Chloride Modulates Central pH Sensitivity and Plasticity of Brainstem Breathing-Related Biorhythms in Zebra Finch Embryos.

All terrestrial vertebrate life must transition from aquatic gas exchange in the embryonic environment to aerial or pulmonary respiration at birth. In addition to being able to breathe air, neonates must possess functional sensory feedback systems for maintaining acid-base balance. Respiratory neurons in the brainstem act as pH sensors that can adjust breathing to regulate systemic pH. The central pH sensitivity of breathing-related motor output develops over the embryonic period in the zebra finch (Taeniopygia guttata). Due to the key role of chloride ions in electrochemical stability and developmental plasticity, we tested chloride's role in the development of central pH sensitivity. We blocked gamma-aminobutyric acid-A receptors and cation-chloride cotransport that subtly modulated the low-pH effects on early breathing biorhythms. Further, chloride-free artificial cerebrospinal fluid altered the pattern and timing of breathing biorhythms and blocked the stimulating effect of acidosis in E12-14 brainstems. Early and middle stage embryos exhibited rebound plasticity in brainstem motor outputs during low-pH treatment, which was eliminated by chloride-free solution. Results show that chloride modulates low-pH sensitivity and rebound plasticity in the zebra finch embryonic brainstem, but work is needed to determine the cellular and circuit mechanisms that control functional chloride balance during acid-base disturbances.

Evidence from an Avian Embryo Model that Zinc-Inducible MT4 Expression Protects Mitochondrial Function Against Oxidative Stress.

BACKGROUND: Metallothioneins (MTs) have a strong affinity for zinc (Zn) and remain at a sufficiently high level in mitochondria. As the avian embryo is highly susceptible to oxidative damage and relatively easy to manipulate in a naturally closed chamber, it is an ideal model of the effects of oxidative stress on mitochondrial function. However, the protective roles and molecular mechanisms of Zn-inducible protein expression on mitochondrial function in response to various stressors are poorly understood. OBJECTIVES: The study aimed to investigate the mechanisms by which Zn-induced MT4 expression protects mitochondrial function and energy metabolism subjected to oxidative stress using the avian embryo and embryonic primary hepatocyte models. METHODS: First, we investigated whether MT4 expression alters mitochondrial function. Then, we examined the effects of Zn-induced MT4 overexpression and MT4 silencing on embryonic primary hepatocytes from breeder hens fed a normal Zn diet subjected to a tert-butyl hydroperoxide (BHP) oxidative stress challenge during incubation. In vivo, the avian embryos from hens fed the Zn-deficient and Zn-adequate diets were used to determine the protective roles of Zn-induced MT4 expression on the function of mitochondria exposed to oxidative stress induced by in ovo BHP injection. RESULTS: An in vitro study revealed that Zn-induced MT4 expression reduced reactive oxygen species accumulation in primary hepatocytes. MT4 silencing exacerbated BHP-mediated mitochondrial dysfunction whereas Zn-inducible MT4 overexpression mitigated it. Another in vivo study disclosed that maternal Zn-induced MT4 expression protected mitochondrial function in chick embryo hepatocytes against oxidative stress by inhibiting the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)/peroxisome proliferators-activated receptor-γ (PPAR-γ) pathway. CONCLUSION: This study underscores the potential protective roles of Zn-induced MT4 expression via the downregulation of the PGC-1α/PPAR-γ pathway on mitochondrial function stimulated by the stress challenge in the primary hepatocytes in an avian embryo model. Our findings suggested that Zn-induced MT4 expression could provide a new therapeutic target and preventive strategy for repairing mitochondrial dysfunction in disease.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species.

BACKGROUND: The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes. METHODS: In this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed. RESULTS: This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines. CONCLUSIONS: An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite-host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.

Effect of four eggshell repair materials on weight loss during incubation of white leghorn chicken eggs.

