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The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro., (© 2024. The Author(s).)

The placenta links feto-maternal circulation for exchanges of nutrients, gases, and metabolic wastes between the fetus and mother, being essential for pregnancy process and maintenance. The allantois and mesodermal components of amnion, chorion, and yolk sac are derived from extraembryonic mesoderm (Ex-Mes), however, the mechanisms contributing to distinct components of the placenta and regulation the interactions between allantois and epithelium during chorioallantoic fusion and labyrinth formation remains unclear. Isl1 is expressed in progenitors of the Ex-Mes and allantois the Isl1 mut mouse line is analyzed to investigate contribution of Isl1 + Ex-Mes / allantoic progenitors to cells of the allantois and placenta. This study shows that Isl1 identifies the Ex-Mes progenitors for endothelial and vascular smooth muscle cells, and most of the mesenchymal cells of the placenta and umbilical cord. Deletion of Isl1 causes defects in allantois growth, chorioallantoic fusion, and placenta vessel morphogenesis. RNA-seq and CUT&Tag analyses revealed that Isl1 promotes allantoic endothelial, inhibits mesenchymal cell differentiation, and allantoic signals regulated by Isl1 mediating the inductive interactions between the allantois and chorion critical for chorionic epithelium differentiation, villous formation, and labyrinth angiogenesis. This study above reveals that Isl1 plays roles in regulating multiple genetic and epigenetic pathways of vascular morphogenesis, provides the insight into the mechanisms for placental formation, highlighting the necessity of Isl1 for placenta formation/pregnant maintenance., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background and Objectives: This study aimed to evaluate the volume, the concentration of steroid hormones, and biochemical composition of the foetal fluids at different gestational ages in dogs and cats., Methods: Following the ovariohysterectomy, the allantoic and amniotic fluid samples were collected from pregnant bitches and queens and were assigned to different groups according to their gestational age., Results: The canine and feline allantoic fluid volume increased during pregnancy, reached its maximum values on days 40-49 and then decreased. The canine and feline amniotic fluid volume increased steadily by the last days of pregnancy. In spite of significant changes of sex hormones in the foetal fluids, their concentration and ratios were not significantly different between male and female fetuses. The canine amniotic cortisol concentration increased until days 40-49 and decreased significantly afterwards. The maximum cortisol concentrations in the feline allantoic and amniotic fluids were observed on days 50-60 and 40-49, respectively. During the canine pregnancy, the concentrations of calcium, phosphorus, chloride, sodium, triglyceride, cholesterol, total protein, albumin and the activities of aminotransferase (AST), alkaline phosphatase (ALP), amylase and gamma-glutamyl transferase (GGT) in the amniotic fluid were higher than the allantoic fluid. The magnesium, potassium, lactate dehydrogenase (LDH) activity, creatine and lipase were higher in the allantoic fluid. In the feline allantoic fluid, potassium, magnesium, phosphorus, creatinine, albumin and glucose concentrations and the activities of creatine kinase (CK), GGT, LDH and lipase were higher. The ALP, AST activities, sodium and calcium concentrations were higher in the amniotic fluid (p < 0.05)., Conclusion: Volume of foetal fluids was determined in dogs and cats. Concentration of sex hormones did not different between male and female fetuses., (© 2024 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

In amniotic vertebrates (birds, reptiles and mammals), an extraembryonic structure called the chorioallantoic membrane (CAM) functions as respiratory organ for embryonic development. The CAM is derived from fusion between two pre-existing membranes, the allantois, a hindgut diverticulum and a reservoir for metabolic waste, and the chorion which marks the embryo's external boundary. Modified CAM in eutherian mammals, including humans, gives rise to chorioallantoic placenta. Despite its importance, little is known about cellular and molecular mechanisms mediating CAM formation and maturation. In this work, using the avian model, we focused on the early phase of CAM morphogenesis when the allantois and chorion meet and initiate fusion. We report here that chicken chorioallantoic fusion takes place when the allantois reaches the size of 2.5-3.0 mm in diameter and in about 6 hours between E3.75 and E4. Electron microscopy and immunofluorescence analyses suggested that before fusion, in both the allantois and chorion, an epithelial-shaped mesothelial layer is present, which dissolves after fusion, presumably by undergoing epithelial-mesenchymal transition. The fusion process per se , however, is independent of allantoic growth, circulation, or its connection to the developing mesonephros. Mesoderm cells derived from the allantois and chorion can intermingle post-fusion, and chorionic ectoderm cells exhibit a specialized sub-apical intercellular interface, possibly to facilitate infiltration of allantois-derived vascular progenitors into the chorionic ectoderm territory for optimal oxygen transport. Finally, we investigated chorioallantoic fusion-like process in primates, with limited numbers of archived human and fresh macaque samples. We summarize the similarities and differences of CAM formation among different amniote groups and propose that mesothelial epithelial-mesenchymal transition mediates chorioallantoic fusion in most amniotic vertebrates. Further study is needed to clarify tissue morphogenesis leading to chorioallantoic fusion in primates. Elucidating molecular mechanisms regulating mesothelial integrity and epithelial-mesenchymal transition will also help understand mesothelial diseases in the adult, including mesothelioma, ovarian cancer and fibrosis. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galβ1-4(SO 3 - -6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galβ1-4(SO 3 - -6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Physiological parturition is characterized by sterile, inflammatory-like processes. During parturition, the placenta expresses various proinflammatory mediators, such as chemokines and IL-17. Nevertheless, inflammatory processes present in the parturient mare are poorly characterized. The aim of this study was to investigate the expression of selected chemokines and IL-17 in the allantochorion and the endometrium of mares that retained fetal membranes (RFM) and expelled them physiologically. We hypothesized that the expression of these mediators may be altered in the placenta of mares with RFM and result in RFM occurrence. Differences in mRNA expression in the placenta of investigated groups of mares were detected for CCL2, CCL3, CCL4, CCL8, CXCL1, CXCL8, CXCL10, CX3CL1 and IL-17. There were no differences in mRNA expression of CCL5 and CXCL6. Gene ontology network analysis showed enrichment in genes related to leukocyte migration, cell chemotaxis and response to chemokine in tissues of RFM mares. Analysis of association network suggested denotations between CXCL6, CXCL8, CXCL1, CCL5, CCL4, CX3CL1 and CXCL10. Moreover, possible inhibition of CXCL10 by IL-17A and prostaglandin peroxide synthase 2 (PTGS2) by CXCL1 was detected. Our results suggest that, based on differences in chemokines and IL-17 expression, recruited subsets of leukocytes might differ between the analyzed groups of mares, which in turn may impair the separation of fetal membranes in the group of RFM mares. In addition, the results of the expression analysis suggest that macrophages might be one of the most abundant cells infiltrating the equine placenta during the expulsion of fetal membranes. Furthermore, we suspect that the synthesis of PTGS2 might be inhibited in mares with RFM., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The extra-embryonic hypoblast/visceral endoderm of Placentalia carries out a variety of functions during gestation, including hematopoietic induction. Results of decades-old and recent experiments have provided compelling evidence that, in addition to its inducing properties, hypoblast/visceral endoderm itself is a source of placental blood cells. Those observations that highlight extra-embryonic endoderm's role as an overlooked source of placental blood cells across species are briefly discussed here, with suggestions for future exploration., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Introduction: Hydrallantois is the excessive accumulation of fluid in the allantoic cavities during the last trimester of pregnancy, leading to abdominal wall hernias, cardiovascular shock, abortion, and dystocia. It has been postulated that hydrallantois is associated with structural and/or functional changes in the chorioallantoic membrane. In the present study, we hypothesized that angiogenesis is impaired in the hydrallantoic placenta., Method: Capillary density in the hydrallantoic placenta was evaluated in the chorioallantois via immunohistochemistry for Von Willebrand Factor. Moreover, the expression of angiogenic genes was compared between equine hydrallantois and age-matched, normal placentas., Results: In the hydrallantoic samples, edema was the main pathological finding. The capillary density was significantly lower in the hydrallantoic samples than in normal placentas. The reduction in the number of vessels was associated with abnormal expression of a subset of angiogenic and hypoxia-associated genes including VEGF, VEGFR1, VEGFR2, ANGPT1, eNOS and HIF1A. We believe that the capillary density and the abnormal expression of angiogenic genes leads to tissue hypoxia (high expression of HIF1A) and edema. Finally, we identified a lower expression of genes associated with steroidogenic enzyme (CYP19A1) and estrogen receptor signaling (ESR2) in the hydrallantoic placenta., Discussion: Based on the presented data, we believe that formation of edema is due to disrupted vascular development (low number of capillaries) and hypoxia in the hydrallantoic placenta. The edema leads to further hypoxia and consequently, causes an increase in vessel permeability which leads to a gradual increase in interstitial fluid accumulation, resulting in an insufficient transplacental exchange rate and accumulation of fluid in the allantoic cavity., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The objective of this study was to describe the dynamic changes in protein composition and protein abundance in amniotic and allantoic fluids from buffaloes during gestation. Amniotic and allantoic fluids were collected during the first, second and third trimesters of gestation. The foetuses were measured and weighed. Fluid samples were centrifuged at 800 g for 10 min and then at 10,000 g for 60 min at 4°C. The supernatant was collected to determine the total protein concentration. Based on total protein concentration, an aliquot (50 μg) was used for in-solution tryptic digestion, and mass spectrometry analysis (nano-LC-MS/MS) was performed. A multivariate statistical analysis of the proteomic data was conducted. Across the different stages of buffalo gestation, fifty-one proteins were found in the amniotic fluid, and twenty-one were found in the allantoic fluid. A total of twelve proteins were common among the stages, and four presented significant differences (VIP score α > 1). Fibronectin and alpha-1-antiproteinase were more abundant in the amniotic fluid than in the allantoic fluid. Alpha-2-macroglobulin and alpha-2-HS-glycoprotein were more abundant in the allantoic fluid than in the amniotic fluid. Alpha-2-macroglobulin participates in remodelling and growth of the uterus at beginning of the gestation (first trimester), and these findings indicate that can serve as a potential tool for the early diagnosis of pregnancy in buffaloes., (© 2019 Blackwell Verlag GmbH.)

