Obstructor A Organizes Matrix Assembly at the Apical Cell Surface to Promote Enzymatic Cuticle Maturation in Drosophila

Assembly and maturation of the apical extracellular matrix (aECM) is crucial for protecting organisms, but underlying molecular mechanisms remain poorly understood. Epidermal cells secrete proteins and enzymes that assemble at the apical cell surface to provide epithelial integrity and stability during developmental growth and upon tissue damage. We analyzed molecular mechanisms of aECM assembly and identified the conserved chitin-binding protein Obst-A (Obstructor A) as an essential regulator. We show in Drosophila that Obst-A is required to coordinate protein and chitin matrix packaging at the apical cell surface during development. Secreted by epidermal cells, the Obst-A protein is specifically enriched in the apical assembly zone where matrix components are packaged into their highly ordered architecture. In obst-A null mutant larvae, the assembly zone is strongly diminished, resulting in severe disturbance of matrix scaffold organization and impaired aECM integrity. Furthermore, enzymes that support aECM stability are mislocalized. As a biological consequence, cuticle architecture, integrity, and function are disturbed in obst-A mutants, finally resulting in immediate lethality upon wounding. Our studies identify a new core organizing center, the assembly zone that controls aECM assembly at the apical cell surface. We propose a genetically conserved molecular mechanism by which Obst-A forms a matrix scaffold to coordinate trafficking and localization of proteins and enzymes in the newly deposited aECM. This mechanism is essential for maturation and stabilization of the aECM in a growing and remodeling epithelial tissue as an outermost barrier.     

Mammary epithelial cell: influence of extracellular matrix composition and organization during development and tumorigenesis

Stromal-epithelial interactions regulate mammary gland development and are critical for the maintenance of tissue homeostasis. The extracellular matrix, which is a proteinaceous component of the stroma, regulates mammary epithelial growth, survival, migration and differentiation through a repertoire of transmembrane receptors, of which integrins are the best characterized. Integrins modulate cell fate by reciprocally transducing biochemical and biophysical cues between the cell and the extracellular matrix, facilitating processes such as embryonic branching morphogenesis and lactation in the mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal responses to regulate processes including branching morphogenesis and alveolar differentiation. Malignant transformation of the breast is also associated with significant matrix remodeling and a progressive stiffening of the stroma that can enhance mammary epithelial cell growth, perturb breast tissue organization, and promote cell invasion and survival. In this review, we discuss the role of stromal-epithelial interactions in normal and malignant mammary epithelial cell behavior. We specifically focus on how dynamic modulation of the biochemical and biophysical properties of the extracellular matrix elicit a dialogue with the mammary epithelium through transmembrane integrin receptors to influence tissue morphogenesis, homeostasis and malignant transformation.     

Intracellular signalling cascades activated by the EGF receptor and/or by integrins, with potential relevance for branching morphogenesis of the fetal mouse submandibular gland

The fetal mouse submandibular gland grown in vitro is a valuable model system to study epithelio-mesenchymal interactions vital for branching morphogenesis. This basic developmental process is dependent upon simultaneous engagement of receptors for growth factors and components of the extracellular matrix. Here we review intracellular signalling cascades mediated by activation of the EGFR in the developing mouse submandibular gland.     

Local requirement of the Drosophila insulin binding protein imp-L2 in coordinating developmental progression with nutritional conditions

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions.     

Collagen in organ development

It is important to know whether microgravity will adversely affect developmental processes. Collagens are macromolecular structural components of the extracellular matrix (ECM) which may be altered by perturbations in gravity. Interstitial collagens have been shown to be necessary for normal growth and morphogenesis in some embryonic organs, and in the mouse salivary gland, the biosynthetic pattern of these molecules changes during development. Determination of the effects of microgravity on epithelial organ development must be preceded by crucial ground-based studies. These will define control of normal synthesis, secretion, and deposition of ECM macromolecules and the relationship of these processes to morphogenesis.     

Control of Airway Tube Diameter and Integrity by Secreted Chitin-Binding Proteins in Drosophila

The transporting function of many branched tubular networks like our lungs and circulatory system depend on the sizes and shapes of their branches. Understanding the mechanisms of tube size control during organ development may offer new insights into a variety of human pathologies associated with stenoses or cystic dilations in tubular organs. Here, we present the first secreted luminal proteins involved in tube diametric expansion in the Drosophila airways. obst-A and gasp are conserved among insect species and encode secreted proteins with chitin binding domains. We show that the widely used tracheal marker 2A12, recognizes the Gasp protein. Analysis of obst-A and gasp single mutants and obst-A; gasp double mutant shows that both genes are primarily required for airway tube dilation. Similarly, Obst-A and Gasp control epidermal cuticle integrity and larval growth. The assembly of the apical chitinous matrix of the airway tubes is defective in gasp and obst-A mutants. The defects become exaggerated in double mutants indicating that the genes have partially redundant functions in chitin structure modification. The phenotypes in luminal chitin assembly in the airway tubes are accompanied by a corresponding reduction in tube diameter in the mutants. Conversely, overexpression of Obst-A and Gasp causes irregular tube expansion and interferes with tube maturation. Our results suggest that the luminal levels of matrix binding proteins determine the extent of diametric growth. We propose that Obst-A and Gasp organize luminal matrix assembly, which in turn controls the apical shapes of adjacent cells during tube diameter expansion.     

