Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with β-d-xyloside

The role of glycosaminoglycans (GAGs) in the branching morphogenesis of embryonic mouse salivary glands was investigated by culturing the glands in the presence of xylose derivatives which stimulate synthesis of the xyloselinked classes of GAGs. Branching morphogenesis is inhibited severely, but reversibly, by 0.5–1.0 mM π-nitrophenyl-β-d-xylopyranoside and the inhibition correlates with a stimulation of incorporation of [³H]glucosamine (1.8-fold) and [³⁵S]sulfate (almost 3-fold) into GAGs. The effect of β-xyloside on accumulation of newly synthesized GAG also occurs in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the production of free GAG chains rather than proteoglycan-associated GAGs is being stimulated. The xyloside effects apparently do not result from general cytotoxicity of the derivatives, since similar concentrations of the α-anomer do not alter salivary branching or GAG synthesis, the rudiments resume morphogenesis when returned to control medium, and the effect on GAG synthesis is stimulatory rather than inhibitory. The study suggests that GAG biosynthesis plays an important role in salivary development, and that xylosides provide useful probes for characterizing the molecular events controlling branching morphogenesis.     

Proteoglycan and glycosaminoglycan synthesis in embryonic mouse salivary glands: effects of beta-D-xyloside, an inhibitor of branching morphogenesis

The proteoglycans and glycosaminoglycans synthesized by embryonic mouse salivary glands during normal morphogenesis and in the presence of beta- xyloside, an inhibitor of branching morphogenesis, have been partially characterized. Control and rho-nitrophenyl-beta-D-xyloside-treated salivary rudiments synthesize proteoglycans that are qualitatively similar, based on mobility on Sepharose CL-4B under dissociative conditions and glycosaminoglycan composition. However, beta-xyloside inhibits total proteoglycan-associated glycosaminoglycan synthesis by 50%, and also stimulates synthesis of large amounts of free chondroitin (dermatan) sulfate. This free glycosaminoglycan accounts for the threefold stimulation of total glycosaminoglycan synthesis in beta- xyloside-treated cultures. Several observations suggest that the disruption of proteoglycan synthesis rather than the presence of large amounts of free glycosaminoglycan is responsible for the inhibition of branching morphogenesis. (a) We have been unable to inhibit branching activity by adding large amounts of chondroitin (dermatan) sulfate, extracted from beta-xyloside-treated cultures, to the medium of salivary rudiments undergoing morphogenesis. (b) In the range of 0.1- 0.4 mM beta-xyloside, the dose-dependent inhibition of branching morphogenesis is directly correlated with the inhibition of proteoglycan synthesis. The stimulation of free glycosaminoglycan synthesis is independent of dose in this range, since stimulation is maximal even at the lowest concentration used, 0.1 mM. The data strongly suggest that the inhibition of branching morphogenesis is caused by the disruption of proteoglycan synthesis in beta-xyloside- treated salivary glands.     

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.     

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.     

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.     

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43^−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43^−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43^−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.     

Interaction of embryonic corneal epithelium with exogenous collagen,laminin, and fibronectin: Role of endogenous protein synthesis

Previously, we have shown that the embryonic corneal epithelium is capable of interacting with exogenous collagen, laminin, and fibronectin in soluble form, each of which causes isolated epithelium cultured on Millipore filter to stop blebbing, reorganize the basal cytoskeleton, and flatten. Here we examine the involvement of endogenously derived extracellular matrix (ECM) molecules in the interaction of the basal epithelial cell surface with the added ECM molecules. We demonstrate here that the isolated avian corneal epithelium cultured on Millipore filter is capable of synthesizing collagens and laminin, but not fibronectin. To examine whether the epithelium is capable of interacting directly with exogenous ECM components or if there is the necessity for production of a linker molecule, epithelial protein synthesis was inhibited with cycloheximide (CHX). The blebbing epithelium in the presence of CHX was then confronted with soluble ECM molecules added to the medium under the filter; such epithelia are able to interact with, and flatten in response to, both collagen and laminin. However, such inhibited epithelia continue to bled in the presence of fibronectin. We next used l-azetidine-4-carboxylic acid (LACA) to interfere with collagen secretion. Epithelia exposed to LACA are still capable of interacting with collagen and laminin, but not fibronectin, indicating a dependence on collagen secretion. These results suggest that fibronectin requires a linker protein, probably collagen, to interact with the basal epithelial surface, whereas both collagen and laminin may interact directly with the cell surface to transform the basal cytoskeleton into the cortical mat typical of differentiating corneal epithelium in situ.     

