Isl Identifies the Extraembryonic Mesodermal/Allantois Progenitors and is Required for Placenta Morphogenesis and Vasculature Formation.

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.

On-field Gross Morphology Evaluation of Dromedary Camel (Camelus dromedarius) Fetal Membranes.

The dromedary camel (Camelus dromedarius) fetal membranes, commonly referred to as "the placenta", are epitheliochorial, diffuse, and microcotyledonary, similarly to the mare's placenta. The evaluation of the placenta is an essential component of the neonatal evaluation in the equine species. However, post-partum or post-abortion placental assessment in dromedary camels is unfortunately too frequently neglected and, to the best of the authors' knowledge, the dromedary camel species lacks a comprehensive description of the normal placenta's gross morphology. In order to facilitate its on-field evaluation, the current study describes the macroscopic features of the placenta of the dromedary camel after full-term pregnancy and spontaneous parturition.

Origin and flow-mediated remodeling of the murine and human extraembryonic circulation systems.

The transduction of mechanical stimuli produced by blood flow is an important regulator of vascular development. The vitelline and umbilico-placental circulations are extraembryonic vascular systems that are required for proper embryonic development in mammalian embryos. The morphogenesis of the extraembryonic vasculature and the cardiovascular system of the embryo are hemodynamically and molecularly connected. Here we provide an overview of the establishment of the murine and human vitelline and umbilico-placental vascular systems and how blood flow influences various steps in their development. A deeper comprehension of extraembryonic vessel development may aid the establishment of stem-cell based embryo models and provide novel insights to understanding pregnancy complications related to the umbilical cord and placenta.

CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development.

Loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT), heterozygous (CDX2-Het), and homozygous null CDX2 (CDX2-KO) genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to single-nucleus RNA and ATAC sequencing, we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile, suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo, we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo.

Intra-allantoic injection of calcium promotes hatching of chick embryos grown in shell-less culture.

The hatch rate of chick embryos cultured outside of the eggshell with 350 mg calcium l-lactate hydrate (CaL) and 3.5 mL water is fourfold greater in cultures in which the chorioallantoic membrane (CAM) surrounds the egg contents by incubation day 17.5 (E17.5) an event which occurs in ovo by E13. It was first investigated whether decreasing the volume of water added with 350 mg CaL would promote CAM expansion due to the smaller volume to enclose. When 350 mg CaL was present, the CAM did not surround the egg contents by E13. By E17.5, the CAM surrounded the egg contents in 53%-74% of cultures; however, CAM expansion was not significantly different when 0, 1, 2, or 3.5 mL water was present. The hatch rate with 2 or 3.5 mL water was greater than 50% but was not improved with less water. Second, it was investigated whether CaL or water inhibits CAM expansion. In the absence of CaL, the CAM surrounded the egg contents in up to two-thirds of cultures by E13, whether 2 mL water was present or not. Thus CaL, but not water, inhibits expansion of the CAM by E13, even though CaL promotes hatching. Finally, it was investigated whether injection of aqueous CaL into the allantoic fluid, in conjunction with not adding CaL to culture hammocks, would promote CAM expansion. Allantoic injection of CaL starting at E13 did not promote CAM expansion at E17.5 but resulted in hatch rates of approximately 30%. Allantoic injection is a novel route for supplementation of calcium in cultured chick embryos.

CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development.

Proper regulation of gene dosage is critical for the development of the early embryo and the extraembryonic tissues that support it. Specifically, loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. In this study, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. We generate an allelic series for CDX2 in human induced pluripotent stem cells (hiPSCs) consisting of WT, heterozygous, and homozygous null CDX2 genotypes, differentiate these cells in a 2D gastruloid model, and subject these cells to multiomic single nucleus RNA and ATAC sequencing. We identify several genes that CDX2 dose-dependently regulate cytoskeletal integrity and adhesiveness in the extraembryonic mesoderm population, including regulators of the VEGF, canonical WNT, and non-canonical WNT signaling pathways. Despite these dose-dependent gene expression patterns, snATAC-seq reveals that heterozygous CDX2 expression is capable of inducing a WT-like chromatin accessibility profile, suggesting accessibility is not sufficient to drive gene expression when the CDX2 dosage is reduced. Finally, because the loss of CDX2 or TBXT phenocopy one another in vivo, we compare differentially expressed genes in our CDX2 knock-out model to those from TBXT knock-out hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability, including ANK3 and ANGPT1. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and suggest these genes may underlie the defects in vascular development and allantoic elongation seen in the absence or reduction of CDX2 in vivo.

