Inhibition of Polarizing Activity in the Anterior Limb Bud Is Regulated by Extracellular Factors

Anterior-posterior patterning of the developing limb is largely viewed as a function of polarizing activity. Recent evidence in polydactylous mutants, however, indicates that development of proper pattern also requires the involvement of inhibitory pathways in the anterior limb that prevent secondary polarizing zone formation, thus limiting the number of digits produced. We report the novel finding that grafts of extracellular matrix from the Mouse Posterior Limb Bud-4 cell line can induce supernumerary digits, including digits with posterior phenotype, from anterior chick limb mesenchyme. Unlike previously described mechanisms of pattern specification during limb development, it is shown that the extracellular matrix effect is not associated with release of an active signal. Rather, evidence is presented suggesting that heparan sulfate moieties in extracellular matrix grafts bind an endogenous, extracellular factor involved in inhibition of anterior polarizing activity, leading to derepression of the anterior limb and induction of polarizing zone marker genes including Sonic hedgehog and Bone morphogenetic protein-2.     

Limb morphogenesis in the branchiopod crustacean, Thamnocephalus platyurus, and the evolution of proximal limb lobes within Anostraca

Crustacean limbs exhibit highly diverse morphologies. One major route of diversification is in the number and position of branches arising from the proximal part of the limb. Here I describe development of larvae of the branchiopod crustacean, Thamnocephalus platyurus and describe in detail the development of the thoracic limbs. The thoracic limbs bear proximal branches both medially and laterally. The most proximal branches on either side (gnathobase and pre-epipod) show a similar developmental history: they develop via fusion of two rudiments into a single adult branch. However, phylogenetic analysis suggests that the developmental fusions have distinct evolutionary histories. In one case (gnathobase), the developmental rudiments reflect the ancestral adult morphology of two distinct branches. In the other (pre-epipod), the rudiments are an apparent novelty within the Anostraca and develop into two adult structures in only a single derived family.     

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.     

Gene Expression Profile Following Stable Expression of the Cellular Prion Protein

1. The cellular prion protein (PrPC) is expressed widely in neural and nonneural tissues at the highest level in neurons in the central nervous system (CNS).2. Recent studies indicated that transgenic mice with the cytoplasmic accumulation of PrPC exhibited extensive neurodegeneration in the cerebellum, although the underlying mechanism remains unknown. To identify the genes whose expression is controlled by overexpression of PrPC in human cells, we have established a stable PrPC-expressing HEK293 cell line designated P1 by the site-specific recombination technique.3. Microarray analysis identified 33 genes expressed differentially between P1 and the parent PrPC-non-expressing cell line among 12,814 genes examined. They included 18 genes involved in neuronal and glial functions, 5 related to production of extracellular matrix proteins, and 2 located in the complement cascade.4. Northern blot analysis verified marked upregulation in P1 of the brain-specific protein phosphatase 2A beta subunit (PPP2R2B), a causative gene of spinocerebellar ataxia 12, and the cerebellar degeneration-related autoantigen (CDR34) gene associated with development of paraneoplastic cerebellar degeneration.5. These results indicate that accumulation of PrPC in the cell caused aberrant regulation of a battery of the genes important for specific neuronal function. This represents a possible mechanism underlying PrPC-mediated selective neurodegeneration.     

Midkine Promotes Selective Expansion of the Nephrogenic Mesenchyme during Kidney Organogenesis

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.     

