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Sir Mac Farlane Burnet was the most honored of all Australian scientists. In 1960, Burnet shared the Nobel Prize for Medicine with Peter Medawar of Britain for the discovery of acquired immunological tolerance. He developed techniques for growing influenza viruses in the chorioallantoic membrane of the chick embryo. This became a standard laboratory practice. He continued to work with chick embryos long after the use of cell cultures had become general. His virology research resulted in significant discoveries concerning the nature and replication of viruses and their interaction with the immune system.

SUMMARY In order to identify the gender of chicken eggs at the early stage of incubation, a machine vision image acquisition system was constructed. Under the light source of LED, the images of 2 batches (186 and 180) of chicken eggs were respectively obtained on d 3, d 4, d 5, d 6, d 8, and d 10 of incubation. Considering the clarity and the integrity of blood vessels in the field of machine vision, the image of d 4 was determined as the basis for gender identification of chick embryos. After image processing, the 11 dimensions of feature parameters depicting the chick's embryonic development were extracted. In this paper, the genetic algorithm (GA) was used to optimize the initial weights and thresholds of backpropagation neural networks (BPNN) with different hidden layers. Then the GA-BPNN with single hidden layer, as well as, double hidden layers was established respectively. According to the research, the comprehensive accuracy of GA-BPNN model with double hidden layers reached 89.74% for the prediction set, which was higher than that of the model with single hidden layer, indicating that optimizing the initial weights and thresholds of BPNN by GA and adding the hidden layer had a certain effect on improving the recognition accuracy. Meanwhile, the results showed that the machine vision technology provided a feasible method for gender identification of chicken eggs at the early stage of incubation.

Three isotopic tracers ([2,3,3-2H3]-L-serine, [2H11]-L-betaine, and [1-13C]-L-methionine) were administered by amnion injection into 18-day-old chick embryos to investigate the kinetics of methionine metabolism. The embryos utilized were from eggs collected from 34-week-old Cobb 500 broiler breeders that were fed either a control diet containing folic acid (1.25 mg/kg diet) and pyridoxine HCl (5 mg/kg diet) or diets devoid of supplemental pyridoxine or folic acid. Intermediate metabolites of methionine metabolism and polyamines were analyzed in 18-day-old chick embryos. There were no differences in hepatic [2H2] methionine or [2H3] cysteine enrichments or in physiological concentrations of sulfur amino acids for chick embryos from breeders fed the control diet and embryos from breeders fed diets containing no pyridoxine or folic acid. Supplementation of B6 or folic acid did not affect the production of methionine and cysteine in chick embryos. However, breeders fed the control diet with both folic acid and pyridoxine supplementation produced embryos with a two-fold reduction of hepatic homocysteine and increased spermine compared with embryos from breeders fed diets containing no supplemental pyridoxine or folic acid (P

In the present study, the effect of manganese (Mn) on antioxidant status and the expression of the manganese superoxide dismutase (MnSOD) gene in cultured primary myocardial cells collected from the chick embryos was investigated. The hypothesis that Mn supplementation would enhance the expression of MnSOD in cultured primary myocardial cells of chick embryos was tested. Eggs collected from Mn-depleted Arbor Acres laying breeder hens were incubated for 10 days and then myocardial cells were isolated and cultivated for 8 days. The embryonic myocardial cells on day 6 were treated with Mn in the cell culture medium at different time points when the proportion of cells showing spontaneous contraction was over 95% after the 3-day primary culture. A completely randomized design involving a 3 Mn levels (0, 0.5 and 1.0 mmol L−1)×3 incubation time points (12, 24 and 48 h) factorial arrangement of treatments (n=6) was used in the current experiment. The results showed that MnSOD activity and mRNA expression level were induced by Mn and increased with incubation time, which supported the hypothesis that Mn would enhance the expression of the MnSOD gene, and thus might protect myocardial cells from oxidative stress during the chick embryonic development.

Temporal expression patterns and activity of two cyclooxygenase (COX-1 and COX-2) isoforms were analysed during early chick embryogenesis to evaluate their roles in development. COX-2 inhibition with etoricoxib resulted in significant structural anomalies such as anophthalmia (born without one or both eyes), phocomelia (underdeveloped or truncated limbs), and gastroschisis (an opening in the abdominal wall), indicating its significance in embryogenesis. Furthermore, the levels of PGE2, PGD2, PGF2α, and TXB2 were assessed using quantitative LC-MS/MS to identify which effector prostanoid (s) had their synthesis initiated by COX-2. COX-2 inhibition was only shown to reduce the level of PGE2 significantly, and hence it could be inferred that the later could be largely under the regulation of activated COX-2 in chick embryos. The compensatory increase in the activity of COX-1 observed in the etoricoxib-treated group helped to maintain the levels of PGD2, PGF2α, and TXB2. Though the roles of these three prostanoids in embryogenesis need to be further clarified, it appears that their contribution to the observed developmental anomalies is minimal. This study has shown that COX-2 is functionally active during chick embryogenesis, and it plays a central role in the structural configuration of several organs and tissues through its downstream effector molecule PGE2.

