Extracellular vesicles from mouse trophoblast cells: Effects on neural progenitor cells and potential participants in the placenta-brain axis†.

The fetal brain of the mouse is thought to be dependent upon the placenta as a source of serotonin (5-hydroxytryptamine; 5-HT) and other factors. How factors reach the developing brain remains uncertain but are postulated here to be part of the cargo carried by placental extracellular vesicles (EV). We have analyzed the protein, catecholamine, and small RNA content of EV from mouse trophoblast stem cells (TSC) and TSC differentiated into parietal trophoblast giant cells (pTGC), potential primary purveyors of 5-HT. Current studies examined how exposure of mouse neural progenitor cells (NPC) to EV from either TSC or pTGC affect their transcriptome profiles. The EV from trophoblast cells contained relatively high amounts of 5-HT, as well as dopamine and norepinephrine, but there were no significant differences between EV derived from pTGC and from TSC. Content of miRNA and small nucleolar (sno)RNA, however, did differ according to EV source, and snoRNA were upregulated in EV from pTGC. The primary inferred targets of the microRNA (miRNA) from both pTGC and TSC were mRNA enriched in the fetal brain. NPC readily internalized EV, leading to changes in their transcriptome profiles. Transcripts regulated were mainly ones enriched in neural tissues. The transcripts in EV-treated NPC that demonstrated a likely complementarity with miRNA in EV were mainly up- rather than downregulated, with functions linked to neuronal processes. Our results are consistent with placenta-derived EV providing direct support for fetal brain development and being an integral part of the placenta-brain axis.

Conditionally mutant animal model for investigating the invasive trophoblast cell lineage.

Placental development involves coordinated expansion and differentiation of trophoblast cell lineages possessing specialized functions. Among the differentiated trophoblast cell lineages are invasive trophoblast cells, which exit the placenta and invade the uterus, where they restructure the uterine parenchyma and facilitate remodeling of uterine spiral arteries. The rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation, and is also amenable to gene manipulation using genome-editing techniques. In this investigation, we generated a conditional rat model targeting the invasive trophoblast cell lineage. Prolactin family 7, subfamily b, member 1 (Prl7b1) is uniquely and abundantly expressed in the rat invasive trophoblast cell lineage. Disruption of Prl7b1 did not adversely affect placental development. We demonstrated that the Prl7b1 locus could be effectively used to drive the expression of Cre recombinase in invasive trophoblast cells. Our rat model represents a new tool for investigating candidate genes contributing to the regulation of invasive trophoblast cells and their roles in trophoblast-guided uterine spiral artery remodeling.

CONDITIONALLY MUTANT ANIMAL MODEL FOR INVESTIGATING THE INVASIVE TROPHOBLAST CELL LINEAGE.

Placental development involves coordinated expansion and differentiation of trophoblast cell lineages possessing specialized functions. Among the differentiated trophoblast cell lineages are invasive trophoblast cells, which exit the placenta and invade into the uterus where they restructure the uterine parenchyma and facilitate remodeling of uterine spiral arteries. The rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation, and is also amenable to gene manipulation using genome editing techniques. In this investigation, we generated a conditional rat model targeting the invasive trophoblast cell lineage. Prolactin family 7, subfamily b, member 1 ( Prl7b1 ) is uniquely and abundantly expressed in the rat invasive trophoblast cell lineage. Disruption of Prl7b1 did not adversely affect placental development. We demonstrated that the Prl7b1 locus could be effectively used to drive the expression of Cre recombinase in invasive trophoblast cells. Our rat model represents a new tool for investigating candidate genes contributing to the regulation of invasive trophoblast cells and their contributions to trophoblast-guided uterine spiral artery remodeling.

Core conserved transcriptional regulatory networks define the invasive trophoblast cell lineage.

