Area Vasculosa Research Articles

Artificial Sweeteners Alter Early Vitelline Vessel Development in Chick Embryo

During early embryonic development, nutrients are transferred to the embryo via vitelline vessels. Disorders that affect vitelline vessel development lead to embryonic lethality or vascular diseases. In recent years, the use of artificial sweeteners has increased and become more widespread, however the potential effects of these compounds on embryonic development remain poorly understood and controversial. This study aimed to establish the effect of commercial artificial sweeteners on early vitelline vessel development in chick embryos. The commercial artificial sweetener used in this study was a mixture of aspartame and acesulfame k. Fertile chicken eggs were divided into four groups: control, sham, and two treated groups that were injected in the air chamber before incubation with 50mg/kg or 100mg/ kg sweetener by body weight. The eggs were then incubated under normal incubation conditions. Embryos were extracted and photographed on days 3 and 4 of incubation, then vitelline vessel measurements were taken (length and width of the vitelline vessels, right and left angles, and number of branches). VEGF concentration within the area vasculosa of 3-day embryos was measured by ELISA. Low- and high- treated embryos showed abnormal vitelline vessel development at 3 and 4 days post-incubation. Some vessels were enlarged while others displayed atrophy. The pattern of branching was also altered compared to controls, and some vessels had hemorrhage. The concentration of VEGF was lower in the treated groups compared to that observed in the control group. This study revealed an association between artificial sweeteners and abnormal vitelline vessel development. These findings could have broad potential implications for human health, as this study used commercial artificial sweeteners that are commonly available in consumer markets.

Cover Image

Primordial germ cells (red) and endothelial cells (green) immunostained by DDX4 and QH1 antibodies in a Hamburger and Hamilton Stage 7 quail embryo. The primordial germ cells and endothelial cells are distributed in the germinal crescent and area vasculosa, respectively. From: Envelopment by endothelial cells initiates translocation of avian primordial germ cell into vascular tissue; Hidetaka Murai, Minami Shibuya, Ryohei Kishita, Chihiro Sunase, Koji Tamura and Daisuke Saito; https://doi.org/10.1002/dvdy.332; DevDyn 250:10; pp.1410–1419.

Temporal dynamics of nitric oxide wave in early vasculogenesis.

Endothelium-derived nitric oxide (NO) is a mediator of angiogenesis. However, NO-mediated regulation of vasculogenesis remains largely unknown. In the present study, we show that the inhibition of NO significantly attenuated endothelial migration, ring formation, and tube formation. The contribution of nitric oxide synthase (NOS) enzymes during early vasculogenesis was assessed by evaluating endothelial NOS (eNOS) and inducible NOS (iNOS) mRNA expression during HH10-HH13 stages of chick embryo development. iNOS but not eNOS was expressed at HH12 and HH13 stages. We hypothesized that vasculogenic events are controlled by NOS-independent reduction of nitrite to NO under hypoxia during the very early phases of development. Semi-quantitative polymerase chain reaction analysis of hypoxia-inducible factor-1α (HIF-1α) showed higher expression at HH10 stage, after which a decrease was observed. This observation was in correlation with the nitrite reductase (NR) activity at HH10 stage. We observed a sodium nitrite-induced increase in NO levels at HH10, reaching a gradual decrease at HH13. The possible involvement of a HIF/NF-κB/iNOS signaling pathway in the process of early vasculogenesis is suggested by the inverse relationship observed between nitrite reduction and NOS activation between HH10 and HH13 stages. Further, we detected that NR-mediated NO production was inhibited by several NR inhibitors at the HH10 stage, whereas the inhibitors eventually became less effective at later stages. These findings suggest that the temporal dynamics of the NO source switches from NR to NOS in the extraembryonic area vasculosa, where both nitrite reduction and NOS activity are defined by hypoxia.

Envelopment by endothelial cells initiates translocation of avian primordial germ cell into vascular tissue.

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. 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. 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.

