Glycocoding as an information management system in embryonic development.

The past 5 years have seen a burgeoning in the amount of data emerging from laboratories studying the early stages of embryogenesis. Much of this data implicates various aspects of glycobiology in the initiation and regulation of these processes. The level of analytical detail coming from these investigations has surpassed our ability to fully understand its overall significance within the context of the interactive/dynamic developmental process. This review proposes a pause in this seemingly endless quest for more detail so that we may take stock of our goals and objectives. The proposed goals include a mechanistic understanding of the process(es) involved in information managed at various 'transition points' during early embryogenesis, and an understanding of the mechanism(s) by which the spatial/temporal regulation of early development are managed. The 'transitions' which occur during cell to cell cluster, cell cluster to early organ architecture, and early organ architecture to functional organ development are fundamental and mirror the evolutionary process of biological information management. All of these 'transitions' involve increasing complexity, the development of hierarchies of information management systems (integrated bidirectionally), and spatial/temporal regulation which relies on historical events to map future structure and function. This review focuses on a relatively small number of studies which highlight these aspects of early development. A mechanism which involves glycocoding, an extension of the Roseman Hypothesis, and its direct use as an information management system is proposed and some supporting experimental evidence is presented. This extension of an existing hypothesis is related to several recent investigations, and is designed to broaden the experimental design of future studies so that these important process issues can be addressed.

Formation and perforation of closing plates in the chick embryo.

The morphology of the closing plates between adjacent pharyngeal arches was examined in chick embryos between stages 11 and 21 (Hamburger-Hamilton). Each closing plate is formed by apposition between the basal surfaces of portions of the pharyngeal pouch endoderm and the ectoderm of the overlying pharyngeal cleft. Initial contact between ectoderm and endoderm occurs at several small points which are separated by regions containing mesenchymal cells and extracellular material. Contact between the opposed epithelia is made by extension of cellular processes through the intervening basal laminae and extracellular space. Endodermal and ectodermal cells then interdigitate to create a cellular layer which rapidly thins. The interposed extracellular material is sequestered into small pools as cellular interdigitation proceeds. Perforations form through certain regions of closing plates 1-3 and persist during the stages studied. Small slit-like depressions appear between cells of the closing plate just prior to perforation. The initial perforations enlarge until they are separated only by thin cellular strands. These strands presumably rupture, leaving small cellular accumulations which persist for a short time marking the junction between ectoderm and endoderm along the walls of adjacent pharyngeal arches. Clear evidence of cell degeneration is rare. These results suggest that cellular reorganization, rather than cell death, is a major mechanism of initial perforation.

Epithelial fusion during early semicircular canal formation in the embryonic zebrafish, Brachydanio rerio.

The developing inner ear of the teleost, Brachydanio rerio, provides an opportunity for observing an epithelial fusion between the apical surfaces of apposed epithelia in a vertebrate embryo in vivo. The developing otocyst was filmed for periods up to 4 days in unanesthetized embryos, and specimens were fixed at intervals and processed for light microscopy, TEM, and SEM. The semicircular canals are formed as a consequence of the union between the tips of three cylindrical projections from the wall of the otocyst, which grow toward corresponding bulges of a projection from the lateral wall. The epithelial cells covering the projections contain extensive rough endopasmic reticulum, exhibit apical junctional complexes, and are not underlain by a basal lamina. The core of each projection contains large amounts of flocculent and fibrillar extracellular material. After a period of growth and elongation, the tip of each projection contacts, and adheres to, the appropriate bulge to create a circular, flattened, bilayered, epithelial plate. Small, focal junctions form between the apposed apical cell surfaces within the plate during this period, but they are not numerous. Junctional complexes do develop, however, between apposed cells at the periphery of the plate. After 1-2 hours, the basal surface of the plate exhibit considerable alteration in contour. Adjacent cells within the plate then separate to allow continuity of the connective tissue components of the two structures. The observations of this study indicate that following an initial period of contact and adhesion, cellular reorientation and changes in junctional contacts between adjacent cells within the epithelial plate, rather than cell degeneration, are responsible for perforation of the plate.

Potential complications in myelography: I. Technical considerations.

Scanning electron microscopy demonstrated starch powder contamination on the stylet tip from a spinal needle after it was lightly touched with nonwashed surgical gloves. A sterile solution of Pantopaque was injected through the spinal needle after the stylet was withdrawn and carried particulate contaminants to a 0.2 micrometer millipore filter. Using both scanning electron microscopy and energy dispersive x-ray analysis, particles of various sized (mostly 5-10 microns) and variable compositions, including starch, talc, and other elements, were identified. Also, glass particles from the Pantopaque vial and plastic particles from the plastic syringe and tubing used in drawing up the Pantopaque were seen. These observations indicate that strict attention to proper technique in myelography is essential in order to eliminate potential cerebrospinal fluid contamination.

