The role of the basal lamina in mouth formation in the embryo of the starfish Pisaster ochraceus

Details of mouth formation in normal and exogastrulated Pisaster ochraceus larvae have been studied by light microscopy and transmission and scanning electron microscopy. As the archenteron begins to bend, the cells in the presumptive mouth region dissociate and migrate into the blastocoele where they become mesenchyme cells. This leaves a defect in the “blind” endodermal tube, which is covered by a basal lamina. Subsequently this exposed basal lamina bulges to form a blister which appears to extend across the blastocoele to make contact with spikelike projections from the future stomodeal region of the ectoderm. Mesenchyme cell processes are associated with both the basal lamina blister and the ectoderm in this region and may provide both motive power and guidance for contact. Shortly after contact is made the blister of basal lamina from the endoderm fuses with the basal lamina of the ectodermal cells and the ectoderm begins to invaginate. At this time the lateral walls of the presumptive oesophagus are largely formed of naked basal lamina with some loosely associated cells on the endodermal side. Eventually the lateral walls of the proximal part of the oesophagus become cellular, giving rise to an epithelium. A cell plug located between the stomodeum and oesophagus persists for some time before finally breaking down to complete the larval digestive tract. Experiments with exogastrulae suggest that many of these developmental patterns are determined before gastrulation.     

Formation of cytoskeletal elements during mouse embryogenesis

Abstract. The ultrastructure of the day 8.5 mouse embryo has been studied by transmission electron microscopy, with special emphasis on the primary mesenchymal cells and their interaction with cells of the embryonic ectoderm and the proximal endoderm. The organization of the two polar epithelial cell layers (embryonic ectoderm and proximal endoderm), the isolated cells of the distal endoderm and the primary mesenchymal cells is described. Primary mesenchymal cells are different from embryonic ectoderm cells, from which they are derived, not only by the absence of desmosomes and intermediate-sized filaments of the cytokeratin type but also by their variable morphology not exhibiting stable polar architecture, and their numerous cytoplasmic processes which make contacts with the basal lamina of the ectoderm, the basal cell surface of the proximal endoderm, and other mesenchymal cells. Over most of the embryo the embryonic ectoderm is covered by a typical basal lamina, except for certain regions that are frequently characterized by cytoplasmic projections ('blebs') from the basal cell surface membrane. In contrast, the basal surface of the proximal endoderm is not covered by a continuous basal lamina and reveals mushroom-like protrusions of the cortical cytoplasm. Junctions between primary mesenchymal cells are numerous and include adhaerens-type formations of various sizes as well as gap junctions. Occasionally, a special type of junction between mesenchymal cells and embryonic ectoderm has been found, resulting in local interruptions of the basal lamina. The observations are discussed in relation to possible mechanisms of mesoderm formation and the drastic changes of cell character that accompany this process, including cytoskeletal changes such as the disappearance of cytokeratin filaments and the expression of vimentin.     

Formation of cytoskeletal elements during mouse embryogenesis. IV. Ultrastructure of primary mesenchymal cells and their cell-cell interactions

The ultrastructure of the day 8.5 mouse embryo has been studied by transmission electron microscopy, with special emphasis on the primary mesenchymal cells and their interaction with cells of the embryonic ectoderm and the proximal endoderm. The organization of the two polar epithelial cell layers (embryonic ectoderm and proximal endoderm), the isolated cells of the distal endoderm and the primary mesenchymal cells is described. Primary mesenchymal cells are different from embryonic ectoderm cells, from which they are derived, not only by the absence of desmosomes and intermediate-sized filaments of the cytokeratin type but also by their variable morphology not exhibiting stable polar architecture, and their numerous cytoplasmic processes which make contacts with the basal lamina of the ectoderm, the basal cell surface of the proximal endoderm, and other mesenchymal cells. Over most of the embryo the embryonic ectoderm is covered by a typical basal lamina, except for certain regions that are frequently characterized by cytoplasmic projections ("blebs') from the basal cell surface membrane. In contrast, the basal surface of the proximal endoderm is not covered by a continuous basal lamina and reveals mushroom-like protrusions of the cortical cytoplasm. Junctions between primary mesenchymal cells are numerous and include adhaerens-type formations of various sizes as well as gap junctions. Occasionally, a special type of junction between mesenchymal cells and embryonic ectoderm has been found, resulting in local interruptions of the basal lamina. The observations are discussed in relation to possible mechanisms of mesoderm formation and the drastic changes of cell character that accompany this process, including cytoskeletal changes such as the disappearance of cytokeratin filaments and the expression of vimentin.     

