The turnover of basal lamina glycosaminoglycan correlates with epithelial morphogenesis

The mouse embryonic submandibular epithelium begins as a single bud from the floor of the mouth which, under the influence of its surrounding mesenchyme, grows and forms lobules that subsequently branch repetitively. The lobular morphology of the 13-day epithelium is maintained by its basal lamina which is a continuous layer on the interlobular clefts but is interrupted on the distal aspects of the lobules. The structural integrity of this lamina is dependent upon its glycosaminoglycan (GAG) which, by histochemistry, is more abundant on the interlobular clefts than on the distal lobules. We have investigated the basis for these regional differences in the lamina by examining the synthesis and degradation of total GAG as well as the accumulation and loss of laminar GAG during the morphogenesis of the 13-day gland. Autoradiography and histochemistry show that laminar GAG is rapidly turning over. Although it is relatively stable in the interlobular clefts, GAG is rapidly degraded on the distal lobules. This difference can account for the regional variation in basal laminar GAG accumulation. The results of incorporation kinetics and precursor pool specific activities of total epithelial GAG show that the rate of GAG synthesis is greater than its rate of degradation in the base of the lobules, which includes the interlobular clefts. In contrast, during morphogenesis, the rate of GAG degradation becomes greater than its rate of replacement in the distal lobules. The epithelial stalk appears to be in the steady state regarding GAG metabolism. We propose (a) that the rapid laminar GAG degradation on the distal lobules produces the interruptions in the lamina, allowing epithelial growth and expansion, and (b) that the metabolic stability of laminar GAG on the interlobular clefts maintains the integrity of this lamina which serves as a cellular constraint. The results are consistent with a model for epithelial morphogenesis in which the mesenchyme remodels the lamina, which in turn, dictates epithelial morphology. Regulation of basal lamina turnover may be a general mechanism for controlling the behavior of epithelial cell populations.     

Mesenchyme cells degrade epithelial basal lamina glycosaminoglycan

We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGs are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.     

Basal lamina of embryonic salivary epithelia. Production by the epithelium and role in maintaining lobular morphology

The role of the basal lamina in maintaining the normal morphology of mouse embryo submandibular epithelia was assessed by examining its production as well as the cellular and organ culture changes associated with its removal and replacement. The lamina was removed from epithelia isolated free of mesenchyme by brief treatment with testicular hyaluronidase in the absence of calcium. The treatment causes rounding- up of the cells, loss of cellular cohesion, appearance of microvilli, and changes in the organization of cytoskeletal structures. The lamina is not removed and the cellular alterations do not occur in the absence of hyaluronidase in calcium-free medium or when both enzyme and calcium are present, possibly because digestion of chondroitin sulfate, a component of the lamina, is inhibited by calcium. Within 2 h after treatment, in the absence of mesenchyme or biological substrata, the epithelia deposits a new lamina, which is identical by several criteria to the preexisting lamina, and reverses the cellular alterations. Epithelia treated with hyaluronidase lose lobular morphology during culture with mesenchyme. Delaying culture with mesenchyme, to allow restoration of the lamina and of normal cellular architecture, prevents the loss of lobular morphology. The results indicate that the basal lamina imposes morphologic stability on the epithelium, while the mesenchyme apparently affects processes involved in changes in morphology, possibly by selective degradation of the basal lamina.     

The basal lamina of the postnatal mammary epithelium contains glycosaminoglycans in a precise ultrastructural organization

The mammary epithelium was investigated to determine whether glycosaminoglycans (GAG) are components of the basal lamina of epithelia undergoing postnatal morphogenesis. Isolated epithelial tissues from midpregnant mice produce substantial amounts of GAG, consisting predominantly of hyaluronic acid and heparan sulfate. The basal surfaces of mammary epithelia at various postnatal developmental stages show GAG, as demonstrated by histochemistry and by autoradiography coupled with enzyme susceptibility. Electron microscopy using ruthenium red staining reveals polyanionic components, presumably GAG, within the epithelial basal lamina. Detailed ultrastructural analyses of tannic acid-treated and ruthenium red-stained material demonstrate that the lamina contains a two-dimensional symmetrical array of tetragonally ordered components colsely associated with the basal plasma membrane. This array is similar to that found in the hyaluronate-containing lamina of embryonic epithelia. A structurally ordered complex of GAG-containing macromolecules may characterize the basal lamina of all epithelia which undergo morphogenetic changes in cell shape.     