A differentiation program induced by bone morphogenetic proteins 4 and 7 in endodermal epithelial cells provides the molecular basis for efficient nutrient transport by the chicken yolk sac.

BackgroundThe mammalian yolk sac provides nutrients for the growing fetus during critical early developmental processes such as neural tube closure, which precedes the functional maturation of the placenta. In contrast, oviparous species such as the chicken rely solely on the yolk sac for transfer of nutrients from the yolk to the developing embryo. However, the molecular mechanisms that provide the yolk sac with nutrient transfer competence remain poorly understood.ResultsWe demonstrate that the chicken endodermal epithelial cells (EEC), which are in close contact with the yolk, gain their nutrient‐transport competence by a paracrine crosstalk with the blood‐vessel forming mesodermal cell layer. Bone morphogenetic proteins (BMP) 4 and 7 produced by ectodermal and mesodermal cell layers likely initiate a differentiation program of EECs during the transition from the area vitellina to the area vasculosa. BMPs, by inducing SMAD signaling, promote the up‐regulation of endocytic receptor expression and thereby provide the EECs with the molecular machinery to produce triglyceride‐rich lipoprotein particles.ConclusionThis paracrine signaling cascade may constitute the basis for the EEC‐mediated mechanism underlying the efficient uptake, degradation, resynthesis, and transfer of yolk‐derived nutrients into the embryonic circulation, which assures proper energy supply and development of the growing fetus.

Morphometric analysis of the branching of the vascular tree in the chick embryo area vasculosa

The chick embryo includes the area vasculosa is subdivided into 2 concentric zones, the inner transparent area pellucida vasculosa and the surrounding less transparent area opaca vasculosa, peripherally limited by the sinus terminalis. In this study, we have analyzed by a modern morphometric approach the total length of the vascular network, the number of vascular branches, of the branching points density, the modality of vessel ramification, and spatial arrangement of the vascular network in four consecutive stages of development of the area vasculosa. The results have shown that there is a significant 15% increase in the total length of the vascular network associated with a progressive increase of the number of vascular branches and of the branching points density. Moreover, the results indicated that vascular spatial disorder significantly decreased during development in area vasculosa, suggesting a more uniform occupancy of the tissue by the vascular pattern. Finally, a more regular pattern of branching was observed, as indicated by the significant decrease of topological disorder of the vascular tree.

Developing a partially perfused liver model using caprine liver explant conditioned to the chicken extra-embryonic perfusion system

The Liver is constantly subjected to mechanical, chemical and pathological insults throughout life, as a result of which there is a common occurrence of various liver diseases. Due to the complex nature of liver architecture, it is not possible to mimic the in-vivo conditions beyond a certain limit. Hence, the development of in-vitro and ex-vivo models to study various liver diseases has gained more importance over the last few decades. The present study aims to develop a semi-perfused liver explant model to give an extended lifetime for studying liver pathology and treatment options. Caprine liver tissue explants were sliced, weighed (25 mg) and placed on the area vasculosa of fertilized chicken eggs. Unfertilized eggs were used as controls. After varying time intervals of incubation on area vasculosa of fertilized chicken eggs, the liver slices were subjected to cell viability assay, lactate dehydrogenase assay and Serum glutamic pyruvic transaminase assays. The results indicate that the viability and functional properties of such semi-perfused liver tissue explants holds good up to 6 h. Finally, the liver tissue explants were pre-treated with FBS, with and without anti-fibrotic drugs, and placed on chick embryo area vasculosa up to 6 h. The anti-fibrotic drug-treated semi perfused liver tissue explant showed a decrease in collagen formation as confirmed by histology and western blot. We deem that the use of extra-embryonic vasculature of chicken bed for extending the life of tissue explants will serve as a cost-effective alternative to animal models to understand disease mechanisms under drug treatments.

