Developmentally regulated conversion of mesenchyme to epithelium

Polarized epithelial cells perform many critical physiological functions in multicellular organisms. Recent embryological studies of the conversion of nonpolar mesenchymal cells to epithelium in the developing mouse kidney have provided vital information on the molecular mechanisms that initiate epithelial cell polarization. To become polar, the cells first attach to the basement membrane that is produced by the developing epithelial cells themselves. Of the basement membrane components, laminin has a key role in the development of epithelial cell polarity. Laminin is a multidomain glycoprotein composed of three subunits: A, B1, and B2. One binding site for epithelial cells is found in the carboxyl-terminal part of the A chain of laminin. Antibodies reacting with this part of laminin inhibit polarization of developing epithelial cells in organ cultures of embryonic kidneys. Expression studies also suggest that the A chain of laminin is important for epithelial cell polarization; the A chain appears when the cells begin to polarize, whereas B chains are expressed at an earlier stage of development. The studies of conversion of mesenchyme to epithelium suggest that morphogenesis can be controlled by differential expression of laminin chains.     

Formation of basement membranes in the embryonic kidney: an immunohistological study

Specific antibodies to laminin, type IV collagen, basement-membrane proteoglycan, and fibronectin have been used in immunofluorescence microscopy to study the development of basement membranes of the embryonic kidney. Kidney tubules are known to form from the nephrogenic mesenchyme as a result of an inductive tissue interaction. This involves a change in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses fibronectin but no detectable laminin, type IV collagen, or basement-membrane proteoglycan. During the inductive interaction, basement-membrane specific components (laminin, type IV collagen, basement membrane proteoglycan) become detectable in the induced area, whereas fibronectin is lost. While the differentiation to epithelial cells of the kidney requires an inductive interaction, the development of the vasculature seems to involve an ingrowth of cells which throughout development deposits basement-membrane specific components, as well as fibronectin. These cells form the endothelium and possibly also the mesangium of the glomerulus, and contribute to the formation of the glomerular basement membrane. An analysis of differentiation of the kidney mesenchyme in vitro in the absence of circulation supports these conclusions. Because a continuity with vasculature is required for glomerular endothelial cell differentiation, it is possible that these cells are derived from outside vasculature.     

Early innervation of the metanephric kidney

During kidney differentiation, the nephrogenic mesenchyme converts into renal tubules and the ureter bud branches to form the collecting system. Here we show that in the early undifferentiated kidney rudiment there is a third cell type present. In whole-mount preparations of cultured undifferentiated metanephric kidneys, neurones can be detected by immunohistochemical means with antibodies against the neurofilament triplet, 13AA8, and against neuronal cell surface gangliosides, Q211. Clusters of neuronal cell bodies can be seen in the mesenchyme close to the ureter bud. The terminal endings of neurites are found around the mesenchymal condensates that later become kidney tubules. A similar distribution of neurites can be revealed in tissue sections of kidney grafts growing in the chicken chorioallantoic membranes. In primary cultures of the ureter bud cells, neurones are constantly present. In another report, we have shown that, in experimental conditions, neurones are involved in regulation of kidney morphogenesis. The present results raise the possibility that neurones of the metanephric kidney may have this function in vivo as well.     

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.     

Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine.

Laminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin alpha2, alpha4, and alpha5 chains in the developing mouse intestine and in in vitro mouse/chick or chick/mouse interspecies hybrid intestines. In situ hybridization and Northern blot analysis revealed that mRNA levels for all three laminin alpha chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin alpha4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin alpha2 and alpha5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin alpha chains occur in the smooth muscle basement membrane, with a differential expression of laminin alpha5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin alpha2 and alpha5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin alpha2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin alpha5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin alpha chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane.     

