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The effects of maternal n-6 and n-3 fatty acid (FA) supplementation on hatched chick tissue FA profile, antioxidant status, and ex vivo eicosanoid production by the cardiac tissue were investigated. Eggs with low, medium, and high levels of n-3 FA were obtained by feeding Cobb breeder hens were fed a corn-soybean meal-based diet containing 3.5% sunflower oil (low n-3), 1.75% sunflower oil plus 1.75% fish oil (medium n-3), or 3.5% fish oil (high n-3). Total n-3 FA in the yolk ranged from 1.8, 10.3, and 13.3% for low, medium, and high n-3 eggs, respectively (P < 0.001). Total long-chain (>20 C) n-6 FA in the egg yolk were 7.4, 2.1, and 1.3 for low n-3, medium n-3, and high n-3 eggs, respectively (P < 0.001). No differences were observed in total fat content of the eggs, which was 33.3, 31.6, and 31.9% for low n-3, medium n-3, and high n-3 eggs, respectively (P > 0.05). Hatchability for the low, medium, and high n-3 eggs was 89, 85, and 83%, respectively (P > 0.05). The total lipid content of chick liver, heart, brain, and lungs can be placed in the following descending order: liver > brain > heart > lung and was not affected by egg FA (P > 0.05). Total n-3 FA were higher in the tissues of medium and high n-3 chicks than in the tissue of low n-3 chicks (P < 0.05). There was no effect of egg FA on docosahexaenoic acid (22:6n-3) in the heart of low, medium, and high n-3 chicks (P > 0.05). There were no differences in total glutathione, glutathione peroxidase, glutathione reductase, or superoxide dismutase activities in the tissues of chicks from low n-3, medium n-3, and high n-3 eggs (P > 0.05). The medium n-3 and high n-3 chicks had lower catalase activity in the heart than did the low n-3 chicks (P = 0.013). The TBA reactive substances were significantly lower in the liver of high n-3 chicks than in that of low and medium n-3 chicks (P < 0.05). Heart tissue prostaglandin E(2) concentration was higher in low n-3 chicks than in those hatched from medium or high n-3 eggs (P < 0.05). Heart tissue thromboxane A(3) was lowest in low n-3 chicks (P < 0.05). There was no effect of yolk FA on ex vivo prostaglandin E(3) or thromboxane A(2) production in cardiac tissue (P > 0.05). These results indicate that modulating egg yolk n-3 FA enhances tissue n-3 FA and reduces proinflammatory cardiac eicosanoid production without affecting hatchability.

Biological bases for sexual differences in the brain exist in a wide range of vertebrate species, including chickens. Recently, the dogma of hormonal dependence for the sexual differentiation of the brain has been challenged. We examined whether sexually dimorphic gene expression in the brain precedes gonadal differentiation. Using the Affymetrix GeneChip Chicken Genome Array, we identified 42 female- and 167 male-enhanced genes that were differentially expressed in sex-specific brains from stage 29 chicken embryos. To confirm the efficacy of the microarray, and to investigate the stage-specific expression patterns of the identified genes, we used quantitative real-time PCR analysis. Our real-time PCR results for the differentially expressed genes agreed well with our microarray results. Thus, we postulate that these genes have potential roles in the sexual differentiation of neural function and development in chickens.

Retinoic acid (RA), an active metabolite of vitamin A, is a diffusible molecule that regulates the expression of several families of genes, playing a key role in specification processes during chordate development. With the aim of defining its possible role in the developing chick inner ear, we obtained in this work a detailed spatiotemporal distribution of the enzymes involved in its synthesis, the retinaldehyde dehydrogenases (RALH1-4). Our results showed that, in contrast to the mouse inner ear, Raldh3 expression was the only Raldh gene detected in the developing chick inner ear, where it appears as early as stage 18. During inner ear morphogenesis, Raldh3 expression was predominantly observed in the endolymphatic system. The Raldh3 expression pattern delimited totally or partially the Bmp4-positive presumptive territories of vestibular sensory epithelia by stage 24 and the basilar papilla at stage 34, suggesting a possible involvement of RA in their specification. In addition, several vestibular sensory areas showed some Raldh3-expressing cells close to the Raldh3-positive domain. These results suggest that the RA signaling pathway may play a role in the initial patterning of the otic epithelium and cell differentiation therein, providing local positional information. Having in mind this Raldh3 expression pattern, we discuss the regulatory interactions among the RA, bone morphogenetic protein, and fibroblast growth factor signaling pathways in the specification of otic sensory elements. Our investigation may underpin further experimental studies aimed at understanding the possible role of signaling pathways in patterning of the developing chick inner ear.

The ontogeny and distribution of gastrin- and serotonin-immunoreactive (IR) cell in the proventriculus of chicks (Gallus gallus domestica, n = 60) in different growth periods was examined immunohistochemically using antisera specific to gastrin and serotonin. Gastrin and serotonin-IR cells were detected in chick proventriculus. Gastrin-IR cells were first evident after 12 days of incubation in lamina epithelialis and compound glands, while serotonin- IR cells were observed only in compound glands at that same time. Gastrin-IR and serotonin-IR cells increased in frequency on incubation day 14 and 16, respectively. Towards the end of incubation, gastrin- and serotonin-IR cell numbers decreased. In adult chicken, both IR cells were present but not lower numbers. The observations demonstrate the presence of gastrin- and serotonin-IR cells in the proventriculus of developing chicks in temporally changing frequencies.

Fetal alcohol syndrome (FAS), a condition occurring in some children of mothers who have consumed alcohol during pregnancy, is characterized by physical deformities and learning and memory deficits. The chick hippocampus, whose functions are controlled by interneurons expressing calcium-binding proteins parvalbumin (PV) and calretinin (CR), is involved in learning and memory mechanisms. Effects on growth and development and hippocampal morphology were studied in chick embryos exposed to 5% and 10% ethanol volume/volume (vol/vol) for 2 or 8 days of gestation. There was a significant dose-dependent reduction (P<.05) in body weight and mean number per section of PV and CR expressing hippocampal neurons in ethanol-exposed chicks, without alterations in neuronal nuclear size or hippocampal volume, compared appropriate controls. Moreover, when chicks exposed to 5% ethanol for 2 and 8 days of gestation were compared, no significant differences were found in body parameters or neuronal counts. Similarly, exposure to 10% ethanol did not induce any significant changes in chicks exposed for 2 or 8 gestational days. Thus, these results suggest that gestational ethanol exposure induces a reduction in the mean number per section of PV and CR expressing hippocampal neurons, and could be a possible mechanism responsible for learning and memory disorders in FAS.

Developmental changes in the levels of the excitatory amino acids L-glutamate (Glu) and L-Aspartate (Asp) and inhibitory amino acids glycine (Gly) and gamma-amino butyric acid (GABA), as well as taurine and its related amino acids L-methionine (Met), L-cysteine (Cys) and L-serine (Ser) in the brain and pectoralis muscle at various embryonic stages and hatch in broiler and layer type chickens were determined. Brain concentrations of Asp, GABA and taurine were higher than those in the muscle, but the difference in the two types was small. The concentrations of the precursors of taurine including Met, Cys and Ser were lower than that of taurine. In conclusion, the synthesis of some amino acids and their metabolites such as Asp, GABA and taurine in the chick embryo is very high in order to support brain development.

