Enhanced branching morphogenesis in mammary glands of mice lacking cell surface beta1,4-galactosyltransferase

Development of the mammary gland is influenced both by the systemic hormonal environment and locally through cell-cell and cell-extracellular matrix (ECM) interactions. We have previously demonstrated aberrant mammary gland morphogenesis in transgenic mice with elevated levels of the long isoform of beta1,4-galactosyltransferase 1 (GalT), a proportion of which is targeted to the plasma membrane, where it plays a role in cell-ECM interactions. Here, we show that mammary glands of mice lacking the long GalT isoform exhibit a complementary phenotype. Cell-surface GalT activity was reduced by over 60%, but because the short GalT isoform is intact, total GalT activity was reduced only slightly relative to wild type. Mammary glands from long GalT-null mice were characterized by excess branching, and this phenotype was accompanied by altered expression of laminin chains. Laminin alpha1 and alpha3 were reduced 2.4- and 3.0-fold, respectively, while expression of laminin gamma2 was elevated 2.3-fold. The expression and cleavage of laminin gamma2 have been correlated with branching and cell migration, and Western blotting revealed an altered pattern in gamma2 cleavage products in long GalT-null mammary glands. We then examined the expression of metalloproteases that cleave laminins or that have been shown to play a role in mammary gland morphogenesis. Expression of MT1-MMP, a membrane-bound protease that can cleave laminin gamma2, was elevated 5.5-fold in the long GalT-nulls. MMP 7 was also elevated 5.1-fold. Our results suggest that expression of surface GalT is important for the proper regulation of matrix expression and deposition, which in turn regulates the proper branching morphogenesis of the mammary epithelial ductal system.     

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.     

HGF/SF-met signaling in the control of branching morphogenesis and invasion

Hepatocyte growth factor/Scatter factor (HGF/SF) is a multifunctional growth factor which can induce diverse biological events. In vitro, these include scattering, invasion, proliferation and branching morphogenesis. In vivo, HGF/SF is responsible for many processes during embryonic development and a variety of activities in adults, and many of these normal activities have been implicated in its role in tumorgenesis and metastasis. The c-Met receptor tyrosine kinase is the only known receptor for HGF/SF and mediates all HGF/SF induced biological activities. Upon HGF/SF stimulation, the c-Met receptor is tyrosine-phosphorylated which is followed by the recruitment of a group of signaling molecules and/or adaptor proteins to its cytoplasmic domain and its multiple docking sites. This action leads to the activation of several different signaling cascades that form a complete network of intra and extracellular responses. Different combinations of signaling pathways and signaling molecules and/or differences in magnitude of responses contribute to these diverse series of HGF/SF-Met induced activities and most certainly are influenced by cell type as well as different cellular environments. In this review, we focus on HGF/SF-induced branching morphogenesis and invasion, and bring together recent new findings which provide insight into how HGF/SF, via c-Met induces this response.     

Changes in adhesive properties of epithelial cells during early morphogenesis of the mammary gland

Previous studies have demonstrated that cell adhesion systems are downregulated in epithelial buds at the earliest stages of submandibular gland and hair follicle development, but are restored at subsequent stages. Here it is shown that epithelial cell adhesion systems are also remodeled during early mammary gland development. Immunofluorescence and electron microscopy of the mouse mammary bud demonstrated that cell-cell adhesion systems were hardly detectable, with significant downregulation of expression of desmosomal molecules, but not of E-cadherin and beta-catenin. Hemidesmosomal structures were also rarely found, although their component molecules were expressed. Differences in cell adhesivity between cells of the mammary bud and those of the overlying epidermis were shown by the finding that the mammary cells formed smaller aggregates than the epidermal cells and were not randomly mixed with the epidermal cells. At subsequent stages, the mammary epithelium restored cell-cell adhesion systems along with de novo expression of tight junction molecules. These data, together with previous findings, indicate that remodeling of epithelial cell adhesion systems is a general feature underlying the early development of several ectoderm-derived organs and support the idea that segregation and rearrangements of cells are involved in early epithelial morphogenesis.     

Mast cells contribute to the stromal microenvironment in mammary gland branching morphogenesis

The stromal microenvironment regulates mammary gland branching morphogenesis. We have observed that mast cells are present in the mammary gland throughout its postnatal development and, in particular, are found around the terminal end buds and ductal epithelium of the pubertal gland. Mast cells contribute to allergy, inflammatory diseases, and cancer development but have not been implicated in normal development. Genetic and pharmacological disruption of mast cell function in the mammary gland revealed that mast cells are involved in rapid proliferation and normal duct branching during puberty, and this effect is independent of macrophage recruitment, which also regulates mammary gland development. For mast cells to exert their effects on normal morphogenesis required activation of their serine proteases and degranulation. Our observations reveal a novel role for mast cells during normal pubertal development in the mammary gland.     

Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co‐culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non‐macromastic epithelial cells when co‐cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia‐derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co‐culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co‐cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy.     

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.     

