Sea urchin arylsulfatase,an extracellular matrix component,is involved in gastrulation during embryogenesis

Arylsulfatases (Arses) have been regarded as lysosomal enzymes because of their hydrolytic activities on synthetic aromatic substrates and their lysosomal localization of their enzymatic activities. Using sea urchin embryos, we previously demonstrated that the bulk of Hemicentrotus Ars (HpArs) does not exhibit enzyme activity and is located on the apical surface of the epithelial cells co-localizing with sulfated polysaccharides. Here we show that HpArs strongly binds to sulfated polysaccharides and that repression of the synthesis by HpArs-morpholino results in retardation of gastrulation in the sea urchin embryo. Accumulation of HpArs protein and sulfated polysaccharides on the apical surface of the epithelial cells in sea urchin larvae is repressed by treatment with β-aminopropionitrile (BAPN), suggesting that deposition of HpArs and sulfated polysaccharides is dependent on the crosslinking of proteins such as collagen-like molecules. We suggest that HpArs functions by binding to components of the extracellular matrix.     

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms

Summary A chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of ³H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of ³H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations. The reverse experiment, in which unlabelled quail hypoblast and possibly some mesoblast have been combined with a tritiated host deprived of its hypoblast, also shows the transfer of label from the host to the cellular surface of the graft. A two-way exchange of glucosamine-containing molecules thus occurs in the blastoderm. It is hypothesized that: (1) low molecular weight compounds, macromolecular material, and/or catabolic products, are exchanged between the different germ layers during gastrulation; (2) the components of the extracellular matrix turn over and are continuously changing; (3) this transfer is a possible mechanism of transmission for developmental or inductive signals during embryonic development. The present results also demonstrate the participation of underlying tissue in the biosynthesis of basement membrane components of an epithelium.     

Bufonid nucleocytoplasmic hybrids arrested at the early gastrula stage lack a fibronectin-containing fibrillar extracellular matrix

Summary Most hybrids betweenBufo bufo andB. calamita obtained by nuclear transplantation become arrested at the early gastrula stage. In both parental controls and the hybrid embryos, the presence and distribution of extracellular matrix was analysed with fluorescent wheat germ agglutinin and by immunolabelling with antibodies directed against fibronectin. InB. bufo andB. calamita gastrulae and in the few hybrids that complete gastrulation, the inner surface of the blastocoel roof is covered by a fibronectin-rich fibrillar matrix. In nucleocytoplasmic hybrids whose development was arrested at the gastrula stage, the fibronectin-containing extracellular matrix was either totally absent or poorly developed and disorganized.     

Extracellular matrix formation by amebocytes during epithelial regeneration in the pearl oysterPinctada fucata

Summary To identify the cells which produce the extracellular matrix during bivalve wound healing, we observed epithelial regeneration inPinctada fucata and evaluated the ability of amebocytes to produce the matrix in vitro. Between days 1 and 3 after an ovary was implanted with abiotic material (a shell ball) via an incision, agranular amebocytes formed a sheath, consisting of 10–20 cell layers, between the implant and incised ovarian tissue. Extracellular matrix was deposited in the spaces between the amebocytes in the sheath. At the incised follicle, gonadal epithelial cells were attached to the newly formed matrix. When a mantle allograft (2 mm square) was implanted with abiotic material to bring them into close contact, epithelial cells emigrated from the allograft along the surface of the abiotic material where they attached to the newly formed matrix at the sheath of amebocytes. In vitro, agranular amebocytes formed a matrix composed of fibrils with a diameter of 20 nm during a 6-day culture period. Pepsin-digested extract of the cell layer forming the matrix gave protein bands with electrophoretic mobilities identical to - and -sized components of a collagen purified from this animal. The matrix exhibited immunoreaction to antiserum raised against the collagen and was stained by alcian bluc. Thus, the agranular amebocyte apparently has the ability to produce an extracellular matrix containing collagen and possibly proteoglycan(s).     

Extracellular matrix formation by epithelial cells from human polycystic kidney cysts in culture

