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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.

Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. We identified 136 cell types plus 14 positional or developmental intermediates distributed among the six classes conserved across vertebrates – photoreceptor, horizontal, bipolar, amacrine, retinal ganglion, and glial cells. To assess morphology of molecularly defined types, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral, and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell, and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse, and primate retinal cell classes and types. Our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system. The evolutionary relationships of organisms and of genes have long been studied in various ways, including genome sequencing. More recently, the evolutionary relationships among the different types of cells that perform distinct roles in an organism, have become a subject of inquiry. High throughput single-cell RNA sequencing is a technique that allows scientists to determine what genes are switched on in single cells. This technique makes it possible to catalogue the cell types that make up a tissue and generate an atlas of the tissue based on what genes are switched on in each cell. The atlases can then be compared among species. The retina is a light-sensitive tissue that animals with a backbone, called vertebrates, use to see. The basic plan of the retina is very similar in vertebrates: five classes of neurons – the cells that make up the nervous system – are arranged into three layers. The chicken is a highly visual animal and it has frequently been used to study the development of the retina, from understanding how unspecialized embryonic cells become neurons to examining how circuits of neurons form. The structure and role of the retina have been studied in many vertebrates, but detailed descriptions of this tissue at the molecular level have been largely limited to mammals. To bridge this gap, Yamagata, Yan and Sanes generated the first cell atlas of the chicken retina. Additionally, they developed a gene editing-based technique based on CRISPR technology called eCHIKIN to label different cell types based on genes each type switched on selectively, providing a means of matching their shape and location to their molecular identity. Using these methods, it was possible to subdivide each of the five classes of neurons in the retina into multiple distinct types for a total of 136. The atlas provided a foundation for evolutionary analysis of how retinas evolve to serve the very different visual needs of different species. The chicken cell types could be compared to types previously identified in similar studies of mouse and primate retinas. Comparing the relationships among retinal cells in chickens, mice and primates revealed strong similarities in the overall cell classes represented. However, the results also showed big differences among species in the specific types within each class, and the genes that were switched on within each cell type. These findings may provide a foundation to study the anatomy, physiology, evolution, and development of the avian visual system. Until now, neural development of the chicken retina was being studied without comprehensive knowledge of its cell types or the developmentally important genes they express. The system developed by Yamagata, Yan and Sanes may be used in the future to learn more about vision and to investigate how neural cell types evolve to match the repertoire of each species to its environment.

Out of the 14 avian β-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian β-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to β-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed β-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-β-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

Morphogenetic flows in developmental biology are characterized by the coordinated motion of thousands of cells that organize into tissues, naturally raising the question of how this collective organization arises. Using only the kinematics of tissue deformation, which naturally integrates local and global mechanisms along cell paths, we identify the dynamic morphoskeletons behind morphogenesis, i.e., the evolving centerpieces of multicellular trajectory patterns. These features are model- and parameter-free, frame-invariant, and robust to measurement errors and can be computed from unfiltered cell-velocity data. We reveal the spatial attractors and repellers of the embryo by quantifying its Lagrangian deformation, information that is inaccessible to simple trajectory inspection or Eulerian methods that are local and typically frame-dependent. Computing these dynamic morphoskeletons in wild-type and mutant chick and fly embryos, we find that they capture the early footprint of known morphogenetic features, reveal new ones, and quantitatively distinguish between different phenotypes.