Ultrastructure and synthesis of the extracellular matrix of Pisaster ochraceus embryos preserved by freeze substitution

When asteroid embryos cryoprotected with propylene glycol are rapidly frozen in liquid propane and freeze substituted with ethanol, preservation of the cells and extracellular matrix (ECM) is excellent. The basal lamina, although thicker and less well defined than in conventionally fixed embryos, demonstrates a region of decreased density just below the cells that corresponds to the lamina lucida and a lamina densa. The former region is often occupied by fibrous material. In addition, as was previously described in conventionally fixed tissues, the basal lamina of the ectoderm is generally thicker and more substantial than that of the endoderm, reinforcing an earlier suggestion that the structure of the basal lamina is different in different regions of the embryo. The ECM of the blastocoel consists of thin “twig-like” elements that form a loose meshwork evenly distributed throughout the blastocoel. Bundles of 20 nm fibers, located within the meshwork, are oriented parallel to the base of the cells of the stomodeum. In the long axis of the embryo, similar fibers are present in the dorsal aspect of the animal between the stomach and the ectoderm and radiate out from the esophagus crossing the region between it and the ectoderm. Immunocytochemical work with three different monoclonal antibodies shows that glycoprotein molecules, synthesized in the Golgi apparatus, are also secreted here and form part of the matrix structure. The results suggest that the blastocoel is filled with a gel-like material reinforced with bundles of 20-nm fibers. The manner in which the observed arrangement could contribute to the development and maintainence of the shape of the embryo is discussed. J Morphol 232:133–153, 1997. © 1997 Wiley-Liss, Inc.     

Behavior of neural crest cells on embryonic basal laminae

Neural crest cells separate from the neural epithelium in a region devoid of a basal lamina and migrate along pathways bordered by intact basal laminae. The distribution of basal laminae suggests that they might have an important role in the morphogenesis of the neural crest by acting as a barrier to migration. The experiments reported here have tested directly whether neural crest cells can penetrate a basal lamina. Isolated neural tubes, neural crest cells cultured for 24 hr, or pigmented neural crest cells were explanted onto human placental amnions from which the epithelium had been removed to expose the basal lamina. In no case did neural crest cells or crest derivatives penetrate the basal lamina to invade the underlying stroma. If crest cells were grown on the stromal side of the amnion, they invaded the connective tissue. Pigmented neural crest derivative and [3H]thymidine-labeled nonpigmented crest cells were also confronted with chick embryonic basal laminae by grafting the cells into the lumen of the neural tube at the axial levels where host crest migration had commenced. Most of the grafted cells invaded the neural epithelium and accumulated after 24 hr at the basal surface of the neural tube. A few crest cells escaped through the dorsal surface of the neural tube and entered the overlying ectoderm, presumably through the wound created during the grafting procedure. Some of these grafted cells, located initially by light microscopy, were examined at the higher magnification and resolution offered by the transmission electron microscope to determine the relationship of the grafted cells to the basal lamina. In 50% (14 total) of the cases, the crest cells never reached the basal lamina of the neural tube, but were trapped by cell junctions between the neural epithelial cells. Of the remaining grafted cells that were relocated in the TEM (50%, total 15) all were spread on the basal lamina, but were not seen penetrating it. Likewise, in the three cases where crest cells were found in the epidermal ectoderm, all were in contact with the basal lamina of the ectoderm but did not have any processes extending through it. In three cases, at the level of the light microscope, crest cells were found to extend through the basal surface of the neural tube. In all these instances, the cells followed the dorsal root nerve exiting through a region of the neural tube that is devoid of a basal lamina.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells

At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.     

