Embryonic Circulation Research Articles

Nicotinamide Adenine Dinucleotide Deficiency and Its Impact on Mammalian Development.

Significance: Nicotinamide adenine dinucleotide (NAD) is an important molecule synthesized from tryptophan or vitamin B3 and involved in numerous cellular reactions. NAD deficiency during pregnancy causes congenital NAD deficiency disorder (CNDD) characterized by multiple congenital malformations and/or miscarriage. Studies in genetically engineered mice replicating mutations found in human patient cases show that CNDD can be prevented by dietary supplements. Recent Advances: A growing number of patient reports show that biallelic loss-of-function of genes involved in NAD de novo synthesis (KYNU, HAAO, NADSYN1) cause CNDD. Other factors that limit the availability of NAD precursors, for example, limited dietary precursor supply or absorption, can cause or contribute to NAD deficiency and result in CNDD in mice. Molecular flux experiments allow quantitative understanding of NAD precursor concentrations in the circulation and their usage by different cells. Studies of NAD-consuming enzymes and contributors to NAD homeostasis help better understand how perturbed NAD levels are implicated in various diseases and adverse pregnancy outcomes. Critical Issues: NAD deficiency is one of the many known causes of adverse pregnancy outcomes, but its prevalence in the human population and among pregnant women is unknown. Since NAD is involved in hundreds of diverse cellular reactions, determining how NAD deficiency disrupts embryogenesis is an important challenge. Future Directions: Furthering our understanding of the molecular fluxes between the maternal and embryonic circulation during pregnancy, the NAD-dependent pathways active in the developing embryo, and the molecular mechanisms by which NAD deficiency causes adverse pregnancy outcomes will provide direction for future prevention strategies. Antioxid. Redox Signal. 39, 1108-1132.

Dynamics of maternal androgens and its metabolites during early embryonic development: embryonic modification of a maternal effect.

In birds, exposure to maternal (yolk) testosterone affects a diversity of offspring post-hatching traits, that eventually affect offspring competitiveness. However, maternal testosterone is heavily metabolized at very early embryonic developmental stages to hydrophilic metabolites that are often assumed to be much less biologically potent. The rapid metabolism could either keep the maternal testosterone from reaching the embryos, opening the possibility for a mother-offspring conflict, or the metabolites may facilitate the uptake of the lipophilic testosterone from the yolk into the embryonic circulation after which they are either converted back to the testosterone or functioning directly as metabolites. To test these possibilities, we injected isotope-labeled testosterone (T-[D5]) into the yolk of freshly laid Rock pigeon (Columba livia) eggs and determined the concentration and distribution of T-[D5] and its labelled metabolites within different egg fractions by LC-MS/MS at day 2, 5 and 10 of incubation. Although under a supraphysiological dosage injection, yolk testosterone decreased within 2 days and was metabolized into androstenedione, conjugated testosterone, etiocholanolone and other components that unidentifiable due to methodological limitation. We show for the first time that testosterone, androstenedione and conjugated testosterone, but not etiocholanolone, reached the embryo including its brain. Their high concentrations in the yolk and extraembryonic membranes suggest that conversion takes place here. We also found no sex-specific metabolism, explaining why maternal testosterone does not affect sexual differentiation. Our findings showed that maternal testosterone is quickly converted by the embryo, with several but not all metabolites reaching the embryo providing evidence for both hypotheses.

Teratogenic Effects of Organohalogen Contaminants Extracted from Whale Bacon in a Whole-Rat-Embryo Culture System.

