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.     

Use of the collagen I-deficient Mov13 mouse mutant to analyse epithelial-mesenchymal tissue interaction

The collagen I-deficient mouse mutant (Mov13 — an embryonic recessive lethal) was used to investigate the function of this major constituent of the extracellular matrix (ECM) in organ development. All epithelial-mesenchymal organs tested as explants (lung, kidney, pancreas, salivary glands, skin) developed normally and, in particular, showed typical branching morphogenesis in the absence of collagen I. It is concluded that the ECM of these organs can organize for proper developmental function in the absence of the major interstitial collagen, but a possible morphogenetic function of other fibrillar collagens (types III and V) cannot be excluded. The only insufficiencies in the mutant were seen in the cornea where deposition and organization of the collagenous stroma was highly inadequate; but even there, development and migration of cells proceeded normally. In summary, the results indicate that ‘cellular’ development in epithelial-mesenchymal organs (including growth, morphogenesis, and differentiation) does not depend on collagen I.     

Adhesion, shape, proliferation, and gene expression of mouse Leydig cells are influenced by extracellular matrix in vitro

Interactions between Leydig cells and the extracellular matrix (ECM) within the interstitial compartment of the mammalian testis have not been characterized. We have examined the influence of ECM on adult mouse Leydig cells by culturing the cells on different ECM substrates. Leydig cells adhere weakly to hydrated gels of type I collagen (including those supplemented with collagen types IV, V, or VIII), or to air-dried films of collagen types I, V, or VIII. In contrast, the cells attach firmly to substrates of purified type IV collagen, fibronectin, or laminin. Leydig cells also attach rapidly and adhere strongly to gelled basement membrane matrix derived from the murine Englebreth-Holm-Swarm sarcoma (Matrigel). Leydig cells assume spherical shapes and form aggregates on thick (1.5-mm) layers of Matrigel; however, on thin (0.1-mm) layers, networks of cell clusters linked by cords of elongated cells are formed within 48 h. Similar networks are formed on thick layers of Matrigel that are supplemented with type I collagen. On substrates with high ratios of collagen I to Matrigel or on untreated tissue culture plastic, Leydig cells flatten and do not aggregate. On substrates that induce rounded shapes, proliferation is inhibited and the cells maintain the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase for as long as 2 wk. Under conditions where Leydig cells are flattened, they divide and cease expressing the enzyme. Proliferating Leydig cells also exhibit elevated levels of mRNA for SPARC (Secreted Protein, Acidic and Rich in Cysteine), a Ca2(+)-binding glycoprotein associated with changes in cell shape that accompany morphogenesis and tissue remodeling. Our results indicate that the shape, association, proliferation, and expression of gene products by Leydig cells can be significantly affected in vitro by altering the composition of the extracellular substratum.     

HGF-Induced Tubulogenesis and Branching of Epithelial Cells Is Modulated by Extracellular Matrix and TGF-β

Beginning with the observation that hepatocyte growth factor (HGF) induces the formation of branching tubular structures in Madin-Darby canine kidney (MDCK) cells cultured in Type I collagen gels but not in basement membrane Matrigel, we examined the individual components within this complex basement membrane extract to determine the effect of these proteins on the morphogenetic changes mediated by HGF. After extraction of several growth factors from Matrigel, HGF was still unable to induce process formation, an early event in tubulogenesis, indicating that one or more of the remaining extracellular matrix (ECM) proteins or growth factors were exerting the inhibitory effect. By individually adding back these components to MDCK cells grown in Type I collagen gels in the presence of HGF, we were able to establish that: (1) certain ECM proteins, such as laminin, entactin, and fibronectln, actually facilitated the formation of branching tubular structures and increased their complexity; (2) other ECM proteins, such as Type IV collagen, heparan sulfate proteoglycan, and vitronectin, caused marked inhibition of HGF-induced morphogenesis; and (3) not only did transforming growth factor-β (TGF-β) inhibit the formation of tubular structures, but those which did form exhibited little branching, thereby suggesting that TGF-β modulates tubulogenesis as well as branching. These results suggest that a tubulogenic morphogen such as HGF and a tubulogenesis-inhibitory morphogen such as TGF-β can, in the context of the dynamic matrix known to exist during epithelial tissue development, modulate the degree of tubule (or ductal) formation, the length of these tubules, and the extent of their arborization. The relevance of these findings to tubulogenesis and branching during kidney development is discussed.     

