Ultrastructure of neural crest formation in the midbrain/rostral hindbrain and preotic hindbrain regions of the mouse embryo

In the mouse embryo, neural crest mesenchyme associated with the first and second pharyngeal arches escapes from the epithelium that forms the tips of the midbrain/rostral hindbrain and preotic hindbrain neural folds. To investigate the ultrastructure of crest formation, embryos with four to eight pairs of somites were processed for transmission electron microscopy. In the earliest event related to crest formation, crest precursors in the midbrain/rostral hindbrain elongated and moved all or most of their contents to the basal region of the epithelium. Elongation was probably mediated by apical bands of microfilaments and longitudinally oriented microtubules. Elongated cells then relinquished apical associations while nonelongated cells maintained those associations and withdrew from the basal lamina. This resulted in an epithelium stratified into apical and basal (crest precursor) layers. The coalescence of enlarging extra-cellular spaces opened a delaminate gap between the two layers. Additional crest precursors entered this gap from the apical layer. From the time crest precursors began moving basally, some formed microfilament- and/or microtubule-containing processes, which penetrated the basal lamina. Some of these cells moved their contents into the larger, microtubule-containing processes, perhaps thereby escaping from the epithelium. Soon after elongating cells appeared, the basal lamina beneath the epithelium began to degrade in a pattern unrelated to process formation. This ultimately resulted in disruption of the lamina, dispersal of the basal layer of the epithelium, and release of the crest precursors in the delaminate gap. Once crest formation was complete, the apical layer reformed a basal lamina on a patch-by-patch, cell-by-cell basis. In the preotic hindbrain, elongating crest precursors apparently forced their basal faces through the basal lamina and then relinquished apical association to escape. As a result, the lamina was disrupted before the epithelium could stratify, and enlarged extracellular spaces appeared among mesenchymal cells rather than creating a delaminate gap. The failure of elongation to disrupt the basal lamina in the midbrain/rostral hindbrain and its success in the preotic hindbrain might be due to less-vigorous, less-concerted elongation in the midbrain/rostral hindbrain or to earlier, more rapid degradation of the lamina in the preotic hindbrain.     

Formation and distribution of neural crest mesenchyme to the first pharyngeal arch region of the mouse embryo

Murine neural crest mesenchyme begins its escape from columnar epithelium near the tips of the midbrain-rostral hindbrain neural folds at 4+ to 5 somites of age. At that time the tip of each fold is located dorsolateral to the pharynx. Once crest formation is complete at this earliest site, it leaves behind both crest mesenchyme and overlying squamous epithelium. Crest formation then progresses medially, into the lateral margin of the neural plate. At the same time, this lateral margin elevates as the tip of the neural fold. By the time crest formation ceases at approximately 10 somites, the result of these simultaneous activities is to passively distribute the earliest mesenchyme, formed from the lateralmost epithelium, dorsolateral to the pharynx and the later, more medially derived mesenchyme lateral to the neural tube. Once formed, the crest mesenchyme dorsolateral to the pharynx is displaced ventromedially in a narrow, transient subectodermal space functionally similar to that observed in the chick embryo. Displacement might result from cell motility or the formation of matrix-filled spaces between cells of the mesenchyme. Displaced cells are closely associated with the overlying columnar epithelium. This association precedes their subsequent induction and may reflect preliminary patterning. The crest mesenchyme passively distributed lateral to the neural tube is subsequently displaced medially. Here the formation of enlarged (matrix-filled?) spaces is clearly involved in the initial displacement. Displaced cells proliferate to form the anlage of the trigeminal ganglion. The other major contributor to this ganglion is the trigeminal placode. The placodal epithelium is located dorsolateral to the pharynx of the 12-somite embryo. If the epithelia of the head maintain their relative positions, this placode is derived from the squamous epithelium formed together with the earliest crest mesenchyme. If not, an alternative source is the columnar epithelium located ventromedial to the tip of the 4+- to 5-somite neural fold.     

An ultrastructural analysis of cell and matrix differentiation during early limb development in Xenopus laevis