Marine foods can be contaminated with organochlorines and the risk to human beings who consume these foods needs to be evaluated. We examined the teratogenic effects of contaminants extracted from whale bacon on rat embryos using a whole-embryo culture system. Embryonic day 11.5 embryos were cultured for 48 h with organohalogens extracted from whale bacon at low (polychlorinated biphenyls (PCBs): 0.32 ppm, dichlorodiphenyltrichloroethanes (DDTs): 0.16 ppm, chlordanes (CHLs): 0.02 ppm) and high (PCBs: 2.15 ppm, DDTs: 1.99 ppm, CHLs: 0.20 ppm) doses. The levels of organohalogen compounds in cultured embryos were determined. The organochlorine contaminants extracted from whale products were readily transferred to the cultured rat embryos. The number of heartbeats, yolk sac circulation score, and embryonic body circulation score of embryos did not change during the culture period in either exposure group. Cultured embryos treated with the low-dose contaminated medium for 48 h showed abnormalities of the mandible, and craniofacial or forelimb hematomas with an incidence of 50%. All embryos treated with the high-dose medium showed craniofacial abnormalities and cleft lip, and limb abnormalities and hematomas. These results indicate that the organohalogen contaminants in whale bacon may be teratogenic in a dose-dependent manner. Further studies are necessary to determine the dose–effect relationship.

Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development

Precursors of the adult hematopoietic system arise from the aorta-gonad-mesonephros (AGM) region shortly after the embryonic circulation is established. Here, we develop a microfluidic culture system to mimic the primitive embryonic circulation and address the hypothesis that circulatory flow and shear stress enhance embryonic blood development. Embryonic (HOXA+) hematopoiesis was derived from human pluripotent stem cells and induced from mesoderm by small-molecule manipulation of TGF-β and WNT signaling (SB/CHIR). Microfluidic and orbital culture promoted the formation of proliferative CD34+RUNX1C-GFP+SOX17-mCHERRY+ precursor cells that were released into the artificial circulation from SOX17+ arterial-like structures. Single-cell transcriptomic analysis delineated extra-embryonic (yolk sac) and HOXA+ embryonic blood differentiation pathways. SB/CHIR and circulatory flow enhance hematopoiesis by the formation of proliferative HOXA+RUNX1C+CD34+ precursor cells that differentiate into monocyte/macrophage, granulocyte, erythrocyte, and megakaryocyte progenitors.

Development and characterization of a chick embryo chorioallantoic membrane (CAM) based platform for evaluation of vasoactive medications.

Among various anti-cancer therapies, tumor vascular disrupting agents (VDAs) play a crucial role, for which their off-targeting effects on normal vessels need also to be investigated. The purpose of this study was to set up an in-ovo platform that combines a laser speckle contrast imaging (LSCI) modality with chick embryo chorioallantoic membrane (CAM) to real-time monitor vascular diameters and perfusion without and with intravascular injection. Two eggshell windows for both observation or measurement and injection were opened. Dynamic blood perfusion images and corresponding statistic graphs were acquired by using a LSCI unit on CAMs from embryo date (ED) 9 to ED15. A dedicated fine needle catheter was made for slow intravascular administration over 30min with simultaneous LSCI acquisition. To verify the connectivity between CAM vessels and the embryonic circulations in the egg, contrast-enhanced 3D micro computed tomography (μCT), 2D angiography and histology were executed. This platform was successfully established to acquire, quantify and demonstrate vascular and hemodynamic information from the CAM. Chick embryos even with air cell opened remained alive from ED9 to ED15. Through collecting LSCI derived CAM vascular diameter and perfusion parameters, ED12 was determined as the best time window for vasoactive drug studies. A reverse correlation between CAM vessel diameter and blood perfusion rate was found (p<0.002). Intravascular infusion and simultaneous LSCI acquisition for 30min in ovo proved feasible. Contrast-enhanced angiography and histomorphology could characterize the connectivity between CAM vasculature and embryonic circulation. This LSCI-CAM platform was proved effective for investigating the in-ovo hemodynamics, which paves the road for further preclinical research on vasoactive medications including VDAs.

