Cellular dynamics during evagination of the thyroid primordium in the chick embryo.

The thyroid forms as an outpouching of the ventral pharynx. Evidence supports the conclusion that formation of the thyroid pit is mediated by changes in the cytoskeleton that cause constriction of cell apices. However, it seems unlikely that a relatively flat epithelial sheet can be converted into a pit without either distortions of the surface or considerable rearrangement of cells to reduce surface area. Possible cellular rearrangements were investigated by tracing the movements of individual cells by using time-lapse video microscopy. Changes in shape of the primordium were investigated by marking with carbon and DiI and by scanning electron microscopy. Cell movements occurred only over short distances, mostly shifts relative to a neighbor, especially at the edge of the pit. Instead, cells rearranged into clusters that piled up at the edge of the pit and then tilted inside. Adjacent rings of pharyngeal cells were annexed by the growing thyroid, undergoing rearrangement into clusters, piling up at the edge, and moving inside the pit. The consequence was the formation of a series of shelf-like extensions within the cavity, representing successive generations of cell rings moving inside. These results have implications for the formation of other organs by evagination.

A role for neural cell adhesion molecule in the formation of the avian inner ear.

The inner ear forms by a series of folds within an ectodermal placode. Previous work has shown that changes in surrounding tissues play a more prominent role in invagination than changes in the cytoskeleton of the primordium. Interference with the integrity of the extracellular matrix causes abnormalities in the folding process, primarily related to abnormalities in the paraxial mesoderm which lies ventral to the placode. In this study, the role of the neural cell adhesion molecule (N-CAM) was investigated, based on the expression of this component of the plasmalemma at the time the otic placode begins to fold. Microinjection of blocking antibodies to N-CAM into the paraxial mesoderm adjacent to the otic placode resulted in two major classes of defects, detachment of the primordium from the neural tube and interference with formation of the folds. Microinjection of saline, control immunoglobulin, or antibody against cytoplasmic domain had no effect. These defects correlate with the pattern of N-CAM expression at the time of injection, along the neural ectoderm and otic epithelium and the mesenchyme cells ventral to the primordium. It seems likely that N-CAM is playing a role in heterophilic associations rather than through the homophilic binding domain during formation of the otic vesicle.

Morphogenesis of the lung: control of embryonic and fetal branching.

Lung development differs in the embryo and fetus with regard to branching pattern and organization of the epithelial cells. The surrounding mesodermal component, the capsule, has long been known to play a role in branching. As a result of recent analyses of distribution of components of the extracellular matrix coupled with interference with their expression, we are beginning to understand how branching is controlled. Insoluble macromolecules of the basal lamina and deeper extracellular matrix may act as physical barriers or traps to sequester soluble components. The soluble growth factors activate genes regulating cell proliferation.

Involvement of extracellular matrix in the formation of the inner ear.

Formation of the inner ear from the optic placode differs from invagination of other cup-shaped organ primordia. Activation of the actin cytoskeleton seems to play a limited role because precocious invagination does not occur upon treatment with activators of a contractile event and cannot be prevented by inhibitors. In this study, the possibility that invagination is mediated by changes in the surrounding mesenchyme was tested by treating embryos with agents which interfere with the integrity of extracellular matrix. Enzymes degrading hyaluronate and/or chondroitin sulfate were microinjected into the otic region prior to folding. Synthesis of chondroitin sulfate proteoglycan was inhibited by microinjection of beta-xyloside. All treatments inhibited otic pit formation by interfering with fold formation within the placode. Immunocytochemical procedures showed depletion of the appropriate extracellular matrix components for a short time period after enzyme treatments and for up to 24 hr after beta-xyloside injection. Invagination of the otic primordium is concluded to be controlled in part by anchorage of the epithelium to adjacent structures and possibly by expansion of the mesenchymal extracellular matrix.

Organization of tracheal epithelium in the cartilaginous portion of adult rabbit and its persistence in organ culture.

