Primitive streak-forming cells of the chick invaginate through a basement membrane

Summary Recently fibronectin was shown to appear in the development of the chick for the first time as a thin band on the epiblastic side facing the hypoblast just prior to primitive streak formation. It was thus suggested that fibronectin might be instrumental in the migration of cells that lead to axis formation during primitive streak formation. In the present work we have examined simultaneously for the presence of fibronectin and the specific basement membrane glycoprotein laminin during primitive streak formation using immunofluorescence methods. Laminin was found to be expressed between the epiblast and the hypoblast of stage XIII¹ chick blastoderms. During the immediately following process of streak formation the laminin was found to be continuously detectable throughout the area covered by the hypoblast, but disrupted on the streak area. Fibronectin was found to co-distribute with laminin in stage XIII and in the early primitive streak chick blastoderms. It is concluded that at stage XIII laminin and fibronectin form part of a basement membrane that is partially disrupted during the immediately following process of primitive streak formation in order to allow the migration of the streak-forming epiblastic cells during this morphogenetic process.     

Activin can generate ectopic axial structures in chick blastoderm explants.

We have recently shown that activin can induce the formation of axial structures from chick blastulae and that activin beta-B is transcribed, in the hypoblast of the chick, at the same stage that axial mesoderm is being induced. It was not clear, however, whether activin was merely allowing the central epiblastic cells to express a differentiated phenotype for which they were already prepared. This report shows that activin-containing medium (ACM) can act as an instructive inductor, which can change the fate of competent cells and bring about the formation of an ectopic embryonic axis. Furthermore, we show data that suggest that during normal development only one axis is obtained as a result of a carefully controlled inhibitory process.     

Fibronectin in the development of embryonic chick eye.

The time course of appearance and distribution of fibronectin in the developing eye have been studied in chick embryos by indirect immunofluorescence. At the 12-somite stage, fibronectin was detected as a layer under the ectodermal cells overlying the forebrain vesicle; it was also present in the head mesenchyme. During formation of the lens placode and its invagination, a zone containing fibronectin persisted around the lens as a component of the capsule. The fibronectin-containing layer was separated from the corneal epithelial cells during the formation of the acellular stroma. The migrating corneal endothelial cells were seen posterior to the fibronectin layer. The secondary stroma was strongly positive for fibronectin. Fibronectin disappeared from the cornea starting from its posterior part along with the corneal condensation. In the newborn chicken cornea, fibronectin was present only in Descemet's membrane. In addition, the embryonic vitreous body had a network of fibronectin-containing material. The distribution of fibronectin in the developing cornea, as well as other data available on this glycoprotein, is consistent with the proposed role of fibronectin in positioning and migration of cells and in organization of the extracellular matrix.     

Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1^lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation.     

Ultrastructural localization of fibronectin in bone marrow of the embryonic chick and its relationship to granulopoiesis

Summary Fibronectin was immunolocated in embryonic chick bone marrow by the use of both a direct peroxidase conjugated antiserum and an indirect Streptavidin bridge technique. Fibronectin is located in the extravascular granulopoietic compartment and, to a lesser extent, in the vascular, erythropoietic compartment. There is no evidence of fibronectin being associated with blood-stromal cell interactions involving either erythropoiesis or thrombopoiesis. However, mature thrombocytes display a substantial surface coat containing fibronectin. Much of the fibronectin appears to be situated on surfaces of those fibroblastic stromal cells which support granulopoiesis. Fibronectin containing extracellular material connects surfaces of developing granulocytes with surfaces of stromal cells. Fibronectin is a surface component of granulocytes as well as nearby stromal cells. However, there appear to be fewer ferritin particles per unit of surface on granulocytic cells. Many of the ferritin particles are not clearly associated with amorphous matrix material at cell surfaces. Immunocytochemical attempts to identify laminin were unsuccessful. These studies indicate that fibronectin is situated at sites where it could mediate adhesive interaction between granulopoietic cells and their stromal cells. Furthermore, cell surface-matrix interaction involving fibronectin could mediate migration of blood cells within the extravascular spaces.     

