The stromatoporoid animal revisited: Building the skeleton

Modern coralline sponges secrete a skeleton by means of a basal pinacoderm, intracellularly, or inside the soft tissue on an organic matrix The examination of terminal growth surfaces of stromatoporoids indicates that soft tissue in laminate and amalgamate forms occupied the upper galleries and that the skeletal elements were secreted within the soft tissue on an organic matrix. The stromatoporellids and clathrodictyids secreted the skeleton in modules that are homologous to the chambers of a sphinctozoan. In stromatoporellids the module was bounded by a floor that formed the upper layer of the tripatite lamina below and a roof that became the lower layer of the next lamina; it further included the intervening pillars. In clathrodictyids the module had only a roof and pillars, and the laminae are single layers. other stromatoporoids may have secreted their skeletons at the base of the soft tissue and had minimal occupation of the skeleton. *** Stromatoporoid, sphinctozoa, sclerospongiae, sponge, paleobiology.     

Morphology of coral desmocytes, cells that anchor the calicoblastic epithelium to the skeleton

 We used light, scanning, and electron microscopy to investigate the ultrastructure of desmocytes in the scleractinian Stylophora pistillata from the Red Sea. Desmocytes are abundant on the calicoblastic epithelium, numbering up to 150 per mm² in the coenosarc. The surface of the skeleton bears shallow pits which may represent desmocyte attachment scars. Previously described as cell remnants or extracellular products, coral desmocytes appear to be bona fide cells as they manifest plasma membranes, organelles, and nuclei. Desmocytes attach to the mesoglea in mortise and tenon fashion. A field of 40 or more tenons protrude fingerlike from the proximal surface of the desmocyte and interdigitate with the mesoglea. Each tenon is coated extracellularly with short fibers which are joined to fibers of the mesoglea. The arrangement resembles previously described “fascial” hemidesmosomes. The short fibers pass through the plasma membrane and connect with relatively long intracellular fibers which occupy the center of each tenon. The long fibers extend distally and attach to structures resembling vertebrate hemidesmosomes. These, in turn, attach to the skeleton. The fiber arrangement and orientation seems designed to resist tensile forces. The dynamic adhesion potentially provided by the distal hemidesmosomes may enable desmocytes to detach and reattach to the skeleton during episodes of mineral accretion. Accepted: 15 April 1997     

Skeletal formation in the modern but ultraconservative chaetetid spongeSpirastrella (Acanthochaetetes) wellsi (demospongiae,porifera)

Summary The modern hadromerid coralline spongeSpirastrella (Acanthochaetetes) wellsi exhibits a unique secondary high-Mg calcite (>19 mol % MgCO₃) basal skeleton. The basal skeleton is constructed of bundles of elongated crystals more or less tangentially orientated. The initial formation of these crystals is controlled by soluble highly acidic aspartic and glutamic-rich (40%) macromolecules. The skeletal mineralization occurs in four different loci: in the top of the calicles, at the tabulae, on collagenous anchor fibres, and within closed spaces between the tabulae. The clicle walls are formed on the uppermost top of the basal skeleton as a continuous process. Based on long term stainings with Ca²⁺-chelating fluorochroms (calcein, chlorotetracyclines) the growth rate of this sponge is extremely low with ca. 50–100μm/a. The skeletal formation takes places outside the sponge, within a narrow zone (300–500 nm) between the basopinacoderm and the mature basal skeleton. The sponge produces thread-like folded templates (‘spaghetti fibres’) of 0,5–2 μm size, the shape controlling insoluble organic matrix. These templates become mineralized in a first step as MgCO₃, then are stretched. A soluble organic matrix is also secreted, and remains are included inside the mineralized skeleton. This organic matrix consists of in a complex mixture containing small very acidic proteins (5, 13, 31 KD; 40% Asp and Glu and therefore most probably Ca²⁺-binding) and high molecular weight glycoproteins among several other organic compounds. The mature crystals are high-Mg calcites. During calcification large cells with large reserve granules (LCG) are always present in a tight connection with the basopinacoderm. These cells form also the collagenous anchor fibres. Primary tabulae are formed by a non-collagenous organic sheet. Calcification happens only when LCG cells are enriched on the organic sheet. Randomly oriented high-Mg calcite crystals are growing on the collagenous anchor fibres. The same type of the mineralization is observed within the spaces of the tabulae. This particular case of mineralization is controlled by decaying sponge tissue (ammonification). The δ¹³C values are in equilibrium with the ambient sea water and vary between +3.2 and +2.8 ‰. The mode of mineralization of the basal skeleton can be described as biologically induced resp. matrix mediated.     

