Ankle restraint modifies motility at E12 in chick embryos

The chick's relationship to its environment changes dramatically over 21 days of embryonic development. At early ages embryos are buoyant; their posture and movements are relatively unconstrained. As embryos grow and fluid level in ovo decreases, movements are increasingly constrained by gravitational forces and reactive forces due to body contact with the shell wall. The issue of how age-related changes in the constraints on movement in ovo may affect embryonic motility is addressed in this paper. Our long-term goal is to determine whether experience imposed by these conditions contributes to development of posthatching motor behaviors. Because previous work indicated that parameters of motility can be modified by a reduction in buoyancy at embryonic day (E) 9, we sought to determine whether a restraint localized to a single joint could also alter either the episodic distribution of activity or the spatiotemporal patterns of limb movement at either E9 or E12. Thus a restraint was applied to the right ankle of embryos prepared for kinematic recordings. Video and kinematic analyses indicated that the restraint had minimal effect at E9, but significantly modified several motility parameters in both the wing and leg at E12. Ankle restraint decreased episode duration. Restraint also decreased most joint excursion parameters, including excursion range, cycles per sequence, and excursion velocity. Restraint increased cycle period duration and signal frequency content under 1.0 Hz. Parameters of intralimb and interlimb coordination exhibited small mixed effects. Results provide support for the hypothesis that environmental conditions contribute to features of embryonic motility. Further, significant modifications of wing excursions in ankle restrained embryos suggest that sensory feedback arising from mechanical perturbations of leg movements may entrain rostral spinal circuits for preservation of interlimb coordination at E12. Potential mechanisms and implications are discussed.     

Teratogenic effects of suramin on the chick embryo

Suramin, a polysulfonated naphthylamine, has been used for the chemotherapy of trypanosomiasis and onchocerciasis since about the 1920s. Currently, it is also being tested as an anticancer agent. It is hoped that suramin might stop the progression of some kinds of cancer since it has been found to inhibit the proliferation and migration of cells and the formation of new blood vessels. These processes are not only essential for the development and progression of cancer, but also for normal embryonic development. Suramin might, therefore, be a potent teratogen. In the literature, however, we have found only scant information on this subject. In the present study, we demonstrate the teratogenic effects of suramin on chick embryos. Suramin was injected into the coelomic cavity of chick embryos on incubation day (ID) 3. Following reincubation until ID 8, suramin-treated embryos ( n=50) were examined for congenital malformations and compared with a control group ( n=30). The survival rate of suramin-treated embryos was markedly reduced compared with controls (50% vs 90%). Among the 25 survivors the following malformations were recorded: caudal dysgenesia (100%), median facial clefts with hypertelorism (92%), malformations of the aortic arch arteries (88%), hypo-/aplasia of the allantoic vesicle (84%), microphthalmia (52%), abnormalities of the great arterial trunks (44%), unilateral or bilateral cleft lips (40%), heart defects with juxtaposition of the right atrial appendage (36%), persistence of the lens vesicle (32%), median clefts of the lower beak (8%), omphalocele (4%), and cloacal exstrophy (4%). These results show that suramin is a potent teratogen. The possible implications of our findings for human beings and the possible teratogenic mechanisms of suramin are discussed. Use of suramin in experimental teratology might help to clarify the morphogenesis of median facial clefts and of some congenital heart defects.     

Transformations in embryonic motility in chick: kinematic correlates of type I and II motility at E9 and E12

