Comparative single cell analysis reveals complex patterns of cell type and cell signaling innovations at the fetal-maternal interface.

The decidual-placental interface is one of the most diverse and rapidly evolving tissues in mammals. Its origin as a chimeric fetal-maternal tissue poses a unique evolutionary puzzle. We present single-cell RNA sequencing atlases from the fetal-maternal interfaces of the opossum, a marsupial, the Malagasy common tenrec, an afrotherian with primitive reproductive features, and mouse, guinea pig, and human. Invasive trophoblast shares a common transcriptomic signature across eutherians, which we argue represents a cell type family that radiated following the evolution of hemochorial placentation. We find evidence that the eutherian decidual stromal cell evolved stepwise from a predecidual state retained in Tenrec , followed by a second decidual cell type originating in Boreoeutheria with endocrine characteristics. We reconstruct ligand-receptor signaling to test evolutionary hypotheses at scale. Novel trophoblast and decidual cell types display strong integration into signaling networks compared to other cells. Additionally, we find consistent disambiguation between fetal and maternal signaling. Using phylogenetic analysis, we infer the cell-cell signaling network of the Placental common ancestor, and identify increased rates of signaling evolution in Euarchontoglires. Together, our findings reveal novel cell type identities and cell signaling dynamics at the mammalian fetal-maternal interface.

A common allele increases endometrial Wnt4 expression, with antagonistic implications for pregnancy, reproductive cancers, and endometriosis.

The common human SNP rs3820282 is associated with multiple phenotypes including gestational length and likelihood of endometriosis and cancer, presenting a paradigmatic pleiotropic variant. Deleterious pleiotropic mutations cause the co-occurrence of disorders either within individuals, or across population. When adverse and advantageous effects are combined, pleiotropy can maintain high population frequencies of deleterious alleles. To reveal the causal molecular mechanisms of this pleiotropic SNP, we introduced this substitution into the mouse genome by CRISPR/Cas 9. Previous work showed that rs3820282 introduces a high-affinity estrogen receptor alpha-binding site at the Wnt4 locus. Here, we show that this mutation upregulates Wnt4 transcription in endometrial stroma, following the preovulatory estrogen peak. Effects on uterine transcription include downregulation of epithelial proliferation and induction of progesterone-regulated pro-implantation genes. We propose that these changes increase uterine permissiveness to embryo invasion, whereas they decrease resistance to invasion by cancer and endometriotic foci in other estrogen-responsive tissues.

Experimental and phylogenetic evidence for correlated gene expression evolution in endometrial and skin fibroblasts.

Gene expression change is a dominant mode of evolution. Mutations, however, can affect gene expression in multiple cell types. Therefore, gene expression evolution in one cell type can lead to similar gene expression changes in another cell type. Here, we test this hypothesis by investigating dermal skin fibroblasts (SFs) and uterine endometrial stromal fibroblasts (ESFs). The comparative dataset consists of transcriptomes from cultured SF and ESF of nine mammalian species. We find that evolutionary changes in gene expression in SF and ESF are highly correlated. The experimental dataset derives from a SCID mouse strain selected for slow cancer growth leading to substantial gene expression changes in SFs. We compared the gene expression profiles of SF with that of ESF and found a significant correlation between them. We discuss the implications of these findings for the evolutionary correlation between placental invasiveness and vulnerability to metastatic cancer.

Evolution of higher mesenchymal CD44 expression in the human lineage: A gene linked to cancer malignancy.

CD44 is an extracellular matrix receptor implicated in cancer progression. CD44 increases the invasibility of skin (SF) and endometrial stromal fibroblasts (ESF) by cancer and trophoblast cells. We reasoned that the evolution of CD44 expression can affect both, the fetal-maternal interaction through CD44 in ESF as well as vulnerability to malignant cancer through expression in SF. We studied the evolution of CD44 expression in mammalian SF and ESF and demonstrate that in the human lineage evolved higher CD44 expression. Isoform expression in cattle and human is very similar suggesting that differences in invasibility are not due to the nature of expressed isoforms. We then asked whether the concerted gene expression increase in both cell types is due to shared regulatory mechanisms or due to cell type-specific factors. Reporter gene experiments with cells and cis-regulatory elements from human and cattle show that the difference of CD44 expression is due to cis effects as well as cell type-specific trans effects. These results suggest that the concerted expression increase is likely due to selection acting on both cell types because the evolutionary change in cell type-specific factors requires selection on cell type-specific functions. This scenario implies that the malignancy enhancing effects of elevated CD44 expression in humans likely evolved as a side-effect of positive selection on a yet unidentified other function of CD44. A possible candidate is the anti-fibrotic effect of CD44 but there are no reliable data showing that humans and primates are less fibrotic than other mammals.

