Planar cell polarity links axes of spatial dynamics in neural-tube closure.

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

Developmental Changes in Patterns of Distribution of Fibronectin and Tenascin-C in the Chicken Cornea: Evidence for Distinct and Independent Functions during Corneal Development and Morphogenesis.

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.

The dependence receptor TrkC triggers mitochondria-dependent apoptosis upon Cobra-1 recruitment.

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

Androgen and mineralocorticoid receptors are present on the germinal disc region in laying hens: Potential mediators of sex ratio adjustment in birds?

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.

In ovo metabolism of estradiol to estrone sulfate in chicken eggs: Implications for how yolk estradiol influences embryonic development.

The steroid 17ß-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.

Hypoxic hypometabolism in chicken embryos: conformism and downregulation.

Postnatally, during hypoxia the decrease in oxygen consumption ( [Formula: see text] ) can exceed what expected from the limitation in O2 availability, meaning that [Formula: see text] -downregulation exceeds O2-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% O2, 37 °C, 30 min), hypoxia in normothermia (Hx-NT, either 18, 15, 12 or 9% O2, 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 [O2]-dependent fashion. The hypoxic drop in [Formula: see text] did not require a post-hypoxic payment of the O2-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 [O2] = 15% or less, as expected by O2-conformism. Differently, with milder hypoxia (18% O2), [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 O2 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.

Testosterone Promotes the Proliferation of Chicken Embryonic Myoblasts Via Androgen Receptor Mediated PI3K/Akt Signaling Pathway.

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.

Avian yolk androgens are metabolized rather than taken up by the embryo during the first days of incubation.

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 5ß-androstane-3α,17ß-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.

Basal and dynamics mRNA expression of muscular HSP108, HSP90, HSF-1 and HSF-2 in thermally manipulated broilers during embryogenesis.

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), 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). 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.

Glycerol in ovo feeding as an energy substrate improves performance of broilers from young breeders.

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.

Transcriptional profiling of liver during the critical embryo-to-hatchling transition period in the chicken (Gallus gallus).

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.

Axin2 overexpression promotes the early epithelial disintegration and fusion of facial prominences during avian lip development.

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 ß-catenin, pGSK3ß, 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.

Role of FGF signalling in neural crest cell migration during early chick embryo development.

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.

Based serum metabolomics analysis reveals simultaneous interconnecting changes during chicken embryonic development.

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.

Mechanisms of Specific Embryonic Effects of Nitrogen Oxide.

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.

Sildenafil therapy for fetal cardiovascular dysfunction during hypoxic development: studies in the chick embryo.

KEY POINTS: Common complications of pregnancy, such as chronic fetal hypoxia, trigger a fetal origin of cardiovascular dysfunction and programme cardiovascular disease in later life. Sildenafil treatment protects placental perfusion and fetal growth, but whether the effects of sildenafil transcend the placenta to affect the fetus is unknown. Using the chick embryo model, here we show that sildenafil treatment directly protects the fetal cardiovascular system in hypoxic development, and that the mechanisms of sildenafil protection include reduced oxidative stress and increased nitric oxide bioavailability; Sildenafil does not protect against fetal growth restriction in the chick embryo, supporting the idea that the protective effect of sildenafil on fetal growth reported in mammalian studies, including humans, is secondary to improved placental perfusion. Therefore, sildenafil may be a good candidate for human translational antioxidant therapy to protect the chronically hypoxic fetus in adverse pregnancy. ABSTRACT: There is a need for developing clinically translatable therapy for preventing fetal origins of cardiovascular disease in pregnancy complicated by chronic fetal hypoxia. Evidence shows that sildenafil protects placental perfusion and fetal growth. However, whether beneficial effects of sildenafil transcend onto the fetal heart and circulation in complicated development is unknown. We isolated the direct effects of sildenafil on the fetus using the chick embryo and hypothesised that sildenafil also protects fetal cardiovascular function in hypoxic development. Chick embryos (n = 11 per group) were incubated in normoxia or hypoxia (14% O2 ) from day 1 and treated with sildenafil (4 mg kg-1  day-1 ) from day 13 of the 21-day incubation. Hypoxic incubation increased oxidative stress (4-hydroxynonenal, 141.1 ± 17.6% of normoxic control), reduced superoxide dismutase (60.7 ± 6.3%), increased phosphodiesterase type 5 expression (167 ± 13.7%) and decreased nitric oxide bioavailability (54.7 ± 6.1%) in the fetal heart, and promoted peripheral endothelial dysfunction (70.9 ± 5.6% AUC of normoxic control; all P < 0.05). Sildenafil treatment after onset of chronic hypoxia prevented the increase in phosphodiesterase expression (72.5 ± 22.4%), protected against oxidative stress (94.7 ± 6.2%) and normalised nitric oxide bioavailability (115.6 ± 22.3%) in the fetal heart, and restored endothelial function in the peripheral circulation (89.8 ± 2.9%). Sildenafil protects the fetal heart and circulation directly in hypoxic development via mechanisms including decreased oxidative stress and enhanced nitric oxide bioavailability. Sildenafil may be a good translational candidate for human antioxidant therapy to prevent fetal origins of cardiovascular dysfunction in adverse pregnancy.

