Methods of In Ovo and Ex Ovo Ostrich Embryo Culture with Observations on the Development and Maturation of the Chorioallantoic Membrane.

Culture of shell-free and windowed eggs for drug testing and other experiments has been perfected for smaller eggs such as those of chickens, where the developing blood vessels of the chorioallantoic membrane (CAM) become accessible for manipulative studies. However, due to the thickness and hardness of the ostrich egg shell, such techniques are not applicable. Using a tork craft mini rotary and a drill bit, we established windowed egg, in-shell-membrane windowed egg, and in-shell-membrane shell-free methods in the ostrich egg, depending on whether the shell membranes were retained or not. Concomitant study of the developing CAM revealed that at embryonic day 16 (E16), the three layers of the CAM were clearly delineated and at E25, the chorionic capillaries had fused with the epithelium while the CAM at E37 had reached maturity and the chorion and the allantois were both 3-4 times thicker and villous cavity (VC) and capillary-covering cells were well delineated. Both intussusceptive and sprouting angiogenesis were found to be the predominant modes of vascular growth in the ostrich CAM. Development and maturation of the ostrich CAM are similar to those of the well-studied chicken egg, albeit its incubation time being twice in duration.

The mouse allantois: new insights at the embryonic-extraembryonic interface.

During the early development of Placentalia, a distinctive projection emerges at the posterior embryonic-extraembryonic interface of the conceptus; its fingerlike shape presages maturation into the placental umbilical cord, whose major role is to shuttle fetal blood to and from the chorion for exchange with the mother during pregnancy. Until recently, the biology of the cord's vital vascular anlage, called the body stalk/allantois in humans and simply the allantois in rodents, has been largely unknown. Here, new insights into the development of the mouse allantois are featured, from its origin and mechanism of arterial patterning through its union with the chorion. Key to generating the allantois and its critical functions are the primitive streak and visceral endoderm, which together are sufficient to create the entire fetal-placental connection. Their newly discovered roles at the embryonic-extraembryonic interface challenge conventional wisdom, including the physical limits of the primitive streak, its function as sole purveyor of mesoderm in the mouse, potency of visceral endoderm, and the putative role of the allantois in the germ line. With this working model of allantois development, understanding a plethora of hitherto poorly understood orphan diseases in humans is now within reach. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

An Improved In Vivo Methodology to Visualise Tumour Induced Changes in Vasculature Using the Chick Chorionic Allantoic Membrane Assay.

BACKGROUND/AIM: Decreasing the vascularity of a tumour has proven to be an effective strategy to suppress tumour growth and metastasis. Anti-angiogenic therapies have revolutionized the treatment of advanced-stage cancers, however there is still demand for further improvement. This necessitates new experimental models that will allow researchers to reliably study aspects of angiogenesis. The aim of this study was to demonstrate an in vivo technique in which the highly vascular and accessible chorioallantoic membrane (CAM) of the chick embryo is used to study tumour-induced changes in the macro and microvessels. MATERIALS AND METHODS: Two cancer cell lines (human melanoma (C8161) and human prostate cancer (PC3)) were selected as model cells. Human dermal fibroblasts were used as a control. One million cells were labelled with green fluorescent protein and implanted on the CAM of the chick embryo at embryonic development day (EDD) 7 and angiogenesis was evaluated at EDDs 10, 12 and 14. A fluorescently-tagged lectin (lens culinaris agglutinin (LCA)) was injected intravenously into the chick embryo to label endothelial cells. The LCA is known to label the luminal surface of endothelial cells, or dextrans, in the CAM vasculature. Macrovessels were imaged by a hand-held digital microscope and images were processed for quantification. Microvessels were evaluated by confocal microscopy. Tumour invasion was assessed by histological and optical sectioning. RESULTS: Tumour cells (C8161 and PC3) produced quantifiable increases in the total area covered by blood vessels, compared to fibroblasts when assessed by digital microscopy. Tumour invasion could be demonstrated by both histological and optical sectioning. The most significant changes in tumour vasculature observed were in the microvascular structures adjacent to the tumour cells, which showed an increase in the endothelial cell coverage. Additionally, tumour intravasation and tumour thrombus formation could be detected in the areas adjacent to tumour cells. The fragility of tumour blood vessels could be demonstrated when tumour cells seeded on a synthetic scaffold were grown on CAM. CONCLUSION: We report on a modification to a well-studied CAM in vivo assay, which can be effectively used to study tumour induced changes in macro and microvasculature.

Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface.

The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization.

Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell-carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell-carrying BALB/c mice. GroEL promoted neovasculogenesis in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E-selectin expression, which is mediated by eNOS production and p38 mitogen-activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel-induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth.

B16-F10 melanoma cells contribute to the new formation of blood vessels in the chick embryo chorioallantoic membrane through vasculogenic mimicry.

Grafting of mammalian cells and tissues to the chick embryo chorioallantoic membrane (CAM) is a well-established experimental system to evaluate many different parameters of tumor growth, and B16-F10 murine melanoma cell line has been successfully used to study metastatic process in the CAM assay. The aim of this study was to demonstrate the capability of B16-F10 melanoma cells to contribute to the new formation of host blood vessels through a vasculogenic mimicry mode. Results have shown that B16-F10 melanoma cells are able to form in 4 days macroscopic tumor masses and induce a strong angiogenic response comparable to that of a well-known angiogenic cytokine, namely fibroblast growth factor-2. Moreover, tumor cells are able to cross the chorionic epithelium, and to move beneath in the mesenchyme to form tumor masses immunoreactive to specific antibodies anti-S100 and anti-MART-1/Melan-A. Finally, we have shown that CAMs new-formed blood vessels are lined by both pigmented melanoma cells and cells immunoreactive to MART-1/Melan-A and PAS, suggesting the occurrence of a vasculogenic mimicry process.

The murine allantois: a model system for the study of blood vessel formation.

The allantois is the embryonic precursor of the umbilical cord in mammals and is one of several embryonic regions, including the yolk sac and dorsal aorta, that undergoes vasculogenesis, the de novo formation of blood vessels. Despite its importance in establishing the chorioallantoic placenta and umbilical circulation, the allantois frequently is overlooked in embryologic studies. Nonetheless, recent studies demonstrate that vasculogenesis, vascular remodeling, and angiogenesis are essential allantois functions in the establishment of the chorioallantoic placenta. Here, we review blood vessel formation in the murine allantois, highlighting the expression of genes and involvement of pathways common to vasculogenesis or angiogenesis in other parts of the embryo. We discuss experimental techniques available for manipulation of the allantois that are unavailable for yolk sac or dorsal aorta, and review how this system has been used as a model system to discover new genes and mechanisms involved in vessel formation. Finally, we discuss the potential of the allantois as a model system to provide insights into disease and therapeutics.

A computational tool for quantitative analysis of vascular networks.

Angiogenesis is the generation of mature vascular networks from pre-existing vessels. Angiogenesis is crucial during the organism' development, for wound healing and for the female reproductive cycle. Several murine experimental systems are well suited for studying developmental and pathological angiogenesis. They include the embryonic hindbrain, the post-natal retina and allantois explants. In these systems vascular networks are visualised by appropriate staining procedures followed by microscopical analysis. Nevertheless, quantitative assessment of angiogenesis is hampered by the lack of readily available, standardized metrics and software analysis tools. Non-automated protocols are being used widely and they are, in general, time--and labour intensive, prone to human error and do not permit computation of complex spatial metrics. We have developed a light-weight, user friendly software, AngioTool, which allows for quick, hands-off and reproducible quantification of vascular networks in microscopic images. AngioTool computes several morphological and spatial parameters including the area covered by a vascular network, the number of vessels, vessel length, vascular density and lacunarity. In addition, AngioTool calculates the so-called "branching index" (branch points/unit area), providing a measurement of the sprouting activity of a specimen of interest. We have validated AngioTool using images of embryonic murine hindbrains, post-natal retinas and allantois explants. AngioTool is open source and can be downloaded free of charge.

Tumor necrosis factor-alpha promotes tumor growth by inducing vascular endothelial growth factor.

Tumor necrosis factor (TNF)-α has been proved as an adjuvant therapy for tumor by FDA. However, the effect of chronic TNF-α expression for tumor is still controversial. In this study, we investigated the effect of low-dose TNF-α on tumor growth. We confirmed that low-dose TNF-α promoted angiogenesis of tumor in vivo, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α, the transcription factor of VEGF, were both upregulated. Our results suggested that low-dose TNF-α was a powerful activator of angiogenesis in tumor and HIF-1α-VEGF pathway seemed to be the most important molecular mechanism.