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.     

The extracellular matrix as an adhesion checkpoint for mammary epithelial function

The development of the mammary gland is spatially regulated by the interaction of the mammary epithelium with the extracellular matrix (ECM). Cells receive cues from the ECM through a family of adhesion receptors called integrins, consisting of alpha- and beta-chain dimers. Integrins assist cells in sensing their appropriate developmental context in response to both hormones and growth factors. Here we argue that cell adhesion to the ECM plays a key role in specific developmental checkpoints, particularly in alveolar survival, morphogenesis and function. Specific ablation of alphabeta1-integrins in the luminal epithelium of the mammary gland shows that this sub-type of receptors is required for proliferation, accurate morphological organisation, as well as milk secretion. Downstream, small Rho GTPases mediate cellular polarisation and differentiation. Current challenges in studying the integration of signals in checkpoints of mammary gland development are discussed.     

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.     

Molecular determinants during dental morphogenesis and cytodifferentiation: a review.

Craniofacial development provides a number of opportunities to investigate the cellular and molecular biology of morphogenesis, cytodifferentiation, tissue-specific extracellular matrix (ECM) formations, and biomineralization. Regulatory processes associated with mandibular morphogenesis and specifically tooth formation are being investigated by the identification of when and where molecular determinants such as cell adhesion molecules (CAMs), substrate adhesion molecules (SAMs), and tissue-specific structural gene products are expressed during sequential developmental stages. Based upon in vitro organotypic culture studies in serumless, chemically defined medium, instructive and permissive signaling has been found to be required for both mandibular and dental morphogenesis and cytodifferentiation. For example, intrinsic developmental instructions (autocrine and paracrine factors), independent of long-range hormonal or exogenous growth factors, mediate morphogenesis from the initiation of the dental lamina through crown and initial root stages of tooth development. This review summarizes recent results using experimental embryology, organ culture, recombinant DNA technology, and immunocytology to elucidate mechanisms responsive to instructive epithelial-mesenchymal interactions associated with mandibular morphogenesis, tooth positional information, and subsequent tooth crown and initial root development.     

Nitric Oxide Synthase Regulates Growth Coordination During Drosophila melanogaster Imaginal Disc Regeneration

Mechanisms that coordinate growth during development are essential for producing animals with proper organ proportion. Here we describe a pathway through which tissues communicate to coordinate growth. During Drosophila melanogaster larval development, damage to imaginal discs activates a regeneration checkpoint through expression of Dilp8. This both produces a delay in developmental timing and slows the growth of undamaged tissues, coordinating regeneration of the damaged tissue with developmental progression and overall growth. Here we demonstrate that Dilp8-dependent growth coordination between regenerating and undamaged tissues, but not developmental delay, requires the activity of nitric oxide synthase (NOS) in the prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone biosynthesis, a requirement for imaginal disc growth during both the regenerative checkpoint and normal development. Therefore, NOS activity in the prothoracic gland coordinates tissue growth through regulation of endocrine signals.     

Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.     

Ontogeny of peptide-producing nerves and endocrine cells of the gastro-duodeno-pancreatic region

Summary The ontogeny of different types of endocrine cells and nerves producing different hormonal peptides was studied by immunocytochemistry. The data indicate that several endocrine cell types are more frequent in the foetal than in the adult gastro-duodeno-pancreatic region. Some cell types were found to show an over-lapping distribution. Such distributional over-laps were more frequent in foetuses and neonates than in adults. Both parallel and non-parallel patterns of development of endocrine cells and peptide-producing nerves were detected. Together with available information on the trophic effects of the gastrointestinal hormonal peptides these findings indicate that foetal and neonatal endocrine cells may participate in the regulation of growth and morphogenesis in the gastrointestinal tract.     

Deciphering the Genetic Programme Triggering Timely and Spatially-Regulated Chitin Deposition

Organ and tissue formation requires a finely tuned temporal and spatial regulation of differentiation programmes. This is necessary to balance sufficient plasticity to undergo morphogenesis with the acquisition of the mature traits needed for physiological activity. Here we addressed this issue by analysing the deposition of the chitinous extracellular matrix of Drosophila, an essential element of the cuticle (skin) and respiratory system (tracheae) in this insect. Chitin deposition requires the activity of the chitin synthase Krotzkopf verkehrt (Kkv). Our data demonstrate that this process equally requires the activity of two other genes, namely expansion (exp) and rebuf (reb). We found that Exp and Reb have interchangeable functions, and in their absence no chitin is produced, in spite of the presence of Kkv. Conversely, when Kkv and Exp/Reb are co-expressed in the ectoderm, they promote chitin deposition, even in tissues normally devoid of this polysaccharide. Therefore, our results indicate that both functions are not only required but also sufficient to trigger chitin accumulation. We show that this mechanism is highly regulated in time and space, ensuring chitin accumulation in the correct tissues and developmental stages. Accordingly, we observed that unregulated chitin deposition disturbs morphogenesis, thus highlighting the need for tight regulation of this process. In summary, here we identify the genetic programme that triggers the timely and spatially regulated deposition of chitin and thus provide new insights into the extracellular matrix maturation required for physiological activity.     

