Chick embryo culture using duck egg shell--first successful hatch

The suitability of duck egg shell (DES) for chick embryo culture was investigated. Chick embryos were transferred into DESs with all egg contents after 3 days of normal incubation and cultured. The vessels made of polyethylene cling film were used for shell-less control. Among 35 embryos cultured in DESs, 21 survived until 16 days of incubation (13 days after transfer) and finally 3 newly hatched chicks were obtained at 22 days of incubation. One of them died 4 days later, but remaining two became full-grown cocks showing normal body weight and production of fertile sperms. Among 37 embryos cultured in polyethylene vessels, none survived over the period of 19 days of incubation. It is suggested that DES culture system may be useful for the various experiments using chick embryos.     

Vasculogenesis of the bursa cloacalis (bursa of Fabricius) of the chick embryo

Vasculogenesis of the bursa cloacalis (bursa of Fabricius) was examined in 10- to 21-day chick embryos and in chicks during the first 5 days post-hatching. The entire circulatory system was injected with India ink, and the bursae were then removed and either cleared for examination in toto or sectioned serially. The bursa was supplied by three pairs of extrinsic blood vessels. At 10 and 11 days of incubation, most intrinsic vessels were arranged in a superficial, hexagonal network. In regions of developing plicae, the hexagonal plexus extended into the core of each plica, forming middle plical vessels. The latter were interconnected across interplical areas by cross-connecting vessels. The middle plical vessels gave rise to small capillary offshoots, which soon increased in complexity, forming delicate loops. Branches extended from these loops through the subepithelial lamina propria to incipient epithelial buds by 12 days of incubation. All epithelial buds were supplied by at least one such branch, and similar branches extended to the basal aspect of the epithelium in areas where epithelial buds had not yet formed. This observation is consistent with the hypothesis that blood vessels induce formation of epithelial buds. At about 15 days of incubation, the cortex and medulla of each developing lymphatic follicle were defined clearly, and an intricate, web-like, capillary network coursed throughout the follicular cortex. The medulla appeared to be devoid of capillaries. The diameters of all intrinsic and extrinsic bursal blood vessels gradually increased throughout development. During post-hatching stages, the diameters of the extrinsic vessels continued to increase, whereas those of the intrinsic vessels were markedly decreased from late pre-hatching stages.     

Vasculogenesis in the chick embryo optic tectum

The initial formation and further development of the intraneural blood vessel network in the tectum opticum of the chick from the 4th to the 14th incubation day have been analyzed and some quantitative data morphometrically recorded. Vessels have been filled by intracardial injection of India ink in vivo. As inferred from our previous investigations on the vasculogenesis of several districts of the central and peripheral nervous system in the chick embryo, also in the developing optic tectum growth and distribution pattern of the vessels seem to unfold step by step under the local influence of earlier occurring morpho-histogenetic processes of the corresponding neural substratum.     

Neural crest origin of cardiac ganglion cells in the chick embryo: Identification and extirpation

Using interspecific grafting of neural crest between quail and chick embryos, it was determined that the cardiac ganglia originate from the cranial region (somites 1–2) of the vagal neural crest (somites 1–7). Neuronal uptake of [³H]choline was used as an index of neuronal development in the chick atrium. Normal uptake was found to be quite high between Days 8 and 14 of incubation. Following extirpation of neural crest over somites 1 to 3 at stages 8 to 10, neuronal uptake in 8-day chick atrium was decreased by 25–60% depending on the stage at which the lesion was performed. It is thought that the residual uptake represents preganglionic terminals from the dorsal motor nucleus of the vagus. Embryos with extirpations of neural crest over somites 1–3 performed at stage 9 showed the greatest decrease of neuronal choline uptake and did not live beyond 11 days of incubation. However, hearts from embryos with partial lesions (performed at stage 10) were treated on incubation Days 12 and 15 for demonstration of acetylcholinesterase in the subepicardial plexus. These hearts showed much less extensive neural plexus with sparse, small cardiac ganglia.     

Vascular system in the developing wing bud of normal and talpid mutant chick embryos

The vascular limb mesoderm probably plays a prominent role in limb pattern formation in both normal and talpid chick embryos. The differential vascularization, or its consequences, is a factor controlling the initial stages of differentiation in the developing bud. The axial artery runs from the subclavian artery to the distal region of the normal limb bud, whereas in the talpid3 only secondary blood vessels develop. In the talpid, the gene permits the chondrogenic regions to grow and at the same time keeps the peripheral regions to a certain size. The mesenchyme tissue lies within the effective range of a metabolic gradient extending from either the ectodermal surface or the peripheral vessels to the limb axis.     

