Intra-amniotic administration of l-glutamine promotes intestinal maturation and enteroendocrine stimulation in chick embryos.

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.

Comparison of the Transcriptomic and Epigenetic Profiles of Gonadal Primordial Germ Cells of White Leghorn and Green-Legged Partridgelike Chicken Embryos.

The Green-legged Partridgelike fowl is a native, dual-purpose Polish chicken. The White Leghorn has been intensively selected for several decades to mainly improve reproductive traits. Primordial germ cells (PGCs) represent the germline stem cells in chickens and are the only cells that can transfer the information stored in the genetic material from generation to generation. The aim of the study was to carry out a transcriptomic and an epigenetic comparison of the White Leghorn and Green-legged Partridgelike gonadal PGCs (gPGCs) at three developmental stages: days 4.5, 8, and 12 of the embryonic development. RNA and DNA were isolated from collected gPGCs. The RNA was further subjected to microarray analysis. An epigenetic analysis was performed based on the global methylation analysis and qMSP method for the particular silenced genes demonstrated in transcriptomic analysis. Statistically significant differences between the gPGCs from both breeds were detected on the day 8 of embryonic development. Global methylation analysis showed significant changes at the methylation level in the White Leghorn gPGCs on day 8 of embryonic development. The results suggest faster development of Green-legged Partridgelike embryos as compared to White Leghorn embryos. Changes in the levels of gene expression during embryonic development are determined by genetic and environmental factors, and this variability is influenced by breed and gender.

Dynamics of primitive streak regression controls the fate of neuromesodermal progenitors in the chicken embryo.

In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers completes by the end of gastrulation. Body formation then proceeds in a head to tail fashion by progressive deposition of lineage-committed progenitors during regression of the primitive streak (PS) and tail bud (TB). The identification by retrospective clonal analysis of a population of neuromesodermal progenitors (NMPs) contributing to both musculoskeletal precursors (paraxial mesoderm) and spinal cord during axis formation challenged these notions. However, classical fate mapping studies of the PS region in amniotes have so far failed to provide direct evidence for such bipotential cells at the single-cell level. Here, using lineage tracing and single-cell RNA sequencing in the chicken embryo, we identify a resident cell population of the anterior PS epiblast, which contributes to neural and mesodermal lineages in trunk and tail. These cells initially behave as monopotent progenitors as classically described and only acquire a bipotential fate later, in more posterior regions. We show that NMPs exhibit a conserved transcriptomic signature during axis elongation but lose their epithelial characteristicsin the TB. Posterior to anterior gradients of convergence speed and ingression along the PS lead to asymmetric exhaustion of PS mesodermal precursor territories. Through limited ingression and increased proliferation, NMPs are maintained and amplified as a cell population which constitute the main progenitors in the TB. Together, our studies provide a novel understanding of the PS and TB contribution through the NMPs to the formation of the body of amniote embryos.

A gradient of Wnt activity positions the neurosensory domains of the inner ear.

The auditory and vestibular organs of the inner ear and the neurons that innervate them originate from Sox2-positive and Notch-active neurosensory domains specified at early stages of otic development. Sox2 is initially present throughout the otic placode and otocyst, and then it becomes progressively restricted to a ventro-medial domain. Using gain- and loss-of-function approaches in the chicken otocyst, we show that these early changes in Sox2 expression are regulated in a dose-dependent manner by Wnt/beta-catenin signalling. Both high and very low levels of Wnt activity repress Sox2 and neurosensory competence. However, intermediate levels allow the maintenance of Sox2 expression and sensory organ formation. We propose that a dorso-ventral (high-to-low) gradient and wave of Wnt activity initiated at the dorsal rim of the otic placode progressively restricts Sox2 and Notch activity to the ventral half of the otocyst, thereby positioning the neurosensory competent domains in the inner ear.

A cell atlas of the chick retina based on single-cell transcriptomics.

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.

New insights into the obligatory nature of cyclooxygenase-2 and PGE2 during early chick embryogenesis.

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 PGE2, PGD2, PGF2α, and TXB2 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 PGE2 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 PGD2, PGF2α, and TXB2. 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 PGE2.

Neither altered incubation temperature during fetal development nor preferred rearing temperature improves leg bone characteristics of broilers.