While recent work demonstrated that enrofloxacin and ciprofloxacin reach the fetoplacental unit without causing obvious lesions in the 9-month-old equine fetus or resulting foal, many practitioners still hesitate to prescribe a fluoroquinolone during pregnancy. Since early gestation is a critical time for fetal skeletal development, if fluoroquinolones are chondrotoxic to the fetus at any point during gestation, this period would be important. The aim of this study was to assess the effects of 2 weeks' exposure to enrofloxacin on the equine fetus between 46 and 60 days gestation. Twelve pregnancies from nine healthy mares were allocated into two groups: untreated (n=7), or treatment (7.5mg/kg enrofloxacin, PO×14days, n=6). Abortion was induced with prostaglandin 24h after the last enrofloxacin dose, or on the equivalent day of gestation for untreated mares. Four of nine mares were rebred for a second cycle and were assigned to the opposite treatment to serve as their own controls. Fetal fluids from treated mares were analysed for enrofloxacin and ciprofloxacin concentrations. Fetal organs (heart, lungs, spleen, kidney, and liver) and limbs were examined histopathologically. Enrofloxacin and ciprofloxacin diffused to the fetal fluids during early gestation and did not result in detectable abnormalities in the fetus after 14 days of treatment. While current research does not determine long-term foal outcomes, enrofloxacin may be useful for select bacterial infections in pregnant mares., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The placenta, responsible for the nutrient and gas exchange between the mother and fetus, is pivotal for successful pregnancy. It has been shown that Rbpj, the core transcriptional mediator of Notch signaling pathway, is required for normal placentation in mice. However, it remains largely unclear how Rbpj signaling in different placental compartments coordinates with other important regulators to ensure normal placental morphogenesis. In this study, we found that systemic deletion of Rbpj led to abnormal chorioallantoic morphogenesis and defective trophoblast differentiation in the ectoplacental cone (EPC). Employing mouse models with selective deletion of Rbpj in the allantois versus trophoblast, combining tetraploid aggregation assay, we demonstrated that allantois-expressed Rbpj is essential for chorioallantoic attachment and subsequent invagination of allantoic blood vessels into the chorionic ectoderm. Further studies uncovered that allantoic Rbpj regulates chorioallantoic fusion and morphogenesis via targeting Vcam1 in a Notch-dependent manner. Meanwhile, we also revealed that trophoblast-expressed Rbpj in EPC facilitates Mash2's transcriptional activity, promoting the specification of Tpbpα-positive trophoblasts, which differentiate into trophoblast subtypes responsible for interstitial and endovascular invasion at the later stage of placental development. Collectively, our study further shed light on the molecular network governing placental development and functions, highlighting the necessity of a spatiotemporal coordination of Rbpj signaling for normal placental morphogenesis.

