Chorionic villus-derived mesenchymal stem cells induce E3 ligase TRIM72 expression and regulate cell behaviors through ubiquitination of p53 in trophoblasts.

Preeclampsia is a significant contributor for maternal or fetal morbidity and mortality, which is characterized by reduced invasion capacity of trophoblasts and is regulated by extracellular matrix (ECM). It is still under investigation whether chorionic villus-derived mesenchymal stem cells (CVMSC) could affect the functionality of trophoblasts. In this study, CVMSC-derived exosomes were isolated; their effect on trophoblasts was investigated based on the CCK8 assay, migration assay, and apoptosis detection. And the underlying mechanism of this effect was investigated using mRNA sequencing, western blot, co-immunoprecipitation, luciferase report assay, and ubiquitination assay. The results show that CVMSC-derived exosomes promote migration and proliferation of trophoblasts, and also reduce cell apoptosis. mRNA sequencing confirmed that after treatment of CVMSC-derived exosomes, Tripartite Motif Containing 72 (TRIM72) expression was upregulated and Tumor Protein P53 (P53) expression was downregulated, both significantly in trophoblasts. Subsequent study confirms that TRM72 can directly interact with P53 and promote P53 ubiquitination and proteasomal degradation, reducing apoptosis rate and elevating proliferation and migration in trophoblasts. Our study confirms that CVMSC-derived exosomes promote trophoblast migration and proliferation by upregulating TRIM72 expression, and subsequently advance P53 ubiquitination and proteasomal degradation.

Placental Villous Explant Culture 2.0: Flow Culture Allows Studies Closer to the In Vivo Situation.

During pregnancy, freely floating placental villi are adapted to fluid shear stress due to placental perfusion with maternal plasma and blood. In vitro culture of placental villous explants is widely performed under static conditions, hoping the conditions may represent the in utero environment. However, static placental villous explant culture dramatically differs from the in vivo situation. Thus, we established a flow culture system for placental villous explants and compared commonly used static cultured tissue to flow cultured tissue using transmission and scanning electron microscopy, immunohistochemistry, and lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) measurements. The data revealed a better structural and biochemical integrity of flow cultured tissue compared to static cultured tissue. Thus, this new flow system can be used to simulate the blood flow from the mother to the placenta and back in the most native-like in vitro system so far and thus can enable novel study designs.

LINE-1 retrotransposon methylation in chorionic villi of first trimester miscarriages with aneuploidy.

PURPOSE: High frequency of aneuploidy in meiosis and cleavage stage coincides with waves of epigenetic genome reprogramming that may indicate a possible association between epigenetic mechanisms and aneuploidy occurrence. This study aimed to assess the methylation level of the long interspersed repeat element 1 (LINE-1) retrotransposon in chorionic villi of first trimester miscarriages with a normal karyotype and aneuploidy. METHODS: The methylation level was assessed at 19 LINE-1 promoter CpG sites in chorionic villi of 141 miscarriages with trisomy of chromosomes 2, 6, 8-10, 13-15, 16, 18, 20-22, and monosomy X using massive parallel sequencing. RESULTS: The LINE-1 methylation level was elevated statistically significant in chorionic villi of miscarriages with both trisomy (45.2 ± 4.3%) and monosomy X (46.9 ± 4.2%) compared with that in induced abortions (40.0 ± 2.4%) (p < 0.00001). The LINE-1 methylation levels were specific for miscarriages with different aneuploidies and significantly increased in miscarriages with trisomies 8, 14, and 18 and monosomy X (p < 0.05). The LINE-1 methylation level increased with gestational age both for group of miscarriages regardless of karyotype (R = 0.21, p = 0.012) and specifically for miscarriages with trisomy 16 (R = 0.48, p = 0.007). LINE-1 methylation decreased with maternal age in miscarriages with a normal karyotype (R = - 0.31, p = 0.029) and with trisomy 21 (R = - 0.64, p = 0.024) and increased with paternal age for miscarriages with trisomy 16 (R = 0.38, p = 0.048) and monosomy X (R = 0.73, p = 0.003). CONCLUSION: Our results indicate that the pathogenic effects of aneuploidy in human embryogenesis can be supplemented with significant epigenetic changes in the repetitive sequences.

Single nuclei RNA-seq of mouse placental labyrinth development.