BACKGROUND: The rabbit trachea provides a model system to test the physiological responses of the airways to various agents. Since three-dimensional organization may affect responses of an organ, an organ culture model was established in serum free medium. METHODS: Tracheas were fixed in situ, at steps in the preparation of organ cultures, and after one day to three weeks in organ culture. Samples were examined by light microscopy and scanning and transmission electron microscopy for surface morphology, distribution of cell types, and characteristics of the epithelial cell layer. RESULTS: The normal tracheal mucosa was discovered to consist of extensive circumferential folds in the cartilaginous portion, which were enhanced upon isolation of the trachea from the animal. The folds consisted principally of differences in epithelial cell height rather than folding of the lamina propria. Enhancement of folding upon removal of the trachea coincided with increased secretion by Clara and possibly mucous cells. In organ culture, epithelial cell height initially increased, producing tall folds and cell types were retained in normal proportions. After prolonged culture, cilia were lost but glandular secretion continued. CONCLUSIONS: Changes in the arrangement of basal cells and secretory activity in isolated trachea and in culture may give insight into the functional significance of the epithelial folds.

Calcium regulation of neural fold formation: visualization of the actin cytoskeleton in living chick embryos.

The involvement of calcium ions during chick neurulation was studied by treating neural plate stage embryos with agonists and antagonists of calcium transport and with inhibitors of calmodulin activity. Organotypic shape changes were examined by light and scanning electron microscopy. Changes in size of cell apices were quantitated by computer image analysis of actin filaments labeled with fluorescent phallicidin in time lapse recordings of living embryos. Both ionomycin and A23187 caused precocious fold elevation around the median hinge point and convergence by bending at the lateral furrows only when Ca2+ was in the external medium. As judged by decreased perimeters of 100 fluorescent apical polygons, cell apices constricted medial to the lateral furrows but did not change significantly within the median hinge point. Pretreatment with dihydrocytochalasin B or cytochalasin D prevented precocious folding and apical constriction. Papaverine and verapamil prevented folding but could be reversed by subsequent ionophore treatment. Calmidazolium and trifluoperazine irreversibly blocked folding. The demonstration in living embryos of constriction of lateral cell apices in a calcium-dependent manner is consistent with a contractile process operating during the formation of neural folds.

Effect of tunicamycin on maturation of fetal mouse lung.

The mesodermal capsule of the fetal lung plays a role in differentiation of the respiratory region. It has been proposed for other epithelial organs that the mesodermal capsule influences development by modifying the basal lamina or the extended extracellular matrix. The effect could be on deposition or turnover of collagens, proteoglycans, and/or glycoproteins. This study tests the role of glycoproteins in differentiation of respiratory endings by inhibiting their synthesis with the antibiotic tunicamycin (TM). Lungs at 16 and 18 days gestation and 3 days after birth were cultured with TM and examined for morphological and biochemical differences from normal controls. With TM, alveolar regions did not expand properly and formed fewer type I pneumocytes, although type II pneumocytes were unaffected. The epithelium of untreated respiratory regions showed greater incorporation of radioactive mannose than the airways region or mesenchyme. This incorporation was diminished in TM, but the pattern persisted. Comparison with the results obtained with beta-xyloside suggested that differentiation of type I and type II pneumocytes is under separate control.

Explant culture of rabbit tracheobronchial epithelium: structure and prostaglandin metabolism.

This study examines the potential usefulness of explant culture of rabbit tracheal epithelium as a model for the study of epithelial function under normal and potentially pathologic conditions. Accordingly, we assessed the structure and prostaglandin E2 (PGE2) release of tracheal epithelial explants obtained from adult pathogen-free rabbits. Epithelial cells attached to their native connective tissue substratum were maintained in culture for 5 days in serum-free medium, under bipolar conditions (air-liquid interface) on a permeable membrane (pore size, 0.2 mm), and nourished from the basolateral surface. At 5 days in culture, scanning and transmission electron microscopy and light microscopy demonstrated a pseudostratified, ciliated columnar epithelium with prominent folds and mucus secretion identical in appearance to the mucosa before culture. On the day of dissection (day 0) and after 4 days in culture (day 4), explants released PGE2 into the medium spontaneously. However, day 4 explants produced 3- to 4-fold greater amounts of PGE2 than day 0 explants. Moreover, day 4 explants demonstrated increased PGE2 release in response to bradykinin, a receptor-dependent agonist, and ionomycin, a calcium ionophore, while day 0 explants did not. Primary tracheal epithelial cell cultures grown to confluence (day 9) on a collagen substrate demonstrated PGE2 responses to bradykinin and ionomycin that qualitatively resembled those of day 4 explants. We conclude that rabbit tracheal explants cultured in vitro under the above conditions maintain cellular differentiation, in situ three-dimensional organization, and PGE2 synthetic pathways over several days in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of basal lamina components during development of chick otic and optic primordia.