TMED2, a member of the transmembrane emp24 domain (TMED) family, is required for transport of cargo proteins between the ER and Golgi. TMED2 is also important for normal morphogenesis of mouse embryos and their associated placenta, and in fact Tmed2 homozygous mutant embryos arrest at mid-gestation due to a failure of placental labyrinth layer formation. Differentiation of the placental labyrinth layer depends on chorioallantoic attachment (contact between the chorion and allantois), and branching morphogenesis (mingling of cells from these two tissues). Since Tmed2 mRNA was found in both the chorion and allantois, and 50% of Tmed2 homozygous mutant embryos failed to undergo chorioallantoic attachment, the tissue-specific requirement of Tmed2 during placental labyrinth layer formation remained a mystery. Herein, we report differential localization of TMED2 protein in the chorion and allantois, abnormal ER retention of Fibronectin in Tmed2 homozygous mutant allantoises and cell-autonomous requirement for Tmed2 in the chorion for chorioallantoic attachment and fusion. Using an ex vivo model of explanted chorions and allantoises, we showed that chorioallantoic attachment failed to occur in 50% of samples when homozygous mutant chorions were recombined with wild type allantoises. Furthermore, though expression of genes associated with trophoblast differentiation was maintained in Tmed2 mutant chorions with chorioallantoic attachment, expression of these genes was attenuated. In addition, Tmed2 homozygous mutant allantoises could undergo branching morphogenesis, however the region of mixing between mutant and wild type cells was reduced, and expression of genes associated with trophoblast differentiation was also attenuated. Our data also suggest that Fibronectin is a cargo protein of TMED2 and indicates that Tmed2 is required cell-autonomously and non-autonomously in the chorion and the allantois for placental labyrinth layer formation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In amniotes, the amniotic fluid is a significant contributor to fetal development and health. While numerous studies have been conducted in mammalian amniotic fluid, the composition of amniotic and other extraembryonic fluids in avian egg along with their physiological functions remain largely unexplored. In such a context, our objective was to characterize the chicken amniotic fluid (AmF) and allantoic fluid (AlF) properties, protein composition, and some associated functions from day 8 to day 16 of incubation. SDS-PAGE combined to mass spectrometry analysis revealed common and specific proteins to each fluid, suggesting distinct properties and functions. Indeed, major AlF proteins are mostly "egg yolk" proteins involved in lipid, vitamin metabolisms, and metal ion transport, while major AmF proteins resemble those of albumen. Drastic changes in the AmF protein profiles were observed during incubation, when the albumen transfers from day 12 onwards, while few changes were detected for the AlF protein profile. The decreases in osmolality (from 231 to 183 mOsm/kg) and pH (from 8.26 to 7.26) observed in the AlF during incubation are associated with water and electrolytes reallocation for the embryo needs. In contrast, AmF pH value remained stable (≈7.5). Active proteolytic enzymes have been identified in the 2 fluids using gelatin zymography, followed by mass spectrometry analysis for protease identification. A total of 12 proteases was detected in the AlF, compared to 5 in the AmF. We have shown that AlF concentrates proteolytic enzymes assumed to participate in digestive processes: aminopeptidase N, dipeptidyl peptidase-4, meprin A, and 72 kDa type IV collagenase preproprotein. The other proteases identified in both fluids also could have a role in morphogenesis (hepatocyte growth factor activator, suppressor of tumorigenicity 14, astacin-like metalloendopeptidase) and hemostasis (prothrombin and coagulation factor X). Altogether, these data suggest that the roles of chicken AlF and AmF are not merely associated with protection of the embryo and regulation of metabolic disposable wastes, but also they could have more sophisticated roles during embryonic development., (© 2017 Poultry Science Association Inc.)

How the fetal-placental arterial connection is made and positioned relative to the embryonic body axis, thereby ensuring efficient and directed blood flow to and from the mother during gestation, is not known. Here we use a combination of genetics, timed pharmacological inhibition in living mouse embryos, and three-dimensional modeling to link two novel architectural features that, at present, have no status in embryological atlases. The allantoic core domain (ACD) is the extraembryonic extension of the primitive streak into the allantois, or pre-umbilical tissue; the vessel of confluence (VOC), situated adjacent to the ACD, is an extraembryonic vessel that marks the site of fetal-placental arterial union. We show that genesis of the fetal-placental connection involves the ACD and VOC in a series of steps, each one dependent upon the last. In the first, Brachyury (T) ensures adequate extension of the primitive streak into the allantois, which in turn designates the allantoic-yolk sac junction. Next, the streak-derived ACD organizes allantoic angioblasts to the axial junction; upon signaling from Fibroblast Growth Factor Receptor-1 (FGFR1), these endothelialize and branch, forming a sprouting VOC that unites the umbilical and omphalomesenteric arteries with the fetal dorsal aortae. Arterial union is followed by the appearance of the medial umbilical roots within the VOC, which in turn designate the correct axial placement of the lateral umbilical roots/common iliac arteries. In addition, we show that the ACD and VOC are conserved across Placentalia, including humans, underscoring their fundamental importance in mammalian biology. We conclude that T is required for correct axial positioning of the VOC via the primitive streak/ACD, while FGFR1, through its role in endothelialization and branching, further patterns it. Together, these genetic, molecular and structural elements safeguard the fetus against adverse outcomes that can result from vascular mispatterning of the fetal-placental arterial connection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The allantois-derived umbilical component of the chorio-allantoic placenta shuttles fetal blood to and from the chorion, thereby ensuring fetal-maternal exchange. The progenitor populations that establish and supply the fetal-umbilical interface lie, in part, within the base of the allantois, where the germ line is claimed to segregate from the soma. Results of recent studies in the mouse have reported that STELLA (DPPA-3, PGC7) co-localizes with PRDM1 (BLIMP1), the bimolecular signature of putative primordial germ cells (PGCs) throughout the fetal-placental interface. Thus, if PGCs form extragonadally within the posterior region of the mammal, they cannot be distinguished from the soma on the basis of these proteins. We used immunohistochemistry, immunofluorescence, and confocal microscopy of the mouse gastrula to co-localize STELLA with a variety of gene products, including pluripotency factor OCT-3/4, mesendoderm-associated T and MIXl1, mesendoderm- and endoderm-associated FOXa2 and hematopoietic factor Runx1. While a subpopulation of cells localizing OCT-3/4 was always found independently of STELLA, STELLA always co-localized with OCT-3/4. Despite previous reports that T is involved in specification of the germ line, co-localization of STELLA and T was detected only in a small subset of cells in the base of the allantois. Slightly later in the hindgut lip, STELLA+/(OCT-3/4+) co-localized with FOXa2, as well as with RUNX1, indicative of definitive endoderm and hemangioblasts, respectively. STELLA was never found with MIXl1. On the basis of these and previous results, we conclude that STELLA identifies at least five distinct cell subpopulations within the allantois and hindgut, where they may be involved in mesendodermal differentiation and hematopoiesis at the posterior embryonic-extraembryonic interface. These data provide a new point of departure for understanding STELLA's potential roles in building the fetal-placental connection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Along the perinatal period, in mammals, cortisol (C) plays a pivotal role in the final intrauterine fetal maturation and in the early neonatal adaptation. Because of the scarce knowledge about canine perinatology, the present study was aimed to assess the C concentrations in amniotic and allantoic fluids collected, without invasiveness, from small-sized, purebred newborn puppies born by elective cesarean section, at term of pregnancy. Possible correlations between fetal fluid C concentrations and maternal parity, litter size, birth weight, Apgar score, were evaluated. In addition, the possible effect of fetal fluid C concentrations on newborn survival at 24 hours of age, and the effect of the litter or the newborn gender on fetal fluid C concentrations were also assessed. The results, obtained from 50 born alive, normal-weight puppies, without gross physical malformation, showed that C concentration was higher in allantoic than in amniotic fluid (P < 0.01), even if a strong positive correlation between the two fluids C concentration was found (P < 0.0001; R = 0.83). Neither amniotic nor allantoic C concentrations were correlated to maternal parity, litter size, birth weight, and Apgar score. Interestingly, higher amniotic (P < 0.05), but not allantoic, C concentrations were found in puppies not surviving at 24 hours after birth. Therefore, it could be suggested that this parameter may be useful for the recognition, at birth, of puppies needing special surveillance during the first day of age. A significant (P < 0.001) effect of the litter in both amniotic and allantoic C concentrations was found. In conclusion, the present results showed that in small-sized purebred puppies, born at term by elective cesarean section, the exact fetal, maternal, or placental source contributing to fetal fluid C concentrations remains to be clarified. From a clinical perspective, however, the evaluation of amniotic C concentration at birth seems useful for the detection of puppies that need special surveillance during the first 24 hours of age, and should be coupled to the early newborn evaluation by Apgar score. However, the small total number of newborns, and especially of the dead puppies enrolled in the present study, suggests that further, more-focused investigations on a large number of subjects are needed before the method could be considered for application in the clinical practices., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Thyroxine (T 4 ) and triiodothyronine (T 3 ) concentrations in pregnant and nonpregnant bitches were measured. The allantoic and amniotic fluid samples were collected separately in the third week of pregnancy, and fetal blood samples were collected in the fourth week of pregnancy. There was no difference between T 4 results in the pregnant and nonpregnant animals, but the measured serum concentrations exceeded the healthy range for normal adults. Serum T 4 concentrations were lower in the fetus than those in adults (P < 0.01). Fetal T 4 concentrations continuously increased and reached 13.38 ± 6.19 nmol/L before birth. The fetal serum T 4 concentrations were lower than the T 4 concentrations in allantoic and amniotic fluid until the seventh week, and the fetal serum T 3 concentrations were lower than those in fetal fluids throughout the pregnancy (P < 0.01). Maximum T 3 concentrations in allantoic and amniotic fluid exceeded the concentrations in the fetal and maternal serum. It is conceivable that the considerable differences between maternal and fetal serum T 4 concentrations in healthy animals are explained by the T 4 impermeability of the placenta. Extremely high maternal T 4 (193.5 nmol/L) in 1 bitch was associated with T 4 concentrations under the detection limit in the fetal fluids and serum suggesting an inhibitory effect. The T 4 concentrations in all the fetal fluids and serum were under the detectable concentration that can be defined by 3.0 nmol/L in that bitch. We have demonstrated that fetal thyroid glands start functioning independently at the same time as thyroid cell formation in the dog, but the overproduction of maternal T 4 may have a suppressive effect on fetal iodothyronine production., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: Chorioallantoic fusion is essential for development of the labyrinth layer of the mouse placenta. However, events that occur after chorioallantoic attachment remain poorly described, partly due to difficulties of conducting ex vivo analysis of the placenta. Herein, we report conditions for ex vivo culture of the developing murine placenta., Methods: Mesometrial halves of decidua containing pre-attachment chorions were cultured alone or with explants of allantoides from stage-matched controls and analyzed by confocal and immunofluorescence microscopy. Expression and levels of marker genes associated with specific placental cell types were measured by in situ hybridization and qRT-PCR, respectively., Results: After 24 h (hr) of co-culture, a mosaic pattern of eGFP + and eGFP - cells were found when explants of pre-attachment chorions from eGFP + embryos were co-cultured with stage-matched allantoides from eGFP - embryos or vice versa. In addition, proliferation increased in the allantoic region and folds formed on the chorionic plate. PECAM positive cells derived from the allantois were found in the chorionic region. Levels of the SynT-II marker, Gcm1, significantly increased at 24 h, although expression of Gcm1, was only found in explants co-cultured with an allantois at 12 h and 24 h. In addition, though levels of Tpbpα was not altered by co-culture with an allantois, Tpbpα was only detected in explants co-cultured with an allantois for 24 h., Discussion: Our data show that chorioallantoic fusion and events associated with initiation of labyrinth layer formation can be modeled ex vivo, and reveal a previously unsuspected requirement of chorioallantoic fusion for Tpbpα expression., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