The placenta is the interface between mother and fetus in all eutherian species. However, our understanding of this essential organ remains incomplete. A substantial challenge has been the syncytial cells of the placenta, which have made dissociation and independent evaluation of the different cell types of this organ difficult. Here, we address questions concerning the ontogeny, specification, and function of the cell types of a representative hemochorial placenta by performing single nuclei RNA sequencing (snRNA-seq) at multiple stages of mouse embryonic development focusing on the exchange interface, the labyrinth. Timepoints extended from progenitor-driven expansion through terminal differentiation. Analysis by snRNA-seq identified transcript profiles and inferred functions, cell trajectories, signaling interactions, and transcriptional drivers of all but the most highly polyploid cell types of the placenta. These data profile placental development at an unprecedented resolution, provide insights into differentiation and function across time, and provide a resource for future study.

Placental delayed villous maturation is associated with evidence of chronic fetal hypoxia.

Background Normal development of the human placenta, referred to as villous tree maturation, entails formation of the vasculosyncytial membranes. These structures develop by the approximation of syncytiotrophoblasts with the villous capillary endothelium and constitute the most efficient sites of gaseous exchange in the placenta. Defective maturation of the villous tree can lead to deficient vasculosyncytial membranes, implicated in the high incidence of hypoxic complications. Hypoxia, in turn, can stimulate production of erythropoietin, whereby increased fetal plasma or amniotic fluid concentrations of this hormone reflect fetal hypoxemia. The current study was undertaken to determine whether delayed villous maturation is associated with changes in amniotic fluid erythropoietin concentrations. Methods Placental histologic examination was performed using hematoxylin and eosin. Subsequent to histologic assessment of delayed villous maturation, the diagnosis was confirmed with CD-15 immunohistochemistry. The controls (n = 61) were pregnancies without villous maturation abnormalities, and cases (n = 5) were pregnancies with delayed villous maturation. Amniotic fluid erythropoietin concentrations were measured using a specific immunoassay. Results Concentrations of erythropoietin in the amniotic fluid (1) of controls were less than the limit of detection and (2) of cases with delayed villous maturation were significantly higher than those of controls (P-value = 0.048). Conclusion Delayed villous maturation is associated with higher concentrations of amniotic fluid erythropoietin.

Investigating a suitable model for the study of vitamin D mediated regulation of human placental gene expression.

Transfer and metabolism of vitamin D across the human placenta is required for fetal development. However, these fundamental mechanisms are not well understood and model systems are required to help understand them. The BeWo choriocarcinoma cell line is derived from extravillous trophoblast but is used as a model for villous syncytiotrophoblast and the placental barrier. Questions have been raised about the suitability of the BeWo cell line as a model for villous trophoblast. This study compares the expression of amino acid transporters and vitamin D related genes in human term placenta with the BeWo and human embryonic kidney (HEK)293 cell lines. HEK293 cells, as transporting epithelium may be more similar to placenta. Gene expression in term placenta was much more similar to HEK293 than BeWo. This study provides further evidence that the BeWo cell line is not an appropriate model for villous trophoblast and a model that more closely represents the human placenta is now required to investigate the effects of vitamin D on the placenta ex-vivo.

Regulation of human trophoblast syncytialization by histone demethylase LSD1.

A successful pregnancy is critically dependent upon proper placental development and function. During human placentation, villous cytotrophoblast (CTB) progenitors differentiate to form syncytiotrophoblasts (SynTBs), which provide the exchange surface between the mother and fetus and secrete hormones to ensure proper progression of pregnancy. However, epigenetic mechanisms that regulate SynTB differentiation from CTB progenitors are incompletely understood. Here, we show that lysine-specific demethylase 1 (LSD1; also known as KDM1A), a histone demethylase, is essential to this process. LSD1 is expressed both in CTB progenitors and differentiated SynTBs in first-trimester placental villi; accordingly, expression in SynTBs is maintained throughout gestation. Impairment of LSD1 function in trophoblast progenitors inhibits induction of endogenous retrovirally encoded genes SYNCYTIN1/endogenous retrovirus group W member 1, envelope (ERVW1) and SYNCYTIN2/endogenous retrovirus group FRD member 1, envelope (ERVFRD1), encoding fusogenic proteins critical to human trophoblast syncytialization. Loss of LSD1 also impairs induction of chorionic gonadotropin α (CGA) and chorionic gonadotropin ß (CGB) genes, which encode α and ß subunits of human chorionic gonadotrophin (hCG), a hormone essential to modulate maternal physiology during pregnancy. Mechanistic analyses at the endogenous ERVW1, CGA, and CGB loci revealed a regulatory axis in which LSD1 induces demethylation of repressive histone H3 lysine 9 dimethylation (H3K9Me2) and interacts with transcription factor GATA2 to promote RNA polymerase II (RNA-POL-II) recruitment and activate gene transcription. Our study reveals a novel LSD1-GATA2 axis, which regulates human trophoblast syncytialization.

