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Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called 'polonaise movements', appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.

Zeolites are crystal structures that have a porous structure and are able to absorb and release substances. In veterinary medicine, zeolites are used as animal feed additives, fishpond ammonia purification, pet odor and moisture control, absorption of mycotoxins and reduction of intestinal pathogens. This research was designed to investigate possible histopathological lesions in the liver and kidney of chickens. 80 fertilized eggs were prepared from Ras 308 chickens and randomly divided into 4 groups of 20 (one control group and three experimental groups). In the control group, 0.3 ml of physiological serum was injected, and in the experimental groups, 0.3 ml of nanozeolite solution (5, 10 and 100 mg/liter) was injected into the air sac of the egg. Then eggs were placed in the hatchery machine and at the end of the 20th day of incubation, liver and kidney samples were taken from the embryos. The histopathological results of the kidney, the group receiving zeolite nanoparticles with a dose of 100 mg/kg had a significant difference with the rest of the groups in terms of pathological symptoms (P<0.05). It was shown that different concentrations of zeolite nanoparticles had an effect on fetal liver tissue and the pathological changes of liver tissue increased with increasing dose (P<0.05). In the immunohistochemistry analysis, no significant difference was observed between the treatment groups with zeolite nanoparticles and the control group (P>0.05). The results of this study showed that excessive use of zeolite nanoparticles in various industries may have long-term harmful effects on living organisms. [ABSTRACT FROM AUTHOR]

Introduction: The therapeutic effect of different doses of the traditional aqueous extract of dried leaves of yerba mate (Ilex paraguariensis A. St.-Hil.) was investigated in an experimental cataract model in chicken embryos. Methods and Results: LC-MS/MS analysis allowed the identification and quantification of 53 metabolites. In the hydrocortisone-induced cataract model, lenses were examined morphologically after treatment and parameters related to oxidative stress (total antioxidant/oxidant status (TAS/TOS), glutathione (GSH), and malondialdehyde (MDA)) were evaluated. Antiproliferative cell nuclear antigen (PCNA) and caspase-3 H-scores were determined and crystallin alpha A (CRYAA) gene expression in the lenses was measured by RT-PCR. The degree of cataract decreased in all treatment groups. While there was no significant difference in TAS levels compared to the negative control, TOS, GSH, and MDA levels were dose-dependently regulated. Treatment groups other than the high-dose group regulated the decrease in PCNA and the increase in caspase-3. CRYAA gene expression increased significantly only at the lowest dose. Conclusion: YM, which is becoming increasingly popular as a traditional tea, showed a therapeutic effect on hydrocortisone-induced cataracts in chicken embryos at relatively low doses. [ABSTRACT FROM AUTHOR]

During vertebrate gastrulation, an embryo transforms from a layer of epithelial cells into a multilayered gastrula. This process requires the coordinated movements of hundreds to tens of thousands of cells, depending on the organism. In the chick embryo, patterns of actomyosin cables spanning several cells drive coordinated tissue flows. Here, we derive a minimal theoretical framework that couples actomyosin activity to global tissue flows. Our model predicts the onset and development of gastrulation flows in normal and experimentally perturbed chick embryos, mimicking different gastrulation modes as an active stress instability. Varying initial conditions and a parameter associated with active cell ingression, our model recapitulates distinct vertebrate gastrulation morphologies, consistent with recently published experiments in the chick embryo. Altogether, our results show how changes in the patterning of critical cell behaviors associated with different force-generating mechanisms contribute to distinct vertebrate gastrulation modes via a self-organizing mechanochemical process.

Abstract Compared to classical techniques of morphological analysis, micro-CT (μ-CT) has become an effective approach allowing rapid screening of morphological changes. In the present work, we aimed to provide an optimized micro-CT dense agent perfusion protocol and μ-CT guidelines for different stages of chick embryo cardiogenesis. Our study was conducted over a period of 10 embryonic days (Hamburger-Hamilton HH36) in chick embryo hearts. During the perfusion of the micro-CT dense agent at different developmental stages (HH19, HH24, HH27, HH29, HH31, HH34, HH35, and HH36), we demonstrated that durations and volumes of the injected contrast agent gradually increased with the heart developmental stages contrary to the flow rate that was unchanged during the whole experiment. Analysis of the CT imaging confirmed the efficiency of the optimized parameters of the heart perfusion.

Introduction: Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are paired somites, which generate the musculoskeletal system, the spinal cord--forming part of the central nervous system, and the notochord, with important patterning functions. The specification of these different cell lineages by key signalling pathways and transcription factors is essential, however, a global map of cell types and expressed genes in the avian trunk is missing. Methods: Here we use high-throughput sequencing approaches to generate a molecular map of the emerging trunk and tailbud in the chick embryo. Results and Discussion: Single cell RNA-sequencing (scRNA-seq) identifies discrete cell lineages including somites, neural tube, neural crest, lateral plate mesoderm, ectoderm, endothelial and blood progenitors. In addition, RNA-seq of sequential tissue sections (RNA-tomography) provides a spatially resolved, genome-wide expression dataset for the avian tailbud and emerging body, comparable to other model systems. Combining the single cell and RNAtomography datasets, we identify spatially restricted genes, focusing on somites and early myoblasts. Thus, this high-resolution transcriptome map incorporating cell types in the embryonic trunk can expose molecular pathways involved in body axis development. [ABSTRACT FROM AUTHOR]

The hatch rate of chick embryos cultured outside of the eggshell with 350 mg calcium l‐lactate hydrate (CaL) and 3.5 mL water is fourfold greater in cultures in which the chorioallantoic membrane (CAM) surrounds the egg contents by incubation day 17.5 (E17.5) an event which occurs in ovo by E13. It was first investigated whether decreasing the volume of water added with 350 mg CaL would promote CAM expansion due to the smaller volume to enclose. When 350 mg CaL was present, the CAM did not surround the egg contents by E13. By E17.5, the CAM surrounded the egg contents in 53%–74% of cultures; however, CAM expansion was not significantly different when 0, 1, 2, or 3.5 mL water was present. The hatch rate with 2 or 3.5 mL water was greater than 50% but was not improved with less water. Second, it was investigated whether CaL or water inhibits CAM expansion. In the absence of CaL, the CAM surrounded the egg contents in up to two‐thirds of cultures by E13, whether 2 mL water was present or not. Thus CaL, but not water, inhibits expansion of the CAM by E13, even though CaL promotes hatching. Finally, it was investigated whether injection of aqueous CaL into the allantoic fluid, in conjunction with not adding CaL to culture hammocks, would promote CAM expansion. Allantoic injection of CaL starting at E13 did not promote CAM expansion at E17.5 but resulted in hatch rates of approximately 30%. Allantoic injection is a novel route for supplementation of calcium in cultured chick embryos. Research Highlights: Injection of calcium l‐lactate hydrate (100 mg/mL in H2O) at incubation days 13 and E17.5 into shell‐less chick cultures resulted in a hatch rate of over 30%.Allantoic injection is a novel route by which calcium can be added to cultured embryos. [ABSTRACT FROM AUTHOR]