Background: Modified Vaccinia virus Ankara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines., Principal Findings: We have developed a genetic complementation system that enables the deletion of essential viral genes from the MVA genome, thereby allowing us to generate MVA vaccine vectors that are antigenically less complex. Using this system, we deleted the essential uracil-DNA-glycosylase (udg) gene from MVA and propagated this otherwise replication-defective variant on a complementing cell line that constitutively expresses the poxvirus udg gene and that was derived from a newly identified continuous cell line that is permissive for growth of wild type MVA. The resulting virus, MVADeltaudg, does not replicate its DNA genome or express late viral gene products during infection of non-complementing cells in culture. As proof-of-concept for immunological 'focusing', we demonstrate that immunization of mice with MVADeltaudg elicits CD8+ T cell responses that are directed against a restricted repertoire of vector antigens, as compared to immunization with parental MVA. Immunization of rhesus macaques with MVADeltaudg-gag, a udg(-) recombinant virus that expresses an HIV subtype-B consensus gag transgene, elicited significantly higher frequencies of Gag-specific CD8 and CD4 T cells following both primary (2-4-fold) and booster (2-fold) immunizations as compared to the udg(+) control virus MVA-gag, as determined by intracellular cytokine assay. In contrast, levels of HIV Gag-specific antibodies were elicited similarly in macaques following immunization with MVADeltaudg-gag and MVA-gag. Furthermore, both udg(-) and udg(+) MVA vectors induced comparatively similar titers of MVA-specific neutralizing antibody responses following immunization of mice (over a 4-log range: 10(4)-10(8) PFU) and rhesus macaques. These results suggest that the generation of MVA-specific neutralizing antibody responses are largely driven by input MVA antigens, rather than those that are synthesized de novo during infection, and that the processes governing the generation of antiviral antibody responses are more readily saturated by viral antigen than are those that elicit CD8+ T cell responses., Significance: Our identification of a spontaneously-immortalized (but not transformed) chicken embryo fibroblast cell line (DF-1) that is fully permissive for MVA growth and that can be engineered to stably express MVA genes provides the basis for a genetic system for MVA. DF-1 cells (and derivatives thereof) constitute viable alternatives, for the manufacture of MVA-based vaccines, to primary CEFs -- the conventional cell substrate for MVA vaccines that is not amenable to genetic complementation strategies due to these cells' finite lifespan in culture. The establishment of a genetic system for MVA, as illustrated here to allow udg deletion, enables the generation of novel replication-defective MVA mutants and expands the repertoire of genetic viral variants that can now be explored as improved vaccine vectors.

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

Background: Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos., Methods: Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine., Principal Findings: Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF(165) to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype., Conclusions/significance: Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

The epibranchial placodes are specialized areas of surface ectoderm that make a vital contribution to the peripheral nervous system, producing sensory neurons of the cranial ganglia. They have long been characterized as a series of patches of thickened ectoderm in the vicinity of each pharyngeal cleft. We have previously demonstrated that Sox3 is not only expressed in these structures but also marks a larger, earlier domain. Here we demonstrate that neurons are produced from the Sox3-positive ectoderm that lies outside of the classically-defined epibranchial placodes. Our data show that these regions contribute neurons to the cranial ganglia, but then cease producing neurons as they lose Sox3 expression. We further demonstrate that the ectoderm in these regions is responsive to extracellular or intracellular stimuli that initiate aspects of neuronal differentiation. This response to neurogenic stimuli is lacking in regions of ectoderm distant from the normal sites of neurogenesis and the response to constitutively active Bmp receptor in particular, disappears coincident with loss of Sox3 expression. Finally, we show that a dominant repressor form of Sox3 blocks the ability of the ectoderm to undergo neurogenesis. Thus, Sox3 appears to be essential for the neurogenic capacity of surface ectoderm exhibited by the epibranchial placodes.

Myostatin is a potent growth and differentiation factor involved in skeletal muscle tissue formation in vertebrates. However, recent studies in chicken embryo suggested that the myostatin was expressed even before the establishment of myogenic lineage. No studies have thus far been reported in birds to define the role of myostatin during the embryonic organogenesis. The present experiment was designed for studying the expression profiles of myostatin mRNA in the chicken liver, heart, brain, and intestine during their morphogenesis, using real-time PCR. The myostatin mRNA expression was significantly upregulated in liver during E15-E18. Similar results were observed during the development of chicken heart. In brain, the expression of myostatin was upregulated from E4 onwards. In intestine, the expression of myostatin was significantly increased many folds on E9-E18. Therefore, the increase in myostatin expression might be related to the growth of liver and heart on days E12-E18; morphogenesis and growth of brain during E15-E18; and morphogenesis and differentiation of intestine during E9-E18. In the present study, the tissue-specific expression of myostatin gene in chicken is similar to fishes, but different from that in mammals. Further, the inspection of chicken genome also suggested that there is no differentiation of GDF-8 and -11. A recent finding suggests that the chicken myostatin gene is closely related to mammals than fishes. Therefore, we propose that the chicken myostatin gene might have diverged in its function between teleosts and mammals. Indeed it is possible that its function might have only become fully differentiated to serve as a control of muscle mass in mammals.

Embryonic cerebrospinal fluid (E-CSF) is involved in the regulation of survival, proliferation and neurogenesis of neuroectodermal progenitor cells, as well as in the control of mesencephalic gene expression in collaboration with the isthmic organizer. Recently, we showed the presence of retinol-binding protein (RBP) within the E-CSF proteome. RBP is an all-trans retinol carrier, a molecule that can be metabolized into retinoic acid, a morphogen involved in central nervous system (CNS) morphogenesis and patterning. Here we demonstrate the presence of all-trans retinol within the E-CSF and analyse the dynamics of RBP and all-trans retinol within this fluid, as well as the expression of retinoic acid-synthesizing enzymes during early CNS development. Our results suggest a relationship between the dynamics of these molecules and the early events of CNS patterning.

Here, we examine the expression and regulation of the gene BAMBI, a kinase-deficient decoy receptor capable of interacting with type I bone morphogenetic protein (BMP) receptors in avian embryos. Initially, expression was limited to the endoderm during neurula and pharyngula stages. From embryonic day 3.5 (stage 20) and onward, BAMBI expression almost perfectly overlapped with known expression patterns for BMP4, particularly in the face and limbs. We performed bead implant experiments in the face to see which signals could be repressing or promoting expression of BAMBI. Our data point to retinoids and BMPs as being major positive regulators of BAMBI expression; however, fibroblast growth factor 2 acts to repress BAMBI. Furthermore, retinoic acid is likely to act directly on BAMBI as induction occurs in the presence of cycloheximide. The data suggested that BAMBI could be used to regulate Bmp signaling during tissue interactions that are an integral part of facial morphogenesis.

