Search

Understanding the mechanism of stem cell maintenance underlies the establishment of long-term and mass culture methods for stem cells that are fundamental for clinical and agricultural applications. In this study, we use chicken primordial germ cell (PGC) as a model to elucidate the molecular mechanisms underlying stem cell maintenance. The PGC is a useful experimental model because it is readily gene-manipulatable and easy to test gene function in vivo using transplantation. Previous studies to establish a long-term culture system have shown that secreted factors such as FGF2 are required to maintain the self-renewal capability of PGC. On the other hand, we know little about intracellular regulators responsible for PGC maintenance. Among representative stem cell factors, we focus on RNA-binding factors LIN28A and LIN28B as possible central regulators for the gene regulatory network essential to PGC maintenance. By taking advantage of the CRISPR/Cas9-mediated gene editing and a clonal culture technique, we find that both LIN28A and LIN28B regulate the proliferation of PGC in vitro. We further showed that colonization efficiency of grafted PGC at the genital ridges, rudiments for the gonads, of chicken embryos were significantly decreased by knockout (KO) of LIN28A or LIN28B. Of note, overexpression of human LIN28 in LIN28-KO PGC was sufficient to restore the low colonization rates, suggesting that LIN28 plays a key role in PGC colonization at the gonads. Transcriptomic analyses of LIN28-KO PGC reveal that several genes related to mesenchymal traits are upregulated, including EGR1, a transcription factor that promotes the differentiation of mesodermal tissues. Finally, we show that the forced expression of human EGR1 deteriorates replication activity and colonization efficiency of PGCs. Taken together, this work demonstrates that LIN28 maintains self-renewal of PGC by suppressing the expression of differentiation genes including EGR1., Competing Interests: Declaration of competing interest No conflicts of interest are declared., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior-posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman's layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles.

Initial nutritional stimulation is a key driving force for small intestinal maturation. In chick embryos, administration of l-glutamine (Gln) into the amniotic fluid stimulates early development of the small intestinal epithelium by promoting enterocyte differentiation. In this study, we evaluated the effects of intra-amniotic administration of Gln on enterocyte morphology and function, and elucidated a potential enteroendocrine pathway through which Gln stimulates small intestinal maturation. Our results show that Gln stimulation at embryonic day 17 significantly increased enterocyte and microvilli dimensions by 10 and 20%, respectively, within 48 h. Post-hatch, enterocytes and microvilli were 20% longer in Gln-treated chicks. Correspondingly, Gln stimulation significantly upregulated mRNA expression of brush border nutrient transporters PepT-1 and SGLT-1 and tight junction proteins TJP-1 and TJP-2, before and after hatch (P < 0.05). Since GLP-2 signaling from intestinal L-cells is associated with enterocyte growth, functionality and integrity, we examined the effects of Gln stimulation on mRNA expression of key hormones and receptors within this enteroendocrine pathway and found significant increases in GLP-2R, IGF-1 and IGF-1R expression before and after hatch (P < 0.05). In conclusion, our findings link primary nutrient stimulation in the developing small intestine with enterocyte morphological and functional maturation and enteroendocrine signaling., (© 2022. The Author(s).)

Dysregulation of the immune system is associated with many pathologies, including cardiovascular diseases, diabetes, and cancer. To date, the most commonly used models in biomedical research are rodents, and despite the various advantages they offer, their use also raises numerous drawbacks. Recently, another in vivo model, the chicken embryo and its chorioallantoic membrane, has re-emerged for various applications. This model has many benefits compared to other classical models, as it is cost-effective, time-efficient, and easier to use. In this review, we explain how the chicken embryo can be used as a model for immune-based studies, as it gradually develops an embryonic immune system, yet which is functionally similar to humans'. We mainly aim to describe the avian immune system, highlighting the differences and similarities with the human immune system, including the repertoire of lymphoid tissues, immune cells, and other key features. We also describe the general in ovo immune ontogeny. In conclusion, we expect that this review will help future studies better tailor their use of the chicken embryo model for testing specific experimental hypotheses or performing preclinical testing., Competing Interests: PG, YW, and JV are employees of Inovotion. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Garcia, Wang, Viallet and Macek Jilkova.)

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galβ1-4(SO 3 - -6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galβ1-4(SO 3 - -6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Temporal expression patterns and activity of two cyclooxygenase (COX-1 and COX-2) isoforms were analysed during early chick embryogenesis to evaluate their roles in development. COX-2 inhibition with etoricoxib resulted in significant structural anomalies such as anophthalmia (born without one or both eyes), phocomelia (underdeveloped or truncated limbs), and gastroschisis (an opening in the abdominal wall), indicating its significance in embryogenesis. Furthermore, the levels of PGE 2 , PGD 2 , PGF 2α , and TXB 2 were assessed using quantitative LC-MS/MS to identify which effector prostanoid (s) had their synthesis initiated by COX-2. COX-2 inhibition was only shown to reduce the level of PGE 2 significantly , and hence it could be inferred that the later could be largely under the regulation of activated COX-2 in chick embryos. The compensatory increase in the activity of COX-1 observed in the etoricoxib-treated group helped to maintain the levels of PGD 2 , PGF 2α , and TXB 2 . Though the roles of these three prostanoids in embryogenesis need to be further clarified, it appears that their contribution to the observed developmental anomalies is minimal. This study has shown that COX-2 is functionally active during chick embryogenesis, and it plays a central role in the structural configuration of several organs and tissues through its downstream effector molecule PGE 2 ., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Human pluripotent stem cells (hPSCs) are commonly kept in a primed state but also able to acquire a more immature naive state under specific conditions in vitro. Acquisition of naive state changes several properties of hPSCs and might affect their contribution to embryonic development in vivo. However, the lack of an appropriate animal test system has made it difficult to assess potential differences for chimera formation between naive and primed hPSCs. Here, we report that the developing chicken embryo is a permissive host for hPSCs, allowing analysis of the pluripotency potential of hPSCs. Transplantation of naive-like and primed hPSCs at matched developmental stages resulted in robust chimerism. Importantly, the ability of naive-like but not of primed hPSCs to form chimera was substantially reduced when injected at non-matched developmental stages. We propose that contribution to chick embryogenesis is an informative and versatile test to identify different pluripotent states of hPSCs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Egg fertilization is a dynamic process, including varieties of biochemical changes. To better understand the molecular mechanisms during the egg embryo development, the objective of this study was to quantify protein expression changes between fertilized and unfertilized Beijing-You chicken eggs using label-free liquid chromatography-tandem mass spectrometry method. The results showed that a total of 1241 proteins were identified from fertilized and unfertilized eggs, 229 proteins were observed difference in fertilized eggs (p < 0.05) compared with that in unfertilized eggs. The expressions of 86 proteins were up-regulated and 48 proteins were down-regulated in fertilized eggs. STRING database analysis and Gene Ontology analysis results showed that these differentially expressed proteins significantly interacted and were involved in lipid transport and inflammatory response biological processes. The mRNA and protein expression levels of most differentially expressed proteins Apolipoprotein B, Fibrinogen alpha chain, Transferrin receptor protein 1, Phospholipid transfer protein and Vimentin were validated by RT-PCR and western blot. These results could provide possible novel insights for the molecular mechanism of egg fertilization., Competing Interests: Declaration of Competing Interest The authors declare that no competing interests., (Copyright © 2019. Published by Elsevier B.V.)

The prethalamic eminence (PThE) is the most dorsal subdomain of the prethalamus, which corresponds to prosomere 3 (p3) in the prosomeric model for vertebrate forebrain development. In mammalian and avian embryos, the PThE can be delimited from other prethalamic areas by its lack of Dlx gene expression, as well as by its expression of glutamatergic-related genes such as Pax6, Tbr2 and Tbr1. Several studies in mouse embryos postulate the PThE as a source of migratory neurons that populate given telencephalic centers. Concerning the avian PThE, it is visible at early embryonic stages as a compact primordium, but its morphology becomes cryptic at perinatal stages, so that its developmental course and fate are largely unknown. In this report, we characterize in detail the ontogeny of the chicken PThE from 5 to 15 days of development, according to morphological criteria, and using Tbr1 as a molecular marker for this structure and its migratory cells. We show that initially the PThE contacts rostrally the medial pallium, the pallial amygdala and the paraventricular hypothalamic alar domain. Approximately from embryonic day 6 onwards, the PThE becomes progressively reduced in size and cell content due to massive tangential migration of many of its neuronal derivatives towards nearby subpallial and hypothalamic regions. Our analysis supports that these migratory neurons from the avian PThE target telencephalic centers such as the commissural septal nuclei, as previously described in mammals, but also the diagonal band and preoptic areas, and hypothalamic structures in the paraventricular hypothalamic area.