Indirect immunofluorescent staining of fibronectin associated with the floor of the foregut during formation and rupture of the oral membrane in the chick embryo.

The distribution of the glycoprotein, fibronectin, within the cranial region of stage 8--16 chick embryos was examined by indirect immunofluorescence using paraffin sections exposed to affinity-purified rabbit anti-human CIG and FITC-conjugated goat anti-rabbit immunoglobulins. Fluorescence was present within the matrix surrounding the cranial mesenchyme, along the basal surfaces of all epithelia, and surrounding the notochord at all stages. Fluorescence associated with the floor of the foregut was particularly intense. The fluorescent layers beneath the ectoderm and endoderm of the oral (oropharyngeal) membrane at stage 8 merged into a single, continuous, intensely fluorescent line as the extracellular space within the oral membrane narrowed during stages 9--12. This line of uniform fluorescence parallels the previously described histological reorganization of the extracellular compartment of the oral membrane, but the ultrastructural localization of this fluorescent material remains unknown. Fluorescence was also intense beneath the foregut endoderm in the presumptive cardiac region caudal to the oral membrane and was continuous with strands of fluorescent material extending into the matrix of the dorsal mesocardium and cardiac jelly of the developing tubular heart. These observations indicate that the extracellular matrix associated with the floor of the entire foregut contains fibronectin during stages encompassing the formation and rupture of the oral membrane. The presence of fibronectin within the oral membrane and dorsal mesocardium, as well as between Rathke's pouch and infundibulum and within the closing plates between ectodermal clefts and endodermal pouches, is consistent with the possibility that this glycoprotein may play a role in adhesion at these sites.

The ultrastructure of oral (buccopharyngeal) membrane formation and rupture in the chick embryo.

The ultrastructure of the oral (buccopharyngeal) membrane was examined by transmission and scanning electron microscopy (SEM) from its initial formation (stage 8) to its complete disappearance (stage 20) in the chick embryo. Thinning of the oral membrane prior to rupture occurs in large measure by increased interdigitation between cells of the stomodeal ectoderm and foregut endoderm coincident with a decrease in the width of the intervening extracellular space. Large numbers of necrotic cells were not observed. Interdigitation of ectodermal and endodermal cells makes it increasingly difficult to discern two discrete epithelia, and no evidence that one germ layer disappears prior to the other was observed. Changes occurred in the fine structure of the extracellular matrix during formation and rupture of the oral membrane, and the organization of this material within the oral membrane differed from that in regions immediately lateral to it. Copious amounts of amorphous, flocculant ("lamina-like") material are present within the oral membrane at all stages. The basal lamina of the ectoderm exhibits small loops or folds at early stages. These decrease in number as the basal lamina becomes discontinuous prior to establishment of direct intercellular contact between cells of the ectoderm and endoderm across the intervening extracellular compartment. Initial perforations of the oral membrane are preceded by clefts between cells on both sides of this structure, and SEM observations suggest that cells of the oral membrane continue to interdigitate, elongate, and change relative positions during the rupture process.

Preparation of embryonic tissues for SEM.

Examination of the SEM literature regarding embryonic stages of a variety of animal species reveals that similar results may be obtained using a range of preparative techniques, but that images of significantly different quality may also be obtained by different investigators studying similar tissues processed by presumably identical methods. This paper provides a discussion of basic procedures which may be used to obtain "good" quality images of embryonic stages of a variety of representative species including insect, marine invertebrate, amphibian, avian and mammalian forms commonly used in developmental biology. It is hoped that such information will serve as an initial starting point for those with little or no previous experience in preparing embryonic specimens for SEM examination. In addition to comments regarding fixation, dehydration, drying and preparation of a conductive surface, representative procedures for procuring, handling and removal of extraembryonic layers are also mentioned. Some of the more common types of artifacts are also considered in an attempt to aid in interpretation of micrographs.

In vitro activation of adenylate cyclase by norepinephrine, parathyroid hormone, calcitonin, and prostaglandins in the developing maxillary process and palatal shelf of the golden hamster.

