Birds have enough conditions to be used as bioindicators for the presence of environmental contaminants. Notably, chlorpyrifos (CPF) remains extensively employed in Argentina, particularly in fruit plantations and livestock settings This study aimed to assess the potential impacts of CPF on common quail (Coturnix coturnix) embryos through external eggshell application during key embryonic stages (days 1, 4, and 14). Concentrations equivalent to those used in fruit applications, 5× and 10 × (38.4, 192, and 384 µg/egg), were employed. A 48% commercial formulation of CPF (Pirfos - Icona®) was utilized. An increase in embryonic deaths, as well as a statistical association between the degree of liver or kidney lesions and CPF concentrations was observed. The results suggest that CPF can induce embryotoxic effects with a single exposure to quail embryos and compromise the viability of the organisms. The study suggests a potential exposure risk for embryos through contact with the insecticide on the eggshell\'s exterior.

BACKGROUND: In avian species, primordial germ cells (PGCs) migrate to the gonadal primordium through the vascular system. Because this mode of migration is reminiscent of cancer metastasis, it would be useful to elucidate the mechanisms underlying PGC migration via the bloodstream. Here, we sought to determine when, where, and how PGCs enter the vascular network by double visualization of PGCs and endothelial cells (ECs) in tie1:H2B-eYFP transgenic quails.
RESULTS: In the left and right lateral germinal crescent regions corresponding to the anterior-most area vasculosa, more than 60% of PGCs were enveloped by differentiating ECs forming blood islands prior to vascular network formation. Cell morphology analysis suggested that the PGC-EC interaction was instructed by differentiating ECs. At a later developmental stage, ECs anastomosed to form a vascular network with a lumen that retained PGCs within it. As a consequence, many PGCs localized within the luminal space of the mature vascular network at later stages.
CONCLUSIONS: Our findings demonstrate that the major type of avian PGC translocation into vascular tissue is not a typical intravasation, as performed by types of metastatic cancer cells, but rather a passive translocation (envelopment) mediated by differentiating ECs during early vasculogenesis.

The material properties of tissues and their mechanical state is an important factor in development, disease, regenerative medicine and tissue engineering. Here we describe a microrheological measurement technique utilizing aggregates of microinjected ferromagnetic nickel particles to probe the viscoelastic properties of embryonic tissues. Quail embryos were cultured in a plastic incubator chamber located at the center of two pairs of crossed electromagnets. We found a pronounced viscoelastic behavior within the ECM-rich region separating the mesoderm and endoderm in Hamburger Hamilton stage 10 quail embryos, consistent with a Zener (standard generalized solid) model. The viscoelastic response is about 45% of the total response, with a characteristic relaxation time of 1.3 s.

Animals developed or in an embryonic stage, are constantly subjected to magnetic pollution generated by electrical and electronic devices. Several researches have used the bird embryo as an experimental model to evaluate the action of magnetic field (MF) and electromagnetic field (EMF). This study proposed to perform a morphometric evaluation in the embryos and in the blood vascular network of the yolk sac membranes (YSM) of Japanese quail (Coturnix japonica) exposed to the 60 Hz MF with two different intensities (0.16 and 0.65 mT). A total of 30 eggs were used, 10 eggs were used for each assay. Each assay formed a group (control group, group submitted to the MF of 0.16 mT and 0.65 mT). The images of the skeletonized vascular network of YSM were evaluated by two methods of fractal dimension: box-counting dimension (Dbc) and information dimension (Dinf). The embryos were evaluated by body mass, percentage cephalic length and body area. The fractal dimensions revealed no difference among groups. There were no significant differences in relation to embryonic body mass among groups. However, the embryos exposed to 0.65 mT MF presented a smaller embryonic body development (body area and percentage cephalic length). In conclusion, 0.16 mT and 0.65 mT magnetic fields were not able to generate significant effects on vasculogenesis and angiogenesis. However, the embryos exposed to 6 h of magnetic field with 0.65 mT intensity and 60 Hz frequency showed a decrease in embryonic body development.

Pathological angiogenesis characterized by uncontrollable vessel growth is an accompanying feature of many diseases. The avian embryo chorioallantoic membrane (CAM) is an excellent model for angiogenesis research. In our study we used a less common Japanese quail CAM model for the testing of angiogenic potential of leptin, high-molecular (heparin sodium) andlow-molecular (nadroparin calcium) heparins. Heparins play a significant role in vascular endothelial cell function, and they are able to modulate the activities of angiogenic growth factors. On embryonic day 7 leptin (5 μg per CAM), heparin sodium (75 IU per CAM) and nadroparin calcium (47.5 IU per CAM) in 500 μl PBS were applied on the CAM surface. After 24 h the fractal dimension (Df) of the vasculature was evaluated. Samples from each group were histologically analyzed and VEGF-A and Quek1 expression were detected by qPCR. Df was significantly increased in the leptin group. A moderate stimulatory effect of heparin sodium and an inhibitory effect of nadroparin calcium were observed. Both leptin and heparin sodium caused a noticeable increase in the CAM thickness compared to the control and nadroparin calcium groups. We observed an increased number of blood vessels and accumulation of fibroblasts. There was no significant impact on gene expression of VEGF-A and Quek1 24 h after treatment, however, trends similar to the changes in Df and CAM thickness were present. The resulting effect of nadroparin administration on Quek1 levels was exactly the opposite to that of leptin (p < 0.05).