Histochemical analysis of extracellular matrix material in embryonic mouse lens morphogenesis

Extracellular matrix material (ECM) present during mouse lens morphogenesis was studied histologically by the periodic acid-Schiff, Alcian blue 8GX, pH 2.5, high iron diamine, and Van Gieson methods, and enzymatically with bovine testicular hyaluronidase, Streptomyces hyaluronidase, malt diastase, and collagenase. The basal lamina of the optic vesicle prior to lens placode formation was found to be higher in glycosaminoglycan (GAG) content than was the ectodermal basal lamina. Upon apposition of the optic vesicle and presumptive lens ectoderm, the ECM plus basal laminae appeared as the equivalent of adding both optic vesicle-associated and ectodermal-associated basal lamina. The proposal is made that the initial triggering mechanism of lens morphogenesis consists of a cross-linking and polymerization of optic vesicle-associated GAG to ectodermal-associated glycoproteins resulting in a firm attachment between the structures. Basal lamina associated with the presumptive pigmented retina and also the more ventral part of the interface matrix were found to change from predominantly GAG in early stages to collagen deposits in more advanced stages, temporally coinciding with the appearance of differentiative markers in each structure. This pattern of GAG turnover and replacement by collagen during the course of development is also seen in mouse salivary gland morphogenesis (M. R. Bernfield, S. D. Banerjee, and R. H. Cohn (1972). J. Cell Biol. 52, 674-686.).     

Pioneer neurones use basal lamina as a substratum for outgrowth in the embryonic grasshopper limb

During axonogenesis, contacts made by the growth cone with its substratum are important in guiding the direction of neurone outgrowth. This study examines the contacts made by the growth cones of pioneer neurones in the embryonic grasshopper limb. Individual pioneer neurones at different stages of development were injected with horseradish peroxidase and the contacts made by the filopodia at the tip of their growth cones were examined by electron microscopy. Filopodia made few contacts with mesodermal cells, some contacts with ectodermal cells and very frequent contacts with basal lamina underlying the ectoderm. Components of the basal lamina may therefore play a role in guiding pioneer axon outgrowth.     

Formation of the Primitive Streak and Mesoderm Cells in Mouse Embryos—Detailed Scanning Electron Microscopical Study

We describe morphological events of the mammalian gastrulation in pre- to middle-primitive-streakstage mouse embryos by using scanning electron microscopy. The first sign of the ingression of the mesodermal cells was disruption of the epithelial structure of ectoderm and the underlying basal lamina, thus forming a semicircular area of the presumptive primitive streak. Then, cells at periphery of the semicircular region spread on the basal lamina by extending many filopodia to it. The majority of the migrating cells formed a loosely arranged cell sheet. We found solitary cells and isolated small groups of cells migrating away from the periphery of the cell sheet. These cells were well spread on the basal lamina, and had large cell processes and many filopodia in the direction of cell migration. Filopodia of these cells were attached to the basal lamina or a meshwork of the extracellular fibrils. These observations suggest that the extracellular matrix serves as the substratum for cell adhesion and migration, and plays an important role in the mammalian gastrulation.     

Ectodermal-mesenchymal interspace during the formation of the chick leg bud

Summary The ectodermal-mesenchymal interspace of the chick leg bud was studied at stages leading to the formation of the apical ectodermal ridge (A.E.R.) (stages 14 to 19 HH), using scanning and transmission electron microscopy. The main findings were: 1. a continuous basal lamina under the ectoderm; 2. extracellular fibrils interconnecting the basal lamina and mesenchymal cell processes; 3. an increase in the number of the fibrils during these stages, with the highest number under the A.E.R.; 4. branching mesenchymal cell processes that spread over the basal lamina, making contact with it in all stages. The morphology of the interspace and the changes in it suggest that extracellular material may be significant in the ectodermal-mesenchymal interactions in the limb bud.     