Isolation and characterization of an endodermally derived, proteoglycan-like extracellular matrix molecule that may be involved in larval starfish digestive tract morphogenesis

A monoclonal antibody, anti-Pisaster matrix-1 (anti-PM1) has been developed against an extracellular matrix antigen, Pisaster matrix-1 (PM1) found in embryos and larvae of the starfish Pisaster ochraceus . Pisaster matrix-1 was first observed in endodermal cells of the early gastrula, and shortly thereafter it was secreted into the blastocoel where it accumulated steadily during gastrulation. During the late gastrula stage it also appeared in the extracellular matrix (ECM) of the gut lumen. Immunogold electron microscopy with anti-PM1 revealed that PM1 was found in condensations of ECM associated with blastocoel matrix fibers, in the trans Golgi network, in Golgi-associated vesicles in endoderm and mesenchyme cells and throughout the ECM lining the digestive tract of late gastrula and bipinnaria larvae. When blastula or early gastrula stage embryos were grown in the presence of the PM1 antibody, archenteron elongation, bending and mouth formation failed to occur. Pisaster matrix-1 stained with alcian blue and its assembly could be disrupted with the common inhibitor of O-linked glycosaminoglycan assembly, β-xyloside but not by tunicamycin. It was not sensitive to enzymes that degrade vertebrate proteoglycans. Pisaster matrix-1 is a large (600 kDa) proteoglycan-like glycosaminoglycan, secreted exclusively by endodermal and/or endodermally derived cells that may be necessary for morphogenesis of the mouth and digestive tract of Pisaster ochraceus embryos/larvae.     

Fibroblast Growth Factor 4 Directs Gap Junction Expression in the Mesenchyme of the Vertebrate Limb Bud

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell–cell communication and polarizing signaling are intimately connected.     

Matrix-cytoskeletal interactions in the developing eye

The embryonic avian corneal epithelium in vitro responds to extracellular matrix (ECM) molecules in either soluble or polymerized form by flattening its basal surface, organizing the basal cortical actin cytoskeleton, and stepping up its production of corneal stroma twofold. Embryonic corneal epithelia, like hepatocytes and mammary gland cells, seem to contain heparan sulfate proteoglycan (HSPG) in their plasmalemma, which may interact with actin on the one hand or underlying collagen on the other. Work on the corneal epithelium suggests that, in addition to HSPG, specific glycoprotein receptors for laminin and collagen exist in the basal plasmalemma and play the critical role in actually organizing the basal epithelial cytoskeleton. As yet, uncharacterized proteins may link such receptors to actin. We suggest that ECM-dependent organization of the cytoskeleton is responsible for ECM enhancement of corneal epithelial differentiation. Cell shape and exogenous ECM also affect mesenchymal cell differentiation. In the case of the corneal fibroblast migrating in collagen gels, an actin cortex present around the elongate cell seems to interact with myosin in the cytosol to bring about pseudopodial extension. Both microtubules and actin microfilaments are involved in fibroblast elongation in collagen gels. It follows from the rules presented in this review that the mesenchymal cell surface is quite different from the epithelial cell surface in its organization. Nevertheless, epithelial cell surface-ECM interaction can be modified in the embryo at particular times to permit predesignated epithelial-mesenchymal transformations, as for example at the primitive streak. Though basal surfaces of definitive, nonmalignant epithelia adhere rather strictly to the rules of epithelium-ECM interaction and do not invade underlying ECM, the environment can be manipulated in vitro to cause these epithelia to send out pseudopodia and give rise aberrantly to mesenchymal cells in collagen gels. Further study of this phenomenon should cast light on the manner in which epithelial and mesenchymal cells organize receptors for matrix molecules on their cell surfaces and develop appropriate cytoskeletal responses to the extracellular matrix.     

Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.     

Epithelial morphogenesis in mouse embryonic submandibular gland: Its relationships to the tissue organization of epithelium and mesenchyme