During avian development the mesodermal layers of the allantois and chorion fuse to form the chorioallantoic membrane (CAM). This structure rapidly expands generating a rich vascular network that provides an interface for gas and waste exchange. The CAM allows to study tissue grafts, tumor growth and metastasis, wound healing, drugs delivery and toxicologic analysis, and angiogenic and anti-angiogenic molecules. The CAM is relatively simple, quick, and low-cost model that allows screening of a large number of pharmacological samples in a short time; does not require administrative procedures for obtaining ethics committee approval for animal experimentation. Moreover, being naturally immunodeficient, the chick embryo may receive transplantations from different tissues and species, without immune responses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The peptide hormone INSL3 is uniquely produced by the fetal testis to promote the transabdominal phase of testicular descent. Because it is fetal sex specific, and is present in only very low amounts in the maternal circulation, INSL3 acts as an ideal biomarker with which to monitor the movement of fetal hormones within the pregnant uterus of a polytocous species, the pig. INSL3 production by the fetal testis begins at around GD30. At GD45 of the ca. 114 day gestation, a time at which testicular descent is promoted, INSL3 evidently moves from male to female allantoic compartments, presumably impacting also on the female fetal circulation. At later time-points (GD63, GD92) there is less inter-fetal transfer, although there still appears to be significant INSL3, presumably of male origin, in the plasma of female fetuses. This study thus provides evidence for substantial transfer of a peptide hormone between fetuses, and probably also across the placenta, emphasizing the vulnerability of the fetus to extrinsic hormonal influences within the uterus.

The aim of the study was an analysis of the peculiarities of morphological changes of the placenta in rats with experimental chronic hepatic lesion. Liver injury was modeled in 3-month-old sexually mature female rats by 2-fold intragastric administration of paracetamol at a dose of 2.5 g/kg body weight (drug group, n=15) and a single intraperitoneal injection of D-galactosamine at a dose of 250 mg/kg of body weight (toxic group, n=15). Intact rats served as a control. The placenta examined at Day 21 of pregnancy using histological and morphometric methods. Рroliferative activity of placental cells was evaluated with the immunocytochemical method using antibodies against Ki-67 antigen. The membrane permeability in different trophoblast compartments was examined. It was found that experimental chronic liver pathology caused morphological changes in the placenta, which were manifested by a decrease in the area of its labyrinthine portion, maternal sinusoids in the basal area, fetal capillaries and maternal lacunae of the labyrinth. In addition, in the experiments with an intraperitoneal injection of trypan blue it was shown that changes in the liver caused increased permeability of the placental barrier, and reduced the proliferative activity of trophoblast cells.

The embryonic origin of umbilical cord vestiges is well documented; however, their immunophenotype is unknown. This study was conducted to determine whether vitelline and allantoic remnants can be differentiated using immunohistochemical markers. All allantoic remnants were stained with p63 and were negative for CDX2, whereas the vitelline remnants stained with CDX2 and were negative for p63. An unexpected finding was a small number of morphologically ambiguous cases that stained with both markers in a complimentary manner. The term "hybrid" remnant is proposed for these remnants.