Cellular origin of fibronectin in interspecies hybrid kidneys

The cellular origin of fibronectin in the kidney was studied in three experimental models. Immunohistochemical techniques that use cross-reacting or species-specific antibodies against mouse or chicken fibronectin were employed. In the first model studied, initially avascular mouse kidneys cultured on avian chorioallantoic membranes differentiate into epithelial kidney tubules and become vascularized by chorioallantoic vessels. Subsequently, hybrid glomeruli composed of mouse podocytes and avian endothelial-mesangial cells form. In immunohistochemical studies, cross-reacting antibodies to fibronectin stained vascular walls, tubular basement membranes, interstitium, and glomeruli of mouse kidney grafts. The species-specific antibodies reacting only with mouse fibronectin stained interstitial areas and tubular basement membranes, but showed no reaction with hybrid glomeruli and avian vascular walls. In contrast, species-specific antibodies against chicken fibronectin stained both the interstitial areas and the vascular walls as well as the endothelial-mesangial areas of the hybrid glomeruli, but did not stain the mouse-derived epithelial structures of the kidneys. In the second model, embryonic kidneys cultured under avascular conditions in vitro develop glomerular tufts, which are devoid of endothelial cells. These explants showed fluorescence staining for fibronectin only in tubular basement membranes and in interstitium. The avascular, purely epithelial glomerular bodies remained unstained. Finally, in outgrowths of separated embryonic glomeruli, the cross-reacting fibronectin antibodies revealed two populations of cells: one devoid of fibronectin and another expressing fibronectin in strong fibrillar and granular patterns. These results favor the idea that the main endogenous cellular sources for fibronectin in the embryonic kidney are the interstitial and vascular cells. All experiments presented here suggest that fibronectin is not synthesized by glomerular epithelial cells in vivo.     

Analysis of the assembly of laminin and the laminin-entactin complex with laminin chain specific monoclonal and polyclonal antibodies

Antibodies specific for the A, B1, and B2 chains of laminin have been obtained and characterized. Lam V, a rat X mouse monoclonal antibody, was obtained by immunizing Lewis rats with the extracellular matrix derived from the mouse endodermal line M1536-B3. The antibody was shown to recognize a conformation-sensitive epitope present on the A chain of laminin. The antibody exhibited high avidity for native laminin and uncomplexed newly synthesized laminin A chains. cDNA clones in the vector lambda-gt11 containing sequences for the B1 and B2 chains of laminin were shown to synthesize beta-galactosidase fusion proteins in the host cells induced with IPTG. The fusion protein F3 contained amino acid residues 822-1765 of the B1 chain of mouse laminin, and the fusion protein E4 contained 219 amino acids at the carboxyl terminus of the B2 chain of rat laminin. These two fusion proteins were used to obtain rabbit polyclonal antibodies which were characterized for their specificity and ability to immunoprecipitate laminin and the B chains of laminin. The chain-specific antibodies were used to analyze the assembly and processing of laminin in the mouse endodermal cell line M1536-B3. The results indicated that the covalent assembly of the A and B chains of laminin was initiated as early as 3 min after labeling cells. At this time point uncomplexed A chain of laminin could be observed even though there was an excess of B1 and B2 chains. As early as 4 min after labeling monomeric, dimeric, and oligomeric forms of the B chains of laminin were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition of extracellular matrix components by neonatal and adult cardiac myocytes

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, Bowman's capsule).     

Mouse heart laminin. Purification of the native protein and structural comparison with Engelbreth-Holm-Swarm tumor laminin

Laminin was selectively extracted from different mouse tissues using EDTA-containing buffer. By immunoblotting with an antiserum raised against mouse Engelbreth-Holm-Swarm (EHS) tumor laminin, such extracts could be shown to contain laminin-like molecules with a low apparent proportion of A chain to B chains. Native laminin was purified from mouse heart tissue and was shown to have an aberrant polypeptide composition as compared to mouse EHS tumor laminin. Most prominently, mouse heart laminin contains an Mr 300,000 polypeptide which is not antigenically related to the A or the B chains. Furthermore, nonreducible polypeptide components were seen with apparent Mr values of 600,000 and 900,000. The Mr 600,000 component contains epitopes shared with both EHS tumor laminin and the Mr 300,000 polypeptide and possibly represents a covalently cross-linked complex of an A or B chain with the Mr 300,000 chain.     