Bone marrow-independent adventitial macrophage progenitor cells contribute to angiogenesis

Pathological angiogenesis promotes tumor growth, metastasis, and atherosclerotic plaque rupture. Macrophages are key players in these processes. However, whether these macrophages differentiate from bone marrow-derived monocytes or from local vascular wall-resident stem and progenitor cells (VW-SCs) is an unresolved issue of angiogenesis. To answer this question, we analyzed vascular sprouting and alterations in aortic cell populations in mouse aortic ring assays (ARA). ARA culture leads to the generation of large numbers of macrophages, especially within the aortic adventitia. Using immunohistochemical fate-mapping and genetic in vivo-labeling approaches we show that 60% of these macrophages differentiate from bone marrow-independent Ly6c+/Sca-1+ adventitial progenitor cells. Analysis of the NCX−/− mouse model that genetically lacks embryonic circulation and yolk sac perfusion indicates that at least some of those progenitor cells arise yolk sac-independent. Macrophages represent the main source of VEGF in ARA that vice versa promotes the generation of additional macrophages thereby creating a pro-angiogenetic feedforward loop. Additionally, macrophage-derived VEGF activates CD34+ progenitor cells within the adventitial vasculogenic zone to differentiate into CD31+ endothelial cells. Consequently, depletion of macrophages and VEGFR2 antagonism drastically reduce vascular sprouting activity in ARA. In summary, we show that angiogenic activation induces differentiation of macrophages from bone marrow-derived as well as from bone marrow-independent VW-SCs. The latter ones are at least partially yolk sac-independent, too. Those VW-SC-derived macrophages critically contribute to angiogenesis, making them an attractive target to interfere with pathological angiogenesis in cancer and atherosclerosis as well as with regenerative angiogenesis in ischemic cardiovascular disorders.

Characterization and functional properties of a novel monoclonal antibody which identifies a B cell subpopulation in bursa of Fabricius

The bursa of Fabricius (BF) plays a central role in the development of B lymphocytes in birds. During embryonic development the BF primordium is colonized by myeloid and lymphoid prebursal stem cells to form the follicle buds, which ultimately develop into lymphoid follicles with a central medullary and an outer cortical region. Lympho-myeloid differentiation within the medulla is fundamental to normal B cell development. In contrast, the complexity of the cellular composition of the follicular cortex and its role in B cell differentiation has only recently begun to be studied. As an effort to characterize the different bursal cells we have produced a large panel of monoclonal antibodies (mAbs) by immunizing mice with a BF cell suspension of guinea fowl (Numida meleagris). One of these antibodies (clone: 7H3) was found to recognize a 80 kDa cell surface antigen expressed first in the yolk sac blood island of 2-day-old guinea fowl and chicken embryos, and later detected in the embryonic circulation and primary lymphoid organs. Double immunofluorescence revealed that chB6+ (Bu-1+) B cells of embryonic BF co-express the 7H3 antigen. 7H3 immunoreactivity of the bursal follicles gradually diminished after hatching and only a subpopulation of cortical B cells expressed the 7H3 antigen. In addition, in post-hatched birds 7H3 mAb recognizes all T lymphocytes of the thymus, peripheral lymphoid organs and blood. Embryonic BF injected with the 7H3 mAb showed a near complete block of lymphoid follicle formationIn conclusion, 7H3 mAb labels a new differentiation antigen specific for avian hematopoietic cells, which migrate through the embryonic mesenchyme, colonize the developing BF lymphoid follicles, and differentiate into a subpopulation of cortical B cells. The staining pattern of the 7H3 mAb and the correlation of expression with cell migration suggest that the antigen will serve as valuable immunological marker for studying the ontogeny of avian B cells.

1529 Management of A Rare Case of Bilateral Persistent Sciatic Artery Aneurysms Causing Critical Limb Ischaemia With Staged Embolisation And Femoropopliteal Bypass: A Case Report