Compared to classical techniques of morphological analysis, micro-CT (μ-CT) has become an effective approach allowing rapid screening of morphological changes. In the present work, we aimed to provide an optimized micro-CT dense agent perfusion protocol and μ-CT guidelines for different stages of chick embryo cardiogenesis. Our study was conducted over a period of 10 embryonic days (Hamburger-Hamilton HH36) in chick embryo hearts. During the perfusion of the micro-CT dense agent at different developmental stages (HH19, HH24, HH27, HH29, HH31, HH34, HH35, and HH36), we demonstrated that durations and volumes of the injected contrast agent gradually increased with the heart developmental stages contrary to the flow rate that was unchanged during the whole experiment. Analysis of the CT imaging confirmed the efficiency of the optimized parameters of the heart perfusion. [ABSTRACT FROM AUTHOR]

Background: Cynometra cauliflora is a traditional folk medicine that is used to treat a variety of unpleasant symptoms without adverse effects. Previous studies have shown that C. cauliflora fruit relieving morning sickness and nausea in pregnant women. However, no research has been performed on the effect of C. cauliflora fruit extract on birth anomalies. Objective: In this study, we aim to investigate the effects of C. cauliflora fruit extract on developing chick embryos. Materials and Methods: Three hundred fertilized eggs were divided into 2 groups; 1) the experimental groups received various doses of C. cauliflora fruit extract at 50, 100, 200, and 400 mg/kg, and 2) the control group received a 0.9 % normal saline solution. One hundred and fifty eggs at embryonic day-3 (ED-3) were observed for LC50, morphology, and histology, while the 150 eggs at embryonic day-6 (ED-6) were investigated for morphology and histology. Results: At a dosage of 291.70 mg/kg, the C. cauliflora fruit extract induced mortality in ED-3 by more than 50 %. Moreover, fruit extraction can also result in anomalies in ED-3 and ED-6, such as anterior neuropore openings, heart tubes, liver and kidney tissue defects. Conclusion: As a result, it was determined that tissue damage and organ malformation followed dose-dependent and all-or-none laws. Despite having a wide range of medical properties, C. cauliflora fruit extracts have been demonstrated to impair early growth and development and cause birth abnormalities in animal studies. This will allow further determination of regulatory criteria for medical use, particularly for pregnant women. [ABSTRACT FROM AUTHOR]

Goal: This study presents a novel MRI coil design approach explicitly tailored for chick embryo measurements, with the primary objective of improving sensitivity and coverage. Methods: The limitations posed by conventional birdcage coils were addressed by introducing a curvature feature into a standard coil. The performance of the modified coil was assessed using EM simulations and experimental evaluations, which were subsequently validated using a 7 T MRI scanner. A comparative analysis was conducted against a standard quadrature low-pass birdcage coil to evaluate key factors. Results: The proposed coil demonstrated improved SNR and uniformity, particularly in the proximity of the end-rings. These results were consistent with the findings obtained from the simulations. Conclusions: The use of our innovative birdcage coil design holds promise and offers practical potential for in ovo studies.

Pesticides have increased crop yield but severely impacted ecosystems and non-target organisms. Flubendiamide, a new generation pesticide, targets insect larvae but also affects non-target organisms. This study examines the effects of lowest observed effect concentration of technical grade flubendiamide (0.5 µg/µL) flubendiamide on chick liver, focusing on cytochrome P450 (CYP) enzyme expression, oxidative stress, and liver damage. Chick embryos treated with flubendiamide showed significant alterations in CYP mRNA and protein levels, indicating increased toxicant accumulation. Elevated CYP3A4, CYP1A1, CYP1A2, and CYP2C19 levels were noted, suggesting enhanced biotransformation and detoxification processes. However, increased oxidative byproducts led to oxidative stress, as evidenced by decreased glutathione (GSH) levels and elevated superoxide dismutase (SOD) and catalase activities. DCFDA staining confirmed increased hydrogen peroxide (H2O2) levels, indicating heightened reactive oxygen species (ROS). Liver function tests revealed significant increases in serum ALP, ALT, and AST levels, indicating acute liver damage. Histopathological analysis showed structural liver damage, including expanded sinusoidal spaces, impaired portal veins, and compromised hepatocyte architecture. These findings underscore flubendiamide's potential hepatotoxicity in non-target organisms, emphasizing the need for cautious pesticide use to minimize environmental impacts.

Background: A hinged jaw that articulates with the skull base is a striking feature of the vertebrate head and has been greatly modified between, and within, vertebrate classes. Genes belonging to the DLX homeobox family are conserved mediators of local signaling pathways that distinguish the dorsal and ventral aspects of the first pharyngeal arch. Specifically, a subset of DLX genes are expressed in the cranial neural crest‐derived mandibular ectomesenchyme in response to ventral endothelin signaling, an important step that confers the first arch with maxillary and mandibular identities. Downstream targets of DLX genes then execute the morphogenetic processes that lead to functional jaws. Identifying lineage‐specific variations in DLX gene expression and the regulatory networks downstream of DLX action is necessary to understand how different kinds of jaws evolved. Results: Here, we describe and compare the expression of all six DLX genes in the chick pharyngeal arches, focusing on the period of active patterning in the first arch. Disruption of endothelin signaling results in the down‐regulation of ventral‐specific DLX genes and confirms their functional role in avian jaw patterning. Conclusions: This expression resource will be important for comparative embryology and for identifying synexpression groups of DLX‐regulated genes in the chick. Key Findings: Systematic comparison of all six DLX genes in chick pharyngeal tissue First description of DLX4 expression in chick Characterization of all DLX gene responses to Endothelin signalling Description of ventral jaw patterning defects in the absence of chick DLX genes [ABSTRACT FROM AUTHOR]

Purpose: Teratological capacities of lambda-cyhalothrin (LCH) and the rescuing abilities of vitamin E and olive oil in ovo exposure were analyzed in the golden black variety of domestic chicken. Methods: The study was conducted on fresh fertilized eggs collected and divided into 5 groups as follows: (1) Vehicle Control group (VCon) which received 0.1mL of 5% dimethyl sulfoxide (DMSO) solution in vegetable oil (2) the Lambdacyhalothrin group (LCH) was administered a 0.1mL of 5% DMSO and 0.01 mg/kg of LCH, all dissolved in vegetable oil. (3) Lambda-cyhalothrin+Vitamin E group (LCHE), was provided with a 0.1mL of 5% DMSO, 0.01mg/kg LCH & 0.1mg/kg of Vitamin E, dissolved in vegetable oil. (4) Lambda-cyhalothrin+ Vitamin E in Olive oil (LCHOE), received 0.1mL of 5% DMSO and 0.01 mg/kg LCH, both dissolved in 0.1mg/kg vitamin E solution (EVOO). Finally, the last group (5) Olive oil +Lambdacyhalothrin (LCHO) were administered 0.1mL of 5% DMSO, 0.01mg/kg LCH & 0.1mg/kg Vitamin E in EVOO. The embryos were removed from egg shells after 14th day of incubation, weighted and disected for the removal of brains for histological outcomes. Results: There was significant decrease in embryonic weight, volume and density of brain in LCH group as compared to vehicle control. LCH treated embryos showed complete cell obliterations, microlesions and partial nuclear disintegration (micronuclear formation). Number of different types of neuronal cells (pyramidal, pyramidal-like, multipolar, local circuit) was also decreased in LCH treated embryos. However, vit E and olive oil treated embryos show rescuing effects towards all above mentioned abnormalities. Conclusions: Type II fluoridated pyrethroid LCH is highly toxic to developing chick brain which may unfortunately be present in chick feed. However, the precious ingredients of olive oil and vitamin E can reduce the risk of neurodevelopmental damages. [ABSTRACT FROM AUTHOR]

