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.

Driven by a global trend of applying replace-reduce-refine or 3Rs' guidance for experimental animals in life sciences, chick embryo and particularly allantois with its chorioallantoic membrane have been increasingly utilized to substitute laboratory animals, which call for more extensive and updated knowledge about this novel experimental setup. In this study, being noninvasive, nonionizing, and super-contrasting with high spatiotemporal resolutions, magnetic resonance imaging (MRI) was chosen as an imaging modality for in ovo monitoring morphologic evolution of the chick embryo, allantois, and chorioallantoic membrane longitudinally throughout embryonic day (ED) 1 until ED20. Cooled in 0°C ice bath for 60 min to reduce MRI motion artifacts, 3 chick embryos (n = 60 in total) on each ED were scanned by a clinical 3.0T MRI scanner to demonstrate 3D images of both T2- and T1-weighted imaging (T2WI, T1WI) sequences at axial, sagittal, and coronal slices. The volumes of both the entire chick embryo and allantois were semi-automatically segmented based on intensity-based thresholding and region-growing algorithms. The morphometries or quantified 3D structures were achieved by refined segmentation, and confirmed by histological analyses (one for each ED). After MRI, the rest of chick embryos (n = 40) continued for incubation. The images from ED2 to ED4 could demonstrate the structural changes of latebra, suggesting its transition into a nutrient supplying channel of yolk sac. The allantois could be recognized by MRI, and its relative volumes on each ED revealed an evolving profile peaked on ED12, with a statistically significant difference from those of earlier and later EDs (P < 0.01). The hypointensity of the yolk due to the susceptibility effect of its enriched iron content overshadowed the otherwise hyperintensity of its lipid components. The chick embryos survived prior cooling and MRI till hatching on ED21. The results could be further developed into a 3D MRI atlas of chick embryo. Clinical 3.0T MRI proved effective as a noninvasive approach to study in ovo 3D embryonic development across the full period (ED1-ED20), which can complement the present knowhow for poultry industry and biomedical science.

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.

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.

Mouse primordial germ cells (PGCs) are specified between embryonic day 6.5 (E6.5) and E7.5, when they have been visualized as an alkaline phosphatase-positive (AP+) cell population in the developing allantois. By E8.5, they are embedded in the hind-gut epithelium. Previous experiments have suggested different sites for PGCs' origin, and it is unclear how they reach the gut epithelium. We have used transgenic mice expressing GFP under a truncated Oct4 promoter to visualize living PGCs. We find GFP+/AP+ cells in the posterior end of the primitive streak as a dispersed population of cells actively migrating into the allantois, and directly into the adjacent embryonic endoderm. Time-lapse analysis shows these cells to be actively migratory from the time they exit the primitive streak.

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.

Many clinically relevant congenital malformations arise during mid to late embryonic stages. This period is challenging to image quantitatively in live embryos, necessitating the use of multiple specimens with increased experimental variability. Here we establish X-ray and blood-pool computed tomography (CT) contrast agent toxicity and teratogenesis thresholds for 3D Micro-CT imaging of live avian embryos. Day 4 chick embryos micro-injected with Visipaque™ (VP) developed for an additional 6 days without defect. X-ray radiation up to 798 mGy was nontoxic. Peak average contrast of 1,060 HU occurred within 1 hr of imaging at 50 μm resolution. VP-enhanced contrast persisted past 24 hr with delayed accumulation in the allantois. Regional volumes of VP-injected embryos were statistically identical to those of fixed embryos perfused with osmium tetroxide. We further quantified longitudinal volumetric morphogenesis of the allantois over 30 hr. These results demonstrate the safety and efficacy of contrast enhanced quantitative micro-CT imaging for live embryos.