Egg weight loss during incubation is a key indicator used to monitor successful egg development and is closely related to hatchability and chick survival. Artificial incubation is one of the most important captive breeding techniques used in conservation efforts to bolster avian populations. To repair damage to the eggshell and ensure embryonic viability during incubation, a variety of repair coverings can be applied. This study tested the impact of four repair materials (nail polish, synthetic glue, medical dressing, and molten wax film) on egg weight loss during incubation. We found no impact on weight loss for coverings smaller than 35% of the eggshell surface, nor did we find any differences between covering types. The average egg weight loss decreased as the coverage area increased, and the weight loss did not differ when blunt versus sharp-end coverings were compared. Given the relative insensitivity of egg weight loss and survival to the type of patch material used, we concluded that the selection of material for the purpose of weight loss management could be based on practical considerations, such as ease of application and availability.

Direct force measurement and loading on developing tissues in intact avian embryos.

Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100 µm-1 mm) in situ, such as in early embryos, require high spatial precision and minimal invasiveness. Here, we introduce a control-based approach, tissue force microscopy (TiFM), that integrates a mechanical cantilever probe and live imaging with closed-loop feedback control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the means to apply stable, minimally invasive and physiologically relevant loads to drive tissue deformation and to follow the resulting morphogenetic progression associated with large-scale cell movements. Together, TiFM allows us to control tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.

Environmental and intrinsic modulations of venous differentiation.

Endothelial cells in veins differ in morphology, function and gene expression from those in arteries and lymphatics. Understanding how venous and arterial identities are induced during development is required to understand how arterio-venous malformations occur, and to improve the outcome of vein grafts in surgery by promoting arterialization of veins. To identify factors that promote venous endothelial cell fate in vivo, we isolated veins from quail embryos, at different developmental stages, that were grafted into the coelom of chick embryos. Endothelial cells migrated out from the grafted vein and their colonization of host veins and/or arteries was quantified. We show that venous fate is promoted by sympathetic vessel innervation at embryonic day 11. Removal of sympathetic innervation decreased vein colonization, while norepinephrine enhanced venous colonization. BMP treatment or inhibition of ERK enhanced venous fate, revealing environmental neurotransmitter and BMP signaling and intrinsic ERK inhibition as actors in venous fate acquisition. We also identify the BMP antagonist Noggin as a potent mediator of venous arterialization.

Avian Embryonic Culture: A Perspective of In Ovo to Ex Ovo and In Vitro Studies.

The avian embryos growing outside the natural eggshell (ex ovo) were observed since the early 19th century, and since then chick embryonic structures have revealed reaching an in-depth view of external and internal anatomy, enabling us to understand conserved vertebrate development. However, the internal environment within an eggshell (in ovo) would still be the ideal place to perform various experiments to understand the nature of avian development and to apply other biotechnology techniques. With the advent of genetic manipulation and cell culture techniques, avian embryonic parts were dissected for explant culture to eventually generate expandable cell lines (in vitro cell culture). The expansion of embryonic cells allowed us to unravel the transcriptional network for understanding pluripotency and differentiation mechanism in the embryos and in combination with stem cell technology facilitated the applications of avian culture to the next levels in transgenesis and wildlife conservation. In this review, we provide a panoramic view of the relationship among different cultivation platforms from in ovo studies to ex ovo as well as in vitro culture of cell lines with recent advances in the stem cell fields.

A Novel Egg-In-Cube System Enables Long-Term Culture and Dynamic Imaging of Early Embryonic Development.

The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O2), which improves to 15 days on switching to a hyperoxic environment of 60% O2. Using transgenic fluorescent quail embryos in the egg-in-cube system, cell movements in the unincubated blastoderm are imaged dynamically using inverted confocal microscopy, which has been challenging to achieve with other culture systems. Apart from these observations, several other imaging applications of the system are described in this work using transgenic fluorescent quail embryos with upright confocal or epifluorescence microscopy. To demonstrate the usefulness of the egg-in-cube system in perturbation experiments, the quail neural tube is electroporated with fluorescent mRNA "in cubo", followed by the incubation of the electroporated embryo and microscopy of the electroporated region with the embryo in the cube. The egg-in-cube culture system in combination with the "in cubo" electroporation and dynamic imaging capabilities described here will enable researchers to investigate several fundamental questions in early embryogenesis with the avian (quail) embryo on its native yolk.

In Vivo and In Vitro Cartilage Differentiation from Embryonic Epicardial Progenitor Cells.

