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.     

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.     

Uptake of fatty acids by cultured cardiac cells from chick embryo: evidence for a facilitation process without energy dependence

Cultured heart cells from chick embryo accumulate fatty acids up to 50 fold at a steady-state level under defined conditions [ref.1]. Studies of fatty acid uptake as a function of different cellular parameters (intracellular ATP and pH, membrane potential and electrochemical gradients for monovalent and divalent cations) show the lack of effect of these factors. The rate of uptake is temperature-dependent. The maximum velocity V is affected with no change in the Km value of the saturable component; the activation energies were found to be 35.5 kJ.mol-1 for palmitate and 42 kJ.mol-1 for oleate. The results are in favour of a facilitation process which leads to an accumulation of fatty acids without energy dependence. The accumulation of fatty acids could be due to their association to intracellular membraneous and/or cytosolic components.     

Metabolism of vitamin D3 in the chick embryo

In agreement with previous reports, chick intestinal calcium-binding protein does not appear in the chick embryo until 1 day after hatching while intestinal alkaline phosphatase begins to appear at 19–20 days of embryonic life. The ability of chick embryo to metabolize vitamin D₃ to 25-hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, and 24,25-dihydroxyvitamin D₃ is present at least by day 18 of embryonic life as demonstrated by in vivo and in vitro techniques. It also illustrates that metabolism of vitamin D₃ was not the limiting factor in the appearance of calcium-binding protein and alkaline phosphatase in intestine. Instead, the uptake of 1,25-dihydroxyvitamin D₃ by the duodenum was very low prior to hatching, even though significant amounts were present in the yolk sac. Injection of a physiological dose of 1,25-dihydroxyvitamin D₃ to chick embryo at 9 days failed to stimulate appearance of calcium binding protein by 18 days of embryonic life. Thus, it appears that either the normal mechanism for transport of 1,25-dihydroxyvitamin D₃ to intestine or its receptors in intestine may not be present prior to day 18–19.A large fraction of radioactive vitamin D₃ injected into the yolk sac was found esterified especially in the embryonic liver. The significance of this is not yet understood.Injection of 1,25-dihydroxyvitamin D₃ at 325 pmoles/per egg at 9 days resulted in 70% mortality of embryos while a 32-pmole dose resulted in no significant increase in mortality. The basis for this toxicity is not yet understood.     

The influence of the metameric pattern in the mesoderm on migration of cranial neural crest cells in the chick embryo

In recent studies of chick embryos, the cranial paraxial mesoblast was found to be patterned into segmental units termed somitomeres. Anterior to the first segmental cleft, seven contiguous segments are aligned, with somitomeric interfaces forming grooves at right angles to the midline. In this study, the morphological relationship between the migratory pathways of cranial neural crest cells and patterned primary mesenchyme was analyzed with the scanning electron microscope, utilizing stereo imaging. In addition, the development of neuromeres in the adjacent neural tube was monitored. It was found that cranial neural crest first appears along the dorsal midline as a ridge of cells which loosens from the wall of the neural tube and migrates laterally as discrete populations. The mesencephalic crest appears first, immediately following neural tube fusion at that level, and migrates over the dorsal surface of the adjacent third somitomere and into the grooves formed by its juncture with the second and fourth somitomeres. Later, the addition of prosencephalic and rostral rhombencephalic crest extends the mesencephalic population to form a shelf of crest which spreads over the dorsal surface of the first four somitomeres. Component cells of this most cranial crest shelf become oriented and mimic the metameric pattern of the subjacent somitomeres. Crest cells adjacent to the fifth somitomeres appear along the midline, but do not migrate, creating a gap anterior to the otic crest. By stage 9, a narrow finger-like segment of the otic crest migrates from a specific neuromere into the grooved interface between the fifth and sixth somitomeres, delimiting the rostral border of the otic placode in the ectoderm above. By the end of stage 9, crest cells delimiting the caudal border of the placode have migrated along the interface of the seventh and eighth somitomeres. The crest cells adjacent to the sixth and seventh somitomeres, between the rostral and caudal otic populations, appear but do not migrate, remaining condensed along the midline. Thus, otic crest cells form a ring which circumscribes the invaginating otic placode. This study suggests that the precise distribution of cranial neural crest cells may result from their introduction at specific times, as specific populations from specific brain regions (neuromeres), onto a patterned mesodermal layer.     

Effect of hydrocortisone on skin development in the chick embryo: ultrastructural, immunohistological, and biochemical analysis

The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.     

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.     