Testosterone (T) is essential for muscle fiber formation and growth. However, the specific mechanism by which T regulates skeletal muscle development in chicken embryos remains unclear. In this study, the role of T in myoblast proliferation both in vivo and in vitro was investigated. Results showed that the T administration significantly increased the ratio of breast muscle and leg muscle. T induced a significant increase in the cross-sectional area (CSA) and density of myofiber and the ratio of PAX7-positive cells in the skeletal muscle. Exogenous T also induced the upregulation of myogenic regulatory factors (MRFs) and cyclin-dependent kinases ( CDK2 ) /Cyclin D1 (CCND1) and protein levels of androgen receptor (AR), p-Akt and PAX7. Furthermore, T treatment significantly promoted myoblasts cultured in vitro entering a new cell cycle and increased PAX7-positive cells. The mRNA and protein expression of AR and PAX7 were upregulated when treated with T compared to that of the control. The addition of T induced proliferation accompanied by increasing AR level as well as PI3K (Phosphoinositide 3-kinase)/Akt activation. However, T-induced proliferation was attenuated by AR, PI3K, and Akt-specific inhibitors. These data indicated that the pro-proliferative effect of T was regulated though AR in response to the activation of PI3K/Akt signalling pathway., Competing Interests: The authors declare no conflict of interest.

The steroid 17β-estradiol (herein "estradiol") is a potent regulator of sexual differentiation that exerts wide-ranging effects on the developing brain and other tissues. The developing gonads are an important source of estradiol but most, if not all, vertebrate embryos are also exposed to maternally derived estradiol during development. In birds, this maternally derived estradiol is present in the egg at the time of oviposition but very little is known about how this source of estradiol influences development. A critical aspect of understanding yolk estradiol effects is deciphering how steroid metabolism may regulate embryonic exposure to yolk estradiol. In this study, we examine the metabolic fate of estradiol during the first five days of incubation in chicken (Gallus gallus) eggs. Using tritiated estradiol to trace the movement and metabolism of estradiol, we demonstrate that estradiol is metabolized to estrone, which is subsequently conjugated to estrone sulfate as the primary metabolite. Estrone sulfate then accumulates in the albumen by day five of incubation. Overall, these findings have important implications for how yolk estradiol may influence development and alter offspring phenotype. Mechanisms through which estradiol, as well as estrone sulfate, might elicit effects are discussed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Female birds skew offspring sex ratios based on environmental and social stimuli; however, the mechanism mediating this phenomenon remains unknown. Growing evidence suggests that testosterone and corticosterone may influence meiosis, as they skew sex ratios when given immediately before chromosomal segregation. It is unclear if these hormones act on the germinal disc (GD) or through a downstream mediator. It is also unknown whether the GD contains receptors for these hormones. If testosterone and/or corticosterone act on the GD to skew sex ratios, then the GD should have receptors for them and that receptor levels should be higher in the GD regions compared to other follicular regions. Furthermore, fluctuations of receptor levels should occur near meiotic segregation. We collected ovarian follicles at 5 h pre-ovulation (just before meiotic segregation) and 20 h pre-ovulation (when sex chromosomes are arrested), and measured androgen receptor (AR) and mineralocorticoid receptor (MR) protein levels via Western blot. ARs and MRs were on the follicle in the GD and non-GD regions, and at 5 h and 20 h pre-ovulation. Both AR and MR protein levels were higher in the GD region than the non-GD region at both time points, but did not differ between time points. These results suggest that hen ovarian follicles have receptors for testosterone and corticosterone, and that the ability for testosterone to respond may be specifically higher in the GD-region, providing further support for the role of testosterone in the alteration of meiotic segregation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

There is limited information on feeding egg-type chick breeders n-3 polyunsaturated fatty acids (PUFA) and its impact on hatching egg quality and embryonic fatty acid (FA) utilization. We investigated the effects of feeding brown and white egg-type chick breeders diets containing sources of n-3 PUFA on egg composition, apparent embryonic FA utilization, and intestinal FA transporter in hatchlings. Twenty-six-week-old ISA brown and Shaver white breeders were fed either 1) control (CON); 2) CON + 1% of microalgae (DMA, Aurantiochytrium limacinum) fermentation product, as a source of docosahexaenoic acid (DHA); or 3) CON + 2.60% of coextruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt) as a source of α-linolenic acid (ALA). Test diets had similar total n-3 and n-6:n-3 ratio. Eggs were hatched, and residual yolk (RY) samples taken for FA analyses. Apparent embryonic FA utilization was calculated by subtracting concentration of FA in RY from concentration of FA in yolk before incubation. There was an interaction between strains and diets (P < 0.05) on DHA in phospholipid and triglyceride fractions of yolk. Both n-3 PUFA sources increased DHA to a greater extent in Shaver white than in ISA brown. The interactive effect of strains and diets (P = 0.019) on embryonic utilization of ALA was such that DMA and FFF reduced ALA utilization, and this pattern was more prevalent in Shaver white birds than in ISA brown birds. There was no interaction between strains and diets on DHA utilization (P > 0.05). Embryos from hens fed n-3 PUFA sources used less total FA in phospholipid fraction (P < 0.001), and they preferentially used more DHA than CON embryos. Shaver white embryos used more (P < 0.05) ALA and DHA than ISA brown embryos. Although data suggested Shaver white had higher propensity of depositing DHA than ISA brown, irrespective of strain, feeding n-3 PUFA modified embryonic pattern of FA utilization toward utilization of DHA., (Copyright © 2019. Published by Elsevier Inc.)