Ontogenetical development of the chick and duck subcommissural organ. An immunocytochemical study

The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day in incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used. In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 blood vessels penetrated the SCO, scarce hypendymal cells appeared, the first signs of ventricular release of IRM were noticed, appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct, day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 hypendymal cells proliferated, developed processes and migrated dorsally, ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. the duck SCO appears to follow a similar pattern of development.     

Vascular abnormalities due to hyperthermia in chick embryos

Intraembryonic vascular abnormalities were studied in chick embryos exposed to temperatures 3 degrees C and 4 degrees C above normal temperature (38 degrees C) from the beginning of incubation. The average duration of hyperthermia was 54 and 53 hours, respectively. Immediately after exposure, the embryos were examined with FITC-Dextran microangiography in vivo. Following hyperthermia various abnormalities in the heart, ventral aortae, aortic arches, omphalomesenteric arteries, and the distal dorsal aortae frequently occurred. There were also significant microvascular changes in the head, in the lateral and caudal parts of the embryos, and in the pellucid area of the yolk sac. In another series incubation at 41 degrees C, hyperthermia of 3 degrees C during the first 3 days of development produced several extraembryonic vascular abnormalities. These included duplication and abnormal branching of the cranial vitelline vein, absence or abnormal course of the omphalomesenteric vessels, aneurysmatic dilatation or abnormal course of the caudal vitelline vein, and aneurysmatic dilatation or occlusion of the abdominal venous sinus. Most frequent findings were blind, congested, and dilated microvascular segments in the pellucid area, commonly associated with an irregular microvascular pattern and perivascular swelling. The abnormalities described are assumed to be caused by the direct effects of hyperthermia upon the developing vessels resulting in microvascular insufficiencies, pathological leakage, and perivascular oedema. Such disturbances may have serious consequences for embryonic vascular development and microcirculation, which in turn may have adverse effects on further embryonic growth and development.     

Emoxipin as an inhibitor of angiogenesis]

The effect of emoxypin on angiogenesis in rabbit cornea in aseptic inflammation induced by intracorneal implantation of a piece of quartz and on the development of the vessels of the chick embryo yolk sac was studied. 1% emoxypin pipetted thrice a day for 10-14 days inhibited corneal neovascularization and reduced the formation of new blood vessels. We observed an inhibitory effect on the development of vascular bed of the embryo yolk sac on incubation hour 64-72. The drug affected neither general growth of the embryos no the number of somites.     

The effects of thiourea and cortisone acetate on duodenal length and diameter and on morphogenesis of duodenal villi in chick embryos

Subnormal growth in length and diameter of the duodenum is encountered both in chick embryos injected with thiourea at 11 days of incubation, and in embryos injected with cortisone acetate at 14 days of incubation. Morphogenesis of previllous ridges and villi is retarded in thiourea-treated embryos. Morphogenesis of previllous ridges and villi in cortisone acetate-treated embryos at first seems to be slightly accelerated over that of controls, but the acceleration does not appear to be maintained, and the hormone may actually be inhibitory to normal morphogenesis of villi by 19 days of incubation. The results indicate that normal levels of endogenous thyroxine are necessary for normal growth in length and diameter of the duodenum and for normal morphogenesis of previllous ridges and villi. The possibility that adrenocorticoids may also play a role in the normal development of these parameters is indicated, although the evidence is inconclusive.     

Vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern

We are using a monoclonal antibody, QH-1, as a label for angioblasts in quail embryos to study vascular development. Our previous experiments showed that major embryonic blood vessels, such as the dorsal aortae and posterior cardinal veins, develop from angioblasts of mesodermal origin that appear in the body of the embryo proper (Coffin and Poole: Development, 102:735-748, '88). We theorized that there are two separate processes for blood vessel development that occur in quail embryos. One mechanism termed "vasculogenesis" forms blood vessels in place by the aggregation of angioblasts into a cord. The other mechanism, termed "angiogenesis," is the formation of new vessels by sprouting of capillaries from existing vessels. Here we report the results of microsurgical transplantation experiments designed to determine the extent of cell migration taking place during blood vessel formation. Comparison of the chimeras to normal embryos suggests that the vascular pattern develops, in part, from the normally restricted points of entry of angioblasts into the head from the ventral and dorsal aortae. Transplantations of quail mesoderm (1-15 somite stage) into the head of 5-15 somite chick hosts resulted in extensive sprouting and in migration of single and small groups of angioblasts away from the graft sites. Transplantations into the trunk resulted in incorporation of the graft into the normal vascular pattern of the host. Lateral plate mesoderm was incorporated into the dorsal aortae and individual sprouts grew between somites and along the neural tube to contribute to the intersomitic and vertebral arteries, respectively.     