The invasive trophoblast cell lineages in rat and human share crucial responsibilities in establishing the uterine-placental interface of the hemochorial placenta. These observations have led to the rat becoming an especially useful animal model for studying hemochorial placentation. However, our understanding of similarities or differences between regulatory mechanisms governing rat and human invasive trophoblast cell populations is limited. In this study, we generated single-nucleus ATAC-seq data from gestation day 15.5 and 19.5 rat uterine-placental interface tissues, and integrated the data with single-cell RNA-seq data generated at the same stages. We determined the chromatin accessibility profiles of invasive trophoblast, natural killer, macrophage, endothelial and smooth muscle cells, and compared invasive trophoblast chromatin accessibility with extravillous trophoblast cell accessibility. In comparing chromatin accessibility profiles between species, we found similarities in patterns of gene regulation and groups of motifs enriched in accessible regions. Finally, we identified a conserved gene regulatory network in invasive trophoblast cells. Our data, findings and analysis will facilitate future studies investigating regulatory mechanisms essential for the invasive trophoblast cell lineage.

Unsupervised contrastive peak caller for ATAC-seq.

The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can access, cut, and ligate adapters to DNA fragments for subsequent amplification and sequencing. These sequenced regions are quantified and tested for enrichment in a process referred to as "peak calling." Most unsupervised peak calling methods are based on simple statistical models and suffer from elevated false positive rates. Newly developed supervised deep learning methods can be successful, but they rely on high quality labeled data for training, which can be difficult to obtain. Moreover, though biological replicates are recognized to be important, there are no established approaches for using replicates in the deep learning tools, and the approaches available for traditional methods either cannot be applied to ATAC-seq, where control samples may be unavailable, or are post hoc and do not capitalize on potentially complex, but reproducible signal in the read enrichment data. Here, we propose a novel peak caller that uses unsupervised contrastive learning to extract shared signals from multiple replicates. Raw coverage data are encoded to obtain low-dimensional embeddings and optimized to minimize a contrastive loss over biological replicates. These embeddings are passed to another contrastive loss for learning and predicting peaks and decoded to denoised data under an autoencoder loss. We compared our replicative contrastive learner (RCL) method with other existing methods on ATAC-seq data, using annotations from ChromHMM genomic labels and transcription factor ChIP-seq as noisy truth. RCL consistently achieved the best performance.

CORE CONSERVED TRANSCRIPTIONAL REGULATORY NETWORKS DEFINE THE INVASIVE TROPHOBLAST CELL LINEAGE.

The invasive trophoblast cell lineage in rat and human share crucial responsibilities in establishing the uterine-placental interface of the hemochorial placenta. These observations have led to the rat becoming an especially useful animal model to study hemochorial placentation. However, our understanding of similarities or differences between regulatory mechanisms governing rat and human invasive trophoblast cell populations is limited. In this study, we generated single-nucleus (sn) ATAC-seq data from gestation day (gd) 15.5 and 19.5 rat uterine-placental interface tissues and integrated the data with single-cell RNA-seq data generated at the same stages. We determined the chromatin accessibility profiles of invasive trophoblast, natural killer, macrophage, endothelial, and smooth muscle cells, and compared invasive trophoblast chromatin accessibility to extravillous trophoblast (EVT) cell accessibility. In comparing chromatin accessibility profiles between species, we found similarities in patterns of gene regulation and groups of motifs enriched in accessible regions. Finally, we identified a conserved gene regulatory network in invasive trophoblast cells. Our data, findings and analysis will facilitate future studies investigating regulatory mechanisms essential for the invasive trophoblast cell lineage.

Unsupervised Contrastive Peak Caller for ATAC-seq.