In vitro attachment of skeletal muscle fibers to a collagen gel duplicates the structure of the myotendinous junction

The myotendinous junction (MTJ) and its associated cells and connective tissue are important structures involved in transmission of contractile force from skeletal muscle to tendon. A model culture system was developed to investigate the formation of the MTJ and its attachment to collagen fibers. Skeletal muscle cells were cultured in a well modeled from two layers of a native gel of type I collagen. Muscle cells cultured in this manner formed attachments to the collagen gel and developed into highly contractile multinucleated muscle fibers with the development of extensive terminal invaginations of the sarcolemma. In addition, the subsarcolemma at the ends of muscle fibers showed areas of increased electron density which corresponded well with the termini of myofibrils. The results indicate that the development of sarcolemmal invaginations at the end of a muscle fiber probably occurs intrinsically during muscle development in vivo. The direct association of collagen fibers with the basal lamina at the end of muscle fibers was only occasionally observed in culture, suggesting that other fibrils or proteins may also be involved in the attachment of collagen fibers to the basal lamina of muscle fibers at the MTJ.     

Evidence for the existence of a nucleolar skeleton attached to the pore complex-lamina in human fibroblasts

This work deals with the types of nuclear skeletal structures obtained from human fibroblast nuclei isolated by different procedures. It is confirmed that, in somatic vertebrate cells, the pore complex-lamina is always observed, whereas the presence of internal nucleolar and extranucleolar residual structures depends upon the method of nuclear isolation used. Furthermore, the results reported here argue for the existence of a nucleolar skeleton different from the nucleolar matrix often observed in different cell types by other investigators. The conditions of nuclear isolation which allow us to visualize this nucleolar skeleton without any other internal residual structures are described. The attachment of the nucleolar skeleton to the lamina suggested by the present data is considered in relation to the in situ position of nucleoli near the nuclear envelope.     

The role of fibronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina

Vascular endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma cells, which do not contain significant amounts of FN, attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-F1) or high (B16-F10) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal lamina and completely abolished attachment of B16 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on B16 cells.     

Proteins and saccharides of the sea urchin organic matrix of mineralization: characterization and localization in the spine skeleton

Properties of the echinoderm skeleton are under biological control, which is exerted in part by the organic matrix embedded in the mineralized part of the skeleton. This organic matrix consists of proteins and glycoproteins whose carbohydrate component is specifically involved in the control mechanisms. The saccharide moiety of the organic matrix of the spines of the echinoid Paracentrotus lividus was characterized using enzyme-linked lectin assays (ELLAs). O-glycoproteins, different types of complex N-glycoproteins, and terminal sialic acids were detected. Sialic acids are known to interact with Ca ions and could play an important role in the mineralization process. Some of the carbohydrate components detected by ELLAs as well as two organic matrix proteins (SM30 and SM50) were localized within different subregions of the spine skeleton using field-emission scanning electron microscopy. The mappings show that some of these components are not homogeneously distributed in the different skeletal subregions. For example, some N-glycoproteins were preferentially located in the putative amorphous subregion of the skeleton, whereas some O-glycoproteins were localized in the subregion where skeletal growth is inhibited. These results suggest that the biological control exerted on the skeletal properties can be partly modulated by local differences in the organic matrix composition.     

Differential expression of insulin-like growth factors in the uterine epithelium and extracellular matrix during early pregnancy

We have studied the simultaneous expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) in the uterine epithelium and extracellular matrix during the time of trophoblast attachment and implantation. These studies reveal that IGF-I and IGF-II display different spatial and temporal patterns of expression during early pregnancy, and suggest a role for them in the process of attachment and implantation. Specifically, IGF-I is strongly expressed in the basal lamina which is the site of trophoblast invasion into the maternal stroma, and also in the apical epithelium, the site of initial trophoblast attachment. IGF-II is expressed to a lesser extent in the basal lamina, lateral plasma membranes and apical epithelium on day 3 but is only prominent apically at the time of implantation, suggesting a role in attachment.     