Immunolocalization of laminin, fibronectin, and type IV collagen was examined during early morphogenetic shape changes of the avian inner ear and eye. The ear was studied from formation of the otic placode to invagination of the otic pit and the eye from the optic vesicle stage to formation of an optic cup. Distribution and intensity of immunoreactivity were compared in the two organ primordia and in adjacent epithelial layers. Laminin formed a continuous layer at the basal surface of the otic ectoderm and adjacent neural tube at all stages. The basal surfaces of the optic and lens epithelia also were continuously covered with laminin throughout development. The otic placode became attached to the neural ectoderm through a single layer of fibronectin and collagen IV between the layers of laminin. The ring-like attachment between the edges of the optic cup and lens primordium had the same structure. In addition, the central regions of the optic and lens primordia were attached by fibrils containing type IV collagen, whereas finer strands containing fibronectin and laminin also connected the otic epithelium and neural tube. The results are discussed in terms of models of invagination for the two primordia.

Developmental changes in tracheal structure.

Mechanical properties of the proximal airways are known to change with development; the highly compliant airways of the immature animal become stiffer and less collapsible with increasing age. Although the relationship between tracheobronchial architecture and function has been described for adult physiology, little is known regarding this relationship during early development. This study was, therefore, designed to test the hypothesis that alterations in tracheal morphometry parallel developmental differences in tracheal functional properties. Tracheal segments obtained from 29 lambs ranging in age from 70% of gestation to full-term newborn lambs up to 6 d old were examined using anatomic, morphometric, and histochemical techniques. The results showed 1) progressive increases in the dimensions of the trachea and the tracheal wall components, 2) alterations in the geometric arrangement of the tracheal ring, and 3) changes in the compositional characteristics of the tracheal cartilage with maturation. These findings demonstrate alterations in tracheal architecture, each of which contribute to the greater stiffness of the trachea, in older animals. When considered together, these factors help explain the differences in tracheal functional characteristics with development.

Altered patterns of proteoglycan deposition during maturation of the fetal mouse lung.

Previous studies have shown that beta-xyloside inhibits maturation of the fetal mouse lung (Smith et al., Dev. Biol. 138, 42-52, 1990). Insofar as this drug inhibits proteoglycan deposition, the present studies were undertaken to examine the chemical composition and tissue distribution of proteoglycans in order to determine, more precisely, their role during lung morphogenesis. Autoradiography of labeled 16- and 19-day embryonic lungs demonstrated greater incorporation over the mesenchyme. Treatment with beta-xyloside did not alter the autoradiographic appearance; however, beta-xyloside treatment followed by nitrous acid digestion, eliminated most silver grains. Isolation of proteoglycans from extracellular, membrane and intracellular pools over the 16- to 19-day interval demonstrated redistribution of heparan sulfate proteoglycan from an intracellular to a membrane location, while chondroitin sulfate proteoglycan redistributed from intracellular to extracellular. Only the synthesis of chondroitin sulfate proteoglycan was inhibited by beta-xyloside. On the basis of these results we suggest that a chondroitin sulfate proteoglycan is required for lung maturation and that inhibition of its synthesis results in inhibition of septa formation and subsequent failure of morphogenesis and differentiation.

Patterns of cell movement in early organ primordia of the chick embryo.

Purse-string constriction of the cytoskeleton at cell poles is generally accepted as the causal mechanism for invagination during early stages of organ formation. However, it is known that other cell movements, including intercalation, play a role in the organotypic shape changes that occur during gastrulation and neurulation. Such cell movements have not been investigated in pouching and branching epithelial primordia. There is reason to suspect that cells within these organ primordia might exchange their neighbors for others, that is, intercalate or translocate, at sites of sharp folding such as borders with the surrounding epithelial sheet or where a bend occurs within the primordium. The greatest difficulty in identifying these movements has been the need to use intact embryos so that the processes are not distorted. This study explores the possibility of using time-lapse video recording to identify cell movement at these locations. Three organ primordia were tested: otic and thyroid placodes, which had not been tested previously, and neural plate as a control, where movements of this sort have been documented. Embryos or parts containing the primordia were immobilized and cell apices visualized with Hoffman modulation contrast optics. Recordings to an optical memory disc recorder were transferred to a microcomputer for image analysis. The viewing procedure allows reasonably clear visualization of cell apices, and image analysis permits tracking of a number of adjacent cell apices over an extended time period. Several types of movement were found to occur within cell sheets, and the relative abundance of each type depends on the specific primordium. In the neural plate, some cells move many cell diameters from their neighbors. In the other two primordia, most cells show limited shifts in position relative to their neighbors except at regions where folds are formed. In other regions, adjacent cells move as a unit. Knowledge of the movements which occur in any particular primordium is essential to an understanding of the mechanisms controlling its formation.