The role of the arginine-glycine-aspartic acid-directed cellular binding to type I collagen and rat mesenchymal cells in colorectal tumour differentiation

The relationship between the adhesion of five human colorectal carcinoma cell lines to extracellular matrix (ECM) proteins, namely type I collagen, type IV collagen, fibronectin, laminin and basement membrane extract (Matrigel), and the ability of these cells to express morphological differentiation when grown in a basement membrane extract (Matrigel) or on normal rat mesenchymal cells has been examined. Two cell lines, SW1222 and HRA-19, organised into glandular structures, with well-defined polarity when cultured on both substrata as well as in three-dimensional (3D) collagen gel culture as previously shown. The remaining three cell lines (SW620, SW480 and HT29) grew as loose aggregates or as they would normally grow on tissue culture plastic. Addition to the culture medium of a hexapeptide, containing the cell-matrix recognition sequence arginine-glycine-aspartic acid (RGD), inhibited attachment and glandular formation of SW1222 and HRA-19 when these cells were grown on living mesenchymal cells, but not in Matrigel. The morphological differentiation of HRA-19 cells in 3D-collagen was also inhibited by the same RGD-containing peptide, as previously shown for SW1222 cells. Attachment of the remaining three cell lines was inhibited on mesenchyme but not in Matrigel, further supporting the specificity of the peptide effect on epithelial-mesenchymal binding. In conclusion we have shown that colorectal tumour cells are able to bind ECM proteins and that the cellular binding is an essential step in the induction of the morphological differentiation seen on living mesenchymal cells, in basement membrane extracts and in type I collagen gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium

Mouse salivary epithelium cannot undergo branching morphogenesis in the absence of the surrounding mesenchyme. To clarify the nature of the mesenchymal influence on the epithelium, we have investigated the culture conditions in which the epithelium could normally branch in the absence of mesenchymal cells. Combination of basement-membrane-like substratum (Matrigel) and epidermal growth factor (EGF) could substitute for the mesenchyme, the epithelium showing typical branching morphogenesis. Transforming growth factor alpha had the same effect as EGF. Matrigel plus basic fibroblast growth factor or transforming growth factor beta 1 and collagen gel plus EGF were not sufficient to support the branching of the epithelium. These results clearly reveal that the role of mesenchyme in salivary morphogenesis is both to provide the epithelium with an appropriate substratum and to accelerate growth of the epithelium.     

Abnormal mucosal extracellular matrix deposition is associated with increased TGF-beta receptor-expressing mesenchymal cells in a mouse model of colitis.

Transforming growth factor-beta (TGF-beta) depresses mucosal inflammation and upregulates extracellular matrix (ECM) deposition. We analyzed TGF-beta receptors RI and RII as well as ECM components using the CD4(+) T-cell-transplanted SCID mouse model of colitis. The principal change in colitis was an increased proportion of TGF-beta RII(+) mucosal mesenchymal cells, predominantly alpha-smooth muscle actin (SMA)(+) myofibroblasts, co-expressing vimentin and basement membrane proteins, but not type I collagen. TGF-beta RII(+) SMA(-) fibroblasts producing type I collagen were also increased, particularly in areas of infiltration and in ulcers. Type IV collagen and laminin were distributed throughout the gut lamina propria in disease but were restricted to the basement membrane in controls. In areas of severe epithelial damage, type IV collagen was lost and increased type I collagen was observed. To examine ECM production by these cells, mucosal mesenchymal cells were isolated. Cultured cells exhibited a similar phenotype and matrix profile to those of in vivo cells. The data suggested that there were at least two populations of mesenchymal cells responsible for ECM synthesis in the mucosa and that ligation of TGF-beta receptors on these cells resulted in the disordered and increased ECM production observed in colitic mucosa.     

Morphogenesis of fingers and branched organs: how collagen and fibroblasts break the symmetry of growing biological tissue

Understanding the growth of branching organs is an important scientific endeavour. It has crucial applications, from saving premature newborns, to repairing or even regenerating organs. Despite differences in timing and shape, branching morphogenesis of all branching organs or glands (lung, kidney, salivary, lachrymal, mammary glands, sebaceous and sweat glands, prostate, guts papillae etc.) is similar: an epithelial sheet of cells, forming a 2D layer, penetrates into a 3D mass of mesenchymal cells. Inside the epithelium, a lumen is filled with fluid. As the epithelium grows, it evolves into a branched structure. The pattern of branches is in some cases stereotypic, deterministic, and it has memory effects. We present a simple line of reasoning that predicts that viscous fingering of biological tissue will exhibit all of these features. The line of reasoning is based on the idea that surface tension selects the shape of a growing branch, as is well known in the context of moving boundary problems, except that in this case, the surface is akin to a liquid-crystal. The anisotropy of the surface tension comes from a symmetry breaking by collagen and fibroblasts. The equilibrium shape of the corresponding boundary is that of an actual fingertip, and the out-of-equilibrium shape is that of branched organs, such as the lung.     