Early development of the hind limb of Xenopus (stages 44–48) has been analyzed at the level of ultrastructure with emphasis on differentiation of extracellular matrix components and intercellular contacts. By stages 44–45, mesenchyme is separated from prospective bud epithelium by numerous adepidermal granules in a subepithelial compartment (the lamina lucida), a continuous basal lamina and several layers of collagen (the basement lamella). Tricomplex stabilization of amphoteric phospholipid demonstrates that each adepidermal granule consists of several membranelike layers (electron-lucent band 25–30 Å; electron-dense band 20–40 Å), which are usually parallel to the basal surface of adjacent epithelial cells. Collagen fibrils are interconnected by filaments (35 Å in diameter) which stain with ruthenium red. Epithelial cells possess junctional complexes at their superficial borders, numerous desmosomes at apposing cell membranes and hemidesmosomes at their basal surface. Mesenchymal cells predominantly exhibit close contacts (100–150 Å separation) with few focal tight junctions at various areas of their surface. By stages 47–48, adepidermal granules are absent beneath bud epithelium and layers of collagen in the basement lamella lose filamentous cross-linking elements. Filopodia of mesenchymal cells penetrate the disorganized matrix and abut the basal lamina. Hemidesmosomes disappear at the basal surface of the epidermis and mesenchymal cells immediately subjacent to epithelium exhibit focal tight junctions and gap junctions at their lateral borders. These structural changes may be instrumental in the epitheliomesenchymal interactions of early limb development. Degradation of oriented collagenous lamellae permits direct association of mesenchymal cell surfaces (filopodia) with surface-associated products of epithelial cells (organized into the basal lamina). Development of structural pathways for intercellular ion and metabolite transport in mesenchyme may coordinate events specific to limb morphogenesis.     

Mesenchyme cells degrade epithelial basal lamina glycosaminoglycan

We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGs are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.     

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.     

Ultrastructural and time-lapse studies of primary mesenchyme cell behavior in normal and sulfate-deprived sea urchin embryos

The mechanism of primary mesenchyme cell migration in the sea urchin, Lytechinus pictus, was studied in normal embryos and in sulfate-deprived embryos in which primary mesenchyme cells do not migrate. Based on scanning electron microscopy (SEM), cell processes were classified into six morphological types. Time-lapse cinematographic studies showed that two types of cell processes, a short finger-like process and a long process which accounted for 40 and 30% of the cell processes formed, respectively, in normal embryos, functioned as kinetic appendages during cell migration. Although the short finger-like process was formed to some extent in sulfate-deprived embryos, these processes were not able to attach to the ectodermal basal lamina, which is the migratory substratum. The long type of cell process was not observed at all in sulfate-deprived embryos. Transmission electron microscopy (TEM) demonstrated that cell processes in normal embryos were associated with 30 nm diameter granules in the basal lamina. Because these granules were absent in sulfate-deprived embryos, it is suggested that a specific component of the basal lamina substratum can be a limiting factor in cell migratory behavior.     

A morphological and experimental study of the mesencephalic neural crest cells in the mouse embryo using wheat germ agglutinin-gold conjugate as the cell marker

The distribution of the mesencephalic neural crest cells in the mouse embryo was studied by mapping the colonization pattern of WGA-gold labelled cells following specific labelling of the neuroectoderm and grafting of presumptive neural crest cells to orthotopic and heterotopic sites. The result showed that (1) there were concomitant changes in the morphology of the neural crest epithelium during the formation of neural crest cells, in the 4- to 7-somite-stage embryos, (2) the neural crest cells were initially confined to the lateral subectodermal region of the cranial mesenchyme and there was minimal mixing with the paraxial mesoderm underneath the neural plate, (3) labelled cells from the presumptive crest region colonized the lateral cranio-facial mesenchyme, the developing trigeminal ganglion and the pharyngeal arch, (4) the formation of neural crest cells was facilitated by the focal disruption of the basal lamina and the cell-cell interaction specific to the neural crest site and (5) the trigeminal ganglion was colonized not only by neural crest cells but also by cells from the ectodermal placode.     

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.     

Invagination of Ectodermal Placodes Is Driven by Cell Intercalation-Mediated Contraction of the Suprabasal Tissue Canopy

Ectodermal organs such as teeth, hair follicles, and mammary glands begin their development as placodes. These are local epithelial thickenings that invaginate into mesenchymal space. There is currently little mechanistic understanding of the cellular processes driving the early morphogenesis of these organs and of why they lead to invagination rather than simple tissue thickening. Here, we show that placode invagination depends on horizontal contraction of superficial layers of cells that form a shrinking and thickening canopy over underlying epithelial cells. This contraction occurs by cell intercalation and is mechanically coupled to the basal layer by peripheral basal cells that extend apically and centripetally while remaining attached to the basal lamina. This process is topologically analogous to well-studied apical constriction mechanisms, but very different from them both in scale and molecular mechanism. Mechanical cell–cell coupling is propagated through the tissue via E-cadherin junctions, which in turn depend on tissue-wide tension. We further present evidence that this mechanism is conserved among different ectodermal organs and is, therefore, a novel and fundamental morphogenetic motif widespread in embryonic development.     

