Structural and functional differentiation of the embryonic chick pineal organ in vivo and in vitro

Summary The development of sensory structures in the pineal organ of the chick was examined by means of scanning electron microscopy from embryonic day 10 through day 12 post-hatching. At embryonic day 10, the wall of the tubules within the pineal primordium is composed of cells with unspecialized luminal surface. Differentiation of sensory structures starts at embryonic day 12 when pinealocytes and supporting cells can be distinguished. Pinealocytes are recognized by virtue of an inner segment only rarely endowed with a cilium, whereas supporting cells exhibit numerous short microvilli. Further differentiation of the sensory apparatus is achieved by development of an oval-shaped, biconcave swelling at the tip of the cilium, 1×2 m in size, and a collar of long microvilli at the base of the inner segment. Membrane specializations of sensory cilia, however, were not detected. Since during embryonic life new tubules and follicles are continuously formed, all stages of differentiation of sensory structures are found in the chick pineal organ during the second half of the incubation period and the first two weeks after hatching. In 200-m-thick Vibratome sections of chick-embryo pineal organs cultured in medium BM 86 Wissler for periods up to 13 days the cytodifferentiation parallels the development in vivo. Using an organ-culture system the 24-h release of melatonin into the culture medium was measured by means of radioimmunoassay after solid-phase extraction. At embryonic day 10, the 24-h secretion of melatonin was at the lower range of detection of the RIA (5 pg). The rapid increase in 24-h secretion in melatonin until hatching (50 g) is approximated by an exponential curve.Preliminary results of this study were reported at the Versammlung der Anatomischen Gesellschaft in Lübeck, 1986 (Möller 1987). Supported by the Deutsche Forschungsgemeinschaft (MO234/9-2)     

Properties and mechanisms of spontaneous activity in the embryonic chick hindbrain

Spontaneous activity regulates many aspects of central nervous system development. We demonstrate that in the embryonic chick hindbrain, spontaneous activity is expressed between embryonic days (E) 6–9. Over this period the frequency of activity decreases significantly, although the events maintain a consistent rhythm on the timescale of minutes. At E6, the activity is pharmacologically dependent on serotonin, nACh, GABA_A, and glycine input, but not on muscarinic, glutamatergic, or GABA_B receptor activation. It also depends on gap junctions, t‐type calcium channels and TTX‐sensitive ion channels. In intact spinal cord‐hindbrain preparations, E6 spontaneous events originate in the spinal cord and propagate into lateral hindbrain tissue; midline activity follows the appearance of lateral activity. However, the spinal cord is not required for hindbrain activity. There are two invariant points of origin of activity along the midline, both within the caudal group of serotonin‐expressing cell bodies; one point is caudal to the nV exit point while the other is caudal to the nVII exit point. Additional caudal midline points of origin are seen in a minority of cases. Using immunohistochemistry, we show robust differentiation of the serotonergic raphe near the midline at E6, and extensive fiber tracts expressing GAD65/67 and the nAChR in lateral areas; this suggests that the medial activity is dependent on serotonergic neuron activation, while lateral activity requires other transmitters. Although there are differences between species, this activity is highly conserved between mouse and chick, suggesting that developmental event(s) within the hindbrain are dependent on expression of this spontaneous activity. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Direct effects of serotonin, and 5-HT2, alpha 1 and H1 receptor-antagonists on embryonic chick skin in vitro: a morphological and functional study

Ketanserin (K), a 5-HT2, alpha 1 and H1 receptor antagonist, at a concentration of 5 micrograms/ml inhibits keratinisation, but increases DNA-, collagen and glycosaminoglycan (GAG) synthesis in embryonic chick skin in vitro. On the other hand, serotonin (S) at a concentration of 10 micrograms/ml stimulates keratinisation and does not enhance DNA, collagen and GAG synthesis. 5 nM doses of ketanserin and serotonin give analogous results. The effects of other 5-HT2-receptor antagonists (e.g. BW 501 and cinanserin) are comparable to those of K, whereas prazosin (an alpha 1 receptor antagonist) and pyrilamin (an H1 receptor antagonist) do not show any effect on embryonic chick skin in vitro.     

