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.

Foxa1 and Foxa2 are essential for sexual dimorphism in liver cancer.

Hepatocellular carcinoma (HCC) is sexually dimorphic in both rodents and humans, with significantly higher incidence in males, an effect that is dependent on sex hormones. The molecular mechanisms by which estrogens prevent and androgens promote liver cancer remain unclear. Here, we discover that sexually dimorphic HCC is completely reversed in Foxa1- and Foxa2-deficient mice after diethylnitrosamine-induced hepatocarcinogenesis. Coregulation of target genes by Foxa1/a2 and either the estrogen receptor (ERα) or the androgen receptor (AR) was increased during hepatocarcinogenesis in normal female or male mice, respectively, but was lost in Foxa1/2-deficient mice. Thus, both estrogen-dependent resistance to and androgen-mediated facilitation of HCC depend on Foxa1/2. Strikingly, single nucleotide polymorphisms at FOXA2 binding sites reduce binding of both FOXA2 and ERα to their targets in human liver and correlate with HCC development in women. Thus, Foxa factors and their targets are central for the sexual dimorphism of HCC.

Foxa2 and H2A.Z mediate nucleosome depletion during embryonic stem cell differentiation.

Nucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions.

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 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.

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.

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.

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.

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.

Early onset preeclampsia in a model for human placental trophoblast.

We describe a model for early onset preeclampsia (EOPE) that uses induced pluripotent stem cells (iPSCs) generated from umbilical cords of EOPE and control (CTL) pregnancies. These iPSCs were then converted to placental trophoblast (TB) representative of early pregnancy. Marker gene analysis indicated that both sets of cells differentiated at comparable rates. The cells were tested for parameters disturbed in EOPE, including invasive potential. Under 5% O2, CTL TB and EOPE TB lines did not differ, but, under hyperoxia (20% O2), invasiveness of EOPE TB was reduced. RNA sequencing analysis disclosed no consistent differences in expression of individual genes between EOPE TB and CTL TB under 20% O2, but, a weighted correlation network analysis revealed two gene modules (CTL4 and CTL9) that, in CTL TB, were significantly linked to extent of TB invasion. CTL9, which was positively correlated with 20% O2 (P = 0.02) and negatively correlated with invasion (P = 0.03), was enriched for gene ontology terms relating to cell adhesion and migration, angiogenesis, preeclampsia, and stress. Two EOPE TB modules, EOPE1 and EOPE2, also correlated positively and negatively, respectively, with 20% O2 conditions, but only weakly with invasion; they largely contained the same sets of genes present in modules CTL4 and CTL9. Our experiments suggest that, in EOPE, the initial step precipitating disease is a reduced capacity of placental TB to invade caused by a dysregulation of O2 response mechanisms and that EOPE is a syndrome, in which unbalanced expression of various combinations of genes affecting TB invasion provoke disease onset.

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.

TissueEnrich: Tissue-specific gene enrichment analysis.

SUMMARY: RNA-Seq data analysis results in lists of genes that may have a similar function, based on differential gene expression analysis or co-expression network analysis. While tools have been developed to identify biological processes that are enriched in the genes sets, there remains a need for tools that identify enrichment of tissue-specific genes. Therefore, we developed TissueEnrich, a tool that calculates tissue-specific gene enrichment in an input gene set. We demonstrated that TissueEnrich can assign tissue identities to single cell clusters and differentiated embryonic stem cells. AVAILABILITY AND IMPLEMENTATION: The TissueEnrich web application is freely available at http://tissueenrich.gdcb.iastate.edu/. The R package is available through Bioconductor at https://bioconductor.org/packages/TissueEnrich. Both the web application and R package are for non-profit academic use under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

Comparative Analysis of the Transcriptome and Proteome during Mouse Placental Development.

The condition of the placenta is a determinant of the short- and long-term health of the mother and the fetus. However, critical processes occurring in early placental development, such as trophoblast invasion and establishment of placental metabolism, remain poorly understood. To gain a better understanding of the genes involved in regulating these processes, we utilized a multiomics approach, incorporating transcriptome, proteome, and phosphoproteome data generated from mouse placental tissue collected at two critical developmental time points. We found that incorporating information from both the transcriptome and proteome identifies genes associated with time point-specific biological processes, unlike using the proteome alone. We further inferred genes upregulated on the basis of the proteome data but not the transcriptome data at each time point, leading us to identify 27 genes that we predict to have a role in trophoblast migration or placental metabolism. Finally, using the phosphoproteome data set, we discovered novel phosphosites that may play crucial roles in the regulation of placental transcription factors. By generating the largest proteome and phosphoproteome data sets in the developing placenta, and integrating transcriptome analysis, we uncovered novel aspects of placental gene regulation.

The enhancer landscape during early neocortical development reveals patterns of dense regulation and co-option.

Genetic studies have identified a core set of transcription factors and target genes that control the development of the neocortex, the region of the human brain responsible for higher cognition. The specific regulatory interactions between these factors, many key upstream and downstream genes, and the enhancers that mediate all these interactions remain mostly uncharacterized. We perform p300 ChIP-seq to identify over 6,600 candidate enhancers active in the dorsal cerebral wall of embryonic day 14.5 (E14.5) mice. Over 95% of the peaks we measure are conserved to human. Eight of ten (80%) candidates tested using mouse transgenesis drive activity in restricted laminar patterns within the neocortex. GREAT based computational analysis reveals highly significant correlation with genes expressed at E14.5 in key areas for neocortex development, and allows the grouping of enhancers by known biological functions and pathways for further studies. We find that multiple genes are flanked by dozens of candidate enhancers each, including well-known key neocortical genes as well as suspected and novel genes. Nearly a quarter of our candidate enhancers are conserved well beyond mammals. Human and zebrafish regions orthologous to our candidate enhancers are shown to most often function in other aspects of central nervous system development. Finally, we find strong evidence that specific interspersed repeat families have contributed potentially key developmental enhancers via co-option. Our analysis expands the methodologies available for extracting the richness of information found in genome-wide functional maps.

Automated discovery of tissue-targeting enhancers and transcription factors from binding motif and gene function data.

Identifying enhancers regulating gene expression remains an important and challenging task. While recent sequencing-based methods provide epigenomic characteristics that correlate well with enhancer activity, it remains onerous to comprehensively identify all enhancers across development. Here we introduce a computational framework to identify tissue-specific enhancers evolving under purifying selection. First, we incorporate high-confidence binding site predictions with target gene functional enrichment analysis to identify transcription factors (TFs) likely functioning in a particular context. We then search the genome for clusters of binding sites for these TFs, overcoming previous constraints associated with biased manual curation of TFs or enhancers. Applying our method to the placenta, we find 33 known and implicate 17 novel TFs in placental function, and discover 2,216 putative placenta enhancers. Using luciferase reporter assays, 31/36 (86%) tested candidates drive activity in placental cells. Our predictions agree well with recent epigenomic data in human and mouse, yet over half our loci, including 7/8 (87%) tested regions, are novel. Finally, we establish that our method is generalizable by applying it to 5 additional tissues: heart, pancreas, blood vessel, bone marrow, and liver.

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.

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.

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.