In human keratinocytes cultured in conditions which allow differentiation and stratification and which are suitable to reconstitute a fully functional epidermis, the integrin alpha-6-beta-4 and two members of the beta-1 integrin family (alpha-2-beta-1 and alpha-3-beta-1) were polarized to the basal and lateral domains of the plasma membrane both in growing colonies and in the reconstituted epidermis. Conversely, alpha-V-beta-5 integrin was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha-6-beta-4 was organized in patches and spots corresponding to F-actin-free submembraneous areas and did not colocalize to focal contacts; moreover, alpha-6-beta-4 colocalized with patches of laminin deposited underneath the ventral membrane of individual cells. The two beta-1 laminin receptor integrins (alpha-2-beta-1 and alpha-3-beta-1) were never found in the basal domain but matched the lateral position of vinculin (but not talin), cingulin and desmoplakins. Only the integrin complex alpha-V-beta-5 was associated with talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The topography of beta-1 and beta-4 integrins reflects a functional role in adhesion and in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta-1 whereas antibodies to beta-4 prevented cell-matrix adhesion.
The spatial relationship of the notochord to the pharyngeal endoderm of 5- to 12-week human embryos was investigated. The light microscopic observations showed a close association of the notochord and endoderm during the 5th embryonic week. Later on, interposition of the mesenchymal cells caused a progressive separation of these two structures. They remained in close apposition only in the area of bursa pharyngea, a deep invagination of the dorsal pharyngeal epithelium. Ultrastructural examination of a 5-week-old embryo revealed cell processes between the juxtaposed notochordal and endodermal cells in the region of the future bursa pharyngea. In already separated areas, mesenchymal cells, well developed basal laminae and small amounts of extracellular matrix were observed in the notochord-endoderm interspace. The observations revealed a sequence of basal lamina formation during notochord-endoderm separation. The stage-dependent lack of basal lamina at the site of the future bursa pharyngea could reflect direct local interactions between notochordal and endodermal cells.
Among 40 notochord cells of an ascidian tadpole larva, 32 notochord cells originate from the anterior-vegetal blastomeres (the A4.1 pair) of an 8-cell embryo and eight cells originate from the posterior-vegetal blastomeres (the B4.1 pair), but the animal blastomeres (the a4.2 and b4.2 pairs) are not engaged in the formation of the notochord. If four pairs of cells, separated from an 8-cell embryo, were allowed to develop into quarter embryos, expression of the notochord-specific antigen was evident in the A4.1 and B4.1 quarter embryos. Embryos, in which cytokinesis had been permanently blocked at the 8-cell and later stages with cytochalasin B, were found to develop the notochord-specific antigen only in the presumptive notochord cells. These findings suggest the developmental autonomy of presumptive notochord cells in the ascidian embryo.
Integrins function at the center of a complex multicomponent system. They act as mechanical connectors linking extracellular molecules to the cytoskeleton and probably also as signal transducers, mediating the transmission of signals from the extracellular matrix to the interior of the cell (for review see Hynes, 1987; Ruoslahti and Pierschbacher, 1987). There is evidence that protein tyrosine and serine/threonine kinases can each modulate integrin function (Hirst et al., 1986; Dustin and Springer, 1990) and it seems likely that other controlling factors will be found. Genetic analysis of such a complex system is likely to prove extremely valuable. I will describe here the early findings of a genetic analysis of the Drosophila PS integrin family. The study has demonstrated important integrin functions during fly development (which I will review briefly) and is beginning to reveal interacting genes which may encode other components of the integrin system or possible constituents of related morphogenetic processes. There are two members of the PS integrin family (Brower et al., 1984) which, like vertebrate integrins, are aft dimers (Wilcox et al., 1984; Leptin et al., 1987). They share a common fl subunit (PSfl) but have different a subunits (PSla and
Sexual cell fusion occurs between HM1 and NC4, heterothallic strains in Dictyostelium discoideum. A membrane component of HM1 cells with a molecular mass of 70 kDa (70K protein) has been shown to be implicated in cell fusion (Urushihara et al. (1988) Cell Differ. Dev. 25, 81-88). In the present study, 70K protein was partially purified using affinity Sepharose on which membrane proteins of NC4 cells were immobilized. Through this process, involvement of Ca2+ in the interaction of 70K protein with its receptor was suggested. Lectin staining of partially purified 70K protein indicated it to be a glycoprotein containing D-mannose and/or D-glucose residues.