During the early incubation period of the duck, from embryonic day 1 to 13, a precise identification of the sex may be difficult. In a preliminary test, we found a defect in the use of the classical P2/P8, 1237L/1272H, and 2550F/2718R primers for chromo-helicase-DNA-binding 1 gene (CHD1) as a PCR-based test to identify sex in ducks. Therefore, universal PCR primers HPF/HPR for sexing ducks were designed. The PCR product was cloned, sequenced, and analyzed using GenBank. The effectiveness of the primers was compared using samples of blood and feathers from adult birds and chorioallantoic membranes and allantoic fluid (AF) of embryos as a source of DNA. The 495-bp CHD1-Z and the 351-bp CHD1-W PCR amplicons could be easily distinguished on a 3% agarose gel, and females (ZW) displayed 2 visible bands whereas only a single band was found in males (ZZ). The results indicated that HPF/HPR primers were highly efficient and more reliable than the classical primers used for sexing ducks. During the design of the new primers, an AF sampling technique was established to collect a very small amount of AF from free-living birds. This technique, which was minimally invasive, had no adverse effects on either embryos or the post-hatching survival of young ducks and could be used in developmental biology research in birds., (© 2015 S. Karger AG, Basel.)

Epidermal growth factor-like domain 7 (Egfl7) expression in the developing embryo is largely restricted to sites of mesodermal progenitors of angioblasts/hemangioblasts and the vascular endothelium. We hypothesize that Egfl7 marks the endothelial lineage during embryonic development, and can be used to define the emergence of endothelial progenitor cells, as well as to visualize newly-forming vasculature in the embryo and during the processes of physiologic and pathologic angiogenesis in the adult. We have generated a transgenic mouse strain that expresses enhanced green fluorescent protein (eGFP) under the control of a minimal Egfl7 regulatory sequence (Egfl7:eGFP). Expression of the transgene recapitulated that of endogenous Egfl7 at sites of vasculogenesis and angiogenesis in the allantois, yolk sac, and in the embryo proper. The transgene was not expressed in the quiescent endothelium of most adult organs. However, the uterus and ovary, which undergo vascular growth and remodeling throughout the estrus cycle, expressed high levels of Egfl7:eGFP. Importantly, expression of the Egfl7:eGFP transgene was induced in adult neovasculature. We also found that increased Egfl7 expression contributed to pathologic revascularization in the mouse retina. To our knowledge, this is the first mouse model that enables monitoring of endothelial cells at sites of active vasculogenesis and angiogenesis. This model also facilitated the isolation and characterization of EGFL7(+) endothelial cell populations by fluorescence activated cell sorting (FACS). Together, our results demonstrate that the Egfl7:eGFP reporter mouse is a valuable tool that can be used to elucidate the mechanisms by which blood vessels form during development and under pathologic circumstances., (© 2014 Wiley Periodicals, Inc.)

Embryonic loss is a major problem in mammals, but there are few effective ways to prevent it. Using a porcine model, we determined effects of dietary L-arginine supplementation between days 14 and 25 of gestation on embryonic growth and survival. Gilts were checked daily for estrus with boars in the morning and bred at onset of the second estrus and 12 h later (the time of breeding = day 0 of gestation). Between days 14 and 25 of gestation, 15 gilts/treatment were housed individually and fed twice daily 1 kg of a corn- and soybean meal-based diet supplemented with 0.0, 0.4, or 0.8 % L-arginine. All diets were made isonitrogenous by addition of L-alanine. On day 25 of gestation, gilts were hysterectomized to obtain conceptuses. Compared with controls, dietary supplementation with 0.4 or 0.8 % L-arginine increased (P ≤ 0.05) arginine concentrations in maternal plasma, total volume of amniotic fluid; total amounts of arginine in allantoic and amniotic fluids; total amounts of fructose and most amino acids in amniotic fluid; placental growth; and the number of viable fetuses per litter by 2. The numbers of total fetuses, fetal weight, corpora lutea, volume of allantoic fluid, maternal circulating levels of progesterone and estrogen, or total amounts of hormones in allantoic fluid did not differ among the three treatment groups. Reproductive performance of gilts did not differ between the 0.4 and 0.8 % L-arginine groups. Thus, dietary supplementation with 0.4 or 0.8 % L-arginine between days 14 and 25 of gestation enhances embryonic/fetal survival in swine.

We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses., (© 2013 Wiley Periodicals, Inc.)

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 chick embryo is as ancient a source of knowledge on animal development as the very beginning of embryology. Already, at the time of Caspar Friedrich Wolff, contemplating the strikingly beautiful scenario of the germ deploying on the yellow background of the yolk inspired and supported the tenants of epigenesis at the expense of the preformation theory. In this article, we shall mention some of the many problems of developmental biology that were successfully clarified by research on chick embryos. Two topics, the development of the neural system and that of blood and blood vessels, familiar to the authors, will be discussed in more detail., (© 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.)

Cdx gene products regulate the extent of axial elongation from the posterior growth zone. These transcription factors sustain the emergence of trunk and tail tissues by providing a suitable niche in the axial progenitor zone, via regulation of Wnt signaling. Cdx genes are expressed in and along the complete primitive streak including its posterior part wherefrom the extraembryonic mesoderm of the allantois emerges. Cdx genes are required for the full development of the allantois and its derivatives in the placental labyrinth. The mouse germ cell lineage also originates from the proximo-posterior epiblast of the primitive streak, and is established within the extraembryonic mesoderm that generates the allantois. We asked whether the expression of Cdx genes around the newly specified PGCs is necessary for the maintenance and expansion of this population, as it is for the allantois and axial progenitors. We observed a significantly lower number of PGCs in Cdx2(null) embryos than in controls. We found that Wnt3a loss of function decreases the PGC population to the same extent as Cdx2 inactivation. Moreover, exogenous Wnt3a corrects the lower PGC number in Cdx2(null) posterior embryonic tissues cultured in vitro. Cdx2 is not expressed in PGCs themselves, and we propose that the expression of Cdx2 in posterior extraembryonic tissues contributes to the proper niche of the germ cell progenitors by stimulating canonical Wnt signaling. Since PGC residence within the posterior growth zone is a mouse-specific feature, our data suggest that mouse PGCs opportunistically became dependent on the axial progenitor niche., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