Live Imaging and Analysis of Vasoactive Properties of Drugs Using an in-ovo Chicken Embryo Model: Replacing and Reducing Animal Testing

Vasodilation occurs as a result of the relaxation of the smooth muscle cells present in the walls of blood vessels. Various suitable models are available for the analysis of the vasoactive properties of drugs with therapeutic applications. But all these models have limitations, such as ethical issues and high cost. The purpose of this study is to develop an alternative model for studying the vasoactive properties of drugs using an in-ovo chicken embryo model. In the preliminary experiment, we used a well-known vasoconstrictor (adrenaline) and a vasodilator (spermine NoNoate) in the chick embryo area vasculosa and evaluated their concentration-response curve. Adrenaline (10 µM) and spermine NoNoate (10 µM) were administered in different arteries and veins and different positions of the right vitelline artery of the chick embryo. Results showed the middle of the vessel bed of the right vitelline artery having the best vasoactive effect compared to others. Finally, anti-hypertensive drugs, calcium channel blockers, and NOS agonists were administered in the chick embryo area vasculosa to validate the model. Results demonstrate that the chick embryo area vasculosa can be an alternative, robust, and unique in-ovo model for screening of anti-hypertensive drugs in real time.

SDF-1/CXCR4 signalling is involved in blood vessel growth and remodelling by intussusception.

The precise mechanisms of SDF‐1 (CXCL12) in angiogenesis are not fully elucidated. Recently, we showed that Notch inhibition induces extensive intussusceptive angiogenesis by recruitment of mononuclear cells and it was associated with increased levels of SDF‐1 and CXCR4. In the current study, we demonstrated SDF‐1 expression in liver sinusoidal vessels of Notch1 knockout mice with regenerative hyperplasia by means of intussusception, but we did not detect any SDF‐1 expression in wild‐type mice with normal liver vessel structure. In addition, pharmacological inhibition of SDF‐1/CXCR4 signalling by AMD3100 perturbs intussusceptive vascular growth and abolishes mononuclear cell recruitment in the chicken area vasculosa. In contrast, treatment with recombinant SDF‐1 protein increased microvascular density by 34% through augmentation of pillar number compared to controls. The number of extravasating mononuclear cells was four times higher after SDF‐1 application and two times less after blocking this pathway. Bone marrow‐derived mononuclear cells (BMDC) were recruited to vessels in response to elevated expression of SDF‐1 in endothelial cells. They participated in formation and stabilization of pillars. The current study is the first report to implicate SDF‐1/CXCR4 signalling in intussusceptive angiogenesis and further highlights the stabilizing role of BMDC in the formation of pillars during vascular remodelling.

Improving colloidal stability of silica nanoparticles when stored in responsive gel: application and toxicity study

When silica nanoparticles (SiNP) are stored in aqueous solution, even for few hours, they have a tendency to form agglomerates and therefore adapt inhomogeneous structures. Here we present a very practical method to store SiNP in responsive hydrogel. We have confirmed that SiNP kept in the responsive hydrogel do not undergo through undesirable morphological changes and while in storage they maintain their excellent colloidal stability. The effect of SiNP hollowing (i.e. dissolution of the core of the particles that leaves empty cavity inside) was significantly inhibited in the hydrogel, which is a critical feature for any nano-medical applications (e.g. controlled drug release). To demonstrate the applicability of the hydrogel-storing concept within a biologically relevant context, in this work we have evaluated the toxicological effects of the responsive SiNP-gel formulation in a model in vitro (human cell line U87GM and hemocompatibility using red blood cells) and ex ovo (hen’s egg test) experiments. Particles stored in the gel as well as the pure gel did not affect the hemocompatibility (hemolysis and erythrocyte aggregation) up to a concentration of 100 µg/mL. Furthermore, systemic injections into the blood circulation of the chick area vasculosa confirmed the biocompatibility in a more complex biological environment. All evaluated toxicological values (hemorrhage, thrombosis, vascular lysis, and lethality) were comparable with the negative control, and no differences in toxicological response could be observed between the SiNP stored in hydrogel and the control nanoparticles stored in the solution.

