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Myc interacting zinc finger protein-1 (Miz1) is a transcription factor known to regulate cell cycle- and cell adhesion-related genes in cancer. Here we show that Miz1 also plays a critical role in neural crest development. In the chick, Miz1 is expressed throughout the neural plate and closing neural tube. Its morpholino-mediated knockdown affects neural crest precursor survival, leading to reduction of neural plate border and neural crest specifier genes Msx-1, Pax7, FoxD3, and Sox10. Of interest, Miz1 loss also causes marked reduction of adhesion molecules (N-cadherin, cadherin6B, and α1-catenin) with a concomitant increase of E-cadherin in the neural folds, likely leading to delayed and decreased neural crest emigration. Conversely, Miz1 overexpression results in up-regulation of cadherin6B and FoxD3 expression in the neural folds/neural tube, leading to premature neural crest emigration and increased number of migratory crest cells. Although Miz1 loss effects cell survival and proliferation throughout the neural plate, the neural progenitor marker Sox2 was unaffected, suggesting a neural crest-selective effect. The results suggest that Miz1 is important not only for survival of neural crest precursors, but also for maintenance of integrity of the neural folds and tube, via correct formation of the apical adhesion complex therein.

The purpose of this study was to investigate the effect of manganese (Mn)-induced cytotoxicity on heat shock proteins in chicken spleen lymphocytes. Lymphocytes were cultured in medium in the absence and presence of MnCl2 (2 × 10(-4), 4 × 10(-4), 6 × 10(-4), 8 × 10(-4), 10 × 10(-4), and 12 × 10(-4) mmol/L) for 12, 24, 36, and 48 h in vitro. Then, the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 were examined by real-time quantitative PCR. The results showed that the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in all treatment groups at all time points, except mRNA levels of HSP27 at 48 h, had the same tendency. As manganese concentration increased, the mRNA expression of the heat shock proteins first increased and then decreased. In other words, we demonstrated that the mRNA expression of the heat shock proteins was induced at lower concentrations of manganese and was inhibited at higher concentrations. Mn had a dosage-dependent effect on HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in chicken spleen lymphocytes in vitro.

In order to degrade aflatoxin B₁ (AFB₁), AFB₁-degrading microbes (probiotics) such as Lactobacillus casei, Bacillus subtilis and Pichia anomala, and the AFB₁-degrading enzyme from Aspergillus oryzae were selected and combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as the basal diet, 400 μg/kg AFB₁ supplement without feed additive, and 200, 400, 800 μg/kg AFB₁ supplement with 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine production performance of broilers. In addition, serum, liver and chest muscle were selected for measuring AFB₁ residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding 0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB₁ on chicken's production performance and nutrient metabolic rates (P<0.05). It could also improve AFB₁ metabolism, hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxidoreductase, apoptosis, cell growth, immune system and metabolic process (P<0.05). It could be concluded that the feed additive was able to degrade AFB₁ and improve animal production., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV) replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg) RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9) and 27 (H3K27) in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.

Recently, we found that ovotransferrin (OTf) undergoes distinct self-cleavage in a redox-dependent process and exhibited in vitro superoxide dismutase (SOD)-like activity. In this study, we explore that the expression of OTf confers high tolerance to oxidative stress in yeast cells. The OTf gene was cloned into the vector pPICZB and was successfully expressed in methylotrophic yeast, Pichia pastoris KM71H. There was no growth difference between the non-transformed strain and recombinant strains harboring a mock vector (pPICZB) or the OTf gene carrying a vector (OTf-pPICZB). Intracellularly expressed OTf was found to undergo self-cleavage, producing a major fragment of 15 kDa, which corresponded to the disulfide kringle domain of the N-terminal lobe. The yeast OTf transformants exhibited strong tolerance to oxidative stress induced by either hydrogen peroxide (H₂O₂) or diethyl maleate (DEM). Further, OTf transformants showed higher intracellular reducing capacity and enhanced cytosolic reductase activity. This study is the first to describe the ability of OTf to confer in vivo antioxidative stress function within a complicated milieu of eukaryotic cells and provide novel insights for the potential of the OTf gene for molecular breeding of industrial yeast strains with high tolerance to oxidative stress.

Scratch proteins are members of the Snail superfamily which have been shown to regulate invertebrate neural development. However, in vertebrates, little is known about the function of Scratch or its relationship to other neural transcription factors. We report the cloning of chicken Scratch2 (cScrt2) and describe its expression pattern in the chick embryo from HH15 through HH29. cScrt2 was detected in cranial ganglia, the nasal placode and neural tube. At all stages examined, cScrt2 expression is only detected within a subregion of the intermediate zone of the neural tube. cScrt2 is also expressed in the developing dorsal root ganglia from HH22-23 onwards and becomes limited to its dorsal medial domain at HH29. phospho-Histone H3 and BrdU-labeling revealed that the cScrt2 expression domain is located immediately external to the proliferative region. In contrast, cScrt2 domain overlapped almost completely with that of the postmitotic neural transcription factor NeuroM/Ath3/NEUROD4. Together, these data define cScrt2-positive cells as a subset of immediately postmitotic neural progenitors. Previous data has shown that Scrt2 is a repressor of E-box-driven transcription whereas NeuroM is an E-box-transactivator. In light of these data, the co-localization detected here suggests that Scrt2 and NeuroM may have opposing roles during definition of neural subtypes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Immune function in the vagina of hen oviduct is essential to prevent infection by microorganisms colonizing in the cloaca. The aim of this study was to determine whether CpG oligodeoxynucleotides (CpG-ODN) stimulate the expression of avian β-defensins (AvBDs) in hen vaginal cells. Specific questions were whether CpG-ODN affects the expression of AvBDs and proinflammatory cytokines and whether the cytokines affect AvBDs expression in vaginal cells. The dispersed vaginal cells of White Leghorn laying hens were cultured and stimulated by different doses of lipopolysaccharide (LPS), CpG-ODN, interleukin 1β (IL1B), or IL6. The cultured cell population contained epithelial cells, fibroblast-like cells, and CD45-positive leukocytes. The immunoreactive AvBD3, -10, and -12 were localized in the mucosal epithelium in the section of the vagina. The expression of AvBDs, IL1B, and IL6 was analyzed by quantitative RT-PCR. RT-PCR analysis showed the expression of AvBD1, -3, -4, -5, -10, and -12 in the cultured vaginal cells without stimulation. Toll-like receptors (TLRs) 4 and 21, which recognize LPS and CpG-ODN respectively and IL1 and IL6 receptors (IL1R1 and IL6R) were also expressed in them. The expression of IL1B, IL6, and AvBD10 and -12 was upregulated by LPS, whereas only IL1B and IL6 were upregulated by CpG-ODN. IL1B stimulation upregulated AvBD1 and -3 expression, whereas IL6 stimulation did not cause changes in AvBDs expression. These results suggest that CpG-ODN derived from microbes upregulates the expression of IL1B and IL6 by interaction with TLR21 and then IL1B induces AvBD1 and -3 to prevent infection in the vagina.