Timed morulae of different stages of development were exposed to cytochalasin B causing depolymerisation of microfilaments and to ECCD-1 antibodies interacting with Ca2+-dependent adhesion molecules or cultured in the absence of calcium. All three treatments decompacted mid-morula-stage embryos within one hour. Late morulae were resistant to ECCD-1 antibody treatment and relatively resistant to calcium-free cultivation, but not to cytochalasin B treatment. Scanning electron microscopy revealed that the decompacting treatments not only loosened the interblastomere contacts but also resulted in rearrangement of the cell surface microvilli. Transmission electron microscopy showed that normal, untreated embryos had specialized membrane junctions in the most apical regions of the interblastomere contacts. Immunoelectron microscopy revealed that these apical junction areas contained vinculin, a protein typical of adherent junctions. Upon decompaction the apical junctions disappeared completely. When transferred back to the normal medium, the embryos rapidly started to recompact. Simultaneously the apical junctions and cell surface microvilli reassumed the organization characteristic of the morula stage. Late morulae that were resistant to treatment had normal apical junctional areas. During subcultivation in the normal medium, the treated morulae developed into morphologically normal blastocysts. These data indicate that adherent-type junctions and cell surface microvilli participate in the initiation and maintenance of compaction of morula-stage embryos.
We have developed and characterized a battery of specific polyclonal antibodies directed against specific portions of the alpha-chain of collagen type IV synthesized in Drosophila by the gene DCg1. Here, we describe the use of these antibodies together with in situ hybridization experiments in an attempt to study the expression and localization of collagen type IV during Drosophila oogenesis and early embryogenesis. The results clearly demonstrate that DCg1 is maternally expressed by follicle cells and that the collagen type IV chain produced is stockpiled in the growing oocyte. During the gastrulation stages, this component of Drosophila basement membranes concentrated on cells involved in the gradual invaginations leading to morphogenetic furrows. The presence of collagen type IV, which is an RGD-bearing molecule, during early stages of Drosophila development is discussed in comparison with the crucial, active role its vertebrate counterpart is supposed to play in morphogenetic processes.
This review discusses some of the recent developmental and molecular biological observations that have been made on laminin expression, distribution and neuronal interactions, in an attempt to delineate what might be the physiological function of laminin in the nervous system.
Considering the initial expression of neurotransmitters and neuropeptides immediately after neural induction in amphibian embryos, we previously pointed out that a neuronal cell population emerges from neural plate (NP) and neural fold (NF) expressing very early specific cholinergic, catecholaminergic, GABAergic and peptidergic traits. The purpose of the present work was to investigate the extent to which the neuroblasts that are present in the neurectoderm immediately after gastrulation are committed to give rise to multiple subsets of neurons containing various combinations of neuroactive transmitters rather than to different subpopulations of neurochemically homogeneous neurons. By means of double immunocytochemical localization with a monoclonal TOH-antibody and polyclonal antibodies against GABA or somatostatin, no coexistence of neurotransmitters and neuropeptide was ever found in neuronal subpopulations arising in vitro from NP or NF. The early emergence, under the same conditions, of distinct neuronal subpopulations as a consequence of neural induction strongly suggests that, at the gastrula stage, the neural precursor population most probably does not constitute a homogeneous set of cells.
Neural-crest-derived melanocytes populate two anatomical sites in the chicken, the epidermis of regenerating feathers and the uveal tract of the eyes. These two anatomical populations of melanocytes differ morphologically and functionally. Morphologically, feather and uveal melanocytes synthesize structurally different pigment granules (melanosomes). Feather melanosomes are rod-shaped, 0.2 x 0.8 micron, whereas uveal melanosomes are larger and more oval, 0.6 x 0.9 micron. Functionally, feather melanocytes continuously synthesize melanosomes during feather regeneration, and transfer these melanosomes to neighboring keratinocytes. Ocular melanocytes, on the other hand, synthesize melanosomes until their cytoplasm becomes congested with melanosomes, at which time the melanocytes become melanogenically dormant and do not transfer granules to neighboring cells. Cultures of melanocytes established from neural tubes of Light Brown Leghorn chick embryos produce two populations of melanocytes containing small (0.45 micron) or larger (0.90 micron) melanosomes which resemble the two types described in situ. Both types of melanocytes emigrate from along the entire length of the neural tube during several embryonic stages. Melanocyte cultures developed from neural tubes of the Recessive White breed of chicken, which has tyrosinase-negative, feather melanocytes and pigmented, functionally normal uveal melanocytes, also develop a mixture of amelanotic and pigmented melanocytes which maintain their respective characteristics even after separation by flow cytometry and reculture. These findings suggest that epidermal and uveal melanocytes are two distinct sub-populations of melanocytes whose commitment to separate lineages can occur in culture in the absence of their respective target tissue environment.
Deletion experiments in neurula stage embryos of Xenopus laevis provide an approximate anuran fate map of the chondrogenic cranial neural crest which is similar to maps produced for other vertebrates. Crest cells in the transverse (rostral) neural fold do not contribute to the skeleton; other cranial crest cells contribute to the larval cranial and visceral skeletons in a rostral to caudal sequence. Grafting experiments show that contact with stomodeal (pharyngeal) endoderm is necessary to elicit chondrogenesis in cranial neural crest. Crest cells in the transverse neural fold, which do not normally form cartilage, formed cartilage in grafts, indicating that they do have the potential to form cartilage.