Collagen involvement in branching morphogenesis of embryonic lung and salivary gland

The possibility that extracellular collagen is involved in branching morphogenesis of mouse embryo lung and salivary glands has been explored duringin vitro organ culture. Control cultures of both rudiment types contain abundant collagen in extracellular spaces between mesenchymal cells and in the epithelial-mesenchymal interface. Branching morphogenesis of lungs and salivary glands is not perturbed by the presence of β-aminopropionitrile, implying that extracellular collagen cross-linking is not required, but is perturbed by α,α′-dipyridyl orl-azetidine-2-car?ylic acid (LACA), agents reported to interfere with collagen synthesis and secretion. Analysis of the structural and biosynthetic effects of LACA revealed a severe inhibition of collagen synthesis, as monitored by hydroxyproline synthesis, and extracellular collagen accumulation. Cell and tissue integrity was not affected, but a slight inhibition of general protein synthesis, protein accumulation, and epithelial expansion was observed. The strong correlations between collagen biosynthesis, extracellular collagen presence, and branching morphogenesis are consistent with an integral role for collagen in embryonic lung and salivary gland morphogenesis.     

Salivary gland branching morphogenesis

Salivary gland branching morphogenesis involves coordinated cell growth, proliferation, differentiation, migration, apoptosis, and interaction of epithelial, mesenchymal, endothelial, and neuronal cells. The ex vivo analysis of embryonic mouse submandibular glands, which branch so reproducibly and beautifully in culture, is a powerful tool to investigate the molecular mechanisms regulating epithelium-mesenchyme interactions during development. The more recent analysis of genetically modified mice provides insight into the genetic regulation of branching morphogenesis. The review begins, as did the field historically, focusing on the role of the extracellular matrix (ECM), and its components such as glycosaminoglycans, collagens, and laminins. Following sections describe the modification of the ECM by proteases and the role of cell-matrix and cell-cell receptors. The review then focuses on two major families of growth factors implicated in salivary gland development, the fibroblast growth factors (FGFs) and the epidermal growth factors (EGFs). The salivary gland phenotypes in mice with genetic modification of FGFs and their receptors highlight the central role of FGFs during salivary gland branching morphogenesis. A broader section mentions other molecules implicated from analysis of the phenotypes of genetically modified mice or organ culture experiments. The review concludes with speculation on some future areas of research.     

Extracellular matrix and cytoskeletal dynamics during branching morphogenesis

Branching morphogenesis is a fundamental developmental process which results in amplification of epithelial surface area for exchanging molecules in organs including the lung, kidney, mammary gland and salivary gland. These complex tree-like structures are built by iterative rounds of simple routines of epithelial morphogenesis, including bud formation, extension, and bifurcation, that require constant remodeling of the extracellular matrix (ECM) and the cytoskeleton. In this review, we highlight the current understanding of the role of the ECM and cytoskeletal dynamics in branching morphogenesis across these different organs. The cellular and molecular mechanisms shared during this morphogenetic process provide insight into the development of other branching organs.     

Extracellular matrix and cytoskeletal dynamics during branching morphogenesis

Branching morphogenesis is a fundamental developmental process which results in amplification of epithelial surface area for exchanging molecules in organs including the lung, kidney, mammary gland and salivary gland. These complex tree-like structures are built by iterative rounds of simple routines of epithelial morphogenesis, including bud formation, extension, and bifurcation, that require constant remodeling of the extracellular matrix (ECM) and the cytoskeleton. In this review, we highlight the current understanding of the role of the ECM and cytoskeletal dynamics in branching morphogenesis across these different organs. The cellular and molecular mechanisms shared during this morphogenetic process provide insight into the development of other branching organs.     