Salmonella enterica serovar enteritidis is an enteric bacterium that can contaminate chicken eggs and meat, resulting in production losses and consumer illness. To provide insight into the systemic metabolic effects of S. enteritidis infection, liver samples were harvested 10-days postinfection from broiler hens. Hepatic global gene expression levels were assessed using a chicken 44K Agilent microarray. Forty-four genes were differentially expressed at a significance level of q value < 0.05. One hundred eighty-three genes were differentially expressed at a suggestive significance level of q value < 0.1. A predominance of downregulation existed among significantly differentially expressed genes. Cell cycle and metabolism networks were created from the differentially expressed genes. Mitochondria-mediated apoptosis, electron transport, peptidase activity, vein constriction, cell differentiation, IL-2 signaling, Jak-Stat signaling, B-cell receptor signaling, GDP/GTP exchange, and protein recycling were among the functions of the differentially expressed genes that were down-regulated in response to S. enteritidis. The effects of S. enteritidis infection on the liver transcriptome profiles of broilers reflect a predominance of downregulation of genes with cell cycle and metabolic functions. The most pronounced response was the downregulation of genes that function in metabolic pathways, inflammation, and mitochondria-mediated apoptosis. These results provide insight into important systemic metabolic mechanisms that are active in the chicken liver in response to S. enteritidis infection at 10-days postinfection., (Copyright © 2012 Wiley Periodicals, Inc.)

Stearoyl-CoA desaturase 1 (SCD1) is a rate limiting enzyme in the biosynthesis of monounsaturated fatty acids. It has been cloned from several species: Rattus norvegicus, Mus musculus, Homo Sapiens and Gallus gallus, but not from Anser anser. This study was conducted to isolate the SCD1 cDNA sequence and investigate the effect of overfeeding on SCD1 gene tissue expression in Landes goose. The complete cDNA is 3294 bp in length, with an ORF of 1.083 bp encoding a predicted polypeptide of 360 amino acids and 5'/3'-UTR of 74 and 2137 bp, respectively. Quantitative real time PCR (qPCR) was used to examine SCD1 expression in heart, liver, spleen, lung, kidney, gizzard, glandular stomach, intestine, crureus, pectoral muscle, hypothalamus and adipose tissue (abdominal fat) in both the overfed and control group. SCD1 mRNA was highly expressed in goose fatty liver, and the expression levels of SCD1 in liver and fat of overfeeding group were more than double that of the control group. During the overfeeding period, SCD1 expression in liver and adipose tissue reached the highest level after 70 days, but declined at 79 days. In the control group, after fasting 24h, the expression level of SCD1 gene in tissues declined sharply. However, SCD1 gene expression in hypothalamus was unaffected. The results of this study provide a theoretical basis to study the relationship between SCD1 gene expression and the formation of fatty liver of Landes goose in response to overfeeding., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

The biological function of selenium (Se) is mainly elicited through Se-containing proteins. Selenoprotein W (SelW), one member of the selenoprotein family, is essential for the normal function of the skeletal muscle system. To investigate the possible relationship of Se in the process of differentiation in chicken myoblasts and the expression of SelW, the cultured chicken embryonic myoblasts were incubated with sodium selenite at different concentrations for 72 h, and then the mRNA levels of SelW and myogenic regulatory factors (MRFs) in myoblasts were determined at 12, 24, 48, and 72 h, respectively. Furthermore, the correlation between SelW mRNA expression and MRF mRNA expression was assessed. The results showed that the sodium selenite medium enhanced the mRNA expression of SelW, Myf-5, MRF4, and myogenin in chicken myoblasts. The mRNA expression levels of MRFs were significantly correlated with those of SelW at 24, 48, and 72 h. These data demonstrate that Se is involved in the differentiation of chicken embryonic myoblasts, and SelW showed correlation with MRFs.

Chronic inflammation has been linked to cancer. Prostaglandin E₂ (PGE₂) is the most pro-inflammatory lipid and one of the downstream products of 2 isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Ovarian cancer is the most lethal gynecological malignancy and mainly occurs in older women. The factors that contribute to the correlation of age and ovarian cancer are unknown. The purpose of this study was to examine the expression of COX enzymes and PGE₂ levels in ovaries and correlate them to ovarian cancer and aging. White Leghorn hens aged 1, 1.5, 2, 2.5, 3 and 3.5 years were used. The incidence of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE₂ concentrations in ovaries were measured using Western blot, quantitative real-time PCR and ELISA, respectively. Our results indicated an increase in ovarian cancer incidence and expression of both COX enzymes in ovaries of older hens. In correlation with ovarian cancer incidence and COX enzymes expression, PGE₂ concentrations were elevated with age. Ovaries with tumor had elevated COX-1 expression and PGE₂ concentration compared to normal ovaries. Our findings suggest that the up-regulation of COX enzymes with age is the main contributing factor in the age associated increase in PGE₂. Furthermore, elevated PGE₂ in ovaries of hens concomitant with age suggests its important role in early stages of ovarian carcinogenesis. These finding may provide the basis for clinical trials utilizing COX specific inhibitors or dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Repulsive guidance molecules (RGM) are high-affinity ligands for the Netrin receptor Neogenin, and they are crucial for nervous system development including neural tube closure; neuronal and neural crest cell differentiation and axon guidance. Recent studies implicated RGM molecules in bone morphogenetic protein signaling, which regulates a variety of developmental processes. Moreover, a role for RGMc in iron metabolism has been established. This suggests that RGM molecules may play important roles in non-neural tissues., Results: To explore which tissues and processed may be regulated by RGM molecules, we systematically investigated the expression of RGMa and RGMb, the only RGM molecules currently known for avians, in the chicken embryo., Conclusions: Our study suggests so far unknown roles of RGM molecules in notochord, somite and skeletal muscle development., (Copyright © 2012 Wiley Periodicals, Inc.)

