Consequences of neural tube and notochord excision on the development of the peripheral nervous system in the chick embryo

Notochordectomy and neuralectomy were carried out either in one- or in two-step experiments on the chick embryo. The aim of this operation was to study the influence of the axial organs (notochord and neural tube) on the development of the ganglia of the peripheral nervous system. The neural crest cells from which most peripheral ganglion cells arise were labeled through the quail-chick marker system and their fate was followed under various experimental conditions. It appeared that the development of the dorsal root and sympathetic ganglia depends on survival and differentiation of somite-derived structures. In the absence of neural tube and notochord, somitic cells die rapidly, and so do the neural crest cells that are present in the somitic mesenchyme at that time. In contrast, those crest cells which can reach the mesenchymal wall of the aorta, the suprarenal glands, or the gut survive and develop normally into nerve and paraganglion cells. Differentiation of the neural crest- and placode-derived sensory ganglia of the head which develop in the cephalic mesenchyme is not affected by removal of notochord and encephalic vesicles. These results show that the peripheral ganglia are differentially sensitive to the presence of the neural tube and the notochord. Among the various ganglia of the peripheral nervous system, spinal and sympathetic ganglia are the only ones which require the presence of these axial structures. The neural tube allows both the spinal and the sympathetic ganglia to develop in the absence of the notochord. In contrast, if the notochord is left in situ and the neural tube removed, the spinal ganglia fail to differentiate and only sympathetic ganglia can develop.     

Choline acetyl transferase activity in chick embryo neuroretinas during development in ovo and in monolayer cultures

The specific activity of the enzyme choline acetyl transferase (CAT) in chick neuroretinas was investigated during in ovo development and in monolayer cultures. The enzyme activity was barely detectable on the 6th day of incubation but increased markedly between the 7th and 11th days. The activity increased sharply between the 15th and 17th days and then slowly until hatching. When cell suspensions from 6- to 7-day neuroretinas were cultured as monolayers, CAT specific activity increased rapidly. After 4–5 days in culture, the activity of the enzyme was identical to that found in the neuroretina on the 11th day of incubation. Cells from 9-day neuroretinas also differentiate in monolayer cultures, but with a more irregular pattern. These data show that cholinergic neurons from chick embryo neuroretina differentiate in monolayer cultures without a lag and at the same rate as in vivo.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

An analysis of the fate of the chick wing bud apical ectodermal ridge in culture

Stages 20 and 25 chick apical ectodermal ridge have been cultured in nutrient medium containing fetal bovine serum and the tissues have been examined for dying cells at 0, 6, 12, 18, and 24 hr. By 12 hr, an average of 43% of the cells were dying. By 24 hr, stage 20 ridge had lost its integrity and stage 25 ridge contained an average of 50% dying cells. These results are in agreement with the observations of R. L. Searls and E. Zwilling (1964, Dev. Biol. 9, 38-55) on isolated stage 20 ridge. In subsequent experiments, ridge ectoderm was cultured in serum-containing medium to which insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenium (5 ng/ml) or insulin (5 micrograms/ml) had been added. Under these conditions the ectoderms remained viable even after 24 hr in vitro.     

Characterization of an alpha-glucan from chick embryo sternum whose synthesis is stimulated by L-3,5,3'-triiodothyronine and human serum.

Incorporation of [³H]glucose into macromolecular components of 12-day chick embryo sternum incubated in vitro was stimulated by both human serum and l-3,5,3′-triiodothyronine. Under all conditions, 65–70% of the radioactivity was incorporated into glycosaminoglycans. About 10% of the radioactivity was incorporated into a fraction separable by ion-exchange chromatography which was stimulated two- to sixfold by addition of 2–10 nm triiodothyronine and 5–20% ($\text{v}\text{v}$) human serum. Further characterization of this fraction by paper electrophoresis at pH 3.5 showed the presence of two components, one apparently anionic and one neutral. All of the increase in incorporation of [³H]glucose was into the former species. Acid hydrolysis of this material showed that it contained only glucose. Treatment with α-amylase released 78% of the label as maltotriose and maltose; digestion with crystalline β-amylase released 75% as maltose; and treatment with glucoamylase and α-amylase released 93% as glucose. There was no incorporation of any amino acid into this fraction, nor could any incorporation of [³²P]phosphate, [³⁵S]sulfate, [³H]uridine, or [³H]acetate be demonstrated. Mild acid hydrolysis (0.1 N HC1, 100 °C, 10–20 min) converted the material to a neutral species with a much lower molecular weight. The results indicate that chick embryo sternum contains a species of glycogen whose synthesis is stimulated by thyroid hormones and other serum factors.     