Abstract Introduction Bilateral persistent sciatic arteries (PSA) are rare with an incidence of 0.001%. They represent a persistence of the embryonic axial limb circulation via the internal iliac artery. Normally the PSA involutes and is replaced by the superficial femoral artery (SFA). Failure of regression leads to a PSA with varying configurations. Up to 50% of PSAs are aneurysmal and may present with rupture or acute/chronic ischaemia, typically at 40-50 years old. Case Report A 74-year-old female presented with limb threatening ischaemia manifesting with rest pain, sensory deficit and early tissue loss (Rutherford IIb). She had a left femoral pulse but absent pulses distally. Pedal pulses were noted on the contralateral limb. CT angiogram revealed bilateral complete PSAs with incomplete hypoplastic SFAs (Pillet-Gauffre 2a). Both PSAs were aneurysmal; the left PSA was acutely occluded distal to the aneurysm. She underwent staged intervention with percutaneous embolisation of the left PSA, followed by femoropopliteal bypass. She was discharged six days later with good symptomatic relief at one month follow up. We are planning to treat the contralateral limb in a similar manner electively in case complications occur. Conclusions PSAs are commonly associated with limb threatening complications but due to their rarity there are limited reports on the management of this condition. Here we can report a good outcome in a late presentation using staged embolisation with open reconstruction.

The method to generate pulsatile circulatory fluid flow using microfluidics

Microfluidic chips provide versatile tools to mimic the biological effect of blood flow on pluripotent stem cells (PSC). This paper presents methods for the use of microfluidics to model embryonic circulation using differentiated PSC. Pulsatile circulatory flow is created with a microfluidics device with pressure-driven microvalves and ventricles. Silicone rubber devices are cast from moulds manufactured using standard and 3D laser lithography. The surface chemistry is modified to support the growth of human umbilical vein endothelial cells and pluripotent stem cells. Pulsatile circulatory fluid flow can be applied at specific stages of cell differentiation with direct observation of cellular responses by time-lapse fluorescent microscopy.•Replicable manufacturing protocol of lab scale microfluidic device generating pulsatile fluid flow mimicry embryonic blood circulation.•Integration of human cell lines on microfluidic chip.

In Xenopus ependymal cilia drive embryonic CSF circulation and brain development independently of cardiac pulsatile forces

BackgroundHydrocephalus, the pathological expansion of the cerebrospinal fluid (CSF)-filled cerebral ventricles, is a common, deadly disease. In the adult, cardiac and respiratory forces are the main drivers of CSF flow within the brain ventricular system to remove waste and deliver nutrients. In contrast, the mechanics and functions of CSF circulation in the embryonic brain are poorly understood. This is primarily due to the lack of model systems and imaging technology to study these early time points. Here, we studied embryos of the vertebrate Xenopus with optical coherence tomography (OCT) imaging to investigate in vivo ventricular and neural development during the onset of CSF circulation.MethodsOptical coherence tomography (OCT), a cross-sectional imaging modality, was used to study developing Xenopus tadpole brains and to dynamically detect in vivo ventricular morphology and CSF circulation in real-time, at micrometer resolution. The effects of immobilizing cilia and cardiac ablation were investigated.ResultsIn Xenopus, using OCT imaging, we demonstrated that ventriculogenesis can be tracked throughout development until the beginning of metamorphosis. We found that during Xenopus embryogenesis, initially, CSF fills the primitive ventricular space and remains static, followed by the initiation of the cilia driven CSF circulation where ependymal cilia create a polarized CSF flow. No pulsatile flow was detected throughout these tailbud and early tadpole stages. As development progressed, despite the emergence of the choroid plexus in Xenopus, cardiac forces did not contribute to the CSF circulation, and ciliary flow remained the driver of the intercompartmental bidirectional flow as well as the near-wall flow. We finally showed that cilia driven flow is crucial for proper rostral development and regulated the spatial neural cell organization.ConclusionsOur data support a paradigm in which Xenopus embryonic ventriculogenesis and rostral brain development are critically dependent on ependymal cilia-driven CSF flow currents that are generated independently of cardiac pulsatile forces. Our work suggests that the Xenopus ventricular system forms a complex cilia-driven CSF flow network which regulates neural cell organization. This work will redirect efforts to understand the molecular regulators of embryonic CSF flow by focusing attention on motile cilia rather than other forces relevant only to the adult.

