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The orphan receptor TIR8, also known as SIGIRR (Single Immunoglobulin IL-1R-Related molecule), belongs to the IL-1R/TLR (TIR) superfamily and plays an important role in the inflammatory responses. The signaling pathways of the receptors belonging to the TIR family are tightly regulated by both extracellular and intracellular mechanisms. TIR8 does not activate the transcription factors NFkB (nuclear factor kB) and IRF3 (interferon regulatory factor 3), although it negatively modulates the inflammatory responses. It acts as an antagonist for the IL-1 receptor family and triggers a negative pathway of the Toll-like/IL-1 receptor system, crucial for dampening inflammation stimuli in the gastrointestinal (GI) tract and in other organs (e.g. lung and kidney). The recent findings of TLRs expression in ovary and embryos of different species (mammals and chickens) are very important for an understanding of reproductive physiology and transovarian pathogen transmission. TIR8 was well characterized in mouse, humans and in other mammalian species, but it is still poorly characterized in the chicken. When TIR8 expression was measured in selected organs of chicken embryos of both broiler and layer types at different time points a unique pattern of expression was observed. Interestingly, TIR8 was detected during the first stages of chicken development (day 1 of incubation), and reached a remarkable level of expression by day 10. We observed this receptor to be ubiquitously expressed in the kidney, GI tract, Bursa of Fabricius, with the highest expression levels in liver and kidney. This pattern was comparable to those observed in post-hatching chickens and in mammals examined to date. No expression differences were observed between the two different chicken breeds (layer- and broiler-type) in the first incubation period (8 days). Whereas in some organs starting from day 10, higher TIR8 expression was observed in broiler-type compared to layer-type. These are the first findings concerning TIR8 expression in developmental stages and therefore they are of comparative value.

In situ hybridization (ISH) in embryos allows the visualization of specific RNAs as a readout of gene expression during normal development or after experimental manipulations. ISH using short DNA probes containing locked nucleic acid nucleotides (LNAs) holds the additional advantage of allowing the detection of specific RNA splice variants or of closely related family members that differ in only short regions, creating new diagnostic and detection opportunities. Here we describe methods for using short (14-24 nt) DNA probes containing LNA nucleotides to detect moderately to highly expressed RNAs in whole chick embryos during the first 5 days of embryonic development. The protocol is easily adaptable for use with embryos of other vertebrate species.

The aim of this study was to evaluate the effect of thermal manipulation (TM) during embryogenesis on hatchability, growth performance and thermotolerance acquisition parameters during thermal challenge (TC). Seven-hundred and fifty fertile chicken eggs were divided randomly into three groups (250 eggs each): control group was maintained at 37.8°C and 56% relative humidity (RH), TM1 was subjected to TM at 38.8°C for 6h and 65% RH during embryonic days (ED)10-18 and TM2 was subjected to TM at 38.8°C for 18 h and 65% RH during ED10-18. Hatched chicks from each treatment group were then randomly divided into two sub-treatment groups (Naive and TC). Chicks in TC groups were subjected to TC by adjusting room temperature to 41.0°C for 6h on days 3, 7, and 42 of age while naïve chicks were kept under regular conditions (25 ± 1°C and 50-60% RH). Percentage of hatched eggs was recorded and post-hatch chick performance was evaluated by recording chick body weight (BW). Chick's response to TC was evaluated by determination of body temperature (T(b)), plasma T3 and T4 levels, and muscle mRNA levels of Hsp70. There was a significant increase in muscle mRNA levels of Hsp70 during embryogenesis and during TC in post-hatch chicks. While hatchability was not adversely affected, the body weight in TM2 chicks was significantly higher at the end of the study period (42 days). Results of this study indicated a long-term enhancement of Hsp70 gene expression associated with improved thermotolerance acquisition in treated chicks without adversely affecting performance., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic "killer" fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1., (Copyright © 2013 Elsevier Inc. All rights reserved.)

NADH dehydrogenase (complex I) catalyzes the transfer of electrons from NADH to ubiquinone with pumping protons across the mitochondrial inner membrane and produces reactive oxygen species as a major source in mitochondria. A missense mutation in the mitochondrially encoded NADH dehydrogenase 2 (MT-ND2) gene, which could produce a change in the protein's secondary structure, has been found in the Tibet chicken breed. In this study, breeding eggs of the Tibet chicken breed with the two genotypes were divided into two groups. One group was incubated in normoxia (20.9% oxygen concentration) and the other in simulated hypoxia (14.5% oxygen concentration). On the 16th day of incubation, complex I activity and mitochondrial reactive oxygen species production in the Tibet chicken embryonic liver with different genotypes in each group were measured. Results showed that: (1) hypoxia reduced complex I activity standardized and mitochondrial reactive oxygen species production significantly compared with normoxia and (2) the missense mutation in the MT-ND2 gene was significantly associated with the production of reactive oxygen species in mitochondria while not associated with the standardized or unstandardized activity of complex I., (© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.)

The present study was designed to investigate the ontogeny and tissue distribution of somatostatin and its five receptor subtypes (SSTR1-5) mRNA expression in embryonic chicken (Gallus gallus). Brain, gonads (male), intestine, kidney, liver, muscle, stomach and yolk sac membrane (YSM) of chicken embryos on the embryonic (E) ages of 10, 16 and 21days (right before hatch) were investigated. Bisulfite sequencing PCR (BSP) was performed to determine the methylation status of the promoter region of all the six genes in the liver. Somatostatin (SST) was predominately expressed in intestine, brain and gonads (male) with different ontogenic patterns. The highest expression in intestine was detected at E10. There was ontogenic shift from intestine to brain as development progressed. Expression pattern of SSTRs in brain, intestine and kidney was similar to human embryonic expression. In liver, the ontogenic expression pattern of SST and its receptors was associated to methylation status of the respective promoters. Methylation of site Sp1 determines expression level of SST, SSTR1, SSTR2 and SSTR3 while site a is important in governing the expression of SSTR4 and SSTR5. The results show that ontogenic expression profile of chicken SST and SSTRs is time and tissue specific., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.

Little is known regarding the diversification of relative low-abundance proteins in hen egg white that underlie the process of chicken embryonic development. The purpose of this study was to identify changes that may be involved in the development of chick embryos. Comparative proteomic analysis was conducive to revealing the potential biological function of low-abundance proteins and embryonic development demand. In addition, those proteins that are increased in abundance seem to have advantages to be targeted as new biomarkers for animal disease or animal production. However, the huge disparity in abundance in chicken egg white and poor performance of affinity depletion methods were specific challenges. This study applied combinatorial peptide ligand library for expanding the deep proteomics. Two-dimensional gel electrophoresis (2-DE) was performed with the hen embryo egg white, which was obtained from embryos at d 0 and 7. The protein spots showing significant increases were selected for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry/mass spectrometry (2 mass analyzers for tandem mass spectrometry). Many precursors were identified as strongly increased after 7 d of incubation. They were ovoinhibitor precursor, clusterin precursor, apolipoprotein D precursor, and extracellular fatty acid-binding protein precursor. Moreover, clusterin precursor were observed concentrated in 2 regions: one is the range of molecular weight increase and the other is the range of high pH. In addition, ovalbumin and ovotransferrin were observed in high pH area in a 2-DE map. These findings will give insight into the changes in fertilized egg white proteins during hatching and provide a better understanding of the biochemical changes in the egg development process.