The present study evaluated whether broiler femoral and tibiotarsal characteristics (as assessed at slaughter age) could be improved if birds were reared under their preferred temperature and whether continuous high or low incubation temperature during the fetal period improves bone characteristics of broilers reared under heat stress or thermal preference. Broiler breeder eggs were incubated from day 13 until hatching under cold (36 °C), control (37.5 °C), or hot (39 °C) temperatures. Under these conditions, the eggshell temperatures were 37.4 ± 0.1°C, 37.8 ± 0.15°C, and 38.8 ± 0.3°C, respectively. Then, broiler chicks were reared under control, preferred (determined previously in thermal preference test), or high temperatures. At day 42 of age, the broilers were weighed and euthanized, and femora and tibiotarsi collected to measure weight, length, diaphysis perimeter, breaking strength, maximum flexion, rigidity, ash, phosphorus, and calcium. Rearing under the preferred temperature did not affect broiler body weight or femoral and tibiotarsal characteristics (P > 0.05). In contrast, high rearing temperature, decreased the body weight, mineral contents of both bones, femoral breaking strength, and tibiotarsal rigidity (P < 0.05). Regarding incubation temperature effects, egg exposure to cold and hot temperatures during the fetal period minimized or avoided a few effects of high rearing temperature, such as those on femoral and tibiotarsal morphological characteristics, mineral composition, and mechanical properties at slaughter age (P < 0.05), but not all. In conclusion, rearing under the preferred broiler temperature did not improve the bone characteristics, and the negative effects of high rearing temperature on bone development were minimized but not completely prevented by high or low temperature incubation during the fetal period.

Bimodal function of chromatin remodeler Hmga1 in neural crest induction and Wnt-dependent emigration.

During gastrulation, neural crest cells are specified at the neural plate border, as characterized by Pax7 expression. Using single-cell RNA sequencing coupled with high-resolution in situ hybridization to identify novel transcriptional regulators, we show that chromatin remodeler Hmga1 is highly expressed prior to specification and maintained in migrating chick neural crest cells. Temporally controlled CRISPR-Cas9-mediated knockouts uncovered two distinct functions of Hmga1 in neural crest development. At the neural plate border, Hmga1 regulates Pax7-dependent neural crest lineage specification. At premigratory stages, a second role manifests where Hmga1 loss reduces cranial crest emigration from the dorsal neural tube independent of Pax7. Interestingly, this is rescued by stabilized ß-catenin, thus implicating Hmga1 as a canonical Wnt activator. Together, our results show that Hmga1 functions in a bimodal manner during neural crest development to regulate specification at the neural plate border, and subsequent emigration from the neural tube via canonical Wnt signaling.

The neural plate is a structure that serves as the basis for the brain and central nervous system during the development of animals with a backbone. In particular, the tissues at the border of the neural plate become the neural crest, a group of highly mobile cells that can specialize to form nerves and parts of the face. The exact molecular mechanisms that allow the crest to emerge are still unknown. The protein Hmga1 alters how genes are packaged and organized inside cells, which in turn influences how genes are switched on and off. Here, Gandhi et al. studied how Hmga1 helps to shape the neural crest in developing chicken embryos. To do so, they harnessed a genetic tool called CRISPR-Cas9, and deleted the gene that encodes Hmga1 at specific developmental stages. This manipulation highlighted two periods where Hmga1 is active. First, Hmga1 helped to define neural crest cells at the neural plate border by activating a gene called pax7. Then, at a later stage, Hmga1 allowed these cells to move to other parts of the body by triggering the Wnt communication system. Failure for the neural crest to develop properly causes birth defects and cancers such as melanoma and childhood neuroblastoma, highlighting the need to better understand how this structure is formed. In addition, a better grasp of the roles of Hmga1 in healthy development could help to appreciate how it participates in a range of adult cancers.

The methylation status of the embryonic limb skeletal progenitors determines their cell fate in chicken.

Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms.

The Wnt/ß-catenin signaling pathway is involved in regulating feather growth of embryonic chicks.