Right ventricle in hypoplastic left heart syndrome exhibits altered hemodynamics in the human fetus

Hypoplastic left heart syndrome (HLHS) represents approximately 9% of all congenital heart defects and is one of the most complex, with the left side of the heart being generally underdeveloped. Numerous studies demonstrate that intracardiac fluid flow patterns in the embryonic and fetal circulation can impact cardiac structural formation and remodeling. This highlights the importance of quantifying the altered hemodynamic environment in congenital heart defects, like HLHS, relative to a normal heart as it relates to cardiac development. Therefore, to study human cardiovascular fetal flow, computational fluid dynamic simulations were performed using 4D patient-specific ultrasound scans in normal and HLHS hearts. In these simulations, we find that the HLHS right ventricle exhibits a greater cardiac output than normal; yet, hemodynamics are relatively similar between normal and HLHS right ventricles. Overall, this study provides detailed quantitative flow patterns for HLHS, which has the potential to guide future prevention and therapeutic interventions, while more immediately providing additional functional detail to cardiologists to aid in decision making.

Abstract 2714: The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models

Abstract Introduction: Progression into metastatic disease is a critical step in most malignancies. Cancer cells may enter circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. CSCs are known to form cell clusters in vitro (spheroids/tumorspheres). We reported that tumorspheres also circulate in blood in vivo. We found that they carry cancer stem cells and strongly correlate with metastatic disease. We collected tumorspheres from blood by filtration. This project aims to test the metastatic potential of tumorspheres by developing an assay using in vivo circulation models. The goal is to establish a novel prognostic test for metastatic predisposition in cancer patients. Methods: To develop an in vivo assay for metastasis we use the chorioallantoic circulation in the chicken embryo, with several advantages: easy access to circulation, lack of immune response, rapid turnaround time (5 to 10 days) and inexpensive and simple technique. For clinical applications we introduced three technical innovations: (i) to make fertilized chicken embryos easily available we worked out a method to store viable embryos beyond 30 days; (ii) we developed a new coverglass chamber technique for micromanipulations; and (iii) for microscopic analysis new over-drained chorioallantois preparations can accommodate high resolution objectives. To test tumorspheres in embryonic circulation we used both highly metastatic (e.g. MDA-MB-436) and non-metastatic (e.g. MCF7) cell lines with GFP label, DiI membrane dye and CFDA-SA dye. For molecular studies DNA was extracted 5 days after cancer spheroid implantation and used qPCR to detect human sequences. Results: In traditional storage few embryos survive beyond 6 days. As fertilized eggs carry pluripotent stem cells, we optimized temperature, gas and humidity combinations to match known stem cell optimums. By suppressing metabolism, blocking embryo adhesion and controlling gas and humidity exchange we extended viable embryo storage beyond 30 days. We modified the traditional window method by forming visually clear microscopic coverglass chambers, and the new over-draining approach lowered the chorioallantois membranes and allowed high resolution imaging. The results showed invasive and hematogenic dissemination of the cancer spheroids. Molecular studies of human markers detected metastatic progression and generated quantitative data. Conclusions: We optimized the in vivo embryonic metastasis assay. We show that it has the potential for a sensitive and quantitative method to diagnose metastatic predisposition in cancer patients. The molecular approach (DNA extraction, PCR markers) is easily up-scalable, the instrumentation is available in most hospitals and offers cost effective and critical early diagnosis of cancer progression. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Kathleen Marinelli, Christine Kyritsis, Viktoria Denes. The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2714.

Is a trident or a bident? Appearance of a primitive trigeminal artery on sagittal views