VEGF-A promotes intussusceptive angiogenesis in the developing chicken chorioallantoic membrane.

OBJECTIVE: To assess the impact of vascular endothelial growth factor (VEGF) on intussusceptive angiogenesis. METHODS AND RESULTS: Polyurethane casts of the microvasculature of chicken chorioallantoic membrane (CAM) were prepared on embryonic days (E) 8, 10, 12, and 14. At light microscopy level, minute holes (<2 microm in diameter) and hollows (>2 microm) were observed in the casts. Transmission electron microscopy disclosed the minute holes to mainly represent transluminal pillars characteristic for intussusceptive angiogenesis. The numerical density of the holes/pillars was highest at an early (E8) and a late (E12-E14) stage. Only mRNA of VEGF-A-122 and VEGF-A-166 isoforms was detected in the CAM. The transcription rate of VEGF-A mRNA peaked on E8/9 and E12, while VEGF-A protein expression increased on E8/9 and E11/12 to rapidly decrease thereafter as determined by immunoblotting. At all time points investigated, VEGF-A immunohistochemical reactivity was restricted to cells of the chorionic epithelium in direct contact to the capillary plexus. When the VEGF-R-inhibitor PTK787/ZK222584 (0.1 mg/mL) was applied on E9 CAM, the microvasculature topology on E12 was similar to that on E10. CONCLUSIONS: The temporal course of intussusception corresponded to the expression of VEGF-A in CAM microvasculature. Inhibition of VEGF-signaling retarded intussusceptive-dependent capillary maturation. These data suggest that VEGF promotes intussusception.

Allantois and placenta as developmental sources of hematopoietic stem cells.

While the aortic region, the para-aortic splanchnopleura/aorta-gonads-mesonephros (P-Sp/AGM) is currently considered as the source of definitive hematopoietic stem cells during development, the mouse placenta has been found to generate large numbers of these cells and to remain functional in this respect for a longer period than the P-Sp/AGM. The fetal component, which derives from the fused allantois and chorion, is responsible for this activity. We and others have shown that the pre-fusion allantois (before the stage of 6 pairs of somites) is able to yield clonogenic progenitors, provided that it is pre-cultured in toto before it is dissociated into single cells and seeded in semi-solid medium. Thus placental hematopoiesis can be concluded to derive from intrinsic precursors. It is similar in this regard to the yolk sac which both produces hematopoietic progenitors and supports their multiplication and differentiation. Hematopoietic activity, detected by in vitro colony assays, has also been recently uncovered in the human placenta. According to the data available, this newly identified source probably provides a large number of HSC during development and must play a foremost role in founding the definitive hematopoietic system.

Impaired vascular development in the yolk sac and allantois in mice lacking RA-GEF-1.

RA-GEF-1 is a guanine nucleotide exchange factor for the small GTPase Rap1. RA-GEF-1 knockout mice show defects in vascular development starting around 7.5days post coitum and die by 9.5days post coitum. Here, we employed in vitro culture systems for allantois explants and endothelial cells to gain insights into the mechanism for RA-GEF-1-mediated regulation of embryonic vascular network formation. The development of the vascular plexus and the accumulation of VE-cadherin at cell-cell junctions were significantly impaired in the RA-GEF-1 knockout allantois and yolk sac. Rap1 activation as visualized by an activation-specific probe was also diminished by RA-GEF-1 knockout. Reduced accumulation of VE-cadherin at cell-cell junctions and defects in blood vessel formation in vitro due to the lack of RA-GEF-1 were suppressed by ectopic expression of constitutively activated Rap1. Overall, these results suggest the involvement of Rap1 downstream of RA-GEF-1 in the regulation of vascular network formation in mouse embryos.

The Allantoic Core Domain: new insights into development of the murine allantois and its relation to the primitive streak.