Acid polysaccharides in the skeletal matrix and calicoblastic epithelium of the stony coral Mycetophyllia reesi

Like many corals the skeletal organic matrix and associated epithelium of Mycetophyllia reesi is physico-chemically unstable to preparative procedures for electron microscopy. Ethanol cryofracture of mineralized and demineralized material is accompanied by delamination of tissue and skeleton. Filamentous algae occur in the interface and account for some but not all of the separation artifact. Transmission microscopy accompanied by decalcification requires embedment in glycerol jelly to preserve the skeletal organic matrix. Even then, the matrix is not fixed and is not retained within the gel using standard double fixation with or without tannic acid as an additive. Ruthenium red, in combination with osmium, prevents the matrix from physical disruption, although positional artifacts relative to the calicoblastic epithelium are still evident. Inclusion of other glycan precipitating agents in the fixative sequence (Alcian blue, iron diamine or the detergent cetylpyridinium chloride) are more useful in preserving an acid polysaccharide-rich, fibrillar, extracellular matrix after demineralization. This material is not observed in SEM preparations. The calicoblast cells appear to be the source of this extracellular material that also appears to contribute to the composition of the mineralizing matrix. Moreover, a hyaluronan-like substance appears to play a significant role in matrix structure as suggested by its degradation by hyaluronidase.     

Localization of calcium and phosphorus in early predentin-matrix components by electron spectroscopic imaging (ESI)-analysis in rat molars

Summary The subcellular distribution of the inorganic elements calcium (Ca) and phosphorus (P) was studied in the first-formed dentin matrix during initial mineralization in neonatal rat molars. This most peripheral matrix region is comprised of a proteoglycan-rich ground substance, interwoven by a collagenous network, matrix vesicles, aperiodic fibrils derived from the dental basal lamina, and apical odontoblastic cell processes. All matrix components may possibly serve as templets for mineral deposition during initial calcification of first-formed mantle dentin and predentin. By means of the very sensitive ESI-analysis we studied the subcellular localization of Ca and P and their possible association with distinct organic extracellular matrix components and odontoblasts. Ca-signals were found in the ground substance, at striated collagen fibrils and plasma membranes of odontoblasts in the cuspal early matrix region, but occurred only sparsely in the ground substance of the more distal matrix region where odontoblast processes attach to aperiodic fibrils of the dental basal lamina. Ca was generally absent in matrix vesicles. In contrast, P-signals were found in matrix vesicles, at aperiodic fibrils and at the plasma membranes of odontoblasts. Ca and P co-localized at striated collagen fibrils (type I or II). These results suggest that striated collagen fibrils might serve as primary deposition sites for calcium phosphate during early biological calcification of organic extracellular macromolecules.     

Intracellular crystal-bearing vesicles in the epidermis of scleractinian corals, Astrangia danae (Agassiz) and Porites porites (Pallas).

Orthorhombic aragonitic crystals, embedded with a granular lipo-protein matrix and surrounded by a trilaminar membrane, are localized in the apical cytoplasm of epidermal cells of Scleractinian corals. Adult specimens of Astrangia danae (Agassiz) and settled planulae of Porites porites (Pallas) contain crystals averaging 0.7 mu by 0.1 mu by 0.3 mu within Golgi-derived vesicles. Short-term labelling with 45Ca reveals distribution of radioactivity amont a basic tissue fraction (92%) an acid tissue fraction (5%) and a skeletal fraction (3%). Identification of the primordial crystal population within membrane-bound visicles provides overwhelming evidence for the intracellular mode of calcification in Scleractinia. Moreover, it permits development of a novel concept of cellular regulation over these dynamic events. The membrane-bound vesicel is a miniature crystal fabrication station and a vehicle responsible for transportation of seed crystals and an organic matrix material to sites of discharge from the cell. The vesicle membrane becomes a probable locus of active transport and enzymatic activity as well as a physical barrier to be penetrated for release of vesicle contents into the extracellular milieu. Contact between the vesicle membrane and the plasmalemma would result in exocytosis and the onset of skeletogenesis. Principles governing crystal growth would prevail from then on. The released crystal becomes a nucleation catalyst and the organic matrix, a supply of ionic calcium for self-limiting crystallization. Crystals are produced by the organism spontaneously and continuously from shortly after larval attachment throughout the life of the polyp. Therefore, these membrane-bound vesicles signal the dynamic process by which initiation, differentiation, growth and limitation of the coral skeleton is regulated.     