Birds have a very different pineal gland structure morphologically and cytologically. The structure of the organ shows significant changes during the incubation periods. This study, which follows the embryological development of the pineal gland and makes histomorphometric measurements of the cellular elements that make up the gland parenchyma, is a current reference for studies in these areas. These brains were taken from 24 Babcock White Leghorn chick embryos on the 10th, 13th, 16th and 21st days of incubation. At 10th embryonic day, the pineal recess was in the structure of an elongated pineal canal. Solid rosette‐shaped cell clusters were transforming into round vesicles with a small lumen. These vesicles had developed into larger, oval‐shaped follicles with a well‐defined central lumen. On 13th day, it was observed that the number and development of follicles increased considerably. The pineal gland showed a follicular‐solid structure in 16th day embryos. While the mean follicle diameter was determined as 123.46 ± 13.28 μm on the 10th embryonic day, the highest value was measured as 187.62 ± 7.37 μm on the hatching day (p < 0.05). While the mean follicle area had the lowest value in the 10th day embryos, it was determined that this value gradually increased compared to the advancing embryonic days (p < 0.05). As conclusion, it is thought that this study provides new data to the literature about pineal gland development by monitoring the histological and histomorphometric developments of chick pineal gland in different incubation periods. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the anti-demineralizing effect of a mouthwash comprising pomegranate peel extract (PPE 3%), sodium trimetaphosphate (TMP 0.3%), and fluoride (F 225 ppm) in an in situ study, and to assess its irritation potential in an ex vivo study. Methods: This double-blind crossover study was conducted in four phases with 7 days each. Twelve volunteers used palatal appliances containing enamel blocks, which were subjected to cariogenic challenges. The ETF formulation (PPE + TMP + F, pH 7.0), TF formulation (TMP + F, pH 7.0), deionized water (W, pH 7.0), and essential oil commercial mouthwash (CM, 220 ppm F, pH 4.3) were dropped onto the enamel twice daily. The percentage of surface hardness loss, integrated loss of subsurface hardness, calcium, phosphorus, and fluoride in enamel and biofilms were determined. In addition, alkali-soluble extracellular polysaccharide concentrations were analyzed in the biofilms. The irritation potential was evaluated using the hen’s egg chorioallantoic membrane test through the vascular effect produced during 300-s of exposure. Results: ETF was the most efficacious in preventing demineralization. It also showed the highest concentrations of calcium and phosphorus in the enamel and in the biofilm, as well as the lowest amount of extracellular polysaccharides in the biofilm. In the eggs, ETF produced light reddening, whereas CM led to hyperemia and hemorrhage. Conclusions: The addition of PPE to formulations containing TMP and F increased its anti-demineralizing property, and this formulation presented a lower irritation potential than the CM. Clinical relevance: ETF can be a promising alternative alcohol-free mouthwash in patients at high risk of caries. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia due to insulin deficiency and/or resistance. Vitamin K (VK) is a group of fat‐soluble molecules, including naturally occurring vitamin K1 (phylloquinone). vitamin K2 (menaquinone), and synthetic vitamin K3 (menadione). Beyond coagulation, the health benefits of VK have been described to play different roles in both physiological and pathological processes such as inflammation, energy metabolism, neuroprotection, cellular growth, and survival. It was aimed to observe the antioxidant and/or neuroprotective activity of vitamin K1 in our model of chick embryo diabetic neuropathy (DN) induced by streptozotocin (STZ). Ninety White Leghorn, fertile and 0‐day‐old SPF (specific pathogen‐free) eggs (57 ± 4 gr) were used in the study. Chick embryo blood brain tissues were taken for biochemical evaluation. Plasma insulin and glucose levels were measured. In addition, brain tissue total antioxidant level (TAS), total oxidant level (TOS), malondialdehyde (MDA), and vascular endothelial growth factor (VEGF) levels were measured. Plasma glucose levels were higher in the STZ‐treated groups and lower in the treatment groups. Plasma insulin levels were observed to be higher in STZ groups in groups treated with high VK. Low TAS, high MDA, TOS, and VEGF levels were recorded in brain tissue STZ groups. Low VEGF, TOS, and MDA levels were recorded in the group treated with the highest VK, while high TAS levels were observed. In our STZ‐induced chick embryo diabetic neuropathy model, we observed that VK1 reduced oxidant damage by showing antioxidant properties or by modulating antioxidant enzymes. [ABSTRACT FROM AUTHOR]

Introduction: Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are paired somites, which generate the musculoskeletal system, the spinal cord—forming part of the central nervous system, and the notochord, with important patterning functions. The specification of these different cell lineages by key signalling pathways and transcription factors is essential, however, a global map of cell types and expressed genes in the avian trunk is missing.Methods: Here we use high-throughput sequencing approaches to generate a molecular map of the emerging trunk and tailbud in the chick embryo.Results and Discussion: Single cell RNA-sequencing (scRNA-seq) identifies discrete cell lineages including somites, neural tube, neural crest, lateral plate mesoderm, ectoderm, endothelial and blood progenitors. In addition, RNA-seq of sequential tissue sections (RNA-tomography) provides a spatially resolved, genome-wide expression dataset for the avian tailbud and emerging body, comparable to other model systems. Combining the single cell and RNA-tomography datasets, we identify spatially restricted genes, focusing on somites and early myoblasts. Thus, this high-resolution transcriptome map incorporating cell types in the embryonic trunk can expose molecular pathways involved in body axis development.