Morphology and behavior of quail neural crest cells in artificial three-dimensional extracellular matrices

Neural crest cells migrate extensively through a complex extracellular matrix (ECM) to sites of terminal differentiation. To determine what role the various components of the ECM may play in crest morphogenesis, quail (Coturnix coturnix japonica) neural crest cells have been cultured in three-dimensional hydrated collagen lattices containing various combinations of macromolecules known to be present in the crest migratory pathways. Neural crest cells migrate readily in native collagen gels whereas the cells are unable to use denatured collagen as a migratory substratum. The speed of movement decreases linearly as the concentration of collagen in the gel increases. Speed of movement of crest cells is stimulated in gels containing 10% fetal calf serum and chick embryo extract, 33 micrograms/ml fibronectin cell-binding fragments, 3 mg/ml chondroitin sulfate, or 3 mg/ml chondroitin sulfate proteoglycan when compared to rates of movement through collagen lattices alone. Low concentrations of hyaluronate (250-500 micrograms/ml) in a 750 micrograms/ml collagen gel do not alter rates of movement over collagen alone, but higher concentrations (4 mg/ml) greatly inhibit migration. Conversely, hyaluronate (250 micrograms/ml) significantly increases speed of movement if the crest cells are cultured in high concentration collagen gels (2.5 mg/ml), suggesting that hyaluronate is expanding spaces and consequently enhancing migration. The morphology and mode of movement of neural crest cells vary with the matrix in which they are grown and can be correlated with their speed of movement. Light and scanning electron microscopy reveal rounded, blebbing cells in matrices associated with slower translocation, whereas rounded cells with branching filopodia or lamellipodia are associated with rapid translocation. Bipolar cells with long processes are observed in cultures of rapidly moving cells that appear to be adhering strongly, as well as in cultures of cells that are stationary for long periods. These data, considered with the known distribution of macromolecules in the early embryo, suggest the following: (1) Both collagen and fibronectin can act as preferred substrata for migration. (2) Chondroitin sulfate and chondroitin sulfate proteoglycan increase speed of movement, but probably do so by decreasing adhesiveness and thereby producing more frequent detachment. In the embryo, crest cells would most likely avoid regions containing high concentrations of chondroitin sulfate. (3) Hyaluronate cannot act as a substratum for migration, but in low concentrations it can open spaces in the matrix and consequently may stimulate movement. The complex interactions of combined matr     

Identification of glycogen as the major xylose acceptor in polysomal preparations from chick embryo cartilage cultures

The capacity of various metal ions to support activation of bovine factor IX, by the coagulant protein of Russell's Viper venom, has been examined. The following metal ions, at concentrations which saturate their effect, promoted activation of factor IX, at approximately equal efficiency: Ca²⁺, Mn²⁺, Sr²⁺, and Co²⁺, Other metal ions, i.e., Ba²⁺, and Mg²⁺, at saturating concentrations, led to a maximum rate of activation of factor IX of 25%, compared to Ca²⁺, The lanthanides, Gd²⁺, and Tb³⁺, also promoted activation in this system, at maximal rates of approximately 15%, compared to Ca²⁺, In this study, it was also discovered that the esterase activity of bovine factor IXa was dependent upon the presence of metal ions. Utilizing α-N-benzoyl-l-arginine ethyl ester as the substrate, steady state kinetic analysis in the absence of metal ion indicated that the K_m and V_max for this substrate was 20 mm and 2.9 μmol substrate cleaved min^?1 mg^?1 of factor IXa, respectively, at pH 8.0 and 30 °C. In the presence of optimal concentrations of Ca²⁺, Mn²⁺, Mg²⁺, Sr²⁺, and Ba²⁺, the V_max values for this same substrate increased to 6.7, 5.9, 5.0, 5.0, and 3.7 μmol cleaved min^?1 mg^?1 of factor IXa, respectively. None of these metal ions had an affect on the K_m value of this substrate.     

Identification of two new collagen alpha-chains in extracts of lathyritic chick embryo tendons

Two new collagen polypeptide chains have been identified in extracts of lathyritic embryonic chick tendons. The electrophoretic migration of these polypeptides in sodium dodecyl sulfate-polyacrylamide gels indicates that they have about 20% greater apparent molecular weights than α₁ and α₂ chains of Type I collagen. These chains are not held by disulfide bonds since reduction does not affect their electrophoretic behavior. Further, they do not represent incompletely cleaved procollagen since their apparent molecular size remains greater than that of Type I collagen polypeptides after limited proteolytic digestion. Because the ratio of these polypeptides in the purified extracts is not 2:1 it appears that they are components of two separate tropocollagen molecules.     

Effects of cultured astroglia on the survival of neonatal rat retinal ganglion cells in vitro

Retinal ganglion cells (RGC), as identified by retrograde horseradish peroxidase (HRP) labeling technique, were cultured in a minimal medium; only 20% of them survived after 16 hr in vitro. However, superior colliculus-conditioned medium was capable of supporting 100% of RGC over this assay time; enhanced neurite expression also was evident. It was decided to investigate whether glial cells within the superior colliculus may provide a soluble factor capable of supporting RGC. Glial-conditioned medium prepared over monolayers of either predominantly flat astrocytes (relatively immature) or predominantly process-bearing (mature) astrocytes failed to maintain RGC. The possibility that astrocytes may provide support for RGC via membrane contact was then investigated. Dissociated retinae were grown on monolayers consisting primarily of either flat or process-bearing astrocytes. Cultures rich in flat astrocytes maintained over 70% of RGC originally present, and many of them exhibited extensive neurite outgrowth and elongation. Process-bearing astrocytes were unable to support RGC survival. Immature astroglial cells may therefore support RGC via glial-neuronal interaction.     