Photodynamic therapy with hypericin (HY-PDT) and hyperforin (HP) could be treatment modalities for colorectal cancer (CRC), but evidence of their effect on angiogenic factors in CRC is missing. Convenient experimental model utilization is essential for angiogenesis research. Therefore, not only 2D cell models, but also 3D cell models and micro-tumors were used and compared. The micro-tumor extent and interconnection with the chorioallantoic membrane (CAM) was determined by histological analyses. The presence of proliferating cells and HY penetration into the tumor mass were detected by fluorescence microscopy. The metabolic activity status was assessed by an colorimetric assay for assessing cell metabolic activity (MTT assay) and HY accumulation was determined by flow cytometry. Pro-angiogenic factor expression was determined by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR). We confirmed the cytotoxic effect of HY-PDT and HP and showed that their effect is influenced by structural characteristics of the experimental model. We have pioneered a method for analyzing the effect of HP and cellular targeted HY-PDT on pro-angiogenic factor expression in CRC micro-tumors. Despite the inhibitory effect of HY-PDT and HP on CRC, the increased expression of some pro-angiogenic factors was observed. We also showed that CRC experimental micro-tumors created on quail CAM could be utilized for analyses of gene and protein expression.

The quail-chick chimera marking system, devised in 1969, gave a new impetus to the analysis of cell migrations and interactions in the developing nervous, immune and hematopoietic systems. The method is based on the observation that the constitutive heterochromatin in all embryonic and adult cells of the quail is condensed in one large mass in the centre of the nucleus and is associated with the nucleolus, making this organelle strongly stained with the Feulgen-Rossenbeck reaction. The association of cells or rudiments from two avian species, advocated as a means to identify cells that migrate during embryogenesis, was rapidly recognized in this context as a useful tool for the study of many developmental biology problems. This article summarizes the fundamental contribution of Nicole Le Douarin to the discovery and the application of this technique over the last 40 years.

Avian primordial germ cells (PGCs) have valuable potentials to cell-based approaches for transgenic bird production. In this regard, improvement of avian PGC expansion in vitro is necessary. Among experimental avian species, quail is a good model for transgenic technology, especially due to its short generation time. In the present study, we have examined the proliferative effects of transforming growth factor β (TGF-β) on the quail PGCs. After isolation of quail PGCs from blood (Hamburger-Hamilton [HH stages 13-15]) and gonads (HH stages 28-30), these cells were cultured on quail embryonic fibroblasts (QEF). Our results indicated th at cultured gonadal-derived PGCs proliferated 400 times in comparison to 100 times for blood PGCs over 40-50 days. Upon in vitro exposure to TGF-β inducers by Activin or the inducer of definitive endoderm 1 (IDE1) small molecule, the number of gonad PGCs significantly increased to 26% and 64%, respectively. In contrast, inhibition of the TGF-β signaling pathway by SB431542 resulted in a significant reduction in the numbers of PGCs (P < 0.001). Moreover, Phosphorylation of SMAD2/3 in the IDE1 group was higher compared to the Activin-treated ones. We confirmed the PGC identification with periodic acid-Schiff (PAS) staining, anti-SSEA1, β-catenin, β-integrin, and Nanog immunofluorescence staining. Exogenously IDE1 treated-PGCs migrated toward the embryonic gonads after transplantation into the heart of the recipient embryo at HH stages 13-15. Our results suggested that the application of IDE1 small molecule into the culture of quail PGCs represented a step toward achieving efficient expansion of the avian PGCs.

Embryonic axis elongation is a complex multi-tissue morphogenetic process responsible for the formation of the posterior part of the amniote body. How movements and growth are coordinated between the different posterior tissues (e.g. neural tube, axial and paraxial mesoderm, lateral plate, ectoderm, endoderm) to drive axis morphogenesis remain largely unknown. Here, we use quail embryos to quantify cell behavior and tissue movements during elongation. We quantify the tissue-specific contribution to axis elongation using 3D volumetric techniques, then quantify tissue-specific parameters such as cell density and proliferation. To study cell behavior at a multi-tissue scale, we used high-resolution 4D imaging of transgenic quail embryos expressing fluorescent proteins. We developed specific tracking and image analysis techniques to analyze cell motion and compute tissue deformations in 4D. This analysis reveals extensive sliding between tissues during axis extension. Further quantification of tissue tectonics showed patterns of rotations, contractions and expansions, which are consistent with the multi-tissue behavior observed previously. Our approach defines a quantitative and multi-scale method to analyze the coordination between tissue behaviors during early vertebrate embryo morphogenetic events.

The vitamin A deficiency-associated lethal syndrome observed in avian embryos may be linked to dysfunction of vitamin A-dependent genes. We tested this hypothesis in a quail embryo model by examining the expression of retinoic acid receptors (RARs) and cytosolic retinoic acid binding protein (CRABP) in normal and vitamin A-deficient embryos during early development. RARα and RARγ mRNA were expressed at the same level in normal and vitamin A-deficient embryos during all stages studied. Expression of CRABP I was low in normal and vitamin A-deficient quail embryos during early development, but increased rapidly at later stages. Two transcripts of RARβ, 3.2 and 3.5 kb, were detected in quail embryos during developmental stages 6-12. In normal emryos the level of the 3.2-kb isoform increased as embryonic development advanced. The expression of the 3.5-kb transcript was significantly decreased in vitamin A-deficient embryos, while the 3.2-kb transcript was undetectable by northern analysis. In situ hybridization of stage 7-8 normal quail embryos using a chicken RARβ2 riboprobe revealed that RARβ2 expression was predominantly associated with the cell populations in heart-forming regions, somites, neural folds, notochord and the presumptive thyroid. In stark contrast, in the vitamin A-deficient quail embryo RARβ2 was not expressed in any of the above cell populations. We conclude that the expressions of RARβ and CRABP I are developmentally regulated. Additionally, the expression of RARβ2 during early embryogenesis is regulated by vitamin A status. We propose that RARβ2 plays an important role in the mechanism of action of retinoids in early avian development. The lack of expression of RARβ2 may be linked to abnormalities of the cardiovascular system.