Background Modern living is awash with low-frequency electromagnetic radiation raising concern over health effects, birth defects, and infant cancers especially leukemias. Medical/scientific opinion is ambivalent, especially regarding possible mechanisms of action despite our bodies׳ many electric currents. Aims Are some cancers induced by morphogenetic changes rather than direct mutation? We wished to see if morphogenetic effects of weak, extremely low-frequency electric (ELF) fields in embryonated hen׳s eggs could induce cancers, knowing that such treatment is usually deleterious. We report a pilot study intended to reveal a promising cell source in which to search for cancer cells by established methods and then to check for DNA damage. Methods Stored (5°C for 1–36 days) fresh, fertile hens׳ eggs were incubated (38°C, total five or six days) in presence or absence of a weak ELF oscillating electric field (1–40V/cm, 1–50Hz and two to six days). Separated embryos were assessed for development stage. Results Storage of untreated eggs (>12 days, 5°C) allows a steady loss of normal embryo formation at 38°C (few viable by 25 days, half-life ~18 days). Surprisingly, incubation in a weak ELF field during the period of declining viability significantly ( P : 0.03–0.0001) improved viability and condition of the embryos (new half-life ~21 days), rather than the expected converse. Thus for a few days, the field could keep viable some embryos that would otherwise not have survived. Conclusions The rescued embryos and their untreated controls seem the most promising place to seek any carcinogenic effects of ELF fields. The nature of the presumed critical component keeping them viable during 5°C storage is at least of equal interest.

Neuroblastoma, an embryonal cancer of neural crest origin, shows metastases frequently at diagnosis. In this issue of Cancer Cell, Delloye-Bourgeois and colleagues demonstrate that neuroblastoma cell lines and patient-derived xenografts engraft and adopt a metastatic program in chick embryos. They identify Sema3C as a candidate switch that regulates metastatic spread.

The objective of the present study was to determine the effect of maternal conjugated linoleic acid (CLA) and soybean oil (SO) dietary on egg hatchability and hepatic lipometabolism in chick embryos. Broiler breeders (36 wk, 3.56 ± 0.12 kg) were randomly assigned to 6 treatments (with 6 replicates per treatment and 25 broiler breeder hens and 1 rooster per replicate) and fed a diet with 0.5% SO (control), or a diet with either 0.5% CLA, or CLA plus 0, 2, 4, 6, or 8% SO for 8 wk, with natural mating. The results showed that 0.5% CLA reduced hatchability of fertile eggs (P

We show that in a shell less culture system, radiated waves from each chick embryo can help us to determine its gender and properties. We also consider thermodynamical evolutions of chick embryos and obtain two different entropies for two chick embryos, one with gender of male and another with gender of female.

Total bacterial counts on hatching eggshell surface were significantly (P

In vertebrates, soluble epoxide hydrolase (sEH) hydrolyzes natural epoxy-fatty acids (EpFAs), which are chemical mediators modulating inflammation, pain, and angiogenesis. Chick embryos are used to study angiogenesis, particularly its role in cardiovascular biology and pathology. To find potent and bio-stable inhibitors of the chicken sEH (chxEH) a library of human sEH inhibitors was screened. Derivatives of 1(adamantan-1-yl)-3-(trans-4-phenoxycyclohexyl) urea were found to be very potent tight binding inhibitors (KI

Background Intestinal damage causes intestinal dysmotility in gastroschisis. Urinary trypsin inhibitor (UTI) has been shown to prevent intestinal damage in chick embryos with gastroschisis. The effect of intra-amniotic administration of UTI on intestinal motility in gastroschisis has not been investigated. Methods Five-day-old fertilized chick embryos were used. Gastroschisis was created through the amniotic cavity without opening the allantoic cavity. There were six groups; control, gastroschisis only, gastroschisis plus meconium and three treatment groups. In the treatment groups, 100IU/mL, 200IU/mL and 400IU/mL UTI were instilled into the amniotic cavity of the gastroschisis plus meconium embryos, respectively. Serosal thickness of the intestines in each group was measured histopathologically. The contractions of the intestines were evaluated by in vitro organ bath technique and the responses were expressed as maximal contraction induced by acetylcholine. Results The serosal thickness was significantly increased in the gastroschisis plus meconium, 100IU/mL, 200IU/mL UTI groups compared to control and gastroschisis only groups. The serosal thickness of the 400IU/mL UTI group was similar to control and gastroschisis only groups. Contractility of the intestines was diminished in the gastroschisis plus meconium, 100IU/mL and 200IU/mL UTI groups. There was no significant difference regarding contractility among control, gastroschisis only and 400IU/mL UTI groups. Conclusion Intra-amniotic administration of UTI preserves intestinal contractility in chick embryos with gastroschisis. However, preservation of intestinal dysmotility by using UTI in the human gastroschisis cases needs further experimental and clinical trials.

SUMMARY From 50,129 fertile eggs, a total of 1,712 dead chick embryos were collected from 5 different strains of chickens: ai-Jiao (233), Xing-Xing (143), HuBei-local (894), Hy-line (212), and Zhu-Si (230). the embryos were collected during the early (1 to 7 d), middle (8 to 14 d), and late (15 to 21 d) stages of incubation. Sex-biased embryonic mortality was analyzed during the different stages. We found that the overall mortality of female embryos was significantly higher than that of males in different stages of incubation (P < 0.05). We then analyzed the data by strain. In Hy-line, HuBei-local, and Zhu-Si chickens, female mortality was significantly higher (P < 0.05) during the early stage. During the middle stage, female mortality in HuBeilocal chickens was significantly greater than that of males (P < 0.005). During the late stage, female mortality was significantly lower (P < 0.05) in ai-Jiao chickens but was significantly higher (P < 0.01) in HuBei-local chickens. thus, more females died during the entire period of incubation, especially in the early and middle stages. Mortality was variable among strains during the late stage of incubation. In strains with smaller egg sizes, more females died, whereas in strains with larger egg sizes, more males died.