Dysmorphogenesis of the inner ear: disruption of extracellular matrix (ECM) formation by an L-proline analog in otic explants

L-azetidine-2-carboxylic acid (LACA), a l-proline analog, disrupts collagen secretion by cells and prevents normal morphogenesis of in vitro developing organ rudiments. Otic explants derived from 10.5-through 14-day-old mouse embryos were continuously exposed to LACA in the nutrient medium at concentrations of 75, 150, and 300 micrograms/ml. LACA disrupted normal in vitro otic morphogenesis in inner ears explanted from embryos of 10.5 through 13 days' gestation. Development of 14-day-old otic explants were not affected by LACA at the concentrations tested. There was a direct correlation between the embryonic age of the explant when exposed to LACA, and the severity of otic dysmorphogenesis. The younger explants (10.5-to 12-day-old) developed abnormalities of both vestibular and auditory structures, but with increasing embryonic age of the explants (12-to 13.5-day-old) abnormalities were confined more to the auditory portion of the inner ear. Disruption of collagen secretion of connective tissue cells of the otic explants are a major teratogenic action of LACA on inner ear development. Disrupted collagen secretion alters otic extracellular matrix production, which in turn affects the tissue interactions that regulate the progressive expression of otic morphogenesis and differentiation.     

Dystroglycan binding to laminin α1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.     

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.     

TGF-β1 Inhibits Growth and Branching Morphogenesis In Embryonic Mouse Submandibular and Sublingual Glands in Vitro

Members of the TGF-β superfamily of polypeptides are key regulators in developmental processes. Several studies have shown that expression of TGF-β mRNA and protein are developmentally regulated and that both are prominently expressed in tissues undergoing epithelial-mesenchymal interactions such as branching morphogenesis. It has been shown that TGF-β1 protein is present in E 14 mouse submandibular glands at a time when branching is already establihsed. Here we demonstrate by RT-PCR and immunofluorescence that both TGF-β1 mRNA and protein are present in E 13 submandibular and sublingual glands at a time when branching is being initiated. Addition of TGF-β1 to E 13 rudiments resulted in reductions in organ size and inhibition of branching. Sensitivity to TGF-β1 depended on the developmental stage of the rudiments (early or late E 13) and the dose of growth factor used. TGF-β1 Also caused epithelial abnormalities, notably treated organs had elongated ducts. The effects were most pronounced in the sublingual gland. Taken together these results suggest a regulatory role for endogenous TGF-β1 in the growth and morphogenesis of mouse salivary glands.     

DEPENDENCE OF SALIVARY EPITHELIAL MORPHOLOGY AND BRANCHING MORPHOGENESIS UPON ACID MUCOPOLYSACCHARIDE-PROTEIN (PROTEOGLYCAN) AT THE EPITHELIAL SURFACE

The morphogenetic role of the acid mucopolysaccharide (glycosaminoglycan) at the epithelial surface of mouse embryo submandibular glands has been studied by comparing the in vitro morphogenesis of epithelia from which the mucopolysaccharide was removed with that of those that retained the mucopolysaccharide. Epithelia isolated free of mesenchyme by procedures which retain the bulk of surface mucopolysaccharide maintain their lobular shape and undergo uninterrupted branching morphogenesis in culture in direct combination with fresh mesenchyme. Under identical culture conditions, epithelia from which surface mucopolysaccharide was removed lose their lobules and become spherical masses of tissue. During continued culture, the spherical epithelia produce outgrowths from which branching morphogenesis resumes. The morphogenetically active mucopolysaccharide is localized within the basal lamina of the epithelial basement membrane and appears to be bound to protein. During culture in combination with mesenchyme, epithelia undergoing uninterrupted morphogenesis show maximal accumulation of newly synthesized surface mucopolysaccharide at the distal ends of the lobules, the sites of incipient branching. In contrast, the material accumulates nearly equivalently over the surface of the spherical epithelia, with the exception that there is greater accumulation of the material at the surfaces of the budding outgrowths, the sites where morphogenesis will resume. Rapidly proliferating cells are localized within the lobules of epithelia undergoing uninterrupted morphogenesis, but are distributed uniformly in the cortex of the spherical epithelia, except for the outgrowths which show a greater localization of proliferating cells. It is concluded that normal salivary epithelial morphology and branching morphegenesis require the presence of acid mucopolysaccharide-protein within the epithelial basal lamina.     