The developmental pattern of protein production in hamster preimplantation embryos was investigated as a preliminary to studying the regulation of gene expression. Optimal radiolabelling of embryonic proteins was achieved by culturing a minimum of 40 embryos in 50 microliters of Hamster Embryo Culture Medium-2 for 2 h with 10 microCi of freshly lyophilized [35S]methionine. Proteins synthesized in vitro by different stages of hamster preimplantation embryos were analysed by one- and two-dimensional polyacrylamide gel electrophoresis followed by autoradiography. There were striking changes in the protein profile following the first cleavage division and also lesser changes after the second cleavage division. There were no detectable qualitative changes in the protein profiles of 4-cell, 8-cell, morula and blastocyst stages although some quantitative difference existed between morula and blastocyst stages. These comparisons of protein profiles during different stages of embryo development indicate that in hamsters the onset of embryonic gene activation occurs during the 2-cell stage.
Tenascin is a large disulfide-linked hexameric extracellular matrix glycoprotein. It is a multidomain protein containing many repeated structural units such as heptad-, EGF-like-, and fibronectin type III repeats, as well as a homology to the globular domains of beta- and gamma-fibrinogen. In the chick embryo three major tenascin variants exist. They arise from one gene by alternative splicing of three of its 11 fibronectin type III repeats. Monoclonal antibodies against the alternatively spliced domains allowed us to study the expression of tenascin variants in tissue sections and in cell cultures. In the gizzard, the largest tenascin variant was only detected in the smooth muscle layer and the connective tissue below the epithelium of the villi, whereas the shortest tenascin variant was predominant in the tendons and the intramuscular connective tissue. Differential expression of tenascin variants was also obtained in cell cultures of chick embryo fibroblasts. Fetal calf serum equally stimulated the accumulation of all three tenascin variants, whereas after transformation with polyomavirus middle-T only the secretion of the largest tenascin variant was greatly enhanced.
Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) invade into a film of the extracellular matrix (ECM) by extending membrane protrusions, termed the invadopodia. The invadopodia share similar cytoskeletal components and membrane receptors for ECM components as adhesion sites. However, the organization of these transmembrane components at invadopodia and adhesion sites differs. In addition, degradation of the ECM occurs at sites of the invadopodia, but not at focal adhesions. Thus, the protease and integrin molecules on invadopodia are available for dynamic interactions with the ECM, cleaving established adhesion complexes as well as reconstituting new adhesion sites.
In the development of secondary bone, mineralization of the cartilage matrix is the first step in endochondral mineralization. The circumstances of cartilage mineralization are not known. Influences of the periosteal tissue have been mentioned. In order to investigate the role of osteoblastic cells in endochondral mineralization, cartilage organoid cultures were induced to mineralize by the addition of beta-glycerophosphate (beta-GP). In cartilage organoid culture, embryonic mouse limb bud mesenchymal cells were grown at high-density. The cells differentiated into mature chondrocytes and produced hyaline cartilage matrix. When cartilage had formed after 6 days in vitro, 10 mM beta-GP was added. The developed mineralized cartilage was investigated by morphological means. Seven days after the addition of beta-GP, the first mineralized spots were visible mainly in the internodular, noncartilage tissue. After 12 to 14 days, large areas of cartilage were mineralized, and after 21 days, nearly the whole culture had been mineralized. Electron microscopic investigations showed a dramatic alteration of the cartilage matrix followed by a homogeneous mineralization of the cartilage matrix. The chondrocytes in the mineralized area died and faded. Typical rod-like apatite crystals were visible at the border between the mineralized and the unmineralized matrix. This result closely resembles the in vivo situation of cartilage mineralization. Addition of osteoblastic calvarial cells enhanced the mineralization process, as did the addition of conditioned medium of calvarial cell monolayers. Under these treatments, mineralization started after 3 days and reached a maximum after 14 days. On the other hand, addition of mouse skin fibroblast-like cells without a direct contact to the cartilage inhibited cartilage mineralization. These results indicate that osteoblastic cells induce endochondral mineralization, whereas fibroblast-like cells inhibit this mineralization via soluble factors.
A cDNA library was constructed from poly(A)+ RNA isolated from slug cells of Dictyostelium discoideum, using λgt11 phage, and screened with an antiserum specific for the spore coat protein sp96. A positive clone was obtained and the gene product was identified as sp96. The sp96 mRNA is 2.2 kb in size, and it starts to accumulate at the tipped aggregate stage only in prespore cells. Southern analysis using nuclear DNA established that the sp96 gene is unique. Two genomic clones containing the sp96 gene were isolated and the sequence of the gene established. The coding region contains a long open reading frame interrupted by a single intron.