[Exploring together with Françoise Dieterlen the origin of hematopoietic stem cells]. / Plongée avec Françoise Dieterlen dans l'origine des cellules souches hématopoïétiques.

This article summarizes Françoise Dieterlen's major scientific discoveries about the hematopoietic and endothelial systems during her 40 years' career. Her most remarkable achievements include notably the demonstration of an intraembryonic source of hematopoietic stem cells, the characterization of the polarization of the aorta, the identification of a hemogenic endothelium as well as that of the allantois as an organ of hematopoietic amplification in the mouse embryo, and the demonstration of the existence of a hemogenic endothelium capable of generating hematopoietic stem cells in the bone marrow of the chicken and mouse embryo. While this last discovery was not made directly by Françoise Dieterlen, it was inspired by the many conversations I have had with her and the lessons she has taught me throughout my career. Her rich career will forever shape the field of hematopoietic development, in which she will remain a guiding figure.

Title: Plongée avec Françoise Dieterlen dans l'origine des cellules souches hématopoïétiques. Abstract: Cet article récapitule les principales découvertes scientifiques réalisées par Françoise Dieterlen sur le système hématopoïétique et endothélial au cours de sa carrière qui s'est déroulée sur plus de 40 années. Ses contributions, toutes majeures, portent notamment sur la démonstration d'une source intra-embryonnaire de cellules souches hématopoïetiques impliquant la polarisation de l'aorte et la formation d'un endothélium homogénique, la mise en évidence de l'allantoïde comme organe d'amplification hématopoïétique chez l'embryon de souris et la démonstration de l'existence d'un endothélium hémogénique capable de générer des cellules souches hématopoïétiques dans la moelle osseuse de l'embryon de poulet et de souris. Cette dernière découverte, bien que n'ayant pas été réalisée directement par Françoise Dieterlen, a été inspirée par les nombreuses discussions que j'ai pu avoir avec elle et les enseignements qu'elle m'a prodigués au début de ma carrière. Les avancées remarquables accomplies par Françoise Dieterlen dans le champ du développement hématopoïétique sont unanimement reconnues par tous les spécialistes pour qui elle reste à jamais l'une des fondatrices de ce domaine de recherche.

Dynamic 3D morphology of chick embryos and allantois depicted nondestructively by 3.0T clinical magnetic resonance imaging.

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.

Evaluation of the Effect of Photodynamic Therapy on CAM-Grown Sarcomas.

Resection margin adequacy plays a critical role in the local control of sarcomas. Fluorescence-guided surgery has increased complete resection rates and local recurrence-free survival in several oncological disciplines. The purpose of this study was to determine whether sarcomas exhibit sufficient tumor fluorescence (photodynamic diagnosis (PDD)) after administration of 5-aminolevulinic acid (5-ALA) and whether photodynamic therapy (PDT) has an impact on tumor vitality in vivo. Sixteen primary cell cultures were derived from patient samples of 12 different sarcoma subtypes and transplanted onto the chorio-allantoic membrane (CAM) of chick embryos to generate 3-dimensional cell-derived xenografts (CDXs). After treatment with 5-ALA, the CDXs were incubated for another 4 h. Subsequently accumulated protoporphyrin IX (PPIX) was excited by blue light and the intensity of tumor fluorescence was analyzed. A subset of CDXs was exposed to red light and morphological changes of both CAMs and tumors were documented. Twenty-four hours after PDT, the tumors were excised and examined histologically. High rates of cell-derived engraftments on the CAM were achieved in all sarcoma subtypes and an intense PPIX fluorescence was observed. PDT of CDXs resulted in a disruption of tumor-feeding vessels and 52.4% of CDXs presented as regressive after PDT treatment, whereas control CDXs remained vital in all cases. Therefore, 5-ALA mediated PDD and PDT appear to be promising tools in defining sarcoma resection margins (PDD) and adjuvant treatment of the tumor bed (PDT).