Since chicken myogenesis is tightly controlled by myogenic regulatory factors (MRFs), the external stimuli (e.g. light or temperature) affecting the proliferation and differentiation of the muscle cells have a primary effect on the gene expression of MRFs. The aim of this study was to analyze the expression of some of MRF genes (MyoD1, myogenin and Myf5) in response to the stimulation of chicken embryos with green light (AL group) or increased temperature (38.5°C; AT group) on day 18 of embryo development (18ED) as well as on days 4 (4PHD) and 8 (8PHD) post hatch. To achieve this goal a quantitative reverse transcription polymerase chain reaction was used. The most prominent differences in gene expression were observed before hatching. Relative expression of MyoD1 on 18ED was higher (p<0.05) in AL and control (AC) groups in comparison to the AT group. Myogenin expression on 18ED was lower (p<0.05) in control chickens than in both treated groups. Light stimulation in ovo decreased (p<0.05) the Myf5 expression on 18ED in comparison to the control group. Green-light illumination applied during in ovo development had more pronounced effects on mRNA level of MRFs genes measured during both the pre- and post hatch development. The elevated temperature applied during embryonic development affected only the 18ED time point. This suggests that the effect of green-light illumination on chicken myogenesis was more prolonged than that of elevated temperature., (Copyright © 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Peptide transporters 1 and 2 (PepT1 and PepT2) and peptide/histidine transporter 1 (PHT1) are all members of the proton-coupled oligopeptide transporter family, which are important for the transport of amino acids in peptide form. The PepT1 acts as a low-affinity/high-capacity transporter and PepT2 as a high-affinity/low-capacity transporter for di- and tri-peptides. The PHT1 transports di- and tri-peptides as well as histidine. The objective of this study was to profile PepT1, PepT2, and PHT1 mRNA expression in the proventriculus, duodenum, jejunum, ileum, ceca, large intestine, brain, heart, bursa of Fabricius, lung, kidney, and liver in layer chicks on embryonic d 18 and 20 and d 1, 3, 7, 10, and 14 posthatch. Absolute quantification real-time PCR was used to measure gene expression. Expression of PepT1 was greatest in the duodenum, jejunum, and ileum. Expression of PepT1 increased in the duodenum, jejunum, and ileum from late embryonic stages to posthatch and in the large intestine from late embryonic stages to d 10 posthatch. In the ceca, PepT1 expression increased from embryonic d 20 to d 1 posthatch and then decreased. Expression of PepT2 was greatest in the brain and kidney. Expression of PepT2 increased from d 10 to 14 in the bursa of Fabricius and decreased in the proventriculus, duodenum, jejunum, and liver from late embryonic stages to posthatch. In the small intestine and liver, PepT2 may function to transport di- and tri-peptides during embryogenesis. The PHT1 was expressed in all tissues analyzed. Expression of PHT1 increased in the jejunum, large intestine, brain, and liver posthatch and decreased in the proventriculus from embryonic stages to posthatch. A tissue × age interaction was observed for all genes. The uptake of peptides in the developing chick is regulated by peptide transporters that are expressed in a tissue- and development-specific manner.

The in vivo chicken embryo model (CEM) demonstrated that gallic acid (GA) induced dysvascularization and hypoxia. Inflammatory edema, Zenker's necrosis, hemolysis, and liposis of cervical muscles were the common symptoms. Levels of the gene hif-1α, HIF-1α, TNF-α, IL-6, and NFκB in cervical muscles were all significantly upregulated, while the vascular endothelial growth factor (VEGF) was downregulated in a dose-responsive manner. Consequently, the cervical muscle inflammation and hemolysis could have been stimulated en route to the tissue TNF-α-canonical and the atypical pathways. We hypothesized that GA could deplete the dissolved oxygen (DO) at the expense of semiquinone and quinone formation, favoring the reactive oxygen species (ROS) production to induce RBC disruption and Fe(2+) ion release. To explore this, the in vitro polyphenolics-erythrocyte model (PEM) was established. PEM revealed that the DO was rapidly depleted, leading to the release of a huge amount of Fe (II) ions and hydrogen peroxide (HPO) in a two-phase kinetic pattern. The kinetic coefficients for Fe (II) ion release ranged from 0.347 h(-1) to 0.774 h(-1); and those for Fe (III) ion production were from 6.66 × 10(-3) h(-1) to 8.93 × 10(-3) h(-1). For phase I HPO production, they ranged from 0.236 h(-1) to 0.774 h(-1) and for phase II HPO production from 0.764 h(-1) to 2.560 h(-1) at GA within 6 μM to 14 μM. Thus, evidence obtained from PEM could strongly support the phenomena of CEM. To conclude, GA tends to elicit hypoxia-related inflammation and hemolysis in chicken cervical muscles through its extremely high prooxidant activity.

RNA interference represents one of the potential mechanisms of regulation of gene expression. Selective downregulation of myostatin (MSTN), a member of transforming growth factor-β (TGF-β) superfamily and a negative regulator of myogenesis, has been demonstrated to enhance skeletal muscle growth. In this study, we studied short hairpin RNA (shRNA)-induced myostatin gene silencing in chicken embryonic myoblast cells using seven different shRNA-expressing constructs by reverse transcription-quantitative real time PCR (RT-qPCR). Myostatin-silencing efficiency of all shRNA constructs were first evaluated in human embryonic kidney cell line 293T (HEK293T) cells, where we observed 30-75.6% reduction in myostatin expression, followed by chicken embryo myoblast cells that revealed up to 55% reduction in myostatin expression along with upregulation of MyoD by 4.65-folds. Consistent with the earlier observations, the transfection of cells with plasmids led to significant increase in interferon responsive genes OAS1 and IFN β (2-112-folds), independent of myostatin silencing in both HEK293T and chicken embryonic myoblast cells. Our study suggests that apart from shRNA sequences, cell type-specific factors may play a significant role in determining the knockdown efficiency of shRNAs., (Copyright © 2013 American Institute of Chemical Engineers.)

Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart and which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2 alpha knockout mice showed that phosphorylation by CK2 is also required for proper targeting of muscle FHOD3 to the myofibrils in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in the adult heart. Following myofibril disassembly, such as that in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3. Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3, and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic of the healthy mature heart and that two distinct phosphorylation events are crucial to regulate the activity of this isoform in thin filament assembly and maintenance.

The neurohypophysis is a crucial component of the hypothalamo-pituitary axis, serving as the site of release of hypothalamic neurohormones into a plexus of hypophyseal capillaries. The growth of hypothalamic axons and capillaries to the forming neurohypophysis in embryogenesis is therefore crucial to future adult homeostasis. Using ex vivo analyses in chick and in vivo analyses in mutant and transgenic zebrafish, we show that Fgf10 and Fgf3 secreted from the forming neurohypophysis exert direct guidance effects on hypothalamic neurosecretory axons. Simultaneously, they promote hypophyseal vascularisation, exerting early direct effects on endothelial cells that are subsequently complemented by indirect effects. Together, our studies suggest a model for the integrated neurohemal wiring of the hypothalamo-neurohypophyseal axis.