The expression dynamics of mechanosensitive genes in extra-embryonic vasculature after heart starts to beat in chick embryo.

Fluid flow plays an important role in vascular development. However, the detailed mechanisms, particularly the link between flow and modulation of gene expression during vascular development, remain unexplored. In chick embryo, the key events of vascular development from initiation of heart beat to establishment of effective blood flow occur between the stages HH10 and HH13. Therefore, we propose a novel in vivo model to study the flow experienced by developing endothelium. Using this model, we aimed to capture the transcriptome dynamics of the pre- and post-flow conditions. RNA was isolated from extra embryonic area vasculosa (EE-AV) pooled from three chick embryos between HH10-HH13 and RNA sequencing was performed. The whole transcriptome sequencing of chick identified up-regulation of some of the previously well-known mechanosensitive genes including NFR2, HAND1, CTGF and KDR. GO analyses of the up-regulated genes revealed enrichment of several biological processes including heart development, extracellular matrix organization, cell-matrix adhesion, cell migration, blood vessel development, patterning of blood vessels, collagen fibril organization. Genes encoding for gap junctions proteins which are involved in vascular remodeling and arterial-venous differentiation, and genes involved in cell-cell adhesion, and ECM interactions were significantly up-regulated. Validation of selected genes through semi quantitative PCR was performed. The study indicates that shear stress plays a major role in development. Through appropriate validation, this platform can serve as an in vivo model to study conditions of disturbed flow in pathology as well as normal flow during development.

Matrix Gla Protein expression pattern in the early avian embryo.

MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-β and BMP activity, and a proposed modulator of cell-matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification.

Delayed vasculogenesis and impaired angiogenesis due to altered Ang-2 and VE-cadherin levels in the chick embryo model following exposure to cadmium

Cadmium (Cd) causes chick embryo malformation and abnormal extra-embryonic vasculature. This study investigates the effect of Cd on vasculogenesis, quantifies extra-embryonic vascular development following exposure to cadmium acetate (CdAc). After 48 or 60h incubation, chicks were explanted and treated with 50µl of 50µM CdAc or equimolar sodium acetate. Embryos were again incubated then re-examined 4, 8, 24 and 48h later. Gross morphological and histological manifestations were noted. Vasculogenesis was assessed by the development of omphalomesenteric vessels from blood islands. Sinus terminalis (ST), area vasculosa (AV), vessel density and embryo crown-rump length (CRL) were measured. Ang-2 and VE-cadherin mRNA expression was analysed by RT-PCR. Vasculogenesis was delayed on gross and histological examination. ST length, AV area, vessel density and CRL were significantly reduced in the Cd group. Ang-2 was increased 4h after exposure to Cd, whereas VE-cadherin was reduced. Cd exposure inhibits normal development of extra-embryonic vasculature in line with growth retardation of the chick embryo in association with altered expression of Ang-2 and VE-cadherin.

Exposure of chick embryos to cadmium changes the extra-embryonic vascular branching pattern and alters expression of VEGF-A and VEGF-R2

Cadmium (Cd) has several industrial applications, and is found in tobacco products, a notable source of human exposure. Vascular endothelial cells are key targets of Cd toxicity. Here, we aim to quantify the alteration to vascular branching pattern following Cd exposure in the chick extra-embryonic membrane (EEM) using fractal analysis, and explore molecular cues to angiogenesis such as VEGF-A and VEGF-R2 expression following Cd treatment. Chicken embryos were incubated for 60h to Hamburger–Hamilton developmental stage 16–17, then explanted and treated with 50μL of 50μmol cadmium acetate (CdAc) or an equivalent volume of equimolar sodium acetate (NaAc). Images of embryos and their area vasculosa (AV) were captured and analyzed at 4 different time points (4, 8, 24 and 48h) following treatment. Vascular branching in the AV was quantified using its fractal dimension (Df), estimated using a box counting method. Gallinaceous VEGF ELISA was used to measure the VEGF-A concentration in the EEM following treatment, with determination of the relative expression of VEGF-A and VEGF-R2 using quantitative real-time RT-PCR. Vascular branching increased monotonically in the control group at all time points. The anti-angiogenic effect of Cd exposure on the AV was reflected by a significant reduction in Df when compared with controls. Df was more markedly reduced in cultures with abnormal embryos. The expression of VEGF-A protein, and VEGF-A and VEGF-R2 mRNA were reduced in Cd-exposed EEMs. Both molecules contribute to growth, vessel sprouting and branching processes, which supports our findings using fractal analysis.