•Recognition of a persistent primitive trigeminal artery (PPTA) is of clinical significance due to its association with aneurysms and several vascular variants in addition to preoperative planning.•Characteristic appearance of PPTA on MR sagittal images of the brain resembles the Greek letter tau (two prongs) and was linked to Neptune’s trident (three prongs).•Use of a bident sign as a more visually and mythologically precise description of the appearance of PPTA. A 50 year old male with an incidental finding on the MRI brain (Fig. 1).Fig. 2A) Lateral digitally subtracted catheter angiogram of a selected internal carotid artery (arrow) injection demonstrating the persistent trigeminal artery (arrowhead) passing posteriorly to join the basilar artery (open arrow). B) Cropped view of the painting “Concilio degli dei” by Raffaello Sanzio da Urbino depicting Hades holding a bident (left), next to his brother Poseidon holding a trident (middle) [10]. Zeus (right) is also pictured, without a spear.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A.fetal posterior cerebral arteryB.persistent trigeminal arteryC.persistent hypoglossal arteryD.persistent otic arteryE.persistent proatlantal artery Answer on page 240 Is a trident or a bident? Appearance of a primitive trigeminal artery on sagittal viewsJournal of Clinical NeuroscienceVol. 75PreviewA persistent primitive trigeminal artery (PPTA) is the remnant of the embryonic arterial circulation in adults and is the most common type of the persistent carotid-vertebrobasilar anastomoses (with frequency 0.1–0.6%); followed by persistent hypoglossal artery (0.02–0.1%), persistent proatlantal artery and otic artery, which existence, however, remains controversial [6,7]. A PPTA arises from the proximal cavernous part of the ICA and courses posteriorly to join the basilar artery. Recognition of a PPTA is of clinical significance due to its association with aneurysms and several vascular variants [1,4]. Full-Text PDF Erratum to “Is a trident or a bident? Appearance of a primitive trigeminal artery on sagittal views – Question and Is a trident or a bident? Appearance of a primitive trigeminal artery on sagittal views – Answer” [J. Clin. Neurosci. 75C (2020) 204–205 and 240–241]Journal of Clinical NeuroscienceVol. 78PreviewThe publisher regrets to inform readers that there was an error in the article title for both the question and answer articles. The correct title is ‘Is it a trident or a bident? Appearance of a persistent primitive trigeminal artery on sagittal views – Question’ and ‘Is it a trident or a bident? Appearance of a persistent primitive trigeminal artery on sagittal views – Answer’ respectively. Full-Text PDF

Brain Parenchymal and Extraparenchymal Macrophages in Development, Homeostasis, and Disease.

Microglia are parenchymal macrophages of the CNS; as professional phagocytes they are important for maintenance of the brain's physiology. These cells are generated through primitive hematopoiesis in the yolk sac and migrate into the brain rudiment after establishment of embryonic circulation. Thereafter, microglia develop in a stepwise fashion, reaching complete maturity after birth. In the CNS, microglia self-renew without input from blood monocytes. Recent RNA-sequencing studies have defined a molecular signature for microglia under homeostasis. However, during disease, microglia undergo remarkable phenotypic changes, which reflect the acquisition of specialized functions tailored to the pathological context. In addition to microglia, the brain-border regions host populations of extraparenchymal macrophages with disparate origins and phenotypes that have recently been delineated. In this review we outline recent findings that provide a deeper understanding of both parenchymal microglia and extraparenchymal brain macrophages in homeostasis and during disease.

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

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

Placental chemokine compartmentalisation: A novel mammalian molecular control mechanism.

Atypical chemokine receptor 2 (ACKR2) is a chemokine-scavenging receptor. ACKR2–/–embryos display a reduction in size of a novel, to our knowledge, embryonic skin macrophage population referred to as ‘intermediate’ cells. CC chemokine receptor 2 (CCR2)–/–embryos display an identical phenotype, indicating that these cells require CCR2 to enable them to populate embryonic skin. Further analysis revealed that ACKR2–/–embryos have higher circulating concentrations of the CCR2 ligand, CC ligand 2 (CCL2); thus, ACKR2 regulates intraembryonic CCL2 levels. We show that ACKR2 is strongly expressed by trophoblasts and that it blocks movement of inflammatory chemokines, such as CCL2, from the maternal decidua into the embryonic circulation. We propose that trophoblastic ACKR2 is responsible for ensuring chemokine compartmentalisation on the maternal decidua, without which chemokines enter the embryonic circulation, disrupting gradients essential for directed intraembryonic cell migration. Overall, therefore, we describe a novel, to our knowledge, molecular mechanism whereby maternal decidual chemokines can function in a compartmentalised fashion without interfering with intraembryonic leukocyte migration. These data suggest similar functions for other atypical chemokine receptors in the placenta and indicate that defects in such receptors may have unanticipated developmental consequences.