Meiosis is a process unique to the differentiation of germ cells and exhibits sex-specific in timing. Previous studies showed that retinoic acid (RA) as the vitamin A metabolite is crucial for controlling Stra8 (Stimulated by retinoic acid gene 8) expression in the gonad and to initiate meiosis; however, the mechanism by which retinoid-signaling acts has remained unclear. In the present study, we investigated the role of the enzyme retinaldehyde dehydrogenase 2 (RALDH2) which catalyzes RA synthesizes by initiating meiosis in chicken ovarian germ cells. Meiotic germ cells were first detected at day 15.5 in chicken embryo ovary when the expression of synaptonemal complex protein 3 (Scp3) and disrupted meiotic cDNA 1 homologue (Dmc1) became elevated, while Stra8 expression was specifically up-regulated at day 12.5 before meiosis onset. It was observed from the increase in Raldh2 mRNA expression levels and decreases in Cyp26b1 (the enzyme for RA catabolism) expression levels during meiosis that requirement for RA accumulation is essential to sustain meiosis. This was also revealed by RA stimulation of the cultured ovaries with the initiation of meiosis response, and the knocking down of the Raldh2 expression during meiosis, leading to abolishment of RA-dependent action. Altogether, these studies indicate that RA synthesis by the enzyme RALDH2 and signaling through its receptor is crucial for meiosis initiation in chicken embryonic ovary.

Sources of Zn, Mn, and Cu (IZMC) as sulfates or as amino acid complexes (OZMC) were used to supplement Cobb 500 breeder hen diets. Experimental treatments consisted of diets supplemented with 1) 100, 100, and 10 mg/kg of Zn, Mn, and Cu, respectively, from IZMC (control); 2) 60, 60, and 3 mg/kg of Zn, Mn, and Cu, respectively, from IZMC plus 40, 40, and 7 mg/kg of Zn, Mn, and Cu, respectively, from OZMC (ISO); and 3) a diet with 100, 100, and 10 mg/kg of Zn, Mn, and Cu, respectively, from IZMC as in control plus 40, 40, and 7 mg/kg of supplemental Zn, Mn, and Cu from OZMC (on top). Ten replications of 20 females and 2 males were used per treatment. Eggs from breeders at 30, 40, 50 and 60 wk of age were incubated, and 5 embryos per replicate were collected at 10 (E10), 14 (E14), and 18 (E18) d of incubation. Midshaft width and calcification were measured for left tibia and femur stained with Alcian Blue and Alizarin Red S. At hatch, the left tibia of 5 chicks per replicate was sampled for histological evaluation of the diaphysis and distal epiphysis. Feeding the ISO treatment compared with the control diet increased the Zn (P < 0.05) but not Mn and Cu content of the yolk and albumen blend. At E14, the ISO and on-top treatments had a trend to increase tibia calcification at the rates of 1.6 and 1%, respectively (P < 0.1). The E18 ISO and on-top treatments had 2% thicker tibia compared with the control, regardless of hen age (P < 0.05). Also, at E18, calcification of tibia and femur was higher from hens fed the on-top treatment (P < 0.05). The chicks from the ISO and on-top groups had increased tibia moment of inertia (P < 0.01) at day of hatch. Broiler breeder hens consuming OZMC associated with IZMC produced embryos and hatching chicks with improvements in selected bone mineralization parameters.

Membrane-type 2 matrix metalloproteinase (MT2-MMP; also called MMP15) is a membrane-bound protease that degrades extracellular matrix and activates proMMPs such as proMMP-2. MMP-2 expression in avian embryos is well documented, but it is not clear how proMMP-2 is activated during avian embryogenesis. Herein, we report that MT2-MMP mRNA is expressed in several tissues including the neural folds and epidermal ectoderm, intermediate mesoderm, pharyngeal arches, limb buds, and dermis. Several, but not all, of these tissues are known to express MMP-2. These observations suggest MT2-MMP may play a role during embryonic development not only through its own proteolytic activity but also by activating proMMP-2., (Copyright © 2012 Wiley Periodicals, Inc.)

1. Two experiments were designed to investigate the effects of mixing eggs of different genotype and incubating with different CO2 concentrations on embryonic parameters. Half of the eggs were incubated in a CO2 controlled incubator (VCO2) during the first 10 d of incubation and the other half was incubated at standard incubation ventilation rate (SV). 2. From 10 to 18 d of incubation, thick albumen and embryos were weighed. Blood samples were collected at d 18 of incubation, at internal pipping and at hatch for determination of triiodothyronine (T3), thyroxine (T4) and corticosterone concentration. During the last 2 d of incubation, hatching events of individual eggs were monitored every 2 h. 3. From 4 to 5 d of incubation in the SV group of Experiment 1, pH values of Ross strain chickens were lower than those of Isa Brown. From d 12 of incubation onwards, Ross embryos grew faster than those of Isa Brown. At 14 and 16 d of incubation, Ross eggs in the CO2 controlled incubator had lower albumen weights compared to all other treatments. 4. T3 concentrations in Ross embryos were higher than those of Isa Brown embryos. Chicken weight at hatch was in the following order: Isa Brown SV < Isa Brown VCO2 = Ross SV < Ross VCO2. 5. In Experiment 2, incubation time of VCO2 eggs until internal pipping, external pipping and hatch was significantly shorter than that of SV eggs. 6. It was concluded that mixing of hatching eggs of differential embryo developmental trajectory affects their hatching process.

It has been suggested that the quantity and quality of nutrients stored in the egg might not be optimal for the fast rate of chicken embryo development in modern broilers, and embryos could be supplemented with nutrients by in ovo injection. Recent experiments showed that in ovo feeding reduces post-hatch mortality and skeletal disorders and increases muscle growth and breast meat yield. Adenosine triphosphate (ATP) is a "ready for use" energetic molecule, while nanoparticles of silver (Nano-Ag) may penetrate tissues as well as cells and localise inside cells. In this investigation, we hypothesised that silver nanoparticles could be used as a protective carrier for ATP as well as an active agent. ATP and/or an ATP complex with Nano-Ag would be delivered to the muscle cells as a gene expression regulator and promoter of growth and development of embryo breast muscle. A collection of 160 broiler eggs was randomly divided into a Control group without injection and injected groups with hydrocolloids of Nano-Ag, ATP or a complex of Nano-Ag and ATP (Nano-Ag/ATP). The embryos were evaluated on day 20 of incubation. The results indicate that the application of ATP to chicken embryos increases expression of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and Na(+)/K(+) transporting ATPase (ATP1A1), which may indicate that an extra energy source can enhance molecular mechanisms of muscle cell proliferation. Nano-Ag also up-regulated expression of FGF2, VEGF, ATP1A1 and, also up-regulated expression of myogenic differentiation 1(MyoD1), affecting cell differentiation. The results indicate that ATP and Nano-Ag may accelerate growth and maturation of muscle cells.