SLC20a1/PiT-1 is required for chorioallantoic placental morphogenesis.

The placenta mediates the transport of nutrients, such as inorganic phosphate (Pi), between the maternal and fetal circulatory systems. The placenta itself also requires high levels of nutrient uptake as it develops to provide critical support for fetal development. This study aimed to determine placental Pi transport mechanisms using in vitro and in vivo models. We observed that Pi (P33) uptake in BeWo cells is sodium dependent and that SLC20A1/Slc20a1 is the most highly expressed placental sodium-dependent transporter in mouse (microarray), human cell line (RT-PCR) and term placenta (RNA-seq), supporting that normal growth and maintenance of the mouse and human placenta requires SLC20A1/Slc20a1. Slc20a1 wild-type (Slc20a1+/+) and knockout (Slc20a1-/-) mice were produced through timed intercrosses and displayed yolk sac angiogenesis failure as expected at E10.5. E9.5 tissues were analyzed to test whether placental morphogenesis requires Slc20a1. At E9.5, the developing placenta was reduced in size in Slc20a1-/-. Multiple structural abnormalities were also observed in the Slc20a1-/-chorioallantois. We determined that monocarboxylate transporter 1 protein (MCT1+) cells were reduced in developing Slc20a1-/-placenta, confirming that Slc20a1 loss reduced trophoblast syncytiotrophoblast 1 (SynT-I) coverage. Next, we examined the cell type-specific Slc20a1 expression and SynT molecular pathways in silico and identified Notch/Wnt as a pathway of interest that regulates trophoblast differentiation. We further observed that specific trophoblast lineages express Notch/Wnt genes that associate with endothelial cell tip-and-stalk cell markers. In conclusion, our findings support that Slc20a1 mediates the symport of Pi into SynT cells, providing critical support for their differentiation and angiogenic mimicry function at the developing maternal-fetal interface.

Mesothelial fusion mediates chorioallantoic membrane formation.

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'.

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

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

The Allantois and Urachus: Histological Study Using Human Embryo and Fetuses.

Relatively little is known about allantois and urachal development in early humans.Serial sagittal histological sections from eight human embryos and fetuses were examined to determine allantois development.At gestational age 6-7 weeks, the primitive allantois consists of an enlarged tube located between the umbilical cord and abdominal cavity, whereas the urachus is not yet developed. At 8 weeks, the allantois gradually withdraws from the distal to the proximal end of the umbilical cord, and both the proximal allantois and the rectum (hindgut) start to develop into the cloaca. At 10 weeks, the allantois was located mostly in the abdominal cavity.The urachus forms from the distal end of the allantois and develops into a closed fibrous cord between the base of the urinary bladder and the umbilicus. The urogenital sinus forms from the proximal end of the allantois.

Peripherally administered persistent organic pollutants distribute to the brain of developing chicken embryo in concentrations relevant for human exposure.

Persistent organic pollutants (POPs) can reach the fetal brain and contribute to developmental neurotoxicity. To explore the distribution of POPs to the fetal brain, we exposed chicken embryos to a POP mixture, containing 29 different compounds with concentrations based on blood levels measured in the Scandinavian human population. The mixture was injected into the allantois at embryonic day 13 (E13), aiming at a theoretical concentration of 10 times human blood levels. POPs concentrations in the brain were measured at 0.5, 1, 2, 4, 6, 24, 48, and 72 h after administration. Twenty-seven of the individual compounds were detected during at least one of the time-points analyzed. Generally, the concentrations of most of the measured compounds were within the order of magnitude of those reported in human brain samples. Differences in the speed of distribution to the brain were observed between the per- and polyfluoroalkyl substances (PFASs), which have protein binding potential, and the lipophilic polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and brominated flame retardants (BFRs). Based on pharmacokinetic modeling, PFASs were best described by a one compartment model. PFASs displayed relatively slow elimination (Kel) and persisted at high levels in the brain. Lipophilic OCPs and PCBs could be fitted to a 2-compartment model. These showed high levels in the brain relative to the dose administrated as calculated by area under the curve (AUC)/Dose. Altogether, our study showed that chicken is a suitable model to explore the distribution of POPs into the developing brain at concentrations which are relevant for humans.