Spatial and temporal variation in the structure of the basal lamina in embryonic grasshopper limbs during pioneer neurone outgrowth

The pioneer neurones of the embryonic grasshopper limb use the basal lamina underlying the limb ectoderm as a substratum over which to grow from the periphery to the CNS (Anderson & Tucker, 1988). In this paper we use transmission electron microscopy to describe the structure of this substratum before, during, and after the time of axon navigation. The organization of the basal lamina varies considerably in different regions and at different times of development of the embryonic limbs, and is unlike that of the fully developed limb at the time of hatching. We suggest that this spatial and temporal variation could play a role in regulating the direction of outgrowth of pioneer neurones.     

Ultrastructural analysis of the apical ectodermal ridge during vertebrate limb morphogenesis: I. The human forelimb with special reference to gap junctions

The fine structure of the human forelimb apical ectodermal ridge of stages 12–19 was examined using techniques of transmission electron microscopy, freeze fracture, and scanning electron microscopy. This paper reports the presence of subcellular structures that distinguish the inductively active apical ectoderm from adjacent dorsal and ventral ectoderms.The apex of the human forelimb begins development with an epithelium of two cell layers (stage 12) which thickens at the distal tip during stages 13 and 14 into a multilayered apical ectodermal ridge. During this transition we have observed that the basal lamina differentiates from a bilayered structure to the definitive single lamina. Some cells in the ectoderm become detached from the basal lamina as stratification begins. At the same time these cells show increased mitotic activity and the developing ridge cells acquire gap junctions. Annular gap junctions are also observed. Gap junctions are not observed in adjacent, presumably noninductive, epithelia. Finally, the ridge cells next to the basal lamina acquire bundles of microfilaments that are oriented in the dorsal-ventral plane in the basal cytoplasm of the cells.The apical ridge reaches its greatest dimensions during stage 15. The number and peripheral extent of gap junctions also appear to be greatest at this same time. At stage 17, cells within the ridge begin to die, and other ridge cells engulf them. By stage 19, gap junctions in the apical epithelium are sparse and are of lesser diameter than in the definitive ridge. In addition, the oriented bundles of microfilaments present at stages 14–17 are absent. Thus, at stage 19 a morphologically distinct apical ectodermal ridge is no longer present. The apex of the limb is covered by two cell layers typical of human embryonic epidermis.     

Structure of the epithelial - mesenchymal interface during early morphogenesis of the chick duodenum.

During the period of early morphogenetic folding of the intestinal epithelium, changes in the epithelial-mesenchymal interface were observed by light microscopy, scanning and transmission electron microscopy. The epithelium in cross-section, appears first as a circle, then an ellipse and finally by a triangle prior to the formation of the first three previllous ridges. The bases of all epithelial cells are flat at the circular stage. At the ellipse and triangle stages the bases of the epithelial cells occupying the sides possess lobopodia that do not penetrate the basal lamina. The immediate mesenchymal cells subjacent to those epithelial cells on the sides of the ellipse and triangle alter their orientation to being rounded-up or perpendicular to the plane of the basal lamina. Large numbers of fine mesenchymal pseudopodia in addition to many extracellular fibrils are revealed by transmission and scanning electron microscopy at the epithelial-mesenchymal interface. The fine mesenchymal pseudopodia come into close contact but do not penetrate the ruthenium red-staining basal lamina. The possible roles of close contact between epithelium and mesenchyme, the alteration in orientation of mesenchyme cells, and of the basal lamina in tissue interaction are discussed.     