Influence of ganglion age,nonneuronal cells and substratum on neurite outgrowth in culture

This study characterizes the outgrowth patterns of superior cervical ganglia (SCG) obtained from embryonic (E15), perinatal (E20–21), and adult (P35) rats when placed in culture on various substrata. Outgrowth morphology, degree of fasciculation, and outgrowth length were examined on collagen (COL), polyornithine (PO), polylysine (PL), fibronectin (FN), and nonneuronal cells (NNCs) from the ganglion. COL and FN supported extensive neuritic outgrowth; PO and PL provided poor support. Outgrowth pattern, degree of fasciculation, neurite growth rate, and the number of NNCs in the outgrowth varied considerably depending upon the COL configuration. When undiluted COL (～5 mg/ml) was air dried, a three-dimensional loose fibrillar network was formed. Upon COL dilution or gelling undiluted COL by ammoniation, an essentially two-dimensional layer was formed. On two-dimensional COL, NNCs were able to proliferate and migrate extensively from ganglia of all ages; their presence influenced the form and extent of neurite growth. E15, E20, and P35 neurites responded differently to their endogenous NNCs. E15 neurites extended in relation to NNC surfaces and were predominantly nonfasciculated. E20 neurites became more fasciculated in the presence of NNCs that exhibited morphological and behavioral differences from those migrating from E15 ganglia. E20 neurite bundles became defasciculated when they extended into E15 outgrowth. Far fewer neurites grew from P35 explants in the presence of their NNCs. Three-dimensional COL greatly slowed NNC migration and thus allowed investigation of neurite outgrowth from ganglia of differing age in the absence of NNCs. We conclude that neuritic outgrowth patterns on varying substrata reflect not only neurite differences depending upon ganglion age but also variation in the behavior of accompanying NNCs.     

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.     

Dissociated limb bud cells of chick embryos can express lens specificity when reaggregated and cultured in vitro

Limb bud cells of chick embryos (stages 23–24) were dissociated into single cells, reaggregated, and cultured in vitro for about a week. δ-Crystallin, generally thought to be a lens-specific protein in the chick, was detected in the aggregates by indirect immunofluorescent staining, double immunodiffusion test, and immunoelectrophoresis with specific antiserum against δ-crystallin. Cells containing δ-crystallin were distributed in epidermal cell clusters and also in mesenchymal tissues surrounding cartilage nodules in the aggregates. Those cells in mesenchymal tissues were shown to have originated from the mesoderm of the limb bud, and those in epidermal cell clusters probably originated from the ectoderm. The possible cellular origin of this appearance of δ-crystallin was discussed.     

Isoform variants of troponin in skeletal and cardiac muscle cells cultured with and without nerves

Immunofluorescence microscopy shows that cultured skeletal and cardiac muscle cells of chicken embryos exhibit the same stainabilities with antibodies against skeletal and cardiac troponin components as do those in embryos. Muscle cells of each type cultured with motor or sympathetic nerves or in medium containing the nerve extract exhibit the same reactivities as do those in adult animals. Cardiac muscle cells incubated in the nerve-conditioned medium also change the form of troponin components to the adult type. It appears that the differentiation of individual muscle fibers to specific types is induced by nerves, and especially by the neurohumoral effect.     

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.     

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.     

Identification of subsets of proliferating low Thy 1 cells in thymus cortex and medulla

Subsets of proliferating thymocytes were identified in the normal mouse thymus by in vivo labeling with [³H]TdR and by cell separation according to relative amounts of Thy 1 antigen. In order to resolve apparent discrepancies in the literature, parenteral and topical application of [³H]TdR were compared as labeling methods for dividing thymocytes, and limited complement lysis and fluorescence-activated cell sorting were compared as separation principles for high Thy 1 and low Thy 1 thymocyte subsets. The separated cells were further characterized by immunofluorescence for terminal deoxynucleotidyltransferase (TdT), which normally is restricted to cortical thymocytes, and for H2 alloantigens, which are preponderant on medullary thymocytes. Four subsets of proliferating cortical thymocytes were identified after application of [³H]TdR to the thymus capsule. The major subset, which comprised about 92% of dividing cortical thymocytes, had a high Thy 1, low H2 phenotype. Most were also TdT + ve. The three minor subsets of proliferating cortical thymocytes each had a low Thy 1 phenotype, but differed according to H2 and TdT markers. Systemic injection of [³H]TdR also labeled the above subsets of dividing cortical thymocytes, but in addition it detected a subset of proliferating low Thy 1, low H2, TdT — ve cells in the thymus medulla. The latter subset comprised about one-third of the pool of proliferating low Thy 1 cells. In their aggregate the four subsets of low Thy 1 cells constituted approximately 13% of total proliferating thymocytes and 1.1% of total thymocytes. The identification of discrete subsets of proliferating low Thy 1 cells in the thymus cortex as well as in the thymus medulla is compatible with the hypotheses that all thymocytes are descended from low Thy 1 precursors and that separate precursor cell subsets exist for cortical and medullary thymocytes.