n reviewing literature, as far back as the 1800s, the use of I heated incubators has improved survival rates of term and preterm infants. The idea of incubation can be traced as far back as to the Egyptians, who used heat to improve the productivity of hatching chicken eggs. As the chick embryos matured, less heat was required because the embryo generated its own heat from an increase in metabolism, related to being more mature and larger. In his travels to Egypt from France in 1799, Napoleon was impressed with the incubation theory and brought it back to France, where it was used in Parisian zoos. A French obstetrician, Tarnier, was the first to apply this idea of incubation to human premature infants in the late 19th century, where he reported improving survival of premature infants using crude incubators to warm them. The survival of these small infants was so impressive that, during this time, it was common to see infants in incubators on display at carnivals and fairs around the world. Over the next 60 years, the survival of small infants weighing less than 2000 grams increased from 38% to 66% when the infants were kept warm with incubators. Numerous studies initiated the technologically advanced development of incubators and radiant warmers used today. The theory of thermoregulation has been studied repeatedly and shown to be an effective intervention in neonatal care. Basic concepts of heat exchange and the physiology of human thermoregulation are crucial points to understand to provide a thermal-neutral environment for the premature infant. To maintain a constant central temperature within narrow limits (36.5°C–37.5°C), very low-birth-weight (VLBW) infants or sick premature infants are typically cared for either in incubators or under radiant warmers. Maintaining a constant temperature requires a balance between heat production and heat loss.

More than a century ago, several embryologists described sites of hematopoietic activity in the vascular wall of mid-gestation vertebrate embryos, and postulated the transient existence of a blood generating endothelium during ontogeny. This hypothesis gained significant attention in the 1970s when orthotopic transplantation experiments between quail and chick embryos revealed specific vascular areas as the site of the origin of definitive hematopoiesis. However, the vascular origin of hematopoietic precursors remained elusive and controversial for decades. Only recently, multiple experimental approaches have clearly documented that during vertebrate development definitive hematopoietic precursors arise from a subset of vascular endothelial cells. Interestingly, this differentiation is promoted by the intravascular fluid mechanical forces generated by the establishment of blood flow upon the initiation of heartbeat, and it is therefore connected with cardiovascular development in several critical aspects. In this review we present our current understanding of the relationship between vascular and definitive hematopoietic development through an historical analysis of the scientific evidence produced in this area of investigation.

Neural crest cells arise fromdorsal regions of the neural tube, delaminate and migrate to many target sites. In the trunk, neural crest cells follow distinct pathways to form multiple derivatives, including neurons and glia in sympathetic and dorsal root ganglia and melanocytes in the skin. The earliest migrating cells proceed ventrally to the sympathetic ganglia, while later migrating cells populate the dorsal root ganglia, and last of all, pre-specified melanoblasts migrate along a dorsolateral pathway. Multiple molecular differences have been identified between neural crest following the dorsolateral pathway compared to those migrating ventrally. Krispin and colleagues examined in detail the emergence of neural crest cells from the dorsal region of the neural tube by labeling and lineage tracing small numbers of cells in the dorsal neural tube (by electroporating a GFP construct or with DiI) at a specified axial level at defined stages of development in chick embryos. Following labeling of dorsal neural tube cells shortly prior to the initial migration of neural crest cells, all of the labeled cells exited the neural tube in ~90% of the experiments. This suggested a ventral to dorsal relocation of cells in the dorsal neural tube, rather than asymmetric cell division in which one daughter cell emigrated as a neural crest cell while the other daughter cell remained within the neural tube. Time lapse imaging of the neural tube opened along themiddle and flattened into a hemi-tube showed movement of labeled cells from ventral to dorsal. When cells at distinct sites within the dorsal 30% of the neural tube were labeled shortly prior to initial migration, a strong correlation was observed between the dorsoventral positionwithin the neural tube and the final localization of the labeled cells. These results suggest that within the neural tube there is a progressive ventral to dorsal relocation of cells prior to neural crest cell delamination, and that the roof plate is formed from the cells that have relocated dorsally after the dorsolaterally migrating melanoblasts have exited the neural tube. The authors concluded that there is a dynamic spatiotemporal fate map in the dorsal neural tube shortly prior to and during neural crest emigration. Finally

The Onecut (OC) transcription factors, namely Hepatocyte nuclear factor 6 (HNF-6), OC-2 and OC-3, are transcriptional activators expressed in liver, pancreas and nervous system during development. Although their expression and roles in endoderm-derived tissues and in the trigeminal ganglia have been investigated, their expression in the CNS has not been described yet. In this study, we report a qualitative and quantitative expression profile of the OC factors in the developing spinal motor neurons (MN). We provide evidence that OC expression is initiated in newly-born MN. At later stages, they are differentially and dynamically expressed in subsets of differentiating motor neuron within the four motor columns. We also show that the expression profile of HNF-6 in spinal MN is conserved in chick embryos. Together, our data unveil a complex and dynamic expression profile of the OC proteins in spinal MN, which suggests that these factors may participate in regulatory networks that control different steps of motor neuron development.

Hyperthermia is a proven teratogen, inducing malformations and embryonic death in humans as well as in laboratory animals. The aim of our study was to define temperatures that are teratogenic after short-term exposure (from 2 to 24 h) on embryonic days 1–7 and to detect critical periods for the origin of structural defects in the chick embryo. Hyperthermia of 41 °C was not embryotoxic, temperatures from 42 to 44 °C induced malformations and embryonic death, while nearly all embryos died even after the shortest exposures to 45 or 46 °C. Among the wide spectrum of observed malformations, only ventricular septal defect (VSD) and caudal regression syndrome (CRS) were present at frequencies significantly different from those seen in controls.