The emergence of larger brains with large numbers of neurons is an evolutionary innovation in mammals and birds. However, the corresponding changes in cortical developmental programmes during amniote evolution are poorly understood. Here we examine the cortical development of Madagascar ground geckos, and report unique characteristics of their reptilian cortical progenitors. The rates of proliferation and neuronal differentiation in the gecko cortex are much lower than those in other amniotes. Notch signalling is highly activated in the gecko cortical progenitors, which provides a molecular basis for the low rate of cortical neurogenesis. Interestingly, multiple neuron subtypes are sequentially generated in the gecko cortex, similar to other amniotes. These results suggest that changes in the regulation of cortical neural progenitors have accelerated neurogenesis and provided encephalization in mammalian and archosaurian lineages. In addition, the temporal regulation for making cortical neuronal subtypes has evolved in a common ancestor(s) of amniotes.

Dorsoventral fibers in the presumptive dermis of the chick limb bud reported first by Hurle's group in 1989 are now revealed as bundles of fibrillin microfibrils (Isokawa et al., 2004). The bundles, which could be called oxytalan fibers at the light microscopic level, are aligned perpendicularly to the overlying ectoderm and form a unique fiber array, originating directly from the basal lamina. This well-oriented organization is beneficial in examining the process of in vivo bundling of microfibrils into oxytalan fibers. In this study, sections through the presumptive limb dermis were preferentially prepared from chick embryos at Days 4-6 (ED4-6). Immunohistochemically, fibrillin-positive dots representing cross-sectioned surfaces of individual fibers, increased in size from ED4 to 6, but their number per unit area remained constant. Ultrastructurally, a single oxytalan fiber at ED4 consisted of ∼15 microfibrils; the latter number increased fourfold from ED4 to 5 and threefold from ED5 to 6. Oxytalan fibers were all closely associated with mesenchymal cell; notably, the fibers at ED5 and 6 were held in a shallow ditch on the cell body or by lamellipodial cytoplasmic protrusion. In the sites of cell-fiber adhesion, microfibrils in the periphery of an oxytalan fiber appeared to adhere directly or by means of short flocculent strands to a nearby cell membrane; the latter showed a thickening of plasmalemma and its undercoat, indicating the presence of adhesive membrane specification. These findings suggest that the bundling of microfibrils is a progressive and closely cell-associated process., (Copyright © 2012 Wiley Periodicals, Inc.)

1. Broiler breeders are unable to self-regulate feed intake to a level that permits reproductive function and restricted feeding is normal for broiler parent stock. The aim of this study was to determine whether myostatin (MSTN) and myogenin (MYOG) mRNA expression could be altered in relation to differences in body composition of the embryos and the hen's diet. 2. A 2 × 2 factorial experiment was conducted with two dietary intake levels (100% and 75% of nutrition recommendation) and two broiler genotypes (Fat line and Lean line). Hens (384 of each line) were randomly divided at 23 weeks of age into 4 treatments, with each treatment represented by 12 replicates of 16 birds each. The experiment started when the rate of lay reached 5% and continued until 54 weeks of age. 3. There were significant effects of line and dietary intake, and interactions between them on MSTN and MYOG mRNA expression levels in embryonic tissues. The trend in MSTN mRNA expression level over the embryonic period was similar between lines. Low daily feed intake did not alter the trend in MSTN mRNA expression levels in either line, but significantly changed the peak values. MYOG mRNA expression varied by line and over time, with a delay in the Lean line. Low daily feed intake influenced the trend in MYOG mRNA expression levels in both lines and delayed its onset.

Background: A primary cutaneous melanoma will not kill the patient, but its metastases. Since in vitro studies on melanoma cells in 2-D cultures do often not reflect reality, 3-D models might come closer to the physiological situation in the patient during cancer initiation and progression., Methodology/principal Findings: Here, we describe the chick embryo model for in vivo studies of melanoma cell migration and invasion. After transplantation of neural crest-derived melanoma cells into the neural tube, the melanoma cells resume neural crest cell migration along the medial and lateral pathways and finally undergo apoptosis in the target areas. Upon transplantation into ectopic areas such as the hindbrain or the optic cup malignant invasion and local tissue destruction occurs. In contrast, melanocytes are not able to spontaneously resume neural crest cell migration. However, malignant invasion can be induced in melanocytes by pre-treatment with the TGF-beta family members bone morphegenetic protein-2 or nodal. Transplantation of MCF7 breast cancer cells yields a different growth pattern in the rhombencephalon than melanoma cells., Conclusions/significance: The chick embryo model is a feasible, cost-effective in vivo system to study invasion by cancer cells in an embryonic environment. It may be useful to study invasive behavior induced by embryonic oncogenes and for targeted manipulation of melanoma or breast cancer cells aiming at ablation of invasive properties.

Vertebrates have achieved great evolutionary success due in large part to the anatomical diversification of their jaw complex, which allows them to inhabit almost every ecological niche. While many studies have focused on mechanisms that pattern the jaw skeleton, much remains to be understood about the origins of novelty and diversity in the closely associated musculature. To address this issue, we focused on parrots, which have acquired two anatomically unique jaw muscles: the ethmomandibular and the pseudomasseter. In parrot embryos, we observe distinct and highly derived expression patterns for Scx, Bmp4, Tgfβ2 and Six2 in neural crest-derived mesenchyme destined to form jaw muscle connective tissues. Furthermore, immunohistochemical analysis reveals that cell proliferation is more active in the cells within the jaw muscle than in surrounding connective tissue cells. This biased and differentially regulated mode of cell proliferation in cranial musculoskeletal tissues may allow these unusual jaw muscles to extend towards their new attachment sites. We conclude that the alteration of neural crest-derived connective tissue distribution during development may underlie the spatial changes in jaw musculoskeletal architecture found only in parrots. Thus, parrots provide valuable insights into molecular and cellular mechanisms that may generate evolutionary novelties with functionally adaptive significance.

Eggs (n = 1,800) obtained from Ross broiler breeders at 32 and 48 wk of age were incubated at either a constant temperature of 37.6°C throughout (T1), or the temperature was reduced for 6 h to 36.6°C each day during embryonic age (EA) 10 to 18 (T2). Yolk sac, liver, and brain fatty acid profiles and oxidant and antioxidant status of liver and brain were measured at EA 14, 19, and day of hatch (DOH). Fatty acid profiles of yolk sac, liver, and brain were influenced by age of breeder with significant breeder hen age × incubation temperature interactions. At EA 14, higher levels of 20:4n-6 and 22:6n-3 had been transferred from the yolk sac to T2 embryos from younger than older breeders, whereas for T1 and T2 embryos, yolk sac 20:4n-6 and 22.6n-3 values were similar for older breeders. Accumulation of 20:4n-6 and 22:6n-3 fatty acids in the liver of T1 and T2 embryos from younger breeders was similar; however, T2 embryos from older breeders had higher liver levels of 20:4n-6 and 22:6n-3 than T1 embryos. At EA 19, liver nitric oxide levels were higher for T2 embryos from younger breeders than those from breeders incubated at T1. Brain catalase levels of T2 embryos from younger breeders were higher than those from older breeders at DOH. Thus, changes in fatty acid profiles and catalase and nitric oxide production of brain and liver tissues resulting from 1°C lower incubation temperature from EA 10 to 18 reflect adaptive changes.