Mixl1 is thought to play important roles in formation of mesoderm and endoderm. Previously, Mixl1 expression was reported in the posterior primitive streak and allantois, but the precise spatiotemporal whereabouts of Mixl1 protein throughout gastrulation have not been elucidated. To localize Mixl1 protein, immunohistochemistry was carried out at 2-4 h intervals on mouse gastrulae between primitive streak and 16-somite pair (s) stages (~E6.5-9.5). Mixl1 localized to the entire primitive streak early in gastrulation. However, by headfold stages (~E7.75-8.0), Mixl1 diminished within the mid-streak but remained concentrated at either end of the streak, and localized throughout midline posterior visceral endoderm. At the streak's anterior end, Mixl1 was confined to the posterior crown cells of Hensen's node, which contribute to dorsal hindgut endoderm, and the posterior notochord. In the posterior streak, Mixl1 localized to the Allantoic Core Domain (ACD), which is the source of most of the allantois and contributes to the posterior embryonic-extraembryonic interface. In addition, Mix1 co-localized with the early hematopoietic marker, Runx1, in the allantois and visceral yolk sac blood islands. During hindgut invagination (4-16s, ~E8.5-9.5), Mixl1 localized to the hindgut lip, becoming concentrated within the midline anastomosis of the splanchnopleure, which appears to create the ventral component of the hindgut and omphalomesenteric artery. Surrounding the distal hindgut, Mixl1 identified midline cells within tailbud mesoderm. Mixl1 was also found in the posterior notochord. These findings provide a critical systematic, and tissue-level understanding of embryonic Mixl1 localization, and support its role in regulation of crucial posterior axial mesendodermal stem cell niches during embryogenesis.

The biochemical composition of uterine and fetal fluids during pregnancy of the grey short-tailed opossum was compared with new and published data on the tammar wallaby. In the grey short-tailed opossum, there are three main phases of embryonic nourishment. During the first phase, the embryo obtains nutrients from uterine secretion transferred into the yolk sac. The amount of uterine secretion declines during the second phase up to the time of shell coat rupture. As a result, the protein concentration in yolk sac fluid also declines. During phase three, which begins with shell coat rupture, nutrients are predominantly available from the maternal blood. In the grey short-tailed opossum that lacks a vesicular, fluid-filled allantois, waste products such as urea are apparently stored in the yolk sac and from there pass into the maternal circulation across the invasive yolk sac placenta. In contrast, in the tammar wallaby, the main source of nutrients available to the late term fetus is glandular secretion that is complemented by substances from the maternal circulation via the chorio-vitelline placenta, and waste products are stored in the large, fluid-filled allantois.

T-box genes encode transcription factors that regulate many developmental processes. We have cloned a novel mouse T-box gene, Tbx12. Tbx12 is the vertebrate homologue of the Drosophila H15 gene and the Caenorhabditis elegans tbx-12 gene. Tbx12 is expressed in extraembryonic tissues such as the amnion and allantois. In the embryo, Tbx12 is strongly expressed in the neural retina and the heart.

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

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.

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

Hypoblast/visceral endoderm assists in amniote nutrition, axial positioning and formation of the gut. Here, we provide evidence, currently limited to humans and non-human primates, that hypoblast is a purveyor of extraembryonic mesoderm in the mouse gastrula. Fate mapping a unique segment of axial extraembryonic visceral endoderm associated with the allantoic component of the primitive streak, and referred to as the "AX", revealed that visceral endoderm supplies the placentae with extraembryonic mesoderm. Exfoliation of the AX was dependent upon contact with the primitive streak, which modulated Hedgehog signaling. Resolution of the AX's epithelial-to-mesenchymal transition (EMT) by Hedgehog shaped the allantois into its characteristic projectile and individualized placental arterial vessels. A unique border cell separated the delaminating AX from the yolk sac blood islands which, situated beyond the limit of the streak, were not formed by an EMT. Over time, the AX became the hindgut lip, which contributed extensively to the posterior interface, including both embryonic and extraembryonic tissues. The AX, in turn, imparted antero-posterior (A-P) polarity on the primitive streak and promoted its elongation and differentiation into definitive endoderm. Results of heterotopic grafting supported mutually interactive functions of the AX and primitive streak, showing that together, they self-organized into a complete version of the fetal-placental interface, forming an elongated structure that exhibited A-P polarity and was composed of the allantois, an AX-derived rod-like axial extension reminiscent of the embryonic notochord, the placental arterial vasculature and visceral endoderm/hindgut.