Postnatally, during hypoxia the decrease in oxygen consumption ( [Formula: see text] ) can exceed what expected from the limitation in O 2 availability, meaning that [Formula: see text] -downregulation exceeds O 2 -conformism. We questioned whether a similar phenomenon could occur prenatally, in chicken embryos at mid- (E11, out of 20.5 days) or near end- (E18) incubation. [Formula: see text] was measured with an open-flow system in the sequence of normoxia-normothermia (21% O 2 , 37 °C, 30 min), hypoxia in normothermia (Hx-NT, either 18, 15, 12 or 9% O 2 , 37 °C, 1 hour), hypoxia in hyperthermia (Hx-HT, up to 43 °C, 1 hour) and return to normoxia-normothermia (30 min). During Hx-NT [Formula: see text] invariably decreased in a [O 2 ]-dependent fashion. The hypoxic drop in [Formula: see text] did not require a post-hypoxic payment of the O 2 -debt, implying that the decrease in [Formula: see text] reflected hypometabolism. [Formula: see text] did not differ significantly between Hx-HT and Hx-NT for [O 2 ] = 15% or less, as expected by O 2 -conformism. Differently, with milder hypoxia (18% O 2 ), [Formula: see text] during Hx-HT significantly exceeded that in Hx-NT, meaning that the value of [Formula: see text] in Hx-NT was not limited by O 2 supply. We conclude that a phenomenon of hypoxic [Formula: see text] downregulation like that observed in postnatal mammals can occur also prenatally, in the chicken embryos. The mechanisms at the basis of the downregulation remain unresolved and could combine physiological and cellular processes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Primordial germ cells (PGCs) are precursors of sperms and oocytes and responsible for passing the genetic information from one generation to the next. Chicken PGCs segregate from somatic cells in early embryo and could be isolated and cultured in vitro, making it a useful tool to produce genetically modified animals. However, the number of PGCs isolated from embryo is limited and these cells are not efficient to proliferation in vitro. GSK-3 plays an important role in multiple intracellular signaling pathways and inhibition of GSK-3-mediated β-catenin phosphorylation is known to reduce apoptosis and promote proliferation in T cells and embryo stem cells (ESC). In this study, we investigate the effect of GSK-3 inhibitor on the proliferation of PGCs in vitro and found significant increases of cell proliferation in the culture supplemented with CHIR. We further found that CHIR regulates PGC cell cycle by activating Wnt signaling and antagonizing the apoptosis of PGCs by inhibition of the expression of caspase-3 and Beclin-1. PGCs treated with CHIR expressed the germ cell-related markers and retain the capability to colonize the embryonic gonad after re-introduction to vasculature of HH stage-15 embryos. These results suggest that GSK-3 is involved in cell renewal and apoptosis in chicken PGCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The effects of maternal conjugated linoleic acid (CLA) on embryonic development and hepatic lipid metabolism were investigated in chick embryos. A total of 180 Arbor Acres female broiler breeders (36 wk old) were randomly divided into the following 3 dietary treatment groups: a basic diet (control), a basic diet containing 0.5% CLA (CLA1), and a basic diet containing 1.0% CLA (CLA2). The females were fed for 8 wk, and the eggs from each group were collected and hatched during the last 2 wk. The results showed that the addition of dietary CLA increased the broken egg rate and reduced the fertilization rate and the egg hatchability (P < 0.05). CLA enrichment decreased the polyunsaturated and monounsaturated fatty acids and increased the saturated fatty acids in the yolk sac (P < 0.05). The yolk sac weight, body weight, and body length had a linear decrease with CLA supplementation (P < 0.05). In the developing chick embryo (at E14) and newly hatched chick (D0), the serum triglyceride concentration decreased with maternal CLA supplementation and was accompanied by a reduction in subcutaneous adipose tissue deposition. In addition, maternal CLA supplementation mediated the hepatic lipid metabolism by decreasing the mRNA expression of sterol regulatory element-binding proteins-1c (SREBP-1c), fatty acid synthase and acetyl-CoA carboxylase, and increasing the mRNA expression of adenosine 5'-monophosphate-activated protein kinase α (AMPKα), peroxisome proliferator-activated receptors α (PPARα), liver fatty acid-binding protein, adipose triglyceride lipase and carnitine palmitoyltransferase in embryonic chick livers (P < 0.05). A drop in SREBP-1c protein expression and an increase in the protein expression of p-AMPKα and PPARα were also observed in the liver of chick embryo (P < 0.05). In conclusion, maternal CLA supplementation regulated the fatty acid composition in the yolk sac, and mediated embryonic chick development and hepatic lipometabolism, and these effects may be related to the AMPK pathway. These findings suggest the potential ability of maternal CLA supplementation to reduce fat deposition in chick embryos., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

To better appreciate the alterations of egg proteins and their modifications during embryonic development, a comparative and quantitative study was performed aimed at chicken egg white and yolk proteome and N -glycoproteome after 12 days of incubation using tandem mass tag (TMT)-labeling technology in conjunction with reversed-phase high-performance liquid chromatography (RP-HPLC). A total of 334 unique N -glycosite-containing peptides from 153 N -glycoproteins were identified, of which 82 N -glycosite-containing peptides showed significant changes after 12 days of incubation. The varied proteome was mainly involved with antibacterial, ionic binding, cell proliferation, and embryonic development, while the different degrading and/or absorbing priorities of egg proteins were proposed. This study provides substantial insight into the effects of N -glycoprotein variations on the utilization of egg proteins by chicken embryo during incubation.

Glycerol is one of the substrates used for glycogen production by the chicken embryo, which is the predominant energy source during the last days of incubation and during hatching. The objective of the present study was to evaluate the in ovo feeding (IOF) of glycerol in the light and heavy broiler eggs derived from breeders of two different ages. Two experiments, with 672 eggs each, were carried out. The only difference between the experiments was breeder age: 32 weeks old in Exp. I and 60 weeks old in Exp. II. A completely randomized experimental design in a 3 × 2 factorial arrangement was applied. Treatments consisted of three glycerol IOF doses (0, 6, or 12 mg/ml) and two egg weights (light or heavy). Incubation parameters, glycogen reserves and live performance parameters (1-7 days of age) were evaluated. Hatch of fertile eggs, embryo mortality after IOF and the number of early-hatching chicks were not affected by the treatments in both experiments. Hatchlings from heavy eggs (68.03 ± 0.64 g) laid by young breeders and receiving 6 mg glycerol/ml showed higher liver glycogen levels than those injected with 0 or 12 mg/ml. Glycerol IOF of embryos from young breeders increased feed intake and weight gain at 7 days of age, independently of egg weight. However, different glycerol dosages had no effect on the performance of the progeny of 60-week-old breeders. These results show that glycerol may be used as an IOF ingredient without affecting incubation parameters. The chickens from young breeders had greater glycogen deposition with inoculation of 6 mg/ml of glycerol and better performance with glycerol administration. However, glycerol IOF did not improve the performance of the progeny of 60-week-old breeders. Therefore, glycogen IOF may be recommended for eggs laid by young breeders., (© 2019 Blackwell Verlag GmbH.)

Small eggs have lesser amounts of nutrients to be used by the embryo, and the yolk glycerol is the main substrate for glycogen production, which is the main energy source in the last days of incubation. Thus, the present study aimed to assess the effect of a glycerol injection in light weight eggs at 2 different days of incubation. To this end, 336 light eggs (55.6 to 58.6 g) from 32-wk-old broiler breeders were incubated. The eggs were divided into 3 treatment groups: 1 group inoculated with saline solution on the 17th d of embryonic development (E17) (control group), the second group injected with a 6 mg glycerol/mL solution at E17, and the third group injected with 6 mg glycerol/mL on the 18th d of incubation (E18). Incubation parameters, liver and muscle glycogen, and broilers performance at 7 d of age were evaluated. Glycerol administration in ovo did not influence hatchability, period of embryonic death or early hatching. Chicks exposed to glycerol in ovo feeding (IOF) used more yolk than birds inoculated with saline solution. Glycerol inoculation at E18 enhanced liver glycogen deposition (P = 0.001) and also improved broilers performance at 7 d, although this improvement in performance and glycogen reserves was not observed when eggs were inoculated at 17 d of incubation. Birds receiving glycerol IOF at E18 showed higher feed intake and body weight gain when compared to the control group and the group inoculated at E17. It was found that glycerol inoculation in light eggs at the 18th d of incubation contributed to raise liver glycerol levels and also to improve broilers performance at 7 d., (© 2019 Poultry Science Association Inc.)

This study aimed to investigate the effect of (-)-hydroxycitric acid ((-)-HCA) on lipid and glucose metabolism, and further analyzed these actions whether associated with modulation of aldehyde dehydrogenase 3 family member A2 (ALDH3A2) expression in chicken embryos. Results showed that (-)-HCA decreased triglyceride content and lipid droplet counts, while these effects induced by (-)-HCA were reversed in chicken embryos pre-transfected with sh4-ALDH3A2. (-)-HCA decreased malic enzyme, acetyl-CoA carboxylase, fatty acid synthase, and sterol regulatory element binding protein-1c mRNA level, while increased carnitine palmitoyl transferase 1A (CPT1A) and peroxisome proliferators-activated receptor α (PPARα) mRNA level; and the action of (-)-HCA on lipid metabolism factors had completely eliminated in embryos pre-transfected with sh4-ALDH3A2. Chicken embryos pre-transfected with sh4-ALDH3A2 had eliminated the increasing of serum glucose and hepatic glycogen content induced by (-)-HCA. (-)-HCA decreased phosphofructokinase-1 and increased G6P, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate carboxylase mRNA level in chicken embryos. Similarly, the effect of (-)-HCA on these key enzyme mRNA level was reversed in embryos pre-transfected with sh4-ALDH3A2. Furthermore, (-)-HCA increased PPAR-γ-coactivator-1α (PGC-1α), PPARα, hepatic nuclear factor-4A, PEPCK, and CPT1A protein level, and these actions of (-)-HCA disappeared in embryos pre-transfected with sh4-ALDH3A2. These results indicated that (-)-HCA reduced fat accumulation and accelerated gluconeogenesis via activation of PGC-1α signaling pathway, and these effects of (-)-HCA might associate with the increasing of ALDH3A2 expression level in chicken embryos., (© 2019 Japanese Society of Animal Science.)