Cells from the cysts of patients with autosomal dominant polycystic kidney disease (PKD) were grown in vitro under standard conditions without the aid of collagen-pretreated surfaces, and both the synthesis and composition of the extracellular matrix were investigated. At confluence, PKD cells presented the typical features of epithelial cells, but showed a different collagen composition from fibroblasts. Compared with normal tubular epithelia (NTE), PKD monolayers produced an excess of extracellular matrix, which accounted for 30% of the total incorporation of [³H] proline, although this value was considerably lower (by a factor of 10) in the case of NTE. Immunohistochemical and electrophoretic techniques revealed a complex collagen composition in the extracellular matrix which included [α(III)]₃ and collagen IV. However, part of the collagen components remained unidentified in spite of the fact that they exhibited a typical M_r of α₁(I) and α₂(I) in the presence of urea. Immunoprecipitation with monospecific antibodies and Northern blotting with specific probes failed to recognize α₁(I) and α₂(I), but demonstrated their presence in fibroblasts. Purification and cyanogen bromide digestion demonstrated a strong interhomology in fingerprint peptide composition among the uncharacterized collagens synthesized by PKD cells, thus suggesting a common identity. These observations document a markedly augmented production of extracellular matrix by PKD cultured cells in vitro, and show the presence of collagens which do not share homologies with the major collagen molecules. A better characterization of extracellular matrix composition is central to any comprehension of the cystogenetic mechanisms in vivo.     

Extracellular matrix control of mammary gland morphogenesis and tumorigenesis: insights from imaging

The extracellular matrix (ECM), once thought to solely provide physical support to a tissue, is a key component of a cell’s microenvironment responsible for directing cell fate and maintaining tissue specificity. It stands to reason, then, that changes in the ECM itself or in how signals from the ECM are presented to or interpreted by cells can disrupt tissue organization; the latter is a necessary step for malignant progression. In this review, we elaborate on this concept using the mammary gland as an example. We describe how the ECM directs mammary gland formation and function, and discuss how a cell’s inability to interpret these signals—whether as a result of genetic insults or physicochemical alterations in the ECM—disorganizes the gland and promotes malignancy. By restoring context and forcing cells to properly interpret these native signals, aberrant behavior can be quelled and organization re-established. Traditional imaging approaches have been a key complement to the standard biochemical, molecular, and cell biology approaches used in these studies. Utilizing imaging modalities with enhanced spatial resolution in live tissues may uncover additional means by which the ECM regulates tissue structure, on different length scales, through its pericellular organization (short-scale) and by biasing morphogenic and morphostatic gradients (long-scale). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A transitional extracellular matrix instructs cell behavior during muscle regeneration

Urodele amphibians regenerate appendages through the recruitment of progenitor cells into a blastema that rebuilds the lost tissue. Blastemal formation is accompanied by extensive remodeling of the extracellular matrix. Although this remodeling process is important for appendage regeneration, it is not known whether the remodeled matrix directly influences the generation and behavior of blastemal progenitor cells. By integrating in vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging, we show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion. These data indicate that both satellite cells and fragmenting myofibers contribute to the regeneration blastema and that the local extracellular environment provides instructive cues for the regenerative process. The fact that amphibian and mammalian myoblasts exhibit similar responses to various matrices suggests that the ability to sense and respond to regenerative signals is evolutionarily conserved.     

Extracellular matrix influences hormone and protein production by human chorionic villi

Summary Increasing evidence confirms that the extracellular matrix greatly influences cell behaviour and function. Collagen and fibrin are in contact with trophoblast throughout pregnancy. To investigate whether these two matrices influence hormon production by the trophoblast, explants from first-trimester chorionic villi were cultured for up to 30 days either a) in medium with agitation, b) embedded in type-I collagen (three-dimensional gels), or c) embedded in fibrin (three-dimensional gels). The supernatant culture medium was changed every 48 h and tested by radioimmunoassay for hCG, progesterone and pregnancy-associated plasma protein A. In addition, after 3, 7, 15, and 30 days of culture villi were fixed and studied by light and electron microscopy. Embedding in the extracellular matrix showed higher and longer-lasting production rates of all measured products and superior structural preservation as compared to cultures with agitation. Collagen matrix proved to be superior to fibrin. As established by several tests, this difference was neither due to thrombin used to polymerize fibrinogen, nor to differences in the diffusion rates through the two different matrices used. We conclude that extracellular matrix, particularly collagen, influences the synthesis of trophoblastic products. Embedding of the villous explants in three-dimensional gels constitutes a new method for long-term cultures of chorionic villi.This study was presented at the workshop Placental-and decidual-specific protein synthesis and secretion: regulation, role and interaction, Zemun, Belgrade, Yugoslavia, 19–20 May, 1988 (Bischof and Castellucci 1988; see also J. Aplin 1989), and at the 11th Rochester Trophoblast Conference, Rochester, N.Y. USA, 9–12 October 1988 (Castellucci et al. 1988)     

Sid4: A secreted vertebrate immunoglobulin protein with roles in zebrafish embryogenesis