New developmental evidence clarifies the evolution of wrist bones in the dinosaur-bird transition.

From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal-anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal-anterior ossification does not support the dinosaur-bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal-posterior ossification develops from a cartilage referred to as "element x," but its position corresponds to distal carpal 3. The proximal-posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal-posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications.

Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds.

Morphological characters are the result of developmental gene expression. The identity of a character is ultimately grounded in the gene regulatory network directing development and thus whole-genome gene expression data can provide evidence about character identity. This approach has been successfully used to assess cell-type identity. Here we use transcriptomic data to address a long-standing uncertainty in evolutionary biology, the identity of avian wing digits. Embryological evidence clearly identifies the three wing digits as developing from digit positions 2, 3 and 4 (ref. 6), whereas palaeontological data suggest that they are digits I, II and III. We compare the transcriptomes of the wing and foot digits and find a strong signal that unites the first wing digit with the first foot digit, even though the first wing digit develops from embryological position 2. Interestingly, our transcriptomic data of the posterior digits show a higher degree of differentiation among forelimb digits compared with hindlimb digits. These data show that in the stem lineage of birds the first digit underwent a translocation from digit position 1 to position 2, and further indicate that the posterior wing digits have unique identities contrary to any model of avian digit identity proposed so far.

Expression of thyroid hormone regulator genes in the yolk sac membrane of the developing chicken embryo.

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.

Regulation of crucial lncRNAs in differentiation of chicken embryonic stem cells to spermatogonia stem cells.

Regulation of crucial lncRNAs involved in differentiation of chicken embryonic stem cells (ESCs) to spermatogonia stem cells (SSCs) was explored by sequencing the transcriptome of ESCs, primordial germ cells (PGCs) and SSCs with RNA-Seq; analytical bioinformatic methods were used to excavate candidate lncRNAs. We detected expression of candidate lncRNAs in ESCs, PGCs and SSCs and forecasted related target genes. Utilizing wego, david and string, function and protein-protein interactions of target genes were analyzed. Finally, based on string analysis, interaction diagrams and relevant signaling pathways were established. Our results indicate a total of 9657 lncRNAs in ESCs, PGCs and SSCs, with 3549 defined as significantly different. We screened 20 candidate lncRNAs, each demonstrating a greater than eight-fold difference in |logFC| value between groups (ESCs vs. PGCs, ESCs vs. SSCs and PGCs vs. SSCs) or specifically expressed in an individual cell type. qRT-PCR results indicated that expression tendencies of candidate lncRNAs were consistent with RNA-Seq. Fifteen cis and four trans target genes were forecasted. Based on wego and string analyses, we found lnc-SSC1, lnc-SSC5, lnc-SSC2 and lnc-ESC2 negatively regulated target genes SUFU, EPHA3, KLF3, ARL3 and TRIM8, whereas SHH, NOTCH, TGF-ß, cAMP/cGMP and JAK/STAT signaling pathways were promoted, causing differentiation of ESCs into SSCs. Our findings represent a preliminary unveiling of lncRNA-associated regulatory mechanisms during differentiation of chicken ESCs into SSCs, filling a research void in male germ cell differentiation related to lncRNA. Our results also provide basic information for improving in vitro induction systems for differentiation of chicken ESCs into SSCs.