Fetal tissues are frequently discarded before (amniocentesis) or after birth, which both facilitates stem cell access and helps to overcome ethical concerns. In the present study, we aimed to isolate and characterize stem cells from the allantoic and amniotic fluids (ALF; AMF) of third trimester canine fetuses. This gestation age has not been previously explored for stem cells isolation. The gestational age, cell culture conditions and method of isolation used in this study allowed for the establishment and efficient expansion of ALF and AMF cells. We showed that the majority of ALF and ALF cells express the stem cell markers, such as vimentin, nestin and cytokeratin 18 (CK18). Under appropriate culture conditions AMF derived cells can undergo differentiation into osteogenic, adipogenic, chondrogenic and neuron-like lineages. ALF derived cells showed adipogenic, and chondrogenic potential. Therefore, ALF and AMF cells derived at the third gestation trimester can be qualified as progenitor stem cells, accordingly referred as (alantoic fluid progenitor/stem) ALF PS cells and (amniotic fluid progenitor/stem) AMF PS cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The developmental relationship between the posterior embryonic and extraembryonic regions of the mammalian gastrula is poorly understood. Although many different cell types are deployed within this region, only the primordial germ cells (PGCs) have been closely studied. Recent evidence has suggested that the allantois, within which the PGCs temporarily take up residence, contains a pool of cells, called the Allantoic Core Domain (ACD), critical for allantoic elongation to the chorion. Here, we have asked whether the STELLA-positive cells found within this region, thought to be specified PGCs, are actually part of the ACD and to what extent they, and other ACD cells, contribute to the allantois and fetal tissues. To address these hypotheses, STELLA was immunolocalized to the mouse gastrula between Early Streak (ES) and 12-somite pair (-s) stages (~6.75-9.0 days post coitum, dpc) in histological sections. STELLA was found in both the nucleus and cytoplasm in a variety of cell types, both within and outside of the putative PGC trajectory. Fate-mapping the headfold-stage (~7.75-8.0 dpc) posterior region, by which time PGCs are thought to be segregated into a distinct lineage, revealed that the STELLA-positive proximal ACD and intraembryonic posterior primitive streak (IPS) contributed to a wide range of somatic tissues that encompassed derivatives of the three primary germ layers. This contribution included STELLA-positive cells localizing to tissues both within and outside of the putative PGC trajectory. Thus, while STELLA may identify a subpopulation of cells destined for the PGC lineage, our findings reveal that it may be part of a broader niche that encompasses the ACD and through which the STELLA population may contribute cells to a wide variety of posterior tissues of the mouse gastrula., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Loss of Tbx4 results in absence of chorio-allantoic fusion and failure of formation of the primary vascular plexus of the allantois leading to embryonic death at E10.5. We reviewed the literature for genes implicated in chorio-allantoic fusion, cavitation and vascular plexus formation, processes affected in Tbx4 mutant allantoises. Using this candidate gene approach, we identified a number of genes downstream of Tbx4 in the allantois including extracellular matrix molecules Vcan, Has2, and Itgα5, transcription factors Snai1 and Twist, and signaling molecules Bmp2, Bmp7, Notch2, Jag1 and Wnt2. In addition, we show that the canonical Wnt signaling pathway contributes to the vessel-forming potential of the allantois. Ex vivo, the Tbx4 mutant phenotype can be rescued using agonists of the Wnt signaling pathway and, in wildtype allantoises, an inhibitor of the canonical Wnt signaling pathway disrupts vascular plexus formation. In vivo, Tbx4 and Wnt2 double heterozygous placentas show decreased vasculature suggesting interactions between Tbx4 and the canonical Wnt signaling pathway in the process of allantois-derived blood vessel formation.

The rodent allantois is thought to be unique amongst mammals in not having an endodermal component. Here, we have investigated the mesothelium, or outer surface, of murine umbilical precursor tissue, the allantois (∼7.25-8.5 days postcoitum, dpc) to discover whether it exhibits the properties of an epithelium. A combination of morphology, challenge with biotinylated dextran amines (BDAs), and immunohistochemistry revealed that the mesothelium of the mouse allantois exhibits distinct regional properties. By headfold stages (∼7.75-8.0 dpc), distal mesothelium was generally squamous in shape, and highly permeable to BDA challenge, whereas ventral proximal mesothelium, referred to as "ventral cuboidal mesothelium" (VCM) for the characteristic cuboidal shape of its cells, was relatively impermeable. Although "dorsal cuboidal mesothelium" (DCM) resembled the VCM in cell shape, its permeability to BDA was intermediate between the other two regions. Results of immunostaining for Zonula Occludens-1 (ZO-1) and Epithelial-cadherin (E-cadherin), together with transmission electron microscopy (TEM), suggested that impermeability in the VCM may be due to greater cellular contact area between cells and close packing rather than to maturity of tight junctions, the latter of which, by comparison with the visceral yolk sac, appeared to be rare or absent from the allantoic surface. Both VCM and DCM exhibited an ultrastructure more favorable for protein synthesis than did the distal squamous mesothelium; however, at most stages, VCM exhibited robust afadin (AF-6), whereas the DCM uniquely contained alpha-4-integrin. These observations demonstrate that the allantoic mesothelium is not a conventional epithelium but possesses regional ultrastructural, functional and molecular differences that may play important roles in the correct deployment of the umbilical cord and its associated vascular, hematopoietic, and other cell types., (Copyright © 2011 Wiley-Liss, Inc.)

Mabuya skinks have the most specialized allantoplacenta and the greatest degree of placentotrophy known among the Reptilia. Their recently ovulated eggs are microlecithal (1-2 mm) and lack fatty yolk platelets; thus, virtually all of the nutrients for embryonic development must pass across the placenta. We quantified the net uptake of nutrients during gestation in an Andean population of Mabuya and compared these results with other matrotrophic skinks and eutherian mammals. Total dry and wet masses, ash and organic content, ions (calcium, potassium, sodium, magnesium, iron), lipids (cholesterol, vitamin E and fatty acids in the major lipid classes-triacylglycerol, phospholipids, cholesteryl ester, free fatty acids), nitrogen (an index of protein) were measured during the different developmental stages throughout gestation, and in neonates. A significant net uptake of inorganic and organic matter was found. This uptake begins slowly during early gestation but accelerates during the last third of gestation when the growth of the embryo is maximal and the allantoplacenta has developed its greatest complexity. The drastic reduction of egg size in this clade is related to the great reduction in the contribution of lecithotrophic nutrients to the embryo, an obligatory placentotrophy from early developmental stages, and the highest placental complexity known in the Reptilia. All of these features converge with features found in eutherian mammals.

Steel factor, the protein product of the Steel locus in the mouse, is a multifunctional signal for the primordial germ cell population. We have shown previously that its expression accompanies the germ cells during migration to the gonads, forming a "travelling niche" that controls their survival, motility, and proliferation. Here we show that these functions are distributed between the alternatively spliced membrane-bound and soluble forms of Steel factor. The germ cells normally migrate as individuals from E7.5 to E11.5, when they aggregate together in the embryonic gonads. Movie analysis of Steel-dickie mutant embryos, which make only the soluble form, at E7.5, showed that the germ cells fail to migrate normally, and undergo "premature aggregation" in the base of the allantois. Survival and directionality of movement is not affected. Addition of excess soluble Steel factor to Steel-dickie embryos rescued germ cell motility, and addition of Steel factor to germ cells in vitro showed that a fourfold higher dose was required to increase motility, compared to survival. These data show that soluble Steel factor is sufficient for germ cell survival, and suggest that the membrane-bound form provides a higher local concentration of Steel factor that controls the balance between germ cell motility and aggregation. This hypothesis was tested by addition of excess soluble Steel factor to slice cultures of E11.5 embryos, when migration usually ceases, and the germ cells aggregate. This reversed the aggregation process, and caused increased motility of the germ cells. We conclude that the two forms of Steel factor control different aspects of germ cell behavior, and that membrane-bound Steel factor controls germ cell motility within a "motility niche" that moves through the embryo with the germ cells. Escape from this niche causes cessation of motility and death by apoptosis of the ectopic germ cells.