The objective of the study was to clone avian beta-defensin (AvBD) 5 gene from pigeon bone marrow tissues and liver tissues, to express the recombinant AvBD5 protein in E. coli, and to determine its antimicrobial activity. The mRNA of duck AvBD5 was cloned from pigeon bone marrow tissues and liver tissues by RT-PCR. In addition, phylogenetic relationships between amino acid sequence of the pigeon AvBD5, AvBDs from other avian species, and some mammalian beta-defensin-5 were analyzed. The cDNA of pigeon AvBD5 was sub-cloned into pGEX-6p-1 vector to construct recombinant plasmid pGEX-pigeon AvBD5. The recombinant protein was expressed into E. coli and purified. Antimicrobial activity and physical-chemical stability of the recombinant fusion protein were measured in vitro. The complete nucleotide sequence of both cDNAs contained 201 bp nucleotides, encoding a polypeptide of 66 amino acids. Both beta-defensins have six conserved cysteines. Phylogenetic relationships were analyzed. Both pigeon AvBDs shared the highest amino acid homology (87.9% and 78.8%) with duck AvBD5. So it was named as pigeon AvBD5alpha (bone marrow) and AvBD5beta (liver). Both recombinant plasmids were transformed into E. coli BL21 and the bacteria were induced with Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). After purification, antibacterial activity of the purified was investigated. In addition, effect of ionic strength on the antibacterial activity, and hemolytic recombinant protein activity of the purified recombinant protein were investigated. A 32 kDa protein was highly expressed. Both purified recombinant pigeon AvBD5alpha and AvBD5beta exhibited extensive antimicrobial activities against 12 bacteria, including Gram-positive and Gram-negative. In high salt ions concentrations, antibacterial activity of both recombinant proteins was decreased. In addition, the hemolysis activity of recombinant protein was extremely low.

Purpose: The molecular mechanisms underlying omphalocele are still largely unknown. Recently, established cadmium (Cd)-induced omphalocele chick model has been used to investigate the pathogenesis of omphalocele. The earliest histologic changes in this model has been observed in somites 4 hours posttreatment, leading us to hypothesize that disruption of migration of somite-derived cells ventrally may cause omphalocele phenotype. Eyes absent (Eya) genes are expressed in the somite (dermomyotome) and play a key role in somitic myogenesis. We designed this study to investigate the hypothesis that Eya1 and Eya2 gene expression is down-regulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model., Methods: After 60 hours of incubation, chicks were exposed to either chick saline or Cd and divided into control and Cd (n = 24 for each group). Chicks were then harvested 1 hour, 4 hours, and 8 hours posttreatment. Real-time quantitative polymerase chain reaction was performed to evaluate gene expression levels of Eya1 and Eya2 in the chick embryo, and they were statistically analyzed. Immunofluorescence confocal microscopy was also performed to evaluate protein expression and distribution pattern of Eya1 and Eya2., Results: At 4 hours posttreatment, the relative messenger RNA expression levels of Eya1 and Eya2 were significantly down-regulated in the Cd group compared with controls (P < .05). The intensity of Eya1 and Eya2 immunofluorescence was also markedly diminished at 4 hours in the Cd-treated embryos, whereas in control embryos, strong intensity of immunofluorescence of them was expressed in the dermomyotomal cells., Conclusion: Down-regulation of Eya genes during the critical period of early embryogenesis may contribute to omphalocele phenotype in the Cd chick model, interfering with migration of embryonic body wall ventrally., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A novel avian β-defensin (AvBD), AvBD10, was discovered in the liver and bone marrow tissues from Chinese painted quail (Coturnix chinensis) in the present study. The complete nucleotide sequence of quail AvBD10 contains a 207-bp open reading frame that encodes 68 amino acids. The quail AvBD10 was expressed widely in all the tissues from quails except the tongue, crop, breast muscle, and thymus and was highly expressed in the bone marrow. In contrast to the expression pattern of AvBD10 in tissues from quail, the chicken AvBD10 was expressed in all 21 tissues from the layer hens investigated, with a high level of expression in the kidney, lung, liver, bone marrow, and Harderian glands. Recombinant glutathione S-transferase (GST)-tagged AvBD10s of both quail and chicken were produced and purified by expression of the two cDNAs in Escherichia coli, respectively. In addition, peptide according to the respective AvBD10s sequence was synthesized, named synthetic AvBD10s. As expected, both recombinant GST-tagged AvBD10s and synthetic AvBD10s of quail and chicken exhibited similar bactericidal properties against most bacteria, including Gram-positive and Gram-negative forms. However, no significant bactericidal activity was found for quail recombinant GST-tagged AvBD10 against Salmonella choleraesuis or for chicken recombinant GST-tagged AvBD10 against Proteus mirabilis., (Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.)

Selenoprotein W (SelW) is an existing form of selenium (Se). Se influences the levels of SelW in mammals. However, little is known about the pattern of SelW expression in the gastrointestinal tract tissue of bird. The present paper describes the effects of different dietary levels of Se on the SelW mRNA expression in the gastrointestinal tract tissue of chicken. The expression levels of SelW mRNA and the Se contents in the gastrointestinal tract tissues (glandular stomach, gizzard, duodenum, small intestine, and rectum) were determined on days 15, 25, 35, 45, and 55, respectively. The results showed that the Se contents and the SelW mRNA expression were significantly higher (p < 0.05) in the high-Se group, and the Se contents and SelW mRNA expression in the low-Se group were significantly lower (p < 0.05) than in the controls. The Se contents were the highest in the duodenum and the lowest in the rectum, while the SelW mRNA expression was the highest in the gizzard and the lowest in the rectum. In addition, the SelW mRNA levels in the gastrointestinal tract tissue were found to increase in a time-dependent manner with increasing feeding time. Furthermore, the expression of the SelW mRNA in the gastrointestinal tract tissues of chickens was found to correlate with the dietary Se concentrations, but not with the tissue Se contents.

The purpose of this study was to investigate the renal and hepatic oxidative damage and toxicity caused by dietary high vanadium in broilers. A total of 420 one-day-old avian broilers were divided into six groups and fed on a corn-soybean basal diet as control diet (vanadium 0.073 mg/kg), and five high vanadium diets (vanadium 5 mg/kg, high vanadium group I; 15 mg/kg, high vanadium group II; 30 mg/kg, high vanadium group III; 45 mg/kg, high vanadium group IV; and 60 mg/kg, high vanadium group V) throughout the experimental period of 42 days. The results showed that the renal and hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, ability to inhibit hydroxy radical, and malondialdehyde (MDA), glutathione, and vanadium contents were not significantly changed in high vanadium group I and II when compared with those of the control groups. However, the SOD and GSH-Px activities, ability to inhibit hydroxy radical, and GSH content were significantly decreased, and the MDA and vanadium contents were markedly increased in high vanadium groups III, IV, and V. At the same time, the lesions were also observed in the kidney and liver of high vanadium groups III, IV, and V. The renal tubular epithelial cells showed granular degeneration and vacuolar degeneration, and hepatocytes showed granular degeneration, vacuolar degeneration, and fatty degeneration. It was concluded that dietary vanadium in the range of 30-60 mg/kg could cause oxidative damage and vanadium accumulation, which induced renal and hepatic toxicity and lesions. The renal and hepatic function was finally impaired in boilers.