We have systematically examined the developmental potential of neural crest stem cells from the enteric nervous system (gut NCSCs) in vivo to evaluate their potential use in cellular therapy for Hirschsprung disease and to assess differences in the properties of postmigratory NCSCs from different regions of the developing peripheral nervous system (PNS). When transplanted into developing chicks, flow-cytometrically purified gut NCSCs and sciatic nerve NCSCs exhibited intrinsic differences in migratory potential and neurogenic capacity throughout the developing PNS. Most strikingly, gut NCSCs migrated into the developing gut and formed enteric neurons, while sciatic nerve NCSCs failed to migrate into the gut or to make enteric neurons, even when transplanted into the gut wall. Enteric potential is therefore not a general property of NCSCs. Gut NCSCs also formed cholinergic neurons in parasympathetic ganglia, but rarely formed noradrenergic sympathetic neurons or sensory neurons. Supporting the potential for autologous transplants in Hirschsprung disease, we observed that Endothelin receptor B (Ednrb)-deficient gut NCSCs engrafted and formed neurons as efficiently in the Ednrb-deficient hindgut as did wild-type NCSCs. These results demonstrate intrinsic differences in the migratory properties and developmental potentials of regionally distinct NCSCs, indicating that it is critical to match the physiological properties of neural stem cells to the goals of proposed cell therapies.

Producción Científica, During early stages of brain development, neuroepithelial stem cells undergo intense proliferation as neurogenesis begins. Fibroblast growth factor 2 (FGF2) has been involved in the regulation of these processes, and although it has been suggested that they work in an autocrine–paracrine mode, there is no general agreement on this because the behavior of neuroepithelial cells is not self-sufficient in explants cultured in vitro. In this work, we show that during early stages of development in chick embryos there is another source of FGF2, besides that of the neuroepithelium, which affects the brain primordium, since the cerebrospinal fluid (E-CSF) contains several isoforms of this factor. We also demonstrate, both in vitro and in vivo, that the FGF2 from the E-CSF has an effect on the regulation of neuroepithelial cell behavior, including cell proliferation and neurogenesis. In order to clarify putative sources of FGF2 in embryonic tissues, we detected by in situ hybridization high levels of mRNA expression in notochord, mesonephros and hepatic primordia, and low levels in brain neuroectoderm, corroborated by semiquantitative PCR analysis. Furthermore, we show that the notochord segregates several FGF2 isoforms which modify the behavior of the neuroepithelial cells in vitro. In addition, we show that the FGF2 ligand is present in the embryonic serum; and, by means of labeled FGF2, we prove that this factor passes via the neuroepithelium from the embryonic serum to the E-CSF in vivo. Considering all these results, we propose that, in chick embryos, the behavior of brain neuroepithelial stem cells at the earliest stages of development is influenced by the action of the FGF2 contained within the E-CSF which could have an extraneural origin, thus suggesting a new and complementary way of regulating brain development. © 2006 Elsevier Inc. All rights reserved.

Dying cells of both chromaffin and cortical cell types were found scattered throughout the adrenal gland of 14-18 day mouse embryos and 17-19 day chick embryos. The ultrastructural appearance of these dying cells was unlike that of cells undergoing apoptosis and there was no evidence of macrophages or other phagocytes removing these cells from the adrenal. Possible morphogenetic functions of cell death in the developing adrenal are discussed.

We tested the hypothesis that exposure of chick embryos to corticosterone leads to increased fear, reduced competitive ability, reduced ability to cross a barrier and reduced growth in juvenile chicks. Behaviour was studied in birds subjected to three different egg injection treatments: a negative control (no treatment of eggs), a positive control (100 μl sesame oil vehicle) and a corticosterone treatment (0.6 μg corticosterone in 100 μl sesame oil). Eggs were injected prior to incubation and the behaviour of chicks was studied during the first 4 weeks of life. Corticosterone treatment increased fear in chicks, as indicated by greater avoidance of an observer in the home pen at 2 weeks of age (P

In a previous study of the embryonic midbrain radial glia in a lizard, we observed that these cells define boundaries and regional subdivisions in a pattern that largely supports the adult model [C. Diaz, C. Yanes, C.M feminine. Trujillo, L. Puelles, Cytoarchitectonic subdivisions in the subtectal midbrain of the lizard Gallotia galloti, J. Neurocytol. 29 (2000) 569-593]. With the goal to check whether the midbrain of chick embryos has a similar pattern, we examined the radial glia distribution in this model using a lipophilic dye (DiI) injected intraventriculary. As in the lizard, chick radial glia distribution and fasciculation defines at least six regional subdivisions in the midbrain, five of which are alar and one basal. Each territory corresponds to a particular cytoarchitectonic area, recognized previously. The rostral mesencephalic limit (m/d) is underlined in the alar zone by a thick band of fasciculated radial glia intercalated between the griseum tectale and the posterior commissure. A fasciculated radial glia band also defines the caudal mesencephalic limit (m/r) across the alar and basal zones. These glial specializations, not described previously in the chick, give physical entity to the limits deduced from gene expression studies and suggested by descriptive cytoarchitectonic analysis. We conclude that the midbrain of sauropsides shares a common subdivision pattern co-defined by radial glia arrangements.

It remained very difficult to manipulate gene expression in chick embryos until the advent of in ovo electroporation which enabled the induction of both gain-of-function, and recently loss-of-function, of a gene of interest at a specific developmental stage. Gain-of-function by electroporation is so effective that it has become widely adopted in developmental studies in the chick. Recently, it became possible to induce loss-of-function by introducing an siRNA expression vector by electroporation. In this review, the methods of electroporation for gain-of-function and for loss-of-function by siRNA are discussed.

The traditional view that all parts of the ribs originate from the sclerotome of the thoracic somites has recently been challenged by an alternative view suggesting that only the proximal rib derives from the sclerotome, while the distal rib arises from regions of the dermomyotome. In view of this continuing controversy and to learn more about the cell interactions during rib morphogenesis, this study aimed to reveal the precise contributions made by somitic cells to the ribs and associated tissues of the thoracic cage. A replication-deficient lacZ-encoding retrovirus was utilized to label cell populations within distinct regions of somites 19–26 in stage 13–18 chick embryos. Analysis of the subsequent contributions made by these cells revealed that the thoracic somites are the sole source of cells for the ribs. More precisely, it is the sclerotome compartment of the somites that contributes cells to both the proximal and distal elements of the ribs, confirming the traditional view of the origin of the ribs. Results also indicate that the precursor cells of the ribs and intercostal muscles are intimately associated within the somite, a relationship that may be essential for proper rib morphogenesis. Finally, the data from this study also show that the distal ribs are largely subject to resegmentation, although cell mixing may occur at the most sternal extremities.