Endocannabinoids are retrograde neurotransmitters, which act upon the presynaptically located, G-protein coupled receptor CB1, to modulate synaptic transmission in the adult brain. Recently, however, a number of lines of evidence have suggested that endocannabinoid signalling may play an important role in early neuronal development. In this study, we show that the CB1 receptor has a wide expression pattern in the developing nervous system and that its expression follows neuronal differentiation in the embryo from the earliest stages. We also show that the enzymes involved in 2-AG synthesis are expressed in an overlapping manner at these stages. We further show that interfering with CB1 function using a pharmacological inhibitor causes problems in axon pathfinding and fasciculation. Similarly, CB1 gene knock down in the zebrafish by morpholino injection results in defects in axonal growth and fasciculation in these embryos. Thus CB1 function is required in the early embryo for axonal growth and fasciculation.

Neural induction is widely believed to be a direct consequence of inhibition of BMP pathways. Because of conflicting results and interpretations, we have re-examined this issue in Xenopus and chick embryos using the powerful and general TGFbeta inhibitor, Smad7, which inhibits both Smad1- (BMP) and Smad2- (Nodal/Activin) mediated pathways. We confirm that Smad7 efficiently inhibits phosphorylation of Smad1 and Smad2. Surprisingly, however, over-expression of Smad7 in Xenopus ventral epidermis induces expression of the dorsal mesodermal markers Chordin and Brachyury. Neural markers are induced, but in a non-cell-autonomous manner and only when Chordin and Brachyury are also induced. Simultaneous inhibition of Smad1 and Smad2 by different approaches does not account for all Smad7 effects, indicating that Smad7 has activities other than inhibition of the TGFbeta pathway. We provide evidence that these effects are independent of Wnt, FGF, Hedgehog and retinoid signalling. We also show that these effects are due to elements outside of the MH2 domain of Smad7. Together, these results indicate that BMP inhibition is not sufficient for neural induction even when Nodal/Activin is also blocked, and that Smad7 activity is considerably more complex than had previously been assumed. We suggest that experiments relying on Smad7 as an inhibitor of TGFbeta-pathways should be interpreted with considerable caution.

Formation of the epicardium requires interactions between alpha(4)beta(1) integrin, and the extracellular matrix. We investigated the role of other integrins expressed by epicardial cells. We detected transcripts for alpha(5), alpha(8), alpha(v), beta(1), beta(3), and beta(5) integrins in the chick proepicardial organ (PE). We demonstrate that alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3) integrins are expressed by chick epicardial mesothelial cells (EMCs). Migration of EMCs in vitro was reduced by RGD-containing peptides. Using adenoviruses expressing an antisense to chick alpha(4) (AdGFPalpha4AS), full-length (Adhalpha4V5), and C-terminal deleted alpha(4) (Adhalpha4DeltaCV5), we found that EMCs were less able to adhere to vitronectin and fibronectin(120) indicating that alpha(4)beta(1) plays a role in regulating EMC adhesion to ligands of alpha(5)beta(1), alpha(8)beta(1), and alpha(v)beta(3). In Adhalpha4DeltaCV5-infected EMCs, alpha(5)beta(1) was diminished in fibrillar adhesions and new FN matrix assembly was abnormal. We propose that cooperation between alpha(4)beta(1) and RGD integrins is important for EMC adhesion and subepicardial matrix formation., ((c) 2008 Wiley-Liss, Inc.)

The extent to which hematocrit (Hct) is regulated and the impact of altered Hct on blood oxygen transport in avian embryos are largely unknown. Consequently, we investigated how acute blood removal or Ringer solution injection modified Hct in day 15 embryos, and how ;blood doping' with erythrocyte-enriched whole blood influenced O(2) consumption in day 15-17 chicken embryos. Mean Hct (+/-s.e.m.) at day 15, 16 and 17 was 26.7+/-0.6%, 28.0+/-0.4% and 30.7+/-0.5%, respectively. Blood withdrawal (19 increments of 125 microl each, separated by 30 min) caused a progressive fall in Hct to approximately 12% at day 15. Hct decline was strictly proportional to the extent of blood withdrawal. Incremental Ringer solution injection over an 8 h period, transiently increasing blood volume up to 85% over initial values, did not decrease Hct, indicating that injected Ringer solution rapidly left the circulating blood compartment. Blood doping with erythrocyte-enriched whole blood artificially elevated Hct from 27% to 38%, but caused no significant change in routine O(2) consumption (0.35-0.39 ml O(2) min(-1) egg(-1)) at any point over the subsequent 6 h period in day 15-17 embryos. We conclude that Hct is not protected acutely in day 15 chicken embryos, with no evidence of erythrocyte sequestration or release. Additionally, at day 15-17, Hct increases of approximately 10% do not enhance embryonic oxygen consumption, suggesting that blood oxygen carrying capacity per se is not limiting to oxygen consumption.

Slit is a secreted protein known to function through the Roundabout (Robo) receptor as a repellent for axon guidance and neuronal migration, and as an inhibitor in leukocyte chemotaxis. We have previously shown that Slit2 is also secreted by a variety of human cancer cells whereby it acts as a chemoattractant to vascular endothelial cells for tumor angiogenesis. We used a blocking antibody to investigate the role of Slit-Robo signaling in tumor angiogenesis during oral carcinogenesis. In this report we undertook a multistage model of 7,12-dimethyl-1,2-benzanthracene-induced squamous cell carcinoma in the hamster buccal pouch. R5, a monoclonal antibody against the first immunoglobulin domain of Robo1, was used to study whether R5 blocks the Slit-Robo interaction and furthermore inhibits tumor angiogenesis and growth in our model. In addition, the expression of Slit2, von Willebrand factor, and vascular endothelial growth factor were examined using human tissue of oral cheek mucosa with oral squamous cell carcinoma. Our data showed that Slit2 was expressed minimally in normal and hyperplastic mucosa, moderately in dysplastic mucosa, and highly in neoplastic mucosa obtained from hamster buccal pouch. We also found that increased Slit2 expression was associated with higher tumor angiogenesis, as reflected by increased vascular endothelial growth factor expression and microvessel density. A similar Slit2 expression profile was found in human tissue. Importantly, interruption of the Slit2-Robo interaction using R5 inhibited tumor angiogenesis and growth in our in vivo model, which indicates that Slit2-mediated tumor angiogenesis is a critical process underlying the carcinogenesis of chemical-induced squamous cell carcinoma. Therefore, targeting Slit-Robo signaling may offer a novel antiangiogenesis approach for oral cancer therapy.

The production of heat (or thermogenesis) and its response to cold improve very quickly around birth in both mammals and birds. The mechanisms for such rapid perinatal development are not fully understood. Previous experiments with hyperoxia suggested that, during the last phases of incubation, eggshell and membranes might pose a limit to oxygen availability. Hence, it was hypothesized that an improvement in oxygenation by opening the eggshell may contribute to the establishment of thermogenesis. Thermogenesis and its response to cold were measured by indirect calorimetry, in warm (38 degrees C) conditions and during 1-h exposure to 30 degrees C. Both improved throughout the various phases of the hatching process. During the latest incubation phases (internal pipping, IP, and star fracture of external pipping, EP), the removal of the eggshell in the region above the air cell raised metabolic rate both in warm and cold conditions (in IP) or the thermogenic response to cold (in EP). Adding hyperoxia after opening the eggshell caused no further increase in the thermogenic response. In cold-incubated embryos thermogenesis during the EP phase was much less than normal; in these embryos, increasing the oxygen availability did not improve thermogenesis. We conclude that oxygenation contributes to the maturation of the thermogenic mechanisms in the perinatal period as long as these mechanisms have initiated their normal developmental process.