The whereabouts and properties of the posterior end of the primitive streak have not been identified in any species. In the mouse, the streak's posterior terminus is assumed to be confined to the embryonic compartment, and to give rise to the allantois, which links the embryo to its mother during pregnancy. In this study, we have refined our understanding of the biology of the murine posterior primitive streak and its relation to the allantois. Through a combination of immunostaining and morphology, we demonstrate that the primitive streak spans the posterior extraembryonic and embryonic regions at the onset of the neural plate stage ( approximately 7.0 days postcoitum, dpc). Several hours later, the allantoic bud emerges from the extraembryonic component of the primitive streak (XPS). Then, possibly in collaboration with overlying allantois-associated extraembryonic visceral endoderm, the XPS establishes a germinal center within the allantois, named here the Allantoic Core Domain (ACD). Microsurgical removal of the ACD beyond headfold (HF) stages resulted in the formation of allantoic regenerates that lacked the ACD and failed to elongate; nevertheless, vasculogenesis and vascular patterning proceeded. In situ and transplantation fate mapping demonstrated that, from HF stages onward, the ACD's progenitor pool contributed to the allantois exclusive of the proximal flanks. By contrast, the posterior intraembryonic primitive streak (IPS) provided the flanks. Grafting the ACD into T(C)/T(C) hosts, whose allantoises are significantly foreshortened, restored allantoic elongation. These results revealed that the ACD is essential for allantoic elongation, but the cues required for vascularization lie outside of it. On the basis of these and previous findings, we conclude that the posterior primitive streak of the mouse conceptus is far more complex than was previously believed. Our results provide new directives for addressing the origin and development of the umbilical cord, and establish a novel paradigm for investigating the fetal/placental relationship.

Cigarette smoke condensate and total particulate matter severely disrupts physiological angiogenesis.

The cigarette smoke condensate (CSC) and total particulate matter (TPM) in cigarette smoke is extremely toxic and may produce several pathologies. In our study, we used a chicken chorioallantoic membrane assay (CAM) to study the toxicological effects of CSC and TPM on different aspects of angiogenesis. CSC and TPM from four different commercial filtered cigarettes were applied to the CAMs on day 6 of incubation. Macroscopic vascular transformations were observed among all treated CAMs. The application of CSC disks caused obliteration of main blood vessels, while the entire architecture of the secondary and tertiary vasculature was completely destroyed. Likewise, the application of TPM from all brands of cigarette caused the disproportionate thinning of all primary and secondary blood vessels. A reduction in the total area and diameter of the primary, secondary, and tertiary blood vessels was observed after treatment with CSC and TPM. Histological evaluations revealed the loss of ectodermal and mesodermal integrity with both types of treatments. We also noted a profound inflammatory reaction restricted to the disk area with a novel filopodial deformity of the endoderm in the CSC treated groups. Scanty capillary plexus formation, deterioration of the extracellular matrix, and delayed migration of blood vessels were prominent findings among all treated groups. Results obtained from the CSC treated groups were more localized, while more generalized results were recorded in the TPM treated groups. Special caution should be taken for the presence of CSC and TPM while smoking during pregnancy or after surgery because it may severely affect the process of angiogenesis, which is vital to the maintenance of pregnancy and wound healing.

VEGF-mediated fusion in the generation of uniluminal vascular spheroids.

Embryonic mouse allantoic tissue (E8.5) was cultured in hanging drops to generate a three-dimensional vascular micro-tissue. The resulting tissue spheroids had an inner network of small diameter vessels expressing platelet endothelial cell adhesion molecule-1 (PECAM-1) and an outer layer of cells expressing SMalphaA, SM22-alpha, and SM-MHC. In a subsequent phase of culture, the fusion-promoting activity of vascular endothelial growth factor (VEGF) was used to transform the inner network of small diameter endothelial tubes into a contiguous layer of cells expressing PECAM-1, CD34, and VE-cadherin that circumscribed a central lumen-like cavity. The blood vessel-like character of the VEGF-treated spheroids was further demonstrated by their physiologically relevant vasodilatory and contractile responses, including contraction induced by KCl and relaxation stimulated by high-density lipoproteins and acetylcholine-induced nitric oxide production.

Vascular sprout formation entails tissue deformations and VE-cadherin-dependent cell-autonomous motility.