Like many other songbird species, male zebra finches learn their song from a tutor early in life. Song learning in birds has strong parallels with speech acquisition in human infants at both the behavioral and neural levels. Forebrain nuclei in the 'song system' are important for the sensorimotor acquisition and production of song, while caudomedial pallial brain regions outside the song system are thought to contain the neural substrate of tutor song memory. Here, we exposed three groups of adult zebra finch males to either tutor song, to their own song, or to novel conspecific song. Expression of the immediate early gene protein product Zenk was measured in the song system nuclei HVC, robust nucleus of the arcopallium (RA) and Area X. There were no significant differences in overall Zenk expression between the three groups. However, Zenk expression in the HVC was significantly positively correlated with the strength of song learning only in the group that was exposed to the bird's own song, not in the other two groups. These results suggest that the song system nucleus HVC may contain a neural representation of a memory of the bird's own song. Such a representation may be formed during juvenile song learning and guide the bird's vocal output.

The avian egg contains all of the necessary nutrients and growth factors required for the developing embryo, including antibodies that are transported from the blood of the hen into the egg yolk to provide immunity to the chick. Since the discovery of egg yolk antibodies, now called immunoglobulin Y (IgY), in the late 1800s, this process has been harnessed to produce antigen-specific yolk antibodies for numerous applications in the medical and research fields, including in areas such as diagnostics and proteomics. However, one of the most valuable and promising areas of IgY research is its use for passive immunization to treat and prevent human and animal diseases. The following review covers the key features and advantages of IgY and the production and purification of IgY from the egg yolk, as well as highlights some of the most promising applications of egg yolk antibodies in human and veterinary medicine.

Peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα) are the master regulators of adipogenesis. The regulatory mechanism of PPARγ and C/EBPα gene expression is clear in mammals, however, little is known in chicken. The aim of the present study was to characterize chicken PPARγ promoter and investigate whether PPARγ could be regulated by C/EBPα in chickens. A 2-kb nucleotide sequence upstream of the start codon of chicken PPARγ gene was cloned and characterized by using bioinformatics and experimental approaches. This 2-kb promoter region exhibited strong promoter activity in DF1 cells. The reporter gene assay showed that the chicken C/EBPα could activate PPARγ gene promoter. Further study by electrophoretic mobility shift assay and mutational analysis revealed that the chicken C/EBPα could directly bind to and regulate the PPARγ gene promoter. Our results demonstrate that PPARγ can be directly regulated by C/EBPα in chickens., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The molecules involved in vertebrate tendon formation during development remain largely unknown. To date, only two DNA-binding proteins have been identified as being involved in vertebrate tendon formation, the basic helix-loop-helix transcription factor Scleraxis and, recently, the Mohawk homeobox gene. We investigated the involvement of the early growth response transcription factors Egr1 and Egr2 in vertebrate tendon formation. We established that Egr1 and Egr2 expression in tendon cells was correlated with the increase of collagen expression during tendon cell differentiation in embryonic limbs. Vertebrate tendon differentiation relies on a muscle-derived FGF (fibroblast growth factor) signal. FGF4 was able to activate the expression of Egr genes and that of the tendon-associated collagens in chick limbs. Egr gene misexpression experiments using the chick model allowed us to establish that either Egr gene has the ability to induce de novo expression of the reference tendon marker scleraxis, the main tendon collagen Col1a1, and other tendon-associated collagens Col3a1, Col5a1, Col12a1, and Col14a1. Mouse mutants for Egr1 or Egr2 displayed reduced amounts of Col1a1 transcripts and a decrease in the number of collagen fibrils in embryonic tendons. Moreover, EGR1 and EGR2 trans-activated the mouse Col1a1 proximal promoter and were recruited to the tendon regulatory regions of this promoter. These results identify EGRs as novel DNA-binding proteins involved in vertebrate tendon differentiation by regulating type I collagen production.

A balance between proteolytic activity and protease inhibition is required to maintain the appropriate function of biological systems in which proteases play a role. The Myeloid and Erythroid Nuclear Termination protein, MENT, is a nonhistone heterochromatin-associated serpin that is an effective inhibitor of the papain-like cysteine proteases. Our laboratories have extensively investigated the dual functions of this protein, namely, chromatin condensation and protease inhibition. Unlike other serpins to date, MENT contains a unique insertion between the C- and D-helices known as the "M-loop." This loop contains two critical functional motifs that allow the nuclear function of MENT, namely, nuclear localization and DNA binding. However, the nuclear function of MENT is not restricted to the activities of the M-loop alone. In vitro, MENT brings about the dramatic remodeling of chromatin into higher-order structures by forming protein bridges via its reactive center loop. Further, we have determined that in a protease-mediated effect, DNA can act as a cofactor to accelerate the rate at which MENT can inhibit its target proteases. In this chapter, we discuss the isolation of MENT from native chicken blood as well as recombinant protein produced in Escherichia coli. Various techniques including in vitro functional assays and biophysical characterization are explained that can be used to elucidate the ability of the protein to interact with DNA and other deoxynucleoprotein complexes. In situ chromatin precipitation using natively purified MENT is also detailed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The transcriptional domain of chicken alpha-globin genes was shown to contain the non-globin coding erythroid-specific TMEM8 gene inducible upon terminal differentiation of erythroblasts. Acquirement by the chicken TMEM8 gene of the erythroid-specific expression correlates with its approachment to the cluster of alpha-globin genes as a result of inversion of a 170-kb chromosomal segment. The human TMEM8 gene is located far from the globin genes and is not expressed in erythroblasts. Transcription of the TMEM8 gene and adult globins in differentiated chicken erythroid cells is controlled by alternative activatory hubs sharing two regulatory elements (including the erythroid enhancer). A conclusion is made that in mature erythroblasts these regulatory elements shuttle between two different activatory hubs.

Reelin-expressing Cajal-Retzius (CR) cells are among the earliest generated cells in the mammalian cerebral cortex and are believed to be crucial for both the development and the evolution of a laminated pattern in the pallial wall of the telencephalon. LIM-homeodomain (LIM-hd) transcription factors are expressed during brain development in a highly restricted and combinatorial manner, and they specify regional and cellular identity. We have investigated the expression of the LIM-hd members Lhx1/Lhx2/Lhx5/Lhx6/Lhx9 in the reelin-expressing cells, the pallium, and the regions of origin of CR cells including the cortical hem of 3 amniote species: the mouse, the chick, and the macaque monkey. We found major differences in the combinatorial LIM-hd expression in the marginal zone as well as in the hem. 1) Lhx5 is a "preferential LIM-hd" for CR cells in mammals but not expressed by these cells in chicks. 2) Lhx2 is expressed in the hem of the chick, whereas it is excluded from this region in mouse. 3) Whereas mouse CR cells express Lhx5/Lhx1, their monkey counterparts express 4 of these factors: Lhx1/Lhx2/Lhx5/Lhx9. We discuss our findings in evolutionary terms for the specification of the midline hem and CR cell type and the emergence of the cortical lamination pattern.

Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be produced as a soluble (H3H4)(2) heterotetrameric complex if they are co-expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)(2) can be purified by simple chromatographic techniques and have similar properties to endogenous histones. Our methods should facilitate histone production for studies of chromatin structure and regulatory proteins that interact with histones. We describe a simple strategy for constructing co-expression plasmids, based on the T7 RNA polymerase system, which is applicable to other systems. It offers several advantages for quickly creating plasmids to express two or more proteins and for testing different combinations of proteins for optimal complex production, solubility or activity., (Copyright 2010 Elsevier Inc. All rights reserved.)

Several perfluoroalkyl compounds (PFCs) are ubiquitous environmental contaminants that can biomagnify in species at high trophic levels including wild birds. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been detected in wild birds and are known to reduce hatching success of laboratory-exposed chicken embryos at environmentally relevant concentrations. Limited toxicity data are available regarding avian exposure to PFCs of chain lengths greater than C(8), which are of increasing environmental relevance following the recent phase-out of PFOS and PFOA. In this study, linear PFOA, perfluoroundecanoic acid (PFUdA) and perfluorodecane sulfonate (PFDS) were injected into the air cell of white leghorn chicken eggs (Gallus gallus domesticus) prior to incubation to determine effects on embryo pipping success. Furthermore, mRNA expression of key genes involved in pathways implicated in PFC toxicity was monitored in liver tissue. PFOA, PFUdA or PFDS had no effect on embryonic pipping success at concentrations up to 10 microg/g. All PFCs accumulated in the liver to concentrations greater than the initial whole-egg concentration as determined by HPLC/MS/MS. Hepatic accumulation was highest for PFOA (4.5 times) compared to PFUdA and PFDS. Cytochrome P450 1A4 and liver fatty acid binding protein mRNA expression increased after exposure to PFUdA but was only statistically significant at 10 microg/g; several orders of magnitude higher than levels found in wild bird eggs. Based on the present results for white leghorn chickens, current environmental concentrations of PFOA, PFUdA and PFDS are unlikely to affect the hatching success of wild birds.

Transforming growth factor beta (TGFbeta) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGFbeta signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGFbetas shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved down-regulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGFbeta signaling up-regulates Sox9 in the in vivo experimental model system in which TGFbeta treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGFbeta signaling, we found that TGFbetas appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGFbeta signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGFbeta signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGFbeta treatments.

In this study we investigated the effect of a single-compound exposure or two compound co-exposure to tetrachlorodibenzo-p-dioxin (TCDD) plus perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA) on the mRNA expression of cytochromes P450 (CYP) 1A4, 4V2 and 3A37, ethoxyresorufin-O-deethylase (EROD) activity and cell viability in chicken (Gallus gallus domesticus) embryo primary hepatocyte cultures. Cell viability after 24 h of incubation was significantly decreased in cells exposed to PFOS at concentrations between 30 microM and 60 microM with or without co-exposure to TCDD (0.3 nM at maximum). PFOA did not decrease cell viability even at maximum concentrations of 60 microM. TCDD induced CYP1A4 mRNA and EROD activity substantially as reported previously. PFOS also increased CYP1A4 mRNA in a concentration-dependent manner. Co-exposure of cells to PFOS plus TCDD did not change CYP1A4 mRNA levels compared to cells treated with TCDD alone. PFOS alone did not induce CYP4V2 mRNA, however 40-50 microM PFOS plus TCDD (0.3 nM) induced CYP4V2 mRNA compared to TCDD alone (P<0.05). This trend was similar to that observed with co-exposure to TCDD plus PFOA, suggesting that PFOA alone did not induce CYP4V2 mRNA, whereas co-exposure to TCDD plus PFOA induced the expression levels. PFOS alone decreased CYP3A37 mRNA by a maximum of 45%, however after co-exposure to TCDD, recovery of mRNA expression to levels measured in DMSO-treated cells was observed. Our data suggest a complex gene response to mixtures of dioxin-like and perfluorinated compounds.

Signal relay by guidance receptors at the axonal growth cone is a process essential for the assembly of a functional nervous system. We investigated the in vivo function of Src family kinases (SFKs) as growth cone guidance signaling intermediates in the context of spinal lateral motor column (LMC) motor axon projection toward the ventral or dorsal limb mesenchyme. Using in situ mRNA detection we determined that Src and Fyn are expressed in LMC motor neurons of chick and mouse embryos at the time of limb trajectory selection. Inhibition of SFK activity by C-terminal Src kinase (Csk) overexpression in chick LMC axons using in ovo electroporation resulted in LMC axons selecting the inappropriate dorsoventral trajectory within the limb mesenchyme, with medial LMC axon projecting into the dorsal and ventral limb nerve with apparently random incidence. We also detected LMC axon trajectory choice errors in Src mutant mice demonstrating a nonredundant role for Src in motor axon guidance in agreement with gain and loss of Src function in chick LMC neurons which led to the redirection of LMC axons. Finally, Csk-mediated SFK inhibition attenuated the retargeting of LMC axons caused by EphA or EphB over-expression, implying the participation of SFKs in Eph-mediated LMC motor axon guidance. In summary, our findings demonstrate that SFKs are essential for motor axon guidance and suggest that they play an important role in relaying ephrin:Eph signals that mediate the selection of motor axon trajectory in the limb.

N-myc downstream-regulated genes 1 and 3 (NDRG1 and NDRG3) are members of the alpha/beta hydrolase superfamily. Phylogenetic analysis of the family demonstrated that human NDRG1 and 3 belong to a subfamily. The mapping and gene expression patterns of these genes represent one step toward further investigation into their possible roles in the chicken (Gallus gallus). To map these genes in the chicken chromosome, a 6000 rads chicken-hamster radiation hybrid panel (ChickRH6) was used. Primers were designed according to the published human sequences for amplification of those two genes. We compared the corresponding human mRNA sequences with the predicted coding sequences of the chicken NDRG1 and 3 genes and found that the assembled contigs shared a high percentage of similarity with the human genes. PCR of samples from ChickRH6 revealed that the locations of NDRG1 and 3 are linked to the markers MYC (58 cRs away, LOD score 4.52) and SEQ0265 (10 cRs away, LOD score 17.81), respectively. This result adds two new markers to the chicken RH map, and it reinforces that the RH technique is indeed a powerful tool for mapping genes due to its rapidity, precision, convenience, and reproducibility. In addition, we detected the gene expression and distribution of chicken NDRG1 and 3 in seven tissues, including heart, liver, spleen, lung, muscle, brain, and thymus, by RT-PCR, and found that NDRG1 is relatively ubiquitously expressed in all the tested tissues and highly expressed in heart and liver, whereas NDRG3 is high in heart, muscle, and brain.