The presence of cartilage tissue in the embryonic and adult hearts of different vertebrate species is a well-recorded fact. However, while the embryonic neural crest has been historically considered as the main source of cardiac cartilage, recently reported results on the wide connective potential of epicardial lineage cells suggest they could also differentiate into chondrocytes. In this work, we describe the formation of cardiac cartilage clusters from proepicardial cells, both in vivo and in vitro. Our findings report, for the first time, cartilage formation from epicardial progenitor cells, and strongly support the concept of proepicardial cells as multipotent connective progenitors. These results are relevant to our understanding of cardiac cell complexity and the responses of cardiac connective tissues to pathologic stimuli.

Exogenous Linoleic Acid Intervention Alters Hepatic Glucose Metabolism in an Avian Embryo Model.

In the present study, developmental changes of gluconeogenesis and glycolysis in an avian model were measured, and then the intervention effects of in ovo feeding (IOF) linoleic acid (LA) on hepatic glucose metabolism were evaluated. In Experiment 1, thirty fertilized eggs were sampled on embryonic days (E) of 16, 19, 22, 25, 28, 31, and thirty newly-hatched ducklings at hatch (E34 and E35). In Experiment 2, a total of 120 fertilized eggs (60 eggs for each group) were injected into the yolk sac with PBS as the control group and LA as the IOF LA group on E25. Twelve eggs were selected for sample collection on E28 and E31. Serum contents of glucose, pyruvate, and lactate increased ( p < 0.05) linearly or quadratically from E16 to hatch, as well as hepatic glycogen and pyruvate contents. Hepatic mRNA expression related to energy homeostasis, gluconeogenesis, and glycolysis increased ( p < 0.05) in embryogenesis, and the plateau period was presented on E25-E31. IOF LA decreased ( p < 0.05) serum contents of glucose, triacylglycerol, cholesterol, and hepatic oleic acid, unsaturated fatty acids on E28, as well as the gene expression relative to gluconeogenesis. IOF LA increased ( p < 0.05) pyruvate content in serum and liver, and hepatic gene expression relative to glycolysis on E31. In summary, hepatic gluconeogenesis and glycolysis were enhanced to meet the increasing energy demands of embryonic development during E25 - hatch. Exogenous LA intervention on E25 could inhibit hepatic gluconeogenesis and enhance glycolysis during the later developmental period, disrupting glucose embryonic homeostasis and energy status.

An Essential Role for Alzheimer's-Linked Amyloid Beta Oligomers in Neurodevelopment: Transient Expression of Multiple Proteoforms during Retina Histogenesis.

Human amyloid beta peptide (Aß) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AßOs), which are known to accumulate in Alzheimer's disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AßO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aß as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AßOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AßO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AßOs, are linked to Alzheimer's disease (AD). To test whether the AßOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AßO-selective antibody to prematurely lower the levels of AßOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AßOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.

Unfolding a sequence of sensory influences and interactions in the development of functional brain laterality.

Evidence of sensory experience influencing the development of lateralized brain and behavior is reviewed. The epigenetic role of light exposure during two specific stages of embryonic development of precocial avian species is a particular focus of the research discussed. Two specific periods of light sensitivity (in early versus late incubation), each depending on different subcellular and cellular processes, affect lateralized behavior after hatching. Auditory and olfactory stimulation during embryonic development is also discussed with consideration of interactions with light-generated visual lateralization.

Putative Role of Ligands of DNIC in the Physiological Action of the Complex.

In a relatively isolated system of avian embryo, the metabolism of NO, a component of the dinitrosyl iron complexes (DNIC), the main NO donor in most tissues, depends on the ligands that make up the complex. This fact corroborates the earlier hypothesis that these ligands perform a regulatory function in NO metabolism. It is also shown that nitrite injected into the embryo is not oxidized to nitrate like NO in DNIC, but is accumulated outside the amniotic sac. Normally, nitrite is present in an embryo in trace amounts. These facts suggest that NO in the embryo is transferred from the donor molecule to a target in the embryo tissues further transformed with minimum oxidation to nitrite.

Mechanical forces in avian embryo development.