Effects of hormones on functional differentiation of mouse respiratory epithelium.

The effects of corticosteroids and thyroxine on morphogenesis and differentiation of fetal respiratory epithelium were tested in serum-free culture. Strips from the margins of 16-day fetal mouse lungs, containing future respiratory branches, were cultured in medium containing growth factors plus different concentrations of dexamethasone and/or thyroxine. After 5 days, some of the cultures were processed for electron microscopy and the rest were used to quantitate unsaturated and saturated phosphatidylcholine. In the absence of thyroxine and dexamethasone, the lung tissue developed the equivalent of one more day in utero during the 5 days of culture, perhaps because of residual hormone in the tissue. Thyroxine and dexamethasone in concentrations compatible with normal physiological conditions permitted normal development from the pseudoglandular stage to the saccular stage, differentiation of respiratory cell types, and normal surfactant accumulation. Thyroxine alone gave essentially the same lack of morphological maturation and accumulation of disaturated phosphatidylcholine as culture in the absence of all hormone, but columnar epithelial cells held increased numbers of lamellar bodies. Dexamethasone alone at 10(-6) M gave essentially normal development. Dexamethasone at 10(-9) M was sufficient to permit almost normal development when combined with 10(-7) M thyroxine. We conclude that dexamethasone is required for normal maturation of the fetal lung and that thyroxine acts synergistically to lower the dexamethasone requirement. The presence of apparently authentic lamellar bodies under some conditions of low disaturated phosphatidylcholine accumulation make this morphological criterion suspect as a test for differentiation of type II cells.

Effects of beta-D-xyloside on differentiation of the respiratory epithelium in the fetal mouse lung.

Differentiation of respiratory endings in the fetal lung appears to be controlled by its surrounding mesodermal capsule. The capsule may exert its influence by controlling the composition of the epithelial basal lamina or of the extended extracellular matrix that is deposited during the period when alveolar sacs are formed. As a first step in testing this hypothesis, the effects of the drug, rho-nitrophenyl-beta-D- xylopyranoside (beta-xyloside), an inhibitor of proteoglycan synthesis, and its inactive alpha anomer (alpha-xyloside) were examined. Lung primordia from mice at 16 days of gestation were tested for inhibition of morphological and functional differentiation as a result of drug treatment. Pseudoglandular lung epithelium did not form respiratory endings, contained fewer specialized cells, and accumulated little additional surfactant when treated with beta-xyloside but developed normally when treated with alpha-xyloside or grown in control medium. The results are interpreted to suggest that deposition of an extracellular matrix rich in proteoglycan is required to support maturation of the respiratory epithelium.

Use of a touch sensitive screen and computer assisted image analysis for quantitation of developmental changes in pulmonary structure.

The extensive changes in pulmonary function occurring during early development may reflect variations in the anatomic structure of the respiratory apparatus during this period. Accurate definition of these alterations could yield important information concerning the structure-function correlations of the respiratory system. To facilitate the acquisition of morphometric data from histologic sections of pulmonary tissues, we propose the use of a computer assisted image analysis system with a touch sensitive screen as an interactive peripheral. This allows planimetric measurements and computation of the dimensions of areas of selected light intensities within an image. We present the description, design, and applications of such an image analysis system and report representative results regarding developmental changes in pulmonary structure. In addition, we correlate these results with previously published information regarding pulmonary mechanics during early development to help clarify the maturational changes in pulmonary structure-function relationships.

Invagination of the otic placode: normal development and experimental manipulation.