The otic placode generates the auditory and vestibular sense organs and their afferent neurons; however, how auditory and vestibular fates are specified is unknown. We have generated a fate map of the otic placode and show that precursors for vestibular and auditory cells are regionally segregated in the otic epithelium. The anterior-lateral portion of the otic placode generates vestibular neurons, whereas the posterior-medial region gives rise to auditory neurons. Precursors for vestibular and auditory sense organs show the same distribution. Thus, different regions of the otic placode correspond to particular sense organs and their innervating neurons. Neurons from contiguous domains rarely intermingle suggesting that the regional organisation of the otic placode dictates positional cues to otic neurons. But, in addition, vestibular and cochlear neurogenesis also follows a stereotyped temporal pattern. Precursors from the anterior-lateral otic placode delaminate earlier than those from its medial-posterior portion. The expression of the proneural genes NeuroM and NeuroD reflects the sequence of neuroblast formation and differentiation. Both genes are transiently expressed in vestibular and then in cochlear neuroblasts, while differentiated neurons express Islet1, Tuj1 and TrkC, but not NeuroM or NeuroD. Together, our results indicate that the position of precursors within the otic placode confers identity to sensory organs and to the corresponding otic neurons. In addition, positional information is integrated with temporal cues that coordinate neurogenesis and sensory differentiation.

The TOJ3 gene was originally identified on the basis of its specific activation in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17). Overexpression of TOJ3 induces cellular transformation of embryonic avian fibroblasts, revealing an intrinsic oncogenic potential. Transforming activity has also been demonstrated for MSP58, the human homolog of TOJ3, and oncogenic cell transformation by MSP58 is specifically inhibited by the tumor suppressor PTEN. To investigate the mechanism of aberrant TOJ3 gene activation in jun-transformed fibroblasts, the entire quail TOJ3 gene including 13 exons and the 5' regulatory region was isolated. Functional analyses of the promoter by transcriptional transactivation assays revealed that the specific induction of TOJ3 is mediated by a cluster of three noncanonical AP-1 binding motifs (5'-CAGCTCA-3' or 5'-CACCTCA-3') which share the 3' half-site with the consensus motif (5'-TGA(C)/(G)TCA-3'). Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses showed that Jun binds to these motifs with an affinity similar to that observed for binding to an AP-1 consensus site. Noncanonical binding sites are also present in the chicken and human TOJ3/MSP58 promoter regions. These results confirm and extend the previous observation that TOJ3 represents an immediate effector gene of Jun and may point to an essential role of TOJ3/MSP58 in carcinogenesis involving aberrant AP-1 expression.

The Caenorhabditis elegans heterochronic gene lin-28 regulates developmental timing in the nematode trunk. We report the dynamic expression patterns of Lin-28 homologues in mouse and chick embryos. Whole mount in situ hybridization revealed specific and intriguing expression patterns of Lin-28 in the developing mouse and chick limb bud. Mouse Lin-28 expression was detected in both the forelimb and hindlimb at E9.5, but disappeared from the forelimb at E10.5, and finally from the forelimb and hindlimb at E11.5. Chicken Lin-28, which was first detected in the limb primordium at stage 15/16, was also downregulated as the stage proceeded. The amino acid sequences of mouse and chicken Lin-28 genes are highly conserved and the similar expression patterns of Lin-28 during limb development in mouse and chicken suggest that this heterochronic gene is also conserved during vertebrate limb development.

Activation-induced deaminase (AID) is thought to initiate somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR) by the transcription-coupled deamination of cytosine residues in Ig genes. Phosphorylation of AID by protein kinase A (PKA) and subsequent interaction of AID with replication protein A (RPA) have been proposed to play important roles in allowing AID to deaminate DNA during transcription. Serine 38 (S38) of mouse AID is phosphorylated in vivo and lies in a consensus target site for PKA, and mutation of this residue interferes with CSR and SHM. In this study, we demonstrate that S38 in mouse and chicken AID is phosphorylated in chicken DT40 cells and is required for efficient GCV and SHM in these cells. Paradoxically, zebra fish AID, which lacks a serine at the position corresponding to S38, has previously been shown to be active for CSR and we demonstrate that it is active for GCV/SHM. Aspartate 44 (D44) of zebra fish AID has been proposed to compensate for the absence of the S38 phosphorylation site but we demonstrate that mutation of D44 has no effect on GCV/SHM. Some features of zebra fish AID other than D44 might compensate for the absence of S38. Alternatively, the zebra fish protein might function in a manner that is independent of PKA and RPA in DT40 cells, raising the possibility that, under some circumstances, AID mediates efficient Ig gene diversification without the assistance of RPA.

In response to overfeeding, geese develop fatty liver. To understand the fattening mechanism, mRNA differential display reverse transcription PCR was used to study the gene expression differences between French Landes grey geese and Xupu white geese in conditions of overfeeding and normal feeding. One gene was found to be up-regulated in the fatty liver in both breeds, and it has a 1797 bp cDNA with 83% identity to chicken SELENBP1. The sequence analysis revealed that its open reading frame of 1413 bp encodes a protein of 471 amino acids, which contains a putative conserved domain of 56 kDa selenium binding protein with high homology to its homologues of chicken (95%), rat (86%), mouse (84%), human (86%), monkey (86%), dog (86%), and cattle (86%). The function of this protein has been briefly reviewed based on published information. In tissue expression analysis, the expression of geese SELENBP1 mRNA was found to be higher in liver or kidney than in other tested tissues. The results showed that overfeeding could increase the mRNA expression level of geese SELENBP1.

Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.

Regulation of VEGFR-2 (Quek1) is an important mechanism during blood vessel formation. In the paraxial mesoderm, Quek1 expression is restricted to the lateral portion of the somite and later to sclerotomal cells surrounding the neural tube. By implanting FGF 8b/8c or SU 5402 beads into the paraxial mesoderm, we show that FGF8 in addition to BMP4 from the intermediate mesoderm (IM) is a positive regulator of VEGFR-2 (Quek1) expression in the quail embryo. The expression of Quek1 in the medial somite half is normally repressed by the notochord and Sfrps-expression in the neural tube. Over-expression of Wnt 1/3a also results in an up-regulation of Quek1 expression in the somites. We also show that up-regulation of FGF8/Wnt 1/3a leads to an increase in the number of endothelial cells, whereas inhibition of FGF and Wnt signaling by SU 5402 and Sfrp-2 results in a loss of endothelial cells. Our results demonstrate that the regulation of Quek1 expression in the somites is mediated by the cooperative actions of BMP4, FGF8 and Wnt-signaling pathways.

Ig hypermutation is limited to a region of approximately 2 kb downstream of the transcription start sites of the Ig loci. The process requires transcription and the presence of Ig enhancer sequences, and is initiated by the activation-induced cytidine deaminase (AID)-mediated deamination of cytidine bases. It remains unknown why AID causes mutations selectively in the Ig genes and not in most other transcribed loci of B cells. In this study, we report that the inactivation of the E2A gene strongly reduces the rate of Ig L chain mutations in the chicken B cell line DT40 without affecting the levels of surface Ig or AID expression. The defect is complemented by the expression of cDNAs corresponding to either of the two E2A splice variants E12 or E47. The results suggest that E2A-encoded proteins enhance Ig hypermutation by recruitment of AID to the Ig loci.