Microwave effects on isolated chick embryo hearts

This study was designed to examine the effects of microwaves on the electric activity of hearts as a means of elucidating interactive mechanisms of nonionizing radiation with cardiac tissue. Experiments were performed on isolated hearts of 9-12-day-old chick embryos placed in small petri dishes. Oxygenated isotonic Ringer's solution at 37 degrees C permitted heart survival. Samples were irradiated at 2.45 GHz with a power density of 3 mW/cm2. The heart signal was detected with a glass micropipet inserted into the sinoatrial node and examined by means of a Berg-Fourier analyzer. Pulsed microwaves caused the locking of the heartbeat to the modulation frequency, whereas continuous wave irradiation might have induced slight bradycardia. Pulsed fields induced stimulation or regularization of the heartbeat in arrhythmia, fibrillation, or arrest of the heart.     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.     

Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro

Summary Chick embryo lumbar sympathetic ganglia (11 day) cultured for three days and uncultured (in vivo) ganglia of comparable age were freeze-dried and processed by the formaldehyde-induced fluorescence technique for the demonstration of biogenic monoamines. The catecholamine levels within principal neurone cell bodies and small intensely fluorescent (SIF) cells were then examined in plastic sections of the in vivo and in vitro ganglia by a quantitative fluorescence method under various experimental conditions. Culture of ganglia for three days in the presence of hydrocortisone acetate (10g/ml) resulted in an increased SIF cell fluorescence (P<0.001 compared to control) and a green to yellow colour shift in the fluorophore of SIF cells. No detectable alteration in the fluorescence level of neurones was observed. When neurones after three days in culture were incubated for 1 h in exogenous catecholamines, a significant increase in fluorescence levels (interpreted as an increase in catecholamine content) occurred with noradrenaline (2×10^–6 M; 2×10^–5 M). SIF cells in ganglia removed directly from 14-day old chicks similarly took up noradrenaline and dopamine, and also adrenaline (2×10^–5 M). Morphological results are presented which indicate that the cellular appearances and architecture of cultured ganglion explants are very similar to those in comparable ganglia in vivo.This work was supported by a grant from the Medical Research Council. We thank Mrs. G. O'Shea, Mr. T.T. Lee and Mr. P.F. Hire for their valuable technical assistance     

Nicotinic and muscarinic responses of embryonic chick ventricular myocardium to acetylcholine: chronotropic and electrophysiological effects

Sensitivity of 7-day-old chick embryo ventricular heart fragments to acetylcholine was investigated. Low doses mainly produced a positive chronotropic effect, whereas high doses of acetylcholine provoked a decrease in the heart beat rhythm. The positive chronotropic effect of acetylcholine was related to the presence of nicotinic receptors that were evidenced within ventricular myocardium by autoradiography. Membrane potential recording showed that acetylcholine hyperpolarizes the diastolic membrane potential when the drug had a negative chronotropic effect. This effect of acetylcholine on the membrane potential was not observed when the drug had a positive chronotropic effect. In many cases, the diastolic membrane potential exhibited spontaneous small depolarizing potentials. Their amplitude was low and their frequency was irregular. These potentials were suppressed by treatment with alpha-bungarotoxin, suggesting that they are triggered by nicotinic receptor activation.     