Calcium waves were first seen about 25 years ago as the giant, 10 micro m/s wave or tsunami which crosses the cytoplasm of an activating medaka fish egg [J Cell Biol 76 (1978) 448]. By 1991, reports of such waves with approximately 10 micro m/s velocities through diverse, activating eggs and with approximately 30 micro m/s velocities through diverse, fully active systems had been compiled to form a class of what are now called fast calcium waves [Proc Natl Acad Sci USA 88 (1991) 9883; Bioessays 21 (1999) 657]. This compilation is now updated to include organisms from algae and sponges up to blowflies, squid and men and organizational levels from mammalian brains and hearts as well as chick embryos down to muscle, nerve, epithelial, blood and cancer cells and even cell-free extracts. Plots of these data confirm the narrow, 2-3-fold ranges of fast wave speeds through activating eggs and 3-4-fold ones through fully active systems at a given temperature. This also indicate Q(10)'s of 2.7-fold per 10 degrees C for both activating eggs and for fully activated cells.Speeds through some ultraflat preparations which are a few-fold above the conserved range are attributed to stretch propagated calcium entry (SPCE) rather than calcium-induced calcium release (CICR).

To determine the effect of skin allograft on open neural tube defects (ONTDs), the neural tube was incised open using Hamburger and Hamilton stage 18 or 19 chick embryos for a length of six somites. Embryos were divided into two groups: graft and control (with and without skin allograft). On postoperative day 5, closure of ONTDs was more frequent in the graft group than in the control group (9/15 versus 0/15), and healing was nearly complete. However, typical lipomatous features were not observed. These results suggest that simple mechanical attachment of skin allograft on ONTDs does not lead to lipomatous malformation in chick embryos. On the other hand, our results support a potential role of skin allograft in the management of prenatal spinal ONTDs.

The role of apoptosis in cardiac morphogenesis has not been directly tested. Cardiomyocyte apoptosis is prevalent during the remodeling of the embryonic chicken cardiac outflow tract (OFT) in the transition from a single to a dual circulation. We tested the hypothesis that OFT cardiomyocyte apoptosis drives the shortening and rotation of the embryonic cardiac OFT and is required to achieve the mature ventriculo-arterial configuration. Chick embryos were treated with the peptide Caspase inhibitors zVAD-fmk or DEVD-cho at HH stages 15–20 (looped heart). Morphology of control and experimental embryos was assessed at HH stage 35, at which time the control hearts have developed a dual circulation. Infection of the hearts with a recombinant adenovirus expressing green fluorescent protein was used to follow the fate of the OFT cardiomyocytes. Affected embryos displayed abnormal persistence of a long infundibulum (OFT myocardial remnant) beneath the great vessels, indicating failure of OFT shortening. In some instances, the infundibulum connected both great vessels to the right ventricle in a side-by-side arrangement with transposition of the aorta, indicating a failure of rotation of the OFT, and modeling human congenital double outlet right ventricle. Defects were also observed at other sites in the heart where apoptosis is prevalent, such as in the formation of the cardiac valves and trabeculae. To more specifically target the apoptosis of the OFT cardiomyocytes, recombinant adenovirus was used to express the X-linked inhibitor of apoptosis protein in these cells. This resulted in an effect on outflow tract shortening and rotation similar to that of the peptide inhibitors, while the effects on the other cardiac structures were not observed. These results demonstrate that elimination of OFT cardiomyocytes by apoptosis is necessary for the proper formation of the ventriculo-arterial connections, and suggest apoptosis as a potential target of teratogens and genetic defects that are associated with congenital human conal heart defects.

We reviewed our ontogenetic studies of thermoregulatory capacity in avian embryos in relation to metabolic responses to egg cooling.Basic understanding of abrupt egg cooling and quasi-equilibrium state of cooling was discussed together with egg's thermal conductance and heat production.Two cooling tests, which were designed to minimize the rate of heat loss during cooling, were conducted to investigate a feeble compensatory metabolic response to cooling in precocial and altricial embryos, and models for early development of homeothermy in birds were suggested.Instantaneous heart rate (IHR) response to cooling was measured in chick embryos. The baseline of IHR rised and became oscillating. HR oscillation may be a phenomenon related to thermoregulation, which needs to be studied.

The expression of inhibin alpha- and beta(A)-subunits was investigated in gonads of male and female chick embryos during the last week of their 21-day incubation period. Fertilized Hisex brown laying hen eggs were incubated at 37.8 +/- 0.2 degrees and 60% relative humidity in an automatic forced-draft incubator with constant lighting. Embryos were killed after 14, 18, and 21 days of incubation, sexed by macroscopical inspection of the gonadal phenotype, and further dissected to obtain the gonads. Total RNA was isolated using the ultraspec RNA method. The expression of alpha- and beta(A)-subunits was evaluated by competitive RT-PCR. Significant differences were found within and between sexes in the expression of the alpha- and beta(A)-subunits. The level of the alpha-subunit in the testis was about 23-fold higher than that in the ovary at all ages. Testicular content of inhibin alpha mRNA levels was similar at days 14 and 18 but declined significantly at day 21 of incubation, whereas no significant differences were observed between the three age groups in the ovary. Testicular and ovarian inhibin beta(A)-subunit increased significantly from day 14 to day 18 followed by a significant decline before hatch. However, inhibin beta(A) level at day 14 was significantly higher in the ovary than in the testis. At days 18 and 21, there were no differences in the levels of the inhibin beta(A) in the sexes. The expression of inhibin beta(A)-subunit in the ovary was significantly higher than that of the alpha-subunit at all ages. In the testis, however, the expression of the beta(A)-subunit was higher at days 18 and 21 than at day 14. The sex difference in gonadal inhibin subunits expression suggests differential roles of inhibin/activin in the development of the chicken gonads. The changing level of expression during incubation also suggests changing biological roles within sexes.