Huntington disease (HD) is an adult-onset neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms that is caused by a CAG expansion in the HTT gene. Palmitoylation is the addition of saturated fatty acids to proteins by DHHC palmitoylacyl transferases. HTT is palmitoylated by huntingtin interacting proteins 14 and 14-like (HIP14 and HIP14L or ZDHHC17 and 13 respectively). Mutant HTT is less palmitoylated and this reduction of palmitoylation accelerates its aggregation and increases cellular toxicity. Mouse models deficient in either Hip14 (Hip14(-/-)) or Hip14l (Hip14l(-/-)) develop HD-like phenotypes. The biological function of HTT palmitoylation and the role that loss of HTT palmitoylation plays in the pathogenesis of HD are unknown. To address these questions mice deficient for both genes were created. Loss of Hip14 and Hip14l leads to early embryonic lethality at day embryonic day 10-11 due to failed chorioallantoic fusion. The chorion is thickened and disorganized and the allantois does not fuse correctly with the chorion and forms a balloon-like shape compared to Hip14l(-/-); Hip14(+/+) littermate control embryos. Interestingly, the Hip14(-/-) ; Hip14(-/-) embryos share many features with the Htt(-/-) embryos, including folding of the yolk sac, a bulb shaped allantois, and a thickened and disorganized chorion. This may be due to a decrease in HTT palmitoylation. In Hip14(-/-); Hip14l(-/-) mouse embryonic fibroblasts show a 25% decrease in HTT palmitoylation compared to wild type cells. This is the first description of a double PAT deficient mouse model where loss of a PAT or multiple PATs results in embryonic lethality in mammals. These results reinforce the physiological importance of palmitoylation during embryogenesis.

The importance of trophic factors, such as nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) during the perinatal period, is now emerging. Through their functional activities of neurogenesis and angiogenesis, they play a key role in the final maturation of the nervous and vascular systems. The present study aims to: (i) evaluate the NGF and VEGF levels obtained at parturition from the mare, foal and umbilical cord vein plasma, as well as in amniotic fluid; (ii) evaluate NGF and VEGF content in the plasma of healthy foals during the first 72 h of life (T0, T24 and T72); (iii) evaluate NGF and VEGF levels at parturition in relation to the selected mares’ and foals’ clinical parameters; (iv) evaluate the relationship between the two trophic factors and the thyroid hormone levels (TT3 and TT4) in the first 72 h of life; (v) assess mRNA expression of NGF, VEGF and BDNF and their cell surface receptors in the placenta. Fourteen Standardbred healthy foals born from mares with normal pregnancies and parturitions were included in the study. The dosage of NGF and VEGF levels was performed using commercial ELISA kits, whereas NGF, VEGF and BDNF placental gene expression was performed using semi-quantitative real-time PCR. In foal plasma, both NGF and VEGF levels decreased significantly over time, from T0 to T24 (p = 0.0066 for NGF; p < 0.0001 for VEGF) and from T0 to T72 (p = 0.0179 for NGF; p = 0.0016 for VEGF). In foal serum, TT3 levels increased significantly over time from T0 to T24 (p = 0.0058) and from T0 to T72 (p = 0.0013), whereas TT4 levels decreased significantly over time from T0 to T24 (p = 0.0201) and from T0 to T72 (p < 0.0001). A positive correlation was found in the levels of NGF and VEGF in foal plasma at each time point (p = 0.0115; r = 0.2862). A positive correlation was found between NGF levels in the foal plasma at T0 and lactate (p = 0.0359; r = 0.5634) as well as between VEGF levels in the foal plasma at T0 and creatine kinase (p = 0.0459; r = 0.5407). VEGF was expressed in all fetal membranes, whereas NGF and its receptors were not expressed in the amnion. The close relationship between the two trophic factors in foal plasma over time and their fine expression in placental tissues appear to be key regulators of fetal development and adaptation to extra-uterine life.

Allantoic endoderm of 3-day chick embryos was combined with pancreatic mesenchyme of 5-day embryos and cultured as chorio-allantoic grafts for a total of 14 days. Recombinations of endoderm and mesenchyme of the pancreas and of the allantois served as controls. The usual types of endocrine cells differentiated in the pancreatic controls, none in the allantoic controls. In experimental grafts simple columnar epithelium with goblet cells and a sucrase-positive brush border developed; a few insulin cells and endocrine cells typical of the intestine differentiated. Hence allantoic endoderm has endocrine potentiality not realised in vivo, where its own mesenchyme may be inhibitory.

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.

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.

The urachus is an embryonic structure that connects the bladder to the allantois during early embryonic development. Occasionally, it fails to disappear at birth, leading to a case of urachal remnant (UR). This study aimed to determine whether our policy for selecting an appropriate UR resection approach is valid. We performed preoperative imaging to examine whether UR continued toward the bladder apex. If so, the UR and bladder apex were excised using the trans-umbilical approach, in addition to laparoscopy, if necessary. If preoperative imaging indicated that the UR ended near the umbilicus, the UR from the umbilicus to the duct end was resected. Pathological evaluations were performed to determine the appropriateness of the surgical approach indicated by preoperative imaging.