Avian feathers have robust growth and regeneration capability and serve as a useful model for decoding hair morphogenesis and other developmental studies. However, the molecular signaling involved in regulating the development of feather follicles is unclear. The purpose of this study was to investigate the role of the Wnt/ß-catenin pathway in regulating feather morphogenesis in embryonic chicks through in ovo injection of different doses of Dickkopf-1 (DKK1, a specific inhibitor of the target of the Wnt/ß-catenin pathway). A total of 120 fertilized embryo eggs were randomly divided into 4 treatments, including a noninjection group (control group) and groups injected with 100 µL of phosphate-buffered saline (PBS)/egg (PBS control group), 100 µL of PBS/egg containing 600-ng DKK1/egg (600-ng DKK1 group), and 100-µL PBS/egg containing 1,200-ng DKK1/egg (1,200-ng DKK1 group). Feathers and skin tissues were sampled on embryonic (E) day 15 and the day of hatching to examine the feather mass, diameter and density of feather follicles, and the protein expression of the Wnt/ß-catenin pathway. The results showed that, compared with CON and PBS treatment, the injection of DKK1 into the yolk sac of chick embryos had no significant effect on the hatching rate and embryo weight (P > 0.05), while it significantly decreased the relative mass of feathers in the whole body (P < 0.05). The high dose of DKK1 (1,200-ng DKK1/egg) decreased the relative mass of feathers on the back, chest, belly, neck, wings, head, and legs, which was more obvious than that in the 600-ng DKK1 group, which presented a dose-dependent effect. In addition, DKK1 injection significantly downregulated the protein expression levels of ß-catenin, transcription factor 4, Cyclin D1, and c-Myc (P < 0.05). The immunofluorescence result of ß-catenin was consistent with the Western blotting assay results. Altogether, these observations suggested that the Wnt/ß-catenin signaling pathway is involved in regulating feather follicle development and feather growth during the embryonic development of chicks.

Comprehensive analysis of miRNAs, lncRNAs, and mRNAs reveals potential players of sexually dimorphic and left-right asymmetry in chicken gonad during gonadal differentiation.

Despite thousands of sex-biased genes being found in chickens, the genetic control of sexually dimorphic and left-right asymmetry during gonadal differentiation is not yet completely understood. This study aimed to identify microRNAs (miRNAs), long noncoding RNAs (lncRNAs), messenger RNAs (mRNAs), and signaling pathways during gonadal differentiation in chick embryos (day 6/stage 29). The left and right gonads were collected for RNA sequencing. Sex-biased, side-biased miRNAs, lncRNAs, mRNAs, and shared differentially expressed miRNAs (DEmiRNA)-differentially expressed mRNAs (DEmRNA)-differentially expressed lncRNAs (DElncRNA) interaction networks were performed. A total of 8 DEmiRNAs, 183 DElncRNAs, and 123 DEmRNAs were identified for the sex-biased genes, and 7 DEmiRNAs, 189 DElncRNAs, and 183 DEmRNAs for the side-biased genes. The results of quantitative real-time PCR were generally consistent with the RNA-sequencing results. The study suggested that miRNAs and lncRNAs regulation were novel gene-specific dosage compensation mechanism and they could contribute to left-right asymmetry of chicken, but sex-biased and side-biased miRNAs, lncRNAs, and mRNAs were independent of each other. The competing endogenous RNA (ceRNA) networks showed that 17 target pairs including miR-7b (CYP19A1, FSHR, GREB1, STK31, CORIN, and TDRD9), miR-211 (FSHR, GREB1, STK31, CORIN, and TDRD9), miR-204 (FSHR, GREB1, CORIN, and TDRD9), and miR-302b-5p (CYP19A1 and TDRD9) may play crucial roles in ovarian development. These analyses provide new clues to uncover molecular mechanisms and signaling networks of ovarian development.

Advances in Isolation and Culture of Chicken Embryonic Stem Cells In Vitro.

Chicken embryonic stem cells (cESCs) isolated from the egg at the stage X hold great promise for cell therapy, tissue engineering, pharmaceutical, and biotechnological applications. They are considered to be pluripotent cells with the capacity to self-renewal and differentiate into specialized cells. However, long-term maintenance of cESCs cannot be realized now, which impedes the establishment of cESC line and limits their applications. Therefore, the separation locations, isolation methods, and culture conditions especially the supplements and action mechanisms of cytokines, including leukemia inhibitory factor, fibroblast growth factor, transforming growth factor beta, bone morphogenic protein, and activin for cESCs in vitro, have been reviewed here. These defined strategies will contribute to identify the key mechanism on the self-renewal of cESCs, facilitate to optimize system that supports the derivation and longtime maintenance of cESCs, establish the cESC line, and develop the biobank of genetic resources in chicken.