Transcriptional profiles in the chicken ductus arteriosus during hatching.

The ductus arteriosus, an essential embryonic blood vessel between the pulmonary artery and the descending aorta, constricts after birth or hatching and eventually closes to terminate embryonic circulation. Chicken embryos have two long ductus arteriosi, which anatomically differ from mammal ductus arteriosus. Each long ductus arteriosus is divided into two parts: the pulmonary artery-sided and descending aorta-sided ductus arteriosi. Although the pulmonary artery-sided and descending aorta-sided ductus arteriosi have distinct functional characteristics, such as oxygen responsiveness, the difference in their transcriptional profiles has not been investigated. We performed a DNA microarray analysis (GSE 120116 at NCBI GEO) with pooled tissues from the chicken pulmonary artery-sided ductus arteriosus, descending aorta-sided ductus arteriosus, and aorta at the internal pipping stage. Although several known ductus arteriosus-dominant genes such as tfap2b were highly expressed in the pulmonary artery-sided ductus arteriosus, we newly found genes that were dominantly expressed in the chicken pulmonary artery-sided ductus arteriosus. Interestingly, cluster analysis showed that the expression pattern of the pulmonary artery-sided ductus arteriosus was closer to that of the descending aorta-sided ductus arteriosus than that of the aorta, whereas the morphology of the descending aorta-sided ductus arteriosus was closer to that of the aorta than that of the pulmonary artery-sided ductus arteriosus. Subsequent pathway analysis with DAVID bioinformatics resources revealed that the pulmonary artery-sided ductus arteriosus showed enhanced expression of the genes involved in melanogenesis and tyrosine metabolism compared with the descending aorta-sided ductus arteriosus, suggesting that tyrosinase and the related genes play an important role in the proper differentiation of neural crest-derived cells during vascular remodeling in the ductus arteriosus. In conclusion, the transcription profiles of the chicken ductus arteriosus provide new insights for investigating the mechanism of ductus arteriosus closure.

Placental formation in early pregnancy: how is the centre of the placenta made?

Correct development of the placenta is critical to establishing pregnancy and inadequate placentation leads to implantation failure and miscarriage, as well as later gestation pregnancy disorders. Much attention has been focused on the placental trophoblasts and it is clear that the trophoblast lineages arise from the trophectoderm of the blastocyst. In contrast, the cells of the placental mesenchyme are thought to arise from the inner cell mass, but the details of this process are limited. Due to ethical constraints and the inaccessibility of very early implantation tissues, our knowledge of early placentation has been largely based on historical histological sections. More recently, stem cell technologies have begun to shed important new light on the origins of the placental mesenchymal lineages. This review aims to amalgamate the older and more modern literature regarding the origins of the non-trophoblast lineages of the human placenta. We highlight ways in which rapidly developing stem cell technologies may shed new light on these crucial peri-implantation events. Relevant articles were identified using the PubMed database and Google Scholar search engines. A pearl growing method was used to expand the scope of papers relevant to the cell differentiation events of non-trophoblast placental lineages. At the start of pregnancy, cells of the extraembyronic mesoderm migrate to underlie the primitive trophoblast layers forming the first placental villi. The mesenchymal cells in the villus core most likely originate from the hypoblast of the embryo, but whether cells from the epiblast also contribute is yet to be determined. This is important because, following the formation of the villus core, vasculogenesis and haematopoiesis take place in the nascent placenta before it is connected to the embryonic circulation, making it likely that haematopoietic foci, placental macrophages, endothelial cells and vascular smooth muscle cells all arise in the placenta de novo. Evidence from the stem cell field indicates that these cells could directly differentiate from the extraembryonic mesoderm. However, the lineage hierarchy involved in cell fate decisions has not been well-established. Mesodermal progenitors capable of differentiating into both vascular and haematopoietic lineages can be derived from human embryonic stem cells, but the identification of such stem cells in the placenta is lacking. Future work profiling rare progenitor populations in early placentae will aid our understanding of early placentation. Understanding the origins of the cell lineages of the normal placenta will help us understand why so many pregnancies fail and address mechanisms that may salvage some of these losses.