Incorporation of a fatty acid containing a photosensitive group into lipids of cultured cardiac cells from chick embryo

Radioactive 12-(4-azido-2-nitrophenoxy)-stearic acid (NAP-stearate) was synthetized; it behaves as a competitive inhibitor of long-chain fatty acids for the entry into cultured cardiac cells from chick embryo. After uptake, [3H] NAP-stearate was incorporated by an energy-dependent process into neutral and polar lipids. Photoactivation as a function of time leads to a covalent labelling of the cells: up to 31 per cent of the radioactivity was recovered in the 105 000 g cell pellet, mainly in proteins. These experiments show that fatty acids containing photosensitive groups would potentially allow to localize the proteins involved in the binding and/or in the transport of fatty acids.     

Epiblast origin and early migration of neural crest cells in the chick embryo

Chick embryos carrying transplants labeled with tritiated thymidine demonstrate that the neural crest originates in the anterior epiblast, at the junction of areas destined for epidermis and neural tube. As the neural tube begins to fold and the axis lengthens, cells along this junction are drawn dorsomedially; at the seven-somite stage they begin to separate from the epithelium of the head, and migrate into the angle between the epidermis and the neural tube. The paraxial mesoderm already populating this angle originates in more posterior and medial portions of the epiblast than do the neural crest cells; after invagination at the primitive streak, it migrates anterolaterally, ventral to the ectoderm layer, until it too is folded dorsomedially into the angle between the epidermis and the neural tube.     

An ultrastructural immunocytochemical localization of hyalin in the sea urchin egg

The fertilized sea urchin egg is invested by the hyaline layer, a thick extracellular coat which is necessary for normal development. On the basis of ultrastructural studies and the fact that hyalin is released during the time of the cortical reaction, it has been generally accepted that hyalin is derived from the cortical granules. However, this has never been proven definitely, and recently, it has been reported that hyalin is a membrane and/or cell surface protein. To determine where hyalin is stored, we carried out an ultrastructural immunocytochemical localization of hyalin in the unfertilized egg. Hyalin purified from isolated hyaline layers was used to immunize rabbits. Antisera so obtained were shown to be hyalin specific following absorption with a combination of sea urchin proteins. Immunocytochemical localizations were carried out on sections of Epon-embedded material using protein A-coated gold particles as an antibody marker. Our results demonstrate that, prior to fertilization, hyalin is stored in the homogeneous component of the cortical granule in Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus. Labeling of small cortical vesicles in both unfertilized and fertilized eggs, suggests that these vesicles may contain a secondary reservoir of hyalin.     

The establishment of vascular-derived microenvironments in the developing chick wing

The results of previous studies on the temporal sequence of limb vascularization suggest that the prospective myogenic and chondrogenic areas of the mesoderm are distinguished by a differential vascularization pattern prior to the overt expression of muscle- and cartilage-specific phenotypes. The experiments presented here are designed to reveal the dynamic aspects of vascular flow in the limb by the observation of how an inert, particular tracer (india ink) is mobilized and dispersed at specific points in the mesoderm. Data are presented as a temporal sequence of fluid flow "maps" which detail both the rate and the direction of vascular flow in the limb. It is proposed that not only does the vasculature compartmentalize the mesoderm into prospective myogenic and chondrogenic zones but also that these broad areas are subcompartmentalized into discrete microenvironments that are spatially distinct with regard to their capacity for transporting the carbon particles. The developmental significance of this observation may be that limb mesodermal cells are granted precise, "positional" information in the form of the specific nutrient and oxygen levels they encounter during critical, or decisional, phases of morphogenesis.     