Segmental independence and age dependence of neurite outgrowth from embryonic chick sensory neurons

Targets in limb regions of the chick embryo are further removed from the dorsal root ganglia that innervate them compared with thoracic ganglion-to-target distances. It has been inferred that axons grow into the limb regions two to three times faster than into nonlimb regions. We tested whether the differences were due to intrinsic properties of the neurons located at different segmental levels. Dorsal root ganglia (DRG) were isolated from the forelimb, trunk, and hind limb regions of stage 25–30 embryos. Neurite outgrowth was measured in dissociated cell culture and in cultures of DRG explants. Although there was considerable variability in the amount of neurite outgrowth, there were no substantive differences in the amount or the rate of outgrowth comparing brachial, thoracic, or lumbosacral neurons. The amount of neurite outgrowth in dissociated cell cultures increased with the stage of development. Overall, our data suggest that DRG neurons express a basal amount of outgrowth, which is initially independent of target-derived neurotrophic influences; the magnitude of this intrinsic growth potential increases with stage of development; and the neurons of the DRG are not intrinsically specified to grow neurites at rates that are matched to the distance they are required to grow to make contact with their peripheral targets in vivo. We present a speculative model based on Poisson statistics, which attempts to account for the variability in the amount of neurite outgrowth from dissociated neurons. © 1995 John Wiley & Sons, Inc.     

Gallera method of chick embryo culture in vitro supports better growth compared with original New method

An avian embryo is a valuable model system for vertebrate embryology. Easy availability, accessibility to various developmental stages and amenability of organ fields makes the chick embryo one of the favored model systems. Seminal discoveries regarding organogenesis and vertebrate morphogenesis have been made using chick embryos cultured in vitro . Dennis A.T. New revolutionized chick embryo culture methodology with his development of a single glass ring explantation technique. Many modifications and/or embellishments were introduced after the New era of embryo culture. A double glass ring method for chick embryo culture introduced by Gallera and Nicolet is compared with the original New method and the EASY method in this study. In addition, a video of culture methods is presented as a valuable tool in learning about and/or teaching techniques of chick embryo culture.     

Perfluorocarbon-based medium for culture of the early chick embryo heart

Summary In an attempt to prolong the survival of the explanted early chick embryo heart, hearts at stages 10 to 28 were cultured in supplemented Dulbecco's modified Eagle's medium with or without the perfluorocarbon, perfluorotributylamine. The perfluorocarbon was added to the standard culture medium in a 50∶50 (vol/vol) mixture. Explants were evaluated daily and were harvested for light microscopy after 2 to 10 d in culture. The tubular shape of the explants was generally maintained for 2 d in culture, after which the hearts became dilated or spherical. Beating was noted in some of the explants on Day 2 in culture but not thereafter. Microscopic evaluation showed patchy areas of necrosis in all explants by Day 3, although large areas of viable epithelioid cells were documented as long as 7 d after explantation. State 16 to 18 hearts cultured in the presence of perfluorocarbon were more likely to maintain tubular architecture on microscopy than hearts cultured in standard medium. Hearts cultured from later stages showed no improvement in appearance with the presence of perfluorocarbon and there was a suggestion of increased necrosis in later-stage explants cultured with pefluorocarbon for 4 d. Further modification of the culture system will be required to prolong explant survival and development beyond 2 d.     

Development changes in regulation of embryonic chick heart gap junctions

Summary Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14-day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of ±100 mV. At all ages, the instantaneous junctional current (or conductance=current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady-state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steadystate junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half-inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue.     

Development of glutamate-induced intracellular Ca2+ rise in the embryonic chick retina

Neurotransmitters affect neuronal development by regulating intracellular Ca²⁺ concentrations. We studied spatiotemporal pattern of the development of glutamate-induced intracellular Ca²⁺ rise in the embryonic chick retina, where developmental changes in mitotic activity, cell death, and synapse formation have been well established. Glutamate was bath-applied to the central part of the retina dissected at embryonic day 3 (E3) to E13, and changes in intracellular Ca²⁺ concentration were measured with Fura-2 fluorescence. The Ca²⁺ rise to glutamate first appeared at E6, reached a maximum at E9–10, and then declined before the appearance of synaptic structures (E12). Ca²⁺ rises to kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) appeared earlier and were larger in amplitude than those to N-methyl-D -aspartic acid. The KA/AMPA receptor of the E9 chick retina was permeable for Ca²⁺, suggesting the functional expression of Ca²⁺-permeable KA/AMPA receptors at the stage of retinal cell death. The Ca²⁺rise to glutamate and KA occurred intensely at the inner plexiform layer, the inner part of inner nuclear layer, and the ganglion cell layer, where the cell death occurs. The Ca²⁺ rise to high K²⁺, in contrast, occurred intensely at the nerve fiber layer and the ganglion cell layer, developing continuously from E3 until E11. Our study shows that the Ca²⁺ rise to glutamate develops with the decline of the mitotic activity of the retinal cells and is transiently enhanced during the period of cell death in the embryonic chick retina. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 113–125, 1998     