Embryonic and fetal vascular sprouts form within constantly expanding tissues. Nevertheless, most biological assays of vascular spouting are conducted in a static mechanical milieu. Here we study embryonic mouse allantoides, which normally give raise to an umbilical artery and vein. However, when placed in culture, allantoides assemble a primary vascular network. Unlike other in vitro assays, allantoic primordial vascular cells are situated on the upper surface of a cellular layer that is engaged in robust spreading motion. Time-lapse imaging allows quantification of primordial vascular cell motility as well as the underlying mesothelial tissue motion. Specifically, we calculate endothelial cell-autonomous motion by subtracting the tissue-level mesothelial motion from the total endothelial cell displacements. Formation of new vascular polygons is hindered by administration of function-blocking VE-cadherin antibodies. Time-lapse recordings reveal that (1) cells at the base of sprouts normally move distally "over" existing sprout cells to form new tip-cells; and (2) loss of VE-cadherin activity prevents this motile behavior. Thus, endothelial cell-cell-adhesion-based motility is required for the advancement of vascular sprouts within a moving tissue environment. To the best of our knowledge, this is the first study that couples endogenous tissue dynamics to assembly of vascular networks in a mammalian system.

IB05204, a dichloropyridodithienotriazine, inhibits angiogenesis in vitro and in vivo.

In the course of a blind screening program for inhibitors of angiogenesis, IB05204 (4,8-dichloro-12-phenylpyrido[5',6':4'',5'';3',2':4,5]dithieno[3'',2''-d':3,2-d]-1,2,3-ditriazine) was selected for its ability to inhibit endothelial tubule-like network formation on Matrigel. IB05204 inhibits the in vivo angiogenesis in the chorioallantoic membrane (CAM) and the mouse Matrigel plug assays. Antiangiogenic activity seems to be highly dependent on the chloro substituents because their removal results in a complete loss of the in vitro inhibitory activity of endothelial differentiation and in vivo antiangiogenic activity in CAM assay. Although IB05204 inhibits the growth of endothelial and tumor cells in culture, its antiangiogenic activity seems to be mainly dependent on the prevention of endothelial capillary-like tube formation and inhibition of endothelial migration because these effects are recorded at lower concentrations. IB05204 treatment inhibits matrix metalloproteinase-2 (MMP-2) production in endothelial and tumor cells, down-regulates endothelial cyclooxygenase-2 expression, and represses phosphorylation of endothelial Akt in response to serum stimulation, suggesting that IB05204 interferes with molecular mechanisms of cell migration and survival. IB05204 induces apoptosis in endothelial cells through cytochrome c release and caspase activation. Data here shown altogether indicate that IB05204 is a compound that interferes with several key steps of angiogenesis, making it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.

Placentation in dolphins from the Amazon River Basin: the Boto, Inia geoffrensis, and the Tucuxi, Sotalia fluviatilis.

A recent reassessment of the phylogenetic affinities of cetaceans makes it timely to compare their placentation with that of the artiodactyls. We studied the placentae of two sympatric species of dolphin from the Amazon River Basin, representing two distinct families. The umbilical cord branched to supply a bilobed allantoic sac. Small blood vessels and smooth muscle bundles were found within the stroma of the cord. Foci of squamous metaplasia occurred in the allanto-amnion and allantochorion. The interhemal membrane of the placenta was of the epitheliochorial type. Two different types of trophoblastic epithelium were seen. Most was of the simple columnar type and indented by fetal capillaries. However, there were also areolar regions with tall columnar trophoblast and these were more sparsely supplied with capillaries. The endometrium was well vascularised and richly supplied with actively secreting glands. These findings are consistent with the current view that Cetacea are nested within Artiodactyla as sister group to the hippopotamids.

[Morphological characteristic of labyrinthine zone of rat allantoic placenta after exposure to vibration of industrial frequency].

The labyrinthine zone of allantoic placenta was studied after dosated exposure to vibration. The study was carried out in pregnant female Wistar rats (n=68). Rats were subjected to short-term vibration from Day 9 to Day 13 and long-term vibration from the Day 9 to Day 18 of pregnancy (taking into account embryogenesis periodization and the development period of extraembryonic organs). Vibration table with fixed parameters (frequency of 32 Hz and acceleration of 50 m/s2) was used. Placental and fetal mass were measured, labyrinthine zone components were evaluated using the morphometric methods, while labyrinthine cord and fetal vessels along Duval's sinus were studied ultrastructurally. Labyrinth was analyzed from the point of view of rapprochement of maternal and fetal bloodstreams. It was found that the exposure to vibration together with the development of compensatory adaptive mechanisms led to chronic placental insufficiency. Experimental group with short-term vibration exposure (Days 9-13) is of particular interest since chronic placental insufficiency in this group should be considered as decompensated.