The chicken embryo has been used as a classical embryological model for studying developmental events because of its ready availability, similarity to the human embryos, and amenability to embryological and surgical manipulations. With the arrival of the molecular era, however, avian embryos presented distinct experimental limitations, largely because of the difficulty of performing targeted mutagenesis or transgenic studies. However, in the last decade and a half, a number of new methods for transient transgenesis have been developed that allow efficient alteration of gene function during early embryonic development. These techniques have made it possible to study the effects of gene inactivation or overexpression on downstream transcriptional regulation as well as on embryonic derivatives. This, together with sequencing of the chicken genome, has allowed the chicken embryo to enter the genomic era. While attempts to establish germ line transgenesis are ongoing, methods for rapid, transient spatiotemporally targeted gene alterations have thus again re-established the chick embryo as an important experimental niche by making it possible to apply genetics in concert with classical embryological techniques. This provides a unique tool to explore the role of developmentally important genes (Ishii and Mikawa, 2005; Itasaki et al., 1999; Krull, 2004; Ogura, 2002; Swartz et al., 2001). Transient transfection methods have allowed for efficient mis- and overexpression of transgenes. For long-term analyses, retrovirally mediated gene transfer has particular advantage. For short-term experiments, electroporation and adenoviral-mediated gene transfer methods provide transient expression, largely because of the short persistence time of the transgene within the cell. More recently, Tol2 transposon-mediated constructs have been employed, allowing for integration into the genome and prolonged expression of the transgene (Sato et al., 2007), see Chapter 14 by Takahashi et al., this volume). These methods today are routinely used for gain-of-function analysis, to overexpress or ectopically express genes of interest (Arber et al., 1999; Barembaum and Bronner-Fraser, 2007; Bel-Vialar et al., 2002). Loss-of-function experiments are also possible using electroporation of dominant-negative constructs that act as competitive inhibitors (Bel-Vialar et al., 2002; Renzi et al., 2000; Suzuki-Hirano et al., 2005), morpholino antisense oligos (Basch et al., 2006; Kos et al., 2001; Sheng et al., 2003) that block translation or splicing, or constructs expressing small interfering or small hairpin RNAs (siRNAs or shRNAs) (Chesnutt and Niswander, 2004; Das et al., 2006; Katahira and Nakamura, 2003). Electroporation as the most popular method of the transient transfection into the chick embryos. Electroporation of chicken embryos involves application of an electric field to the exposed tissue that transiently disrupts the stability of the cell plasma membrane, creating reversible pores through which nucleic acids or their analogues can be readily transported into the cytosol. The use of this method for transfection into the vertebrate embryos has been facilitated by adapting the voltage parameters and the type and the duration of the electric pulse. By applying several successive square pulses at a very low voltage, with long rest periods in between, one can successfully deliver a DNA construct or another small charged particle into the cytoplasm, with minimal cell death, high efficiency of the uptake and good embryonic survival rate. The size limit of the DNA molecule that can be transfected in such a way is not yet known, though it is more likely that the size limitation in this procedure (if any) lies within the practical problems of cloning large fragments into the plasmid. We routinely overexpress constructs containing 3-4 kb inserts and coharboring a GFP or RFP reporter whose translation is initiated from an internal ribosomal entry site (IRES), thus allowing easy detection of the electroporated cells.

Formation of the primitive streak is one of the key events in the early development of amniote embryos. The streak is the site where during gastrulation the mesendoderm cells ingress to take up their correct topographical positions in the embryo. The process of streak formation can be conveniently observed in the chick embryo, where the streak forms as an accumulation of cells in the epiblast in the posterior pole of the embryo and extends subsequently in anterior direction until it covers 80% of the epiblast. A prerequisite for streak formation is the differentiation of mesoderm, which is induced in the epiblast at the interface between the posterior Area Opaca and Area Pellucida in a sickle shaped domain overlying Koller's sickle. Current views on the molecular mechanisms of mesoderm induction by inducing signals from the Area Opaca and inhibitory signals from the hypoblast are briefly discussed. During streak formation the sickle of mesoderm cells transforms into an elongated structure in the central midline of the embryo. We discuss possible cellular mechanisms underlying this process, such as oriented cell division, cell-cell intercalation, chemotactic cell movement in response to attractive and repulsive signals and a combination of chemotaxis and contact following. We review current experimental evidence in favor and against these different hypotheses and outline some the outstanding questions. Since many of the interactions between cells signaling and moving are dynamic and nonlinear in nature they will require detailed modeling and computer simulations to be understood in detail.

During gastrulation in vertebrates, mesenchymal cells at the anterior end of the presomitic mesoderm (PSM) periodically compact, transiently epithelialize and detach from the posterior PSM to form somites. In the prevailing clock-and-wavefront model of somitogenesis, periodic gene expression, particularly of Notch and Wnt, interacts with an FGF8-based thresholding mechanism to determine cell fates. However, this model does not explain how cell determination and subsequent differentiation translates into somite morphology. In this paper, we use computer simulations of chick somitogenesis to show that experimentally-observed temporal and spatial patterns of adhesive N-CAM and N-cadherin and repulsive EphA4-ephrinB2 pairs suffice to reproduce the complex dynamic morphological changes of somitogenesis in wild-type and N-cadherin (-/-) chick, including intersomitic separation, boundary-shape evolution and sorting of misdifferentiated cells across compartment boundaries. Since different models of determination yield the same, experimentally-observed, distribution of adhesion and repulsion molecules, the patterning is independent of the details of this mechanism.

During somite maturation, the ventral half of the epithelial somite disintegrates into the mesenchymal sclerotome, whereas the dorsal half forms a transitory epithelial sheet, the dermomyotome, lying in between the sclerotome and the surface ectoderm. The dermomyotome is the source of the majority of the mesodermal tissues in the body, giving rise to cell types as different as muscle, connective tissue, endothelium and cartilage. Thus, the dermomyotome is the most important turntable of mesodermal cell fate choice in the vertebrate embryo. Sclerotome development is characterized by a cranio-caudal polarization, resegmentation and axial identity. Its formation is controlled by signals from the notochord, the neural tube, the lateral plate mesoderm and the myotome. These signals and cross-talk between somite cells lead to the separation of various subdomains, like the central, ventral, dorsal and lateral sclerotome. Here, we discuss the current knowledge on the formation of the dermomyotome and the mechanisms leading to the development of the various dermomyotomal derivatives, with special emphasis on the development of musculature and dermis. We further discuss the molecular control of sclerotomal subdomain formation and cell type specification.

