We here studied the distribution of five different laminin α chains during tooth development. We show that both epithelial and mesenchymal cells produce laminin α chains. The mRNAs of three laminin α chains, α1, α2, and α4, were expressed in the tooth mesenchyme, whereas two, the α3 and α5 chain mRNAs, were found in epithelium. Drastic changes in the expression patterns of the two epithelial chains were found during development. The α5 mRNA was widely expressed in tooth epithelia, and the corresponding protein was evenly distributed along the tooth basement membrane throughout embryonic development. This suggests a role for α5 as a major laminin α chain in tooth basement membrane during embryonic stages. The subsequent disappearance of α5 and the drastic increase in α3A mRNA expression during terminal ameloblast differentiation and enamel secretion suggest that α3A acts as an important chain in the enamel matrix after degradation of tooth basement membrane. These studies show that laminin networks in tooth epithelia form as a result of epithelial–mesenchymal interactions and that the molecular composition of the laminin networks varies drastically during development of tooth. Dev. Dyn. 1997;210:206–215.

Development of kidney epithelial cells

Epithelial cells constitute the basic functional units of the kidney. The renal epithelia are responsible for the selective transport either into or out of the tubular fluid. The proper execution of these vectorial transport processes depends on the polar nature of the epithelial cells. The functional importance of the polar nature of the epithelial cells is well illustrated in the kidney but has considerably broader physiological significance, as reviewed in other chapters of this book. In view of the significant roles of epithelial cells in the body, it is of primary importance to investigate the molecular basis of epithelial cell polarization. The developing tubular and glomerular epithelium of the kidney are particularly interesting cell types for such studies because they develop during embryogenesis from non-polarized mesenchymal cells. We will here review current knowledge suggesting that cell—matrix interactions could be important for the development of the kidney tubules and the glomerular epithelium.