In the early embryo, the brain initially forms as a relatively straight, cylindrical epithelial tube composed of neural stem cells. The brain tube then divides into three primary vesicles (forebrain, midbrain, hindbrain), as well as a series of bulges (rhombomeres) in the hindbrain. The boundaries between these subdivisions have been well studied as regions of differential gene expression, but the morphogenetic mechanisms that generate these constrictions are not well understood. Here, we show that regional variations in actomyosin-based contractility play a major role in vesicle formation in the embryonic chicken brain. In particular, boundaries did not form in brains exposed to the nonmuscle myosin II inhibitor blebbistatin, whereas increasing contractile force using calyculin or ATP deepened boundaries considerably. Tissue staining showed that contraction likely occurs at the inner part of the wall, as F-actin and phosphorylated myosin are concentrated at the apical side. However, relatively little actin and myosin was found in rhombomere boundaries. To determine the specific physical mechanisms that drive vesicle formation, we developed a finite-element model for the brain tube. Regional apical contraction was simulated in the model, with contractile anisotropy and strength estimated from contractile protein distributions and measurements of cell shapes. The model shows that a combination of circumferential contraction in the boundary regions and relatively isotropic contraction between boundaries can generate realistic morphologies for the primary vesicles. In contrast, rhombomere formation likely involves longitudinal contraction between boundaries. Further simulations suggest that these different mechanisms are dictated by regional differences in initial morphology and the need to withstand cerebrospinal fluid pressure. This study provides a new understanding of early brain morphogenesis.

Neural crest cells (NCC) are a transient, embryonic cell population characterized by unusual migratory ability and developmental plasticity. To annotate and characterize cis-regulatory elements utilized by the human NCC, we coupled a hESC differentiation model with genome-wide profiling of histone modifications and of coactivator and transcription factor (TF) occupancy. Sequence analysis predicted major TFs binding at epigenomically annotated hNCC enhancers, including a master NC regulator, TFAP2A, and nuclear receptors NR2F1 and NR2F2. Although many TF binding events occur outside enhancers, sites coinciding with enhancer chromatin signatures show significantly higher sequence constraint, nucleosomal depletion, correlation with gene expression, and functional conservation in NCC isolated from chicken embryos. Simultaneous co-occupancy by TFAP2A and NR2F1/F2 is associated with permissive enhancer chromatin states, characterized by high levels of p300 and H3K27ac. Our results provide global insights into human NC chromatin landscapes and a rich resource for studies of craniofacial development and disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Birth dating neurons with bromodeoxyuridine (BrdU) labeling is an established method widely employed by neurobiologists to study cell proliferation in embryonic, postnatal, and adult brain. Birth dating studies in the chick dorsal telencephalon and the mammalian striatum have suggested that these structures develop in a strikingly similar manner, in which neurons with the same birth date aggregate to form "isochronic clusters." Here we show that isochronic cluster formation in the chick dorsal telencephalon is an artifact. In embryos given standardly employed doses of BrdU, we observed isochronic clusters but found that clusters were absent with BrdU doses close to the limits of detection. In addition, in situ hybridization experiments established that neurons in the clusters display errors in cell type specification: BrdU cell clusters in nidopallium adopted a mesopallial neuronal fate, mesopallial clusters were misspecified as nidopallial cells, and in some instances, the BrdU clusters failed to express neuronal differentiation markers characteristic of the dorsal telencephalon. These results demonstrate that the chick dorsal telencephalon does not develop by isochronic cluster formation and highlight the need to test the integrity of BrdU-treated tissue with gene expression markers of regional and cell type identity.

Housekeeping genes are widely used as internal controls for gene expression normalization for western blotting, northern blotting, RT-PCR, etc. They are generally thought to be expressed in all cells of the organism at similar levels because it is assumed that these genes are required for the maintenance of basic cellular function as constitutive genes. However, real-time RT-PCR experiments revealed that their expression may vary depending on the developmental stage, type of tissue examined, experimental condition, and so on. To date, no histological data on their expression are available for embryonic development. In the present study, we compared the histological expression profile of two commonly used housekeeping genes, GAPDH and beta-actin, in the developing chicken embryo by using section and whole mount in situ hybridization supplemented by RT-PCR. Our results show that neither GAPDH mRNA nor beta-actin mRNA is expressed in all cell types or tissues at high levels. Strikingly, expression levels are very low in some organs. Moreover, the two genes show partially complementary expression patterns in the liver, the vascular system and the digestive tract. For example, GAPDH is more strongly expressed in the liver than beta-actin, but at lower levels in the arteries. Vice versa, beta-actin is more strongly expressed in the gizzard than GAPDH, but it is almost absent from cardiac muscle cells. Researchers should consider these histological results when using GAPGD and beta-actin for gene expression normalization in their experiments.

Aims: In this study, we evaluated the ability of differentiating embryonic chick DRG neurons to release and respond to acetylcholine (ACh). In particular, we investigated the neuronal soma and neurites as sites of ACh release, as well as the mechanism(s) underlying this release., Main Methods: ACh release from DRG explants in the Campenot chambers was measured by a chemiluminescent assay. Real-time PCR analysis was used to evaluate the expression of ChAT, VAChT, mediatophore and muscarinic receptor subtypes in DRGs at different developmental stages., Key Findings: We found that ACh is released both within the central and lateral compartments of the Campenot chambers, indicating that ACh might be released from both the neuronal soma and fibers. Moreover, we observed that the expression of the ChAT and mediatophore increases during sensory neuron differentiation and during the post-hatching period, whereas VAChT expression decreases throughout development. Lastly, the kinetics of the m2 and m3 transcripts appeared to change differentially compared to the m4 transcript during the same developmental period., Significance: The data obtained demonstrate that the DRG sensory neurons are able to release ACh and to respond to ACh stimulation. ACh is released both by the soma and neurite compartments. The contribution of the mediatophore to ACh release appears to be more significant than that of VAChT, suggesting that the non-vesicular release of ACh might represent the preferential mechanism of ACh release in DRG neurons and possibly in non-cholinergic systems., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In many regions of the nervous system, the combinatorial action of transcriptional factors specifies the individual fate of neuronal subtypes. Contrary to this, we report that a single transcriptional factor controls a phenotype shared by different subtypes of neurons, namely the expression of a neurotrophic factor receptor in the spinal cord. Along the dorsoventral axis of the chick embryo spinal cord, the expression pattern of a specific receptor for glial cell line derived-neurotrophic factor (GDNF family of receptors α1: GFRα1) was related to that of two basic helix-loop-helix (bHLH) transcriptional factors (NeuroM and Neurogenin2: Ngn2). In ovo electroporation in the chick embryo revealed that the overexpression of NeuroM alone was sufficient to induce ectopic GFRα1 expression without overt neuronal differentiation, whereas the suppression of NeuroM activity resulted in the specific loss of GFRα1 expression, indicating that NeuroM may act as a differentiation factor for GFRα1 expression. Ngn2 overexpression was also sufficient to induce precocious GFRα1 expression. However, the forced expression of both obligate suppressor and activator forms of Ngn2 also induced aberrant GFRα1 expression. Thus, any deviation from an optimum level of Ngn2 expression resulted in aberrant GFRα1 expression. Consistent with this, manipulation of Ngn2 expression levels by other bHLH factors also resulted in ectopic GFRα1 expression. For example, the downregulation by Ascl1 and the upregulation by Ptf1a induced ectopic GFRα1 expression, irrespective of endogenous expression patterns of Ascl1 and Ptf1a (Ascl1/Ptf1) in the spinal cord. The suppression of Ascl1/Ptf1a activities abolished Ngn2 and GFRα1 expression, even in Ascl1/Ptf1a-negative regions. These data indicate the presence of a distinct regulatory sequence for a determinant of GFRα1 expression, in which Ascl1/Ptf1a may competitively intervene to stochastically modulate default Ngn2 expression levels. Thus, Ngn2 together with NeuroM serves as readout to regulate GFRα1 expression, which occurs in multiple subtypes of spinal neurons., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The dermomyotome is the dorsal compartment of the somite which gives rise to multiple cell fates including skeletal muscle, connective tissue, and endothelia. It consists of a pseudostratified, roughly rectangular epithelial sheet, the margins of which are called the dermomyotomal lips. The dermomyotomal lips are blastema-like epithelial growth zones, which continuously give rise to resident dermomyotomal cells and emigrating muscle precursor cells, which populate the subjacent myotomal compartment. Wnt signaling has been shown to regulate both dermomyotome formation and maintenance of the dermomyotomal lips. Whereas the epithelialization of the dermomyotome is regulated via canonical, β-catenin-dependent Wnt signaling, the downstream signaling mechanisms suppressing epithelial-mesenchymal transition (EMT) in the mature dermomyotomal lips have been unknown. Here, we present evidence that dermomyotomal lip sustainment is differentially regulated. Whereas the dorsomedial dermomyotomal lip is maintained by canonical Wnt signaling, development of the ventrolateral dermomyotomal lip is regulated by non-canonical, PCP-like Wnt signaling. We discuss our results in the light of the different developmental prerequisites in the dorsomedial and ventrolateral lips, respectively, thus providing a new perspective on the regulation of dermomyotomal EMT.

Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption of nutrients posthatch. The uptake of nutrients is mediated by a variety of membrane-bound transporter proteins. The objective of this study was to determine the expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and embryonic intestine of egg-laying (Leghorn) and meat-producing (Cobb) chickens derived from 22 to 30 wk (young) and 45 to 50 wk (old) breeder flocks. Transporters examined included the peptide transporter PepT1, the glutamate/aspartate (EAAT3), cationic (CAT-1) and neutral (B0AT) amino acid transporters, and the fructose (GLUT5) and glucose (SGLT1) transporters. Digestive enzymes included aminopeptidase N (APN) and sucrase-isomaltase (SI). Expression of these genes was assessed by real-time PCR using the absolute quantification method in YSM at embryonic day (E) 11, 13, 15, 17, 19, 20, and 21 and intestine at E15, 17, 19, 20, and 21. The PepT1 and APN gene expression in the YSM increased until E15 and then decreased until E21, whereas expression in the intestine increased from E15 to E21. The B0AT showed a similar pattern, with greatest expression in the YSM occurring at E17/E19. The CAT1 and GLUT5 genes showed decreased expression in the YSM and increased expression in the intestine until E17/E19 and then a decrease until E21. Expression of SGLT1 and EAAT3 showed increased gene expression over time in both the intestine and YSM. Expression of SI showed little to no gene expression in the YSM, whereas the intestine exhibited consistently high levels of gene expression. In YSM and intestine, SI expression was greater in Leghorn than Cobb, whereas CAT1 and GLUT5 expression was greater in Cobb than Leghorn. Expression of the APN, CAT1, and SI genes was greater in embryos from young flocks than old flocks in YSM and intestine. These results demonstrate that the YSM expresses many of the digestive enzymes and nutrient transporters typically associated with the intestine and that these genes show tissue- and development-specific patterns of expression.

Your brain and spinal cord began as a flat sheet, which narrowed, elongated, and rolled up to form a tube. A new study identifies a key molecular link underlying vertebrate neural tube formation, connecting planar cell polarity patterning to contraction of specific cell-cell junctions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Perfluoroalkyl acids (PFAAs) such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are developmental toxicants in various animal classes, including birds. Both compounds interact with peroxisome proliferator-activated receptors (PPARs), but it is not known whether activation of PPARs is involved in their embryo toxicity in birds. We exposed chicken embryos via egg injection at a late developmental stage to GW7647, a potent PPARα agonist in mammals, and to PFOS or PFOA. Mortality was induced by PFOS and PFOA but not by GW7647. Transcripts of a number of genes activated by PPARα agonists in mammals were analyzed in liver and kidney of 18-day-old embryos. Several of the genes were induced in both liver and kidney following exposure to GW7647. Treatment with PFOA resulted in induction of acyl-coenzyme A oxidase mRNA in liver, whereas none of the genes were significantly induced by PFOS treatment. No up-regulation of gene transcription was found in kidney following treatment with PFOS or PFOA. Principal component analysis showed that PFOA caused an mRNA expression pattern in liver more similar to the pattern induced by GW7647 than PFOS did. Our findings do not support that the embryo mortality by PFOS and PFOA in chicken embryos involves PPARα activation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neural-tube closure is a critical step of embryogenesis, and its failure causes serious birth defects. Coordination of two morphogenetic processes--convergent extension and neural-plate apical constriction--ensures the complete closure of the neural tube. We now provide evidence that planar cell polarity (PCP) signaling directly links these two processes. In the bending neural plates, we find that a PCP-regulating cadherin, Celsr1, is concentrated in adherens junctions (AJs) oriented toward the mediolateral axes of the plates. At these AJs, Celsr1 cooperates with Dishevelled, DAAM1, and the PDZ-RhoGEF to upregulate Rho kinase, causing their actomyosin-dependent contraction in a planar-polarized manner. This planar-polarized contraction promotes simultaneous apical constriction and midline convergence of neuroepithelial cells. Together our findings demonstrate that PCP signals confer anisotropic contractility on the AJs, producing cellular forces that promote the polarized bending of the neural plate., (Copyright © 2012 Elsevier Inc. All rights reserved.)

During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node., (Copyright © 2012. Published by Elsevier Inc.)