Experimental Models to Study Skin Wound Healing with a Focus on Angiogenesis.

A large number of models are now available for the investigation of skin wound healing. These can be used to study the processes that take place in a phase-specific manner under both physiological and pathological conditions. Most models focus on wound closure, which is a crucial parameter for wound healing. However, vascular supply plays an equally important role and corresponding models for selective or parallel investigation of microcirculation regeneration and angiogenesis are also described. In this review article, we therefore focus on the different levels of investigation of skin wound healing (in vivo to in virtuo) and the investigation of angiogenesis and its parameters.

Phylogeny and evolutionary history of the amniote egg.

We review morphological features of the amniote egg and embryos in a comparative phylogenetic framework, including all major clades of extant vertebrates. We discuss 40 characters that are relevant for an analysis of the evolutionary history of the vertebrate egg. Special attention is given to the morphology of the cellular yolk sac, the eggshell, and extraembryonic membranes. Many features that are typically assigned to amniotes, such as a large yolk sac, delayed egg deposition, and terrestrial reproduction have evolved independently and convergently in numerous clades of vertebrates. We use phylogenetic character mapping and ancestral character state reconstruction as tools to recognize sequence, order, and patterns of morphological evolution and deduce a hypothesis of the evolutionary history of the amniote egg. Besides amnion and chorioallantois, amniotes ancestrally possess copulatory organs (secondarily reduced in most birds), internal fertilization, and delayed deposition of eggs that contain an embryo in the primitive streak or early somite stage. Except for the amnion, chorioallantois, and amniote type of eggshell, these features evolved convergently in almost all major clades of aquatic vertebrates possibly in response to selective factors such as egg predation, hostile environmental conditions for egg development, or to adjust hatching of young to favorable season. A functionally important feature of the amnion membrane is its myogenic contractility that moves the (early) embryo and prevents adhering of the growing embryo to extraembryonic materials. This function of the amnion membrane and the liquid-filled amnion cavity may have evolved under the requirements of delayed deposition of eggs that contain developing embryos. The chorioallantois is a temporary embryonic exchange organ that supports embryonic development. A possible evolutionary scenario is that the amniote egg presents an exaptation that paved the evolutionary pathway for reproduction on land. As shown by numerous examples from anamniotes, reproduction on land has occurred multiple times among vertebrates-the amniote egg presenting one "solution" that enabled the conquest of land for reproduction.

Analysis of Placental Arteriovenous Formation Reveals New Insights Into Embryos With Congenital Heart Defects.

The placental vasculature provides the developing embryo with a circulation to deliver nutrients and dispose of waste products. However, in the mouse, the vascular components of the chorio-allantoic placenta have been largely unexplored due to a lack of well-validated molecular markers. This is required to study how these blood vessels form in development and how they are impacted by embryonic or maternal defects. Here, we employed marker analysis to characterize the arterial/arteriole and venous/venule endothelial cells (ECs) during normal mouse placental development. We reveal that placental ECs are potentially unique compared with their embryonic counterparts. We assessed embryonic markers of arterial ECs, venous ECs, and their capillary counterparts-arteriole and venule ECs. Major findings were that the arterial tree exclusively expressed Dll4, and venous vascular tree could be distinguished from the arterial tree by Endomucin (EMCN) expression levels. The relationship between the placenta and developing heart is particularly interesting. These two organs form at the same stages of embryogenesis and are well known to affect each other's growth trajectories. However, although there are many mouse models of heart defects, these are not routinely assessed for placental defects. Using these new placental vascular markers, we reveal that mouse embryos from one model of heart defects, caused by maternal iron deficiency, also have defects in the formation of the placental arterial, but not the venous, vascular tree. Defects to the embryonic cardiovascular system can therefore have a significant impact on blood flow delivery and expansion of the placental arterial tree.