Mammary gland morphogenesis in vitro: Extracellular requirements for the formation of tubules in collagen gels by a cloned rat mammary epithelial cell line

Summary The mechanism of induction of tubular outgrowths in vitro on floating collagen gels and the influence of extracellular factors on this process have been investigated using the clonal rat mammary epithelial cell line, Rama 25. Growth of Rama 25 on such floating gels causes their contraction. Contraction of the gel is accompanied by a 10-fold increase in the number of cells per unit area, a change in cell shape, and a convolution of the epithelial cell sheet. Gels folded over manually show an 11-times higher incidence of tubules along the folds than on the flat surface. Tubular formation begins when cords of cells develop from local proliferations of the cell sheet and become canalized. Tubules follow wrinkles in the gel and branch to yield monopodial, dichotomous, or lobular architecture. Hydrocortisone and insulin, in the presence of serum, stimulate both narrow and thick tubular structures on folded gels, whereas extra additions of 1 ng/ml cholera toxin or 100 ng/ml epidermal growth factor preferentially stimulate thick tubular structures. Floating glutaraldehyde-fixed gels, very thick collagen gels, and collagen gels prepared on the top of rigid steel grids fail to support the formation of tubules, suggesting that flexibility and access of the medium to basal surfaces are important to their genesis. Incorporation of hyaluronic acid into the gel matrix preferentially inhibits the thick tubular outgrowths. Thus, the branching tubular structures generated by Rama 25 can be influenced in different ways by various extracellular factors in the medium and in the gel. During the course of this work E. J. Ormerod was in receipt of a Ludwig Research Studentship.     

Hormonal control of shoot branching

Shoot branching is the process by which axillary buds, located on the axil of a leaf, develop and form new flowers or branches. The process by which a dormant bud activates and becomes an actively growing branch is complex and very finely tuned. Bud outgrowth is regulated by the interaction of environmental signals and endogenous ones, such as plant hormones. Thus these interacting factors have a major effect on shoot system architecture. Hormones known to have a major influence are auxin, cytokinin, and a novel, as yet chemically undefined, hormone. Auxin is actively transported basipetally in the shoot and inhibits bud outgrowth. By contrast, cytokinins travel acropetally and promote bud outgrowth. The novel hormone also moves acropetally but it inhibits bud outgrowth. The aim of this review is to integrate what is known about the hormonal control of shoot branching in Arabidopsis, focusing on these three hormones and their interactions.     

The control of branching morphogenesis

Many organs of higher organisms are heavily branched structures and arise by an apparently similar process of branching morphogenesis. Yet the regulatory components and local interactions that have been identified differ greatly in these organs. It is an open question whether the regulatory processes work according to a common principle and how far physical and geometrical constraints determine the branching process. Here, we review the known regulatory factors and physical constraints in lung, kidney, pancreas, prostate, mammary gland and salivary gland branching morphogenesis, and describe the models that have been formulated to analyse their impacts.     

Colony stimulating factor-1 is required to recruit macrophages into the mammary gland to facilitate mammary ductal outgrowth

Mammary gland development initiates postnatally with the development of terminal end buds (TEBs) at the end of the rudimentary ducts. These grow out through the fat pad and bifurcate to lay down the rudimentary ductal tree. At the initiation of their development, TEBs recruit to their surrounding stroma a substantial population of macrophages. Using mice homozygous for a null mutation in the gene for the macrophage growth factor, colony stimulating factor-1 (CSF-1), that are severely depleted in macrophages, we demonstrated that CSF-1-regulated macrophages are required for normal branching morphogenesis in the mammary gland. However, these mice have a pleiotropic phenotype as a result of the generalized macrophage deficiency. To test that the effect of the mutation observed in the mammary gland was organ-autonomous, we developed a tetracycline-binary system whereby CSF-1 was specifically expressed in the mammary epithelium under the regulation of the MMTV-promoter. This restored mammary macrophage populations but not those in other tissues and corrected the branching morphogenesis defect. Inhibition of CSF-1 expression by tetracycline treatment for varying periods suggested that CSF-1-regulated macrophages are required throughout early mammary gland development. These data show that macrophages acting locally are required for branching morphogenesis of the mammary gland.     

The specificity of chitosan in promoting branching morphogenesis of progenitor salivary tissue

Chitosan has been shown to be effective in regulating progenitor salivary tissue morphogenesis, however, the specificity of chitosan effects remains unclear. To assess the regulatory ability of chitosan in salivary gland morphogenesis, progenitor salivary tissue from embryonal submandibular gland (SMG) was cultured in chitosan-containing medium. It was found that soluble chitosan was able to promote SMG branching in a dose-dependent manner. The effect was chitosan-specific and was not reproduced by substrates with similar chemical structures or other polymeric molecules of natural or synthetic origin. Furthermore, the branch-promoting effects were molecular weight-dependent. In addition, following digestion with lysozyme, chitinase, or chitosanase, digested chitosan was unable to reproduce the similar effects. In all, this study clarifies the specificity and preferential activity of chitosan in enhancing branching morphogenesis of progenitor salivary tissue and highlights its potential utility for application in salivary tissue regeneration.     