Research using avian embryos has led to major conceptual advances in developmental biology, virology, immunology, genetics and cell biology. The avian embryo has several significant advantages, including ready availability and ease of accessibility, rapid development with marked similarities to mammals and a high amenability to manipulation. As mechanical forces are increasingly recognised as key drivers of morphogenesis, this powerful model system is shedding new light on the mechanobiology of embryonic development. Here, we highlight progress in understanding how mechanical forces direct key morphogenetic processes in the early avian embryo. Recent advances in quantitative live imaging and modelling are elaborating upon traditional work using physical models and embryo manipulations to reveal cell dynamics and tissue forces in ever greater detail. The recent application of transgenic technologies further increases the strength of the avian model and is providing important insights about previously intractable developmental processes.

Symmetry and fluctuation of cell movements in neural crest-derived facial mesenchyme.

In the face, symmetry is established when bilateral streams of neural crest cells leave the neural tube at the same time, follow identical migration routes and then give rise to the facial prominences. However, developmental instability exists, particularly surrounding the steps of lip fusion. The causes of instability are unknown but inability to cope with developmental fluctuations are a likely cause of congenital malformations, such as non-syndromic orofacial clefts. Here, we tracked cell movements over time in the frontonasal mass, which forms the facial midline and participates in lip fusion, using live-cell imaging of chick embryos. Our mathematical examination of cell velocity vectors uncovered temporal fluctuations in several parameters, including order/disorder, symmetry/asymmetry and divergence/convergence. We found that treatment with a Rho GTPase inhibitor completely disrupted the temporal fluctuations in all measures and blocked morphogenesis. Thus, we discovered that genetic control of symmetry extends to mesenchymal cell movements and that these movements are of the type that could be perturbed in asymmetrical malformations, such as non-syndromic cleft lip. This article has an associated 'The people behind the papers' interview.

Transient increase in VEGF-A leads to cardiac tube anomalies and increased risk of congenital heart malformations.

Vascular endothelial growth factor (VEGF) plays a critical role during early heart development. Clinical evidence shows that conditions associated with changes in VEGF signaling in utero are correlated with an increased risk of congenital heart defects (CHD) in newborns. However, how malformations develop after abnormal VEGF exposure is unknown. During embryogenesis, a primitive heart, consisting of an endocardial tube enveloped by a myocardial mantle, is the first organ to function. This tubular heart ultimately transforms into a four-chambered heart. To determine how a transient increase in VEGF prior to heart tube formation affects heart development leading to CHD, we applied exogenous VEGF or a control (vehicle) solution to quail embryos in ovo at Hamburger-Hamilton (HH) stage 8 (28-30 hr of incubation), right before heart tube formation. Light microscopy analysis of embryos re-incubated after treatment for 13 hrs (to approximately HH11/HH12) showed that increased VEGF leads to impaired heart tube elongation accompanied by diameter expansion. Micro-CT analysis of embryos re-incubated for 9 days (to approximately HH38), when the heart is fully formed, showed that VEGF treatment increased the rate of cardiac malformations in surviving embryos. Despite no sex differences in survival, female embryos were more likely to develop cardiac malformations. Our results further suggest that heart tube malformations after a transient increase in VEGF right before heart tube formation may be reversible, leading to normal hearts.

Ex Ovo Culture System for Avian Embryos and its Application.

Ex ovo culture of avian embryos can be applied not only to embryology but also to various fields of basic research such as embryo manipulation, toxicology, and regenerative medicine. The windowing method, which facilitates various manipulations and observations by opening a hole in one part of the eggshell, and culture systems using surrogate eggshells, are widely used. Despite this, biology lessons in high schools cover shell-less culture systems, which involve the development of avian embryos in artificial vessels, such as rice bowls, without using surrogate eggshells. However, as embryo development stops at its early stages in this method, it is not possible to continuously observe the development of the embryo. This led to attempts to develop an embryo culture method using a complete artificial culture vessel that does not use surrogate eggshells, and Kamihira et al. (1998) succeeded in hatching quail embryos in an artificial culture vessel using polytetrafluoroethylene membranes. In addition, Tahara succeeded in hatching chick embryos in artificial culture vessels that used cling film made of polymethylpentene and reported their detailed methodology (Tahara and Obara, 2014). These technologies are being applied not only to school education but also to various fields of research.

Characterization of muscle development and gene expression in early embryos of chicken, quail, and their hybrids.