Epithelial-Directed Mesenchyme Differentiation in vitro

To assess the requirement for specific or possibly non-specific epithelial instructions for mesenchymal cell differentiation, we designed studies to evaluate and compare homotypic with heterotypic tissue recombinations across vertebrate species. These studies further tested the hypothesis that determined dental papilla mesenchyme requires epithelial-derived instructions to differentiate into functional odontoblast cells using a serumless, chemically-defined medium. Theiler stage 25 C57BL/6 or Swiss Webster cap stage mandibular first molar tooth organs or trypsin-dissociated, homotypic epithelial-mesenchymal tissue recombinants resulted in the differentiation of odontoblasts within 3 days. Epithelial differentiation into functional ameloblasts was observed within 7 days. Trypsin-dissociated and isolated mesenchyme did not differentiate into odontoblasts under these experimental conditions. Heterotypic recombinants between quail Hamburger-Hamilton stages 22–26 mandibular epithelium and Theiler stage 25 dental papilla mesenchyme routinely resulted in odontoblast differentiation within 3 days in vitro. Odontoblast differentiation and the production of dentine extracellular matrix continued throughout the 10 days in organ culture. Ultrastructural observations of the interface between quail and mouse tissues indicated the reconstitution of the basal lamina as well as the maintenance of an intact basal lamina during 10 days in vitro. Quail epithelial cells did not differentiate into ameloblasts and no enamel extracellular matrix was observed. These results show that quail mandibular epithelium can provide the required developmental instructions for odontoblast differentiation in the absence of serum or other exogenous humoral factors in a chemically-defined medium. They also suggest the importance of reciprocal epithelial-mesenchymal interactions during epidermal organogenesis.     

Dynamic formation of microenvironments at the myotendinous junction correlates with muscle fiber morphogenesis in zebrafish

Muscle development involves the specification and morphogenesis of muscle fibers that attach to tendons. After attachment, muscles and tendons then function as an integrated unit to transduce force to the skeletal system and stabilize joints. The attachment site is the myotendinous junction, or MTJ, and is the primary site of force transmission. We find that attachment of fast-twitch myofibers to the MTJ correlates with the formation of novel microenvironments within the MTJ. The expression or activation of two proteins involved in anchoring the intracellular cytoskeleton to the extracellular matrix, Focal adhesion kinase (Fak) and beta-dystroglycan is up-regulated. Conversely, the extracellular matrix protein Fibronectin (Fn) is down-regulated. This degradation of Fn as fast-twitch fibers attach to the MTJ results in Fn protein defining a novel microenvironment within the MTJ adjacent to slow-twitch, but not fast-twitch, muscle. Interestingly, however, Fak, laminin, Fn and beta-dystroglycan concentrate at the MTJ in mutants that do not have slow-twitch fibers. Taken together, these data elucidate novel and dynamic microenvironments within the MTJ and indicate that MTJ morphogenesis is spatially and temporally complex.     

Composition, synthesis, and assembly of the embryonic chick retinal basal lamina

To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly.     