Rhythmic pineal melatonin biosynthesis develops in chick embryos incubated under a light (L)-dark (D) cycle of polychromatic white light. The spectral sensitivity of the embryonic pineal gland is not known and was investigated in this study. Broiler breeder eggs (Ross 308, n=450) were incubated under white, red, green or blue light under the 12L : 12D cycle. Melatonin was measured in extracts of pineal glands by radioimmunoassay. The daily rhythm of pineal melatonin levels in 20-day-old chick embryos was confirmed during the final stages of embryonic life under all four wavelengths of light with expected higher concentrations during dark- than light-times. The highest pineal melatonin levels were determined in chick embryos incubated under red and white light and lower levels under green light. The incubation under blue light resulted in the lowest melatonin biosynthesis. Pineal melatonin concentrations increased substantially on post-hatching day two compared with pre-hatching levels and we did not find differences between birds incubated and kept in either white or green light. Our results demonstrate a selective sensitivity of the chick embryo pineal gland to different wavelengths of light. Rhythmic melatonin production is suggested as a possible mechanism, which transfers information about the quality of ambient light to the developing avian embryo.

The current research aimed to explore the impact of (-)-hydroxycitric acid (HCA) on fat metabolism and investigate whether this action of (-)-HCA was associated with modulation of glucose-6-phosphote isomerase (GPI) expression in chicken embryos. We constructed a recombinant plasmid (sh2-GPI) to inhibit GPI expression, and then embryos were treated with (-)-HCA. Results showed that (-)-HCA reduced lipid droplet accumulation, triglyceride content, and lipogenesis factors mRNA level and increased lipolysis factors mRNA expression, while this effect caused by (-)-HCA was markedly reversed when the chicken embryos were pretreated with sh2-GPI. (-)-HCA increased phospho (p)-acetyl-CoA carboxylase, enoyl-CoA hydratase short chain-1, carnitine palmitoyl transferase 1A, p-AMP-activated protein kinase, and peroxisome proliferators-activated receptor α protein expression, and this action of (-)-HCA also dispelled when the chicken embryos were pretreated with sh2-GPI. These data demonstrated that (-)-HCA decreased fat deposition via activation of the AMPK pathway, and the fat-reduction action of (-)-HCA was due to the increasing of GPI expression in chicken embryos.

Effects of 3 eggshell temperatures (EST; 36.7. 37.8, and 38.9°C) in 2 genetic laying hen crossbreds (AB and BB; same hen line, different rooster line) on embryonic metabolism and hatchling quality were investigated. EST were applied from day 14.5 of incubation (E14.5) until hatching. The experiment consisted of 6 consecutive batches with eggs weighing between 59 and 61 g. Heat production was determined continuously from E14.5 onward. In fresh eggs, yolk weight tended to be higher (Δ = 0.28 g; P = 0.08) in the AB crossbred than in the BB crossbred. At E14.5 and E18.5, yolk-free body mass (YFBM) and residual yolk (RY) weight did not differ between genetic crossbred and EST. Hatching time after the start of incubation was not affected by genetic crossbred, but was longer in the 36.7°C (517 h) than in the 38.9°C (505 h), with 37.8°C in between (506 h). At 6 h after hatching, no differences between crossbreds were found for chicken quality parameters, such as chicken weight, chicken length, RY, YFBM, and organ weights, but heart weight was higher in the 36.7°C EST than in the other 2 EST (Δ = 0.24 to 0.30% of YFBM, P = 0.005). Intestinal weight was higher at 36.7°C EST than at 38.9°C EST (Δ = 0.79% of YFBM; P = 0.02), with 37.8°C EST in between. Heat production between E14.5 and E18.5 was higher in the AB crossbred than in the BB crossbred (Δ = 2.61%, P < 0.001) and regardless of crossbred higher at an EST of 38.9°C than at other 2 EST (Δ = 3.59% on average; P < 0.001). Hatchling quality determined at pulling (E21.5) was not affected by EST, but AB chickens were lighter (Δ = 0.46 g; P = 0.03), had less red hocks (Δ = 0.03; P = 0.02), more red beaks (Δ = 0.10; P < 0.001), and a higher (worse) navel score (Δ = 0.11; P < 0.001) than BB chickens. It can be concluded that not only incubation temperature, but also the rooster line appears to play a role in layer crossbred embryo metabolism and hatchling quality., (© The Author 2019. Published by Oxford University Press on behalf of Poultry Science Association.)

Several studies show effects of yolk androgens in avian eggs on the phenotype of the offspring. Yolk hormone concentrations decline strongly within the first few days of incubation. Although early embryonic uptake of yolk androgens is suggested by the presence of radioactivity in the embryo when eggs are injected with radiolabelled androgens, these studies do not verify the chemical identity of radioactive compound(s), although it is known that these androgens can be metabolized substantially. By using stable isotope-labelled testosterone and androstenedione in combination with mass spectrometry, enabling verification of the exact molecular identity of labelled compounds in the embryo, we found that after 5 days of incubation the androgens were not taken up by the embryo. However, their concentrations in the entire yolk albumen homogenates declined strongly, even when corrected for dilution by albumen and water. Our results indicate metabolism of maternal androgens, very likely to 5β-androstane-3α,17β-diol, etiocholanolone and their conjugated forms. The results imply that the effects of increased exposure of the embryo to maternal androgens take place either before this early conversion or are mediated by these metabolites with an as yet unknown function, opening new avenues for understanding hormone-mediated maternal effects in vertebrates., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The ductus arteriosus, an essential embryonic blood vessel between the pulmonary artery and the descending aorta, constricts after birth or hatching and eventually closes to terminate embryonic circulation. Chicken embryos have two long ductus arteriosi, which anatomically differ from mammal ductus arteriosus. Each long ductus arteriosus is divided into two parts: the pulmonary artery-sided and descending aorta-sided ductus arteriosi. Although the pulmonary artery-sided and descending aorta-sided ductus arteriosi have distinct functional characteristics, such as oxygen responsiveness, the difference in their transcriptional profiles has not been investigated. We performed a DNA microarray analysis (GSE 120116 at NCBI GEO) with pooled tissues from the chicken pulmonary artery-sided ductus arteriosus, descending aorta-sided ductus arteriosus, and aorta at the internal pipping stage. Although several known ductus arteriosus-dominant genes such as tfap2b were highly expressed in the pulmonary artery-sided ductus arteriosus, we newly found genes that were dominantly expressed in the chicken pulmonary artery-sided ductus arteriosus. Interestingly, cluster analysis showed that the expression pattern of the pulmonary artery-sided ductus arteriosus was closer to that of the descending aorta-sided ductus arteriosus than that of the aorta, whereas the morphology of the descending aorta-sided ductus arteriosus was closer to that of the aorta than that of the pulmonary artery-sided ductus arteriosus. Subsequent pathway analysis with DAVID bioinformatics resources revealed that the pulmonary artery-sided ductus arteriosus showed enhanced expression of the genes involved in melanogenesis and tyrosine metabolism compared with the descending aorta-sided ductus arteriosus, suggesting that tyrosinase and the related genes play an important role in the proper differentiation of neural crest-derived cells during vascular remodeling in the ductus arteriosus. In conclusion, the transcription profiles of the chicken ductus arteriosus provide new insights for investigating the mechanism of ductus arteriosus closure., Competing Interests: The authors have declared that no competing interests exist.