We previously identified and cloned a neurite outgrowth promoting protein, Neurocrescin (NC), from the extract of the chick denervated leg muscles. In this study, we explored the active region of NC for neurite outgrowth. Using the deletion mutants of NC, we tested their neurite outgrowth activity in the cultured telencephalic neurons of E5 chick embryos. We found three regions which independently had significant neurite outgrowth activity comparable with that of the extract of the chick denervated leg muscles. These regions were not homologous to any well-known active sites such as the laminin active region, IKVAV. In parallel, searching the endogenous deletion mutants of NC in the rat brain, we cloned a mutant in which the region including the larger part of one of the three active regions was deleted. The neurite outgrowth activity of the mutant was significantly lower than that of normal NC. These results suggest the physiological significance of these active regions.

According to the optimization principle known as Murray's law, the blood vessel geometry at a bifurcation satisfies the relation alpha = (D3(1) + D3(2))/D3(0) = 1, where D0, D1, and D2 are the diameters of the parent and two daughter vessels, respectively. Previous investigations have shown that mature blood vessels adhere to this law fairly closely. The purpose of this study was to test Murray's law in the developing extraembryonic blood vessels of 2-4 day-old chick embryos. Vessel diameters were measured manually using image analysis software. The measurements for the group of all vessels at all studied stages (n = 449) gave alpha = 1.01+/-0.34 (mean +/- SD), and the value of alpha is similar at all stages. These results indicate that Murray's law holds in the chick embryo, even before medial smooth muscle becomes functional, suggesting that blood vessels follow the same basic morphogenetic rules throughout life.

To investigate the re-closure capacity of surgically induced spinal open neural tube defects (ONTD) and its correlation to the cell proliferation activity in embryonic stages, longitudinal incisions of 3- or 6-somite lengths were made in neural tubes of chick embryos at Hamburger and Hamilton stage 18-19, and the length of the ONTDs was measured on postoperative days 2, 3, 5, 7, and 10. The results demonstrated that re-closure occurred more actively when the incision length was shorter. The chronological changes in the re-closure capacity of the ONTDs paralleled the proliferative activity of the neural tube. We conclude that surgically induced spinal ONTDs may be remodeled in early chick embryos and that manipulation of cell dynamics can enhance the re-closure process in the embryonic spinal cord tissue.

We previously studied the response of mammalian cultured cells to weak, 60 Hz-electromagnetic (EM) fields. Two time constants, similar to those observed in chemotaxis, were found to govern the cellular response to the field. We concluded that a system of temporal sensing, similar to that employed in chemotaxis by motile bacteria, was operative. We termed the shorter time (approximately 0.1 s) the "sensing" time, and the longer time (approximately 10 s) the "memory" time. To investigate the possibility that temporal sensing was a general property of EM field-cell interaction, the temporal properties of another EM field-induced effect was studied. The EM field-induced protection against the effects of extreme hypoxia was examined in chick embryos. Embryos were exposed to 60 Hz-magnetic fields, the amplitudes of which were regularly altered throughout the 20-min exposure. Alteration was accomplished either by turning the field off and on at regular intervals (1-50 s), or by introducing brief (10 or 100 ms), zero amplitude gaps, once each second, throughout exposure. When the field was turned on and off at 0.1 s intervals, the protective effect conferred by a constant field was lost. At progressively longer on/off intervals, protection was progressively restored, maximizing at intervals of 10-30 s. Gapping the magnetic field for 10 ms, each second of exposure conferred the same protection as that observed for an uninterrupted field, but gapping the field at 100 ms each second produced a significant reduction in protection. These data exhibit remarkable consistency with those obtained in similar temporal studies of the magnetic field-induced enhancement of ornithine decarboxylase activity in L929 fibroblasts. It appears that temporal sensing is a general feature of the EM field-cell interaction.

We have recently reported that weak electromagnetic (EM) field exposure of chick embryos induces a response that can be used to protect against subsequent hypoxic insult. This work is continued here with an exposure response study using 20-min exposure to 60 Hz magnetic fields over a range of 2-10 microT. Once again, the biomarker used was induction of hypoxia protection. A sigmoidal response curve was found, with exposures to magnetic field strengthsor = 4 microT inducing maximum hypoxia protection (68% survival). We also attempted to determine whether the magnetic or induced electric component of the EM field was responsible for the observed protection. This was accomplished by making measurements with two different orientations of the magnetic fields (perpendicular and parallel to the major axis of the egg). Owing to the configuration of the embryo in the egg, the induced electric field at the embryo was lower when the magnetic field was parallel to the major axis even though the magnetic field strength was the same for each orientation. Exposure of the embryos to the parallel orientation resulted in a reduced protective response. An exposure-response curve generated for this orientation of the field also showed a more "drawn-out" appearance, consistent with the observed distribution of embryo positions within the egg. Our results suggest that the induced electric, not the applied magnetic field, plays a primary role in the protective effect observed in this chick embryo model.