Controlling embryonic growth Most animal embryos grow through cell accumulation in a posterior growth zone, but the morphogenic forces that control the formation and directionality of the growth are unknown. Based on a study of axis elongation during formation of the trunk and tail structures in the chicken embryo, Bénazéraf et al . propose that tissue elongation in response to signalling mediated by fibroblast growth factor is a property emerging from the collective regulation of graded, random cell motion rather than by the regulation of directionality of individual cellular movements. Most animal embryos grow through cell accumulation in a posterior growth zone, but the underlying forces are unknown. It is now proposed that posterior elongation in chicken embryos is an emergent property that arises from graded cell motility in random directions (as opposed to directed movement). This occurs in response to signalling through the fibroblast growth factor. Vertebrate embryos are characterized by an elongated antero-posterior (AP) body axis, which forms by progressive cell deposition from a posterior growth zone in the embryo. Here, we used tissue ablation in the chicken embryo to demonstrate that the caudal presomitic mesoderm (PSM) has a key role in axis elongation. Using time-lapse microscopy, we analysed the movements of fluorescently labelled cells in the PSM during embryo elongation, which revealed a clear posterior-to-anterior gradient of cell motility and directionality in the PSM. We tracked the movement of the PSM extracellular matrix in parallel with the labelled cells and subtracted the extracellular matrix movement from the global motion of cells. After subtraction, cell motility remained graded but lacked directionality, indicating that the posterior cell movements associated with axis elongation in the PSM are not intrinsic but reflect tissue deformation. The gradient of cell motion along the PSM parallels the fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK) gradient 1 , which has been implicated in the control of cell motility in this tissue 2 . Both FGF signalling gain- and loss-of-function experiments lead to disruption of the motility gradient and a slowing down of axis elongation. Furthermore, embryos treated with cell movement inhibitors (blebbistatin or RhoK inhibitor), but not cell cycle inhibitors, show a slower axis elongation rate. We propose that the gradient of random cell motility downstream of FGF signalling in the PSM controls posterior elongation in the amniote embryo. Our data indicate that tissue elongation is an emergent property that arises from the collective regulation of graded, random cell motion rather than by the regulation of directionality of individual cellular movements.

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.

Embryo development and heat production (HP) were studied in eggs of similar size (60 to 65 g) that were incubated at normal (37.8°C) or high (38.9°C) eggshell temperature (EST) and exposed to low (17%), normal (21%), or high (25%) O₂ concentration from d 9 through 19. High EST initially increased HP, but gradually O₂ became more important for HP than EST. Finally,HP was highest for the combination of high EST with high O₂ and lowest for the combination of high EST with low O₂. High EST decreased hatch time, BW, yolk free BW, and relative heart weight. The EST had no effect on residual yolk weight, chick length, or relative liver weight. Increased O₂ increased yolk free BW and chick length and decreased residual yolk weight at hatch. No interactions between EST and O₂ were observed with regard to embryo development and hatchling characteristics. If embryo development is reflected by HP, it can be concluded that high EST primarily increased embryonic development until the second week of incubation. During the third week of incubation, O₂ had a greater effect in determining embryo development than EST.

Early embryonic exposure to maternal glucocorticoids can broadly impact physiology and behaviour across phylogenetically diverse taxa. The transfer of maternal glucocorticoids to offspring may be an inevitable cost associated with poor environmental conditions, or serve as a maternal effect that alters offspring phenotype in preparation for a stressful environment. Regardless, maternal glucocorticoids are likely to have both costs and benefits that are paid and collected over different developmental time periods. We manipulated yolk corticosterone (cort) in domestic chickens (Gallus domesticus) to examine the potential impacts of embryonic exposure to maternal stress on the juvenile stress response and cellular ageing. Here, we report that juveniles exposed to experimentally increased cort in ovo had a protracted decline in cort during the recovery phase of the stress response. All birds, regardless of treatment group, shifted to oxidative stress during an acute stress response. In addition, embryonic exposure to cort resulted in higher levels of reactive oxygen metabolites and an over-representation of short telomeres compared with the control birds. In many species, individuals with higher levels of oxidative stress and shorter telomeres have the poorest survival prospects. Given this, long-term costs of glucocorticoid-induced phenotypes may include accelerated ageing and increased mortality.

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.

This protocol describes how to process samples potentially containing influenza A virus (IAV), amplify the samples in chicken eggs or mammalian cells and identify whether and which IAV is present. Influenza A viruses (IAVs) cause epidemics and pandemics that result in considerable financial burden and loss of human life. To manage annual IAV epidemics and prepare for future pandemics, an improved understanding of how IAVs emerge, transmit, cause disease and acquire pandemic potential is urgently needed. Fundamental techniques essential for procuring such knowledge are IAV isolation and culture from experimental and surveillance samples. Here we present a detailed protocol for IAV sample collection and processing, amplification in chicken eggs or mammalian cells, and identification from samples containing unknown pathogens. This protocol is robust, and it allows for the generation of virus cultures that can be used for downstream analyses. Once experimental or surveillance samples are obtained, virus cultures can be generated and the presence of IAVs can be verified in 3–5 d via reverse-transcription (RT)-PCR or hemagglutination assay. Increased time frames may be required for less experienced laboratory personnel, or when large numbers of samples will be processed.