Recognition specificities, development, and possible biological function of natural killer cells in the mouse. I. Spleen focus forming assay for natural killer activity and analysis of lectin-like recognition structures on the surface of murine natural killer cells

A number of simple sugars have been tested and found to be effective in blocking lysis of YAC-1 tumor target cells by nonimmune murine natural killer (NK) effector cells. Using a spleen fragment culture system an assay has been developed which allows us to compare the inhibition of lysis observed in replicate culture wells prepared from cells contained in one spleen fragment (less than or equal to 1 X 10(6) cells). The inhibition pattern of any well was found to fall naturally into 1 of 25 (of the total 128 possible tested) patterns. Using this panel analysis of NK activity in individual mice of the same or different strain has been compared. Our data suggest that within any given strain the inhibition pattern of NK effector cells is quite uniform. Consistent differences are seen between strains which are interpreted in terms of a genetic control of the final expression of the NK recognition repertoire. In adult F1 hybrid individuals the pattern of recognition by NK cells is best considered a result of the codominant expression of genes contributed by each parent.     

A genetic analysis of visual system development in Drosophilia melanogaster.

The eyes and optic lobes of adult Drosophila melanogaster comprise a highly organized system of interconnected neurons. The eye and optic lobe primordia are physically separate during the embryonic and larval stages of development, and these tissues do not come into contact until the third larval instar, as a consequence of axons growing from the receptor cells of the developing eyes to the primordial optic lobes. After this contact, the axons of the eyes arrange themselves into their complex and orderly adult pattern. Simultaneously, the optic lobe cells begin elaborating axons which organize into their precise adult array. One question posed by this system is: Does cellular pattern formation in either the eyes or optic lobes depend on eye-brain interactions, or do the two tissues organize autonomously? To answer this question, mutations were found which cause abnormal ommatidial array in the eyes and which also perturb the normal adult axon array in the optic lobes. By means of X ray-induced somatic recombination and by genetically controlled mitotic chromosome loss (gynandromorph formation), flies mosaic for genotypically mutant and normal tissue were constructed. Analysis of the neuronal array in mosaic flies in which eye and optic lobe tissue differed genotypically showed that the axon array phenotype of the optic lobe depends on the genotype of the eye tissue innervating that lobe, while the eye phenotype does not depend on optic lobe genotype. Thus, the axonal organization of the D. melanogaster optic lobe has been shown to depend on the transmission of information from the eyes to the optic lobes.     

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.     

Is the onset of actin histidine methylation under developmental control in the chick embryo

It had previously been reported (B. Krzysik, J. P. Vergnes, and I. R. McManus (1971) Arch. Biochem. Biophys., 146, 34–45) that prior to day 11 of embryonic life chick skeletal muscle actin contained little or no 3-methylhistidine, and that between Day 11 and 18, the degree of actin histidine methylation increased until it leveled off at 1 mol of 3-methylhistidine/mol actin. This is the value seen in adult muscle and nonmuscle actins so far analyzed. To determine whether this delayed onset of actin methylation occurred simultaneously throughout the organism or differed from tissue to tissue, the 3-methylhistidine content of cardiac muscle actin from Day 2 of embryonic life to hatching and of brain actin at Days 9, 11, and 14 were analyzed. These results, obtained by analyzing unlabeled actin samples as well as samples labeled in vivo with [³H]histidine, showed that at all stages, 1 mol of 3-methylhistidine was present per mol of actin. When skeletal muscle samples obtained from Day 11 to 18 embryos were analyzed 1 mol of 3-methyl histidine/mol of actin was observed. Thus, in the chick embryo, contrary to those reports published earlier, it was found that actin histidine methylation is not under developmental control.     

Effect of lectins and sugars on primary sperm attachment in the horseshoe crab, Limulus polyphemus L

The in vitro differentiation of quail neural crest cells into serotoninergic neurons is reported. Serotoninergic neurons were identified by two independent methods, formaldehyde-induced histofluorescence and indirect staining with antiserotonin antibodies. Serotonin-positive cells first appeared on the third day in culture, simultaneously, or slightly prior to the first pigmented cells and adrenergic neurons. Comparable numbers of serotoninergic cells were found in crest cell cultures derived from vagal, thoracic/upper lumbar, and lumbosacral levels of the neuraxis. The neural crest origin of the serotonin neurons was further corroborated by the demonstration that cultures of somites, notochords, and neural tubes (three tissues adjacent to the neural crest and thus the most likely contaminants of crest cell cultures) did not contain serotonin-producing cells, and that mast cells were absent in crest cell cultures. The identification of serotoninergic neurons in quail neural crest cell cultures makes an important addition to the number of neural crest derivatives that are capable of differentiating in culture. Furthermore, it suggests that the in vitro culture system will prove a valid approach to the elucidation of the cellular and molecular mechanisms that govern neural crest cell differentiation.     