Soon after hatching, chicks exhibit an array of adaptive, coordinated behaviors. Chick embryos also acquire nearly 18 days of movement experience, referred to as embryonic motility, before hatching. The chick expresses three forms of motility, types I, II, and III, and each emerges at a different stage of embryonic development. Although much is known about the mechanisms associated with motility at early embryonic stages and at the onset of hatching, the transformations in behavior and underlying mechanisms are not fully understood. Thus the purpose of this study was to determine how motility is modified during the first expected transformation, from type I to type II. It was hypothesized that kinematic features for motility at embryonic day 12 (E12) would differ significantly from features at E9 because type II motility emerges during E11. Embryos were video taped for extended intervals in ovo at E9 or E12 and entire sequences of motility were computer digitized for kinematic analyses. Results reported here indicate that several of the kinematic features characteristic of motility at E9 are also reliable features at E12. On the basis of these findings, a kinematic definition of type I motility is posed for use in subsequent behavioral studies. Several parameters distinguished motility at E12 from E9. The most notable difference between ages was the less regular timing of repetitive limb movements at E12, a finding consistent with recent reports suggesting early motility is an emergent product of a transient neural network rather than a specialized pattern generator. As predicted from established definitions for type II motility, startle-like movements were common at E12; however, they also were present in many kinematic plots at E9, suggesting the discreet age-dependent boundaries in the established definition for type II motility may require modification. Some age-related differences, such as increased intralimb coordination and excursion velocity, may be prerequisites for adaptive behavior after hatching.     

Developmental effects of lead acetate on the chick embryo metanephros

The developmental effects of lead acetate were studied in the chick embryo metanephros, the third renal rudiment that acquires morphological characteristics of functioning kidney already during the prenatal life. Lead exposure was obtained by applying a lead acetate solution on the chick embryo chorioallantoic membrane at the days 9, 10 and 11 of incubation. Quantitative evaluation of the lead concentration assessed by furnace atomic absorption spectrophotometry at the days 14 and 21 of incubation demonstrated metal presence both in the chorioallantoic membrane (CAM) and in metanephros (MN). The lead concentration was higher in CAM than in MN; the metal amount was similar in the CAM of 14 and 21 day embryos, but significantly higher in the 14day embryo MN than in the 21 day embryo MN. Morphological observations on metanephros tissue of control and lead-treated embryos were performed under light, electron transmission and electron scanning microscopes. Peculiar attention was devoted to the expression of the junctional protein connexin 43, the major component of the gap junctions in the renal cells. The results indicated that lead treatment does not intervene in the general differentiation of the metanephric nephrons. The lead is reabsorbed by the proximal tubule cells that are engulfed by endocytotic vacuoles and metal deposits and show long term degenerative changes. Expression of Cx43 protein and ultrastructure of gap junctions between proximal tubule cells appeared to be unchanged. The morphological aspects of the MN corpuscles and tubules agree with the suggestion of a lead cytotoxic effect but do not corroborate, at least in this experimental model, the view of primary damage exerted by lead on the gap junctions of the renal epithelial cells.     

Fibronectin distribution in the chick embryo during formation of the blastula

Summary The distribution of the fibronectin-rich extracellular matrix (ECM) in the chick embryo during formation of the blástula has been evaluated semiquantitatively using an electron microscopical immunogold staining technique. During the first 10 h of postlaying development, fibronectin was found in both embryonic area pellucida and extra-embryonic area opaca of the blastoderm. In the area pellucida, the fibronectin was (1) associated with the basal lamina of the epiblast, (2) present between epiblastic and hypoblastic cells and (3) occasionally internalized in hypoblastic cells. Along the embryonic axis, a transient and high density of ECM was associated with the front of the anteriorly and rapidly expanding hypoblast. Very high density of fibronectin was observed in the marginal zone of the area pellucida, where the epiblastic and deeper cell layers show contacts and intense re-arrangements. In the area opaca, fibronectin was at first found only sporadically between contacting cells, but its density increased steadily and markedly during the first day of development. These rapid and significant changes in the regional distribution of fibronectin-rich ECM are discussed with respect to the early morphogenesis of the chick embryo.     