Rhus Toxicodendron (RT) extract has been used in homeopathic and phytomedicines for a few centuries. RT chiefly comprises a bioactive chemical named Urushiol. Studies showed RT extracts have potential immunomodulatory, anti-inflammatory, and anti-arthritic activities. In this experiment, we studied whether ethanolic extracts of RT can inhibit Delta SARS-Cov-2 spike protein RBD-induced inflammation, leading to cytokine inequity in fertilized chick embryos (Gallus gallus domesticus). Inoculation of Delta SARS-CoV2 spike RBD protein was done in 14th-day-old chick embryos along with control, pre-, and post-treatment sets consisting of ultra-diluted RT extract. Allantoic fluids from the eggs were collected and preserved at -80 ºC after 48 h to study different cytokines. Dissect ion was done, and the liver of each animal was collected and sent for histological study. The most prominent result was up-regulation in the expression of Interferon alpha and Interleukin-10 genes in RT 6 CH challenged, pre-treatment, and post-treatment experimental sets. However, IL 6, IL 8, IL1B, IFN-beta, IFN-gamma, and TGF beta expressions were insignificant in all the other experimental sets. The histopathological result showed that embryos in the pre-treatment experimental set prevented pathological changes. This study indicated the ethanolic extract of RT can up-regulate the expression of Interferon-α genes and the anti-inflammatory cytokine IL-10 gene. Therefore, it prevents the spike protein of the delta SARS-CoV-2-induced pathological changes in fertilized chick embryos.

Eye deformity, a noteworthy embryopathy, is intricately linked with maternal diabetes, frequently resulting in embryonic malformation or even death. In this investigation, a hyperglycemic chick embryo model was established to delve into the protective potential of tamarind shell extract (TSE) on eye development. Chick embryos were administered with TSE (at dosages of 1, 2, and 4 µg/egg) through air sac injection on embryo development day 0 (EDD0) and later exposed to D-glucose (0.4 mmol/egg) on EDD1. To validate our findings, we replicated hyperglycemic environment using D-glucose in ARPE-19 cell model. Our research unveiled that TSE proficiently counters O-GlcNAcylation of proteins in the presence of D-glucose, thereby reinstating levels of key proteins involved in eye development, including Pax6, Daam1, mTOR, and Cdc42. Furthermore, TSE was pinpointed to curtail the D-glucose concentration within the embryo eye, considerably decreasing the accumulation of reactive oxygen species and malondialdehyde accumulation in both the ARPE-19 cells and chick embryos. Beyond its fundamental role in boosting the cellular antioxidative activity, the improved expression of apoptosis-related proteins like Caspase-3, BAX, BCL-2 and other like PI3K and AKT were observed. This suggests a probable activation of the PI3K-AKT-mTOR pathway on ARPE-19 cells by TSE, thereby downregulating apoptotic proteins in ARPE-19 cells during oxidative stress. TSE may become a potent natural countermeasure against D-glucose-induced eye malformation.

Introduction: The therapeutic effect of different doses of the traditional aqueous extract of dried leaves of yerba mate (Ilex paraguariensis A. St.-Hil.) was investigated in an experimental cataract model in chicken embryos. Methods and Results: LC-MS/MS analysis allowed the identification and quantification of 53 metabolites. In the hydrocortisone-induced cataract model, lenses were examined morphologically after treatment and parameters related to oxidative stress (total antioxidant/oxidant status (TAS/TOS), glutathione (GSH), and malondialdehyde (MDA)) were evaluated. Antiproliferative cell nuclear antigen (PCNA) and caspase-3 H-scores were determined and crystallin alpha A (CRYAA) gene expression in the lenses was measured by RT-PCR. The degree of cataract decreased in all treatment groups. While there was no significant difference in TAS levels compared to the negative control, TOS, GSH, and MDA levels were dose-dependently regulated. Treatment groups other than the high-dose group regulated the decrease in PCNA and the increase in caspase-3. CRYAA gene expression increased significantly only at the lowest dose. Conclusion: YM, which is becoming increasingly popular as a traditional tea, showed a therapeutic effect on hydrocortisone-induced cataracts in chicken embryos at relatively low doses.

Metabolism of nitric oxide (NO) donors: dinitrosyl iron complexes (DNIC), nitrosothiols (RSNO), and nitroprusside was studied on a chick embryo model. The obtained results give reason to assume that DNIC constituting the main pool of nitroso compounds in the vast majority of tissues are NO donors immediately interacting with the physiological target of NO, and other NO donors can perform this function after their transformation into DNIC. NO is released from DNIC not spontaneously, but under a joint influence of a factor destroying the complex and a target having chemical affinity for NO. A similar mechanism is apparently implicated in NO passage through the cell membrane. [ABSTRACT FROM AUTHOR]

Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands increased temporal resolution for studies of the brain at this stage, necessitating precise and unbiased staging. Here, we investigated whether we could train a deep convolutional neural network to sub-stage HH10 chick brains using a small dataset of 151 expertly labelled images. By augmenting our images with biologically informed transformations and datadriven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (269) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effects of in ovo injection of soy isoflavones (ISF) on hatchability, body weight, antioxidant status and intestinal development of newly hatched broiler chicks. One hundred and eighty fertile eggs were divided as follows: the control group, 3 mg/egg ISF (low dose) and 6 mg/egg ISF (high dose) on the 18th day of incubation. The results demonstrated that in ovo inclusion of 6 mg of ISF significantly increased hatchability and hatch weight. Both doses of ISF inclusion elevated the serum glutathione peroxidase and slightly decreased malondialdehyde compared to the control group. The high dose of ISF brings higher villus height and a higher villus/crypt ratio in chicks. Moreover, the mRNA levels of tumour necrosis factor‐ α and interferon‐gamma in the spleen were significantly decreased. The ISF treatments showed an improvement in intestinal enzyme expression levels of sucrose isomaltase and mucin 2 as well as tight junction protein (TJ) mRNA expression of claudin‐1 at high doses of ISF (p < 0.05) when compared with the other groups. Furthermore, the mRNA level of IGF‐1 was increased in the high doses of ISF compared to the control. Overall, these findings indicate that in ovo administration of ISF on the 18th day of incubation enhances hatchability, antioxidant status and intestinal morphometrics in hatched chicks and modulates the expression of proinflammatory cytokines, TJs and insulin‐like growth factor. In addition, the sustainability of antioxidants and other positive effects of ISF may increase chick viability and growth performance. [ABSTRACT FROM AUTHOR]

Background: Sugammadex is a modified gamma‐cyclodextrin that has been developed with the goal of reversing the steroidal neuromuscular blocking agents. The aim of the present study is to investigate the effects of different sugammadex doses on embryologic and neural tube development in an early‐stage chick embryo model. Methods: A total of 100 specific pathogen‐free, fertilized domestic chicken eggs were randomly divided into five groups (n = 20, each), and placed in an automatic cycle incubator. The eggs in the "control (C)" group were incubated without administration of any drug till the end of the experiment. Sub‐blastodermic administration of 0.9% NaCl as vehicle control (VC) and different doses of sugammadex solutions prepared with the latter [2 mg/mL (LD), 4 mg/mL (MD), 16 mg/mL (HD)] were performed at 30 hr of incubation. All embryos were removed from the eggs at 72 hr when they were expected to reach Hamburger‐Hamilton (HH) stages 19–20, then they were fixed, and evaluated histo‐morphologically. Results: Embryonic development was not observed in 11 eggs (1 in C, 1 in VC; 3 in LD, 3 in MD, and 3 in HD). All the developed embryos were compatible with the HH stages 19–20. A neural tube closure defect was detected in one embryo in the HD group. No statistically significant difference was found between the groups in terms of embryonic and neural tube developments. Conclusions: No significant association was found between the drug and adverse outcomes; however, a trend with dosing was seen. Further studies are required before conclude on safety and extrapolate these results to human beings. [ABSTRACT FROM AUTHOR]