Background: Limited data are available about the kinetics of mRNA expressions of Heat shock proteins (HSPs) and heat shock factors (HSFs) in the thermally manipulated (TM) broiler chicks during acute heat stress. Therefore, this study aimed to investigate effects of thermal manipulation (TM) of broiler chicken during embryonic days (ED) 12-18 on the basal and dynamics mRNA expression of heat shock proteins (HSP108 and HSP90) and heat shock factors (HSF-1 and HSF-2) in the muscle tissue during late embryogenesis, first week of life and during heat stress (HS) on post-hatch days 14 and 28. One thousand and five hundred fertile Ross 315 broiler eggs were randomly allocated to five groups: control group (37.8 °C), TM 1 (38.5 °C for 18 h), TM 2 (39 °C for 18 h), TM 3 (39.5 °C for 18 h) and TM 4 (40 °C for 18 h). Chicks from each treatment group were then randomly sub-divided into two further treatment groups, naïve and thermal challenged (TC). On post-hatch days 14 and 28, thirty chicks from each TC group were subjected to heat stress (41 °C for 6 h), while naïve chicks of each group (n = 30) were kept under regular conditions. The response of chicks to heat stress was investigated by evaluating the change in mRNA expressions of HSP108, HSP90, HSF-1 and HSF-2 in muscle tissue after 1, 3 and 5 h of heat stress., Results: When compared to the control group, TM resulted in significant increases in the basal mRNA expression of HSPs and HSFs during embryogenesis and altered their dynamic expressions in the muscle tissue after heat stress on post-hatch days 14 and 28., Conclusion: the current study indicated short- and long-term enhancement of HSPs and HSFs gene expression which was associated with acquisition of improved thermotolerance in thermally manipulated chicks.

A total of 864 settable Cobb 500 eggs were used to explore changes in yolk mineral content during incubation. Eggs were individually weighed and then placed in a commercial incubator. On embryonic day (ED) 0, 6.5, 13.5, and 17.5, 36 eggs were sampled and yolk weight and mineral content were determined. The concentration of iron (Fe), phosphorus (P), and zinc (Zn) declined (P < 0.05) from ED0 to ED17.5. The concentration of calcium (Ca), magnesium (Mg), and strontium (Sr) increased (P < 0.05) from ED0 to ED17.5. The concentration of copper (Cu), potassium (K), and sodium (Na) increased initially (ED0 to ED6.5) but declined thereafter. There was no change (P > 0.05) in the concentration of yolk manganese (Mn) from ED0 to ED17.5. Substantial changes in yolk mineral concentration occur during incubation and are presumably associated with mobilization of shell reserves and flux between albumen and yolk. These data may be useful in designing in ovo interventions, optimizing meat chicken breeder premix formulation or assembly of suitable neonatal or pre-starter diets for meat chicken chicks., (© The Author 2018. Published by Oxford University Press on behalf of Poultry Science Association.)

There are indications that lighting schedules applied during incubation can affect leg health at hatching and during rearing. The current experiment studied effects of lighting schedule: continuous light (24L), 12 hours of light, followed by 12 hours of darkness (12L:12D), or continuous darkness (24D) throughout incubation of broiler chicken eggs on the development and strength of leg bones, and the role of selected hormones in bone development. In the tibiatarsus and femur, growth and ossification during incubation and size and microstructure at day (D)0, D21, and D35 post hatching were measured. Plasma melatonin, growth hormone, and IGF-I were determined perinatally. Incidence of tibial dyschondroplasia, a leg pathology resulting from poor ossification at the bone's epiphyseal plates, was determined at slaughter on D35. 24L resulted in lower embryonic ossification at embryonic day (E)13 and E14, and lower femur length, and lower tibiatarsus weight, length, cortical area, second moment of area around the minor axis, and mean cortical thickness at hatching on D0 compared to 12L:12D especially. Results were long term, with lower femur weight and tibiatarsus length, cortical and medullary area of the tibiatarsus, and second moment of area around the minor axis, and a higher incidence of tibial dyschondroplasia for 24L. Growth hormone at D0 was higher for 24D than for 12L:12D, with 24L intermediate, but plasma melatonin and IGF-I did not differ between treatments, and the role of plasma melatonin, IGF-I, and growth hormone in this process was therefore not clear. To conclude, in the current experiment, 24L during incubation of chicken eggs had a detrimental effect on embryonic leg bone development and later life leg bone strength compared to 24D and 12L:12D, while the light-dark rhythm of 12L:12D may have a stimulating effect on leg health., Competing Interests: Authors Carla W. van der Pol, Inge A. M. van Roovert-Reijrink, Conny M. Maatjens, and Jan Wijnen are employed by HatchTech B.V. This does not alter our adherence to PLOS ONE policies on sharing data and materials. No consultancy, marketed products, patents, or products in development related to the materials discussed in this manuscript exist at the time of writing.

Fibroblast Growth Factors (FGFs) play several important roles during organ morphogenesis and act as multi-functional growth factors that bind to their membrane bound receptors (FGFRs) and activate further downstream signalling pathways. Several studies have investigated the function and expression of FGF/FGFRs in endochondral bone development, however, we know little about their role in the development of neural crest derived intramembranous bones. Here, we investigate the expression of 'b' and c' isoforms of FGFRs 1-3 during the development of the scleral ossicles, a ring of neural crest derived intramembranous bones in the chicken eye. These bones are induced by conjunctival papillae. We identified the expression of both 'b' and 'c' isoforms of FGFRs1-3 during phase 1 of ossicle development when conjunctival papillae development takes place. In contrast, during phase 2, when skeletal condensations are induced in the mesenchyme, all isoforms were downregulated. This data shows for the first time the presence of FGFRs in the chicken sclera, thus implicating FGFs as a signalling pathway potentially involved in scleral ossicle development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

SummaryFibroblast growth factor (FGF) signalling acts as one of modulators that control neural crest cell (NCC) migration, but how this is achieved is still unclear. In this study, we investigated the effects of FGF signalling on NCC migration by blocking this process. Constructs that were capable of inducing Sprouty2 (Spry2) or dominant-negative FGFR1 (Dn-FGFR1) expression were transfected into the cells making up the neural tubes. Our results revealed that blocking FGF signalling at stage HH10 (neurulation stage) could enhance NCC migration at both the cranial and trunk levels in the developing embryos. It was established that FGF-mediated NCC migration was not due to altering the expression of N-cadherin in the neural tube. Instead, we determined that cyclin D1 was overexpressed in the cranial and trunk levels when Sprouty2 was upregulated in the dorsal neural tube. These results imply that the cell cycle was a target of FGF signalling through which it regulates NCC migration at the neurulation stage.

The objective of this study was to evaluate the comparison of different brown and white layers in embryonic development and uptake of nutrients in the egg. A total of 360 fertilized eggs obtained from two brown (Atak-S and Brown Nick) and two white (Atabey and Nick) layer breeders at 28 wk old. Hatching eggs from each genotype were examined on the day of setting for fresh egg analysis and then at the beginning of the embryonic day (E19) and embryonic day (E21) for egg, embryo and villus analysis. Differences in egg weight, shell percentages, relative weight of yolk and albumen, relative weight and length of embryo, villus height, some values of shell, yolk and albumen and relative chick weight in examined hybrids were significant. Yolk sac utilization of embryos during the incubation in the white layer hybrids was greater than that in the brown layer hybrids. Villus heights in the duodenum, jejenum and ileum of embryos in the brown layer hybrids was greater than that in the white layer hybrids. Genotype is important parameter to determine the egg composition at the same age and in animals being fed the same diet. It was observed that the consumption of yolk and shell nutrients from the embryos during the incubation was not related to whether embryos were from the brown or white layer hybrids. Only uptake of the yolk sac and villus height in the embryo among examined variables varied depending on whether the embryos were from the brown or white layer hybrids., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Metabolic disorder is a major health problem and is associated with a number of metabolic diseases. Due to native hyperglycaemia and resistance to exogenous insulin, chickens as a model had used in the studies of adipose tissue biology, metabolism and obesity. But no detailed information is available about the comprehensive changes of serum metabolites at different stages of chicken embryonic development. This study employed LC/MS-QTOF to determine the changes of major functional metabolites at incubation day 14 (E14d), 19 (E19d) and hatching day 1 (H1d), and the associated pathways of differential metabolites during chicken embryonic development were analysed using Metabolite Set Enrichment Analysis method. Results showed that 39 metabolites were significantly changed from E14d to E19d and 68 metabolites were significantly altered from E19d to H1d in chicken embryos. Protein synthesis was promoted by increasing the concentrations of L-glutamine and threonine, and gonadal development was promoted through increasing oestrone content from E14d to E19d in chicken embryos, which indicated that serum glutamine, threonine and oestrone contents may be considered as the candidate indicators for assessment of early embryonic development. 2-oxoglutaric acid mainly contributed to enhancing the citric cycle, and it plays an important role in improving the growth of chicken embryos at the late development; the decreasing of L-glutamine, L-isoleucine and L-leucine contents from E19d to H1d in chicken embryonic development implied their possible functions as the feed additive during early posthatch period of broiler chickens to satisfy the growth. These results provided insights into understand the roles of serum metabolites at different developmental stages of chicken embryos, it also provides available information for chicken as a model to study metabolic disease or human obesity., (© 2018 Blackwell Verlag GmbH.)