Previous investigations have shown that an ex vivo model on the basis of the incubated hen's egg represents a potential alternative to animal experimentation for xenobiotic metabolism studies. This model is characterized by administration of the xenobiotic into the yolk sac and identification of metabolites in the allantoic fluid. In the present study, gentisic acid (2,5-dihydroxybenzoic acid, 2,5-DHBA, CAS 490-79-9) was used as a test substance. The oxidative fate of this salicylate metabolite in human and animal species could not be completely elucidated until now. Inoculation of 2,5-DHBA into the incubated hen's egg in concentrations up to 8 mg/egg did not affect embryo viability. For the metabolism studies a dose of 2 mg/egg was chosen and 2,5-DHBA was identified as the main metabolite in both the free and conjugated form (glucuronidel/sulfate). Two minor metabolites were detected in addition. The first of these could be identified as 2,3,5-trihydroxybenzoic acid (2,3,5-THBA) and the second one as 2-hydroxy-5-methoxybenzoic acid (2-H-5-MBA). 2,3,5-THBA has been characterized as a metabolite of 2,5-DHBA in a vertebrate species for the first time. Since 2,3,5-THBA can be methylated to the guaiacol derivative, 2,5-dihydroxy-3-methoxybenzoic acid (2,5-DH-3-MBA) can not to be excluded as a further possible metabolite. The metabolites were identified by comparative TLC with the authentic reference substances as well as additionally by GC and GC-MS analysis of the trimethylsilyl derivatives. The structures of the synthesized reference substances were confirmed by MS, 1H and 13C NMR spectral data.

Vertebrates have a metameric bodyplan that is based on the presence of paired somites. Somites develop from the segmental plate in a cranio-caudal sequence. At the same time, new material is added from Hensen's node, the primitive streak and the tailbud. In this way, the material residing in the segmental plate remains constant and comprises 12 prospective somites on each side. Prospective segment borders are not yet determined in the caudal segmental plate. Prior to segmentation, the cranial segmental plate undergoes epithelialization, which is controlled by signals from the neural tube and ectoderm. The bHLH transcription factor Paraxis is critically involved in this process. Formation of a new somite from the cranial end of the segmental plate is a highly controlled process involving complex cell movements in relation to each other. Hox genes specify regional identity of the somites and their derivatives. In the chicken a transposition of thoracic into cervical vertebrae has occurred as compared to the mouse. Transcription factors of the bHLH and homeodomain type also specify the cranio-caudal polarity and that of particular cell groups within the somites. According to segmentation models, somitogenesis is under the control of a "segmentation clock" in combination with a morphogen gradient. This hypothesis has recently found support from molecular data, especially the cycling expression of genes such as cHairy1 and Lunatic Fringe, which depend on the Notch/Delta pathway of signal transduction. FGF8 has been described to be distributed along a cranio-caudal gradient. The first oscillating gene described shown to be independent of Notch is Axin2, encoding a negative regulator of the canonical Wnt pathway and a target of Wnt3a. Wnt3a and Axin2 show a similar distribution as FGF8 with high levels in the tailbud. The chick embryo has recently become accessible to molecular approaches such as overexpression by electroporation and RNA interference which can be expected to help elucidating some of the still open questions concerning somitogenesis.

The effect of genetic strain (Ross 308; Cobb 500) and parent flock age [young (29 wk), peak (Ross = 34 wk; Cobb = 36 wk), postpeak (40 wk), mature (45 wk), old (55 wk), and very old (59 wk)] on eggshell conductance and embryonic metabolism were examined. At each flock age, eggs from each strain were incubated for 21.5 d in individual metabolic chambers to measure embryonic O(2) intake and CO(2) output. From these data, the respiratory quotient (RQ) and metabolic heat production were calculated. Data were analyzed by the GLM procedure of SAS at P < or = 0.05. Neither strain nor flock age influenced conductance. Total embryonic O(2) consumption, CO(2) output, RQ, and metabolic heat production over the entire incubation period were not affected by strain. Daily differences existed between strains for embryonic O(2) intake (1, 7, 16, 17, 19, 20 d of incubation), CO(2) output (1 to 4, 16 to 20 d of incubation), and heat production (4, 7, 16 to 19 d of incubation). Embryos from young, mature, old, and very old flocks produced significantly more total embryonic heat over the entire 21 d (1,712, 1,677, 1,808, and 1,832, respectively) than embryos from peak (1,601) and postpeak (1,693) flocks. Average RQ for the entire incubation period was higher in embryos from mature flocks compared with all other flock ages. Daily differences among embryos from different flock ages were shown for O(2) consumption (all but d 8 of incubation), CO(2) production (all but d 7 and 9 of incubation), and heat output. The results showed that genetic strain and parent flock age influence daily embryonic metabolism, especially during the early and latter days of incubation. These daily differences coincide with the days of incubation having a higher incidence of embryonic mortality; these 2 factors may be related. Further investigation into the relationship between embryonic metabolic heat production and mortality during incubation may lead to the development of specific incubation conditions for different genetic strains and flock ages.

Prolactin (PRL) is a pituitary hormone with multiple homeostatic roles among vertebrates. Although it has mainly been studied in relation to its role during the initiation and maintenance of incubation behavior in avian species, it has also been shown to act on the immune system. In this study, levels of PRL receptor (PRLR) mRNA were quantified by real-time PCR, and tissue expression was localized by in situ hybridization in primary and secondary lymphoid organs. Prolactin receptor was shown to be expressed in the bursa follicles, thymus lobules, and splenic pulp at all stages of development examined. Levels of PRLR expression were consistently higher in the bursa of Fabricius when compared with other lymphoid organs, suggesting that PRL acts primarily on bursal development. Furthermore, levels of PRLR mRNA appeared to fluctuate during embryogenesis, with a significant increase observed at embryonic day 19 in the bursa, at 7 d of age in the thymus, and on hatching day in the spleen. Thus, PRL might play an important role during the development of the immune system in chickens.

Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.

Embryo development and heat production (HP) were studied in eggs of similar size (60 to 65 g) that were incubated at normal (37.8 degrees C) or high (38.9 degrees C) eggshell temperature (EST) and exposed to low (17%), normal (21%), or high (25%) O(2) concentration from d 9 through 19. High EST initially increased HP, but gradually O(2) became more important for HP than EST. Finally,HP was highest for the combination of high EST with high O(2) and lowest for the combination of high EST with low O(2). High EST decreased hatch time, BW, yolk free BW, and relative heart weight. The EST had no effect on residual yolk weight, chick length, or relative liver weight. Increased O(2) increased yolk free BW and chick length and decreased residual yolk weight at hatch. No interactions between EST and O(2) were observed with regard to embryo development and hatchling characteristics. If embryo development is reflected by HP, it can be concluded that high EST primarily increased embryonic development until the second week of incubation. During the third week of incubation, O(2) had a greater effect in determining embryo development than EST.