High resolution assessment of cardiac functional parameters is crucial in translational animal research. The chick embryo is a historically well‐used in vivo model for cardiovascular research due to its many practical advantages, and the conserved form and function of the chick and human cardiogenesis programs. This review aims to provide an overview of several different technical approaches for chick embryo cardiac assessment. Doppler echocardiography, optical coherence tomography, micromagnetic resonance imaging, microparticle image velocimetry, real‐time pressure monitoring, and associated issues with the techniques will be discussed. Alongside this discussion, we also highlight recent advances in cardiac function measurements in chick embryos. Key Findings: Studying the physiology of the chick embryo is critical to understand many diseases such as CHDs.Advancements have been made in techniques to measure the blood flow velocity such as using Doppler Electrocardiography, Optical Coherence Tomography and micro‐MRI.Measurement of Blood pressure is equally important and techniques have been developed to measure the blood pressure through direct measurement and through servo null micropressure technique. [ABSTRACT FROM AUTHOR]

Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.

The cerebellum which is widely used in studies related to the motor system is responsible for maintaining balance, muscle tone and coordination. In present study, we aimed to evaluate the cerebellum at different incubation periods in terms of stereology, embryology and histomorphometry. Twenty-four Babcock White Leghorn chick embryos were used. All data regarding embryo, egg and cerebellum were measured and calculated. All histologic and histomorphometric examinations were evaluated on the preparations stained with Crossmon"s trichrome stain, Kluver-Barrera stain, and silver stain. It was determined that the cerebellum weight, which was 0.0191±0.0064 g on the 10th day of incubation, increased gradually towards to hatching day. On the 10th day, it was seen that a four-layered primitive substantia grisea structure began to form. While primary foliation seen on the 10th day, secondary foliation started on the 13th day. On the 16th day, Purkinje cells forming the stratum gangliosum were arranged almost in a single row. At day 21th, the general structure of the cerebellum was almost similar to that of the adult cerebellum. While the stratum moleculare and granulosum thicknesses were measured in all embryonic periods, the stratum gangliosum thickness could be measured on the 16th and hatching days. As a result, it was thought that the data obtained from this study might be a reference for studies on the cerebellum and especially on motor control disorders.

To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1, 2NYT4 and 18NGT20, in all AIV sequences and studied their impact on H4N2 virus fitness. Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry.

BMP signaling plays iterative roles during vertebrate neural crest development from induction through craniofacial morphogenesis. However, far less is known about the role of BMP activity in cranial neural crest epithelial-to-mesenchymal transition and delamination. By measuring canonical BMP signaling activity as a function of time from specification through early migration of avian midbrain neural crest cells, we found elevated BMP signaling during delamination stages. Moreover, inhibition of canonical BMP activity via a dominant negative mutant Type I BMP receptor showed that BMP signaling is required for neural crest migration from the midbrain, independent from an effect on EMT and delamination. Transcriptome profiling on control compared to BMP-inhibited cranial neural crest cells identified novel BMP targets during neural crest delamination and early migration including targets of the Notch pathway that are upregulated following BMP inhibition. These results suggest potential crosstalk between the BMP and Notch pathways in early migrating cranial neural crest and provide novel insight into mechanisms regulated by BMP signaling during early craniofacial development.

Objective(s): Demineralized bone matrix (DBM) is an allograft bone composed of native insoluble bone morphogenetic proteins and plays important roles in skeletal development, osteogenesis, and differentiation of mesenchymal stem cells. The osteoinductive capabilities of Allogenic DBM make it a potential drug delivery system for preventive treatment in various anatomical sites. In this study, the cytotoxic and teratogenic effects of DBM nanoparticles, on Wharton’s jelly mesenchymal stem cells and chicken embryos were evaluated. Materials and Methods: DBM nanoparticles were injected into fertile eggs at doses of 10, 20, 40, 80, and 100 µM / egg. Then morphological, histological, and skeletal malformations were evaluated. Cytotoxic effects of DBM nanoparticles on Wharton’s jelly mesenchymal stem cells were also assessed using MTT test.Results: Results showed that the fetal growth abnormality occurred only in embryos treated at the highest dose tested (i.e., 100 µM / egg) and MTT test showed no cytotoxicity in low concentration. Conclusion: These results indicated that nanoparticles do not have significant toxic effects on chick embryos and cultured stem cells. Only high doses of DBM nanoparticles reduce growth in embryos and cultured cells.

Purpose: This study aims to analyze the effect of acrylamide (AC) on hepatic cells of fertilized eggs. The subclasses of glutathione S-transferases and histological variations in serum and liver samples were analyzed in AC-induced chick embryos. Results: Our results showed that the levels of alanine aminotransferase, serum aspartate aminotransferase, and alkaline phosphatase were significantly (p < 0.05) elevated in the liver of AC-treated chick embryo groups compared to control. In addition, AC-treated groups exhibited a significant (p < 0.05) decreased levels of glutathione content (GSH), glutathione reductase, GSTs, and increased malondialdehyde (MDA levels) in hepatic tissue of chick embryos. Furthermore, liver GSTs binding to various substrates such as CDNB, EPNP, pNPA, BSP, pNBC, ∆5-androstene-3, 17-dione, and EA was significantly (p < 0.05) decreased in AC-treated chick embryo groups compared to control. Histopathology examination (H&E staining) of the AC-treated chick embryo liver showed liver damage to a larger extent, specifically cytoplasmic vacuolation, pyknotic nuclei and blood vein congestion with patchy degeneration were observed. Further, the obtained in vitro results of α-GST, μ-GST, and π-GST were subjected to rigid receptor docking and quantum-polarized-ligand docking, which predicted that the in silico binding affinities of these classes of GSTs are in good correlation with strong hydrogen and van der Waal interactions to AC with −32.760, −26.254, −33.594 and −36.287, −33.980 and −36.363 kcal/mol MMGBSA (ΔGbind) scores respectively. The long-range molecular dynamics (50 ns) simulations have shown more consistency by AC. Conclusion: The in vivo and in silico findings conclude that AC resulted in the dysfunction of the liver by altering its biochemical markers, α-GST, μ-GST, and π-GST, as well as by histological changes, and thus, AC is suggested as a more harmful agent in developing chick embryo. [ABSTRACT FROM AUTHOR]