This study investigated the effects of thermal manipulation (TM) during embryonic days 12 to 18 on the cloacal temperature (Tc) and the kinetics of muscle mRNA levels of heat shock proteins 70 and 60 (Hsp70 and Hsp60) and heat shock factors 3 and 4 (HSF3 and HSF4) during the first week of life and during thermal stress (TS). One thousand five hundred fertile chicken eggs were randomly divided into 5 groups: control group (37.8°C), TM1 (38.5°C for 18 h), TM2 (39°C for 18 h), TM3 (39.5°C for 18 h), and TM4 (40°C for 18 h). On post-hatch days 14 and 28 of age, 30 randomly selected chicks from each group were thermally stressed at 41.0°C for 6 h, while another 30 randomly selected chicks from each group were kept under thermo-neutral conditions. The Tc of TM chicks was only numerically lower than that of the control during the study period. However, during TS at days 14 and 28 of age, the Tc of TM chicks was significantly lower than that of the controls. On post-hatch days 14 and 28, the basal mRNA levels of Hsps and HSFs were significantly higher than those of the control. Furthermore, during TS, rapid increases in the mRNA levels of Hsps and HSFs in the TM groups were observed. These results indicate that, as well as altering their basal mRNA levels during the first week post-hatch, TM also altered the dynamics of the mRNA expression of HSPs and HSFs, which was associated with improved acquisition of thermotolerance during TS.

Background: Although hatching is perhaps the most abrupt and profound metabolic challenge that a chicken must undergo; there have been no attempts to functionally map the metabolic pathways induced in liver during the embryo-to-hatchling transition. Furthermore, we know very little about the metabolic and regulatory factors that regulate lipid metabolism in late embryos or newly-hatched chicks. In the present study, we examined hepatic transcriptomes of 12 embryos and 12 hatchling chicks during the peri-hatch period-or the metabolic switch from chorioallantoic to pulmonary respiration., Results: Initial hierarchical clustering revealed two distinct, albeit opposing, patterns of hepatic gene expression. Cluster A genes are largely lipolytic and highly expressed in embryos. While, Cluster B genes are lipogenic/thermogenic and mainly controlled by the lipogenic transcription factor THRSPA. Using pairwise comparisons of embryo and hatchling ages, we found 1272 genes that were differentially expressed between embryos and hatchling chicks, including 24 transcription factors and 284 genes that regulate lipid metabolism. The three most differentially-expressed transcripts found in liver of embryos were MOGAT1, DIO3 and PDK4, whereas THRSPA, FASN and DIO2 were highest in hatchlings. An unusual finding was the "ectopic" and extremely high differentially expression of seven feather keratin transcripts in liver of 16 day embryos, which coincides with engorgement of liver with yolk lipids. Gene interaction networks show several transcription factors, transcriptional co-activators/co-inhibitors and their downstream genes that exert a 'ying-yang' action on lipid metabolism during the embryo-to-hatching transition. These upstream regulators include ligand-activated transcription factors, sirtuins and Kruppel-like factors., Conclusions: Our genome-wide transcriptional analysis has greatly expanded the hepatic repertoire of regulatory and metabolic genes involved in the embryo-to-hatchling transition. New knowledge was gained on interactive transcriptional networks and metabolic pathways that enable the abrupt switch from ectothermy (embryo) to endothermy (hatchling) in the chicken. Several transcription factors and their coactivators/co-inhibitors appear to exert opposing actions on lipid metabolism, leading to the predominance of lipolysis in embryos and lipogenesis in hatchlings. Our analysis of hepatic transcriptomes has enabled discovery of opposing, interconnected and interdependent transcriptional regulators that provide precise ying-yang or homeorhetic regulation of lipid metabolism during the critical embryo-to-hatchling transition.

Our previous study showed that ethanol exposure inhibited embryonic angiogenesis mainly due to the excessive stimulation of reactive oxygen species (ROS) production. In this study, we investigated whether sulforaphane (SFN), a known dietary bioactive compound, could ameliorate ethanol-suppressed angiogenesis using chick embryo angiogenesis models. Using chick yolk sac membrane (YSM) and chorioallantoic membrane (CAM) models, we demonstrated that administration of low concentrations of SFN (2.5-10 μM) alone increased angiogenesis, but high concentrations of SFN (20-40 μM) inhibited angiogenesis. SFN administration alleviated ethanol-suppressed angiogenesis and angiogenesis-related gene expression in both angiogenesis models. Ethanol exposure caused cell apoptosis in chick CAM, and the cell apoptosis could be remitted by administration of SFN. Subsequently, we demonstrated that the ethanol-induced increase in production of ROS and reduction of antioxidant enzymes' activity were partially rescued by SFN. Similar results were obtained in endoplasmic reticulum (ER) stress determination, indicated by ATF6 and GRP78 expression or thapsigargin-induced ER stress in the presence or absence of SFN. Taken together, our experiments show that SFN administration can ameliorate ethanol-suppressed embryonic angiogenesis, and this is mainly achieved by alleviating excessive ROS production and ER stress. This study suggests that SFN, in appropriate concentrations, could be a potential candidate compound for preventing the negative impact of alcohol on angiogenesis.

The epithelial disintegration and the mesenchymal bridging are critical steps in the fusion of facial prominences during the upper lip development. These processes of epithelial-mesenchymal transition and programmed cell death are mainly influenced by Wnt signals. Axis inhibition protein2 (Axin2), a major component of the Wnt pathway, has been reported to be involved in lip development and cleft pathogenesis. We wanted to study the involvement of Axin2 in the lip development, especially during the epithelial disintegration of facial prominences. Our results show that Axin2 was expressed mainly in the epithelium of facial prominences and decreased when the prominences were about to contact each other between Hamburger-Hamilton stages 27 and 28 of chicken embryos. The epithelial integrity was destructed or kept intact by the local gain or loss of Axin2 expression, resulting in morphological changes in the facial processes and their skeletal derivatives including the maxilla, nasal, premaxilla bone, and their junctions without cleft formation. These changes were related to expression changes in nuclear β-catenin, pGSK3β, Slug, Smad3, E-cadherin, and p63. All these data indicate that Axin2 participates in the regulation of epithelial integrity and fusion by promoting epithelial disassociation, basement membrane breakdown, and seam loss during the fusion of facial prominences in lip development.

The study of NO metabolism in chicken embryos showed that the intensity of oxidation of both endogenous and exogenous for the embryo NO donors to nitrate is determined by the presence or state of NO targets, rather than donor concentration. The mechanism of this oxidation and its physiological role are discussed. It was also shown that oxidation product nitrate is actively eliminated from the amnionic sac.

Broiler strain, maternal age, and incubation temperature influence embryo metabolism. Hatching eggs were obtained from young (Y; 28 to 34 wk, $\bar{\rm x}$ = 31.2 wk), mid (M; 36 to 45 wk, $\bar{\rm x}$ = 40.5 wk) and old (O; 49 to 54 wk, $\bar{\rm x}$ = 51.4 wk) Ross 708 (n = 88; Experiment 1) and Ross 308 [(n = 45; Experiment 2: (Y; 25 to 34 wk, $\bar{\rm x}$ = 30.5 wk), (M; 35 to 44 wk, $\bar{\rm x}$ = 40.2 wk), and (O; 49 to 54 wk, $\bar{\rm x}$ = 51.6 wk)] breeders. Eggs were stored for 2 to 4 d (18°C, 73% RH), and incubated for 14 d at 37.5°C and 56% RH. At 15 d (E15), 8 fertile eggs per flock age were incubated in individual metabolic chambers at 36.0, 36.5, 37.0, or 37.5°C until E21.5. Each temperature was repeated one additional time. O2 consumption and CO2 production were used to calculate embryonic heat production (EHP). Embryo temperature was measured as eggshell temperature (EST). Initial egg weight was used as a covariate; significance was assessed at P < 0.05. In Ross 708, daily EHP tended to be higher in M and O than Y treatments at E16; EHP of M was higher than Y and O eggs at E18; M and O were higher than O eggs at E19. Incubation at 37.0°C resulted in the highest EHP from E15 to E21, except at E17. Embryos at 37.5°C had reduced EHP beyond E17. Daily EST from E15 to E21 was higher at 37.5 and 37.0°C than at 36.0 and 36.5°C. In Ross 308, daily EST was highest at 37.5°C except at E20. Incubation temperature and EST were highly correlated (R2 = 0.90 to 0.89; P < 0.001). Ross 708 chicks were longer and hatched earlier at 37.0°C than at 36.0 and 37.5°C. EST and EHP increased with incubation temperature in Ross 708. In Ross 308, maternal flock age and incubation temperature did not impact EHP. However, EST was highest at 37.5°C except at E20. Ross 708 was more sensitive to incubation temperature than Ross 308.