Normal epithelial branching morphogenesis in the absence of collagen I

Interstitial collagens are thought to mediate epithelial-mesenchymal interactions during organogenesis. We have used the collagen I-deficient mouse mutant Mov13 to directly investigate the role of this major representative of the interstitial collagens in epithelial branching morphogenesis. Since homozygous embryos die at midgestation, we have studied the development of organ rudiments from Mov13 homozygous (i.e., collagen I-deficient), heterozygous, and wild-type embryos in culture. Development of all explants, including lung, kidney, salivary glands, pancreas, and skin, was normal by light and electron microscopic criteria and was independent of the genotype of the donor embryo. Metabolic labeling and immune staining verified the complete absence of collagen I in homozygous explants while revealing substantial production of collagens III and V in explants of all three genotypes. These results indicate either that collagen I has no role in the morphogenesis of these organs, or that its function is shared, or can be substituted for, by other fibrillar collagens.     

Epithelial-mesenchymal interactions in tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors.

Morphogenesis and cell differentiation in the developing tooth are controlled by a series of reciprocal interactions between the epithelial and mesenchymal tissues. The exact molecular mechanisms operating in these interactions are unknown at present, but both structural components of the extracellular matrix (ECM) and diffusible growth factors have been suggested to be involved. In this review article we summarize our findings on the distribution patterns of three ECM molecules and two cell surface receptors during tooth morphogenesis through bud, cap, and bell stages of development. The examined molecules include fibronectin, type III collagen, and tenascin, which all represent components of the mesenchymal ECM, the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix, and the cell surface receptor for epidermal growth factor. Based on the observed changes in distribution patterns and on experimental evidence, roles are suggested for these molecules in epithelial-mesenchymal interactions during tooth development. Fibronectin is suggested to be involved in the cell-matrix interaction that controls odontoblast differentiation. Epidermal growth factor and its receptors are suggested to be involved in a paracrine fashion in the epithelial-mesenchymal interactions regulating morphogenesis of bud- and cap-stage teeth. Tenascin and syndecan are accumulated in the dental mesenchyme during the bud stage of development, and it is suggested that they represent a couple of a cell surface receptor and its matrix ligand and that they are involved in mesenchymal cell condensation during the earliest stages of tooth morphogenesis.     

Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix

Summary The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells. This work was supported by grants HL35684 and SCOR HL14212 from the National Institutes of Health, Bethesda, MD.     

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells

Cultured hepatic stellate cells (HSCs) are known to change their morphology and function with respect to the production of extracellular matrices (ECMs) and matrix metalloproteinases (MMPs) in response to ECM components. We examined the regulatory role of the native form of type I collagen fibrils in pro-MMP-2 production and activation in cultured HSCs. Gelatin zymography of the conditioned media revealed that pro- and active form of MMP-2 was increased in the HSCs cultured on type I collagen gel but not on type I collagen-coated surface, gelatin-coated surface, type IV collagen-coated surface, or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 production and activation. The induction of active MMP-2 by extracellular type I collagen was suppressed by the blocking antibody against integrin beta1 subunits, indicating the involvement of integrin signaling in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, membrane type-1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA levels were elevated in HSCs cultured on type I collagen gel. The increased MT1-MMP proteins were localized on the cell surface of HSCs cultured on type I collagen gel. In contrast to the expression of MMP-2, HSCs showed a great decline in MMP-13 expression in HSCs cultured on type I collagen gel. These results indicate that the native fibrillar (polymerized) but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs, suggesting an important role of HSCs in ECM remodeling in the hepatic perisinusoidal spaces.     

Lefty contributes to the remodeling of extracellular matrix by inhibition of connective tissue growth factor and collagen mRNA expression and increased proteolytic activity in a fibrosarcoma model

Homeostasis of the extracellular matrix (ECM) of tissues is regulated by controlling deposition and degradation of ECM proteins. The breakdown of ECM is essential in blastocyst implantation and embryonic development, tissue morphogenesis, menstrual shedding, bone formation, tissue resorption after delivery, and tumor growth and invasion. TGF-beta family members are one of the classes of proteins that actively participate in the homeostasis of ECM. Here, we report on the effect of lefty, a novel member of the TGF-beta family, on the homeostasis of extracellular matrix in a fibrosarcoma model. Fibroblastic cells forced to express lefty by retroviral transduction lost their ability to deposit collagen in vivo. This event was associated with down-regulation of the steady-state level of connective tissue growth factor that induces collagen type I mRNA. In addition, lefty transduction significantly decreased collagen type I mRNA expression and simultaneously increased collagenolytic, gelatinolytic, elastolytic, and caseinolytic activities in vivo by the transduced fibroblasts. These findings provide a new insight on the actions of lefty and suggest that this cytokine plays an active role in remodeling of the extracellular matrix in vivo.     

Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel,collagen, fibrin,and plasma clot

Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of³H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis. This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes of Health, Bethesda, MD.     

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.