Systematization, Description and Territory of The Middle and Rostral Cerebral Arteries in Broad-Snouted Caimans (Caiman latirostris)

Background: Information on the arterial vascularization of the broad-snouted caimans’ brain (Caiman latirostris), a specie from the Brazilian fauna, is inexistent and the literature consulted yielded only one description about the development of embryonic circulation and post-birth cerebrovascular alterations in the brain of three caymans. The objective of this study was to describe and systematize the cerebral arterial vascularization of this reptile, by providing a standard irrigation model with its major variations, in addition to determining its territorial vascular areas. Materials, Methods & Results: Thirty heads of male broad-snouted caimans (Caiman latirostris), with the neck segment, were used in the study. Additionally, two whole female and male specimens, with approximately 3 years of age and weighing between 8 to 10 Kg were also used. The animals were sacrificed according to routine slaughterhouse norms. The heads were sectioned at the third cervical vertebra level and the azygos carotid artery was cannulated. The arterial system was washed with 0.9% saline solution and filled with red-colored latex. The pieces were fixed in formaldehyde and the encephalon removed through a window made in the cranial vault, which was followed by removal of the dura mater and dissection of the arteries. The internal carotid arteries displayed an intercarotic anastomosis in the samples examined, continuing rostrally in 100% of the cases, exiting from the skull as the internal ophthalmic artery. The internal carotid arteries divided into a rostral branch and a short caudal branch at the hypophysis level, which continued naturally as the caudal cerebral artery. The rostral branch from both right and left antimeres was shown as a single branch in 86.7% and as double branch in 13.3% of the samples, and it formed the middle cerebral artery network. The rostral cerebral artery represented the terminal branch of the internal carotid artery’s rostral branch, and an osculum anastomosis between its terminal end and its contralateral homologous artery occurred, forming the rostral communicating artery and thus closing the cerebral artery circle rostrally. Discussion: To support the results of the discussion, we used information available on the encephalic vascularization of turtles, lizards, iguanas and crocodiles, due to the absence of important publications about Caiman latirostris. One single source of blood supply, solely originated from the left aortic arch, was observed in the two whole specimens. This source was called the azygos carotid artery. Such denomination was given because these species did not show a typical common carotid artery, as the ventral part of the neck, up to the mandible, was vascularized by branches from the subclavian arteries. Therefore, we named this single and unique source as the azygos carotid artery. The cerebral arterial circle was rostrally closed by an anastomosis between the terminal ends of the rostral cerebral artery. Consequently, the blood supply to the brain was made exclusively by the carotid system. The main differences of the cerebral vascularization observed in caymans were: a network formation of the middle cerebral artery.

Surface temperatures of albatross eggs and nests

ABSTRACTKnowledge of thermal traits is essential for understanding and modelling physiological responses to environmental change. Egg temperatures are poorly studied in most tubenose species. We employed a contactless infrared thermometer to measure egg and nest surface temperatures throughout the incubation period for four albatross species at Bird Island, South Georgia. The observed mean warm-side temperature of 33.4°C for Wandering Albatross (Diomedea exulans) was similar to measurements obtained from this species using dummy eggs elsewhere. Observed mean warm-side temperatures for Black-browed Albatross (Thalassarche melanophris), Grey-headed Albatross (Thalassarche chrysostoma), and Light-mantled Albatross (Phoebetria palpebrata), reported here for the first time, were 30.7–31.5°C, which is lower than the egg temperatures reported for most Procellariiformes. Temperature gradients across viable eggs declined by up to 9°C during incubation, reflecting increased embryonic circulation and metabolic heat production. This suggests that bioenergetic models should not assume constant egg temperatures during embryo development. Non-viable (addled) eggs could be identified by large temperature gradients in late incubation, indicating that infrared thermometry can be used to determine whether the embryo has died or the egg is infertile in monitoring and managed breeding (e.g. translocation) programmes. Egg temperatures were correlated with ground temperatures, indicating that incubated eggs are vulnerable to environmental variability.