Disconnect between the developing eye and craniofacial prominences in the avian embryo.

In the amniote embryo, the upper jaw and nasal cavities form through coordinated outgrowth and fusion of craniofacial prominences. Adjacent to the embryonic prominences are the developing eyes, which abut the maxillary and lateral nasal prominences. The embryos of extant sauropsids (birds and nonavian reptiles) develop particularly large eyes in comparison to mammals, leading researchers to propose that the developing eye may facilitate outgrowth of prominences towards the midline in order to aid prominence fusion. To test this hypothesis, we performed unilateral and bilateral ablation of the developing eyes in chicken embryos, with the aim of evaluating subsequent prominence formation and fusion. Our analyses revealed minor interaction between the developing craniofacial prominences and the eyes, inconsequential to the fusion of the upper beak. At later developmental stages, the skull exhibited only localized effects from missing eyes, while geometric morphometrics revealed minimal effect on overall shape of the upper jaw when it develops without eyes. Our results indicate that the substantial size of the developing eyes in the chicken embryo exert little influence over the fusion of the craniofacial prominences, despite their effect on the size and shape of maxillary prominences and components of the skull.

Longitudinal developmental analysis of prethalamic eminence derivatives in the chick by mapping of Tbr1 in situ expression.

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.

A reviewed chronology of primordial germ cells migration in the chick embryo.

INTRODUCTION: Primordial Germ Cells (PGCs) are present in all sexually reproducing animals. They differentiate into spermatozoa or oocytes and are therefore responsible for the transmission of genetic and epigenetic information across generations. In birds, PGCs are first observed in the center of the blastodisc at stage Eyal-Giladi X. With the formation of the primitive streak, germ cells are translocated anteriorly to the germinal crescent. At stage Hamburger- Hamilton 10-12, they enter the vasculature before migrating through the dorsal mesentery towards the genital ridges. MATERIAL AND METHODS: Embryos from stages Hamburger-Hamilton (HH) 16 to 22 were collected. Blood samples were taken from the dorsal aorta and from the heart in order to perform blood smears and PAS staining. Embryos were dissected and fixed in Serra's medium. Sections were placed on slides for PAS staining. A sample of each embryo was collected for DNA extraction and PCR in order to determine the sex of the embryos. RESULTS: PGCs were observed in blood circulation until stage HH 20 on blood smears and until stage HH 19 on histological sections. The first PGCs arrived in the genital ridges were observed from stage HH 17. A few germ cells were still migrating in the dorsal mesentery at stage HH 22. The aim of this study was to review the chronology of the migration of PGCs in chick embryos.

Mitotic granule cell precursors undergo highly dynamic morphological transitions throughout the external germinal layer of the chick cerebellum.

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL). The EGL is comprised solely of granule cell precursors, whose progeny migrate inwardly to form the internal granule cell layer. While a range of cell morphologies in the EGL has long been known, how they reflect the cells' differentiation status has previously only been inferred. Observations have suggested a deterministic maturation from outer to inner EGL that we wished to test experimentally. To do this, we electroporated granule cell precursors in chick with plasmids encoding fluorescent proteins and probed labelled cells with markers of both proliferation (phosphohistone H3) and differentiation (Axonin1/TAG1 and NeuroD1). We show that granule cell precursors can display a range of complex forms throughout the EGL while mitotically active. Overexpression of full length NeuroD1 within granule cell precursors does not abolish proliferation, but biases granule cells towards precocious differentiation, alters their migration path and results in a smaller and less foliated cerebellum. Our results show that granule cells show a greater flexibility in differentiation than previously assumed. We speculate that this allows the EGL to regulate its proliferative activity in response to overall patterns of cerebellar growth.

Differential proteomic analysis revealed crucial egg white proteins for hatchability of chickens.