Better therapeutic potential of bone marrow-derived mesenchymal stem cells compared with chorionic villi-derived mesenchymal stem cells in airway injury model.

Aim: To determine the efficiency of mesenchymal stem cells (MSCs) of different sources on airway epithelial cells regeneration and track where and to what extent transplanted MSCs home to injured tissues. Materials & methods: We performed DiO-labeled human bone marrow-derived MSCs (hBMSCs) or human chorionic villi-derived MSCs transplantation studies using naphthalene-induced airway injury animal models. Results: Compared with human chorionic villi-derived MSCs, hBMSCs facilitated airway epithelium regeneration faster and better from day 5 after transplantation; moreover, more transplanted hBMSCs distributed in injured lung tissues at the early stage of postinjury, which was mediated by C-X-C motif chemokine ligand 12. Conclusion: hBMSCs possessed better potential of migration to the damaged lung and promoting the repair of the injured airway epithelium.

Villi development core-related gene expression associated with lung squamous cancer prognosis.

Similarities between embryonic development and tumorigenesis are reflected in biological behavior and gene expression. Although the gene signature during development and the clinical phenotype of different cancers show certain correlation pattern, the correlation between early embryo development and cancer remains largely unexplored. To compare the gene expression profile between development and cancer, our study analyzed the gene expression of chorionic villi samples at different gestational ages (6, 7, 8, 9, 10, 40 weeks) obtained from gene expression omnibus (GEO) datasets using correlation test. Then the villi development-related genes that gradually showed a positive correlation (upregulated) (n = 394) or negative correlation (downregulated) (n = 325) with time were used to construct protein-protein interaction (PPI) networks. Three subnetworks among the gradually upregulated genes and 3 subnetworks among the downregulated genes were identified using the molecular complex detection (MCODE) plugin in Cytoscape software. The most significant GO terms for villi-correlated genes were immune, inflammatory response and cell division. These gene clusters were also dysregulated in lung squamous cell carcinoma (SCC). Moreover the prognostic value of the gene clusters was then analyzed with TCGA lung SCC data, which showed 4 clusters that were associated with prognosis. Our results demonstrate the gene expression similarity between development and lung SCC and identified development-associated gene clusters that could contain prognostic information for lung SCC patients.

Investigating human placentation and pregnancy using first trimester chorionic villi.

Chorionic villus sampling (CVS), routinely used for prenatal diagnosis of cytogenetic disorders, also possesses great potential for the study of placentation. To better understand villus biology, human placentation, and how these relate to pregnancy outcomes, we examined the morphology and transcriptomes of villi obtained via CVS from 10 to 14 weeks of pregnancy and correlated these with pregnancy attributes and clinical outcomes. First, we established a morphological scoring system based on three main villus features: branching, budding and vascularization. We then tested whether morphology scores were predictive of pregnancy attributes and clinical outcomes. Finally, we used RNA sequencing to assess the transcriptional basis of villus morphology and tested the hypothesis that gene expression may predict pregnancy outcomes. We demonstrate that villus morphology varies tremendously between patients, irrespective of gestational age, and that transcriptional differences are highly predictive of villus morphology. We show that pre-eclampsia markers are associated with villi with low morphology scores. Additionally, we identify SVEP1 as a possible biomarker for defining gestational age. Overall, chorionic villi in the first trimester remain one of the few means to correlate placental function with pregnancy outcome and these samples are a valuable and increasingly rare resource.

Antenatal corticosteroid treatment and placental pathology, with a focus on villous maturation.

INTRODUCTION: Mothers at risk of preterm birth are treated with antenatal corticosteroids, which have advantageous effects for prematurely born infants. Accelerated villous maturation in the placenta is also associated with improved perinatal outcome. The primary aim of this study was to examine the association between antenatal corticosteroids and accelerated villous maturation. The secondary aim was to study associations with other placental pathologies. MATERIAL AND METHODS: A retrospective cohort study including 105 women who had (n = 75) or had not (n = 30) been treated with antenatal corticosteroids. The women gave birth between 22+0 and 26+6  weeks of gestation in Stockholm County between 1 April 2004 and 31 March 2007. A pathologist blinded to all clinical data except gestational age examined the placental slides to identify pathology parameters. The outcomes were correlated with antenatal corticosteroid treatment, and confounding factors were adjusted using logistic regression. RESULTS: Accelerated villous maturation was significantly higher in the group treated with corticosteroids (odds ratio 16, 95% CI 2.4-690, p = 0.0005). After adjustment for gestational age and preeclampsia, the difference remained significant (odds ratio 8.9, 95% CI 1.2-389, p = 0.021). No significant associations were found regarding the secondary outcome variables, after adjusting for possible confounders. CONCLUSIONS: Antenatal corticosteroid treatment before preterm birth is associated with accelerated villous maturation. This could be one of the pathways by which corticosteroids are beneficial for preterm infants.