During skeletal development, bone growth and mineralization require transport of substantial amounts of calcium, while maintaining very low concentration. How an organism overcomes this major logistical challenge remains mostly unexplained. To shed some light on the dynamics of this process, cryogenic focused ion beam‐scanning electron microscopy (cryo‐FIB/SEM) is used to image forming bone tissue at day 13 of a chick embryo femur. Both cells and matrix in 3D are visualized and observed as calcium‐rich intracellular vesicular structures. Counting the number of these vesicles per unit volume and measuring their calcium content based on the electron back‐scattering signal, the intracellular velocity at which these vesicles need to travel to transport all the calcium required for the mineral deposited in one day within the collagenous tissue can be estimated. This velocity at 0.27 µm s−1 is estimated, which is too large for a diffusion process and rather suggests active transport through the cellular network. It is concluded that calcium logistics is hierarchical and based on several transport mechanisms: first through the vasculature using calcium‐binding proteins and the blood flow, then active transport over tens of micrometers through the network of osteoblasts and osteocytes, and finally diffusive transport over the last one or two microns. [ABSTRACT FROM AUTHOR]

The cerebellum which is widely used in studies related to the motor system is responsible for maintaining balance, muscle tone and coordination. In present study, we aimed to evaluate the cerebellum at different incubation periods in terms of stereology, embryology and histomorphometry. Twenty-four Babcock White Leghorn chick embryos were used. All data regarding embryo, egg and cerebellum were measured and calculated. All histologic and histomorphometric examinations were evaluated on the preparations stained with Crossmon's trichrome stain, Kluver-Barrera stain, and silver stain. It was determined that the cerebellum weight, which was 0.0191±0.0064 g on the 10th day of incubation, increased gradually towards to hatching day. On the 10th day, it was seen that a four-layered primitive substantia grisea structure began to form. While primary foliation seen on the 10th day, secondary foliation started on the 13th day. On the 16th day, Purkinje cells forming the stratum gangliosum were arranged almost in a single row. At day 21th, the general structure of the cerebellum was almost similar to that of the adult cerebellum. While the stratum moleculare and granulosum thicknesses were measured in all embryonic periods, the stratum gangliosum thickness could be measured on the 16th and hatching days. As a result, it was thought that the data obtained from this study might be a reference for studies on the cerebellum and especially on motor control disorders. [ABSTRACT FROM AUTHOR]

Objective : Chiari II malformation (CM II) is still the main cause of severe morbidity and mortality in children with open neural tube defects (ONTDs). The goal of this study was to validate a CM II model in late-stage chick embryos with surgically induced ONTDs. Methods : To make the chick embryo model of ONTD, their neural tubes were opened for a length of 5–6 somites at the thoracic level in Hamburger and Hamilton stage 18 chick embryos (n=150). They were reincubated in ovo up to a total age of 17–21 days. A total of 19 embryos survived and were assigned to either the postoperative day (POD) 14–15 group (n=6) or the POD 17–18 group (n=13). Magnetic resonance imaging (MRI) and histopathologic findings of embryo heads with spinal ONTDs were compared with age-matched normal chick embryos. Results : The chick embryos with ONTDs demonstrated definite and constant structural changes, such as downward displacement of the cerebellum to just above the foramen magnum and narrow and small cerebrospinal fluid spaces in the crowded small posterior fossa. These morphologic features were more prominent in the POD 17–18 group than in the POD 14–15 group. Conclusion : This is the first description of CM II with spinal ONTD in a late-stage chick embryo model with MRI and histopathological analysis. The morphological changes of the posterior fossa in this study mimic those of CM II associated with spinal ONTD in humans. This model will facilitate investigation of the pathogenesis of CM II. [ABSTRACT FROM AUTHOR]

Monosodium glutamate (MSG), commonly used as a food enhancer, has been reported to have teratogenicity during the first 3 days of development. Furthermore, the neural crest cells (NCCs) are crucial for embryonic development during organogenesis. The present study aimed to investigate the treatment effect of folic acid (FA) on MSG-induced teratogenicity, focusing on the toxicity and teratogenic effects on somatic and neural crest cells in chick embryos as models. Six hundred and fifty fertilized eggs were divided into control, FA, MSG, and MSG with FA groups. The chemicals were administered, and the results were investigated after 3 days of incubation. The morphology and histology were studied using stereomicroscopy and hematoxylin and eosin staining, respectively. The NCC population was confirmed by the presence of HNK-1 using immunohistochemistry. The finding showed that the MSG at 2 mg/kg of egg weight induced retardation, tissue malformation, craniofacial, and heart defects, whereas the FA alleviated those adverse effects and reduced the MSG-induced NCCs destruction in the eyes, heart, stomach, and nerves. In conclusion, although MSG harms embryos, FA effectively diminished its teratogenicity in the chick embryo model. These experimental protocols are beneficial for teratogenic studies on preventing birth defects that are harmful to the embryo. [ABSTRACT FROM AUTHOR]

Peripheral and intraspinal feedback is required to shape and update the output of spinal networks that execute motor behavior. We report that lumbar dI2 spinal interneurons in chicks receive synaptic input from afferents and premotor neurons. These interneurons innervate contralateral premotor networks in the lumbar and brachial spinal cord, and their ascending projections innervate the cerebellum. These findings suggest that dI2 neurons function as interneurons in local lumbar circuits, are involved in lumbo-brachial coupling, and that part of them deliver peripheral and intraspinal feedback to the cerebellum. Silencing of dI2 neurons leads to destabilized stepping in posthatching day 8 hatchlings, with occasional collapses, variable step profiles, and a wide-base walking gait, suggesting that dI2 neurons may contribute to the stabilization of the bipedal gait.

Abstract During skeletal development, bone growth and mineralization require transport of substantial amounts of calcium, while maintaining very low concentration. How an organism overcomes this major logistical challenge remains mostly unexplained. To shed some light on the dynamics of this process, cryogenic focused ion beam‐scanning electron microscopy (cryo‐FIB/SEM) is used to image forming bone tissue at day 13 of a chick embryo femur. Both cells and matrix in 3D are visualized and observed as calcium‐rich intracellular vesicular structures. Counting the number of these vesicles per unit volume and measuring their calcium content based on the electron back‐scattering signal, the intracellular velocity at which these vesicles need to travel to transport all the calcium required for the mineral deposited in one day within the collagenous tissue can be estimated. This velocity at 0.27 µm s−1 is estimated, which is too large for a diffusion process and rather suggests active transport through the cellular network. It is concluded that calcium logistics is hierarchical and based on several transport mechanisms: first through the vasculature using calcium‐binding proteins and the blood flow, then active transport over tens of micrometers through the network of osteoblasts and osteocytes, and finally diffusive transport over the last one or two microns.