This investigation evaluated the effects of nanoparticles of silver (AgNano) and gold (AuNano) on metabolic rate (O(2) consumption, CO(2) production and heat production-HP) and the development of embryos from different breeds of broiler and layer chicken. Gaseous exchange was measured in an open-air-circuit respiration unit, and HP was calculated for 10, 13, 16 and 19-day-old embryos. Relative chick and muscle weights were used as a measure of growth rate and development. AgNano but not AuNano increased the rates of O(2) consumption and HP of the layer embryos. The metabolic rate of broiler embryos was not affected by either of the treatments, but it was significantly higher compared to the layer embryos. Neither of the nanoparticles promoted nor depressed growth and development of the embryos, irrespective of breed. Although the metabolic rate of AgNano-injected layer embryos was significantly increased, their BW and muscle weights at hatching were similar to those of the control group, which suggests that the concentration of AgNano used was adequate for increasing the metabolic rate but not enough to affect growth and development. The results show that AgNano could be a potential metabolic modifier for layer embryos; however, the exact mechanism of action should be elucidated in future research., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Glycogen and glucose concentrations (mg/g of tissue) and amounts (mg) were determined in the yolks of fertile eggs on the day of set and in the yolk sac (YS) and liver of broiler chick embryos between 11 and 21 embryonic days of age (E). On the day of set, the yolk contained 50 mg of glucose (0.31% of yolk) but did not contain glycogen. During incubation, the amount of glucose in the YS increased from 20 mg on E11 to 60 mg on E19. A parallel increase in YS and liver glycogen concentrations (mg/g) during the last week of incubation implied a similar capacity for glycogen synthesis per gram of tissue. However, due to its larger size, the YS capacity for glycogen storage far exceeded that of the liver, which stored less than 12 mg of glycogen up to E19, as compared with more than 200 mg in the YS. Between E19 and 21, liver and YS glycogen amounts decreased by 10 mg and 100 mg, respectively. These results indicated that the YS is a glycogenic and perhaps gluconeogenic organ. We therefore evaluated the gene expression of glycogen synthase and glycogen phosphorylase as well as gluconeogenic enzymes (fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase) in the YS membrane and liver by real-time reverse-transcription PCR. Although the YS membrane and liver displayed different patterns of mRNA abundance, the high abundance of fructose 1,6-bisphosphatase mRNA in the YS membrane between E11 and 15, and the expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, supported the postulated gluconeogenic abilities of the YS membrane and indicated its role in providing glucose to the embryo. Thus, glucose is probably synthesized in the YS, stored in the form of glycogen, and toward hatch, the YS may have the potential to transfer 10 times more glycogen-derived glucose to the embryo as compared with the liver. As such, the YS may play a major role in the synthesis and storage of glucose and its supply to the chick embryo toward hatch.

Estrogen has been reported to act on B cell genesis in the bursa of Fabricius of chick embryos. In this study, we attempted to demonstrate the hypothesis that B cell genesis is controlled by estrogen receptor (ER) in the bursal cells and steroidogenic enzymes synthesized in the bursa. We previously reported the presence of estrogen receptor α (ERα) in the bursa during the late stage of embryogenesis and an increase in the expression of ERα messenger RNA (mRNA) between the 13th day and 16th day. The number of ER-positive cells was maximal on the 16th day. In the present study, ER-positive cells in the bursa during the late stage of embryogenesis increased 4 h after β-estradiol treatment on the 14th to 18th day. The concentration of β-estradiol in the embryonic bursa increased. These results suggest that this stage of embryogenesis is critical in B cell development in the bursa in connection with the effect of estrogen treatment. Our findings also showed that the mRNA expression of five steroidogenic enzymes occurred in the bursa of chick embryos. These results suggest that estrogen is synthesized in the embryonic bursa and estrogen acts on the bursal cells in a paracrine fashion., (© 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.)

The comprehensive profiles of the internal and external temperatures of embryonated Ross × Ross 708 broiler hatching eggs during incubation were determined using temperature transponders, and eggshell water vapor conductance (G(H2O)), specific G(H2O) (g(H2O); G(H2O) adjusted to a 100 g set egg weight basis), and G(H2O) constants (K(H2O)) were calculated. On each of 8 replicate tray levels of an incubator, 2 nonembryonated and 4 embryonated eggs were each implanted with a transponder on d 10.5 of incubation for the determination of internal (air cell) temperatures of nonembryonated (T(nem)) and embryonated (T(emb)) eggs, respectively. In addition, 2 water-filled vials, each containing a transponder, were used on each tray level for the determination of the external microenvironment temperatures (T(ext)) of the embryonated and nonembryonated eggs. Between 10.5 and 18 d of incubation, incubator data logger temperatures were determined every 5 min; and incubator dry bulb temperature, T(ext), T(nem), T(emb), and the difference between T(emb) and T(nem) (T) were determined every 12 h. Over the days of incubation, regression coefficients for T(emb) and T were positive, whereas the regression coefficient for T(nem) was negative. There was a significant day of incubation × type of temperature measurement (T(ext), T(nem), and T(emb)) interaction for temperature. Between 13 and 18 d of incubation, mean values of T(emb) readings that were recorded every 12 h were consistently higher than those of T(ext) and T(nem), indicating the importance of air cell transponder implantation for the efficient estimation of broiler embryo temperature. Furthermore, mean values of the percentage of daily incubational egg weight loss, G(H2O), g(H2O), and K(H2O) of the embryonated eggs were 0.54 ± 0.019%, 14.4 ± 0.56 mg of H₂O/d per Torr, 25.0 ± 0.96 mg of H₂O/d per Torr per 100 g, and 5.20 ± 0.205, respectively. The results suggest that transponders may be implanted in the air cells of broiler hatching eggs to detect incubational variations in T(emb) and to subsequently calculate G(H2O), g(H2O), and K(H2O).

The proneural gene Atoh1 is crucial for the development of inner ear hair cells and it requires the function of the transcription factor Sox2 through yet unknown mechanisms. In the present work, we used the chicken embryo and HEK293T cells to explore the regulation of Atoh1 by Sox2. The results show that hair cells derive from Sox2-positive otic progenitors and that Sox2 directly activates Atoh1 through a transcriptional activator function that requires the integrity of Sox2 DNA binding domain. Atoh1 activation depends on Sox transcription factor binding sites (SoxTFBS) present in the Atoh1 3' enhancer where Sox2 directly binds, as shown by site directed mutagenesis and chromatin immunoprecipitation (ChIP). In the inner ear, Atoh1 enhancer activity is detected in the neurosensory domain and it depends on Sox2. Dominant negative competition (Sox2HMG-Engrailed) and mutation of the SoxTFBS abolish the reporter activity in vivo. Moreover, ChIP assay in isolated otic vesicles shows that Sox2 is bound to the Atoh1 enhancer in vivo. However, besides activating Atoh1, Sox2 also promotes the expression of Atoh1 negative regulators and the temporal profile of Atoh1 activation by Sox2 is transient suggesting that Sox2 triggers an incoherent feed-forward loop. These results provide a mechanism for the prosensory function of Sox2 in the inner ear. We suggest that sensory competence is established early in otic development through the activation of Atoh1 by Sox2, however, hair cell differentiation is prevented until later stages by the parallel activation of negative regulators of Atoh1 function.

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

Planar cell polarity (PCP) is the coordinate organization of cells within the plane of a tissue. PCP is essential for tissue function, such as for proper hearing in the vertebrate ear or for accurate vision in the Drosophila eye. Using the chick embryo, we have recently shown that during early muscle formation, the first formed muscle fibres utilize the PCP pathway to orient parallel to a WNT11 source present in the medial border of somites. Our results further establish that WNT11 acts as a directional cue to regulate this process. To perform this study, two major techniques have been utilized, the gene loss-of-function using a vector-based shRNAmir expression and confocal videomicroscopy of fluorescent gene reporters targeted in specific cell subpopulations by in vivo electroporation. Here we describe the two techniques.

Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in vitro under iron-limiting conditions. The 1.8 Å crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding copurified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cadherins are adhesion molecules that play a crucial role in tissue morphogenesis. Studies on N-cadherin and E-cadherin in the ovary of fetal hamster suggest that these adhesion molecules are involved in primordial follicle formation. In chicken embryo, present results demonstrate that N-cadherin is located on the surface epithelium and in the cortical cords of the ovary. Moreover, N-cadherin is identified in germ cells on day 14 of chicken embryo development. Quantification of mRNA of N-cadherin and E-cadherin demonstrates that treatments with follicle-stimulating hormone (FSH) or human chorionic gonadotropin (hCG), increase N-cadherin expression. Whereas, E-cadherin expression is decreased by gonadotropin treatments. The negative correlation between both cadherins expression is demonstrated after 18 h of hormonal treatment. Regulation of cadherin expression by gonadotropins and the presence of N-cadherin in the ovarian cortex suggest that these adhesion molecules are involved in ovarian morphogenesis in the chicken embryo., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The objective was to determine the contributions of glucose to glycogen synthesis and glycerol to glycogen, glucose, and nonessential AA (NEAA) synthesis on embryonic day (e) 14/15 and e19/20. Chicken embryos from small (56.6 ± 0.88 g) and large eggs (71.7 ± 1.09 g) were repeatedly dosed with either [(13)C(3)]glycerol (14 mg/d for 4 d) or [(13)C(6)]glucose (15 mg/d for 3 d) into the chorio-allantoic fluid before blood and tissue collection. (13)C-Mass isotopomer enrichments in blood glucose, liver, and muscle glycogen, and blood and tissue NEAA were analyzed by mass spectrometry. Glucose metabolism did not differ between small- and large-egg embryos. Although glucose entry was 60% less for e20 compared with e15 embryos, e20 embryos conserved glucose more efficiently as a result of 2- to 3-fold greater (P < 0.001) rates of glucose carbon recycling. Importantly, the direct contribution of glucose to liver glycogen synthesis was minimal on e15, and on e20 direct incorporation of glucose into liver glycogen was only 17%. By comparison, [(13)C(3)]glycerol dosing led to the appearance of [M + 1], [M + 2], and [M + 3] isotopomers in blood glucose and in liver and muscle glycogen on e14 and e19. Here, the (13)C-isotopomer enrichments in blood glucose were ~2-fold greater (P < 0.05) in small- than in large-egg embryos on e14 and e19. Furthermore, [(13)C(3)]glycerol dosing led to substantial labeling of [M + 1], [M + 2], and [M + 3] isotopomers of alanine, aspartate, and glutamate in blood and in tissues where (13)C enrichments were greater (P < 0.05) in liver of small-egg embryos. In summary, this study provides unequivocal evidence that glycerol is a precursor for glucose and NEAA synthesis. Furthermore, glycerol, but not egg-derived glucose, is a major substrate for synthesis of liver and muscle glycogen and is an important anaplerotic substrate for the tricarboxylic acid cycle of embryos during later development.

This study was conducted to investigate the gene expression of cationic and neutral amino acid (AA) transporters in the small intestine of chick embryos with different genetic backgrounds [Wenshi Yellow-Feathered chick (WYFC) and White Recessive Rock chick (WRRC)]. The study also investigated the correlation between the abundance of AA transporter mRNA and the AA content of fertilized eggs. Intestinal samples were collected on embryonic d 9, 12, 14, 17, and 19 and the day of hatch. The results showed that, before incubation, the AA content of WRRC eggs was lower (P < 0.05) than the AA content of WYFC eggs. In WYFC, the mRNA abundance of CAT-1 [solute carrier (SLC) family 7 member 1], CAT-4 (SLC family 7 member 4), rBAT (SLC family 3 member 1), y(+)LAT-1 (SLC family 7 member 7), y(+)LAT-2 (SLC family 7 member 6), LAT-4 (SLC family 43 member 2), and SNAT-2 (SLC family 38 member 2), as detected by real-time reverse transcriptase PCR, was greater (P < 0.05) than the mRNA abundance detected in the WRRC samples. The mRNA abundance of all measured AA transporters was affected (P < 0.05) by embryonic age. Sex had the largest effect (P < 0.05) on the mRNA expression of CAT-1, CAT-4, y(+)LAT-2, and LAT-4 in WYFC and on CAT-4 and B(0)AT-1 (SLC family 6 member 19) mRNA expression in WRRC. In WYFC, only CAT-1 mRNA expression was negatively correlated (r = -0.68 to -0.84, P < 0.05) with all AA content. However, few correlations were detected between AA content and the mRNA expression of multiple transporters in WRRC. These findings provide a comprehensive profile of the temporal and spatial mRNA expression of AA transporters in the small intestine of chick embryos. Few correlations were detected between the AA content of the eggs and mRNA expression of specific AA transporters in the small intestine.

To evaluate the effect of breeder age and egg size on yolk absorption and embryo development, a total of 4,800 Ross 308 hatching eggs were subjected to 4 treatments arranged in a 2 × 2 factorial randomized complete block design using 2 breeder ages (29 and 53 wk of age, or young and old) and 2 egg sizes (57-61 g and 66-70 g, or small and large). A significant interaction between breeder age and egg size was found for egg composition. Yolk weight increased with flock age, whereas a larger egg size resulted in higher albumen content. A significant interaction between breeder age and egg size was found for yolk-free body (YFB) weight only at d 7. Until the fourteenth day of incubation, eggs from the old flock yielded greater YFB weight than did eggs from the young flock. At hatch, chicks of both age groups had comparable wet YFB weight, chick weight, wet and dry residual yolk weight, and chick length. Dry YFB weight of chicks from the old flock was higher than that of chicks from the young flock. Compared with the small eggs, embryos and chicks of the large eggs had greater YFB weight from d 14 to hatching. At hatch, these chicks were also heavier, longer, and had higher wet and dry YFB and residual yolk weight. Yolk absorption at d 18 and at hatch of embryos and chicks of the old flock was higher than that of the young flock, both in absolute values and percentages. Rates of absolute and percentage yolk absorption through d 18 and percentage yolk absorption at hatch were higher in the small eggs than in the large eggs. It can be concluded that egg size influences chick length at hatch and embryo development when expressed in terms of total and YFB weight. Although yolk availability and rate of absorption may have influenced dry YFB weight, they did not influence hatching chick length or total and YFB weight.

We present a broadly applicable procedure for whole-mount imaging of antibody probes in embryonic tissues at microscopic resolutions based on combining a metal-based immunodetection scheme with x-ray microtomography (microCT). The method is generally accessible, relying on standard enzyme-conjugated secondary antibodies and other readily available reagents, and is demonstrated here with microCT visualizations of acetylated α-tubulin in the chick nervous system and of type II collagen in developing limbs. The tomographic images offer complete three-dimensional representations of molecular patterns obtained with immunostaining methods at the level of organ development, with added possibilities to quantify both spatial distributions and varying densities of gene products in situ. This imaging modality bridges a crucial gap in three-dimensional molecular imaging by combining the histological resolutions of confocal microscopy with a greater specimen size range than optical projection tomography, and thus enables a powerful new approach to long-standing issues of skeletogenic pattern formation in vertebrate limbs., (Copyright © 2011 Wiley-Liss, Inc.)