Bruce J. Crawford

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.

A fine structural study of the testicular wall and spermatogenesis in the crinoid, Florometra serratissima (Echinodermata)

The testicular wall and the process of spermatogenesis in the crinoid, Florometra serratissima, has been studied at the fine structural level. The testicular wall is composed of three layers: a perivisceral layer consisting of nerve processes, muscle fibers, and epithelial cells; a haemal sinus containing haemal fluid, collagen‐like fibers, and haemocytes; and a germinal layer consisting of germinal and interstitial cells.

Morphogenesis of the digestive system during metamorphosis of the nudibranch Doridella steinbergae (Gastropoda): Conversion from phytoplanktivore to carnivore

The structural changes undergone by the digestive system of a phytoplanktotrophic nudibranch larva during metamorphosis into a benthic carnivore are described using histological and electron microscopic techniques. The relative positions of the stomach, digestive gland, and distal end of the intestine are rearranged at metamorphosis by the actions of the larval retractor muscle and the accessory pedal retractor muscle. Although the anus and distal end of the intestine are secondarily displaced to the posterior end of the gastropod, the stomach undergoes further torsional displacement at metamorphosis. The tissues of the larval stomach and distal end of the larval esophagus undergo drastic alteration at metamorphosis. The larval stomach consists of a ciliated vestibule, which receives the openings of the esophagus and left digestive gland, a gastric shield, a style sac, and an intestinal groove. All of these areas, except the vestibule, are destroyed by cell dissociation at metamorphosis. The vestibule becomes the ventral stomach of the benthic stage and the proximal end of the intestine becomes enlarged and muscularized to form the dorsal ‘stomach’ of the benthic stage. The metamorphic changes involving the distal end of the esophagus include the continued development of the radula and oral lip glands, which both appear in rudimentary form during the larval stage, and differentiation of the buccal pump, salivary glands, and oral lips.

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.

Changes in the arrangement of the extracellular matrix, larval shape, and mesenchyme cell migration during asteroid larval development

When fixed in the presence of alcian blue, extracellular matrix (ECM) in the embryonic asteroid blastocoel can be visualized by light and electron microscopy as a fibrous meshwork encrusted with alcianophilic material. In early to mid‐gastrulae, the ECM is associated with the basal laminae underlying the ectoderm and endoderm. It also forms a fibrous meshwork between them in the posterior part of the blastocoel. In early larvae, when mesenchyme cells arrive at the esophagus to differentiate into smooth muscle, very little ECM is associated with the stomach region. In contrast, a meshwork of long ECM strands radiates from the esophageal basal lamina which connects to a dense ECM web associated with the inner aspect of the dorsal ectoderm. This dorsal web is associated, in turn, with numerous long ECM strands which run parallel to the stomodeum. The strands located between the esophagus and the ectoderm appear when the mouth and coeloms form and may be responsible for a constriction of the ectoderm that forms in this region. During late gastrula one population of mesenchyme cells becomes associated with the esophageal region and differentiates into muscle. Most of the other mesenchyme cells stop migrating through the esophageal web at this time. Less alcianophilic material is associated with the esophageal basal lamina, and the ECM adjacent to the esophagus in the late gastrula and early bipinnaria larvae. The arrangement of the ECM elements suggests that they could be involved in controlling the migration of mesenchyme cells, particularly those destined for the esophagus.

A simple ethanol-based freeze-substitution technique for marine invertebrate embryos which allows retention of antigenicity

A simple technique has been developed for flash freezing and freeze substituting small (0.5 mm) marine embryos, which effectively preserves both cellular and extracellular components, using inexpensive equipment that is readily available in most laboratories. To achieve this, embryos of the starfish, Pisaster ochraceus, were isolated on copper freeze‐fracture EM grids. The embryos were then rapidly frozen by plunging the grids into a supercooled liquid cryogen, and stored in liquid nitrogen. Freeze substitution was carried out by placing the specimens in sealed vials containing anhydrous ethanol at −90°C for 4–5 days. Following substitution, the specimens were passively warmed to −20°C over 2 h and then to room temperature over a further 2 h. They were then embedded in either JB4 for light microscopy or Epon or LR White resins for transmission electron microscopy. Four different liquid cryogens, Freon 12, ethane, propane, and nitrogen slush, were tested. Freezing in propane, the best cryogen of the four, gave good preservation of the embryonic cells but poor preservation of the extracellular matrix (ECM). To overcome this, the embryos were exposed to four cryoprotective agents, dimethylsulphoxide, glycerol, ethylene glycol and propylene glycol prior to freezing, and the results were assessed. The experiments demonstrated that good preservation of both cells and ECM could be achieved by adding 15% propylene glycol in sea water to the embryos prior to freezing in propane. Material preserved in this manner not only gave excellent morphological results, but the antigenicity of both native antigens of ECM components and antibodies to which the animals had been exposed in vivo were retained. The application of this technique to other tissues and embryos should prove useful in many future studies.