Investigation of Effects of Essential Oil from Berries of Juniperus excelsa Bieb. Subsp excelsa (Cupressaceae) on Angiogenesis in Shell-less Chick Embryo (CAM) Culture

The aim of this study was to investigate the effect of essential oil of the berries from Juniperus excelsa Bieb. subsp. excelsa (Cupressaceae) to the vascularization systems of the chick embryos in a chorioallantoic membrane model. The berries of J. excelsa were subjected to hydrodistillation. 50 µl of essential oil were applied to the center of the blastodisc using a precision micropipette prior to the transfer of shell-less cultures to the incubator. Antiangiogenic effect of essential oil of the study plant was determined with the use of the chick chorioallantoic membrane model. Numbers of main, branching and capillary blood vessels were counted. In the statistical analysis, when the groups were compared with regard to the main blood vessel (MBV) and branch blood vessel (BBV) development, no significant difference (p>0.05) was observed. However, when the groups were compared with regard to capillary vessel (CV) development, the difference between the data of the groups was found significant (p<0.05). This study examined the effect of exposure J. excelsa essential oil on extraembryonic vascular development in the chick embryo area vasculosa (AV) in shell-less culture for the first time. Essential oil of the berries from J. excelsa has antiangiogenic effect.

Avian area vasculosa and CAM as rapid in vivo pro-angiogenic and antiangiogenic models.

Angiogenesis, the development of new blood vessels from preexisting ones, is driven by coordinated signaling pathways governed by specific molecules, hemodynamic forces, and endothelial and periendothelial cells. The processes involve adhesion, migration, and survival machinery within the target endothelial and periendothelial cells. Factors that interfere with any of these processes may therefore influence angiogenesis either positively (pro-angiogenesis) or negatively (antiangiogenesis). The avian area vasculosa (AV) and the avian chorioallantoic membrane (CAM) are two useful tools for studying both angiogenesis and antiangiogenesis since they are amenable to both intravascular and topical administration of target, agents, are relatively rapid assays, and can be adapted very easily to study angiogenesis-dependent processes, such as tumor growth. Both models provide a physiological setting that permits investigation of pro-angiogenic and antiangiogenic agent interactions in vivo.

Embryonic development in the ostrich (Struthio camelus) during the first 7 days of artificial incubation

1. Early development of ostrich embryos was investigated in relation to time of egg collection and genotype.2. A total of 321 ostrich eggs were collected during the 2008 and 2009 breeding seasons and the development of the embryo for up to the first 168 h of incubation was described and analysed. A sample of the incubated eggs was weighed and opened daily to investigate developmental changes.3. In fresh eggs, the blastoderm contained a round, translucent dark area pellucida (AP) in the centre, with a surrounding thin white ring, likely to be the beginning of the area opaca (AO). Fresh eggs were considered infertile if the blastoderm was absent and instead numerous white droplets were present surrounded by vacuoles.4. The average blastoderm area of a fresh fertile egg was 15.8 mm2, increasing to 143.3 mm2 after 2 d of incubation. By 72 h of incubation the area vasculosa (AV) was discernible in the posterior half of the blastoderm.5. At 48 h of incubation the blastoderm area in eggs from the South African Black genotype (SAB) × Zimbabwean Blue genotype (ZB) crosses (104.5 ± 18.6 mm2) was lower than the pure SAB (141.0 ± 10.5 mm2), ZB (161.7 ± 13.5 mm2) and ZB × SAB crosses (166.1 ± 14.2 mm2).6. Embryo length was 5.01 mm after 72 h of incubation and 14.5 mm after 168 h of incubation. At 168 h of incubation AV lengths for both ZB × SAB (53.2 mm) and SAB × ZB crosses (54.1 mm) were longer than in embryos from the pure breeds.7. Results from this study can be put to practical use when determining whether eggs are infertile or fertile and also in investigating the age of early embryonic mortalities.