Extragonadal distribution of primordial germ cells in the early chick embryo

Chick primordial germ cells (PGCs), after separation from the endoderm in early embryonic development, temporarily circulate via the blood vascular system and finally migrate into the gonadal anlagen. It has been noted by some authors that some PGCs are present in extragonadal sites in some vertebrates. In the present study, we examined the distribution and localization of PGCs in extragonadal sites in the chick embryo. PGCs were identified by periodic acid-Schiff staining with light microscopy. In embryos at stages 20-24 (PGCs are in the settlement stage in the gonadal primordium), approximately 20% of the total number of PGCs were observed in extragonadal regions. Approximately 90% of these ectopic PGCs were found in the head, mainly in the mesenchyme surrounding the neural tube. Even at stage 14 when PGCs were usually circulating in the blood vessels, some of the PGCs had emerged from the blood vessels and were detected in the extragonadal site. This pattern of distribution of ectopic PGCs in the head area is probably attributed to the earlier, dominant development of the capillary network, and to the sluggish capillary blood flow in that region, which allows intravascular PGCs to escape into the tissue.     

Migration and distribution of circumpharyngeal crest cells in the chick embryo

The cardiac neural crest is located in a transitional area on the neuraxis between trunk and cephalic regions and gives rise to both the dorsolateral and ventrolateral crest cell populations. Around stage 18 of chick development, a mass of E/C8⁺ cells surrounds the postotic pharyngeal arches and forms a crescent-shaped arch, termed the circumpharyngeal ridge. Using immunohistochemistry and quail-chick chimeras, it was determined that the E/C8⁺ cell mass located in the circumpharyngeal ridge derives from the dorsolateral component of the cardiac neural crest. The ventrolateral cell population of the cardiac crest is located more medially and shows long-persistent HNK-1 immunoreactivity dorsolateral to the foregut. The crest cells that populate the gut arise from the caudal portion of the circumpharyngeal crest and are always located caudal to the caudalmost pharyngeal ectomesenchyme. Circumpharyngeal crest cells continuously populate the pharyngeal arch ectomesenchyme and enteric nervous system on the lateral side of the foregut wall, as well as the hypoglossal pathway which develops within the ventral portion of the circumpharyngeal ridge. E/C8 and HNK-1 immunoreactivity are associated with the cells migrating via the dorsolateral (circumpharyngeal) and ventrolateral pathways, respectively, with one exception: there is a population of putative crest cells along the proximal course of the vagal intestinal branch that shows both immunoreactivities around stage 20. Dil labeling of the cells in the circumpharyngeal ridge suggests that the cells are contributed from the circumpharyngeal ridge to this population. Thus, the distribution of the circumpharyngeal crest cells and their derivatives coincides with the peripheral branch distribution of the cranial nerves IX, X, and XII, whose development is selectively affected in the absence of the cardiac neural crest, the source of the circumpharyngeal crest.© Willey-Liss, Inc.     

Teratogenic effects of coherent light on chick embryos

In this work we study the possible teratogenic effects of laser light with very low surface power densities. In all the cases we confirmed embryonic anomalies that were more manifest and generalized as earlier the stage where the embryos were exposed to irradiation.     

Light exposure of chick embryo influences lateralized recall of imprinting memory

Environmental cues, such as light during the later part of incubation, are known to establish lateralization of some forms of visually guided behaviors in birds. The authors investigated the effect of light on lateralized recall of imprinting memory in chicks. On Day E19 of incubation, one eye was occluded for 24 hr. The other eye received stimulation by light. Chicks were imprinted and then tested for their imprinting preferences after administration of a low dose (500 ng) of glutamate into either hemisphere. Chicks that had the right eye exposed to light during incubation showed recall of the imprinting stimulus after injection of the left hemisphere but not after injection into the right hemisphere. The reverse was found for chicks that had the left eye exposed to light. Hence, the hemisphere ipsilateral to the eye exposed to light before hatching became essential for recall of imprinting memory. The hemisphere used in recall of imprinting memory received indirect visual inputs and was determined by environmental stimulation (asymmetrical light input).     