Epithelial tissues in various organ rudiments undergo extensive shape changes during their development. The processes of epithelial shape change are controlled by tissue interactions with the surrounding mesenchyme which is kept in direct contact with the epithelium. One of the organs which has been extensively studied is the mouse embryonic submandibular gland, whose epithelium shows the characteristic branching morphogenesis beginning with the formation of narrow and deep clefts as well as changes in tissue organization. Various molecules in the mesenchyme, including growth factors and extracellular matrix components, affect changes of epithelial shape and tissue organization. Also, mesenchymal tissue exhibits dynamic properties such as directional movements in groups and rearrangement of collagen fibers coupled with force-generation by mesenchymal cells. The epithelium, during early branching morphogenesis, makes a cell mass where cell-cell adhesion systems are less developed. Such properties of both the mesenchyme and epithelium are significant for considering how clefts, which first appear as unstable tiny indentations on epithelial surfaces, are formed and stabilized.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Cross-talk between mesenchyme and epithelium increases H19 gene expression during scattering and morphogenesis of epithelial cells

The H19 gene is an imprinted gene expressed from the maternal allele. It is known to function as an RNA molecule. We previously reported that in breast adenocarcinoma, H19 is often overexpressed in stromal cells and preferentially located at the epithelium/stroma boundary, suggesting that epithelial/mesenchymal interactions can control H19 RNA expression. In some cases of breast adenocarcinoma with poor prognosis, H19 is overexpressed in epithelial cells. Therefore we examined whether mesenchymal factors can induce H19 expression in epithelial cells. Using quantitative RT-PCR and in situ hybridization, we found that when mammary epithelial cells were cultured in collagen gels, H19 expression was strongly up-regulated compared to when cells were cultured on plastic. Collagen gels allow three-dimensional growth of epithelial cells and morphogenetic responses to soluble factors. A conditioned medium from MRC-5 fibroblasts caused branching morphogenesis of HBL-100 cells and invasive growth of MDA-MB-231 cells, whereas MCF-7 cells were unresponsive. Induction of H19 expression correlated with morphological changes in HBL-100 and in MDA-MB-231 cells, whereas H19 expression was not induced in MCF-7 cells. Using a blocking antibody, HGF/SF was identified as the fibroblast-derived growth factor capable of inducing H19 expression and cell morphogenesis. We further demonstrated that H19 promoter activity was stimulated by various growth factors using transient transfection in MDCK epithelial cells. HGF/SF was more efficient than EGF or FGF-2 in transactivating the H19 promoter, whereas IGF-2, TGFbeta-1, and TNF-alpha were ineffective. This activation by HGF/SF was prevented by pharmacological inhibition of MAP kinase or of phospholipase C. We conclude that H19 is a target gene for HGF/SF, a known regulator of epithelial/mesenchymal interactions, and suggest that the up-regulation of H19 may be implicated in morphogenesis and/or migration of epithelial cells.     

Interdigital cell death in the embryonic limb is associated with depletion of Reelin in the extracellular matrix

Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which has important functions in the developing nervous system, including neuronal survival; however, the significance of Reelin in other systems has received very little attention. We show that reelin expression becomes intensely downregulated in both the chick and mouse interdigits preceding the establishment of the areas of interdigital cell death. Furthermore, fibroblast growth factors, which are cell survival signals for the interdigital mesoderm, intensely upregulated reelin expression, while BMPs, which are proapototic signals, downregulate its expression in the interdigit. Gene silencing experiments of reelin gene or its intracellular effector Dab-1 confirmed the implication of Reelin signaling as a survival factor for the limb undifferentiated mesoderm. We found that Reelin activates canonical survival pathways in the limb mesoderm involving protein kinase B and focal adhesion kinase. Our findings support that Reelin plays a role in interdigital cell death, and suggests that anoikis (apoptosis secondary to loss of cell adhesion) may be involved in this process.     

Expression and Role of Integrins in Invasive Activity of Oncotransformed Fibroblasts Differing in Spontaneous Metastasizing

Four closely related lines of RSV-transformed Syrian hamster fibroblasts differing drastically in their spontaneous metastatic capacity were investigated for the surface expression of integrins, in vitro invasion, and production of MMP-2 collagenase. The highly metastasizing HET-SR-2SC-LNM cells differ from the lowly metastasizing parental HET-SR cells in a high level of the surface expression of the collagen-specific alpha1beta1, alpha2beta1, and alphavbeta3 integrins, a high invasive activity, and an increased production of MMP-2. The same properties are characteristic for the actively metastasizing cells of the independent HET-SR-1 line. The lowly metastasizing fibroblasts that are derived from HET-SR-2SC-LNM retain a high level of the expression of the alpha1beta1 and alpha2beta1 integrins, but, unlike the parental line, they exhibit a decreased expression of the alphavbeta3 integrin, invasion in Matrigel, and MMP-2 production. Substrate stimulation of the signal function of the collagen-specific integrins increases the production of MMP-2 by the metastatically active fibroblasts. Inhibition of the signal activity of the integrins by RGD-containing pentapeptide or by genistein reduces markedly in vitro invasion in Matrigel and MMP-2 production. The role of specific properties of the extracellular matrix surrounding tumor cells and of specific surface integrins expressed in these cells in developing of the malignant phenotype is discussed.     

Localization of transforming growth factor beta receptor II interacting protein-1 in bone and teeth: implications in matrix mineralization

Transforming growth factor beta receptor II (TGFβR-II) interacting protein 1 (TRIP-1) is a WD-40 protein that binds to the cytoplasmic domain of the TGF-β type II receptor in a kinase-dependent manner. To investigate the role of TRIP-1 in mineralized tissues, we examined its pattern of expression in cartilage, bone, and teeth and analyzed the relationship between TRIP-1 overexpression and mineralized matrix formation. Results demonstrate that TRIP-1 was predominantly expressed by osteoblasts, odontoblasts, and chondrocytes in these tissues. Interestingly, TRIP-1 was also localized in the extracellular matrix of bone and at the mineralization front in dentin, suggesting that TRIP-1 is secreted by nonclassical secretory mechanisms, as it is devoid of a signal peptide. In vitro nucleation studies demonstrate a role for TRIP-1 in nucleating calcium phosphate polymorphs. Overexpression of TRIP-1 favored osteoblast differentiation of undifferentiated mesenchymal cells with an increase in mineralized matrix formation. These data indicate an unexpected role for TRIP-1 during development of bone, teeth, and cartilage.     

Development of the pseudothumb in frogs

Frogs have highly conserved hand and foot morphology, possessing four fingers and five toes. As an exception, two Japanese ranid frog species, the Otton frog Babina subaspera and the dagger frog Babina holsti, possess a unique thumb-like structure (the pseudothumb) in the forelimb, giving an appearance of a total of five fingers on the hand. To obtain insights into the developmental mechanisms that generate this novel character, we investigated the hand morphogenesis of the Otton frog. The unique morphological pattern of the pseudothumb was already established in juveniles. Surprisingly, the bud-like structure, which is similar to the area of inductive activity (e.g. feather buds in birds and the carapacial ridge in turtles), was detected over the site where the future prepollex develops in larvae. By contrast, this bud-like structure was not found in larvae of other ranid species. We discuss possible scenarios that would favour the evolution of this very unusual trait in frogs.     

法乐氏四联症右室流出道心肌间质胶原表达改变

摘要：探讨法乐氏四联症患者右室流出道I、Ⅲ型胶原蛋白mRNA与蛋白质合成的表达改变,以期进一步阐明法乐氏四联症的发病机制．用RT—PCR方法检测32例法乐氏四联症患者与同期17例小室缺患者右室流出道Ⅰ、Ⅲ型胶原mRNA的表达差异,并分析了不同氧饱和度对法乐氏四联症胶原蛋白mRNA表达的影响．同时应用免疫组化图像分析及Western blot检测右室流出道Ⅰ、Ⅲ型胶原蛋白含量并作对比分析．研究发现法乐氏四联症患者右室流出道Ⅰ、Ⅲ型胶原蛋白mRNA与蛋白质合成均较对照组增高并且出现胶原比值改变,缺氧程度与胶原合成关系密切．     

Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.