The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can access, cut, and ligate adapters to DNA fragments for subsequent amplification and sequencing. These sequenced regions are quantified and tested for enrichment in a process referred to as "peak calling". Most unsupervised peak calling methods are based on simple statistical models and suffer from elevated false positive rates. Newly developed supervised deep learning methods can be successful, but they rely on high quality labeled data for training, which can be difficult to obtain. Moreover, though biological replicates are recognized to be important, there are no established approaches for using replicates in the deep learning tools, and the approaches available for traditional methods either cannot be applied to ATAC-seq, where control samples may be unavailable, or are post-hoc and do not capitalize on potentially complex, but reproducible signal in the read enrichment data. Here, we propose a novel peak caller that uses unsupervised contrastive learning to extract shared signals from multiple replicates. Raw coverage data are encoded to obtain low-dimensional embeddings and optimized to minimize a contrastive loss over biological replicates. These embeddings are passed to another contrastive loss for learning and predicting peaks and decoded to denoised data under an autoencoder loss. We compared our Replicative Contrastive Learner (RCL) method with other existing methods on ATAC-seq data, using annotations from ChromHMM genome and transcription factor ChIP-seq as noisy truth. RCL consistently achieved the best performance.

Identifying novel regulators of placental development using time-series transcriptome data.

The placenta serves as a connection between the mother and the fetus during pregnancy, providing the fetus with oxygen, nutrients, and growth hormones. However, the regulatory mechanisms and dynamic gene interaction networks underlying early placental development are understudied. Here, we generated RNA-sequencing data from mouse fetal placenta at embryonic days 7.5, 8.5, and 9.5 to identify genes with timepoint-specific expression, then inferred gene interaction networks to analyze highly connected network modules. We determined that timepoint-specific gene network modules were associated with distinct developmental processes, and with similar expression profiles to specific human placental cell populations. From each module, we identified hub genes and their direct neighboring genes, which were predicted to govern placental functions. We confirmed that four novel candidate regulators identified through our analyses regulate cell migration in the HTR-8/SVneo cell line. Overall, we predicted several novel regulators of placental development expressed in specific placental cell types using network analysis of bulk RNA-sequencing data. Our findings and analysis approaches will be valuable for future studies investigating the transcriptional landscape of early development.

Conservation at the uterine-placental interface.

The hemochorial placentation site is characterized by a dynamic interplay between trophoblast cells and maternal cells. These cells cooperate to establish an interface required for nutrient delivery to promote fetal growth. In the human, trophoblast cells penetrate deep into the uterus. This is not a consistent feature of hemochorial placentation and has hindered the establishment of suitable animal models. The rat represents an intriguing model for investigating hemochorial placentation with deep trophoblast cell invasion. In this study, we used single-cell RNA sequencing to characterize the transcriptome of the invasive trophoblast cell lineage, as well as other cell populations within the rat uterine-placental interface during early (gestation day [gd] 15.5) and late (gd 19.5) stages of intrauterine trophoblast cell invasion. We identified a robust set of transcripts that define invasive trophoblast cells, as well as transcripts that distinguished endothelial, smooth muscle, natural killer, and macrophage cells. Invasive trophoblast, immune, and endothelial cell populations exhibited distinct spatial relationships within the uterine-placental interface. Furthermore, the maturation stage of invasive trophoblast cell development could be determined by assessing gestation stage-dependent changes in transcript expression. Finally, and most importantly, expression of a prominent subset of rat invasive trophoblast cell transcripts is conserved in the invasive extravillous trophoblast cell lineage of the human placenta. These findings provide foundational data to identify and interrogate key conserved regulatory mechanisms essential for the development and function of an important compartment within the hemochorial placentation site that is essential for a healthy pregnancy.

The product of BMP-directed differentiation protocols for human primed pluripotent stem cells is placental trophoblast and not amnion.

The observation that trophoblast (TB) can be generated from primed pluripotent stem cells (PSCs) by exposure to bone morphogenetic protein-4 (BMP4) when FGF2 and ACTIVIN signaling is minimized has recently been challenged with the suggestion that the procedure instead produces amnion. Here, by analyzing transcriptome data from multiple sources, including bulk and single-cell data, we show that the BMP4 procedure generates bona fide TB with similarities to both placental villous TB and TB generated from TB stem cells. The analyses also suggest that the transcriptomic signatures between embryonic amnion and different forms of TB have commonalities. Our data provide justification for the continued use of TB derived from PSCs as a model for investigating placental development.