Origin of the dorsal surface of the neural tube by progressive delamination of epidermal ectoderm and neuroepithelium: implications for neurulation and neural tube defects

Knowledge of the morphogenetic events involved in the development of the dorsal portion of the neural tube is important for understanding neural tube closure, neural crest cell formation and emigration, and the origin of neural tube defects. Here, I characterize the progressive development of the tips of the neural folds during fold elevation in the trunk of mouse and chick embryos and the events leading to formation of the dorsal portion of the neural tube as the epidermal ectoderm (EE) and neuroepithelium (NE) separate from each other. The nature and timing of appearance of collagen IV, laminin and fibronectin were analysed by immunofluorescent and immunogold labelling, and ruthenium red and tannic acid were used to enhance staining for proteoglycans and glycosaminoglycans. As the neural folds elevate, the NE and EE delaminate progressively beginning at the basal surface of the lateral extremes of the neural plate. Nevertheless, the two epithelia remain connected across the zone of delamination by their previously existing basal laminae. In each fold, proteoglycan granules appear at the interface between the NE and EE before delamination begins, and then an (interepithelial) space begins to open and propagate dorsally. Other extracellular matrix (ECM) molecules appear within the space a short distance behind its tip and basal lamina deposition begins shortly thereafter. As fusion occurs, the interepithelial spaces of the two folds coalesce and the final separation of the EE from the NE is accomplished. These observations suggest that the previously recognized delay in deposition of ECM and basal lamina on the dorsal portion of the neural tube and on the overlying EE is a direct consequence of the delamination of the two epithelia and the establishment of two new basal surfaces. The observation that the surface of the dorsal third of the neural tube forms by delamination rather than by juxtaposition of previously existing basal surfaces of the two epithelial is discussed in terms of possible implications for models of neurulation and the origin of neural tube defects.     

Ultrastructure of oral (buccopharyngeal) membrane formation and rupture in the hamster embryo

The ultrastructure of the oral (buccopharyngeal) membrane was examined during normal development in embryos removed from pregnant hamsters at intervals from 7.5–10 days postcoitum. The oral membrane is represented at 7.5 days by a region of close approximation between endoderm and surface ectoderm anterior to the neural folds. A distinct basal lamina develops subjacent to each epithelial germ layer, and the narrow extracellular space separating the epithelia contains patches of fibrillar and flocculent material. Cell processes extend from cells of one epithelium across the extracellular space to make direct contact with cells of the opposing germ layer by 7.75 days. Increased intermingling of cells subsequently occurs within the oral membrane, and some cells extend the entire width of the membrane with surfaces exposed to both foregut and stomodeum. Accumulations of presumed basal lamina and extracellular material are observed at intervals within the oral membrane, but a continuous intercellular space is no longer present. Many of these accumulations are encompassed by processes of adjacent cells containing dense intracellular bodies, indicating active phagocytosis of this material by the epithelial cells. Rupture of the oral membrane begins between 8.25 and 8.75 days, and all remnants are removed by Day 9. Possible factors involved in rupture are discussed.     

Permeability of the haemolymph-neural interface in the terrestrial snail Megalobulimus abbreviatus (Gastropoda, Pulmonata): an ultrastructural approach

The aim of this study was to investigate the ultrastructure of the interface zone between the nervous tissue and the connective vascular sheath that surround the central ganglia of the terrestrial snail of Megalobulimus abbreviatus and test its permeability using lanthanum as an electron dense tracer. To this purpose, ganglia from a group of snails were fixed by immersion in a 2% colloidal lanthanum solution, and a second group of animals was injected in the foot with either a 2%, 10% or 20% lanthanum nitrate solution and then sacrificed 2 or 24 h after injection. Ganglia from both groups were processed for transmission electron microscopy. The vascular endothelium, connective tissue and basal lamina of variable thickness that ensheathe the nervous tissue and glial cells of the nervous tissue constitute the interface zone between the haemolymph and the neurones. The injected lanthanum reached the connective tissue of the perineural capsule; however, it did not permeate into the nervous tissue because the basal lamina interposed between both tissues interrupted this passage. Moreover, the ganglia fixed with colloidal lanthanum showed electron dense precipitates between the glial processes in the area adjacent to the basal lamina. It can be concluded from these findings that, of the different components of the haemolymph-neuronal interface, only the basal lamina, between the perineural capsule and the nervous tissue, limits the traffic of substances to and from the central nervous system of this snail.     