Myogenesis is a complex, regulated process that involves myoblast proliferation, migration, adhesion, and fusion into myotubes. To investigate early development of embryonic muscles and the expression of regulatory genes during myogenesis in chicken, quail and their hybrids, meat-breeding cocks and egg-breeding cocks were selected as male parents, quails were used as female parents. Their offspring were meat and egg hybrids via Artificial insemination. We measured expression of MUSTN1, IGF-1, and PDK4 using qRT-PCR. We examined muscle fiber diameter using scanning electron microscopy. The results showed that muscle development was two days slower in chicken, egg hybrid, and meat hybrid than in quail. Muscle fiber spacing was the largest in chicken, followed by meat hybrid, egg hybrid, and quail. A similar trend was obtained for muscle fiber diameter. Additionally, muscle fiber diameter increased with embryogenesis. The sarcomere was present on day 17 of incubation in quail, but not in the other species. MUSTN1 could up-regulated IGF-1 by activating PI3K/Akt. IGF-1 expression was consistent with myoblast proliferation and myotube fusion. PDK4 was expressed from E7 to E17. The first peak was reached on E10, egg hybrid and meat hybrid reached their peak at E15. PDK4 is involved in the early proliferation and differentiation of muscle, thereby affecting muscle growth and development. Our findings demonstrated that MUSTN1, IGF-1 and PDK4 genes are expressed to varying levels in breast muscle of chicken, quail, egg hybrid and meat hybrid during the embryonic period. Interestingly, with increasing embryonic age, muscle development was approximately 48 h faster in quail than in other species. We speculated that MUSTN1, IGF-1 and PDK4 genes may be the main candidate genes that cause differences in poultry muscle traits, but the molecular regulation mechanisms need to be further studied. Our findings shed some light on the avian embryo muscle formation and molecular breeding of poultry muscle traits, which provide theoretical basis for poultry breeding.

A Bird's Eye View on the Origin of Aortic Hemogenic Endothelial Cells.

During early embryogenesis, the hemogenic endothelium of the developing dorsal aorta is the main source of definitive hematopoietic stem cells (HSCs), which will generate all blood cell lineages of the adult organism. The hemogenic endothelial cells (HECs) of the dorsal aorta are known to arise from the splanchnic lateral plate mesoderm. However, the specific cell lineages and developmental paths that give rise to aortic HECs are still unclear. Over the past half a century, the scientific debate on the origin of aortic HECs and HSCs has largely focused on two potential and apparently alternative birthplaces, the extraembryonic yolk sac blood islands and the intraembryonic splanchnic mesoderm. However, as we argue, both yolk sac blood islands and aortic HECs may have a common hemangioblastic origin. Further insight into aortic HEC development is being gained from fate-mapping studies that address the identity of progenitor cell lineages, rather than their physical location within the developing embryo. In this perspective article, we discuss the current knowledge on the origin of aortic HECs with a particular focus on the evidence provided by studies in the avian embryo, a model that pioneered the field of developmental hematopoiesis.

Establishing Primary Cultures of Trunk Neural Crest Cells.

Neural crest cells constitute a unique population of progenitor cells with extensive stem cell capacities able to navigate throughout various environments in the embryo and are a source of multiple cell types, including neurons, glia, melanocytes, smooth muscles, endocrine cells, cardiac cells, and also skeletal and supportive tissues in the head. Neural crest cells are not restricted to the embryo but persist as well in adult tissues where they provide a reservoir of stem cells with great therapeutic promise. Many fundamental questions in cell, developmental, and stem cell biology can be addressed using this system. During the last decades there has been an increased availability of elaborated techniques, animal models, and molecular tools to tackle neural crest cell development. However, these approaches are often very challenging and difficult to establish and they are not adapted for rapid functional investigations of mechanisms driving cell migration and differentiation. In addition, they are not adequate for collecting pure populations of neural crest cells usable in large scale analyses and for stem cell studies. Transferring and adapting the neural crest system in tissue culture may then represent an attractive alternative, opening up numerous prospects. Here we describe a simple method for establishing primary cultures of neural crest cells derived from trunk neural tubes using the avian embryo as a source of cells. This protocol is suited for producing pure populations of neural crest cells that can be processed for cytological, cellular, and functional approaches aimed at characterizing their phenotype, behavior, and potential. © 2020 Wiley Periodicals LLC. Basic Protocol: Primary cultures of avian trunk neural crest cells Support Protocol: Adaptations for immunofluorescence labeling and videomicroscopy.