For healthy development, an avian embryo needs the nutritional and functional molecules maternally deposited in avian eggs. Egg white not only provides nutritional components but also exhibits functional properties, such as defenses against microbial invasion. However, the roles of the more detailed messages in embryo development remain unclear. In this study, a tandem mass tag labeling quantitation approach was used to innovatively identify the differential proteins in the egg whites of fresh eggs produced by hens with divergent high/low hatchability and in the egg whites of embryonated eggs with healthy and dead embryos. A total of 378 proteins were quantified in egg white, which is the most complete proteome identified for egg white to date, and up to 102 differential proteins were identified. GO enrichment, pathway, and hierarchical clustering analysis revealed some of the differential proteins that are the main participants in several biological processes, including blood coagulation, intermediate filament, antibacterial activity, and neurodevelopment. A list of 11 putative protein biomarkers, such as keratin (KRT19, KRT12, KRT15, and KRT6A), which is involved in cell architecture, and fibrinogen (fibrinogen alpha chain, fibrinogen beta chain, and fibrinogen gamma chain), which is related to blood coagulation, were ultimately screened. The current study screened egg white proteins that can predict low hatchability and embryonic death and deciphered the role of these proteins in embryonic development, which is meaningful for the comprehensive understanding of embryonic growth.

Migration of chicken egg-white protein ovalbumin-related protein X and its alteration in heparin-binding affinity during embryogenesis of fertilized egg.

Ovalbumin-related protein X (OVAX) is a 50 kDa egg-white protein which has heparin-binding affinity. In this study, migration of OVAX and its heparin-binding performance during embryogenesis of fertilized egg were investigated to explore a possible involvement in chick embryo development. Western blotting of egg yolks at different stages using an anti-OVAX antibody showed that OVAX accumulates in yolk during incubation of fertilized egg. Immunohistochemical analysis of embryo resided in 10-day-incubated eggs showed that OVAX existed in almost all tissues of the embryo. These suggest that OVAX is incorporated from egg white into the embryo through yolk sac. Heparin-sepharose chromatography, isothermal titrating calorimetry using fondaparinux as a ligand, and zeta potential measurement indicated that OVAX retained the heparin-binding affinity (Kd = 0.185 ± 0.037) even after 10 D incubation of fertilized egg, although the affinity was slightly decreased during egg incubation because of acidification of molecular surface charge. In conclusion, although heparin-binding ability of egg-white OVAX slightly decreases during embryogenesis, OVAX incorporated into embryo can retain heparin-binding affinity. Our findings provide a new insight that OVAX participates in the functions of heparin during embryogenesis.

Embryonic Chicken (Gallus gallus domesticus) as a Model of Cardiac Biology and Development.

Cardiovascular disease remains one of the top contributors to morbidity and mortality in the United States. Increasing evidence suggests that many processes, pathways, and programs observed during development and organogenesis are recapitulated in adults in the face of disease. Therefore, a heightened understanding of cardiac development and organogenesis will help increase our understanding of developmental defects and cardiovascular diseases in adults. Chicks have long served as a model system in which to study developmental problems. Detailed descriptions of morphogenesis, low cost, accessibility, ease of manipulation, and the optimization of genetic engineering techniques have made chicks a robust model for studying development and make it a powerful platform for cardiovascular research. This review summarizes the cardiac developmental milestones of embryonic chickens, practical considerations when working with chicken embryos, and techniques available for use in chicks (including tissue chimeras, genetic manipulations, and live imaging). In addition, this article highlights examples that accentuate the utility of the embryonic chicken as model system in which to study cardiac development, particularly epicardial development, and that underscore the importance of how studying development informs our understanding of disease.

The morphological basis of the development of the chick embryo immune system.

The chick immune system is a fundamental model in basic immunology. In birds, the bone marrow derived pluripotent stem cells after entering the circulation, migrate to bursa of Fabricius to benefit from a microenvironment which supports the differentiation and maturation of B lymphocytes by the help of its resident cells and tissues. Delivering sufficient functional B cells is required to maintain their peripheral population and normal peripheral humoral responses. Additionally, bursa acts as an active site for the generation of antibody diversity through gene conversion. Being consisted of 98% B lymphocytes, the organ is occupied by other cell types including T cells, macrophages, eosinophils and mast cells. Thymus, which is an epithelial organ is the main site of T cell development where positive and negative selections contribute to the development of functional and not autoreactive T cell repertoire. Bursectomy and thymectomy are surgical exercises through which the involvement of cells of specific immunity including B cells and T cells can be determined.