The impact of bisphenol A on the placenta†.

Bisphenol A (BPA), an endocrine-disrupting chemical, is used to produce a wide variety of plastic and common house-hold items. Therefore, there is potential continual exposure to this compound. BPA exposure has been linked to certain placenta-associated obstetric complications such as preeclampsia, fetal growth restriction, miscarriage, and preterm birth. However, how BPA exposure results in these disorders remains uncertain. Hence, we have herein summarized the reported impacts of BPA on the morphology and metabolic state of the placenta and have proposed mechanisms by which BPA affects placentation, potentially leading to obstetric complications. Current findings suggest that BPA induces pathological changes in the placenta and disrupts its metabolic activities. Based on exposure concentrations, BPA can elicit apoptotic or anti-apoptotic signals in the trophoblasts, and can exaggerate trophoblast fusion while inhibiting trophoblast migration and invasion to affect pregnancy. Accordingly, the usage of BPA products by pregnant women should be minimized and less harmful alternative chemicals should be explored and employed where possible.

Transcriptional regulation of Satb1 in mouse trophoblast stem cells.

SATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages that emerge during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain the trophoblast stem-state. However, the molecular mechanisms that regulate TS-specific Satb expression are not yet known. We identified Satb1 variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated an active state of the promoter. A novel cis-regulatory region with active histone marks was identified ∼21 kbp upstream of the variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreased Satb1 expression in TS cells and chromosome conformation capture analysis confirmed looping of this distant regulatory region into the proximal promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close proximity to SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulated Satb1 expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specific Satb1 expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins.

Mapping cis-regulatory elements in the midgestation mouse placenta.

The placenta is a temporary organ that provides the developing fetus with nutrients, oxygen, and protection in utero. Defects in its development, which may be caused by misregulated gene expression, can lead to devastating outcomes for the mother and fetus. In mouse, placental defects during midgestation commonly lead to embryonic lethality. However, the regulatory mechanisms controlling expression of genes during this period have not been thoroughly investigated. Therefore, we generated and analyzed ChIP-seq data for multiple histone modifications known to mark cis-regulatory regions. We annotated active and poised promoters and enhancers, as well as regions generally associated with repressed gene expression. We found that poised promoters were associated with neuronal development genes, while active promoters were largely associated with housekeeping genes. Active and poised enhancers were associated with placental development genes, though only active enhancers were associated with genes that have placenta-specific expression. Motif analysis within active enhancers identified a large network of transcription factors, including those that have not been previously studied in the placenta and are candidates for future studies. The data generated and genomic regions annotated provide researchers with a foundation for future studies, aimed at understanding how specific genes in the midgestation mouse placenta are regulated.

Single Nucleus RNA Sequence (snRNAseq) Analysis of the Spectrum of Trophoblast Lineages Generated From Human Pluripotent Stem Cells in vitro.

One model to study the emergence of the human trophoblast (TB) has been the exposure of pluripotent stem cells to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB; A83-01 and FGF2; PD173074 (BAP), which generates a mixture of cytotrophoblast, syncytiotrophoblast, and cells with similarities to extravillous trophoblast. Here, H1 human embryonic stem cells were BAP-exposed under two O2 conditions (20% and 5%, respectively). At day 8, single nuclei RNA sequencing was used for transcriptomics analysis, thereby allowing profiling of fragile syncytial structures as well as the more resilient mononucleated cells. Following cluster analysis, two major groupings, one comprised of five (2,4,6,7,8) and the second of three (1,3,5) clusters were evident, all of which displayed recognized TB markers. Of these, two (2 and 3) weakly resembled extravillous trophoblast, two (5 and 6) strongly carried the hallmark transcripts of syncytiotrophoblast, while the remaining five were likely different kinds of mononucleated cytotrophoblast. We suggest that the two populations of nuclei within syncytiotrophoblast may have arisen from fusion events involving two distinct species of precursor cells. The number of differentially expressed genes between O2 conditions varied among the clusters, and the number of genes upregulated in cells cultured under 5% O2 was highest in syncytiotrophoblast cluster 6. In summary, the BAP model reveals an unexpectedly complex picture of trophoblast lineage emergence that will need to be resolved further in time-course studies.