The effects of exogenous nutrients on growth of chick embryo brain

The effect of an early addition of exogenous nutrients on brain growth has been investigated in chick embryo. The nutrients were introduced onto chorioallantoic membrane at day 6 or 7 of embryonal life, and the cerebral hemispheres examined at the end of neuronal proliferation for the following parameters: weight, DNA content (index of cell number) and protein content. L-Tryptophan produced significant inhibition, probably by creating amino acid imbalance and interference with the transport of other amino acids. D-Tryptophan (slower transport) was inactive. 5-Methyltryptophan produced significant inhibition, probably by causing deficiency of tryptophan utilization and of production of serotonin, which is a growth factor for early brain. Glycine stimulated brain growth, probably by conversion to glucose which is the main energy source for the embryo in this period. Brain weight and DNA were found to be significantly correlated with blood glucose level. It is concluded that, within genetic limits, early brain growth might be manipulated in both directions (inhibition or stimulation) by addition of proper nutrients during sensitive period of neuronal proliferation.     

Neurite-promoting effects of 12-O-tetradecanoylphorbol-13-acetate on chick embryo neurons

The addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to defined growth medium enhanced the survival of ciliary neurons in dissociated cultures and promoted the rapid development of neurites from sympathetic and parasympathetic ganglion explants. Explants of the central nervous system including the cerebral cortex and spinal cord of chick embryos were, however, unresponsive to either 10 or 100 ng/ml of TPA.     

A comparison of mutagenic and embryotoxic effects of cyclophosphamide on the chick embryo

The cytogenetic and embryotoxic effects of cyclophosphamide (CP) after injection on day 3 of single doses ranging from 0.1 to 30 micrograms were investigated in the chick embryo. The occurrence of embryotoxicity was checked on incubation day 8; clastogenic effects as well as mitotic indices were evaluated in primitive blood cells 6, 12, 18, 24 and 30 hr after the administration of CP. Teratogenic doses of CP induced high percentages of aberrant cells with many unstable aberrations. Mitotic activity appeared to be inhibited at the early collection intervals. Nonembryotoxic doses of CP also increased the incidence of aberrant cells without any substantial effect upon mitotic activity. Although the clastogenic effects of CP occur at lower doses than the teratogenic effects, the thresholds for both effects lie in the lower part of the embryotoxicity dose range, as estimated by the Chick Embryotoxicity Screening Test (CHEST).     

A comparison of mutagenic and snbryotoxic effects of cyclophosphamide on the chick embryo

The cytogenetic and embryotoxic effects of cyclophosphamide (CP) after injection on day 3 of single doses ranging from 0.1 to 30 μg were investigated in the chick embryo. The occurrence of embryotoxicity was checked on incubation day 8; clastogenic effects as well as mitotic indices were evaluated in primitive blood cells 6, 12, 18, 24 and 30 hr after the administration of CP. Teratogenic doses of CP induced high percentages of aberrant cells with many unstable aberrations. mitotic activity appeared to be inhibited at the early collection intervals. Non-embryotoxic doses of CP also increased the incidence of aberrant cells without any substantial effect upon mitotic activity. Although the clastogenic effects of CP occur at lower doses than the teratogenic effects, the thresholds for both effects lie in the lower part of the embryotoxicity dose range, as estimated by the Chick embryotoxicity Screening Test (CHEST).     

Pathway formation and the terminal distribution pattern of the spinocerebellar projection in the chick embryo

Summary Pathway formation and the terminal distribution pattern of spinocerebellar fibers in the chick embryo were examined by means of an anterograde labelling technique with wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP).Spinocerebellar fibers, which originate in the lumbar spinal cord and are located in the marginal layer of the spinal cord, reach the corsal part of the cerebellar plate on embryonic day (E)8. On the way to the cerebellum the fibers form one distinct bundle, that suggests that gross projection errors probably do not occur during the formation of the spinocerebellar pathway.On E10, labelled fibers are located mostly in the medullary zone of the anterior lobe. By E12, the number of labelled fibers increases greatly in the inner granular and molecular layers. In transverse sections labelling was distributed throughout the mediolateral extent of the medullary zone. By E14, sagittal strips of labelling were clearly recognized in lobules II–IV; however, labelled terminals were present throughout lobule I. Although the adult pattern of terminal distribution is attained by E14, the mossy fiber terminals are still quite immature. The density of labelling decreased greatly by E16, and small terminal varicosities were first recognized. Structural differentiation of mossy fiber terminals continues to the end of the embryonic or the newly posthatched period.