The small members of the immunoglobulin superfamily (IGSF) are a molecularly diverse group of proteins composed solely of immunoglobulin domains. They may be secreted or tethered to the cell mebrane via GPI linkages and are proposed to have important functions in vivo. However, very few small IGSFs have been functionally characterized. During an ongoing in situ hybridization analysis of expressed sequence tags in zebrafish we identified secreted immunoglobulin domain 4 (sid4), a gene encoding a soluble vertebrate protein composed solely of four immunoglobulin domains. Throughout development, sid4 is expressed in regions of the embryo undergoing active cell division and migration. Functional analysis using morpholino antisense oligonucleotides demonstrates that timing of gene expression is normal in morphants, but these embryos are smaller and exhibit defects in epiboly and patterning of axial and prechordal mesoderm. Analyses of chordin, pax2, krox20, and dlx2 expression in morphants demonstrate that early brain patterning is normal but later organization of hindbrain neurons and development of cranial neural crest are perturbed. Levels of apoptosis in morphants were normal prior to 90% epiboly, but were elevated after 10 h post-fertilization (hpf). Apoptosis does not account for early patterning defects of axial mesoderm, but likely contributes to overall reduction in embryo size. Phylogenetic analysis demonstrates that Sid4 is strikingly similar to the fibronectin binding Ig domains of Perlecan/HSPG2. Overall, our data demonstrate a fundamental role for sid4, possibly as a co-factor in extracellular matrix (ECM) interactions, in processes underlying tissue patterning and organogenesis in a vertebrate.     

A dynamic spatiotemporal extracellular matrix facilitates epicardial-mediated vertebrate heart regeneration

Unlike humans, certain adult vertebrates such as newts and zebrafish possess extraordinary abilities to functionally regenerate lost appendages and injured organs, including cardiac muscle. Here, we present new evidence that a remodeled extracellular matrix (ECM) directs cell activities essential for cardiac muscle regeneration. Comprehensive mining of DNA microarrays and Gene Ontology term enrichment analyses for regenerating newt and zebrafish hearts revealed that distinct ECM components and ECM-modifying proteases are among the most significantly enriched genes in response to local injury. In contrast, data analyses for mammalian cardiac injury models indicated that inflammation and metabolic processes are the most significantly activated gene groups. In the regenerating newt heart, we show dynamic spatial and temporal changes in tenascin-C, hyaluronic acid, and fibronectin ECM distribution as early as 3 days postamputation. Linked to distinct matrix remodeling, we demonstrate a myocardium-wide proliferative response and radial migration of progenitor cells. In particular, we report dramatic upregulation of a regeneration-specific matrix in the epicardium that precedes the accumulation and migration of progenitor cells. For the first time, we show that the regenerative ECM component tenascin-C significantly increases newt cardiomyocyte cell cycle reentry in vitro. Thus, the engineering of nature-tested extracellular matrices may provide new strategic opportunities for the enhancement of regenerative responses in mammals.     

Extracellular matrix in ovarian follicular development and disease

The ovarian follicle contains several different cell types and separate compartments and undergoes substantial development during its growth and maturation. Extracellular matrix (ECM) could be expected to play a major role in these processes. Most research on ECM in follicles has focused on the follicular basal lamina and its changing composition during folliculogenesis and on the specialised matrix formed at ovulation by the cumulus cells surrounding the oocyte and the zona pellucida. We review these aspects. Few naturally occurring gene mutations have identified unique roles for ECM molecules in follicular function. Presumably, any mutations leading to reduced fertility are eliminated quickly by natural selection and, when mutations are not eliminated, considerable redundancy occurs to ensure successful reproduction. In mice, in which the genome can be easily manipulated, the modification of matrix components associated with cumulus and oocytes has often resulted in partial infertility, suggesting redundancy. We provide an update of basal lamina components focusing on newer discoveries. In addition, we review matrix associated with the occyte and cumulus cells (excluding the zona pellucida) and other components of ECM. Where possible, we examine evidence for the role of the ECM in follicular development and diseases.Research in the authors' laboratories has been supported by the National Health and Medical Research Council of Australia, The University of Adelaide, Wellcome Foundation, and the Clive and Vera Ramaciotti Foundation.     

Extracellular matrix fibrils and cell contacts in the chick embryo

Summary The migration of neural crest and sclerotome cells and the extension of ventral root axons in chick embryos at stages 16–20 were studied by light microscopy as well as scanning and transmission electron microscopy at the leg bud level of fixed specimens. Extensive cellular movements take place in association with an extracellular matrix consisting of microfibrils. The neural crest and sclerotome cells migrate into the large matrix-filled extracellular space surrounding the neural tube and notochord, apparently using microfibril bundles as substratum. The cells exhibit pseudopodia which are closely associated with the matrix fibrils. The fibrils around the notochord show a spatial arrangement indicating that the sclerotome cells are contact-guided to their subsequent positions. Mutual cell contacts, including those established by cell processes, frequently show cytoplasmic electron dense plaques at adjacent membranes. These small plaque contacts might be correlated to contact inhibition of locomotion between the cells and participate in the guidance of cells. The growth cones of extending axons exhibit filopodia contacting both surrounding mesenchyme cells and extracellular fibrils. The orientation of the axons might thus be affected by contacts with cell surfaces as well as with extracellular material.Technical assistance was given by Mrs. Kerstin Ahlfors, Mrs. Charlotte Fällström, Mrs. Annika Kylberg and Mrs. Stine SöderströmSupported by grants from The Swedish Natural Science Research Council     

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.     