The regional localization of hormonally sensitive adenylate cyclase within the maxillary-palatal process complex was examined in tissue homogenates at different stages during the development of the secondary palate in the golden hamster. The most potent agents capable of activating adenylate cyclase were parathyroid hormone (PTH) and calcitonin (CT). Highest activities were observed in the intact maxillary process-palatal shelf complex and the isolated maxillary process prior to and during fusion of the palate. Thereafter, neither hormone displayed a remarkable capacity to elevate enzyme activity. Parathyroid hormone and CT exhibited a similar, but considerably reduced, capacity for enzyme stimulation in the isolated palatal shelf. 1'-Norepinephrine also increased adenylate cyclase activity in both the palatal shelf and the maxillary process at the earlier stages. Prostaglandins (PG) E1, E2, and F2 alpha stimulated adenylate cyclase activity within the intact palate, the maxillary process, and the palatal shelf primarily at the earlier stages. The adenylate cyclase from the isolated palatal shelf was more sensitive to stimulation by the PGs than that from either the intact palate samples or isolated maxillary processes. The findings imply that the fusion process of the secondary palate is under a highly sensitive hormonal control mechanism.

In vitro activation of adenylate cyclase by parathyroid hormone and calcitonin during normal and hydrocortisone-induced cleft palate development in the golden hamster.

An adenylate cyclase highly responsive to stimulation by parathyroid hormone (PTH) and calcitonin (CT) in vitro was observed at certain times during normal prenatal development of the maxillary-palatal process complex in the golden hamster. Responses of the enzyme to these hormones were barely detectible at the earliest stage examined (day 10/20). The enzyme became extremely sensitive to activation by either hormone during the time of rapid growth of the palatal processes (day 11/20) and during fusion between the palatal processes (day 12/20). Thereafter, responses were greatly diminished and little or no activation of adenylate cyclase was observed until birth. Adenylate cyclase from fetuses in which clefts of the secondary palate were induced by maternal treatment with hydrocortisone (50 mg) on day 11/3 also displayed an enhanced sensitivity to PTH and CT on day 11/20, but the sensitivity of the enzyme was greatly decreased from that in normal animals during the normal time of palatal fusion (day 12/20) and was barely detectible or absent at the remaining time periods studied (days 13/20 and 14/20). Addition of hydrocortisone to the incubation mixture, either separately or in combination with PTH or CT, did not remarkably affect the response of adenylate cyclase to these hormones. Moreover, the appearance of the adenylate cyclase sensitive to hormonal activation did not result from changes in phosphodiesterase activity during palatal maturation.

Catecholamine-sensitive adenylate cyclase in the developing golden hamster palate.

The present study was initiated to determine whether specific hormones would influence adenylate cyclase activity within the maxillary-palatal complex during formation of the hamster secondary palate. Stages from initial appearance of the palatal processes to shortly after birth were studied. Highest basal adenylate cyclase activities occurred during the earliest periods of palate development. This basal enzyme activity began to diminish as palatal fusion occurred and remained lowered until birth. Activation of adenylate cyclase by fluoride was maximal at concentrations of 5-10 mM, and was observed throughout the span of palatal development. Fluoride activation of adenylate cyclase was greatest prior to fusion of the palatal processes, then decreased until birth when a slightly increased enzymatic stimulation was seen. Norepinephrine and epinphrine were the catecholamines most capable of inducing increased activation of adenylate cyclase at most periods of palatal growth. Increased enzyme activity in the presence of norepinephrine was more susceptible to antagonism by the beta adrenergic agent, propranolol, than to the alpha adrenergic agent, phentolamine. The remaining catecholamines, namely isoproterenol and dopamine, displayed a lesser ability to activate the enzyme, and adenylate cyclase was not equally responsive to these catecholamines at identical developmental stages. Other hormones, i.e. histamine, serotonin, thyrotropin, growth hormone, thyroxine and glucagon were generally ineffective in activating the enzyme. Phosphodiesterase activity was not detected until shortly before birth.

Topographical changes along the neural fold associated with neurulation in the hamster and mouse.

The topography of the ectoderm was examined by scanning electron microscopy during neurulation in hamster and mouse embryos. Stages from the appearance of the neural folds to closure of the posterior neuropore were studied. Progressive development of a zone of altered cellular morphology was observed along the crests of the neural folds. This zone evolved from an abrupt transition between surface and neural regions of the ectoderm to a narrow band of flattened cells which exhibited numerous membranous "ruffles" in the mouse, or blebs and presumably degenerating cells in the hamster, immediately prior to contact between the folds. These alterations were more prominent along the anterior than the posterior portions of the folds. Contact of the folds occurred first between the flattened cells with subsequent union of the surface cells. Stages of neural crest cell formation was observed subjacent to the zone of alterations in histological sections. It is suggested that the observed surface alterations may reflect changes in the membrane properties of the altered cells which are correlated with both neural crest formation and initial adhesion between the folds.