Gene markers of normal villous maturation and their expression in placentas with maturational pathology.

INTRODUCTION: The placenta demonstrates a recognized sequence of histomorphologic maturation throughout pregnancy, and in some cases, shows abnormally advanced (AVM) or delayed (DVM) villous maturation. While AVM and DVM have important clinical implications, it is unknown whether they truly represent a state of accelerated/delayed normal maturation or a state of pathological maldevelopment. The purpose of our study is, therefore, to address this challenge via a genome-wide search for expression markers of normal villous maturation (NM) and the assessment of these genes in cases of maturational pathology. METHODS: A total of 142 placentas, previously evaluated by gene expression microarray, were reviewed histologically and classified as NM, AVM, or DVM. Expression data from healthy NM placentas underwent Pearson correlations with gestational age (GA) and network/pathway analysis to identify candidate gene markers. Candidates were then validated in an independent microarray dataset and used to calculate "molecular GAs" of placentas with maturational pathology. RESULTS: Analysis of NM placentas yielded 17 candidate markers of normal villous maturation, of which 11 were independently validated. Genes with expression increasing across gestation were associated with transcription and metabolism, while those demonstrating decreasing expression were involved in cell cycle and division. Molecular GA was 5.3 weeks older than true GA among AVM placentas (p < 0.001), and 1.1 weeks younger among DVM placentas (p = 0.149). DISCUSSION: We have found evidence of advanced molecular GA in AVM placentas, while molecular alterations in DVM placentas were merely suggestive of delayed maturation. In the future, these findings will need to be validated with additional techniques such as in situ hybridization or immunohistochemistry.

Comparative Analysis of the Proliferative Potential of Human Mesenchymal Stromal Cells from Extraembryonic Organs, Endometrium, and Adipose Tissue.

Proliferative activity of mesenchymal stromal cells isolated from five sources (chorionic villi, Wharton's jelly, amnion, endometrium, and adipose tissue) was compared by flow cytometry and real-time PCR (by the content of mRNA of genes encoding of cell cycle regulators). Mesenchymal stromal cells derived from the endometrium demonstrated maximum stability and high proliferative potential.

Isolation and characterisation of a novel trophoblast side-population from first trimester placentae.

The placenta is responsible for all nutrient and gas exchange between mother and baby during pregnancy. The differentiation of specialised placental epithelial cells called trophoblasts is essential for placental function, but we understand little about how these populations arise. Mouse trophoblast stem cells have allowed us to understand many of the factors that regulate murine trophoblast lineage development, but the human placenta is anatomically very different from the mouse, and it is imperative to isolate a human trophoblast stem cell to understand human placental development. Here we have developed a novel methodology to isolate a Hoechst side-population of trophoblasts from early gestation placentae and compared their transcriptome to differentiated trophoblast populations (cytotrophoblasts and extravillous trophoblasts) using microarray technology. Side-population trophoblasts clustered as a transcriptomically distinct population but were more closely related to cytotrophoblasts than extravillous trophoblasts. Side-population trophoblasts up-regulated a number of genes characteristic of trophectoderm and murine trophoblast stem cells in comparison to cytotrophoblasts or extravillous trophoblasts and could be distinguished from both of these more mature populations by a unique set of 22 up-regulated genes, which were enriched for morphogenesis and organ development and the regulation of growth functions. Cells expressing two of these genes (LAMA2 and COL6A3) were distributed throughout the cytotrophoblast layer at the trophoblast/mesenchymal interface. Comparisons to previously published trophoblast progenitor populations suggest that the side-population trophoblasts isolated in this work are a novel human trophoblast population. Future work will determine whether these cells exhibit functional progenitor/stem cell attributes.

Ontogenic Changes of Villus Growth, Lactase Activity, and Intestinal Glucose Transporters in Preterm and Term Born Calves with or without Prolonged Colostrum Feeding.