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal. Received: 19 August 1997 / Accepted: 11 December 1997     

Actin dynamics at sites of extracellular matrix degradation

The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.     

Extracellular matrix-dependent myosin dynamics during G1-S phase cell cycle progression in hepatocytes

Cell spreading and proliferation are tightly coupled in anchorage-dependent cells. While adhesion-dependent proliferation signals require an intact actin cytoskeleton, and some of these signals such as ERK activation have been characterized, the role of myosin in spreading and cell cycle progression under different extracellular matrix (ECM) conditions is not known. Studies presented here examine changes in myosin activity in freshly isolated hepatocytes under ECM conditions that promote either proliferation (high fibronectin density) or growth arrest (low fibronectin density). Three different measures were obtained and related to both spreading and cell cycle progression: myosin protein levels and association with cytoskeleton, myosin light chain phosphorylation, and its ATPase activity. During the first 48 h in culture, corresponding with transit through G1 phase, there was a six-fold increase in both myosin protein levels and myosin association with actin cytoskeleton. There was also a steady increase in myosin light chain phosphorylation and ATPase activity with spreading, which did not occur in non-spread, growth-arrested cells on low density of fibronectin. Myosin-inhibiting drugs blocked ERK activation, cyclin D1 expression, and S phase entry. Overexpression of the cell cycle protein cyclin D1 overcame both ECM-dependent and actomyosin-dependent inhibition of DNA synthesis, suggesting that cyclin D1 is a key event downstream of myosin-dependent cell cycle regulation.     

Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5 h, while a significant inhibition was recorded at 24 h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.     

Fibronectin alters the rate of formation and structure of the fibrin matrix

Plasma fibronectin is a vital component of the fibrin clot; however its role on clot structure is not clearly understood. The goal of this study was to examine the influence of fibronectin on the kinetics of formation, structural characteristics and composition of reconstituted fibrin clots or fibrin matrices. Fibrin matrices were formed by adding thrombin to 1, 2 or 4 mg/ml fibrinogen supplemented with 0–0.4 mg/ml fibronectin. The rate of fibrin matrix formation was then monitored by measuring light absorbance properties at different time points. Confocal microscopy of fluorescein conjugated fibrinogen was used to visualize the structural characteristics of fibrin matrices. The amount of fibronectin in fibrin matrices was determined through electrophoresis and immunoblotting of solubilized matrices. Fibronectin concentration positively correlated with the initial rate of fibrin matrix formation and with steady state light absorbance values of fibrin matrices. An increase in fibronectin concentration resulted in thinner and denser fibers in the fibrin matrices. Electrophoresis and immunoblotting showed that fibronectin was covalently and non-covalently bound to fibrin matrices and in the form of high molecular weight multimers. The formation of fibronectin multimers was attributed to cross-linking of fibronectin by trace amounts Factor XIIIa. These findings are novel because they link results from light absorbance studies to microcopy analyses and demonstrate an influence of fibronectin on fibrin matrix structural characteristics. This data is important in developing therapies that destabilize fibrin clots.     

Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2

Extracellular matrix protein 1 (ECM1), a widely expressed glycoprotein, has been shown to harbor mutations in lipoid proteinosis (LP), an autosomal recessive disorder characterized by profound alterations in the extracellular matrix of connective tissue. The biological function of ECM1 and its role in the pathomechanisms of LP are unknown. Fibulins comprise a family of extracellular matrix components, and the prototype of this family, fibulin-1, is expressed in various connective tissues and plays a role in developmental and pathologic processes. In this study, we demonstrate that ECM1, and specifically the second tandem repeat domain which is alternatively spliced, interacts with the C-terminal segments of fibulins 1C and 1D splice variants which differ in their C-terminal domain III. The interactions were detected by yeast two-hybrid genetic system and confirmed by co-immunoprecipitations. Kinetics of the binding between ECM1 and fibulin-1D, measured by biosensor assay, revealed a K(d) of 5.71 x 10(-8) M, indicating a strong protein-protein interaction. Since distinct splice variants of ECM1 and fibulin-1 have been shown to be co-expressed in tissues affected in LP, we propose that altered ECM1/fibulin-1 interactions may play a role in the pathogenesis of this disease as well as in a number of processes involving the extracellular matrix of connective tissues.