Morphology of the midgut trunk in the penaeid shrimp, Sicyonia ingentis, highlighting novel nuclear pore particles and fixed hemocytes

The morphology of the midgut trunk (MGT) in the penaeid shrimp Sicyonia ingentis was examined by light and scanning and transmission electron microscopy. Although the function of the MGT is poorly understood, it is not involved with the digestion and absorption of nutrients, and it appears to be the surface of a shrimp least protected from penetration by potential pathogens. As described for other decapod crustaceans, the MGT in shrimp is composed of a simple columnar epithelium separated from a layer of connective tissue by a thick basal lamina. Beneath the basal lamina is a previously unreported layer of hemocytes, exclusively of the granulocyte variety, embedded in a matrix continuous with the basal lamina and extending into the connective tissue. This layer was observed in four other species of penaeid shrimp. Granulocytes in circulation can phagocytose and encapsulate foreign material and the granules contain antibacterial molecules, lysosomal enzymes, and prophenoloxidase. We suggest that the granulocytes associated with the basal lamina have matured at this site and are well positioned to fight potential pathogens that have penetrated the epithelial layer of the MGT. A second observation is the presence of clusters of cylinders bound to the nuclear pores of the epithelial cells. The possibility that these clusters are viruses, organelles, or abnormal organelles induced by disease or toxic materials is discussed. These unique particles were observed in S. ingentis but none of the other penaeid shrimp we examined.     

Biologically controlled mineralization in the hypercalcified sponge Petrobiona massiliana (Calcarea, Calcaronea)

Hypercalcified sponges, endowed with a calcium carbonate basal skeleton in addition to their spicules, form one of the most basal metazoan group engaged in extensive biomineralization. The Mediterranean species Petrobiona massiliana was used to investigate biological controls exerted on the biomineralization of its basal skeleton. Scanning and transmission electron microscopy (SEM, TEM) confirmed that basopinacocytes form a discontinuous layer of flattened cells covering the skeleton and display ultrastructural features attesting intense secretory activity. The production of a highly structured fibrillar organic matrix framework by basopinacocytes toward the growing skeleton was highlighted both by potassium pyroantimonate and ruthenium red protocols, the latter further suggesting the presence of sulfated glycosaminoglycans in the matrix. Furthermore organic material incorporated into the basal skeleton was shown by SEM and TEM at different structural levels while its response to alcian blue and acridine orange staining might suggest a similar acidic and sulfated chemical composition in light microscopy. Potassium pyroantimonate revealed in TEM and energy electron loss spectroscopy (EELS) analysis, heavy linear precipitates 100-300 nm wide containing Ca(2+) and Mg(2+) ions, either along the basal cell membrane of basopinacocytes located toward the decalcified basal skeleton or around decalcified spicules in the mesohyl. Based on the results of the previous mineralogical characterization and the present work, an hypothetical model of biomineralization is proposed for P. massiliana: basopinacocytes would produce an extracellular organic framework that might guide the assemblage of submicronic amorphous Ca- and Mg-bearing grains into higher structural units.     

Role of muscle fibroblasts in the deposition of type-IV collagen in the basal lamina of myotubes

In cell cultures of quail, chick, or mouse skeletal muscle, both myogenic and fibrogenic cells synthesize and secrete type-IV collagen, a major structural component of the basal lamina. Type-IV collagen, together with laminin, forms characteristic patches and strands on the surface of developing myotubes, marking the onset of basement-membrane formation. The pattern for type-IV collagen and laminin is unique to these proteins and is not paralleled by other matrix proteins, such as fibronectin or type-I or -III collagen. In the present study, we used species-specific antibodies to either mouse or chick type-IV collagen to demonstrate the ability of fibroblast--derived type-IV collagen to incorporate in the basal lamina of myotubes. In combination cultures of embryonic quail skeletal myoblasts and mouse muscle fibroblasts, antibodies specific for mouse type-IV collagen revealed the deposition of type-IV collagen on the surface of quail myotubes in the pattern typical of the beginning of basement-membrane formation. Control cultures consisting of only quail muscle cells containing myoblasts and fibroblasts demonstrated no such reaction with these antibodies. Deposits of mouse type-IV collagen were also observed on the surface of quail myotubes when conditioned medium from mouse muscle fibroblasts was added to quail myoblast cultures. Similarly, in combination cultures of mouse myoblasts and chick muscle fibroblasts, chick type-IV-collagen deposits were identified on the surface of mouse myotubes. These results indicate that type-IV collagen synthesized by muscle fibroblasts may be incorporated into the basal lamina forming on the plasmalemma of myotubes, and may explain ultrastructural studies by Lipton on the contribution of fibroblasts to the formation of basement membranes in skeletal muscle.     

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.