Composition, synthesis, and assembly of the embryonic chick retinal basal lamina

To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly.     

Differential sensitivity of cholinergic and GABAergic neurons in chick embryos treated intracerebrally with ethanol at 8 days of embryonic age

We have shown that in embryos treated with ethanol in ovo during days 1–3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferse (ChAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in both cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergic neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick.     

Comparison between the in vivo and in vitro expression of three adhesion-signaling proteins in embryonic cells

We have examined the relationship between the in vivo and in vitro expression of three adhesion-signaling proteins (FAK, PYK2 and Paxillin), using cells of the early chick embryo, where pure cell populations may be isolated and cultured, and in which epithelial-to-mesenchymal transformation is occurring. Focal Adhesion Kinase (FAK) and Proline-rich Tyrosine Kinase-2 (PYK2) are related in molecular structure, and may have some overlapping functions in signal transduction associated with cell-substratum adhesion. Paxillin, a cytoskeletal protein, is also localized to focal adhesions. We show that the immunocytochemical detection of these molecules in vivo does not reflect their in vitro localization. Focal Adhesion Kinase showed a developmentally regulated localization to the cytoplasm, but not to sites of adhesion, in epithelial cells in vivo, while Paxillin was associated with migrating mesoderm cells. Proline-rich Tyrosine Kinase-2 was undetectable in vivo. The level of expression of these molecules was compared under in vivo and in vitro conditions. While the expression of Focal Adhesion Kinase showed a tissue-specific regulation of expression with the change to in vitro conditions, Proline-rich Tyrosine Kinase-2 showed a more uniform and less tissue-specific up-regulation. Levels of Paxillin expression also showed an increase with this change in conditions. We conclude that despite the structural and functional relationships between these three molecules in the developing embryo, the expression and localization of each is independently regulated. We suggest that this provides these cells with the adaptability that they require in order to respond to the changing extracellular environment in the early embryo, and to undergo epithelial-to-mesenchymal transformation.     

Expression of Eph receptor tyrosine kinases and their ligands in chick embryonic motor neurons and hindlimb muscles

Evidence is accumulating that Eph receptor tyrosine kinases and their ligands regulate cell migration and axonal guidance during development. It was previously found that one of the Eph receptors, EphA4, is transiently expressed in subsets of chick embryonic motor neurons. Here, the expression of EphA and ephrin-A subfamily members was further examined, and the dynamic patterns of expression in chick embryonic motor neurons found. EphA3, EphA4, ephrin-A2, and ephrin-A5 were also expressed in the connective tissues of limb muscles and EphA3 and EphA4 expressing motor neurons innervated EphA3 and EphA4 expressing limb muscles, respectively. These spatiotemporal expression patterns suggest that EphA and ephrin-A proteins play important roles in muscle patterning and motor axonal guidance.     

Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

Gene manipulation of chick embryos in vitro, early chick culture, and long survival in transplanted eggs

We introduce a revolutionary gene transfer system in chick: transfect chick embryos at early developmental stage by electroporation in vitro, Early Chick (EC) culture, and transplant to the egg to let the embryo survive until E5.5. Referring to the fate map, we could target the tissues of transfection, or transfect large areas of the embryo. We could get tissue-specific expression of a transgene by tissue-specific promoter. This method is very convenient and rapid, but allows us to get stable expression of the transgene in combination with transposon system.