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40 % increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80 % increase in vascular density and pillar number augmentation by 420 %. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturation.

Inhibition of Notch signalling induces extensive intussusceptive neo‐angiogenesis by recruitment of mononuclear cells

Objectiveto evaluate the effects of Notch inhibition on the vasculature using chick area vasculosa and MxCre Notch1(lox/lox) mice and to test to which extent this effect is mediated by ephrinB2/EphB4.Methods and ResultsInhibition of Notch signaling by Y secretase 1 induced significantly intussusceptive angiogenesis (IA) forming large immature capillary plexus. The latter it is characterized by 40% increased vascular density, pericyte detachment, increased transpermeability and recruitment of mononuclear cells to the sites of new formed transluminal tissue pillars (sign of IA). The process was dramatically enhanced when Notch inhibition was combined with the injection of chicken or mouse bone marrow‐derived mononuclear cells. Along with the morphological alterations of IA, we observed down‐regulation of ephrinB2 mRNA levels. Inhibition of ephrinB2 and EphB4 signaling induces pericytes detachment and significantly upregulation of VEGFR1 but without angiogenic response and recruitment of mononuclear cells. Remarkable, the chemotactic factors SDF‐1 and CXCR4 were up‐regulated only due to the Notch inhibition.ConclusionsOur results indicated that Notch inhibition disturbs vessel stability, induces extravasation of mononuclear cells of bone marrow origin by up‐regulation SDF‐1/CXCR4 chemotactic factors. Recruited cells contributed to formation of transluminal tissue pillars i.e. intussusceptive neo‐angiogenesis, triggering in this way the augmentation of the capillary plexus but without the accompanying vascular maturation and remodeling. .

The Developing Chicken Yolk Sac Acquires Nutrient Transport Competence by an Orchestrated Differentiation Process of Its Endodermal Epithelial Cells

During chicken yolk sac (YS) growth, mesodermal cells in the area vasculosa follow the migrating endodermal epithelial cell (EEC) layer in the area vitellina. Ultimately, these cells form the vascularized YS that functions in nutrient transfer to the embryo. How and when EECs, with their apical aspect directly contacting the oocytic yolk, acquire the ability to take up yolk macromolecules during the vitellina-to-vasculosa transition has not been investigated. In addressing these questions, we found that with progressive vascularization, the expression level in EECs of the nutrient receptor triad, LRP2-cubilin-amnionless, changes significantly. The receptor complex, competent for uptake of yolk proteins, is produced by EECs in the area vasculosa but not in the area vitellina. Yolk components endocytosed by LRP2-cubilin-amnionless, preformed and newly formed lipid droplets, and yolk-derived very low density lipoprotein, shown to be efficiently endocytosed and lysosomally processed by EECs, probably provide substrates for resynthesis and secretion of nutrients, such as lipoproteins. In fact, as directly demonstrated by pulse-chase experiments, EECs in the vascularized, but not in the avascular, region efficiently produce and secrete lipoproteins containing apolipoprotein A-I (apoA-I), apoB, and/or apoA-V. In contrast, perilipin 2, a lipid droplet-stabilizing protein, is produced exclusively by the EECs of the area vitellina. These data suggest a differentiation process that orchestrates the vascularization of the developing YS with the induction of yolk uptake and lipoprotein secretion by EECs to ensure embryo nutrition.