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.

Treatment with the progestin altrenogest is widely used in pregnant mares. The fact that foals born from healthy mares treated with altrenogest until term suffered from neonatal problems raises the question of direct effects of altrenogest on vital functions in the neonate. We have therefore investigated altrenogest concentrations in maternal and neonatal blood plasma and in fetal fluids. Pregnant mares were treated with altrenogest orally once daily (0,088 mg/kg bodyweight, n = 7) or left untreated (n = 8) from 280 d of gestation until foaling. Altrenogest concentration was determined in plasma of the mares, their foals and in amniotic and allantoic fluid. The concentration of altrenogest in plasma from treated mares (2.6 +/- 1.0 ng/mL) was significantly lower than in plasma from their foals immediately after birth (5.6 +/- 1.9 ng/mL; p < 0.05), but was significantly higher than in their fetal fluids (amniotic fluid: 0.4 +/- 0.1 ng/mL; p < 0.05; allantoic fluid: 3.0 +/- 1.5 ng/mL). Altrenogest was undetectable in maternal and fetal plasma and fetal fluids of control pregnancies at all times. Altrenogest concentration in plasma of foals from treated mares was strongly correlated to the altrenogest concentration in plasma of their dams (r = 0.938, p < 0.001) and in amniotic (r = 0.886, p < 0.001) and allantoic fluid (r = 0.562, p < 0.05). A significant decrease in altrenogest concentration between the time periods 0-15 min, 30-120 min, and 180-360 min after parturition was seen in the plasma from foals born to altrenogest-treated mares. In conclusion, our data demonstrate that altrenogest reaches the equine fetus at high concentrations., (Copyright 2010 Elsevier Inc. All rights reserved.)

Members of the bone morphogenetic protein (BMP) family have been implicated in the development and maintenance of vascular systems. Whereas members of the BMP-2/4 and osteogenic protein-1 groups signal via activin receptor-like kinase (ALK)-2, ALK-3 and ALK-6, BMP-9 and BMP-10 have been reported to bind to ALK-1 in endothelial cells. However, the roles of BMP-9-ALK-1 signaling in the regulation of endothelial cells have not yet been fully elucidated. Here, using various systems, we examined the effects of BMP-9 on the proliferation of endothelial cells. Vascular-tube formation from ex vivo allantoic explants of mouse embryos was promoted by BMP-9. BMP-9, as well as BMP-4 and BMP-6, also induced the proliferation of in-vitro-cultured mouse embryonic-stem-cell-derived endothelial cells (MESECs) by inducing the expression of vascular endothelial growth factor receptor 2 and Tie2, a receptor for angiopoietin-1. A decrease in ALK-1 expression or expression of constitutively active ALK-1 in MESECs abrogated and mimicked the effects of BMP-9 on the proliferation of MESECs, respectively, suggesting that BMP-9 promotes the proliferation of these cells via ALK-1. Furthermore, in vivo angiogenesis was promoted by BMP-9 in a Matrigel plug assay and in a BxPC3 xenograft model of human pancreatic cancer. Consistent with these in vivo findings, BMP-9 enhanced the proliferation of in-vitro-cultured normal endothelial cells from dermal tissues of adult mice and of tumor-associated endothelial cells isolated from tumor xenografts in host mice. These findings suggest that BMP-9 signaling activates the endothelium tested in the present study via ALK-1.

A body of evidence suggests that the murine allantois contains a stem cell niche, the Allantoic Core Domain (ACD), that may contribute to a variety of allantoic and embryonic cell types. Given that extracellular matrix (ECM) regulates cell fate and function in niches, the allantois was systematically examined for Collagen type IV (ColIV) and Perlecan, both of which are associated with stem cell proliferation and differentiation. Not only was localization of ColIV and Perlecan more widespread during gastrulation than previously reported, but protein localization profiles were particularly robust and dynamic within the allantois and associated visceral endoderm as the ACD formed and matured. We propose that these data provide further evidence that the ACD is a stem cell niche whose activity is synchronized with associated visceral endoderm, possibly via ECM proteins., ((c) 2009 Wiley-Liss, Inc.)

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.

Steel factor is an essential survival and proliferation factor for primordial germ cells (PGCs) during their migration in the early mouse embryo. PGCs arise during gastrulation, and migrate into the posterior endoderm that becomes the hindgut. Previous reports have suggested that PGCs become dependent on Steel factor when they colonize the hindgut. However, in the absence of a good marker for living PGCs, their behavior before hindgut colonization has not been previously studied. We report here the normal behavior of PGCs in live embryos before hindgut colonization, and the roles of Steel factor, using a reporter line in which GFP is driven by the promoter of the Stella gene, whose activation accompanies the initial specification of PGCs. We show first that PGCs are surrounded by Steel factor-expressing cells from their first appearance in the allantois to the time they enter the genital ridges. Second, fewer PGCs are found in the allantois in Steel-null embryos, but this is not due to a failure of PGC specification. Third, the analysis of cultured Steel-null early embryos shows that Steel factor is required for normal PGC motility, both in the allantois and in the hindgut. Germ cells migrate actively in the allantois, and move directionally from the allantois into the proximal epiblast. In the absence of Steel factor, caused by either null mutation or antibody blockade, PGC motility is dramatically decreased, but directionality is maintained, demonstrating a primary role for Steel factor in PGC motility. This was found both before and after colonization of the hindgut. These data, together with previously published data, show that PGCs are Steel factor dependent from their initial specification until they colonize the genital ridges, and suggest the existence of a ;spatio-temporal niche' that travels with this important pluripotential cell population in the embryo.

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., ((c) 2009 Wiley-Liss, Inc.)