Contact-Triggered Lipofection from Multilayer Films Designed as Surfaces for in Situ Transfection Strategies in Tissue Engineering.

Biomaterials, which release active compounds after implantation, are an essential tool for targeted regenerative medicine. In this study, thin multilayer films loaded with lipid/DNA complexes (lipoplexes) were designed as surface coatings for in situ transfection applicable in tissue engineering and regenerative medicine. The film production and embedding of lipoplexes were based on the layer-by-layer (LbL) deposition technique. Hyaluronic acid (HA) and chitosan (CHI) were used as the polyelectrolyte components. The embedded plasmid DNA was complexed using a new designed cationic lipid formulation, namely, OH4/DOPE 1/1, the advantageous characteristics of which have been proven already. Three different methods were tested regarding its efficiency of lipid and DNA deposition. Therefore, several surface specific analytics were used to characterize the LbL formation, the lipid DNA embedding, and the surface characteristics of the multilayer films, such as fluorescence microscopy, surface plasmon resonance spectroscopy, ellipsometry, zeta potential measurements, atomic force microscopy, and scanning electron microscopy. Interaction studies were conducted for optimized lipoplex-loaded polyelectrolyte multilayers (PEMs) that showed an efficient attachment of C2C12 cells on the surface. Furthermore, no acute toxic effects were found in cell culture studies, demonstrating biocompatibility. Cell culture experiments with C2C12 cells, a cell line which is hard to transfect, demonstrated efficient transfection of the reporter gene encoding for green fluorescent protein. In vivo experiments using the chicken embryo chorion allantois membrane animal replacement model showed efficient gene-transferring rates in living complex tissues, although the DNA-loaded films were stored over 6 days under wet and dried conditions. Based on these findings, it can be concluded that OH4/DOPE 1/1 lipoplex-loaded PEMs composed of HA and CHI can be an efficient tool for in situ transfection in regenerative medicine.

The mouse fetal-placental arterial connection: A paradigm involving the primitive streak and visceral endoderm with implications for human development.

In Placentalia, the fetus depends upon an organized vascular connection with its mother for survival and development. Yet, this connection was, until recently, obscure. Here, we summarize how two unrelated tissues, the primitive streak, or body axis, and extraembryonic visceral endoderm collaborate to create and organize the fetal-placental arterial connection in the mouse gastrula. The primitive streak reaches into the extraembryonic space, where it marks the site of arterial union and creates a progenitor cell pool. Through contact with the streak, associated visceral endoderm undergoes an epithelial-to-mesenchymal transition, contributing extraembryonic mesoderm to the placental arterial vasculature, and to the allantois, or pre-umbilical tissue. In addition, visceral endoderm bifurcates into the allantois where, with the primitive streak, it organizes the nascent umbilical artery and promotes allantoic elongation to the chorion, the site of fetal-maternal exchange. Brachyury mediates streak extension and vascular patterning, while Hedgehog is involved in visceral endoderm's conversion to mesoderm. A unique CASPASE-3-positive cell separates streak- and non-streak-associated domains in visceral endoderm. Based on these new insights at the posterior embryonic-extraembryonic interface, we conclude by asking whether so-called primordial germ cells are truly antecedents to the germ line that segregate within the allantois, or whether they are placental progenitor cells. Incorporating these new working hypotheses into mutational analyses in which the placentae are affected will aid understanding a spectrum of disorders, including orphan diseases, which often include abnormalities of the umbilical cord, yolk sac, and hindgut, whose developmental relationship to each other has, until now, been poorly understood. This article is categorized under: Birth Defects > Associated with Preimplantation and Gastrulation Early Embryonic Development > Gastrulation and Neurulation.

TMED2/emp24 is required in both the chorion and the allantois for placental labyrinth layer development.

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.