Tenascins are glycoproteins found primarily in the embryonic extracellular matrix. Here we have characterized the fourth and final member of the tenascin family in birds: tenascin-W. Avian tenascin-W has 3.5 epidermal growth factor-like repeats, 6 fibronectin type III domains, and a C-terminal fibrinogen-related domain. Immunohistochemistry reveals that avian tenascin-W is expressed transiently in developing smooth muscle, tendons, and ligaments, but the primary site of tenascin-W expression during development is in the extracellular matrix of bone and the cellular periosteum. In bony matrix, tenascin-W-coated fibrils partly overlap with fibrils that contain tenascin-C. The anti-tenascin-W also labels fibrils in cultures of osteogenic embryonic chicken calvarial cells. Primary calvarial cells cultured on purified tenascin-W become rounded, and fewer of these cells spread on fibronectin when tenascin-W is added to the medium when compared with calvarial cells cultured on fibronectin alone. Moreover, tenascin-W reduces the adhesion of calvarial cells to collagen type I in a shear force assay. We conclude that tenascin-W is likely to play a phylogenetically conserved role in developing bone and that it shares some of the basic anti-adhesive and matrix modulatory properties as tenascin-C., (Developmental Dynamics 235:1532-1542, 2006. (c) 2006 Wiley-Liss, Inc.)

Development of the lip and palate involves a complex series of events that are frequently disturbed resulting in the congenital anomalies cleft lip and cleft palate. Van der Woude syndrome (VWS) is an autosomal dominant disorder that is characterised by cleft lip, cleft palate, lower lip pits, and hypodontia. VWS arises as the result of mutations in the gene encoding interferon regulatory factor 6 (IRF6). To provide insights into the role of IRF6 during embryogenesis, we have analysed the expression of this molecule during mouse and chick facial development. Irf6 was expressed in the ectoderm covering the facial processes during their fusion to form the upper lip and primary palate in both mouse and chick. However, while Irf6 was expressed in the medial edge epithelia of the developing secondary palate of the mouse, which fuses as in man, Irf6 was not expressed in the medial edge epithelia of the naturally cleft chick secondary palate. Similarly, Irf6 was found to be down-regulated in the medial edge epithelia of transforming growth factor beta3-null mice, which also exhibit cleft palate. Together, these results support a role for IRF6 during the fusion events that occur during development of the lip and palate., ((c) 2005 Wiley-Liss, Inc.)

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, yet there is no understanding of the role of BMP4 in this process. The present work was aimed at exploring the effects of BMP-signaling on the development of hair-cells. For this purpose, we studied gene expression, cell proliferation and cell death in isolated chick otic vesicles that were grown in vitro in the presence of recombinant BMP4 or the BMP-inhibitor Noggin. Cath1 was used as a marker for hair-cell specification. BMP4 reduced the number of Cath1-cells and, conversely, Noggin increased the size of the sensory patches and the number of Cath1-positive cells. The effect of BMP4 was irreversible and occurred before hair-cell specification. Lfng and Fgf10 were expressed in the prosensory domain before Cath1, and their expression was expanded by Noggin. At these stages, modifications of BMP activity did not respecify non-sensory epithelium of the otic vesicle. The expression of Bmp4 at sensory patches was suppressed by BMP4 and induced by Noggin suggesting an autoregulatory loop. Analysis of BrdU incorporation during 6 and 18 h indicated that the effects of BMP4 were due to its ability to reduce the number of actively proliferating progenitors and inhibit cell fate specification. BMP4 induced cell death within the prosensory domain of the otic vesicle, along with the expression of Msx1, but not Msx2. On the contrary, BMP-inhibition with Noggin favored hair-cell specification without changes in the overall cell proliferation. We propose that about the stage of terminal division, the balance between BMP and BMP-inhibitory signals regulates survival and specification of hair-cell precursors, the final number of sensory hair-cells being limited by excess levels of BMPs. The final size of sensory patches would hence depend on the balance between BMP4 and opposing signals.

Tetrapod limbs, forelimbs and hindlimbs, emerge as limb buds during development from appropriate positions along the rostro-caudal axis of the main body. In this study, tissue interactions by which rostro-caudal level-specific limb initiation is established were analyzed. The limb bud originates from the lateral plate located laterally to the paraxial mesoderm, and we obtained evidence that level-specific tissue interactions between the paraxial mesoderm and the lateral plate mesoderm are important for the determination of the limb-type-specific gene expression and limb outgrowth. When the wing-level paraxial mesoderm was transplanted into the presumptive leg region, the wing-level paraxial mesoderm upregulated the expression of Tbx5, a wing marker gene, and down regulated the expression of Tbx4 and Pitx1, leg marker genes, in the leg-level lateral plate. The wing-level paraxial mesoderm relocated into the leg level also inhibited outgrowth of the hindlimb bud and down regulated Fgf10 and Fgf8 expression, demonstrating that the wing-level paraxial mesoderm cannot substitute for the function of the leg-level paraxial mesoderm in initiation and outgrowth of the hindlimb. The paraxial mesoderm taken from the neck- and flank-level regions also had effects on Tbx5/Tbx4 expression with different efficiencies. These findings suggest that the paraxial mesoderm has level-specific abilities along the rostro-caudal axis in the limb-type-specific mechanism for limb initiation.

Gallinacins (Gal) are antimicrobial peptides that play significant roles in innate immunity in chickens. The aim of this study was to examine whether age of birds and egg-laying activity (laying and non-laying caused by feed-regulation) affect the mRNA expression of Gal-1, -2, and -3 in the vagina of hens, and whether their expressions are changed in response to the stimulation with Salmonella enteritidis (SE) and lipopolysaccharide (LPS). White Leghorn hens were divided into groups of young and old laying hens, and groups of laying and non-laying hens after feed-regulation. Vaginal cells were cultured and stimulated with SE or LPS. Expressions of Gal-1, -2, and -3 mRNA in their vaginal mucosa and cultured cells were examined by quantitative real-time RT-PCR. The expressions of Gal-1, -2, and -3 of the vaginal mucosa were significantly greater in old birds than in young birds. Expression of these Gals in the vagina were decreased in the regressed oviduct of non-laying birds compared with laying birds. The expressions of Gal-1, -2, and -3 in the cultured vaginal cells were increased by stimulation with SE or LPS within 24 h. These results suggest that the mRNA expressions of Gal-1, -2 and -3 in the vagina of laying hens increased with age, whereas they decreased in the regressed oviduct during the non-laying phase. Also, synthesis of these antimicrobial peptides in the vagina may increase in response to SE and LPS to eliminate SE bacteria.