Research focus: Chlorpyrifos is an organophosphate insecticide used primarily to control pests on a variety of food and feed crops. Humans are directly or indirectly exposed to this pesticide through food, air, and occupation. The ill effects of chlorpyrifos on various organ systems of human has been widely documented, but less is known about its influence on human bones. Aim: To analyze the effect of chlorpyrifos and its metabolites 3,5,6‐trichloro‐2‐pyridinol (TCPy) on the skeletal system of the chick embryo. Materials and Methods: Fertilized chick eggs were exposed to different concentrations of chlorpyrifos and its metabolite 3,5,6‐TCPy on 1.5 days of incubation. The proximal phalanx of 18‐day‐old embryos was analyzed for defects in growth and ossification through histopathology, immunohistochemistry, angiogenesis assay, and gene expression study. Results: Dose‐dependent variations in developing bone of chick embryo were observed. Histochemical and histomorphometry studies of proximal phalanx showed increased in the growth plate length (F(9, 59) = 228.9509, p =.00001) with a reduction in the total length of the phalanx (F(9, 59) = 109.9905, p =.00001), decreased mineralization (F(9, 59) = 224.6872, p =.00001), decreased blood islands in the bone marrow (F(9, 59) = 7.7083, p =.0001) of chlorpyrifos, and 3,5,6‐TCPy‐exposed group. Significant downregulations in the expression patterns of the transcription factors, such as SOX9, RUNX2, and ALP, were also observed. Conclusion: Chlorpyrifos and its metabolite 3,5,6‐TCPy exposure alters the chondrogenesis in the growth plate cartilage of long bone in chick embryo. The pesticide and its metabolite also interfere in ossification. [ABSTRACT FROM AUTHOR]

Background: Tramadol hydrochloride or tramadol is an opioid analgesic that acts on the central nervous system. The pregnancy category of tramadol is determined as "C" according to the Food and Drug Administration. There are no adequate and well‐controlled studies in pregnant women. In this study, we aimed to reveal the effects of tramadol on neural tube (midline) closure by analyzing morphologically, histologically and genetically in chick embryos. Methods: Ninety White Leghorn species, fertile and 0‐day‐old specific pathogen‐free eggs (60 ± 5 g) were used in the study. Eggs were divided into a total of six groups (control, sham, and drug groups). Four different doses of tramadol (1, 2.5, 5, and 7.5 mg/egg) were administered subblastodermically at the 28th hour of the incubation. All eggs were opened at the 48th hour of incubation and evaluated. Results: Embryos in the control group according to Hamburger–Hamilton classification were compatible with stages 13 and 14. In the groups treated with tramadol, it was determined that the embryos had neural tube closure defects (such as neck, tail regions) and some embryos showed developmental retardation due to the increase in the drug dose. In the statistical analysis performed, a significant difference was found between the control group and the group receiving the highest dose of tramadol in terms of crown‐rump length and number of somites (p <.05). The brain and reproductive expression gene expression was upregulated in embryos at each of tramadol doses compared to control group. Conclusions: It was determined that tramadol causes neural tube closure defects in embryos depending on the dose. [ABSTRACT FROM AUTHOR]

Areca nut (AN) and smokeless tobacco (SLT) are indiscriminately consumed among the populations of Southeast and South Asian countries, even by women during the gestational period. This study aimed to investigate the genotoxic and cytotoxic potentials of AN and Sadagura (SG), a unique homemade SLT preparation, alone and in combination in early chick embryos. Fertile white leghorn chicken eggs were randomly divided into five treatment groups: vehicle control, positive control (Mitomycin C, 20 μg/egg), AN, SG, and AN+SG. AN, SG, and AN+SG were given at dosages of 0.125, 0.25, and 0.5 mg/egg. The hen's egg test for micronucleus induction (HET‐MN) was performed in chick embryos to evaluate the genotoxic potential of the test agents. Furthermore, the cytotoxic potential was assessed by studying erythroblast cell populations and the polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) ratio. Our results indicated a significant increase (p <.001) in MN frequency and other nuclear abnormalities, suggesting the potential of AN and SG to cause genotoxicity. Also, AN and SG exposure alone and in combination considerably altered the erythroblast cell population (%) and the PCE to NCE ratio in all the treatment periods. Our findings established the genotoxic and cytotoxic potential of both AN and SG alone and in combination during early embryonic development in the chick embryo. [ABSTRACT FROM AUTHOR]

In amniote vertebrates, the definitive dorsal aorta is formed by the fusion of two primordial aortic endothelial tubes. Formation of the definitive dorsal aorta requires extensive cellular migrations and rearrangements of the primordial tubes in order to generate a single vessel located at the embryonic ventral midline. This study examines the role of VEGF signaling in the generation of the definitive dorsal aorta. Through gain- and loss-of-function studies in vivo in the chick embryo, we document a requirement for VEGF signaling in growth and remodeling of the paired primordia. We find that regions of the aorta are differentially sensitive to levels of VEGF signaling, and present evidence that areas of low blood flow are more sensitive to the loss of VEGF signaling. We also find that VEGF signaling regulates the intracellular distribution between membrane and cytoplasm of the cell-cell adhesion molecule VE-cadherin in aortic endothelial cells in vivo. Together, these finding identify mechanisms that likely contribute to the dynamic behavior of endothelial cells during aorta morphogenesis. [Display omitted] • Lowering VEGF levels causes defects in chick aorta morphogenesis. • Regions of the aorta are differentially sensitive to lowering of VEGF levels. • VEGF-sensitive regions are areas of lower blood flow. • VEGF causes redistribution of VE-Cadherin from membrane to cytoplasm. [ABSTRACT FROM AUTHOR]

Since 2014, methods have been described to hatch chick embryos from shell‐less culture after egg contents are first incubated within shells for 55–70 h. The present report describes for the first time a shell‐less culture system for chick embryos from the blastoderm stage to hatching. For the first 69–70 h, egg contents suspended in polymethylpentene kitchen wrap (F.O.R. Wrap, Riken Fabro, Tokyo, Japan) supported in 6.35 or 6.67 cm inside diameter tripods and covered with a disc of immobilized Milli‐Wrap, were rotated back and forth through 90° at 16 or 22 cycles per minute (CPM). Subsequently, the Milli‐Wrap disc was removed and culture tripods were transferred to environmental chambers, which were rocked ±20° through incubation day 8.5 (E8.5). From E9, environmental chambers were maintained in the horizontal position through to hatching with controlled O2 and CO2. To provide supplemental calcium, an aqueous solution containing 100 mg/mL of calcium l‐lactate hydrate was injected through the plastic wrap into the albumen at E9 (2.5 mL) and at E13 (1.0 mL) or E15 (1.0 mL). After incubation for 69–70 h at 16 or 22 CPM, 80%–83% of previously unincubated egg contents yielded apparently normal embryos. Hatch rate of normal embryos resulting from turntable incubation at 16 or 22 CPM was approximately 43%. Of note, egg contents remained in the same culture tripod from blastoderm stage to hatching. This technique may find use as an educational tool and in basic investigations of early embryogenesis, teratogenesis, and gene transfer experiments. Research Highlights: •A novel technique is described for shell‐less culture of chick embryos from the blastoderm stage to hatching.•For the first 69–70 h, egg contents suspended in plastic wrap are covered with a disc of immobilized Milli‐Wrap, then rotated back and forth through 90° at 16 or 22 cycles per minute. [ABSTRACT FROM AUTHOR]