Mammalian models are frequently used for preclinical evaluation of new drug delivery systems (DDS). However, valid mammalian models are expensive, time-consuming, and not easy to set up and evaluate. Furthermore, they are often linked to administrative burden with respect to ethical and legal aspects. The present review outlines the possibilities and limitations of using the hen's embryo, and specifically its chorioallantoic membrane (CAM), as an alternative to mammalian models for the evaluation of DDS. Features of the CAM, the anatomy of the embryo, and the blood were investigated to assess properties of the drug carriers such as toxicity and biocompatibility, as well as the activity, toxicity, biodistribution and pharmacokinetics of the drug. The simplicity, rapidity, and low cost of the different assays that can be performed with chick embryos strengthen the interest of routinely using this model in pharmaceutical technology research. It is concluded that there is a big potential for using chick embryos in screening procedures of formulation candidates, thus establishing an intermediate step between in vitro cellular tests and preclinical mammalian models.

In differentiating red blood cells (RBCs) of the chick embryo, the synthesis of carbonic anhydrase (CAII) and pyrimidine 5'-nucleotidase (P5N-I) is triggered by the hypoxic mediators norepinephrine and adenosine via receptor-mediated cAMP formation. The process is accompanied by the induction of IFR1 and TOB which are putative regulators of transcription or translation in different cell types. The present investigation studied the erythroid TOB and IFR1 expression: mRNA and protein are up-regulated in post-mitotic RBCs from D11-19 treated with cAMP-elevating agonists. In contrast, immature RBCs of early embryos (D5-7) fail to synthesize a significant amount of IFR1/TOB. In D11 RBCs, TOB and IFR1 are cytosolic proteins with different half-lives (TOB<4h, IFR1>12h). Cytosolic fractionation characterized TOB as a free soluble protein while the abundant IFR1 (c(max) approximately 3microM) is completely associated with the ribosomal fraction. A putative function of both proteins as translational regulators is discussed.

In order to study the mechanism of DHEA (Dehydroepiandrosterone) in reducing fat in broiler chickens during embryonic development, fertilized eggs were administrated with DHEA before incubation and its effect on lipid metabolism and expression of hepatic lipogenetic genes was investigated. The mRNA levels of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), apolipoprotein B100 (apoB100) and sterol regulator element binding protein-1c (SREBP-1c) were determined using real time quantitative PCR. Samples of livers were collected from the chickens on days 9, 14, and 19 of embryonic development as well as at hatching. Blood samples were extracted on days 14, 19 of incubation and at hatching. The results showed that DHEA decreased the concentration of triacyglycerol in the blood and the content in liver, and the mRNA levels of ACC, FAS, ME, SREBP-1c and apoB. This suggested that DHEA decreased the expression of hepatic lipogenetic genes and suppressed triglycerols transport, by which it reduced the deposition of fat in adipose tissue in broiler chickens during embryonic development and hatching.

Cytokine gene expression in the chicken spleen during embryogenesis and the early posthatch period was investigated in the present study. The constitutive expression of interleukin-4, interleukin-10, interleukin-18, and interferon-gamma genes was detectable as early as embryonic day 12. Expression of cytokine genes was higher in the spleen of posthatch chickens compared with chick embryos. There was a gradual increase in expression of all the cytokine genes in the spleen, which peaked by d 7 posthatch. This expression pattern coincided with the completion of T-cell colonization and structural development of the spleen during the early posthatch period. It is therefore possible that the cytokines examined in the present study are involved in the maturation of colonized T cells and in shaping the spleen microenvironment.

With sequencing of the chicken genome largely completed, significant effort is focusing on gene annotation, including acquiring information about the patterns of gene expression. The chicken embryo is ideally suited to provide detailed temporal and spatial expression information through in situ hybridization gene expression analysis in vivo. We have developed the Gallus expression in situ hybridization analysis (GEISHA) database and user interface (http://geisha.arizona.edu) to serve as a centralized repository of in situ hybridization photos and metadata from chicken embryos. This report describes the design and implementation the GEISHA database and Web site and illustrates its usefulness for researchers in the biomedical and poultry science communities. Results from a recent comprehensive expression analysis of microRNA expression in chicken embryos are also presented.

Changes in circulating levels of prolactin (PRL) and tissue content of PRL receptor (PRLR) messenger RNA (mRNA) in the liver, pancreas, kidney, and gonads (testis/ovary) were measured in turkey and chicken embryos from embryonic day (ED) 21 or ED15, respectively, to 1 d after hatch by real-time PCR. There were no differences between the sexes in chickens or turkeys. Both species had very similar patterns of PRL release and expression of PRLR mRNA, and no major differences were observed between turkey or chicken embryos. Plasma levels of PRL increased from low levels during the last week of embryonic development and were at significantly higher levels (about 4-fold) by 1 d after hatch. Similarly, in all tissues the content of PRLR mRNA was minimal at the outset and increased to reach maxima about the time of hatch. In both species, the highest levels of transcript were observed in the kidney followed by the gonad, liver, and pancreas. The tissue content of PRLR was correlated (0.6 to 0.8 dependent on the tissue) to circulating levels of PRL, which suggested that PRL may be associated with an increase in its receptor number around the time of hatch. Because levels of PRL and tissue content of PRLR mRNA increased around the time of hatch, this suggests that these tissues may be targets for PRL and may be involved in the physiologic changes occurring in embryos around the time of hatching.

Myogenic differentiation is a multistep process that begins with the commitment of mononucleated precursors that withdraw from cell cycle. These myoblasts elongate while aligning to each other, guided by the recognition between their membranes. This step is followed by cell fusion and the formation of long and striated multinucleated myotubes. We have recently shown that cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) induces myogenic differentiation by enhancing myoblast recognition and fusion. Here, we further studied the signaling pathways responsible for early steps of myogenesis. As it is known that Wnt plays a role in muscle differentiation, we used the chemical MbetaCD to deplete membrane cholesterol and investigate the involvement of the Wnt/beta-catenin pathway during myogenesis. We show that cholesterol depletion promoted a significant increase in expression of beta-catenin, its nuclear translocation and activation of the Wnt pathway. Moreover, we show that the activation of the Wnt pathway after cholesterol depletion can be inhibited by the soluble protein Frzb-1. Our data suggest that membrane cholesterol is involved in Wnt/beta-catenin signaling in the early steps of myogenic differentiation.

Adequate energy status in lymphocytes is vital for their development. The ability of developing chicken lymphocytes to acquire and metabolize energy substrates was determined during embryonic days (e) and neonatal days (d) of life when primary-energy substrate metabolism is altered at the whole-animal level. In 3 experiments, bursacytes and thymocytes were isolated on e17, e20, d1, d3, d7, or d14 to analyze markers associated with glucose, glutamine, and lipid metabolism. Bursacyte glucose transporter-3 (Glut-3) mRNA abundance increased from d1 to d14 and hexokinase-1 (HK-1) mRNA abundance was maximum on e20 (P<0.05). Thymocyte Glut-1, Glut-3, and HK-1 mRNA abundance increased from e17 to d14 (P<0.05). HK enzyme activity increased from e20 to d3 in bursacytes and d3 to d7 in thymocytes (P<0.05). Glucose uptake by bursacytes and thymocytes was greater on d14 compared to d1 and d7 (P<0.05). Bursacyte and thymocyte sodium coupled neutral amino acid transporter-2 and glutaminase (GA) mRNA abundance increased from e20 to d7 (P<0.05). GA enzyme activity increased from e20 to d7 in bursacytes (P<0.05) and did not change in thymocytes. Carnitine palmitoyl transferase enzyme activity did not change over time in either cell type. These studies suggest that developing B and T lymphocytes adapt their metabolism during the first 2 wk after hatch. Developing lymphocytes increase glucose metabolism with no change in fatty acid metabolism and bursacytes, but not thymocytes, increase glutamine metabolism. Understanding the factors that regulate lymphocyte development in neonatal chicks may help promote their adaptive immune responses to pathogens in early life.