Ultrastructure of the basal lamina and its relationship to extracellular matrix of embryos of the starfish Pisaster ochraceus as revealed by anionic dyes

The ultrastructure of the 51/2–6‐day‐old embryonic asteroid basal lamina (BL) was studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and after treatment with anionic dyes. Conventional fixation in glutaraldehyde and osmium reveals a BL consisting of a lamina densa separated from the basal cell surface by a lamina lucida. Little or no reticular lamina is present. Material similar in appearance to the basal lamina extends into the blastocoel, forming an extracellular matrix (ECM). Following fixation in the presence of the dye ruthenium red, proteoglycan (PG) granules are visible in the lamina lucida and immediately beneath the lamina densa. The ECM consists of granules of a similar appearance, which are associated with fibers of an intermediate electron density resembling invertebrate collagen. After fixation in the presence of alcian blue under polyanionic conditions, all aspects of the basal lamina and the ECM stain very densely. The use of alcian blue in 0.3 M MgCl2 (monoanionic condition) or in low concentrations reveals a lamina densa consisting of a fine feltwork and tubule‐like structures. A meshwork composed of thick, densely stained and thinner, intermediately stained strands is embedded in the inner aspect (that adjacent to the blastocoel) of the ectodermal lamina densa. Similar elements are present in the endodermal BL, but the dense material is represented by short regions that do not form a meshwork. The dense and intermediate strands of both basal laminae also extend into the blastocoel as ECM. The tubule‐like structures extend from the dense material of the inner meshwork into the lamina densa. They also cross both the lamina densa and lucida to associatee with the basal cell membranes. The fact that the basal cell surfaces are often puckered outward at the points of contact suggests that this configuration might be providing a means whereby forces can be transferred from the ECM through the basal lamina to the cells.

Regional Ultrastructural Differences in Basal Laminae Isolated from the Starfish Pisaster ochraceus

The ultrastructure of different regions of the basal laminae isolated from 5‐1/2‐6 day‐old embryos of the starfish, Pisaster ochraceus, has been described after fixation in the presence of anionic dyes. Isolated basal laminae from all regions of the embryo exhibit a lamina lucida and lamina densa. No lamina fibroreticularis is present. Instead, a coarse meshwork of thick densely stained and thinner intermediately stained fibers is embedded in the lamina densa and extends into the blastocoel forming the extracellular matrix. The coarse meshwork associated with the ectodermal basal lamina consists primarily of thick densely stained fibers with a small number of intermediate ones while that associated with the endodermal one contains much less densely stained material. These structures were morphologically identical to those found in control embryos. Examination of different regions of the endodermal basal lamina shows that the amount of dense material varies from region to region. These differences in dense material may reflect biochemical differences, particularly of proteoglycans, which could provide positional information to migrating mesenchyme cells.

Ultrastructural aspects of mouth formation in the starfish Pisaster ochraceus

Early events during mouth formation in embryos of the starfish Pisaster ochraceus have been studied with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Embryos examined by TEM were fixed in glutaraldehyde with Alcian blue, a dye which preserves extracellular materials. Initially, mesenchyme cells migrate off the tip of the archenteron, leaving a defect in the cell layer that is covered by the basal lamina. This region of “naked” basal lamina bulges into the blastocoele and forms a hemispherical blister. At the same time that this is occurring, filamentous and conical processes extend from the inner surface of the presumptive stomodeal ectoderm cells which are located directly opposite the bulge of basal lamina. These processes penetrate the ectodermal basal lamina and project “naked” plasmalemma into the blastocoele. Shortly after this, the blister of endodermal basal lamina becomes irregular in shape, and scattered cells are found both within the blister and between it and the presumptive stomodeal ectoderm cells. Processes of endodermal cells appear to make contact with the filamentous processes of the stomodeal ectoderm cells. In other embryos which appear to be at a slightly later stage, the free basal lamina is conical in shape and is associated with at least one conical ectodermal process. In yet other embryos, the free endodermal basal lamina is either in contact with several other large processes forming a circular region of contact, or the free endodermal and ectodermal basal laminae are fused at the edge of the circle. Degeneration of both the ectodermal and endodermal basal laminae located within the circle, and subsequent invagination of the stomodeal ectoderm, appear to complete this process. The pulsations of stomodeal ectodermal cells seen throughout early stages in mouth formation may be involved in these events.

A technique for processing mucous coated marine invertebrate spermatozoa for scanning electron microscopy

A technique for preparing heavily mucous coated marine invertebrate spermatozoa for scanning electron microscopy (SEM) is described. This technique involves washing in 1500 NF units/ml hyaluronidase in millipored sea water to remove mucus, followed by fixation in glutaraldehyde and osmium tetroxide. Following primary fixation, spermatozoa are enclosed in Nuclepore membrane bags positioned within Teflon specimen capsules allowing them to be processed and critical point dried without excessive mechanical damage or loss.