Immunohistochemical study of the appearance of somatotropic hormone in the adenohypophysis of chick embryos

The differentiation of somatotropocytes was studied in the Leghorn chick embryos during 11 to 18 days of incubation and in chickens during the first week of life using the immunohistochemical method of nonlabelled antibodies with PAP complex and antiserum against human somatotropic hormone (STH). Unlike in humans, the fixation of pituitaries in the Carnoy mixture is optimal for STH to be immunohistochemically estimated in the chickens and chick embryos. STH was found in adenohypophysis from 12-13 days of development. Besides predominant localization of somatotropocytes in the adenohypophysis caudal lobe, individual STH-positive cells are also present in the cephalic lobe of chickens and chick embryos. The results obtained suggest a relatively late appearance of STH during histotypical development of adenohypophysis in chick embryos.     

Cultured chick yolk sac epithelium: structure and IgG surface binding

The chick yolk sac endoderm transports maternal immunoglobulin G (IgG) from the yolk into the embryo during development, providing the newly hatched chick with passive immunity until it becomes immunocompetent. To study this transport process, chick yolk sac endodermal cells isolated from embryos of 6 to 18 days of incubation were grown in vitro on a collagen substrate. The cultured cells possessed a remarkable structural similarity to the in vivo tissue and reformed a polarized confluent epithelium with tight junctions and desmosomes joining the cells at their apical margins. In addition, the cells exhibited apical microvilli, numerous phagolysosomes in the cytoplasm and retained the expression of the yolk sac endoderm-specific enzyme marker, cysteine lyase. Importantly, the cultured cells retained the ability to specifically bind IgG as demonstrated by indirect immunofluorescence. Chicken IgG bound to the cultured cells at 4 degrees C in a diffuse pattern that clustered into a punctate pattern when a second antibody was used. Cultures from yolk sacs of day 6 through day 18 of development all demonstrated this immunofluorescent labeling for at least 14 days in culture. These results demonstrate that cultured yolk sac endoderm maintains its differentiated morphology and ability to bind IgG.     

Mechanisms in the development of retinotectal projections in the chick embryo studied by surgical deflection of the retinal pathway

Retinotopic analysis of the pathways of normal and aberrant retinal axons within the tectum of developing chick embryos was performed by selective labeling of retinal axons with a fluorescent dye, rhodamine-B isothiocyanate. To produce aberrant retinal axons, the presumptive optic chiasma was surgically disorganized at the 3rd day of incubation. At the 11th and 13th days of incubation, more than half of the operated embryos exhibited several aberrant retinal axons which reached ectopic parts of the tectum. The pathways of these aberrant axons within the tectum depended on the position of their initial invasion into the tectum at the diencephalotectal junction, and not on their position of origin within the retina. The aberrant retinal axons did not show any sign of correction of their pathways toward their normal sites of innervation within the tectum. As development proceeded, elimination of the aberrant retinal axons occurred. By the 16th day of incubation, almost all operated embryos lacked aberrant retinal axons and although the total number of axons often appeared reduced, a nearly normal topography of retinotectal projections was established. These findings indicate that the initial invasion of the retinal axons into the tectum is conducted predominantly by nonspecific mechanisms and, thereafter, a selective maintenance of appropriate retinal axons occurs.     

Opioid elements in development of the spontaneous motility of chick embryos

The effects of the acute and chronic administration of a pure opioid antagonist--naltrexone--was studied in chick embryos from the 4th to the 19th day of incubation. In acute administration, naltrexone (40 mg/kg egg weight) induced paroxysmal activation of spontaneous motility in both normal and spinal embryos from the 13th-15th day of incubation. Activation attained 3- to 4-fold the resting activity of chick embryos of the same ages. The chronic administration of naltrexone (7.46 +/- 1.18 mg/kg e.w. per 24 h) from the 4th to the 16th day of incubation was not manifested either in the embryos' somatic development or in the weight of the brain hemispheres, but it depressed the development of spontaneous motility to 26.1-75.8% of the activity of the control embryos. This developmental effect was not demonstrably correlated either to the length of time for which naltrexone was administered, or to when, in the course of incubation, it was administered to the chick embryos. The results are evaluated as evidence of the participation of opioid elements in the development and effectuation of central motor input functions in the early stages of ontogenetic development.     