Spatial transcriptomics analysis of uterine gene expression in enhancer of zeste homolog 2 conditional knockout mice†.

Histone proteins undergo various modifications that alter chromatin structure, including addition of methyl groups. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that methylates lysine residue 27, and thereby suppresses gene expression. EZH2 plays integral roles in the uterus and other reproductive organs. We have previously shown that conditional deletion of uterine EZH2 results in increased proliferation of luminal and glandular epithelial cells, and RNA-seq analyses reveal several uterine transcriptomic changes in Ezh2 conditional (c) knockout (KO) mice that can affect estrogen signaling pathways. To pinpoint the origin of such gene expression changes, we used the recently developed spatial transcriptomics (ST) method with the hypotheses that Ezh2cKO mice would predominantly demonstrate changes in epithelial cells and/or ablation of this gene would disrupt normal epithelial/stromal gene expression patterns. Uteri were collected from ovariectomized adult WT and Ezh2cKO mice and analyzed by ST. Asb4, Cxcl14, Dio2, and Igfbp5 were increased, Sult1d1, Mt3, and Lcn2 were reduced in Ezh2cKO uterine epithelium vs. WT epithelium. For Ezh2cKO uterine stroma, differentially expressed key hub genes included Cald1, Fbln1, Myh11, Acta2, and Tagln. Conditional loss of uterine Ezh2 also appears to shift the balance of gene expression profiles in epithelial vs. stromal tissue toward uterine epithelial cell and gland development and proliferation, consistent with uterine gland hyperplasia in these mice. Current findings provide further insight into how EZH2 may selectively affect uterine epithelial and stromal compartments. Additionally, these transcriptome data might provide mechanistic understanding and valuable biomarkers for human endometrial disorders with epigenetic underpinnings.

PlacentaCellEnrich: A tool to characterize gene sets using placenta cell-specific gene enrichment analysis.

Single-cell RNA-Sequencing (scRNA-Seq) has improved our understanding of individual cell types in the human placenta. However, placental scRNA-Seq data is not readily accessible when trying to understand how expression patterns in model systems correspond to those from first trimester human placenta. Therefore, we developed PlacentaCellEnrich, a tool that takes a gene set as input, and then reports if the input set is enriched for genes with placenta cell-specific expression patterns, based on human placenta scRNA-Seq data. The PlacentaCellEnrich tool is freely available at https://placentacellenrich.gdcb.iastate.edu/ for non-profit academic use under the MIT license.

Transcription Factor PLAGL1 Is Associated with Angiogenic Gene Expression in the Placenta.

During pregnancy, the placenta is important for transporting nutrients and waste between the maternal and fetal blood supply, secreting hormones, and serving as a protective barrier. To better understand placental development, we must understand how placental gene expression is regulated. We used RNA-seq data and ChIP-seq data for the enhancer associated mark, H3k27ac, to study gene regulation in the mouse placenta at embryonic day (e) 9.5, when the placenta is developing a complex network of blood vessels. We identified several upregulated transcription factors with enriched binding sites in e9.5-specific enhancers. The most enriched transcription factor, PLAGL1 had a predicted motif in 233 regions that were significantly associated with vasculature development and response to insulin stimulus genes. We then performed several experiments using mouse placenta and a human trophoblast cell line to understand the role of PLAGL1 in placental development. In the mouse placenta, Plagl1 is expressed in endothelial cells of the labyrinth layer and is differentially expressed in placentas from mice with gestational diabetes compared to placentas from control mice in a sex-specific manner. In human trophoblast cells, siRNA knockdown significantly decreased expression of genes associated with placental vasculature development terms. In a tube assay, decreased PLAGL1 expression led to reduced cord formation. These results suggest that Plagl1 regulates overlapping gene networks in placental trophoblast and endothelial cells, and may play a critical role in placental development in normal and complicated pregnancies.