Oil spills on birds and other organisms have focused primarily on direct effects of oil exposure through ingestion or direct body fouling. Little is known of indirect effects of airborne volatiles from spilled oil, especially on vulnerable developing embryos within the bird egg. Here a technique is described for exposing bird embryos in the egg to quantifiable amounts of airborne volatile toxicants from Deepwater Horizon crude oil. A novel membrane inlet mass spectrometry system was used to measure major classes of airborne oil-derived toxicants and correlate these exposures with biological endpoints. Exposure induced a reduction in platelet number and increase in osmolality of the blood of embryos of the chicken (Gallus gallus). Additionally, expression of cytochrome P4501A, a protein biomarker of oil exposure, occurred in renal, pulmonary, hepatic and vascular tissues. These data confirm that this system for generating and measuring airborne volatiles can be used for future in-depth analysis of the toxicity of volatile organic compounds in birds and potentially other terrestrial organisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Avian species have a unique integument covered with feathers. Skin morphogenesis is a successive and complex process. To date, most studies have focused on a single developmental point or stage. Fewer studies have focused on whole transcriptomes based on the time-course of embryo integument development. To analyze the global changes in gene expression profiles, we sequenced the transcriptome of chicken embryo skin samples from day 6 to day 21 of incubation and identified 5830 differentially expressed genes (DEGs). Hierarchical clustering showed that E6 to E14 is the critical period of feather follicle morphogenesis. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs, two kinds of Wnt signaling pathways (a canonical pathway and a non-canonical pathway) changed during feather follicle and feather morphogenesis. The gene expression level of inhibitors and ligands related to the Wnt signaling pathway varied significantly during embryonic development. The results revealed a staggered phase relationship between the canonical pathway and the non-canonical pathway from E9 to E14. These analyses shed new light on the gene regulatory mechanism and provided fundamental data related to integument morphogenesis of chickens.

This study was conducted to determine the impact of (-)-hydroxycitric acid ((-)-HCA) on biochemical indices and lipid metabolism parameters in chicken embryos. Two hundred and forty fertilized eggs were divided into six groups and injected with (-)-HCA at concentrations of 0, 0.1, 0.5, 1.0, 10.0 and 50 mg/kg (n = 40). After 19 days of incubation, serum and liver were collected for analysis of biochemical indices and lipid metabolism parameters. Results showed no significant differences on serum biochemical indices: 1-50 mg/kg (-)-HCA significantly increased serum glucose and hepatic glycogen contents (P < 0.05). Oil Red O staining analysis showed total area, counts of lipid droplets and hepatic triglyceride content were significantly decreased (P < 0.01), meanwhile hepatic lipase and lipoprotein lipase activity were significantly increased (P < 0.05). ACLY, ME1, SREBP-1c messenger RNA (mRNA) levels in 0.5-10 mg/kg groups and FAS mRNA level in 1-10 mg/kg groups were significantly decreased (P < 0.05), while PPARα mRNA level, serum adiponectin content and AdipoR1 mRNA level were significantly increased in 0.5-50 mg/kg groups (P < 0.05). These results indicated (-)-HCA treatment inhibited triglyceride synthesis via decreasing lipogenesis-related factors, mRNA expression level and accelerated lipolysis by enhancing lipoprotein lipase and hepatic lipase activity, which finally reduced lipid droplet accumulation, and this action may be associated with activating the adiponectin signaling pathway., (© 2017 Japanese Society of Animal Science.)

Chicken embryos are used widely in the fields of developmental biology and neurobiology. The chicken embryo also serves as a model to analyze gene expression and function using in ovo electroporation. Plasmids may be injected into the spinal cord or tectum of the chicken central nervous system by microinjection for electroporation. Here, we developed a novel method that combines in ovo electroporation and neuronal culturing to study gene function in the chicken tectum during embryo development. Our method can be used to study in-vivo and in-vitro exogenous genes' function. In addition, live cell imaging microscopy, immunostaining, and transfection can be used with our method to study neuronal growth, development, neurite growth and retraction, and axonal pathfinding. Our result showed that axons were present in isolated neurons after culturing for 24 h, and cell debris was low after replacing the media at 48 h. Many GFP-expressing neurons were observed in the cultured cells after 48 h. We successfully cultured the neurons for 3 weeks. Together, this method combines in ovo electroporation and neuronal culturing advantages and is more convenient for the gene function analysis.

Thyroid hormones (THs) are essential for the correct development of nearly every structure in the body from the very early stages of development, yet the embryonic thyroid gland is not functional at these stages. To clarify the roles of the egg yolk as a source of THs, the TH content in the yolk and the expression of TH regulator genes in the yolk sac membrane were evaluated throughout the 21-day incubation period of chicken embryos. The yolk TH content (22.3 ng triiodothyronine and 654.7 ng thyroxine per total yolk on day 4 of incubation) decreased almost linearly along with development. Real-time PCR revealed gene expression of transthyretin, a principal TH distributor in the chicken, and of a TH-inactivating iodothyronine deiodinase (DIO3), until the second week of incubation when the embryonic pituitary-thyroid axis is generally thought to start functioning. The TH-activating deiodinase (DIO2) and transmembrane transporter of thyroxine (SLCO1C1) genes were expressed in the last week of incubation, which coincided with a marked increase of circulating thyroxine and a reduction in the yolk sac weight. DIO1, which can remove iodine from inactive THs, was expressed throughout the incubation period. It is assumed that the chicken yolk sac inactivates THs contained abundantly in the yolk and supplies the hormones to the developing embryo in appropriate concentrations until the second week of incubation, while THs may be activated in the yolk sac membrane in the last week of incubation. Additionally, the yolk sac could serve as a source of iodine for the embryo.

Limb muscles derive from pax3 expressing precursor cells that migrate from the hypaxial somite into the developing limb bud. Once there they begin to differentiate and express muscle determination genes such as MyoD. This process is regulated by a combination of inductive or inhibitory signals including Fgf18, retinoic acid, HGF, Notch and IGFs. IGFs are well known to affect late stages of muscle development and to promote both proliferation and differentiation. We examined their roles in early stage limb bud myogenesis using chicken embryos as an experimental model. Grafting beads soaked in purified recombinant IGF-I, IGF-II or small molecule inhibitors of specific signaling pathways into developing chick embryo limbs showed that both IGF-I and IGF-II induce expression of the early stage myogenic markers pax3 and MyoD as well as myogenin. Their effects on pax3 and MyoD expression were blocked by inhibitors of both the IGF type I receptor (picropodophyllotoxin, PPP) and MEK (U0126). The PI3K inhibitor LY294002 blocked IGF-II, but not IGF-I, induction of pax3 mRNA as well as the IGF-I, but not IGF-II, induction of MyoD mRNA. In addition SU5402, an FGFR/ VEGFR inhibitor, blocked the induction of MyoD by both IGFs but had no effect on pax3 induction, suggesting a role for FGF or VEGF signaling in their induction of MyoD. This was confirmed by in situ hybridization showing that FGF18, a known regulator of MyoD in limb myoblasts, was induced by IGF-I. In addition to their well-known effects on later stages of myogenesis via their induction of myogenin expression, both IGF-I and IGF-II induced pax3 and MyoD expression in developing chick embryos, indicating that they also regulate early stages of myogenesis. The data suggests that the IGFs may have slightly different effects on IGF1R signal transduction via PI3K and that their stimulatory effects on MyoD expression may be indirect, possibly via induction of FGF18 expression.

Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin-associated matrix protein-1 (hLAMP-1) is a matrix protein expressed within Hensen's node at Hamburger-Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5-6 and enhanced within the left pre-cardiac field at HH stage 7. At HH stages 15-16, hLAMP-1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP-1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8-14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8-14 with antibodies specific to hLAMP-1. Our results showed prominent expression of hLAMP-1-positive particles in the extracellular matrix associated with the pre-cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8-9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP-1-stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP-1 may be involved in the formation of the endocardial tube., (© 2017 Blackwell Verlag GmbH.)

The yolk sac and small intestine are 2 important organs responsible for the digestion and absorption of nutrients in chickens during the embryonic and posthatch periods, respectively. The peptide transporter PepT1 is expressed in both the yolk sac and small intestine and plays an important role in the transport of amino acids as short peptides. The objective of this study was to profile the spatial transcriptional patterns of PepT1 mRNA in the yolk sac and small intestine from embryonic and posthatch broilers. The distribution of PepT1 mRNA was investigated by in situ hybridization at embryonic (e) d 11, 13, 15, 17, 19 and day of hatch (doh) in the yolk sac and at e19, doh, and d 1, d 4, and d 7 posthatch in the small intestine. PepT1 mRNA was expressed in the endodermal cells of the yolk sac. PepT1 mRNA was barely detectable at e11, increased from e11 to e13, e15, and e17, and then gradually decreased from e19 to doh. In the small intestine, there was a rapid increase in expression of PepT1 mRNA in the enterocytes from e19 to doh, with expression relatively constant from d 1 to d 7. In addition, there was a differential increase in the heights of the villi in different parts of the small intestine from d 1 to 7, which may partially explain the temporal increase in PepT1 mRNA detected by qPCR. The villi in the duodenum showed the earliest increase in villus height and ultimately resulted in the highest villi at d 7. These results demonstrate that there are temporal changes in PepT1 mRNA expression in the yolk sac and the small intestine, which correspond with their expected role in nutrient uptake during the embryonic and posthatch periods., (© 2017 Poultry Science Association Inc.)

Breeder age and broiler strain influence the availability of nutrients and oxygen through yolk size and eggshell conductance, and the effects of these egg characteristics on nutrient metabolism might be influenced by eggshell temperature (EST). This study aims to determine effects of breeder age, strain, and EST on nutrient metabolism of embryos. A study was designed as a 2 × 2 × 2 factorial arrangement using four batches of in total 4,464 hatching eggs of 2 flock ages at 29 to 30 wk (young) and 54 to 55 wk (old) of Ross 308 and Cobb 500. EST of 37.8 (normal) or 38.9°C (high) was applied from incubation day 7 (E7) until hatching. Wet yolk weight was determined mainly by breeder age (P = 0.043). Energy content in yolk (P = 0.004) and albumen + yolk (P = 0.005) were higher in old flock eggs than in young flock eggs, but did not differ between broiler strains. Eggshell conductance was higher in Ross 308 eggs than in Cobb 500 eggs (P < 0.001). Old flock embryos used more energy (P = 0.046) and accumulated more energy into yolk free body mass (YFBM; P = 0.030) than young flock embryos, whereas heat production (HP), energy lost, and efficiency of converting energy used to YFBM (EYFB) did not differ. Ross 308 embryos used more energy (P = 0.006), had a higher energy lost (P = 0.010), and a higher HP between E15 to E18 (P < 0.05) than Cobb 500 embryos. Energy content in YFBM did not differ between strains and EYFB (P = 0.024) was lower in Ross 308 than in Cobb 500. High EST resulted in higher HP than low EST from E11 to E15 (P < 0.05), but not after E15. Amount of energy used (P = 0.006) and energy accumulated in the YFBM (P < 0.001) was lower for embryos incubated at an EST of 38.9 than that of 37.8°C, whereas EYFB did not differ. In conclusion, breeder age, broiler strain, and EST differentially influence embryonic metabolism and particularly the availability of oxygen could have contributed to these differences., (© 2016 Poultry Science Association Inc.)

We investigated effects of eggshell temperature (EST) of 35.6, 36.7, 37.8, or 38.9°C applied from d of incubation (E) 15, E17, or E19 onward on chicken embryo physiology. A total of 2,850 first-grade eggs of a 43-week-old Ross 308 broiler breeder flock were incubated at an EST of 37.8°C until E15. From E15, E17, or E19 onward, eggs were incubated at an EST of 35.6, 36.7, 37.8, or 38.9°C. Plasma glucose, uric acid, and lactate concentrations, and hepatic glycogen amount and concentration were measured at E15, E17, E19, internal pipping (IP), external pipping (EP), and hatch.An EST of 38.9°C applied from E15 onward decreased the amount of hepatic glycogen from E19 to IP and resulted in a lower glycogen amount at IP compared to all other EST. At EP, when oxygen (O2) becomes largely available, an EST of 38.9°C resulted in a higher glycogen amount and concentration compared to IP, which suggests that plasma glucose between IP and EP might be used for building up hepatic glycogen reserves. However, hepatic glycogen levels remained considerably lower at IP, EP, and hatch at an EST of 38.9°C, compared to an EST of 35.6 and 36.7°C.Opposite to an EST of 38.9°C, from IP onward, an EST of 35.6°C resulted in a higher glycogen amount and concentration compared to all other EST, which might be caused by the higher O2 availability relative to the lower metabolic rate, which provided time to build up glycogen stores from excessive glucose. A higher availability of hepatic glycogen might contribute to an improved physiological status of the broiler chicken embryo toward hatch. Hepatic gluconeogenesis is crucial for developing embryos, as glucose is the major energy source from IP until hatch. At hatch, no effect of EST was found for glucose, uric acid, or lactate.Results of this study emphasize that EST of 35.6 and 36.7°C from E15 onward appear to be beneficial for chicken embryo physiology., (© 2016 Poultry Science Association Inc.)

The development of vertebrate appendages, especially the limb and feather buds are orchestrated by numerous secreted signalling molecules including Sonic Hedgehog, Bone Morphogenetic Proteins, Fibroblast Growth Factors and Wnts. These proteins coordinate the growth and patterning of ectodermal and mesenchymal cells. The influence of signalling molecules is affected over large distances by their concentration (morphogen activity) but also at local levels by the presence of proteins that either attenuate or promote their activity. Glypicans are cell surface molecules that regulate the activity of the major secreted signalling molecules expressed in the limb and feather bud. Here we investigated the expression of all Glypicans during chick limb and feather development. In addition we profiled the expression of Notum, an enzyme that regulates Glypican activity. We show that five of the six Glypicans and Notum are expressed in a dynamic manner during the development of limbs and feathers. We also investigated the expression of key Glypicans and show that they are controlled by signalling molecules highlighting the presence of feedback loops. Lastly we show that Glypicans and Notum are expressed in a tissue specific manner in adult chicken tissues. Our results strongly suggest that the Glypicans and Notum have many as yet undiscovered roles to play during the development of vertebrate appendages., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vertebrate development is orchestrated by secreted signalling molecules that regulate cell behaviour and cell fate decisions during early embryogenesis. The activity of key signalling molecules including members of Hedgehog, Bone Morphogenetic Proteins and Wnt families are regulated by Glypicans, a family of GPI linked polypeptides. Glypicans either promote or inhibit the action of signalling molecules and add a layer of complexity that needs to be understood in order to fully decipher the processes that regulate early vertebrate development. Here we present a detailed expression profile of all six Glypicans and their modifying enzyme Notum during chick embryogenesis. Our results strongly suggest that these proteins have many as yet undiscovered roles to play during early embryogenesis. Finally, we have taken an experimental approach to investigate their role during the patterning of a key embryonic structure - the neural tube. In particular, we show that over-expression of Notum leads to the dorsalisation of this structure., (Copyright © 2017 Elsevier B.V. All rights reserved.)