Extraembryonic Blood Vessels Research Articles

Automated non-invasive laser speckle imaging of the chick heart rate and extraembryonic blood vessels and their response to nifedipine and amlodipine drugs.

Non-invasive Laser Speckle Contrast Imaging (LSCI) of the chick chorioallantoic membrane (CAM) in whole incubated eggsSimultaneous recording images of the CAM, dynamics of blood flow, and heart rateLive view mode to identify size, heart position, and location of the embryo in the eggAutomated system for data acquisition and analysisLongitudinal quantification of the impact of a calcium channel antagonists, nifedipidine and amlodipine on the embryonic heart rate, CAM's blood flow, size and number of vessels.

Non-invasive laser speckle contrast imaging (LSCI) of extra-embryonic blood vessels in intact avian eggs at early developmental stages.

Imaging blood vessels in early-stage avian embryos has a wide range of practical applications for developmental biology studies, drug and vaccine testing, and early sex determination. Optical imaging, such as brightfield transmission imaging, offers a compelling solution due to its safe non-ionizing radiation, and operational benefits. However, it comes with challenges, such as eggshell opacity and light scattering. To address these, we have revisited an approach based on laser speckle contrast imaging (LSCI) and demonstrated a high-quality, comprehensive, and non-invasive visualization of blood vessels in few-days-old chicken eggs, with blood vessels as small as 100 µm in diameter (with LSCI profile full-width-at-half-maximum of 275 µm). We present its non-invasive use for monitoring blood flow, measuring the embryo's heartbeat, and determining the embryo's developmental stages using machine learning with 85% accuracy from stage HH15 to HH22. This method can potentially be used for non-invasive longitudinal studies of cardiovascular development and angiogenesis, as well as egg screening for the poultry industry.

Exploring developmental MeHg impact on extraembryonic and cardiac vessels and its effect on cardiomyocyte contractility.

The toxicity of methylmercury (MeHg) during embryonic development is a relevant issue that remains unclear and deserves investigation. In this sense, there is evidence that links the intake of contaminated food with cardiovascular pathologies in human adults and children. Thus, this study aimed to verify the impact of MeHg on the structure and integrity of extraembryonic and cardiac blood vessels and the contractile function of cardiomyocytes, also evaluating embryonic weight and the cardiosomatic index (CSI). Thus, chicken embryos, used as an experimental model, were exposed to a single dose of 0.1μg MeHg/50 μl saline at E1.5 and analyzed at E10. After exposure, an increase in the number of extraembryonic blood vessels and the veins of the cardiac tissue was observed. These increases were accompanied by a reduction in the content of VEGF and VCAM proteins related to vessel growth and adhesiveness. Together, these results were related to reduced nitrite (NOx) levels. Furthermore, MeHg reduces the number of sarcomeres and increases the content of cardiac troponin I (cTnI), a protein that regulates contraction. In general, exposure to MeHg affected the integrity of extraembryonic and cardiac vessels and the contractile function of cardiomyocytes, which had a systemic impact evidenced by the reduction in embryonic weight gain and CSI.

Developmental toxicity in early chicken embryos on exposure to an organophosphorus flame retardant, tris(2-chloroisopropyl) phosphate

Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage avian embryos are vulnerable to the toxic effects of chemicals. However, studies on the specific effects of TCIPP on avian embryonic development are limited. We aimed to investigate the toxicity of TCIPP in early chicken embryos using a previously developed shell-less incubation system. Fertilized chicken (Gallus gallus domesticus) eggs (n = 220) were exposed to 50 or 500 nmol TCIPP/(g egg) or dimethyl sulfoxide (DMSO) as a vehicle control on Day 0 of incubation. Development of 198 embryos was monitored from Days 3–9 of incubation, and 22 embryos on Day 4 and 74 embryos on Day 9 were dissected. Messenger RNA expression levels for several genes were measured in embryos on Day 4. Both TCIPP-exposed groups showed a significant reduction in survival rate. Imaging analyses revealed significant decreases in body length, head and bill length, eye diameter, and forelimb and hindlimb length in both TCIPP-treated groups. TCIPP exposure significantly impaired the development of extraembryonic blood vessels and the production of red blood cells. A TCIPP-dose-dependent decreasing trend in heart rate was observed on Days 4–7. The somitic angle increased significantly on Days 4–6, and embryos with curved somites showed cleavage in the back and gaps between somites, resulting in asymmetrical somite formation. A significant correlation was found between the somitic angle and FGF8 expression levels, suggesting that TCIPP exposure affects somite formation through an altered FGF-signaling pathway. Embryos with somitic deformities in TCIPP-exposed groups had significantly reduced survival rates, indicating that abnormal segment formation directly increased mortality. Finally, eye weight and ocular luminosity values were significantly reduced, suggesting that TCIPP may also affect eye development. Overall, these findings highlight severe toxic effects of TCIPP on avian embryonic development, including in vascularization, cardiac function, and somite and ocular development.