Maternal oxycodone treatment causes pathophysiological changes in the mouse placenta.

INTRODUCTION: Pregnant women are increasingly being prescribed and abusing opioid drugs. As the primary communication organ between mother and conceptus, the placenta may be vulnerable to opioid effects but also holds the key to better understanding how these drugs affect long-term offspring health. We hypothesized that maternal treatment with oxycodone (OXY), the primary opioid at the center of the current crisis, deleteriously affects placental structure and gene expression patterns. METHODS: Female mice were treated daily with 5 mg OXY/kg or saline solution (Control, CTL) for two weeks prior to breeding and until placenta were collected at embryonic age 12.5. A portion of the placenta was fixed for histology, and the remainder was frozen for RNA isolation followed by RNAseq. RESULTS: Maternal OXY treatment reduced parietal trophoblast giant cell (pTGC) area and decreased the maternal blood vessel area within the labyrinth region. OXY exposure affected placental gene expression profiles in a sex dependent manner with female placenta showing up-regulation of many placental enriched genes, including Ceacam11, Ceacam14, Ceacam12, Ceacam13, Prl7b1, Prl2b1, Ctsq, and Tpbpa. In contrast, placenta of OXY exposed males had alteration of many ribosomal proteins. Weighted correlation network analysis revealed that in OXY female vs. CTL female comparison, select modules correlated with OXY-induced placental histological changes. Such associations were lacking in the male OXY vs. CTL male comparison. DISCUSSION: Results suggest OXY exposure alters placental histology. In response to OXY exposure, female placenta responds by upregulating placental enriched transcripts that are either unchanged or downregulated in male placenta. Such changes may shield female offspring from developmental origins of health and disease-based diseases.

Bisphenol A and bisphenol S disruptions of the mouse placenta and potential effects on the placenta-brain axis.

Placental trophoblast cells are potentially at risk from circulating endocrine-disrupting chemicals, such as bisphenol A (BPA). To understand how BPA and the reputedly more inert bisphenol S (BPS) affect the placenta, C57BL6J mouse dams were fed 200 µg/kg body weight BPA or BPS daily for 2 wk and then bred. They continued to receive these chemicals until embryonic day 12.5, whereupon placental samples were collected and compared with unexposed controls. BPA and BPS altered the expression of an identical set of 13 genes. Both exposures led to a decrease in the area occupied by spongiotrophoblast relative to trophoblast giant cells (GCs) within the junctional zone, markedly reduced placental serotonin (5-HT) concentrations, and lowered 5-HT GC immunoreactivity. Concentrations of dopamine and 5-hydroxyindoleacetic acid, the main metabolite of serotonin, were increased. GC dopamine immunoreactivity was increased in BPA- and BPS-exposed placentas. A strong positive correlation between 5-HT+ GCs and reductions in spongiotrophoblast to GC area suggests that this neurotransmitter is essential for maintaining cells within the junctional zone. In contrast, a negative correlation existed between dopamine+ GCs and reductions in spongiotrophoblast to GC area ratio. These outcomes lead to the following conclusions. First, BPS exposure causes almost identical placental effects as BPA. Second, a major target of BPA/BPS is either spongiotrophoblast or GCs within the junctional zone. Third, imbalances in neurotransmitter-positive GCs and an observed decrease in docosahexaenoic acid and estradiol, also occurring in response to BPA/BPS exposure, likely affect the placental-brain axis of the developing mouse fetus.