Cells from Early Chick Embryos in Culture

Just prior to streak formation (Stage XIII) the two layered chick blastoderm is formed by the one layer epiblast which needs the influence of the hypoblastic layer to develop an embryonic axis. A study has been made of this latest possible stage in the development of the chick in which one cell population, the epiblast, is still totipotential. The intention being to examine in particular the differentiation capacities of these cells in culture and at the same time to compare them with hypoblastic cells. In studying differentiation we have attempted to minimize heterogeneity of the starting cell population by culturing either hypoblastic cells or epiblastic cells. The epiblastic cells were derived from epiblasts deprived of the marginal zone and of the area opaca. Hypoblastic cells formed a one cell thick characteristic epithelium. Epiblastic cells in culture were found to evolve from a homogenous sheet to clearly demarcated areas to dome structures which resemble embryoid bodies from teratocarcinomas. Histologically three main tissue types were found in the epiblastic cultures. Sometimes the borderline between two of the tissue types was found to be clearly demarcated by a basement membrane. Both hypoblastic and epiblastic cells produced a basement membrane-like structure when cultured in vitro. The appearance of mesoderm in the epiblastic cultures was particularly interesting and it was evident by the appearance of blood islands and clearly defined endothelial-lined cavities. No complex organized embryonic structures of any kind were found in the cultures.     

Mesoblastic Cells in the Early Phase of Primitive Streak Formation in Chick Embryos. Observations by Scanning Electron Microscopy in ovo and time-Lapse Cinematography in vitro.

During primitive streak formation in the chick embryo, mesoblastic cells were observed by SEM after removal of the hypoblast layer. Before the primitive streak began to develop, numbers of bleb cells and bleb-like protrusions were seen on the ventral surface of the epiblast. From optical observation on the process of change of epiblastic cells into bleb cells in vitro , it was concluded that cells that had elongated became bleb cells when they emerged from the epiblast. Cell behavior during primitive streak formation is discussed on the basis of these findings.     

The accumulation of basement membrane components during the onset of chondrogenesis and myogenesis in the chick wing bud

The study describes the distribution of several basement membrane molecules in the embryonic chick wing bud from stages 23 to 26, during the onset of myogenesis and chondrogenesis, and then later at stage 28. Laminin is localized as early as stage 23, prior to the onset of myogenesis, in regions corresponding to the position of the future dorsal and ventral myogenic areas. Other matrix components, including fibronectin, do not differentially accumulate in these same regions. Fibronectin, basement membrane heparan sulphate proteoglycan and type IV collagen are more widespread in their distribution than laminin, and are even present between mesenchymal cells. These results suggest a role for laminin in the initial differentiation of the muscle masses and emphasize that components of basement membrane can also be associated with mesenchymal cells.     

Expression of the cell surface-associated glycoprotein, fibronectin, in the early mouse embryo.

The expression of the cell surface-associated glycoprotein fibronectin was studied by indirect immunofluorescence in the early stages of mouse embryogenesis. Fibronectin was not detectable in early preimplantation embryos. Trace amounts of the protein were first found between the cells of the inner cell mass of late blastocysts. In implanted early egg cylinders, fibronectin was deposited between the ectoderm and endoderm of the inner cell mass and in the nascent Reichert's membrane. With development, the visceral and the parietal endoderm cells became positive for the protein, but no fibronectin was detected in ectoderm cells. During segregation of mesoderm from ectoderm, fibronectin appeared in mesoderm cells and as a band between the two germ layers. In the developing amnion and chorion, the protein was localized between the ectodermal and mesodermal cell layers. The results indicate that fibronectin is an early differentiation market for the stage of endoderm formation in the inner cell mass of the mouse blastocyst. It is also a marker of mesoderm appearance and seems to be associated with the accumulating extracellular matrix material in the developing embryo.     