An autoradiographic method for labelling β1- and β2-adrenoceptors using [ 3 H ]CGP 12177 as a radioligand is described as well as the procedure for an autoradiographic saturation kinetic study. The method afforded higher quality autoradiographs as well as an improvement in the tissue preservation when assayed in birds and chick embryos. The results confirmed the Kd values previously reported for membrane homogenate binding. The use of different radioligands to characterise β-adrenoceptors, the higher Bmax values found with autoradiography than those obtained by the membrane homogenate binding method and the typical errors in quantifying autoradiography are discussed. It is concluded that the method described here considerably improves autoradiographic β-adrenergic characterisation. Themes: Neurotransmitters, modulators, transporters and receptors Topics: Catecholamine receptors

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricular myocardium did not produce factors capable of supporting myocardialization. Copyright 1999 Academic Press.

Monitoring the migrations of cells during embryonic development requires a system in which cells can be identified in situ during locomotion. One promising system involves the generation of chimeras by transplanting mouse cells into chick embryos in ovo to exploit the wealth of mouse genetic variants. The success of this technique relies on the ability to detect individual mouse cells in a chick environment with high specificity. The murine B2 family of short interspersed elements is present in the mouse genome at copy numbers in excess of 10(5), whereas this sequence is absent in the chick genome based on hybridization techniques. This differential of five orders of magnitude produces signals in mouse cells that are easily identified, even in an environment that is predominantly chick. Thus, the B2 repeat probe is highly effective for the purpose of identifying mouse cells in mouse-chick chimeras.

In a study on the embryotoxicity of pineal indoles on developing chick embryos in vivo, the pineal indoles—namely, melatonin (MEL), methoxytryptamine (MTA) and methoxytryptophol (MTP)—were injected into the yolk sacs of the chick embryos through the air chambers of the eggs on the 4th day of incubation. The eggs were opened and the embryos examined after 6, 10 or 14/15 days of incubation. Abnormalities were found to occur mainly in the 6- and 10-day-old embryos, which exhibited external malformations such as twisted vertebral column, abdominal hernia, exteriorization of heart and viscera, defects of eye, beak and limb. From the results obtained from embryos on the 14th or 15th day of incubation, MEL was found to be the most toxic indole in regard to the mortality induced, whereas MTA had the highest teratogenicity because of the frequent incidence of abnormal embryos. Effect of MTP treatment on the development of chick embryos varied greatly between doses, and there were no abnormal embryos found on the 14th or l5th day of incubation.

In this study we examined the Golgi apparatus of avian notochord transplants excised from 2-day-old (E2) chick embryos and grafted isochronically into a chick host either in a medial-ventral position, next to the host notochord, or in a superficial position under the ectoderm laterally or dorsally to the neural tube. The operated embryos were examined from E2 to E8. The diameters, the cytoplasmic vacuolization and the immunostained Golgi apparatus were identical between the endogenous and ventrally grafted notochords, as well as between host-and superficially transplanted notochords when observed at E2. In contrast, from E4 to E8, the size of the notochords grafted dorsally or laterally to the neural tube was significantly smaller than the host, while the cytoplasmic vacuolization and the degree of fragmentation of the Golgi apparatus were significantly less than in the host notochords. These results show that environmental and position-specific factors influence the developmental program and the secretory activity of the notochordal cells.

Depriving developing neurons of retrograde trophic support may disrupt their development and often causes them to die. We here report the effects, in chick embryos, of eliminating retrograde support in the isthmo-optic projection by blocking axoplasmic transport in the terminal parts of the axons, which is known ultimately to kill the isthmo-optic neurons. Within only 9 h, this had perturbed the process of cellular reorganisation that eventually leads to the laminated appearance of the mature isthmo-optic nucleus. Neuron survival in the isthmo-optic nucleus was affected even more quickly, but the earliest change, occurring in as little as 3 h, was not an increase in the number of dying neurons, but a decrease below control values. This novel effect was still present at 6 and 9 h after the injection, but at longer survival times the number of dying neurons increased well above control values as expected. Our interpretation of the transient decrease in neuronal death is that retrograde trophic signals include both death-promoting and life-promoting components, and that the former act faster in this system. © 1997 Elsevier Science B.V. All rights reserved.

Members of theHoxgene family appear to regulate anterior–posterior (A–P) regionalization in embryos. Genetic manipulation of numerousHoxgenes in the developing trunk region of vertebrates results in changes in the morphology of individual vertebrae. We have used virally mediated, targeted misexpression to ectopically express the Hoxc-6 protein in chick embryos.Hoxc-6has an anterior border of expression at the cervical–thoracic transition in tetrapods. Misexpression of this gene in the cervical mesoderm of chick embryos results in dramatic truncations of the ventral rami of cervical spinal nerves in the infected region. These data point to a role forHoxc-6in axon guidance, and suggest that in addition to regulating proliferative rates,Hoxgene expression provides positional information utilized in producing domain-specific extracellular signals.

The preventive effect of SA3443 [(4R)-hexahydro-7,7-dimethyl-6-oxo-1,2,5-dithiazocine-4-carboxylic acid] against glucocorticoid-induced cataract of developing chick embryos was studied. When hydrocortisone succinate sodium (HC: 0.25 mumol per egg) was administered to 15-day-old embryos, almost all lenses became opaque (stage I:O%, II: 2.5 +/- 4.6%, III: 5 +/- 5.4%, IV-V 92.5 +/- 7.1%) at 48 hr after the treatment. However, a double application of SA3443 (10 mumol per egg) at 3 and 10 hr after HC treatment effectively prevented the cataract formation (stage I: 52.8 +/- 13.7%, II: 11.6 +/- 6.3%, III: 22.9 +/- 8.9%, IV-V: 13.9 +/- 11.0%) and diminished the decline in glutathione in the lens at 48 hr and in the liver at 24 hr after HC administration. The cleavage of the cyclic disulfide bond of SA3443 did not occur in the lens homogenate but in the liver homogenate. These results suggest that the appearance of sulfhydryl residue in the liver may contribute to the anticataract effects by representing radical scavenger activities.