Effects of hepatocyte growth factor (HGF) and activin a on the morphogenesis of rat submandibular gland-derived epithelial cells in serum-free collagen gel culture

Summary To study the mechanisms of morphogenesis in salivary gland regeneration, we have established the RSMG-1 cell line derived from submandibular gland (SMG) of 10-wk-old Wistar female rats in serum-free culture. Our finding that RSMG-1 cells originated from duct cells was based on morphology and immunohistochemical results. In three-dimensional serum-free collagen gel culture, HGF induced branching morphogenesis of RSMG-1 cells. Histological examination revealed that HGF-induced branching structure exhibited well-formed lumina. This morphology closely resembles that found in vivo. The cells also expressed activin A. Exogenously added activin A at a high concentration reduced HGF-induced branching morphogenesis. These findings suggest that the morphogenesis of the salivary gland is modulated by HGF and activin A. Our results show that the RSMG-1 cell line may be useful in studies of salivary gland regeneration.     

Signaling pathways activated by epidermal growth factor receptor or fibroblast growth factor receptor differentially regulate branching morphogenesis in fetal mouse submandibular glands

Although growth factor signaling is required for embryonic development of organs, individual signaling mechanisms regulating these organotypic processes are just beginning to be defined. We compared signaling activated in fetal mouse submandibular glands (SMGs) by three growth factors, epidermal growth factor (EGF), fibroblast growth factor (FGF) 7, or FGF10, and correlated it with specific events of branching morphogenesis. Immunoblotting showed that EGF strongly stimulated phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and weakly stimulated phosphorylation of phospholipase C γ 1 (PLC γ 1) and phosphatidylinositol-3 kinase (PI3K) in cultured E14 SMG. However, FGF7 and FGF10 stimulated phosphorylation of both PLC γ 1 and PI3K, but elicited only minimal phosphorylation of ERK-1/2. Morphological study of mesenchyme-free SMG epithelium cultured in Matrigel revealed that EGF induced cleft formation of endpieces, that FGF7 stimulated both cleft formation and stalk elongation, but that FGF10 induced only stalk elongation. In mesenchyme-free SMG epithelium cultured with EGF, FGF7 and FGF10, U0126 (MEK inhibitor) completely blocked cleft formation, whereas U73122 (PLC γ 1 inhibitor) suppressed stalk elongation. These finding suggest that EGF stimulates cleft formation and drives branch formation via ERK-1/2, and that FGF7 stimulates both cleft formation and stalk elongation via PLC γ 1 and partly via ERK-1/2, but that FGF10 stimulates stalk elongation mainly via PLC γ 1.     

Sucrose stimulates branching morphogenesis of embryonic mouse lung in vitro: a problem of osmotic balance between lumen fluid and culture medium

In organ cultures of lung rudiments from 11-day mouse embryos, it was found that addition of sucrose to the culture medium stimulated branching morphogenesis and reduced lumen distension. Two possible roles of sucrose were postulated: one as a nutrient and another as a generator of osmotic pressure inducing osmosis of water from the lumen fluid to the culture medium across a simple columnar epithelial cell layer. To assess which was the case, branching morphogenesis was investigated in lung rudiments cultured in medium in which osmotic pressure was increased by the addition of lactose or NaCl rather than sucrose: similar acceleration of branching was observed in both. In another experiment, lumen fluid of cultured lung rudiments was mechanically drained each day, and significantly stimulated branching morphogenesis was observed even when sucrose was not added to the culture medium. Heparin is known to induce abnormal lumen distension and inhibits branching morphogenesis. Heparin-induced abnormal morphogenesis was prevented either by the addition of sucrose to the culture medium or by the mechanical drainage of lumen fluid. These results suggest that lumen distension caused by the accumulation of lumen fluid disrupts lung branching morphogenesis in vitro, even when the mechanism of branching morphogenesis is intact.     

Canonical Wnt signaling negatively regulates branching morphogenesis of the lung and lacrimal gland

Key gene families such as FGFs and BMPs are important mediators of branching morphogenesis. To understand whether Wnt genes, and in particular, the canonical Wnt signaling pathway also function in the branching process, we have used a combination of experimental and genetic gain and loss of function approaches to perturb the levels of canonical Wnt signaling in two arborized structures, the lung and the lacrimal gland. Here, we show that the addition of Wnt3a conditioned medium or LiCl strongly represses growth and proliferation of the lung and lacrimal gland, a result that was confirmed in vivo using a dominant stable mutation of beta-catenin conditionally expressed in the lacrimal gland epithelium. In agreement with these data, knockdown of Wnt signaling with beta-catenin morpholinos results in a greater number of branches and increased cell proliferation. In addition, we show that canonical Wnt signaling is able to modulate the levels of Fgf10 and suppress BMP-induced proliferation in the lacrimal gland. Thus, canonical Wnt signaling negatively regulates branching morphogenesis providing a balance to FGFs and BMPs which positively regulate this process. This multilayered control of growth and proliferation ensures that branched structures attain the morphology required to function efficiently.