Developmental stage dependent expression of the endothelial stress fibers and organization of fibronectin fibrils in the aorta of chick embryos

Organizational relationships between endothelial stress fibers and fibronectin fibrils in the developing chick abdominal aorta, from 5th day embryos to 3rd day young chicks, were studied with immunofluorescence and electron microscopy. Stress fibers, axially aligned parallel to the longitudinal cell axis, were expressed in the largely elongated endothelial cells, in embryos older than 8th day of incubation. Fibronectin fibrils in the aortic basal lamina, changed its organizational pattern from the network-like form to the straight bundles arranged parallel to the vessel's longitudinal axis after 9th day of incubation. Such axial alignment was dominant in the matrix beneath the elongated cells containing stress fibers, suggesting the existence of stress fibers may possibly modify the fibronectin's organizational pattern. The vinculin-containing dense plaque, which shaped like as the adhesion plaque in the cultured cells, was located at the ends of or lateral associating sites of stress fibers in embryos older than 8th day stage. The expression of stress fibers, as well as the formation of stress fiber's end plaques, may closely relate to the alignment between the stress fiber and fibronectin fibrils in the extracellular matrix.     

Fibronectin has a dual role in locomotion and anchorage of primary chick fibroblasts and can promote entry into the division cycle

Fibronectin (FN), which is already known to be a natural factor for fibroblast spreading on substrata, has now been shown to be essential for two distinct types of adhesion with different biological functions in chick heart fibroblasts, namely adhesion directed toward locomotion and toward stationary anchorage for growth. Manipulation of culture conditions and the use of antisera of differing specificities has demonstrated that both exogenous and cell-derived FN are important in each process. The organization of the fibronectin-containing matrix differs between the two states. Immunoelectron microscopy with a colloidal gold marker reveals the presence of small membrane-associated plaques of fibronectin in motile cells with associated submembranous specialization. A fibrillar matrix containing fibronectin is dominant in nonmotile, growing fibroblasts. The development of focal adhesions for stationary anchorage can be dramatically enhanced by addition of cell-derived FN at an appropriate stage, and this promotes entry into the growth cycle. New macromolecular synthesis in addition to FN is necessary for focal adhesion development but not for locomotion.     

Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ.

The distribution of the matrix protein fibronectin was studied by indirect immunofluorescence in differentiating mouse molars from bud stage to the stage of dentin and enamel secretion, and compared to that of collagenous proteins procollagen type III and collagen type I. Fibronectin was seen in mesenchymal tissue, basement membranes, and predentin. The dental mesenchyme lost fibronectin staining when differentiating into odontoblasts. Fibronectin was not detected in mineralized dentin. Epithelial tissues were negative except for the stellate reticulum within the enamel organ. Particularly intense staining was seen at the epithelio-mesenchymal interface between the dental epithelium and mesenchyme. Fibronectin may here be involved in anchorage of the mesenchymal cells during their differentiation into odontoblasts. Procollagen type III was lost from the dental mesenchyme during odontoblast differentiation but reappeared with advancing vascularization of the dental papilla. Similarly, procollagen type III present in the dental basement membrane during the bud and cap stages disappeared from the cuspal area along with odontoblast differentiation. Weak staining was seen in predentin but not in mineralized dentin. The staining with anti-collagen type I antibodies was weak in dental mesenchyme but intense in predentin as well as in mineralized dentin.     

Fibronectin in evolution: presence in invertebrates and isolation from Microciona prolifera

Fibronectin is found in the tissues of a series of vertebrates and invertebrates which suggests its appearance with the simplest multicellular organisms. Fibronectin is specifically localized on the surface and on the substrate in the immediate vicinity of some, but not all, dissociated Microciona prolifera cells, suggesting that the expression of fibronectin in this organism might be dependent on cell type and/or developmental stage. Fibronectin has been partially purified and characterized from intact Microciona prolifera tissue on the basis of its immunological and biochemical properties.     

Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen

The trunk neural crest originates by transformation of dorsal neuroepithelial cells into mesenchymal cells that migrate into embryonic interstices. Fibronectin (FN) is thought to be essential for the process, although other extracellular matrix (ECM) molecules are potentially important. We have examined the ability of three dimensional (3D) ECM to promote crest formation in vitro. Neural tubes from stage 12 chick embryos were suspended within gelling solutions of either basement membrane (BM) components or rat tail collagen, and the extent of crest outgrowth was measured after 22 hr. Fetal calf serum inhibits outgrowth in both gels and was not used unless specified. Neither BM gel nor collagen gel contains fibronectin. Extensive crest migration occurs into the BM gel, whereas outgrowth is less in rat tail collagen. Addition of fibronectin or embryo extract (EE), which is rich in fibronectin, does not increase the extent of neural crest outgrowth in BM, which is already maximal, but does stimulate migration into collagen gel. Removal of FN from EE with gelatin-Sepharose does not remove the ability of EE to stimulate migration. Endogenous FN is localized by immunofluorescence to the basal surface of cultured neural tubes, but is not seen in the proximity of migrating neural crest cells. Addition of the FN cell-binding hexapeptide GRGDSP does not affect migration into either the BM gel or the collagen gel with EE, although it does block spreading on FN-coated plastic. Thus, although crest cells appear to use exogenous fibronectin to migrate on planar substrata in vitro, they can interact with 3D collagenous matrices in the absence of exogenous or endogenous fibronectin. In BM gels, the laminin cell-binding peptide, YIGSR, completely inhibits migration of crest away from the neural tube, suggesting that laminin is the migratory substratum. Indeed, laminin as well as collagen and fibronectin is present in the embryonic ECM. Thus, it is possible that ECM molecules in addition to or instead of fibronectin may serve as migratory substrata for neural crest in vivo.     

Adhesion to extracellular materials by neural crest cells at the stage of initial migration

Summary Trunk-level neural anlagen bearing neural crest cells at the stage of initiation of migration were isolated from chick embryos and explanted in serum-free medium onto glass substrates which had previously been treated with extracellular materials. After 0.5–2 h incubation, the expiants were dislodged with a stream of culture medium and the substrate examined for adherent crest cells. Crest cells adhered to collagen gels, and adhered to and spread on adsorbed fibronectin; antiserum to fibronectin prevented adhesion to fibronectin but not to collagen gels. Air-dried collagen gels and collagen solutions were less adhesive, the adhesivity declining with longer drying time and lower collagen concentration. Crest cells adhered poorly to dried gelatin and not at all to adsorbed collagen. Fibronectin increased the adhesion to dried collagen and gelatin. Pretreatment of collagen gels with hyaluronate retarded adhesion. Hyaluronate pretreatment also retarded adhesion to adsorbed fibronectin but only when adsorbed collagen was also present. Pretreatment of collagen gels with the proteoglycan monomer from bovine nasal cartilage had no effect of the adhesion of crest cells, but the proteoglycan almost completely inhibited adhesion to adsorbed fibronectin, but only when absorbed collagen was also present. The results are discussed in terms of the control of migration of neural crest cells by extracellular materials.     

Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis

We have previously characterized monoclonal antibodies against chick brain cells. One of them (14-2B2) brightly stained all capillaries in frozen sections of chick brain. Here we show that this antibody is directed against chick fibronectin. Using this antibody and polyclonal antibodies against laminin, we have studied the development of the vascular extracellular matrix. Vasculogenesis, the development of capillaries from in situ differentiating endothelial cells, was studied in yolk sac blood islands and intraembryonic dorsal aorta. Blood islands produced high levels of fibronectin but not laminin. Early intraembryonic capillaries all expressed fibronectin but little if any laminin. The dorsal aorta of a 6-day-old chick embryo has several layers of fibronectin-producing cells, but is devoid of laminin. Laminin expression commenced at Day 8 and by Day 10 an adult-like distribution was found in the aortic vascular wall. Angiogenesis, the formation of capillaries from preexisting vessels, was studied during brain development. Capillary sprouts invading the neuroectoderm at Embryonic Day 4 migrated in a fibronectin-rich matrix devoid of laminin. Ultrastructural immunolocalization demonstrated the presence of fibronectin exclusively on the abluminal site of the endothelial cells. Beginning on Day 6, laminin codistributed with fibronectin in brain capillaries. We conclude that immature capillaries migrate and proliferate in a fibronectin-rich extracellular matrix, which is subsequently remodeled acquiring basement membrane-like characteristics. We suggest that laminin expression is an early indication of vascular maturation.     

Fibronectin fibril formation involves cell interactions with two fibronectin domains

Fibronectin fragments and domain-specific antibodies have been used to study the mechanism by which cells reorganize exogenous fibronectin substrata into fibrils. Fibroblasts prevented from protein synthesis, and hence not secreting endogenous fibronectin or other matrix components, reorganized exogenous fibronectin substrata into arrays resembling the matrix of normally cultured cells. Cells also formed fibrils from substrata containing mixtures of cell- and either of two different heparin-binding fibronectin fragments but not from either fragment alone. The gelatin-binding fragment alone or in conjunction with the cell-binding fragment did not promote fibril formation. Antibodies recognizing cell- and either heparin- or the gelatin-binding domains labeled fibrils formed by cells under normal culture conditions or when a substratum of intact fibronectin was used as the sole exogenous source. However, only antibodies recognizing the cell- or either heparin-binding fragment reduced fibrillogenesis from intact fibronectin substrates when added during cell spreading. These data suggest that formation of fibronectin fibrils can occur at the cell surface and that membrane components recognizing the cell- and the heparin-binding domains in fibronectin may cooperate in the assembly process     

Appearance of fibronectin during the differentiation of cartilage, bone, and bone marrow

Fibronectin has been localized by indirect immunofluorescence during the various phases of endochondral bone formation in response to subcutaneously implanted demineralized bone matrix. Its histologic appearance has been correlated with results of biosynthetic experiments. (a) The implanted collagenous bone matrix was coated with fibronectin before and during mesenchymal cell proliferation. (b) During proliferation of mesenchymal precursor cells, the newly synthesized extracellular matrix exhibited a fibrillar network of fibronectin. (c) During cartilage differentiation, the fibronectin in the extracellular matrix was apparently masked by proteoglycans, as judged by hyaluronidase treatment. (d) Differentiating chondrocytes exhibited a uniform distribution of fibronectin. (e) Fibronectin was present in a cottony array around osteoblasts during osteogenesis. (f) The developing hematopoietic colonies revealed fibronectin associated with them. Therefore, it appears that fibronectin is ubiquitous throughout the development of endochondral bone and bone marrow.     

Fibronectin in the area opaca of the young chick embryo

Summary Distribution of fibronectin-like immunoreactivity was studied in the area opaca of the young chick embryo (stages 4–6 HH) by use of the immunofluorescence and protein A-coupled to colloidal gold techniques. Fibronectin, associated to the basement membrane, formed a fibrillar network, the pattern of which changed from the centre to the periphery of the area opaca. At the ultrastructural level, differences in fibronectin distribution were found between non-moving and moving cells. The epithelial-like cells presented fibronectin staining exclusively on their basal side. Actively migrating cells (edge and mesodermal cells) showed immunoreactive material localized around their entire surface and within the cytoplasm. The fibronectin distribution is discussed in relation to three important phenomena taking place during the early growth of the area opaca: (i) anchorage and migration of the edge cells, (ii) modification of cell shape in relation to mechanical tension, and (iii) expansion of the area vasculosa.