Y-27632 Impairs Angiogenesis on Extra-Embryonic Vasculature in Post-Gastrulation Chick Embryos

Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in various embryonic developmental processes, including angiogenesis, by controlling actin cytoskeleton assembly and cell contractility. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos, leading to ventral body wall defects (VBWDs). Impaired angiogenesis has been hypothesized to contribute to VBWDs. ROCK is essential in transmitting signals downstream of vascular endothelial growth factor (VEGF). VEGF-mediated angiogenesis induces gene expressions and alterations of the actin cytoskeleton upon binding to VEGF receptors (VEGFRs). The aim of this study was to investigate effects of Y-27632 on angiogenesis in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Y-27632-treated embryos showed reduced extra-embryonic blood vessel formation with impaired circulation of the yolk sac, confirmed by fractal analysis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated myosin light chain. Interestingly, RT-PCR demonstrated increased gene expression of VEGF and VEGFR-2 1 h post-treatment. Protein levels of VEGF were higher in Y-27632-treated embryos at 8 h following treatment, whereas no difference was seen in membranes. We hypothesize that administration of Y-27632 impairs vessel formation during angiogenesis, which may contribute to failure of VWB closure, causing VBWDs.

Effects of tris(2-chloroethyl) phosphate exposure on chicken embryos in a shell-less incubation system

Tris(2-chloroethyl) phosphate (TCEP) is an organophosphate flame retardant that used in textiles, industrial materials, and furniture to delay the spread of fire after ignition. TCEP has been detected in the tissues and eggs of fish and birds. However, there are no studies regarding the effects of TCEP on avian embryos. In the present study, we investigated the developmental toxicity of TCEP exposure on chicken embryos in a shell-less incubation system, which enables in situ observation. Chicken embryos were treated with graded doses of TCEP (50, 250, and 500 nmol/g egg) on incubation day 0. The survival rate, morphological biometrics, heart rate, and length and branch number of extraembryonic blood vessels were measured on incubation days 3–9. Survival rates were reduced from incubation day 3 and were significantly decreased until day 9. Body length, head + bill length and eye diameter were significantly reduced by TCEP exposure. Regarding skeletal effects, spine length was decreased in a dose-dependent manner on day 9. Body weight on day 9 significantly reduced in all TCEP treatment groups. These results suggest that TCEP exposure to >50 nmol/g egg retards development in chicken embryos. TCEP exposure to 500 nmol/g egg significantly increased heart weight to body weight ratio in the embryos. More than 250 nmol/g egg of TCEP significantly reduced the heart rate of embryos in the early developmental stage. The formation of extraembryonic blood vessels and the number of erythrocytes were significantly reduced even with 50 nmol/g egg of TCEP. These findings suggest that TCEP exposure specifically affects the cardiovascular system in chicken embryos, which leads to developmental delay. The results of this study also demonstrate that the shell-less incubation system can be used to continuously monitor the effects of chemicals on developing avian embryos.

Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

Egg-in-cube: design and fabrication of a novel artificial eggshell with functionalized surface.

An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research.

Moderate Calcium Channel Dysfunction in Adult Mice with Inducible Cardiomyocyte-specific Excision of the cacnb2 Gene

The major L-type voltage-gated calcium channels in heart consist of an α1C (Ca(V)1.2) subunit usually associated with an auxiliary β subunit (Ca(V)β2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of Ca(V)β2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the Ca(V)β2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, Ca(V)β2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include Ca(V)β2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other Ca(V)β proteins or changes of Ca(V)1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, Ca(V)β2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, Ca(V)β2 down-regulation in cardiomyocytes is well tolerated by the adult mice.

A point mutation in Arid1a reveals an essential role for this SWI/SNF subunit in extraembryonic blood vessel and trophoblast development

A point mutation in Arid1a reveals an essential role for this SWI/SNF subunit in extraembryonic blood vessel and trophoblast development

A follistatin-BMP7 feedback circuit controls taste papillae development and patterning in mouse tongue

A follistatin-BMP7 feedback circuit controls taste papillae development and patterning in mouse tongue

Loss of Mouse Ikbkap, a Subunit of Elongator, Leads to Transcriptional Deficits and Embryonic Lethality That Can Be Rescued by Human IKBKAP

Familial dysautonomia (FD), a devastating hereditary sensory and autonomic neuropathy, results from an intronic mutation in the IKBKAP gene that disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP) in the nervous system. To better understand the roles of IKAP in vivo, an Ikbkap knockout mouse model was created. Results from our study show that ablating Ikbkap leads to embryonic lethality, with no homozygous Ikbkap knockout (Ikbkap(-)(/)(-)) embryos surviving beyond 12.5 days postcoitum. Morphological analyses of the Ikbkap(-)(/)(-) conceptus at different stages revealed abnormalities in both the visceral yolk sac and the embryo, including stunted extraembryonic blood vessel formation, delayed entry into midgastrulation, disoriented dorsal primitive neural alignment, and failure to establish the embryonic vascular system. Further, we demonstrate downregulation of several genes that are important for neurulation and vascular development in the Ikbkap(-)(/)(-) embryos and show that this correlates with a defect in transcriptional elongation-coupled histone acetylation. Finally, we show that the embryonic lethality resulting from Ikbkap ablation can be rescued by a human IKBKAP transgene. For the first time, we demonstrate that IKAP is crucial for both vascular and neural development during embryogenesis and that protein function is conserved between mouse and human.

Micro-PIV measurements of blood flow in extraembryonic blood vessels of chicken embryos

The hemodynamic characteristics of blood flow are important in the diagnosis of circulatory diseases, since such diseases are related to wall shear stress of cardiovascular vessels. In chicken embryos at early stages of development, it is possible to directly visualize blood flow inside blood vessels. We therefore employed a micro-PIV technique to assess blood flow in extraembryonic venous and arterial blood vessels of chicken embryos, using red blood cells (RBCs) as tracers and obtaining flow images of RBCs using a high-speed CMOS camera. The mean velocity field showed non-Newtonian flow characteristics. The blood flow in two venous vessels merged smoothly into the Y-shaped downstream vein without any flow separation or secondary flow. Vorticity was high in the inner regions, where the radius of curvature varied greatly. A periodic variation of temporally resolved velocity signals, due to beating of the heart, was observed in arterial blood vessels. The pulsating frequency was obtained by fast Fourier transform analysis using the measured velocity data. The measurement technique used here was useful in analyzing the hemodynamic characteristics of in vivo blood flow in chicken embryos.

Reduced Cardiac L-Type Ca 2+ Current in Ca v β 2 −/− Embryos Impairs Cardiac Development and Contraction With Secondary Defects in Vascular Maturation

Cardiac myocyte contraction depends on transmembrane L-type Ca2+ currents and the ensuing release of Ca2+ from the sarcoplasmic reticulum. Here we show that these L-type Ca2+ currents are essential for cardiac pump function in the mouse at developmental stages where the functional significance of the heart becomes imperative to blood flow and to the continuing growth and survival of the embryo. Disruption of the Ca(V)beta2 gene, which encodes for the predominant ancillary beta subunit of cardiac Ca2+ channels, resulted in diminished L-type Ca2+ currents in cardiomyocytes of embryonic day 9.5 (E9.5). This led to a functionally compromised heart, causing defective remodeling of intra- and extraembryonic blood vessels and embryonic death following E10.5. The defects in vascular remodeling were also observed when the Ca(V)beta2 gene was selectively targeted in cardiomyocytes, demonstrating that they are secondary to cardiac failure rather than a result of the lack of Ca(V)beta2 proteins in the vasculature. Partial rescue of the Ca2+ channel currents by a Ca2+ channel agonist significantly postponed embryonic death in Ca(V)beta2-/- mice. Taken together, these data strongly support the essential role of L-type Ca2+ channel activity in cardiomyocytes for normal heart development and function and that this is a prerequisite for proper maturation of the vasculature.

유정란 배아 혈액유동의 in vivo 계측

To analyze in-vivo blood flow characteristics in a chicken embryo, in-vivo experiment was carried out using micro-PIV technique. Because endothelial cells in blood vessels are subject to shear stress of blood flow, it is important to get velocity field information of the placental blood flow. Instantaneous velocity fields of an extraembryonic blood vessel using a high-speed camera and intravital microscope. The flow images of RBCs were obtained with a spatial resolution of 20×20 ㎛ in the whole blood vessels. The mean velocity field data confirm that the blood flow does show non-Newtonian fluid characteristic. The blood in a branched vessel merged smoothly without any flow separation into the main blood vessel with the presence of a slight bump. This in-vivo micro-PIV measurement technique can be used as a powerful tool in various blood flow researches.

Comparison of maf gene expression patterns during chick embryo development

Maf proteins are basic-leucine zipper transcription factors belonging to the AP1 superfamily. Several developmental processes require Maf proteins yet, the redundancy or complementarity of their respective roles in common processes has been only partially investigated. We present for the first time a complete comparative analysis of maf gene expression patterns in vertebrates. Expression of c- maf, mafB/ kreisler, mafA/L- maf, mafF, mafG and mafK was analyzed by whole-mount in situ hybridization within chick embryos and their extraembryonic tissues ranging from embryonic day (E) 1 to 7. We carefully examined the extent of overlap between distinct maf genes and report that the developing lens, kidney, pancreas and apoptotic zones of limb buds show sustained co-expression of large maf genes. Small maf genes also exhibit overlap, for example in the dermomyotome. We also describe so far unidentified sites of maf gene expression. mafA is found in the developing neural tube and dorsal root ganglia. c- maf hybridization is detected in the neuroretina, the notochord and the endothelium of extraembryonic blood vessels.

Opposing functions of the Ets factors NERF and ELF-1 during chicken blood vessel development.

The purpose of this study was to evaluate the role of the Ets factor NERF in the regulation of the Tie1 and Tie2 genes during chicken blood vessel development. We have isolated the full-length cDNA for the chicken homologue of the human Ets factor NERF2 (cNERF2). Northern blot analysis and in situ hybridization demonstrate that cNERF2 is enriched in the developing blood vessels of the chicken chorioallantoic membrane. Interestingly, cNERF2 functions as a competitive inhibitor of a highly related Ets factor cELF-1, which we have previously shown to be enriched in chicken blood vessel development. Although in vitro-translated cELF-1 and cNERF2 can bind equally well to conserved Ets binding sites in the promoters of the Tie1 and Tie2 genes, cELF-1 preferentially binds to the Ets sites in these promoters during early stages of chicken blood vessel development, suggesting that cNERF may bind during later stages of blood vessel development and vascular remodeling. cNERF2 is enriched during embryonic and extraembryonic blood vessel development in the chicken and facilitates tight control of Tie1 and Tie2 gene regulation.

Investigating Murray's law in the chick embryo

According to the optimization principle known as Murray's law, the blood vessel geometry at a bifurcation satisfies the relation α=( D 1 3+ D 2 3)/ D 0 3=1, where D 0, D 1, and D 2 are the diameters of the parent and two daughter vessels, respectively. Previous investigations have shown that mature blood vessels adhere to this law fairly closely. The purpose of this study was to test Murray's law in the developing extraembryonic blood vessels of 2–4 day-old chick embryos. Vessel diameters were measured manually using image analysis software. The measurements for the group of all vessels at all studied stages ( n=449) gave α=1.01±0.34 (mean ± SD), and the value of α is similar at all stages. These results indicate that Murray's law holds in the chick embryo, even before medial smooth muscle becomes functional, suggesting that blood vessels follow the same basic morphogenetic rules throughout life.

In vivo investigations of vascular density and local mitochondrial metabolism after irradiation.

It is obvious that irradiation increases local oxygen uptake. Because the oxygen supply is not altered, a decrease in local oxygen partial pressure may develop. This state may be called relative hypoxia. If the metabolism is altered for a period of at least 24 hrs an increase in angiogenic mitogens, inducing angiogenesis will occur. Presumably this is the mechanism underlying the therapeutic effect of radiotherapy in benign disease.

Endothelial cell origin and migration in embryonic heart and cranial blood vessel development

Using the QH-1 monoclonal antibody as a marker for quail endothelium, blockage and transplant experiments were carried out to construct fate maps for the embryonic endocardium, to determine whether preendocardial angioblasts are migratory, and, if these cells are migratory, to outline the pathways that they use for directed migration in embryonic blood vessel development. Recent descriptive studies using QH-1 to make immunofluorescent whole mounts have described a sequence of events leading to the establishment of the embryonic heart tube. These reports suggest that the pattern for the endocardium and cranial vasculature is established by migrating angioblasts that form vascular cords which mature into blood vessels. Blockage experiments showed that the ventrolateral edge of the anterior intestinal portal serves as a substrate for the directed migration of pre-endocardial angioblasts and that the pattern of the cranioventral vasculature forms independent of the source of angioblasts. Transplant experiments showed that the origin for endocardial angioblasts lies in mesodermal tissue just anterior to Henson's node, that these cells undergo directed migration to the pericardial area, and that angioblasts are pluripotent with the ability to form different blood vessels. The transplant studies also showed that the embryonic mesoderm may contribute to extraembryonic blood vessels on the embryonic yolksac. These results support the hypothesis that embryonic blood vessels may develop by either the vasculogenesis or by the angiogenesis mechanism, and show that the endocardium of the primitive heart tube forms by vasculogenesis.