Calcium regulation in the embryonic chick. II. Ultrastructure of the parathyroid glands in shell-less and in ovo embryos

The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell-less culture (after removal of the eggshell). Shell-less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell-less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12-day shell-less embryos. However, the parathyroids of the 15-day shell-less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of the two groups is indistinguishable, both appearing to be more active than the 15-day shell-less group. Thus, protein synthetic activity of the parathyroid glands, as detected by ultrastructural alterations of the chief cells, normally appears to be initiated during the latter part of embryogenesis (by approximately 15 days incubation) and its onset can be stimulated at least 3 days prematurely by hypocalcemia.     

Origin of cutaneous smooth muscles in birds (author's transl)

The origin of smooth muscles in the skin of bird embryos has been analyzed in heterospecific quail/chick recombinants. The somitic mesoderm of the wing level of 2-day chick embryos was replaced by homotopic or heterotopic somitic mesoderm from quail embryos. The cellular constitution of tissues was observed in twelve recombinant embryos at 17 or 18 days of incubation. Results show that feather smooth muscles and vascular smooth muscles have the same origin as the cutaneous mesenchyme in which they differentiate. They are of somatopleural origin in the wing integument and of somitic (dermatomal) origin in the dorsal integument. This study further reveals that the muscular and connective tissue wall of blood vessels does not have the same embryonic origin as the endothelium. It is suggested that the latter originates from the primitive aorta.     

Ontogeny of Glucose Metabolism in Sympathetic Ganglia of Chickens. Changes in the Carbon Fluxes to CO2, Lactate, and Tissue Constituents from 8 to 19 Days of Embryonic Age

Chains of sympathetic ganglia were excised from the lumbar region of white Leghorn chicken embryos, 8-19 days of age. The chains were incubated for 5 h at 36 degrees C in a bicarbonate-buffered physiological salt solution containing 5.55 mM unlabeled glucose and tracer amounts of glucose labeled either uniformly or at carbon-1 or carbon-6. Glucose uptake and labeled lactate output were both highest in ganglia from the youngest embryos studied and declined progressively with increasing age. The output of labeled CO2 rose to a peak rate at an incubation age of 10-12 days in the presence of either [U-14C]glucose or [1(-14)C]glucose, but changed relatively little with age in the presence of [6(-14)C]glucose. The incorporation of 14C into tissue constituents was fastest at 10-12 days with all three labeled glucoses. It is concluded that the hexosemonophosphate shunt is most active at an incubation age of 10-12 days, after glycolysis has greatly slowed. The literature on morphological and biochemical changes in the sympathetic ganglia during development is briefly reviewed and discussed in relation to the observed metabolic changes. The early high glycolytic rate may be related to the normal developmental delay in vascularization of the sympathetic chains.     

Effects of chronic hypoxia on microcirculation of the central nervous system during embryonic development

In the attempt to know the factors influencing the vasculogenesis and to verify whether the vessel formation and growth are influenced, during different ontogenetic periods, by oxygen deficiency, the intraneural vascular network has been morphometrically analyzed in chicken embryo optic tectum under conditions of aerogenic hypoxia. Chicken eggs, incubated under routine conditions, have been half-painted with melted wax at the 2nd incubation day (i.d.). Fragments of optic tectum, isolated from living embryos at the 8th, 14th and 17th i.d., have been fixed and embedded according to the usual E/M procedures. A parallel series of normally developed embryos of 8, 14 and 17 incubation days has been likewise prepared. On semithin sections from the hypoxic specimens and normal control embryos the area occupied by vessels (Av), the number of vessels (Nv) and the diameter of the radially directed ones (Dv) have been evaluated. The preliminary results indicate that hypoxia evokes, from the 8th to the 17th i.d., an increment of the Av parameter, due first to microvessel neoformation and enlargement from the 8th to the 14th i.d., then from the 14th to the 17th i.d. only to a growth of new capillary branches. The response of the vascular network to the hypoxic condition is more marked from the 8th to the 14th i.d., t.i. when, in relation to the differentiation and the stratification of the neurons, the metabolic requirements of the developing tectum are presumably increased. O2 deficiency causes severe developmental disorders of the cyto- and mieloarchitecture of the tectum: the vascular response can apparently prevent actual damages only when the hypoxic condition lasts for a relatively short time.     

Ontogenetical development of the chick and duck subcommissural organ

Summary The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day of incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used.In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 i) blood vessels penetrated the SCO, ii) scarce hypendymal cells appeared, iii) the first signs of ventricular release of IRM were noticed, iv) appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct; day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 i) hypendymal cells proliferated, developed processes and migrated dorsally, ii) ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. The duck SCO appears to follow a similar pattern of development.Supported by Grant I/60 935 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile. M.H. was recipient of a personal grant from JNO (29-5-54), which is gratefully acknowledged     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.