EGF-receptor regulates salivary gland branching morphogenesis by supporting proliferation and maturation of epithelial cells and survival of mesenchymal cells

Epidermal growth factor receptor (EGF-R) regulates epithelial morphogenesis during development and is important for the proper branching of the lung, mammary gland, and pancreas. We analyzed the salivary gland phenotype of EGF-R-deficient mice and showed impaired growth, branching, and maturation of the epithelium. Furthermore, treatment of wild-type E13 salivary glands with gefitinib, a small molecular inhibitor of EGF-R, led to apoptosis of the mesenchyme. Interestingly, MMP2 and plasminogen activators were upregulated upon inhibition of EGF-R signaling. To summarize, we show that EGF-R is a physiological regulator of salivary gland development and its main function is to support the proliferation and maturation of the epithelium and the survival of the mesenchyme.     

Cellular events associated with lung branching morphogenesis including the deposition of collagen type IV

In this study mouse lung development was examined using an in vitro model system. The culture system permitted examination of a morphogenic process that eventually led to the formation of presumptive alveoli (terminal sacs). The observations included changes in epithelial cell morphology (transition from a columnar to a spindle shape), and evidence for motile activity on the part of primitive airway epithelial cells. The importance of Type IV collagen to the cellular events associated with branching morphogenesis was investigated by immunolocalization. In addition, we assessed the similarity of normal lung development to in vitro development by comparing cultured lungs with equivalent stages of embryonic and fetal mouse lungs. The results show that cultured embryonic lung explants proceed along a morphogenic pathway that parallels normal lung development; that primitive pulmonary epithelial cells engage in motile activity and transiently acquire an extended cell shape both in vitro and in vivo; that, as suggested by others, the pattern of late branching morphogenesis is not dichotomous, but irregular; and that short wisplike fibers of Type IV collagen are present in developing embryonic and fetal lung mesenchyme. Taken together, the results show that early and late lung branching patterns differ significantly, and suggest that later stages of lung branching involve distinct epithelial cell shape transitions. The immunofluorescence data suggest that fibrous Type IV collagen may be the extracellular matrix scaffold within which early epithelial cells accomplish lung branching morphogenesis.     

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.     

Action of L-azetidine in in vitro culture on the formation of uropygial invaginations and the branching of glandular buds in the duck embryo]

Azetidine-2-carboxylic acid, a proline analogue which depresses collagen deposition in extracellular spaces, stops uropygial invaginations morphogenesis and epithelial buds branching in vitro. The results show that uninterrupted collagen synthesis is necessary for preen gland morphogenesis.     

Protein Patterns and Synthetic Profiles during Distal Regeneration in Hydra oligactis

Protein patterns and synthetic profiles were examined during distal regeneration in Hydraoligactis . Electrophoretic and radioactive tracer analyses revealed qualitative changes in the general protein profile during regeneration, with a heightened period of protein synthesis between 27–30 hr of regeneration, immediately preceding emergence of the first pair of tentacles. Following this, an increase in collagen-rich mesogleal protein secretion was observed coincident with tentacle initiation. Inhibition of collagen secretion with the proline analog L-azetidine-2-carboxylic acid (LACA) inhibited tentacle formation, and resulted in the development of unique "hypostome buds" at the distal regeneration surface. At the cellular level LACA did not inhibit the nerve cell differentiation that normally precedes tentacle growth, although some predicted decline in cnidocyte production was noted. It is proposed that mesogleal collagen secretion and structural organization may play a major role in the mechanical aspects of Hydra tentacle morphogenesis.     

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.     

Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung

Hox proteins control structural morphogenesis, pattern formation and cell fate in the developing embryo. To determine if Hoxb-5 participates in patterning of early airway branching during lung morphogenesis, gestational day 11.5 embryonic lung cultures were treated with retinoic acid (RA) to up-regulate and antisense oligonucleotides to down-regulate Hoxb-5 protein expression. RA (10^?6 M) and Hoxb-5 antisense oligonucleotide (20 μM) treatment each significantly decreased branching morphogenesis (P<0.001), but the morphology of branching under these conditions was very different. RA-treated lungs had elongated primary branches but decreased further branching with increased Hoxb-5 immunostaining in subepithelial regions underlying these elongated airways. Western blots confirmed that Hoxb-5 protein was increased by 189±20% (mean±S.E.M., P<0.05) in RA-treated lungs compared to controls. In contrast, lungs treated with Hoxb-5 antisense oligos plus RA had foreshortened primary branches with rudimentary distal clefts resulting in decreased numbers of primary and subsequent branches. Immunohistochemistry confirmed that Hoxb-5 antisense oligos inhibited Hoxb-5 protein expression even in the presence of RA. We conclude that regional and quantitative changes in Hoxb-5 protein expression influence morphogenesis of the first airway divisions from the mainstem bronchi. RA-induced alterations in branching are mediated in part through regulated Hoxb-5 expression.