Three-dimensional culture models of mammary gland

The mammary gland is a complex tissue comprised of a branching network of ducts embedded within an adipocyte-rich stroma. The ductal epithelium is a bi-layer of luminal and myoepithelial cells, the latter being in contact with a basement membrane. During pregnancy, tertiary branching occurs and lobuloalveolar structures, which produce milk during lactation, form in response to hormonal and cytokine signals. Postlactational regression is characterized by extensive cell death and tissue remodeling. These complex developmental events have been difficult to mimic in cell culture although many useful culture models exist. Recently, considerable advances in three-dimensional modelling of the mammary gland have been made with the use of collagen and other biomaterials for the study of branching morphogenesis and tumorigenesis, techniques which may enable rapid advances in our understanding of both basic biology and the study of cancer therapeutics.Key words: mammary gland, models, extracellular matrix, cell culture, cell-lines, scaffolds, tissue engineering, epithelium, adipocytes     

Drosophila convoluted/dALS Is an Essential Gene Required for Tracheal Tube Morphogenesis and Apical Matrix Organization

Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.     

Some old concepts and new findings on hormonal control of insect morphogenesis

By means of the artificially induced heterochronic developmental deviations represented by local prothetelies and metathetelies it has been possible to investigate the individual developmental fates of ontogenetically different tissues, such as larval, pupal, and adult epidermal cells, in one and the same body and under the identical concentration of juvenile hormone (JH) in the haemolymph.In contrast to the widely accepted hormonal theories which claim that the kind of morphogenesis is determined by large, intermediate, and low titres of JH, the heterochronic character of the tissues never developed into a uniform population of homomorphic epidermal cells. Instead, in the presence of effective amounts of JH the heterochronic pattern has been fully preserved and carried on into the next developmental instar. Moreover, in the absence of the effective JH amounts the ontogenetically different tissues, such as larval and pupal epidermal cells, simultaneously undergo their respective morphogenetic developments, i.e. larval-pupal and pupal-adult morphogenesis in the same hormonal milieu. It is concluded that the selective factor in determination of the kind of morphogenetical changes is not an altered JH titre but the extant, previously attained degree of ontogenetic structural differentiation. It has been demonstrated that JH can temporarily and reversibly inhibit the morphogenetic progress at quite different ontogenetic levels but it cannot cause a ‘reversal of metamorphosis’ at any of these levels.Under specific experimental conditions the larval epidermal cells can undergo pupal and adult morphogenesis without secreting the pupal cuticle. However, the pupal morphogenetic interstage, whether with the cuticle or without the pupal cuticle, constitutes an obligatory developmental step. Further, it appears that an absence of JH may represent an important condition but not a real cause of insect metamorphosis, as presumed in some other hormonal concepts. Thus, chromosomal duplications or cellular divisions in the absence of JH have not committed the cells to morphogenesis unless provided by an additional stimulus of endogenous prothoracic gland hormone or exogenous ecdysterone. An important factor in understanding the hormonal control of insect morphogenesis is the critical timing of the respective morphogenetic steps. This corresponds closely with the duration of the pharate phases in insect development. Possible hormonal mechanisms concerned in the regulation of morphogenesis in endopterygote insects have been outlined.     

Microphthalmia Gene Product as a Signal Transducer in cAMP-Induced Differentiation of Melanocytes

Islets of Langerhans are microorgans scattered throughout the pancreas, and are responsible for synthesizing and secreting pancreatic hormones. While progress has recently been made concerning cell differentiation of the islets of Langerhans, the mechanism controlling islet morphogenesis is not known. It is thought that these islets are formed by mature cell association, first differentiating in the primitive pancreatic epithelium, then migrating in the extracellular matrix, and finally associating into islets of Langerhans. This mechanism suggests that the extracellular matrix has to be degraded for proper islet morphogenesis. We demonstrated in the present study that during rat pancreatic development, matrix metalloproteinase 2 (MMP-2) is activated in vivo between E17 and E19 when islet morphogenesis occurs. We next demonstrated that when E12.5 pancreatic epithelia develop in vitro, MMP-2 is activated in an in vitro model that recapitulates endocrine pancreas development (Miralles, F., P. Czernichow, and R. Scharfmann. 1998. Development. 125: 1017–1024). On the other hand, islet morphogenesis was impaired when MMP-2 activity was inhibited. We next demonstrated that exogenous TGF-β1 positively controls both islet morphogenesis and MMP-2 activity. Finally, we demonstrated that both islet morphogenesis and MMP-2 activation were abolished in the presence of a pan-specific TGF-β neutralizing antibody. Taken together, these observations demonstrate that in vitro, TGF-β is a key activator of pancreatic MMP-2, and that MMP-2 activity is necessary for islet 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.