Purpose: To elucidate the early cellular events that take place during induction of retina regeneration in the embryonic chick, focusing on the relationship between fibroblast growth factor (FGF) signaling and the regulation of Pax6 and Mitf., Methods: The retina of embryonic day 4 (E4) chicks was removed and a heparin coated bead soaked in fibroblast growth factor 2 (FGF2) was placed into the optic cup. The pharmacological inhibitor PD173074 was used to inhibit FGF receptors, PD98059 was used to inhibit MAP kinase-kinase/extracellular signal-regulated kinase (MEK/Erk) signaling. Retroviral constructs for paired box 6 (Pax6), MEK, and microphthalmia (Mitf) were also used in overexpression studies. Immunohistochemistry was used to examine pErk, Pax6, Mitf, and melanosomal matrix protein 115 (MMP115) immunoreactivity and bromodeoxyuridine (BrdU) incorporation at different time points after removing the retina., Results: The embryonic chick has the ability to regenerate a new retina by the process of transdifferentiation of the retinal pigment epithelium (RPE). We observed that during the induction of transdifferentiation, downregulation of Mitf was not sufficient to induce transdifferentiation at E4 and that FGF2 was required to drive Pax6 protein expression and cell proliferation, both of which are necessary for transdifferentiation. Furthermore, we show that FGF2 works through the FGFR/MEK/Erk signaling cascade to increase Pax6 expression and proliferation. Ectopic Mitf expression was able to inhibit transdifferentiation by acting downstream of FGFR/MEK/Erk signaling, likely by inhibiting the increase in Pax6 protein in the RPE., Conclusions: FGF2 stimulates Pax6 expression during induction of transdifferentiation of the RPE through FGFR/MEK/Erk signaling cascade. This Pax6 expression is accompanied by an increase in BrdU incorporation. In addition, we show that Mitf is spontaneously downregulated after removal of the retina even in the absence of FGF2. This Mitf downregulation is not accompanied by Pax6 upregulation, demonstrating that FGF2 stimulated Pax6 upregulation is required for transdifferentiation of the RPE. Furthermore, we show that ectopic Mitf expression is able to protect the RPE from FGF2 induced transdifferentiation by inhibiting Pax6 upregulation.

The study of development has been greatly aided by the use of the chick embryo as an experimental model. The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation. These experiments have yielded important data on the development of the central and peripheral nervous systems. For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth. The embryo can be examined at successive developmental stages by re-opening the eggshell. While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times. In this method, the egg rests on its side and a small window is cut in the shell. After the experimental procedure, the shell is used to cover the egg for the duration of its development. Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period. This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

Left-right asymmetry of internal organs is widely distributed in the animal kingdom. The chick and mouse embryos have served as important model organisms to analyze the mechanisms underlying the establishment of the left-right axis. In the chick embryo many genes have been found to be asymmetrically expressed in and around the node, while the same genes in the mouse show symmetric expression patterns. In the mouse there is strong evidence for an establishment of left-right asymmetry through nodal cilia. In contrast, in the chick and in many other organisms left-right asymmetry is probably generated by an early-acting event involving membrane depolarization. In both birds and mammals a conserved Nodal-Lefty-Pitx2 module exists that controls many aspects of asymmetric morphogenesis. This review also gives examples of divergent mechanisms of establishing asymmetric organ formation. Thus there is ample evidence for conserved and non-conserved strategies to generate asymmetry in birds and mammals., (Copyright 2007 S. Karger AG, Basel.)

SCO-spondin is a multidomain glycoprotein secreted by the subcommissural organ (SCO). It belongs to the thrombospondin type 1 repeat superfamily and has been identified in several vertebrate species. We report the cloning of the chick SCO-spondin ortholog and examine its temporal and spatial expression during early embryogenesis from Hamburger and Hamilton (HH) stage 12 to HH stage 21. Chick SCO-spondin cDNA contains a long open reading frame encoding a predicted protein of 5255 amino acids. Northern blot analysis has revealed SCO-spondin mRNA as a band of about 15 kb. Many conserved domains have been identified, including 27 thrombospondin type 1 repeats, 13 low-density lipoprotein receptor type A domains, one EMI domain (a cysteine-rich domain of extracellular proteins), three von Willebrand factor type D domains, and one cystine knot C-terminal domain. Whole-mount in situ hybridization enabled the first signal of mRNA expression to be detected at HH stage 17, exclusively in a thin area of the prosencephalon roof plate. During the following stages of development, SCO-spondin expression remained restricted to this region. The multidomain structure of SCO-spondin and its early expression suggest that it plays a role in developmental processes in the central nervous system.

Perlecan is a major heparan sulfate proteoglycan that binds growth factors and interacts with various extracellular matrix proteins and cell surface molecules. The expression and spatiotemporal distribution of perlecan was studied by RT-PCR, immunoprecipitation and immunofluorescence in the chick embryo from stages X (morula) to HH17 (29 somites). Combined RT-PCR and immunohistochemistry demonstrated the expression of perlecan as early as stage X and its presence may be fundamental to the first basement membrane assembly on the epiblast ventral surface at stage XIII (blastula). Perlecan fluorescence was intense in the cells ingressing through the primitive streak and was strong lining the epiblast ventral surface lateral to the streak at stage HH3-4 (gastrula). At stage HH5-6 (neurula), perlecan fluorescence was low in the neuroepithelium and stronger in the apical surface of the neural plate. At stage HH10-11 (12 somites), perlecan fluorescence was intense in the neuroepithelium and was then essentially nondetectable in the neuroepithelium, and the intensity had shifted to the basement membranes of encephalic vesicles by stage HH17. Perlecan immunofluorescence was intense in neural crest cells, strong in pharyngeal arches, intense in thymus and lung rudiments, intense in aortic arches and in dorsal aorta, strong in lens and retina and intense in intraretinal space and in optic stalk, strong in the dorsal mesocardium, myocardium and endocardium, strong in dermomyotome, low in sclerotome in somites, intense in mesonephric duct and tubule rudiments, intense in the lining of the gut luminal surface. Inhibition of the function of perlecan by blocking antibodies showed that perlecan is crucial for maintaining basement membrane integrity which mediates the epithelialization, adhesive separation and maintenance of neuroepithelium in brain, somite epithelialization, and tissue architecture during morphogenesis of the heart tube, dorsal aorta and gut. An intriguing possibility is that perlecan, as a signaling molecule that modulates the activity of growth factors and cytokines, participates in the signaling pathways that guide gastrulation movements and neural crest cell migration, proliferation and survival, cardiac cell proliferation and paraxial mesoderm (somitic) cell proliferation and segmentation., ((c) 2007 S. Karger AG, Basel.)

MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function., ((c) 2006 Wiley-Liss, Inc.)