Oral glucose supply is important for neonatal calves to stabilize postnatal plasma glucose concentration. The objective of this study was to investigate ontogenic development of small intestinal growth, lactase activity, and glucose transporter in calves (n = 7 per group) that were born either preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery) or spontaneously born and fed colostrum for 4 days (TC). Tissue samples from duodenum and proximal, mid, and distal jejunum were taken to measure villus size and crypt depth, protein concentration of mucosa and brush border membrane vesicles (BBMV), total DNA and RNA concentration of mucosa, mRNA expression and activity of lactase, and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter 2 (GLUT2) in mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and immunochemical localization of GLUT2 in the enterocytes were determined. Villus height in distal jejunum was lower in TC than in T. Crypt depth in all segments was largest and the villus height/crypt depth ratio in jejunum was smallest in TC calves. Concentration of RNA was highest in duodenal mucosa of TC calves, but neither lactase mRNA and activity nor SGLT1 and GLUT2 mRNA and protein expression differed among groups. Localization of GLUT2 in the apical membrane was greater, whereas in the basolateral membrane was lower in TC than in T and PT calves. Our study indicates maturation processes after birth for mucosal growth and trafficking of GLUT2 from the basolateral to the apical membrane. Minor differences of mucosal growth, lactase activity, and intestinal glucose transporters were seen between PT and T calves, pointing at the importance of postnatal maturation and feeding for mucosal growth and GLUT2 trafficking.

Energy status and HIF signalling in chorionic villi show no evidence of hypoxic stress during human early placental development.

Early human placental and embryonic development occurs in a physiologically low oxygen environment supported by histiotrophic secretions from endometrial glands. In this study, we compare the placental metabolomic profile in the first, second and third trimesters to determine whether the energy demands are adequately met in the first trimester. We investigated whether hypoxia-inducible factors, HIF-1α and/or HIF-2α, might regulate transcription during the first trimester. First and second trimester tissue was collected using a chorionic villus sampling-like (CVS) technique. Part of each villus sample was frozen immediately and the remainder cultured under 2 or 21% O2 ± 1 mM H2O2, and ±the p38 MAPK pathway inhibitor, PD169316. Levels of HIF-1α were assessed by western blotting and VEGFA, PlGF and GLUT3 transcripts were quantified by RT-PCR. Term samples were collected from normal elective Caesarean deliveries. There were no significant differences in concentrations of ADP, NAD(+), lactate, and glucose, and in the ATP/ADP ratio, across gestational age. Neither HIF-1α nor HIF-2α could be detected in time-zero CVS samples. However, culture under any condition (2 or 21% O2 ± 1 mM H2O2) increased HIF-1α and HIF-2α. HIF-1α and HIF-2α were additionally detected in specimens retrieved after curettage. HIF-1α stabilization was accompanied by significant increases in VEGFA and GLUT3 and a decrease in PlGF mRNAs. These effects were suppressed by PD169316. In conclusion, our data suggest that first trimester placental tissues are not energetically compromised, and that HIF-1α is unlikely to play an appreciable role in regulating transcriptional activity under steady-state conditions in vivo. However, the pathway may be activated by stress conditions.

A novel regulator of human villous trophoblast fusion: the Krüppel-like factor 6.

Cell-cell fusion is an essential event during life. Throughout human pregnancy, the syncytiotrophoblast (STB) layer of the placenta is formed by continuous fusion of the underlying villous cytotrophoblasts, thus maintaining placental functionality. Defects in this process are associated with pathologies like pre-eclampsia and intrauterine growth restriction. Krüppel-like factor 6 (KLF6) is a transcription factor highly expressed in human and murine placenta. However, KLF6 functions in trophoblast cells remain largely unexplored. The aim of this work was to address the role of KLF6 during STB formation. KLF6 knockdown through small interfering RNA experiments hindered cell-cell fusion revealed by immunofluorescence microscopy in human primary villous cytotrophoblast as well as in the human placental-derived BeWo cell line. Furthermore, KLF6 silencing led to a decrease in the expression of the fusogenic protein Syncytin-1 and the cell cycle regulator p21 CIP1/WAF1: measured by quantitative RT-PCR and western blot assays. On the contrary, transcript levels of genes that encode for proteins involved in STB formation such as Syncytin-1, Syncytin-2, Connexin-43 and Zonula Occludens-1 increased when KLF6 was overexpressed in differentiating villous cytotrophoblasts and in non-fusing placental-derived JEG-3 cells. Interestingly, the expression of two trophoblast biochemical differentiation markers, ßhCG and PSG3, were not reduced after KLF6 silencing in differentiating trophoblast cells. Present results support the notion that KLF6 is a relevant participant in cytotrophoblast fusion.