The influence of substitution at C24 on the calcium-binding protein-stimulating activity of vitamin D metabolites in chick embryonic duodenum

The production of calcium-binding protein, in vitro, by embryonic chick duodenum has been used to assess the potency of vitamin D compounds. The introduction of an hydroxyl on 1-, 25-, or 24R-position enhanced biological activity while the introduction of both 1α- and 25-hydroxyls produced maximal activity. However 24R-hydroxylation of 1,25-dihydroxyvitamin D₃ diminished activity. The vitamin D₂ side chain on 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D did not greatly diminish activity in contrast to the fact that the vitamin D₂ compounds are 10% as active as the vitamin D₃ compounds in vivo in the chick. These results support the idea that the target organs of the chick do not discriminate against the vitamin D₂ side chain and that the discrimination in this species is at the level of metabolism.     

Ionic dependence and transmission of epidermal action potentials in a newt embryo

The ionic dependency and transmission of epidermal action potentials have been examined from tailbud to hatching stages of newt embryos. Previously we have reported that the epidermal action potential is composed of a fast- and slow-action component; only the slow-action component, however, is transmitted to other cells. We address in this report the mechanism by which these responses are mediated. The slow-action potential is not produced in Na⁺-free saline, tricaine saline, or following the application of TTX, and thus appears to be Na⁺ dependent. The fast-action potential on the other hand is blocked by application of Co²⁺ and verapamil saline and thus appears to be Ca²⁺ dependent. The slow-action potentials appear to be chemically transmitted since they are transmitted even to those cells which are electrically uncoupled at low intracellular pH (NaHCO₃ + HCl, pH 6.2). Furthermore 1 μM curare and atropine are inhibitory to transmission of the slow potential. Epidermal cells of the newt embryo are sensitive to acetylcholine (ACh) applied by hydrostatic ejection through a micropipet. The latter observation further suggests that propagation of the slow-action potential is, in part, a chemical event.     

Presence of nerve growth factor receptors and catecholamine uptake in subpopulations of chick sympathetic neurons: correlation with survival factor requirements in culture

The presence of nerve growth factor receptors and the imipramine-sensitive uptake of catecholamines in sympathetic neurons of chick embryos were investigated by autoradiography. Neurons were dissociated from paravertebral ganglia of different embryonic ages and receptors and catecholamine uptake were then determined both at the beginning of culture and after 2 days in culture, at which time the number of surviving neurons is determined by the survival factors present. It was found that while essentially all the neurons specifically bound 125I-NGF both after dissociation and at the end of the culture period, only 60% of the neurons take up [3H]norepinephrine after dissociation, and this proportion remained constant through the culture period under conditions where all the neurons survived. All of the neurons maintained by NGF in culture (35% of the total) displayed this uptake, and in contrast, only one-quarter of those maintained by heart cell-conditioned medium alone (60% of the total) took up catecholamines. The uptake was shown to be neither induced by NGF nor suppressed by heart cell-conditioned medium. These results support the hypothesis that chick sympathetic ganglia contain discrete subpopulations of neurons which may be selected in culture by virtue of their different requirements for survival factors.     

Inhibition of precartilaginous chick somites by oncogenic virus

Infection of embryonic chicken notochord-somite explants with Rous sarcoma virus inhibited the in vitro differentiation of somites into cartilage. Visual inspection of the explants revealed that viral infection reduced the size of cartilage nodule formation. Formation of the complex of sulfated proteoglycans with hyaluronic acid was inhibited by RSV infection, and sedimentation analysis of the sulfated proteoglycans showed that very little fast sedimenting proteoglycans were synthesized by RSV-infected explants. The infected explants primarily synthesize a slowly sedimenting sulfated proteoglycan which was chondroitinase resistant. These slow-sedimenting sulfated proteoglycans lack the ability to associate with hyaluronic acid and appear to be noncartilaginous. These effects of RSV are apparently due to the src gene of this virus since the mutant td108, which lacks part of the src gene, has no detectable influence on the chondrogenic differentiation of somite explants. Similarly, infection with RAV-2 as well as with uv-irradiated virus had no detectable effect. The inhibition of synthesis of fast sedimenting proteoglycans was observed at 41 degrees C with explants infected with tsNY68, suggesting that residual activity of transforming gene of this virus at the non-permissive temperature is sufficient for this inhibition in the explants.