Objective: Numerous researchers have evaluated the insulin-like growth factors (IGF) influence on mammalian fetal development. Although IGF has been explored in the avian system, questions remain on the role of IGF in avian development. Therefore, the current study evaluated the mRNA abundance of IGF in the amnion and allantoic membranes and developing small intestine in the chicken, duck, and turkey during the incubation and post-hatch period., Design: Broiler, duck, and turkey eggs were incubated with small intestinal, allantoic, and amniotic membranes collected in the final days of incubation and 1 week post-hatch. RNA was extracted using Trizol and qRT-PCR was utilized to compare differences during embryo development within and across species., Results: The expression of the IGF mRNA varied between species in the final days of incubation in the amniotic and allantoic membranes. The turkey had higher (0.38-1.72 log) transcript abundance of IGF-I and IGF-II in the amnion and allantois compared to the chicken and duck. Evaluating the mRNA abundance within the chicken duodenum, jejunum, and ileum, the duodenum had the lowest expression of IGF-I and IGF-II (P<0.05) at day -4 of incubation compared to the jejunum and ileum. Focusing on differences in jejunal IGF expression among the three species, the turkey had the lowest IGF-I abundance at day -4 of incubation and highest IGF-I abundance at day of hatch (P<0.05). Transcript abundance of both IGF-II and IGF-R was highest in the turkey at day of hatch and day 1 post-hatch compared to the duck and chicken. The whole tissue versus the mucosal expression of the IGF mRNA abundance was evaluated during the post-hatch period. Duodenal, jejunal, and ileal segments had higher IGF-I transcript abundance (P<0.05) at day 1, day 3, and day of hatch, respectively. No differences were observed between segment and mucosa for IGF-II in the post-hatch period. The duodenal and jejunal mucosa IGF-R transcript abundance was greater (P<0.05) at day of hatch compared to the intestinal segment. The duck IGF mRNA in the jejunal mucosa was higher than the whole segment and decreased from day of hatch to day 3 post-hatch while the IGF mRNA abundance increased in the whole segment during the same time period. The turkey IGF-I transcript abundance decreased in both the segment and mucosa following hatch while the IGF-II mRNA expression increased by 1.5 logs from hatch to day 1 post-hatch., Conclusion: The transcript abundance of the IGF axis in the extra-embryonic membranes and gastrointestinal tissue of the developing chicken, duck, and turkey are influenced by embryonic age and species. A better understanding of the IGF axis in the small intestine during embryonic development may allow for increasing the optimal growth of both the gastrointestinal tract and the neonate.

Background: In mice the exchange of oxygen and nutrients between mother and fetus occurs in the chorioallantoic placenta where fetal capillaries come in close proximity with maternal blood perfusing trophoblast-lined sinusoids. Despite its critical importance, quantitative in vivo gene expression over the initial stages of chorioallantoic placental development has not been described, nor are there in vitro systems recapitulating the critical syncytiotrophoblast differentiation step in its formation. Here we describe molecular events that occur during the onset of chorioallantoic morphogenesis in mice in vivo, and in placental explant and whole conceptus cultures in vitro., Results: Chorioallantoic morphogenesis began immediately following allantoic fusion with the chorion in vivo, and was associated with significant upregulation of syncytiotrophoblast associated mRNA (Gcm1 and Syncytin A). However mouse placentas with chorioallantoic point attachment cultured with the allantois or as whole conceptuses did not upregulate Gcm1 and/or Syncytin A, suggesting that syncytiotrophoblast differentiation did not occur in vitro. Failure of morphogenesis appeared to be due to failure to sustain in vitro the chorionic trophoblast cells from which the syncytiotrophoblast cells are derived. In vitro culture conditions did support the upregulation of ectoplacental cone marker Tpbpalpha, maintenance of giant cell marker Pl1, and maintenance of Fgfr2 expression; all of which mimicked in vivo events observed over this developmental interval., Conclusions: We conclude that chorionic trophoblast maintenance and the early events that occur in vivo between chorioallantoic point attachment and primary villous formation are dependent on undefined intrauterine factors that were not present in the in vitro culture system. Nevertheless, in vitro culture conditions were appropriate to reproduce in vivo expression levels of Fgfr2, Pl1, and Tpbpalpha in placental explants.

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., (Copyright (c) 2008 Wiley-Liss, Inc.)

Proteomic analysis of bovine conceptus fluid proteins during early pregnancy has the potential to expose protein species indicative of both the overall health of the fetal-maternal environment and fetal developmental status. In this study, we examined the differential abundance of bovine conceptus fluid proteins (5-50 kDa fraction) from naturally conceived, in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT)-derived pregnancies at days 45 and 90 of gestation. In day 45 allantoic fluid (AllF) samples, an atypical cluster of low molecular weight ( approximately 14-16 kDa), low pI (between 3.0 and 4.5 pH units) protein species was increased in three of four IVF samples (30-100-fold increase in protein spot volumes compared to normal). These proteins were identified as paralogs of the bovine cathelicidin antimicrobial protein (CAMP) by MALDI-TOF MS peptide mass fingerprint and MALDI-TOF MS/MS peptide sequence analysis. Peptidoglycan recognition protein and serine (or cysteine) proteinase inhibitor clade B1, were also significantly increased in the corresponding IVF samples. In two of four SCNT AllF samples, a 2-10-fold increase in CAMP protein spot volumes were detected. No aberrant abundance levels of individual protein species were observed in amniotic fluid samples, or in day 90 IVF AllF samples. Identification of unique protein species present in the normal bovine AllF proteome at day 45 is also reported.

VE-cadherin is an endothelial-specific transmembrane protein concentrated at cell-to-cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE-cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE-cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE-cadherin expression and junctional clustering are required for optimal transforming growth factor-beta (TGF-beta) signalling in endothelial cells (ECs). TGF-beta antiproliferative and antimigratory responses are increased in the presence of VE-cadherin. ECs lacking VE-cadherin are less responsive to TGF-beta/ALK1- and TGF-beta/ALK5-induced Smad phosphorylation and target gene transcription. VE-cadherin coimmunoprecipitates with all the components of the TGF-beta receptor complex, TbetaRII, ALK1, ALK5 and endoglin. Clustered VE-cadherin recruits TbetaRII and may promote TGF-beta signalling by enhancing TbetaRII/TbetaRI assembly into an active receptor complex. Taken together, our data indicate that VE-cadherin is a positive and EC-specific regulator of TGF-beta signalling. This suggests that reduction or inactivation of VE-cadherin may contribute to progression of diseases where TGF-beta signalling is impaired.

Slit is a secreted protein known to function through the Roundabout (Robo) receptor as a repellent for axon guidance and neuronal migration, and as an inhibitor in leukocyte chemotaxis. We have previously shown that Slit2 is also secreted by a variety of human cancer cells whereby it acts as a chemoattractant to vascular endothelial cells for tumor angiogenesis. We used a blocking antibody to investigate the role of Slit-Robo signaling in tumor angiogenesis during oral carcinogenesis. In this report we undertook a multistage model of 7,12-dimethyl-1,2-benzanthracene-induced squamous cell carcinoma in the hamster buccal pouch. R5, a monoclonal antibody against the first immunoglobulin domain of Robo1, was used to study whether R5 blocks the Slit-Robo interaction and furthermore inhibits tumor angiogenesis and growth in our model. In addition, the expression of Slit2, von Willebrand factor, and vascular endothelial growth factor were examined using human tissue of oral cheek mucosa with oral squamous cell carcinoma. Our data showed that Slit2 was expressed minimally in normal and hyperplastic mucosa, moderately in dysplastic mucosa, and highly in neoplastic mucosa obtained from hamster buccal pouch. We also found that increased Slit2 expression was associated with higher tumor angiogenesis, as reflected by increased vascular endothelial growth factor expression and microvessel density. A similar Slit2 expression profile was found in human tissue. Importantly, interruption of the Slit2-Robo interaction using R5 inhibited tumor angiogenesis and growth in our in vivo model, which indicates that Slit2-mediated tumor angiogenesis is a critical process underlying the carcinogenesis of chemical-induced squamous cell carcinoma. Therefore, targeting Slit-Robo signaling may offer a novel antiangiogenesis approach for oral cancer therapy.

A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy.