scholarly journals Angiogenic Effect of Leptin in the Quail Chorioallantoic Membrane

2010 ◽  
Vol 79 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Pavel Výboh ◽  
Michal Zeman ◽  
Boris Bilčík ◽  
Božena Šárniková ◽  
Ľubor Košťál
Keyword(s):  
Embryonic Development ◽  
Growth And Development ◽  
Chorioallantoic Membrane ◽  
Nutrient Utilization ◽  
Avian Embryo ◽  
In Ovo ◽  
Angiogenic Effect ◽  
Vessel Development ◽  
Stimulation Of

Leptin, the product ofobgene, beside its key role in the control of body weight and food consumption, can be involved in the control of embryonic development. Leptin administrationin ovoaccelerated the embryonic and post-embryonic development in Japanese quail. Although the mechanisms of leptin effects on growth and development acceleration are not clear, stimulation of angiogenesis represents one of plausible explanations. Therefore, the aim of the present study was to investigate the pro-angiogenic effect of leptinin vivoin the quail chorioallantoic membrane (CAM). The recombinant murine leptin (10, 100, and 1000 ng) was applied eitherex ovoon the CAM surface ofex ovoincubated embryos at embryonic day 7 (ED7) orin ovointo the egg albumen at ED5. Changes in blood vessels were quantified by the fractal analysis providing the fractal dimension (Df) estimate. Leptin administeredin ovowas more efficient in stimulation of angiogenesis than theex ovotreatment, since 10 ng dose elicited significantly higher (P< 0.001) stimulation of vessel development of the CAM under the air cell than it did afterex ovocultivation. Our study confirmed that exogenously applied leptin was able to stimulate angiogenesis in CAM. Leptin-mediated stimulation of angiogenesis may improve nutrient utilization from the yolk and explain at least partially the accelerating effect of leptin on avian embryo growth and development.

scholarly journals The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma

2020 ◽  
Vol 21 (15) ◽  
pp. 5499
Author(s):  
Hannah L. Smith ◽  
Stephen A. Beers ◽  
Juliet C. Gray ◽  
Janos M. Kanczler
Keyword(s):  
Three Dimensional ◽  
Chorioallantoic Membrane ◽  
3D Models ◽  
In Ovo ◽  
Future Directions ◽  
3 Dimensional ◽  
In Vivo Animal Models

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

scholarly journals In vivo regulation of cell death by embryonic (pro)insulin and the insulin receptor during early retinal neurogenesis

Development ◽  
2000 ◽  
Vol 127 (8) ◽  
pp. 1641-1649
Author(s):  
B. Diaz ◽  
J. Serna ◽  
F. De Pablo ◽  
E.J. de la Rosa
Keyword(s):  
Cell Death ◽  
Embryonic Development ◽  
Insulin Receptor ◽  
Neural Development ◽  
Developmental Process ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
In Ovo ◽  
Retinal Neurogenesis

Programmed cell death is an established developmental process in the nervous system. Whereas the regulation and the developmental role of neuronal cell death have been widely demonstrated, the relevance of cell death during early neurogenesis, the cells affected and the identity of regulatory local growth factors remain poorly characterized. We have previously described specific in vivo patterns of apoptosis during early retinal neurogenesis, and that exogenous insulin acts as survival factor (Diaz, B., Pimentel, B., De Pablo, F. and de la Rosa, E. J. (1999) Eur. J. Neurosci. 11, 1624–1632). Proinsulin mRNA was found to be expressed broadly in the early embryonic chick retina, and decreased later between days 6 and 8 of embryonic development, when there was increased expression of insulin-like growth factor I mRNA, absent or very scarce at earlier stages. Consequently, we studied whether proinsulin and/or insulin ((pro)insulin) action in prevention of cell death has physiological relevance during early neural development. In ovo treatment at day 2 of embryonic development with specific antibodies against (pro)insulin or the insulin receptor induced apoptosis in the neuroretina. The distribution of apoptotic cells two days after the blockade was similar to naturally occurring cell death, as visualized by TdT-mediated dUTP nick end labeling. The apoptosis induced by the insulin receptor blockade preferentially affected to the Islet1/2 positive cells, that is, the differentiated retinal ganglion cells. In parallel, the insulin survival effect on cultured retinas correlated with the activation of Akt to a greater extent than with the activation of MAP kinase. These results suggest that the physiological cell death occurring in early stages of retinal development is regulated by locally produced (pro)insulin through the activation of the Akt survival pathway.

Nerve-dependent accumulation of myosin light chain 3 in developing limb musculature

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 673-684
Author(s):  
P.A. Merrifield ◽  
I.R. Konigsberg
Keyword(s):  
Neural Tube ◽  
Light Chain ◽  
Limb Development ◽  
Myosin Light Chain ◽  
Chorioallantoic Membrane ◽  
In Ovo ◽  
Fast Myosin ◽  
Limb Musculature

Myosin alkali light chain accumulation in developing quail limb musculature has been analysed on immunoblots using a monoclonal antibody which recognizes an epitope common to fast myosin light chain 1 (MLC1f) and fast myosin light chain 3 (MLC3f). The limb muscle of early embryos (i.e. up to day 10 in ovo) has a MLC profile similar to that observed in myotubes cultured in vitro; although MLC1f is abundant, MLC3f cannot be detected. MLC3f is first detected in 11-day embryos. To determine whether this alteration in MLC3f accumulation is nerve or hormone dependent, limb buds with and without neural tube were cultured as grafts on the chorioallantoic membrane of chick hosts. Although differentiated muscle develops in both aneural and innervated grafts, innervated grafts contain approximately three times as much myosin as aneural grafts. More significantly, although aneural grafts reproducibly accumulate normal levels of MLC1f, they fail to accumulate detectable levels of MLC3f. In contrast, innervated grafts accumulate both MLC1f and MLC3f, suggesting that the presence of neural tube in the graft promotes the maturation, as well as the growth, of muscle tissue. This is the first positive demonstration that innervation is necessary for the accumulation of MLC3f that occurs during normal limb development in vivo.

scholarly journals Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Biology Open ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. bio055343 ◽  
Author(s):  
Daniel Chu ◽  
An Nguyen ◽  
Spenser S. Smith ◽  
Zuzana Vavrušová ◽  
Richard A. Schneider
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Inducible Promoter ◽  
In Ovo ◽  
Control Of Gene Expression ◽  
Promoter Construct ◽  
Avian Development ◽  
Spatiotemporal Control ◽  
Promoter System

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

scholarly journals Developmental regulation of the multiple myogenic cell lineages of the avian embryo.

1986 ◽  
Vol 103 (6) ◽  
pp. 2197-2208 ◽  
Author(s):  
J B Miller ◽  
F E Stockdale
Keyword(s):  
Developmental Regulation ◽  
High Density ◽  
Fiber Formation ◽  
Myogenic Cell ◽  
Avian Embryo ◽  
In Ovo ◽  
Cell Lineages ◽  
Clonal Cultures ◽  
Later Phase

The developmental regulation of myoblasts committed to fast, mixed fast/slow, and slow myogenic cell lineages was determined by analyzing myotube formation in high density and clonal cultures of myoblasts isolated from chicken and quail embryos of different ages. To identify cells of different myogenic lineages, myotubes were analyzed for content of fast and slow classes of myosin heavy chain (MHC) isoforms by immunocytochemistry and immunoblotting using specific monoclonal antibodies. Myoblasts from the hindlimb bud, forelimb bud, trunk, and pectoral regions of the early chicken embryo and hindlimb bud of the early quail embryo (days 3-6 in ovo) were committed to three distinct lineages with 60-90% of the myoblasts in the fast lineage, 10-40% in the mixed fast/slow lineage, and 0-3% in the slow lineage depending on the age and species of the myoblast donor. In contrast, 99-100% of the myoblasts in the later embryos (days 9-12 in ovo) were in the fast lineage. Serial subculturing from a single myoblast demonstrated that commitment to a particular lineage was stably inherited for over 30 cell doublings. When myoblasts from embryos of the same age were cultured, the percentage of muscle colonies of the fast, fast/slow, and slow types that formed in clonal cultures was the same as the percentage of myotubes of each of these types that formed in high density cultures, indicating that intercellular contact between myoblasts of different lineages did not affect the type of myotube formed. An analysis in vivo showed that three types of primary myotubes--fast, fast/slow, and slow--were also found in the chicken thigh at day 7 in ovo and that synthesis of both the fast and slow classes of MHC isoforms was concomitant with the formation of primary myotubes. On the basis of these results, we propose that in the avian embryo, there is an early phase of muscle fiber formation in which primary myotubes with differing MHC contents are formed from myoblasts committed to three intrinsically different primary myogenic lineages independent of innervation and a later phase in which secondary myotubes are formed from myoblasts in a single, secondary myogenic lineage with maturation and maintenance of fiber diversity dependent on innervation.

scholarly journals Circulation Index as a Quantitative Indicator of Angiogenesis in Chorioallantoic Membrane of Chicken Broilers

2018 ◽  
Vol 67 (2) ◽  
pp. 164-170
Author(s):  
Zoran Ružić ◽  
Zdenko Kanački ◽  
Dragan Žikić ◽  
Gordana Ušćebrka ◽  
Jovan Mirčeta
Keyword(s):  
Embryonic Development ◽  
Blood Vessels ◽  
Chorioallantoic Membrane ◽  
New Method ◽  
In Vivo Studies ◽  
Circulation Index ◽  
Fast Method ◽  
Angiogenic Response ◽  
Embryonic Life

Summary Chorioallantoic membrane (CAM) is an extraembryonic membrane very frequently used for in vivo studies in various researches. Since researches require a fast method for quantifying the CAM angiogenic response, there is a need to develop a new precise and unbiased method of quantification of angiogenesis in CAM, which would be easy to perform and suitable for analysis of a large number of samples. The objective of this paper is to apply a new method of quantification of angiogenesis in investigation of the development of blood vessels in the CAM, in particular days of embryonic life considered essential for CAM development. The present research included 75 fertilized eggs of heavy hybrid Ross 308. CAM sampling for stereological analyses was in key phases of embryonic development, namely on the 12th, 15th and 19th day. The results of the present investigation show that the increase in embryonic age results in increase in circulation index, which is also an indicator of angiogenic processes developing in CAM. The lowest value of circulation index (0.1952) was recorded on the first sampling day (E12), while the highest value (0.2666) was recorded on the last sampling day (E19). This method may be applied in researching different factors which affect angiogenesis in CAM.

scholarly journals Extending the Applicability of In Ovo and Ex Ovo Chicken Chorioallantoic Membrane Assays to Study Cytostatic Activity in Neuroblastoma Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Miguel Angel Merlos Rodrigo ◽  
Berta Casar ◽  
Hana Michalkova ◽  
Ana Maria Jimenez Jimenez ◽  
Zbynek Heger ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Neuroblastoma Cells ◽  
Chorioallantoic Membrane ◽  
Stage Iv ◽  
Metastatic Potential ◽  
Significant Inhibition ◽  
Cam Assay ◽  
Anticancer Activities ◽  
In Ovo

PurposeThe chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential.MethodsThe Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed.ResultsEx ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p &lt; 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM.ConclusionsTaken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

scholarly journals The Chorioallantoic Membrane Assay in Nanotoxicological Research—An Alternative for In Vivo Experimentation

Nanomaterials ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2328
Author(s):  
Christoph R. Buhr ◽  
Nadine Wiesmann ◽  
Rachel C. Tanner ◽  
Jürgen Brieger ◽  
Jonas Eckrich
Keyword(s):  
Imaging Techniques ◽  
Chorioallantoic Membrane ◽  
Drug Carriers ◽  
Human Organism ◽  
Cam Assay ◽  
In Ovo ◽  
In Vivo Models ◽  
Toxicological Profile ◽  
Application Fields

Nanomaterials unveil many applicational possibilities for technical and medical purposes, which range from imaging techniques to the use as drug carriers. Prior to any human application, analysis of undesired effects and characterization of their toxicological profile is mandatory. To address this topic, animal models, and rodent models in particular, are most frequently used. However, as the reproducibility and transferability to the human organism of animal experimental data is increasingly questioned and the awareness of animal welfare in society increases at the same time, methodological alternatives are urgently required. The chorioallantoic membrane (CAM) assay is an increasingly popular in ovo experimental organism suitable for replacement of rodent experimentation. In this review, we outline several application fields for the CAM assay in the field of nanotoxicology. Furthermore, analytical methods applicable with this model were evaluated in detail. We further discuss ethical, financial, and bureaucratic aspects and benchmark the assay with other established in vivo models such as rodents.

scholarly journals Differential regulation of Igf1 and Igf2 mRNA levels in tilapia hepatocytes: effects of insulin and cortisol on GH sensitivity

2011 ◽  
Vol 211 (2) ◽  
pp. 201-210 ◽  
Author(s):  
Andrew L Pierce ◽  
Jason P Breves ◽  
Shunsuke Moriyama ◽  
Tetsuya Hirano ◽  
E Gordon Grau
Keyword(s):  
Growth And Development ◽  
Mrna Level ◽  
Differential Regulation ◽  
Postnatal Life ◽  
Mrna Levels ◽  
Primary Cultured Hepatocytes ◽  
Mammalian Liver ◽  
Growth Promoting ◽  
Stimulation Of

Igf1 and Igf2 stimulate growth and development of vertebrates. In mammals, liver-derived endocrine Igf1 mediates the growth promoting effects of GH during postnatal life, whereas Igf2 stimulates placental and fetal growth and is not regulated by GH. Insulin enhances Igf1 production by the mammalian liver directly, and by increasing hepatocyte sensitivity to GH. We examined the regulation ofigf1andigf2mRNA levels by GH, insulin, and cortisol, and the effects of insulin and cortisol on GH sensitivity in primary cultured hepatocytes of tilapia, a cichlid teleost. GH increased mRNA levels of bothigf1andigf2in a concentration-related and biphasic manner over the physiological range, with a greater effect onigf2mRNA level. Insulin increased basaligf2mRNA level, and strongly increased GH-stimulatedigf2mRNA level, but slightly reduced basaligf1mRNA level and did not affect GH-stimulatedigf1mRNA level. Cortisol inhibited GH stimulation ofigf1, but increased GH stimulation ofigf2mRNA level. The synergistic effect of insulin and GH onigf2mRNA level was confirmedin vivo. These results indicate that insulin and cortisol differentially modulate the response ofigf1andigf2mRNA to GH in tilapia hepatocytes, and suggest that the regulation of liver Igf2 production differs between fish and mammals. Regulation of liver Igf2 production in fish appears to be similar to regulation of liver Igf1 production in mammals.

scholarly journals Selective Binding of Lectins to Embryonic Chicken Vasculature

2003 ◽  
Vol 51 (5) ◽  
pp. 597-604 ◽  
Author(s):  
Shahla M. Jilani ◽  
Thomas J. Murphy ◽  
Shelley N.M. Thai ◽  
Anne Eichmann ◽  
Jackelyn A. Alva ◽  
...  
Keyword(s):  
Lens Culinaris ◽  
Developmental Stages ◽  
Chorioallantoic Membrane ◽  
Experimental System ◽  
In Ovo ◽  
Additional Advantage ◽  
Chicken Embryos ◽  
Lens Culinaris Agglutinin ◽  
Embryonic Chicken

Chicken embryos are an excellent model system for studies related to vascular morphogenesis. Development in ovo allows manipulations otherwise difficult in mammals, and the use of chicken-quail chimeras offers an additional advantage to this experimental system. Furthermore, the chicken chorioallantoic membrane has been extensively used for in vivo assays of angiogenesis. Surprisingly, few markers are available for a comprehensive visualization of the vasculature. Here we report the use of lectins for identification of embryonic chicken blood vessels. Nine lectins were evaluated using intravascular perfusion and directly on sections. Our results indicate that Lens culinaris agglutinin, concanavalin A, and wheat germ agglutinin can be used effectively for visualization of vessels of early chicken embryos (E2.5-E4). At later developmental stages, Lens culinaris agglutinin is a better choice because it displays equal affinity for the endothelia of arteries, veins, and capillaries. The findings presented here expand our understanding of lectin specificity in the endothelium of avian species and provide information as to the use of these reagents to obtain comprehensive labeling of the embryonic and chorioallantoic membrane vasculature.

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