Significant role of laminin-1 in branching morphogenesis of mouse salivary epithelium cultured in basement membrane matrix

Mouse submandibular epithelium shows branching morphogenesis in mesenchyme-free conditions when covered with a basement membrane matrix (Matrigel) in medium supplemented with epidermal growth factor. In the present study, the role of laminin-1 (LN1), a major glycoprotein of Matrigel, in this culture system was defined. When the epithelium was cultured in a LN1-nidogen gel, the epithelium showed much branching, comparable to that observed with Matrigel. By electron microscopy, only a felt-like matrix was formed on the epithelial surface in the LN1-nidogen gel cultures, while an organized basal lamina structure was formed on the epithelial surface in direct or transfilter recombination cultures with mesenchyme. Next, the epithelium covered with Matrigel was cultured in medium containing either biologically active peptides from LN1, IKVAV-including peptide (2097-2108), AG10 (2183-2194), AG32 (2370-2381) or AG73 (2719-2730) from the alpha1 chain, or YIGSR-including peptide (926-933) from the beta1 chain. Only AG73 (RKRLQVQLSIRT from the alpha1 chain carboxyl-terminal globular domain) inhibited the epithelial branching in Matrigel. These results suggest that LN1-nidogen can support the branching morphogenesis of submandibular epithelium even if LN1-nidogen is not assembled into an intact basal lamina, and that the AG73 sequence is an important site on LN1, which interacts with submandibular epithelial cells.     

Antibodies to cell surface ganglioside GD3 perturb inductive epithelial-mesenchymal interactions

Most epithelial sheets emerge during embryogenesis by a branching and growth of the epithelium. The surrounding mesenchyme is crucial for this process. We report that branching morphogenesis and the formation of a new epithelium from the mesenchyme in the embryonic kidney can be blocked by a monoclonal antibody reacting with a surface glycolipid, disialoganglioside GD3. In contrast, a more than 10-fold excess of antibodies to adhesive glycoproteins (N-CAM, L-CAM, fibronectin) fails to inhibit morphogenesis. Although the anti-GD3 antibody affected epithelial development, the disialoganglioside GD3 was expressed not in the epithelium, but in the mesenchyme surrounding the developing epithelia. The data raise the intriguing possibility that the anti-GD3 antibody inhibits epithelial development by interfering with epithelial-mesenchymal interactions.     

ACID MUCOPOLYSACCHARIDE (GLYCOSAMINOGLYCAN) AT THE EPITHELIAL-MESENCHYMAL INTERFACE OF MOUSE EMBRYO SALIVARY GLANDS

Acid mucopolysaccharide (glycosaminoglycan) has been demostrated at the epithelial-mesenchymal interface of mouse embryo submandibular glands by (a) specific staining for polymeric sulfate with Alcian blue 8 GX at various magnesium concentrations, (b) specific staining for polymeric uronic acid by selective oxidation of these residues to Schiff-reactive compounds, (c) electron microscope localization of ruthenium red staining, (d) radioautographic localization of glucosamine-³H and ³⁵SO₄, and (e) by susceptibility of the glucosamine radioactivity at the interface to digestion with protease-free hyaluronidase. Moreover, material labeled with glucosamine-³H and ³⁵SO₄ and with chemical characteristics identical with those of acid mucopolysaccharide were isolated from the glands. Acid mucopolysaccharide is distributed over the entire epithelial surface. The amount of acid mucopolysaccharide, as revealed by the staining procedures, is nearly equivalent at all sites. In contrast, the rate of accumulation of glucosamine-labeled mucopolysaccharide is greater at the surface of the distal ends of the growing and branching lobules. This distribution of newly synthesized acid mucopolysaccharide at the sites of incipient cleft formation suggests that surface-associated acid mucopolysaccharide is involved in the morphogenetic process. A mechanism of branching morphogenesis is proposed which accounts for the distribution of collagen fibers and total and newly synthesized acid mucopolysaccharide at the epithelial surface.     

Epithelial shape change in mouse embryonic submandibular gland: modulation by extracellular matrix components

Early morphogenesis of mouse submandibular gland provides an excellent model for the formation of epithelial lobules as a consequence of epithelial-mesenchymal interactions. Both proteoglycans and a glycosaminoglycan, high molecular weight components which contain amino-sugars and hexuronic acids, seem to be important in maintaining the lobular structure through the formation of epithelial basal lamina. Collagen also appears to play a crucial role in this morphogenesis. By visualizing the distribution of collagen fibrils and by changing the concentration of collagen in the gland, we have developed a new hypothesis which emphasizes the mechanical role of mesenchyme in epithelial cleft formation. Precise mechanisms for the involvement of these molecules have not been elucidated, yet it is now clear that knowledge of the function of the extracellular matrix components is a prerequisite for understanding the epithelial-mesenchymal interactions.     

