Peter Ekblom

A new nomenclature for the laminins

The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively.

Role of laminin a chain in the development of epithelial cell polarity

Kidney organ culture was used to study the conversion of embryonic mesenchymal cells into a polarized, differentiated kidney epithelium. We examined the expression of laminin, a basement membrane glycoprotein, during this conversion. The B chains of laminin were constitutively expressed, whereas the appearance of the A chain of laminin was dependent on embryonic induction and coincided with the onset of cell polarization. Antisera against the carboxy-terminal end of laminin inhibited polarization but did not affect the developmental events that precede polarization. Antisera against N-terminal parts of laminin failed to inhibit morphogenesis. Since the fragments at the carboxy-terminal end contain parts of the A chain, we suggest that the appearance of this chain is fundamental for initiation of cell polarity.

Transient and locally restricted expression of laminin a chain mRNA by developing epithelial cells during kidney organogenesis

Three polypeptide chains, A, B1, and B2, have been described for mouse laminin, a basement membrane protein. We studied expression of laminin A, B1, and B2 mRNA in the developing mouse kidney. Induction of kidney mesenchyme differentiation in vitro led to an increased expression of B1 and B2 chain mRNA on day 1 of development. In contrast, expression of A chain mRNA increased on day 2, when epithelial cell polarization begins. Laminin A mRNA and polypeptide were expressed only by epithelia during in vivo development as well. Some polarized cell types producing basement membrane (endothelium, some adult epithelia) lacked the A chain mRNA and polypeptide, although they did express B chains. Laminin with the 400 kd A chain is therefore a transient form appearing at specific sites of kidney morphogenesis, whereas isoforms with a different A chain or without it have a more widespread distribution.

Differentiation and vascularization of the metanephric kidney grafted on the chorioallantoic membrane.

The origin and development of mouse kidney vasculature were examined in chorioallantoic grafts of early kidney rudiments and of experimentally induced explants of separated metanephric mesenchymes. Whole kidney rudiments developed into advanced stages, expressed the segment-specific antigenic markers of tubules and the polyanionic coat of the glomeruli. In contrast to development in vitro, these grafts regularly showed glomeruli with an endothelial component and a basement membrane expressing type IV collagen and laminin. The glomerular endothelial cells in these grafts were shown to carry the nuclear structure of the host. This confirms the outside origin of these cells and the true hybrid nature of the glomeruli. When in vitro induced mesenchymes were grafted on chorioallantoic membranes, abundant vascular invasion was regularly found but properly vascularized glomeruli were exceptional. Uninduced, similarly grafted mesenchymal explants remained avascular as did the undifferentiated portions of partially induced mesenchymal blastemas. It is concluded that the stimulation of the host endothelial cells to invade into the differentiating mesenchyme requires the morphogenetic tissue interaction between the ureter bud and the mesenchyme. The induced metanephric cells presumably start to produce chemoattractants for endothelial cells at an early stage of differentiation. Kidney development thus seems to require an orderly, synchronized development of the three cell lineages: the branching ureter, the induced, tubule-forming mesenchyme, and the invading endothelial cells of outside origin.

Expression and biological role of laminin-1

Of the approximately 15 laminin trimers described in mammals, laminin-1 expression seems to be largely limited to epithelial basement membranes. It appears early during epithelial morphogenesis in most tissues of the embryo, and remains present as a major epithelial laminin in some adult tissues. Previous organ culture studies with embryonic tissues have suggested that laminin-1 is important for epithelial development. Recent data using genetically manipulated embryonic stem (ES) cells grown as embryoid bodies provide strong support for the view of a specific role of laminin-1 in epithelial morphogenesis. One common consequence of genetic ablation of FGF signaling, beta1-integrin or laminin gamma1 chain expression in ES cells is the absence of laminin-1, which correlates with failure of BM assembly and epiblast differentiation. Partial but distinct rescue of epiblast differentiation has been achieved in all three mutants by exogenously added laminin-1. Laminin-1 contains several biologically active modules, but several are found in beta1 or gamma1 chains shared by at least 11 laminins. However, the carboxytermini of the alpha chains contain five laminin globular (LG) modules, distinct for each alpha chain. There is increasing evidence for a particular role of alpha1LG4 binding to its receptors for epithelial tubulogenesis. The biological roles of this and other domains of laminin-1 are currently being explored by genetic means. The pathways controlling laminin-1 synthesis have remained largely unknown, but recent advances raise the possibility that laminin-1 and collagen IV synthesis can be regulated by pro-survival kinases of the protein kinase B/Akt family.

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.

Cell-adhesion molecule uvomorulin during kidney development

We studied the expression of a cell adhesion molecule during morphogenesis of the embryonic kidney. The 120-kDa glycoprotein, called uvomorulin, is known to be present on a number of epithelia. During the development of the kidney, a mesenchyme is converted into an epithelium when it is properly induced. The uninduced mesenchyme did not express uvomorulin, as judged by immunofluorescence and immunoblotting using previously characterized antibodies. Uvomorulin does not appear in the mesenchyme as a direct consequence of induction. Rather it becomes detectable approximately 12 hr after completion of induction, at 30-36 hr in vitro when the cells adhere to each other. Distinct differences in uvomorulin expression were seen in the different parts of the nephron. In the mesenchymally derived epithelia (glomeruli, tubules), uvomorulin could be detected only in the tubules, whereas the epithelium of the glomeruli remained negative at all stages of development. Our embryonic studies show that these differences arise very early, as soon as the different parts of the nephron can be distinguished morphologically. It is likely that uvomorulin plays a role in the initial adhesion of the differentiating tubule cells. However, we failed to disrupt histogenesis by applying antibodies to the organ cultures of developing tubules although the antibodies penetrated the tissues well and bound to the differentiating cells.

Laminin isoforms and epithelial development

ABSTRACT: 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 α, β, and γ 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 α‐chains are typically made by epithelial, α1, α3, and α5. Three major epithelial heterotrimers can at present be distinguished‐laminin‐1 (α1β1γ1), laminin‐5 (α3β3γ2), and laminin‐10 (α5β1γ1)‐but other heterotrimers may exist in epithelia. Laminins containing either α1 or α3 chains are largely limited to epithelia, whereas the α5 is also found in endothelial and muscle basement membranes, particularly in the adult. Some epithelial cell types express several laminin α‐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 these protein‐protein interactions are important for epithelial morphogenesis.

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.

Dual origin of glomerular basement membrane.

The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowmans capsule).