Four-jointed is a type II transmembrane protein that is thought to be cleaved to give rise to a secreted protein. In Drosophila, four-jointed controls outgrowth, vein patterning, and bristle polarity in the developing limb together with the polarity of the ommatidia in the developing eye. In Drosophila and mice, Fj is regulated by notch signaling. Here, we have determined the expression of the chick four-jointed (fjx) homologue during embryonic development. We show that fjx is expressed in the limb bud; facial primordia; the proliferating zone of the lens, feather buds, the neural tube; and neural crest derivatives such as the dorsal root ganglia. Analysis of the fjx expression in the developing limb bud showed that initially fjx is expressed throughout the limb bud, but as the limb develops, highest levels of fjx transcripts are found distally. However, by stage 27, fjx expression is predominantly found in the central core of the limb bud. Finally, fjx expression becomes confined to the developing tendons, ligaments, articular cartilage, and arteries but not the veins. Comparison with scleraxis (scx), a marker of tendons and ligaments, revealed that they are coexpressed in the majority of tendons but that fjx is expressed after scx, when the tendons have begun to differentiate. These data suggest that fjx has two roles during limb development: the first controlling outgrowth and the second tissue differentiation., ((c) 2006 Wiley-Liss, Inc.)

Drosophila teashirt (tsh) is involved in the patterning of the trunk identity together with the Hox genes. In addition, it is also a player in the Wingless and the Hedgehog pathways. In birds and mammals, three Tshz genes are identified and the expression patterns for mouse Tshz1 and Tshz2 have been reported during embryogenesis. Recently, we showed that all three mouse Tshz genes can rescue the Drosophila tsh loss-of-function phenotype, indicating that the function of the teashirt genes has been conserved during evolution. Here we describe the expression pattern of chick TSHZ3 during embryogenesis. Chick TSHZ3 is expressed in several tissues including mesodermal derivatives, the central and peripheral nervous systems. Emphasis is laid on the dynamic expression occurring in regions of the somites and limbs where tendons develop. We show that TSHZ3 is activated in the somites by FGF8, a known inducer of the tendon marker SCX.

Odd-skipped genes encode zinc-finger transcription factors with widespread roles in embryonic development. In Drosophila, odd-skipped acts as a pair-rule gene, while its orthologous gene in Caenorhabditis elegans is involved in gut development. In mammals two paralogs exist, Osr1 and Osr2, with functions described in heart and urogenital, and in secondary palate development, respectively. As the chicken embryo is a widely used system for analysing gene function in vivo, we determined the expression pattern of the two chicken orthologues, cOsr1 and cOsr2, during embryonic development. We demonstrate expression of both genes in a variety of organs and structures, such as kidney, eye, branchial arches and dermis. Both genes show a highly dynamic expression pattern with partially overlapping, but mostly distinct domains of expression. Special emphasis in this study was laid on the investigation of cOsr1 and cOsr2 in limb development, where we compared their expression pattern with the expression of Osr1 and Osr2 in the mouse.

Three classes of signaling molecule, VG1, WNT and BMP, play crucial roles in axis formation in the chick embryo. Although VG1 and WNT signals have a pivotal function in inducing the primitive streak and Hensen's node in the embryo midline, their action is complemented by that of BMP antagonists that protect the prospective axial tissue from the inhibitory influence of BMPs secreted from the periphery. We have previously reported that a secreted factor, chick Tsukushi (TSK), is expressed in the primitive streak and Hensen's node, where it works as a BMP antagonist. Here, we describe a new crucial function for TSK in promoting formation of the primitive streak and Hensen's node by positively regulating VG1 activity. We provide evidence that TSK directly binds VG1 in vitro, and that TSK and VG1 functionally interact in axis formation, as shown by biological assays performed in chick and Xenopus embryos. Furthermore, we show that alternative splicing of TSK RNA leads to the formation of two isoforms (TSKA, originally designated as TSK, and TSKB) that differ in their C-terminal region. Biochemical and biological assays indicate that TSKB is a much weaker BMP antagonist than TSKA, although both isoforms efficiently interact with VG1. Remarkably, although both TSKA and TSKB are expressed throughout the early extending primitive streak, their expression patterns diverge during gastrulation. TSKA expression concentrates in Hensen's node, a well-known source of anti-BMP signals, whereas TSKB accumulates in the middle primitive streak (MPS), a region known to work as a node-inducing center where VG1 expression is also specifically localized. Loss-of-function experiments demonstrate that TSKB, but not TSKA, function is required in the MPS for induction of Hensen's node. Taken together, these results indicate that TSK isoforms play a crucial role in chick axis formation by locally modulating VG1 and BMP activities during gastrulation.

Most post-crossing commissural axons turn into longitudinal paths to make synaptic connections with their targets. Mechanisms that control their rostrocaudal turning polarity are still poorly understood. We used the hindbrain as a model system to investigate the rostral turning of a laterally located commissural tract, identified as the caudal group of contralateral cerebellar-projecting second-order vestibular neurons (cC-VC). We found that the caudal hindbrain possessed a graded non-permissive/repulsive activity for growing cC-VC axons. This non-permissiveness/repulsion was in part mediated by glycosyl-phosphatidylinositol (GPI)-anchored ephrin A. We further demonstrated that ephrin A2 was distributed in a caudal-high/rostral-low gradient in the caudolateral hindbrain and cC-VC axons expressed EphA receptors. Finally, perturbing ephrin A/EphA signalling both in vitro and in vivo led to rostrocaudal pathfinding errors of post-crossing cC-VC axons. These results suggest that ephrin A/EphA interactions play a key role in regulating the polarity of post-crossing cC-VC axons as they turn into the longitudinal axis.

Modification of the chicken germline has been difficult, because it has been challenging to fractionate sufficient numbers of primordial germ cells for manipulation and implantation into developing embryos. A technique to enrich cell suspensions for primordial germ cells, using fluorescence-activated cell sorting (FACS), has recently been developed. The objective of the current study was to demonstrate that the FACS-enriched early embryonic gonocytes could fully participate in development of the germline. Therefore, cells were disassociated from stage 27 gonads, incubated with mouse anti-stage-specific embryonic antigen-1, which was detected with goat-antimouse IgM-fluorescein isothiocyanate, and the fluorescently labeled cells were sorted from the unlabeled cells using FACS. The isolated gonocyte population was injected into the blastoderm of unincubated stage X embryos, the germinal crescent of 3-d embryos, and into the circulation of stage 17 embryos that were pretreated with busulfan. Barred Plymouth Rock gonocytes were implanted exclusively into recipient White Leghorn embryos, and White Leghorn gonocytes were implanted exclusively into Barred Plymouth Rock recipient embryos. Embryos were cultured until hatch, and male putative chimeras were reared to sexual maturity. Germline chimerism was evaluated by observing feather color of the progeny. All injection methods resulted in germline chimeras demonstrating that FACS-sorted gonocytes can fully participate in development. Moreover, it was demonstrated that gonocytes isolated from stage 27 embryonic gonads can be introduced into embryos at an earlier stage of development, and the introduced gonocytes can fully participate in germline development.