The inner ear forms from paired ectodermal primordia that lie to either side of the developing hindbrain. Initially each primordium forms a shallow depression in the ectodermal surface. Invagination to form an otic pit coincides with the formation of several deep folds in the epithelial surface. An initial fold appears parallel to the embryonic axis and at the junction of the rhombencephalon with somitomeric mesoderm. This is followed by formation of cranial and caudal folds perpendicular to the axis and minor folds that are within the pit formed by earlier folding. The central region of the otic primordium remains in close apposition to the lateral surface of the neural tube during the process of fold formation, until the otic pit becomes quite deep. At that time, mesenchymal cells penetrate between the two layers. Experimental analysis of invagination supports the conclusion that otic invagination is controlled differently from that of similar organ primordia, such as the eye and thyroid. Whereas these other primordia can be stimulated to undergo normal morphogenetic shape changes precociously by treatments that presumably activate motile processes in the cytoskeleton, the same conditions have little effect on the otic placode. Similarly, neither inhibitors of calcium transport nor inactivators of calmodulin activity prevent otic pit formation, while these drugs block invagination of other primordia. These results suggest that otic invagination may be caused by changes in the surrounding tissues rather than by an activation of motility within the primordium.

Accumulation and distribution of sulfated materials in the maturing mouse lens capsule.

Lenses of late gestational and postnatal normal-eyed mice were tested for accumulated sulfated materials by using Spicer's high-iron-diamine staining method and also for newly incorporated sulfate autoradiographically following administration of 35SO4 either in vivo or in isolated and organ-cultured lenses. Accumulated and newly incorporated sulfate was observed in all lenses for each age group tested. Discrete regional differences were seen in histochemical staining patterns for sulfate on the lens capsule in specimens of all ages, and distinct laminar zonations were seen in the various regions of the capsule in older specimens. Typically, the anterior and equatorial regions of the capsule demonstrated three histochemically distinct laminar zones while the posterior capsule usually demonstrated two laminar zones. Autoradiographic results indicated that sulfate was indeed being incorporated into these regions, and in the same general pattern as seen with histochemistry. The materials were largely insensitive to testicular hyaluronidase but were preferentially sensitive to nitrous acid digestion, indicating the presence of capsular heparan sulfates. Autoradiographic results from organ-cultured lenses indicated that this tissue itself is a primary source of these materials.

The effect of culture conditions on cytodifferentiation of fetal mouse lung respiratory passageways.

Differentiation of the respiratory region of fetal mouse lungs was investigated in serum-free medium supplemented with growth factors and hormones. Terminal buds from the margins of a lobe were removed from 16-day fetuses and organ cultures prepared either in submersion culture or at the air-medium interface. It was found that glycyl-L-histidyl-L-lysine, transferrin, and somatostatin were sufficient to promote branching in the absence of serum. However, type II pneumocytes containing lamellar bodies formed only in the presence of thyroxine or dexamethasone. At concentrations of these hormones slightly above the physiological range most of the cells became cuboidal and contained lamellar bodies; at lower concentrations regions of flattened cells appeared. In submersion culture a large, central cavity surrounded by saccules was formed rather than a branched tree. Thus, the pattern of differentiation is significantly influenced by culture conditions.

Cell proliferation in condensing scleral ectomesenchyme associated with the conjunctival papillae in the chick embryo.

The role of cell proliferation in the formation of scleral ectomesenchymal condensations underlying the conjunctival papillae was examined with in vivo tritiated thymidine labelling in chick embryos ranging in age from 8 days 0 h to 10 days 12 h. Percentages of labelled nuclei were determined in both ectomesenchyme and the deeper fibrous sclera for short-term and continuous tritiated thymidine incubations. During formation of the ectomesenchymal condensations the percentages of labelled nuclei were consistently higher within the condensations than in corresponding non-condensing ectomesenchyme between papillae. The consistent differences of labelling percentages observed within the condensing versus non-condensing ectomesenchyme were not found in the fibrous sclera at any stage. All areas of both the ectomesenchyme and fibrous sclera showed decreases in the percentages of labelled nuclei from 8 days 0 h to 10 days 12 h, although the decline in the ectomesenchymal condensations beneath papillae occurred more slowly than in areas between papillae. The data suggest that the conjunctival papillae directly influence the proliferation in the subjacent condensing ectomesenchyme but have no effect on the ectomesenchyme between papillae or any region of the deeper fibrous sclera. The observations of this investigation are discussed in relation to other studies of the development of the pre-ossicular mesenchyme.