Lung organoid culture

An organoid culture system for lung cells is described in which morphogenesis of lung histotypic structures and differentiation of both pneumocytes type II and mesenchyme occur. The principle of this technique is the culture of mouse fetal lung cells at high density on a membrane filter at the medium/air interface. In the course of cultivation, cell sorting-out, epithelial cell aggregation, formation of an alveolar-like lumen in the organoids and formation of a basal lamina occur. Epithelial differentiation culminates in the production of lamellar bodies, and the mesenchyme develops into mature connective tissue. Morphogenesis and differentiation depend on the stage of fetal development from which the lung cells were derived but appear independent of the formation of a basal lamina. Various drugs have been tested for their effects on morphogenesis and differentiation in this lung organoid culture: some of them inhibit differentiation or damage the mesenchyme, others stimulate surfactant production. Due to the quite complex morphogenetic and cellular events occurring in lung organoid culture, it may be an applicable tool for alternative in vitro screening methods.     

Basal lamina of embryonic salivary epithelia. Nature of glycosaminoglycan and organization of extracellular materials

The ultrastructural organization and the composition of newly synthesized glycosaminoglycan (GAG) in the epithelial basal lamina of mouse embryo submandibular glands were assessed. The labeled GAG accumulating in the lamina is distinct from that in its tissue of origin, the epithelium, or from that in the surrounding mesenchyme. In the lamina, hyaluronic acid accounts for approximately 50% of the labeled GAG, chondroitin-4-sulfate is twice the chondroitin-6-sulfate, and there is a low proportion of chondroitin. This composition is constant regardless of whether the lamina is labeled by whole glands or, in the absence of mesenchyme, by isolated epithelia retaining a lamina and by isolated epithelia generating a lamina de novo. The results andicate that the labeled GAG are bona fide components of the lamina, and suggest that laminar GAG is deposited in units of constant composition. Ultrastructural observations following ruthenium red staining or tannic acid fixation extablish that the lamina is a highly ordered specialization of the basal cell surface. Discrete structures in macroperiodic arrays apparently attached to the plasmalemma are visualized. This organization is seen in intact glands and in the laminae produced by epithelia in the absence of mesenchyme or biological substrate. The data are interpreted as indicating that the basal lamina contains supramolecular complexes of hyaluronic acid and proteoglycan which are organized into an extracellular scaffolding which imposes structural form on the epithelium.     

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity^1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood ⁴. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo⁵. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111^6,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands⁸. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.     

Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 microns. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.     

Isolation,culture, and differentiation of echinoid primary mesenchyme cells

Summary Methods are described for isolation and culture of primary mesenchyme cells from echinoid embryos. Ninety-five percentpure primary mesenchyme cells were isolated from early gastrulae ofStrongylocentrotus purpuratus, exploiting the biological segregation of these cells within the blastocoel. When cultured, more than 90% of the isolated cells reached the differentiated state, spicule formation, in synchrony with in vivo controls. Isolated primary mesenchyme cells were cultured with and without various cellular and acellular components of normal embryos in order to study the potential involvement of these components in the morphogenesis of the primary mesenchyme. Our data indicate that: 1. primary mesenchyme cells lack the ability to form the annular pattern of the primary mesenchymal ring autonomously; 2. they autonomously produce spicules of a characteristic morphology that differs from that of embryonic spicules; 3. morphogenesis of the primary mesenchyme is not affected by association with embryonic basal lamina, blastocoel matrix, or loosely aggregated epithelial cells, or by close confinement of each set of primary mesenchyme cells within the blastocoelar space; and 4. reaggregated, tightly associated epithelial cells can promote normal primary mesenchyme ring formation, and modify the primary mesenchyme-intrinsic spicule pattern to produce more normal spicule forms.     

Thyroid hormone affects embryonic mouse lung branching morphogenesis and cellular differentiation

Although thyroid hormone (T(3)) influences epithelial cell differentiation during late fetal lung development, its effects on early lung morphogenesis are unknown. We hypothesized that T(3) would alter embryonic lung airway branching and temporal-spatial differentiation of the lung epithelium and mesenchyme. Gestational day 11.5 embryonic mouse lungs were cultured for 72 h in BGJb serum-free medium without or with added T(3) (0.2, 2.0, 10.0, or 100 nM). Evaluation of terminal bud counts showed a dose- and time-dependent decrease in branching morphogenesis. Cell proliferation was also significantly decreased with higher doses of T(3). Morphometric analysis of lung histology showed that T(3) caused a dose-dependent decrease in mesenchyme and increase in cuboidal epithelia and airway space. Immunocytochemistry showed that with T(3) treatment, Nkx2.1 and surfactant protein SP-C proteins became progressively localized to cuboidal epithelial cells and mesenchymal expression of Hoxb5 was reduced, a pattern resembling late fetal lung development. We conclude that exogenous T(3) treatment during early lung development accelerated epithelial and mesenchymal cell differentiation at the expense of premature reduction in new branch formation and lung growth.     