Angiogenin expression during early human placental development; association with blood vessel formation.

The placenta is a transient organ essential for fetal development. During human placental development, chorionic villi grow in coordination with a large capillary network resulting from both vasculogenesis and angiogenesis. Angiogenin is one of the most potent inducers of neovascularisation in experimental models in vivo. We and others have previously mapped angiogenin expression in the human term placenta. Here, we explored angiogenin involvement in early human placental development. We studied, angiogenin expression by in situ hybridisation and/or by RT-PCR in tissues and primary cultured trophoblastic cells and angiogenin cellular distribution by coimmunolabelling with cell markers: CD31 (PECAM-1), vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGF-R2), Tie-2, von Willebrand factor, CD34, erythropoeitin receptor (Epo-R), alpha-smooth muscle actin, CD45, cytokeratin 7, and Ki-67. Extravillous and villous cytotrophoblasts, isolated and differentiated in vitro, expressed and secreted angiogenin. Angiogenin was detected in villous trophoblastic layers, and structured and nascent fetal vessels. In decidua, it was expressed by glandular epithelial cells, vascular cells and macrophages. The observed pattern of angiogenin expression is compatible with a role in blood vessel formation and in cross-talk between trophoblasts and endothelial cells. In view of angiogenin properties, we suggest that angiogenin may participate in placental vasculogenesis and organogenesis.

Role of IGF2BP3 in trophoblast cell invasion and migration.

The insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) is a member of a highly conserved protein family that is expressed specifically in placenta, testis and various cancers, but is hardly detectable in normal adult tissues. IGF2BP3 has important roles in RNA stabilization and translation, especially during early stages of both human and mouse embryogenesis. Placenta is an indispensable organ in mammalian reproduction that connects developing fetus to the uterine wall, and is responsible for nutrient uptake, waste elimination and gas exchange. Fetus development in the maternal uterine cavity depends on the specialized functional trophoblast. Whether IGF2BP3 plays a role in trophoblast differentiation during placental development has never been examined. The data obtained in this study revealed that IGF2BP3 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells (CTBs) and trophoblast column, but a much lower level of IGF2BP3 was detected in the third trimester placental villi. Furthermore, the expression level of IGF2BP3 in pre-eclamptic (PE) placentas was significantly lower than the gestational age-matched normal placentas. The role of IGF2BP3 in human trophoblast differentiation was shown by in vitro cell invasion and migration assays and an ex vivo explant culture model. Our data support a role of IGF2BP3 in promoting trophoblast invasion and suggest that abnormal expression of IGF2BP3 might be associated with the etiology of PE.

A positive feedback loop involving Gcm1 and Fzd5 directs chorionic branching morphogenesis in the placenta.

Chorioallantoic branching morphogenesis is a key milestone during placental development, creating the large surface area for nutrient and gas exchange, and is therefore critical for the success of term pregnancy. Several Wnt pathway molecules have been shown to regulate placental development. However, it remains largely unknown how Wnt-Frizzled (Fzd) signaling spatiotemporally interacts with other essential regulators, ensuring chorionic branching morphogenesis and angiogenesis during placental development. Employing global and trophoblast-specific Fzd5-null and Gcm1-deficient mouse models, combining trophoblast stem cell lines and tetraploid aggregation assay, we demonstrate here that an amplifying signaling loop between Gcm1 and Fzd5 is essential for normal initiation of branching in the chorionic plate. While Gcm1 upregulates Fzd5 specifically at sites where branching initiates in the basal chorion, this elevated Fzd5 expression via nuclear ß-catenin signaling in turn maintains expression of Gcm1. Moreover, we show that Fzd5-mediated signaling induces the disassociation of cell junctions for branching initiation via downregulating ZO-1, claudin 4, and claudin 7 expressions in trophoblast cells at the base of the chorion. In addition, Fzd5-mediated signaling is also important for upregulation of Vegf expression in chorion trophoblast cells. Finally, we demonstrate that Fzd5-Gcm1 signaling cascade is operative during human trophoblast differentiation. These data indicate that Gcm1 and Fzd5 function in an evolutionary conserved positive feedback loop that regulates trophoblast differentiation and sites of chorionic branching morphogenesis.