Serum is believed to harbor thousands of distinct proteins that are either actively secreted or leak from various blood cells or tissues. Exploring protein composition in serum may accelerate the discovery of novel protein biomarkers for specific economic traits in livestock species. This study analyzed serum protein composition to establish a 2-DE reference map, and monitored protein dynamics of single-comb White Leghorn hens at 8, 19 and 23 weeks after hatching. A total of 119 CBB-stained and 315 silver-stained serum protein spots were analyzed by MALDI-TOF MS. Of these, 98 CBB-stained and 94 silver-stained protein spots were significantly matched to existing chicken proteins. The identified spots represented 30 distinctive proteins in the serum of laying hens. To compare protein expression during development, expression levels of 47 protein spots were quantified by relative spot volume with Melanie 3 software. Ten protein spots increased and 3 protein spots decreased as hen age increased. Previous research has suggested that some of these proteins play critical roles in egg production. The differentially expressed proteins with unknown identities will be valuable candidates for further explorations of their roles in egg production of laying hens.

Photooxidized tryptophan (TRP) in tissue culture medium elicits a transient cytochrome P450 (CYP1) induction response in cultured cells. We show here that exposure of TRP to window sunlight (aTRP) greatly increased the potency, efficacy, and duration of CYP1A induction by TRP in primary chick embryo hepatocytes and in vivo. Aqueous TRP exposed to sunlight for 7 days exhibited a 100-fold or greater increase in potency over TRP in medium. The induction response was sustained for at least 48 h and was comparable in efficacy to 2,3,7,8-tetrachlorodibenzo-p-dioxin. In hepatocytes, increases in mRNAs for CYP1A4 and CYP1A5, chick orthologs of mammalian CYP1A1 and 1A2, preceded increases in CYP1A proteins and enzyme activities, 7-ethoxyresorufin deethylase (EROD) for CYP1A4 and arachidonic acid epoxygenation for CYP1A5, consistent with a transcriptional mechanism. Aryl hydrocarbon receptor (AhR) dependence was evidenced by aTRP induction of EROD in wild-type Hepa1c1c7 cells but not in AhR-defective (c35) mutants. Preparations of aTRP were stable for many months at 4 degrees C and were relatively resistant to metabolism by hepatocytes or liver microsomes. Fractionation of aTRP by HPLC analysis coupled with EROD assays showed that aTRP contained multiple photoproducts and CYP1A inducing components, which varied in sensitivity to metabolism by hepatocytes. The previously identified TRP photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ), was one component, but FICZ was not required for CYP1A induction by the aTRP mixture. These findings identify the indoor environment, and window sunlight in particular, as a new source of CYP1A inducers. Further, the evidence that biologically active metabolites of an endogenous substrate, arachidonic acid, are formed by aTRP-induced CYP1A provides a pathway by which TRP photoproducts, like toxic xenobiotics, could have significant physiologic effects.

An increasing number of axon guidance cues have been shown recently to play important roles in the development of non-neural tissues. Semaphorins comprise one of the largest conserved families of axon guidance factors. We analyzed the expression patterns of Sema3D, Sema3F, and Sema5A genes in the chick embryonic heart by in situ hybridization. All three genes are expressed in the cardiac cushion regions, both in the mesenchymal cells, and epithelial cells in the endocardial layer, during the period of cardiac remodeling. In addition to the overlapping expression patterns in the cardiac cushion regions, these genes also exhibit distinct expression patterns in the developing heart: Sema3D is additionally expressed in the tips of the ventricular trabeculae; Sema3F is expressed in a subset of cells scattered throughout the ventricles; and Sema5A is expressed in the newly formed atrioventricular valves. The overlapping and distinct expression patterns of these genes suggest that they may play important roles in heart development., (2005 Wiley-Liss, Inc.)

The archetypal genomic arrangement of vertebrate Dlx genes is as three bigene clusters (Dlx1/2, Dlx3/4, Dlx5/6). Phylogenetic sequence analysis of mouse and zebrafish Dlx clusters supports the notion that the Dlx3/4 cluster is more derived and the absence of expression of either Dlx3 or Dlx4 in the central nervous system, as reported to date, is consistent with this. Together, these observations have prompted a model in which cis-regulatory elements, responsible for directing Dlx gene transcription in the forebrain, were lost from the Dlx3/4 bigene cluster prior to the divergence of tetrapods from fish. Here, we describe Dlx3 expression in the forebrain of chicken embryos; this constitutes the first documented evidence of expression of either Dlx3 or Dlx4 in the central nervous system of a vertebrate. Our observations have implications for models of the evolutionary history of the Dlx gene family, for the genomic organization of Dlx genes in birds and for functional redundancy of Dlx gene function during avian forebrain development.

Nkx2-5 gene mutations cause cardiac abnormalities, including deficits of function in the atrioventricular conduction system (AVCS). In the chick, Nkx2-5 is elevated in Purkinje fiber AVCS cells relative to working cardiomyocytes. Here, we show that Nkx2-5 expression rises to a peak as Purkinje fibers progressively differentiate. To disrupt this pattern, we overexpressed Nkx2-5 from embryonic day 10, as Purkinje fibers are recruited within developing chick hearts. Overexpression of Nkx2-5 caused inhibition of slow tonic myosin heavy chain protein (sMHC), a late Purkinje fiber marker but did not affect Cx40 levels. Working cardiomyocytes overexpressing Nkx2-5 in these hearts ectopically up-regulated Cx40 but not sMHC. Isolated embryonic cardiomyocytes overexpressing Nkx2-5 also displayed increased Cx40 and suppressed sMHC. By contrast, overexpression of a human NKX2-5 mutant did not effect these markers in vivo or in vitro, suggesting one possible mechanism for clinical phenotypes. We conclude that a prerequisite for normal Purkinje fiber maturation is precise regulation of Nkx2-5 levels., (2005 Wiley-Liss, Inc.)

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent OFT septation are critical events during vertebrate heart development. We previously had performed four modified differential display (DD) screens in the chick embryo to identify genes that may be involved in CNC and heart development. Full-length sequence of one of the DD clones has been obtained and identified as chick PINCH-1. This particularly interesting new cysteine-histidine-rich protein contains five protein-binding LIM domains (five double zinc fingers), a nuclear localization signal, and a nuclear export signal, allowing it to participate in integrin and growth factor signaling and possibly act as a transcription factor. We show here for the first time that chick PINCH-1 is expressed in neural crest cells, both in the neural fold and cardiac OFT, and is also expressed in mesoderm derived-structures, including the myocardium, during avian embryogenesis. The normal expression pattern and overexpression in neural crest cell explants suggest that PINCH-1 may be a regulator of neural crest cell adhesion and migration., (2005 Wiley-Liss, Inc.)