Ultrastructure of the pre-implantation shark yolk sac placenta

During ontogeny, the yolk sac of viviparous sharks differentiates into a yolk sac placenta which functions in gas exchange and hematrophic nutrient transport. The pre-implantation yolk sac functions in respiration and yolk absorption. In a 10.0 cm embryo, the yolk sac consists of six layers, viz. (1) somatic ectoderm; (2) somatic mesoderm; (3) extraembryonic coelom; (4) capillaries; (5) endoderm; and (6) yolk syncytium. The epithelial ectoderm is a simple cuboidal epithelium possessing the normal complement of cytoplasmic organelles. The endoplasmic cisternae are dilated and vesicular. The epithelium rests upon a basal lamina below which is a collagenous stroma that contains dense bodies of varying diameter. They have a dense marginal zone, a less dense core, and a dense center. The squamous mesoderm has many pinocytotic caveolae. The capillary endothelium is adjacent to the mesoderm and is delimited by a basal lamina. The endoderm contains yolk degradation vesicles whose contents range from pale to dense. The yolk syncytium contains many morphologically diverse yolk granules in all phases of degradation. Concentric membrane lamellae form around yolk bodies as the main yolk granules begin to be degraded. During degradation, yolk platelets exhibit a vesicular configuration.     

Origin and Role of Collagen in the Embryo

The first collagen recognizable in the embryo is in the formof an incomplete basal lamina under the epiblast and hypoblast.We suggest that this collagen acts as a railroad track to guidethe migration of the primitive streak mesenchyme. The mesenchymeaggregates into chordamesoderm, a layer which is said to "induce"the overlying epiblast (now ectoderm) to develop into neuralfolds. This tissue interaction may be mediated by the formationof complete basal laminas separating the two tissues and bydeposition of sulfated mucopolysaccharides in the interveningextracellular space. At the very least, the collagenous basallamina serves to give the elongating cells of the developingneural tube a firm foothold. The fully formed neural tube andadjacent notochord are said to induce the sclerotome of thesomite to migrate medially and differentiate into cartilage.Notochord and neural tube basal lamina and collagen fibrilsmay play a role by guiding the migrating cells and stabilizingthe already existing chondrogenic bias of the cells. We wereunable to prove this hypothesis directly (by adding collagento somite cultures), because in our hands the somites died invitro even in the presence of neural tube and notochord. Wedid obtain direct evidence, however, that the basal lamina ofthe lens can promote the differentiation of the cornea in vitro.     

FORMATION AND ORIGIN OF BASAL LAMINA AND ANCHORING FIBRILS IN ADULT HUMAN SKIN

The purpose of this investigation was to study the formation and origin of basal lamina and anchoring fibrils in adult human skin. Epidermis and dermis were separated by "cold trypsinization." Viable epidermis and viable, inverted dermis were recombined and grafted to the chorioallantoic membrane of embryonated chicken eggs for varying periods up to 10 days. Basal lamina and anchoring fibrils were absent from the freshly trypsinized epidermis before grafting although hemidesmosomes and tonofilaments of the basal cells remained intact. Basal lamina and anchoring fibrils were absent from freshly cut, inverted surface of the dermis. Beginning 3 days after grafting, basal lamina was noted to form immediately subjacent to hemidesmosomes of epidermal basal cells at the epidermal-dermal interface. From the fifth to the seventh day after grafting, basal lamina became progressively more dense and extended to become continuous in many areas at the epidermal-dermal interface. Anchoring fibrils appeared first in grafts consisting of epidermis and viable dermis at five day cultivation and became progressively more numerous thereafter. In order to determine the epidermal versus dermal origin of basal lamina and anchoring fibrils, dermis was rendered nonviable by repeated freezing and thawing 10 times followed by recombination with viable epidermis. Formation of basal lamina occurred as readily in these recombinants of epidermis with freeze-thawed, nonviable dermis as with viable dermis, indicating that dermal viability was not essential for synthesis of basal lamina. This observation supports the concept of epidermal origin for basal lamina. Anchoring fibrils did not form in recombinants containing freeze-thawed dermis, indicating that dermal viability was required for anchoring fibrils formation. This observation supports the concept of dermal origin of anchoring fibrils.