Genes for basement membrane proteins are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues

We have obtained cDNA clones coding for the A, B1, and B2 chains of laminin by screening a cDNA library prepared from mouse EHS tumor poly(A)RNA in the lambda gt11 expression vector with polyclonal antibody against denatured laminin. These cDNA clones were used in combination with a cDNA clone coding for the alpha 1 type IV collagen chain to study the regulation of genes for these basement membrane proteins in retinoic acid-induced differentiating mouse F9 teratocarcinoma cells and in various adult murine tissues. The levels of mRNA for the laminin A, B1, and B2 chains and for the alpha 1 type IV collagen chain were increased simultaneously and reached a maximum at almost the same time during the differentiation of F9 cells, suggesting coordinate expression in these cells. The tissue levels of mRNA encoding for the basement membrane components, however, varied considerably. The highest level of the B1 chain mRNA was observed in kidney, whereas, the levels of mRNA for A and B2 chains were highest in heart. Almost the same levels of expression of the alpha 1(IV) collagen mRNA were found in kidney, lung, and heart. The results indicate that the expression of genes for the basement membrane proteins is not coordinately regulated in these tissues. It is thus possible that different subunit structures of the laminin molecule may exist in tissues.     

Extracellular matrix and capillary ingrowth in interspecies chimeric kidneys

The migration of capillaries into mouse embryonic kidneys grafted on quail chorioallantoic membrane (CAM) was analyzed by two monoclonal antibodies against quail endothelial and haematopoietic cells. As shown by immunohistochemistry, the quail chorioallantoic vessels invaded the kidney explant. Initially, the capillaries were detected in the interstitial stroma and, soon thereafter, tightly adjacent to the branches of the ureteric bud. The induced mesenchymal cell condensates, the prospective nephric vesicles, were avascular, but when the early S-shaped body was formed, the capillaries invaded its lower crevice. Finally chimeric glomeruli consisting of mouse podocytes and quail endothelial cells, were formed and, contemporarily, the capillaries ceased to migrate. Within the endothelial-mesangial area of the chimeric glomeruli, all cells expressed the quail-type nuclear structure and were stained by the quail endothelial-specific antibodies. The pattern of migrating capillaries was compared to the distribution of the extracellular matrix (ECM) molecules by double staining with polyclonal antibodies against laminin or fibronectin, and monoclonal quail endothelial-specific antibodies. Initially, the capillaries migrated in a fibronectin-rich matrix, devoid of laminin, but when the epithelial kidney tubules formed, some capillaries attached to the newly formed epithelial basement membrane. At no stage were the capillaries seen to penetrate the epithelial basement membrane. The orderly branching of the ureteric bud, followed by the formation of nephrons and the shift in the ECM, might create pathways for an oriented capillary migration. The fibronectin-rich areas could be a scaffold for the capillary migration, and the attachment to the basement membranes a means for their cessation.     

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.     

In situ hybridization reveals temporal and spatial changes in cellular expression of mRNA for a laminin receptor, laminin, and basement membrane (type IV) collagen in the developing kidney

The appearance of extracellular matrix molecules and their receptors represent key events in the differentiation of cells of the kidney. Steady-state mRNA levels for a laminin receptor, the laminin B1, B2, and A chains, and the alpha 1-chain of collagen IV (alpha 1[IV]), were examined in mouse kidneys at 16 d gestation and birth, when cell differentiation is active, and 1-3 wk after birth when this activity has subsided. Northern analysis revealed that mRNA expression of laminin receptor precedes the alpha 1(IV) and laminin B chains whereas laminin A chain mRNA expression was very low. In situ hybridization reflected this pattern and revealed the cells responsible for expression. At 16 d gestation, laminin receptor mRNA was elevated in cells of newly forming glomeruli and proximal and distal tubules of the nephrogenic zone located in the kidney cortex. These cells also expressed mRNA for alpha 1(IV) and laminin chains. At birth, mRNA expression of receptor and all chains remained high in glomeruli but was reduced in proximal and distal tubules. At 1 wk after birth, expression was located in the medulla over collecting ducts and loops of Henle. Little expression was detectable by 3 wk. These results suggest that cellular expression of steady-state mRNA for laminin receptor, laminin, and collagen IV is temporally linked, with laminin receptor expression proceeding first and thereafter subsiding.     