Patterns of cells and extracellular material of the sea urchin Lytechinus variegatus (Echinodermata; Echinoidea) embryo,from hatched blastula to late gastrula

Scanning electron microscopy of six stages of Lytechinus variegatus embryos from hatching through gastrulation reveals changes in the shapes of the ectodermal cells and morphological changes in the extracellular material (ECM) in relation to the locations and migratory activities of mesenchyme cells. The classical optical patterns in the blastular wall (Okazaki patterns) are due to differential orientations of the cells, which bend and extend sheet-like lamellipodia over adjoining cells toward the eventual location of the primary mesenchymal ring. The blastocoelic surfaces of the blastomeres become covered with a thin basal lamina (BL) composed of fibers and nonfibrous material. During primary mesenchyme cell (PMC) ingression, a web-like ECM is located in the blastocoel overlying the amassed PMCs. This ECM becomes sparse in migratory mesenchyme blastulae, and is confined to the animal hemisphere. Localized regions of intertwining basal cell processes in the blastular wall are also present during PMC migration. While a distinct BL is present during early and midgastrulation, blastocoelic ECM is absent. Late gastrulae, on the other hand, have an abundance of blastocoelic ECM concentrated near secondary mesenchyme cell protrusive activity. ECM appearing at both the early mesenchyme and late gastrula stages are probably remnants of degraded BL and intercellular matrix preserved by fixation for SEM. Thus, early mesenchyme ECM is formed of BL material whose degradation is necessary for entry of PMCs into the blastocoel. Late gastrula ECM is apparently a degradation product of BL and intercellular material whose destruction is required for fusion of the gut with oral ectoderm in formation of the mouth.     

Migratory and invasive behavior of pigment cells in normal and animalized sea urchin embryos

Pigment cell precursors in the vegetal plate of late mesenchyme blastulae of the sea urchin Strongylocentrotus purpuratus begin to express a cell surface epitope recognized by the monoclonal antibody SP-1/20.3.1. When one-quarter gastrulae are dissociated into ectodermal and mesenchymal fractions, most SP-1/20.3.1 immunoreactive cells separate into the mesenchymal fraction, whereas at the full gastrula and all later stages almost all epitope-bearing cells are in the ectodermal fraction. Exposure of embryos to sulfate-free seawater p-nitrophenyl beta-D-xyloside, and tunicamycin, all of which prevent primary mesenchyme migration, does not inhibit SP-1/20.3.1 immunoreactive cells from distributing similarly to those in controls, although pigment synthesis is completely inhibited in sulfate-free conditions. Time-lapse video sequences reveal that pigment cells, and a small set of rapidly migrating, SP-1/20.3.1 immunoreactive amoeboid cells that appear in the pluteus, remain closely associated with the ectodermal epithelium during most of larval development. Transmission electron microscopy observations of plutei show pigment cells tightly apposed to the ectodermal epithelium at discontinuities in the basal lamina and sandwiched between the basal lamina and the epithelial cells. It is concluded that SP-1/20.3.1 immunoreactive mesenchymal cells invade the ectodermal epithelium and may use migratory substrates other than those used by primary mesenchymal cells.     

Retention of epithelial basal lamina allows isolated mandibular mesenchyme to form bone

The initiation of bone formation in the avian mandible requires that neural crest-derived cells undergo an inductive interaction with mandibular epithelium. To examine the role of the epithelial basal lamina in that interaction, mandibles were separated into their epithelial and mesenchymal components following exposure to the chelating agent, EDTA. Transmission and scanning electron microscopy was used to show that the basal lamina was retained as a continuous layer over the mesenchyme. Osteogenesis was initiated when such EDTA-isolated mesenchyme was grafted to the chorioallantoic membranes of host embryos. In contrast, mesenchyme isolated using trypsin and pancreatin failed to form bone. It is concluded that the property of mandibular epithelium which permits osteogenesis resides within the basal lamina.     

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.     

Ultrastructural changes in rat palatal epithelium after beta-aminopropionitrile.

Beta-Aminopropionitrile (BAPN) retarded or suppressed epithelial changes in the medial edge of the palatal process in later stages of gestation in rats. Programmed cell death did not follow the usual pattern, and only a few lysosomes were observed on day 18 of gestation. The sensitivity of the medial epithelium to BAPN appeared to be different in various areas of the palatal epithelium; the epithelium on the anterior region of the palatal process was hypertrophied and keratinized, while posteriorly the medial or neighboring epithelium was very thin and, in neonatal rats, the covering was absent. A basal lamina was distinct in the anterior region and indistinct or fragmented posteriorly. Collagen fibers did not develop adjacent to the basal lamina, and an amorphous material was scattered throughout the mesenchymal tissue. These findings suggest that BAPN decreases the "connecting capacity" between mesenchyme and epithelium, and results in a modification of epithelial changes.     