Although the use of mammalian embryos must remain the ideal for most teratological studies, non-mammalian embryos offer certain advantages. Their independence from the mother makes it possible to study the effects of reagents directly on the embryo without having to take possible placental effects into consideration, and it enables manipulations to be carried out more easily. In the early embryonic stages the cells of Xenopus and the chick are larger than those of the mouse, and these non-mammalian embryos tend to be bigger than those of the mouse at comparable stages of development. Attention is focused on the chick embryo as a useful experimental animal in the study of teratological effects. Consideration is given to some of the critical stages in embryogenesis as well as to the culture techniques available.

There will be ongoing refinement in the implementation of 3-D methods, and some day the 2-D methods might indeed need to be scrapped. Until the ‘practical’ accuracy of the disector method is shown to apply not only after retrofitting existing protocols with new restrictions 7xAnalysis of cell death in the trochlear nucleus of chick embryos: Calibration of the optical disector counting technique reveals systematic bias. Hatton, W.J and von Bartheld, C.S. J. Comp. Neurol. 1999; 409: 169–186Crossref | PubMed | Scopus (120)See all References, 8xVerification of the disector method for counting neurons, with comments on the empirical method. Pover, C.M and Coggeshall, R.E. Anat. Rec. 1991; 231: 573–578Crossref | PubMedSee all References, 9xReliability and validity of the physical disector method for estimating neuron number. Popken, G.J and Farel, P.B. J. Neurobiol. 1996; 31: 166–174Crossref | PubMed | Scopus (33)See all References, we should hold on to methods which have not yet outlived their usefulness in quantitative neuromorphology. The debate about 2-D versus 3-D counting will probably continue for decades to come. We should use both types of methods with prudence and common sense, avoid blatant mistakes such as the ‘reference trap’ 2xHoward, C.V and Reed, M.G. : 246See all References, 19xMethods for determining numbers of cells and synapses: a case for more uniform standards of review. Coggeshall, R.E and Lekan, H.A. J. Comp. Neurol. 1996; 364: 6–15Crossref | PubMed | Scopus (838)See all References, and, when using 2-D methods, we should choose an appropriate section thickness relative to particle heights to minimize biases 12xHow inaccurate is the Abercrombie correction factor for cell counts?. Clarke, P.G.H. Trends Neurosci. 1992; 15: 211–212Abstract | Full Text PDF | PubMed | Scopus (64)See all References, 13xAn unbiased correction factor for cell counts in histological sections. Clarke, P.G.H. J. Neurosci. Methods. 1993; 49: 133–140Crossref | PubMed | Scopus (35)See all References, and clearly distinguish between counts of profiles and estimates of cell numbers. We should calibrate when absolute numbers are needed or if there is even a remote chance that the conclusions of the study might be altered by hidden biases – regardless of the type of method. Scenarios must be avoided in which 3-D studies with potentially huge biases receive the stamp of approval as ‘unbiased’, while 2-D studies with minimal biases are rejected as flawed. Finally, we should appreciate that there are choices of methods and use them to the advantage of our studies, and let's stop squabbling over minimal biases when larger sources of error are not even known or understood yet.

RBC velocity was determined in the major blood vessels of the extraembryonic circulation of early chick embryos between Day 4 and Day 6 of development using fluorescent-labeled erythrocytes. Measurements were performed by applying a digital frame-by-frame video technique. The expenditure of operator interaction was minimized by computer support. Velocity measurements of more than 15,000 labeled blood cells were evaluated for mean RBC velocity and volume flow of 354 venous blood vessel segments. Linear regression for the power function of the calculated volume flow vs the vessel diameter yielded an exponent of 2.77 at Day 4, increasing to 2.96 by Day 6. Applying Murray's model of energetic cost, these data indicate that in the course of development the newly formed extraembryonic vascular system is optimized in terms of minimizing cardiac work. The total extraembryonic blood flow as calculated from the sum of the volume flows of the main veins was 656 ± 218 and 1169 ± 409 nl/sec at Day 4 and Day 6, respectively. Using previously determined values of blood oxygen concentration, embryonic oxygen uptakes of 9.6 nl/sec (Day 4) and 40.2 nl/sec (Day 6) were calculated.

This article examines the effects of experimental prenatal intestinal obstruction on the growth and blood composition of chick embryos. Intestinal atresia (IA) was produced by bipolar bowel electrocoagulation in fertile eggs on the 14th day of incubation. The chicks killed on the 19th day were measured, weighed, and blood-sampled. Twenty-three control, 10 sham-operated, and 11 IA chicks were studied. Animals with IA were severely undernourished by weight (43.4 +/- 4.7 v 70.3 +/- 7.6% of egg weight, P.001) and length (15.3 +/- 1.1 v 18.1 +/- 0.9 mm tibial length, P.001) in comparison with sham-operated ones. Their hematocrit was slightly lower, and total protein increased. Prealbumin was absent in their sera and albumin, alpha and beta globulins were significantly decreased, whereas gamma-globulin was greatly increased. Sodium, potassium chloride, urea, and glucose remained within normal limits. The lack of placenta in the avian embryo precludes any supply of nutrients by this route and the ingestion of amniotic fluid, which is protein-rich after the 13th day of incubation, when the opening of the seroamniotic connection allows albumen to be mixed with it, becomes the main source of nutrients until hatching. Obstruction of the main incoming avenue by IA induces severe malnutrition in this model which relies on this route to a greater extent than the human fetus. In spite of the obvious biological differences between the avian embryo and the human fetus, the present evidence supports the hypothesis that prenatal interruption of the amniotic fluid transit contributes to fetal undergrowth in IA.