LEF1 identifies androgen-independent epithelium in the developing prostate

Lymphoid enhancer-binding factor (LEF)1 is a major mediator and a target in canonical Wnt/β-catenin pathway. Interactions between the androgen receptor (AR) and canonical Wnt pathways have been implicated in the development of the genitourinary organs. Here, we investigated the localization and role of LEF1-positive cells during development of the prostate gland in human and in the murine model. We show that during human prostate development, LEF1 is restricted to the basal epithelial layer of the urogenital sinus. During mouse development, Lef1 is also present in the urogenital mesenchyme in addition to the basal epithelial layer of the urogenital sinus. In the course of elongation and branching of the prostatic ducts, Lef1 is localized to the proliferating epithelium at the distal tips of the buds. Notably, during branching morphogenesis, domains of Lef1 and AR are mutually exclusive. We further employed the TOPGAL reporter strain to examine the dynamics of Wnt signaling in the context of prostate regression upon a 7-d treatment with a competitive AR inhibitor, bicalutamide. We found that Wnt/Lef1-positive basal cells are not dependent upon androgen for survival. Furthermore, upon bicalutamide treatment, Wnt/Lef1-positive basal progenitors repopulated the luminal compartment. We conclude that Wnt/Lef1 activity identifies an androgen-independent population of prostate progenitors, which is important for embryonic development and organ maintenance and regeneration in the adult.     

The morphology of the basal lamina and periepithelial collagen during growth and branching of the embryonic lung of mice (day 14 to 16)

In order to be able to interpret the developmental mechanism of the epithelial branching pattern, we investigated lung development of mouse embryos of gestational days 14 to 16 electron microscopically. Various fixation techniques (Karnovsky, tannic acid, ruthenium red) were employed. Four regions could be distinguished in a growing and branching epithelial bud: 1) the epithelial tube before the site of branching; 2) the actual site and gap of branching; 3) the already formed part of the new buds and 4) the actual leading, i.e., the growing tip. Regions 1 and 3 were characterized by a continuous basal lamina and a more (1) or less (3) thick sheath of accompanying collagen. The site of branching (2) showed a pronounced folding of the basal lamina to which numerous collagenous fibrils were attached. At the distal, i.e., growing site (4) the basal lamina was characterized by numerous interruptions; accompanying collagen was missing. Immunomorphological investigations of regions 1 to 3 revealed all components of a basal lamina and collagen types I, III and V and fibronectin at the border between epithelium and connective tissue. However, this amount was clearly reduced at the growing tip, and collagen type I was missing. These findings help to understand and elucidate the importance of the mesenchyme for the growth and branching process and the development of the branching pattern.     

Retention of epithelial basal lamina allows isolated mandibular mesenchyme to form bone

The initiation of bone formation in the avian mandible requires that neural crest-derived cells undergo an inductive interaction with mandibular epithelium. To examine the role of the epithelial basal lamina in that interaction, mandibles were separated into their epithelial and mesenchymal components following exposure to the chelating agent, EDTA. Transmission and scanning electron microscopy was used to show that the basal lamina was retained as a continuous layer over the mesenchyme. Osteogenesis was initiated when such EDTA-isolated mesenchyme was grafted to the chorioallantoic membranes of host embryos. In contrast, mesenchyme isolated using trypsin and pancreatin failed to form bone. It is concluded that the property of mandibular epithelium which permits osteogenesis resides within the basal lamina.     

Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis

Branching morphogenesis is central to epithelial organogenesis. In the developing kidney, the epithelial ureteric bud invades the metanephric mesenchyme, which directs the ureteric bud to undergo repeated branching. A soluble factor(s) in the conditioned medium of a metanephric mesenchyme cell line is essential for multiple branching morphogenesis of the isolated ureteric bud. The identity of this factor had proved elusive, but it appeared distinct from factors such as HGF and EGF receptor ligands that have been previously implicated in branching morphogenesis of mature epithelial cell lines. Using sequential column chromatography, we have now purified to apparent homogeneity an 18 kDa protein, pleiotrophin, from the conditioned medium of a metanephric mesenchyme cell line that induces isolated ureteric bud branching morphogenesis in the presence of glial cell-derived neurotrophic factor. Pleiotrophin alone was also found to induce the formation of branching tubules in an immortalized ureteric bud cell line cultured three-dimensionally in an extracellular matrix gel. Consistent with an important role in ureteric bud morphogenesis during kidney development, pleiotrophin was found to localize to the basement membrane of the developing ureteric bud in the embryonic kidney. We suggest that pleiotrophin could act as a key mesenchymally derived factor regulating branching morphogenesis of the ureteric bud and perhaps other embryonic epithelial structures.     

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.