Pioneer neuron pathfinding from normal and ectopic locations in vivo after removal of the basal lamina

The contribution of the basal lamina to Ti1 pioneer axon guidance in grasshopper limb buds was investigated by allowing growth cones to migrate in 30%-31% stage limbs from which the basal lamina had been removed by enzymatic treatment. When the Ti1 axons extended from their normal location, the pathways established in the absence of basal lamina were normal. This indicates that the basal lamina is not required for initial proximal axon outgrowth, recognition of limb segment boundaries, or selective interaction with neuronal somata. Removal of the basal lamina from slightly older (32% stage) embryos resulted in displacement of the Ti1 somata to ectopic locations in approximately 50% of the limbs. Pathfinding from ectopic locations was aberrant in 45% of the cases observed. This demonstrates that if orienting information is present in the basal lamina-free epithelium at this stage, it is not the predominant factor in determining growth cone orientation from ectopic locations.     

Fine-grained fate maps for the ophthalmic and maxillomandibular trigeminal placodes in the chick embryo

Vertebrate cranial ectodermal placodes are transient, paired thickenings of embryonic head ectoderm that are crucial for the formation of the peripheral sensory nervous system: they give rise to the paired peripheral sense organs (olfactory organs, inner ears and anamniote lateral line system), as well as the eye lenses, and most cranial sensory neurons. Here, we present the first detailed spatiotemporal fate-maps in any vertebrate for the ophthalmic trigeminal (opV) and maxillomandibular trigeminal (mmV) placodes, which give rise to cutaneous sensory neurons in the ophthalmic and maxillomandibular lobes of the trigeminal ganglion. We used focal DiI and DiO labelling to produce eight detailed fate-maps of chick embryonic head ectoderm over approximately 24 h of development, from 0-16 somites. OpV and mmV placode precursors arise from a partially overlapping territory; indeed, some individual dyespots labelled both opV and mmV placode-derived cells. OpV and mmV placode precursors are initially scattered within a relatively large region of ectoderm adjacent to the neural folds, intermingled both with each other and with future epidermal cells, and with geniculate and otic placode precursors. Although the degree of segregation increases with time, there is no clear border between the opV and mmV placodes even at the 16-somite stage, long after neurogenesis has begun in the opV placode, and when neurogenesis is just beginning in the mmV placode. Finally, we find that occasional cells in the border region between the opV placode and mmV placode express both Pax3 (an opV placode specific marker) and Neurogenin1 (an mmV placode specific marker), suggesting that a few cells are responding to both opV and mmV placode-inducing signals. Overall, our results fill a large gap in our knowledge of the early stages of development of both the opV and mmV placodes, providing an essential framework for subsequent studies of the molecular control of their development.     

Hyaluronan Disappears Intercellularly and Appears at the Basement Membrane Region during Formation of Embryonic Epithelia

The distribution of tissue hyaluronan has been assessed in the neuraxial region of 8.5 to 10.5 day mouse embryos using a fragment of bovine nasal cartilage proteoglycan that binds specifically to hyaluronan. Hyaluronan is abundant in all mesenchymal tissues, predominantly intercellularly, but markedly diminishes when mesenchymal cells organize into epithelia, as in the formation of somites. Hyaluronan reappears in abundance when epithelia (e.g. sclerotome) disperse into mesenchyme. Hyaluronan is present between cells of early epithelia (e.g. neural plate), but is lost during their subsequent development when it becomes abundant at their basement membrane regions. These results show for the first time changes in hyaluronan distribution during the development of embryonic epithelia. The hyaluronan distribution found is consistent with the functions proposed for hyaluronan in embryonic mesenchyme: intercellular hyaluronan would allow the epithelial cells to move and reduced hyaluronan would allow the cells to associate. The absence of intercellular hyaluronan in later epithelia would allow increased membrane contacts that lead to the formation of intercellular junctions. The restriction of hyaluronan to basement membrane regions in later epithelia further substantiates the suggestion that hyaluronan is a bona fide component of the basal lamina and that it is involved in maintaining epithelial morphology.     

Fine structure of the epidermis of the optic tentacle in a slug, Limax flavus L.

The epidermis at the tip of the optic tentacle in Limax flavus is constructed of columnar epithelial cells, distal processes of nerve cells, and scattered processes of the collar cells. The epithelial cells extend stout microvilli called plasmatic processes by Wright perpendicularly from the free surface. Each plasmic process branches into a few terminal twigs embedded in a fuzzy filamentous substance. Most nerve cells have their nuclei under the basal lamina. The distal processes of these nerve cells reach the free surface and send long microvilli to form the spongy layer under a filamentous covering. At the side surface of the tentacle the epithelial cells are cuboidal or squamous and the neural elements are fewer. Here, no spongy layer is formed; and the collar cell processes are replaced by the lateral cell processes. Peculiar secretion granules are contained in the lateral and collar cell processes as well as in their cell bodies situated beneath the basal lamina.