During development, the hypothalamus emerges from the ventral diencephalon and is regionalized into several distinct functional domains. Each domain is characterized by a different combination of transcription factors, including Nkx2.1, Nkx2.2, Pax6, and Rx, which are expressed in the presumptive hypothalamus and its surrounding regions, and play critical roles in defining each area. Here, we recapitulated the molecular networks formed by the gradient of Sonic Hedgehog (Shh) and the aforementioned transcription factors. Using combinatorial experimental systems of directed neural differentiation of mouse embryonic stem (ES) cells, as well as a reporter mouse line and gene overexpression in chick embryos, we deciphered the regulation of transcription factors by different Shh signal intensities. We then used CRISPR/Cas9 mutagenesis to demonstrate the mutual repression between Nkx2.1 and Nkx2.2 in a cell-autonomous manner; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region. Our findings suggest that Shh signaling and its downstream transcription network are required for hypothalamic regionalization and establishment. [ABSTRACT FROM AUTHOR]

Highly dynamic oxygen gradients occur within tumors that can result in a hypoxic response, contributing to tumor progression and metastasis. Evidence in uveal melanoma (UM) suggests an upregulated hypoxia response in some poor prognosis UM characterized by HIF1α signaling. We aimed to investigate the effects of exposure to hypoxia on tumor growth and dissemination in the chick embryo chorioallantoic membrane (CAM) model. UM cell lines (MP41, 92.1, MP46, and OMM1) were grown in two-dimensional culture and pre-exposed to hypoxic (1% O2) conditions for 72 h. The effects of this hypoxia pre-conditioning on cell number and clonogenicity as compared with 21% O2 ("normoxia") were investigated prior to transplantation of the cells onto the CAM. Nodule-forming efficiency (NFE), nodule size, and the presence/absence of tumor cell dissemination were determined macroscopically and histologically. Exposure of UM cell lines to hypoxia upregulated HIF1α expression compared to cells cultured in normoxia. A 72-h pre-exposure to hypoxia significantly reduced cell number and clonogenicity in the MP41 and OMM1 cell lines while it had little effect in 92.1 and MP46 cells. When 72-h hypoxia pre-conditioned cells were grown in three-dimensions on the CAM, a reduction in NFE and nodule size was observed when compared with normoxic UM cells. All nodules were composed of proliferating (Ki-67+) Melan-A + cells and displayed chick blood vessel recruitment. Spread of UM cells into the adjacent CAM was observed; however, dissemination to the chick liver was only seen with 92.1 cells grown under normoxia. Hypoxia pre-conditioning does not appear to drive a metastatic phenotype in UM; however, further understanding of how oxygen dynamics within the tumor microenvironment regulates HIF1 signaling is needed to determine whether inhibitors of HIF signaling represent a therapeutic option in metastatic UM. [ABSTRACT FROM AUTHOR]

Objective(s): Demineralized bone matrix (DBM) is an allograft bone composed of native insoluble bone morphogenetic proteins and plays important roles in skeletal development, osteogenesis, and differentiation of mesenchymal stem cells. The osteoinductive capabilities of Allogenic DBM make it a potential drug delivery system for preventive treatment in various anatomical sites. In this study, the cytotoxic and teratogenic effects of DBM nanoparticles, on Wharton's jelly mesenchymal stem cells and chicken embryos were evaluated. Materials and Methods: DBM nanoparticles were injected into fertile eggs at doses of 10, 20, 40, 80, and 100 µM / egg. Then morphological, histological, and skeletal malformations were evaluated. Cytotoxic effects of DBM nanoparticles on Wharton's jelly mesenchymal stem cells were also assessed using MTT test. Results: Results showed that the fetal growth abnormality occurred only in embryos treated at the highest dose tested (i.e., 100 µM / egg) and MTT test showed no cytotoxicity in low concentration. Conclusion: These results indicated that nanoparticles do not have significant toxic effects on chick embryos and cultured stem cells. Only high doses of DBM nanoparticles reduce growth in embryos and cultured cells. [ABSTRACT FROM AUTHOR]

A method is described for culturing 64–70 h‐old chicken embryos and egg contents outside of the eggshell through to hatching. Cultured egg contents were suspended in polymethylpentene kitchen wrap (F.O.R. wrap; Riken Fabro) supported in polyvinyl chloride tripods. Tripods were incubated in Plexiglas environmental chambers which were rocked automatically through an angle of ±20°. The concentration of CO2 was maintained at 2% throughout incubation, while that of O2 was increased from ambient to 50%, and relative humidity was decreased from 90%–92% to 83%–84% at incubation Day 9. Cultured embryos not supplemented with calcium did not hatch. The Hatch rate increased when supplemental calcium L‐lactate hydrate was increased between 250 and 350 mg. A maximal hatch rate of 54.8% was achieved when cultures were supplemented with 350 mg of calcium L‐lactate hydrate and 3.5 ml of sterile water. Adding 400 or 450 mg of calcium L‐lactate hydrate did not increase the hatch rate further. The mass of cultured hatchlings (including the retracted yolk) and yolk‐free carcass wet and dry mass and length of the right third toe were significantly less than the corresponding parameters observed in hatchlings in ovo. No statistically significant differences in hatchling mass, yolk‐free carcass wet or dry mass, or length of the right third toe were noted among cultured hatchlings supplemented with 250–450 mg of calcium L‐lactate hydrate. Failure to completely absorb albumen was the most common abnormality observed in cultures which failed to hatch. The present technique allows a unique approach to study the physiology of the developing chicken embryo. Research Highlights: Supplementing shell‐less cultures of chick embryos with calcium L‐lactate hydrate and water resulted in a maximal hatch rate of 54.8%.Cultured hatchling yolk‐free dry mass was about 77% that of in ovo hatchings and was not increased with more calcium. [ABSTRACT FROM AUTHOR]

A histological examination is an important tool in embryology, developmental biology, and correlated areas. Despite the amount of information available about tissue embedding and different media, there is a lack of information regarding best practices for embryonic tissues. Embryonic tissues are considered fragile structures, usually small in size, and frequently challenging to position correctly in media for the subsequent histological steps. Here, we discuss the embedding media and procedures that provided us with appropriate preservation of tissue and easier orientation of embryos at early development. Fertilized Gallus gallus eggs were incubated for 72 h, collected, fixed, processed, and embedded with paraplast, polyethylene glycol (PEG), or historesin. These resins were compared by the precision of tissue orientation, the preview of the embryos in the blocks, microtomy, contrast in staining, preservation, average time, and cost. Paraplast and PEG did not allow correct embryo orientation, even with agar–gelatin pre-embedded samples. Additionally, structural maintenance was hindered and did not allow detailed morphological assessment, presenting tissue shrinkage and disruption. Historesin provided precise tissue orientation and excellent preservation of structures. Assessing the performance of the embedding media contributes significantly to future developmental research, optimizing the processing of embryo specimens and improving results. [ABSTRACT FROM AUTHOR]