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.     

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.     

The extracellular matrix glycoproteins BM-90 and tenascin are expressed in the mesenchyme at sites of endothelial-mesenchymal conversion in the embryonic mouse heart

Abstract. BM-90 is a novel glycoprotein initially isolated from the extracellular matrix of a mouse tumor. We here studied the expression of BM-90 during embryonic development of the mouse heart and compared its expression pattern with that of tenascin and laminin. Distribution was studied by immunofluorescence using antibodies specifically raised against mouse BM-90, laminin and tenascin. Some expression of BM-90 was seen in myocardial basement membranes at early developmental stages, but expression abruptly decreased from these sites at day 12 of embryogenesis. Laminin B chains were also found in the muscle basement membranes early but did not decrease with advancing development. The most striking observation was the markedly enriched expression of BM-90 in the endocardial cushion tissue (ECT). The ECT is derived from mesenchymal cells converted from endothelium and they will form the cardiac valves and septa. In the ECT, BM-90 showed considerable co-distribution with tenascin, but tenascin expression was more focal and did not mark all areas of the ECT. Northern blot data show that BM-90 and tenascin were produced by the developing heart. With antibodies detecting A, B1 and B2 chains of mouse laminin, no immunoreactivity was seen in the ECT. Our data thus show clear-cut differences in the molecular composition of the ECT and muscle basement membranes in the developing heart. The focal expression of BM-90 in the ECT suggests that BM-90 could be involved in epithelial-mesenchymal transitions.     

Tenascin during gut development: appearance in the mesenchyme, shift in molecular forms, and dependence on epithelial-mesenchymal interactions [published erratum appears in J Cell Biol 1989 Mar;108(3):following 1175]

Tenascin, an extracellular matrix protein, is expressed in the mesenchyme around growing epithelia in the embryo. We therefore investigated whether epithelial cells can stimulate expression of tenascin in embryonic mesenchyme. Mesenchyme from the presumptive small intestine was used because it is known that reciprocal epithelial- mesenchymal interactions are important for gut morphogenesis. Rat monoclonal antibodies against mouse tenascin were raised and were found to react specifically with mouse tenascin in ELISA. In supernatants of cultured fibroblasts, the antibodies precipitated two peptides of Mr 260 and 210 kD. One of the antibodies also reacted with these tenascin chains in immunoblots of tissue extracts. We found that tenascin was absent during early stages of gut development, at stages when the mesenchyme is already in contact with the stratified epithelium of the endoderm. Rather, it appeared in the mesenchyme when the homogenous endodermal epithelium differentiated into the heterogenous absorptive epithelium. Tenascin remained present in the stroma of the adult gut, close to the migration pathways of the continuously renewing epithelium. When first detected during intestinal differentiation, the 210-kD component was predominant but at birth the relative amount of the 260-kD component had increased. The expression data suggested that the appearance of tenascin in the mesenchyme was dependent on the presence of epithelium. To test this, isolated gut mesenchymes from 13- d-old mouse embryos were cultured for 24 h either alone or together with epithelial and nonepithelial cells. Whereas mesenchyme cultured alone or in the presence of nonepithelial B16-F1 melanoma cells produced only trace amounts of tenascin, expression was strongly stimulated by the epithelial cell line, Madin-Darby canine kidney (MDCK). We propose that growing and differentiating epithelia produce locally active factors which stimulate synthesis of tenascin in the surrounding mesenchyme.     

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.