Proteotype coevolution and quantitative diversity across 11 mammalian species.

Evolutionary profiling has been largely limited to the nucleotide level. Using consistent proteomic methods, we quantified proteomic and phosphoproteomic layers in fibroblasts from 11 common mammalian species, with transcriptomes as reference. Covariation analysis indicates that transcript and protein expression levels and variabilities across mammals remarkably follow functional role, with extracellular matrix-associated expression being the most variable, demonstrating strong transcriptome-proteome coevolution. The biological variability of gene expression is universal at both interindividual and interspecies scales but to a different extent. RNA metabolic processes particularly show higher interspecies versus interindividual variation. Our results further indicate that while the ubiquitin-proteasome system is strongly conserved in mammals, lysosome-mediated protein degradation exhibits remarkable variation between mammalian lineages. In addition, the phosphosite profiles reveal a phosphorylation coevolution network independent of protein abundance.

Tracing the cis-regulatory changes underlying the endometrial control of placental invasion.

Among eutherian (placental) mammals, placental embedding into the maternal endometrium exhibits great differences, from being deeply invasive (e.g., humans) to noninvasive (e.g., cattle). The degree of invasion of placental trophoblasts is positively correlated with the rate of cancer malignancy. Previously, we have shown that fibroblasts from different species offer different levels of resistance to the invading trophoblasts as well as to cancer cell invasion. Here we present a comparative genomic investigation revealing cis-regulatory elements underlying these interspecies differences in invasibility. We identify transcription factors that regulate proinvasibility and antiinvasibility genes in stromal cells. Using an in vitro invasibility assay combined with CRISPR-Cas9 gene knockout, we found that the transcription factors GATA2 and TFDP1 strongly influence the invasibility of endometrial and skin fibroblasts. This work identifies genomic mechanisms explaining species differences in stromal invasibility, paving the way to therapies targeting stromal characteristics to regulate placental invasion, wound healing, and cancer dissemination.

Molecular Evolution of CatSper in Mammals and Function of Sperm Hyperactivation in Gray Short-Tailed Opossum.

Males have evolved species-specifical sperm morphology and swimming patterns to adapt to different fertilization environments. In eutherians, only a small fraction of the sperm overcome the diverse obstacles in the female reproductive tract and successfully migrate to the fertilizing site. Sperm arriving at the fertilizing site show hyperactivated motility, a unique motility pattern displaying asymmetric beating of sperm flagella with increased amplitude. This motility change is triggered by Ca2+ influx through the sperm-specific ion channel, CatSper. However, the current understanding of the CatSper function and its molecular regulation is limited in eutherians. Here, we report molecular evolution and conservation of the CatSper channel in the genome throughout eutherians and marsupials. Sequence analyses reveal that CatSper proteins are slowly evolved in marsupials. Using an American marsupial, gray short-tailed opossum (Monodelphis domestica), we demonstrate the expression of CatSper in testes and its function in hyperactivation and unpairing of sperm. We demonstrate that a conserved IQ-like motif in CatSperζ is required for CatSperζ interaction with the pH-tuned Ca2+ sensor, EFCAB9, for regulating CatSper activity. Recombinant opossum EFCAB9 can interact with mouse CatSperζ despite high sequence divergence of CatSperζ among CatSper subunits in therians. Our finding suggests that molecular characteristics and functions of CatSper are evolutionarily conserved in gray short-tailed opossum, unraveling the significance of sperm hyperactivation and fertilization in marsupials for the first time.

Evolution of Embryo Implantation Was Enabled by the Origin of Decidual Stromal Cells in Eutherian Mammals.

Mammalian pregnancy evolved in the therian stem lineage, that is, before the common ancestor of marsupials and eutherian (placental) mammals. Ancestral therian pregnancy likely involved a brief phase of attachment between the fetal and maternal tissues followed by parturition-similar to the situation in most marsupials including the opossum. In all eutherians, however, embryo attachment is followed by implantation, allowing for a stable fetal-maternal interface and an extended gestation. Embryo attachment induces an attachment reaction in the uterus that is homologous to an inflammatory response. Here, we elucidate the evolutionary mechanism by which the ancestral inflammatory response was transformed into embryo implantation in the eutherian lineage. We performed a comparative uterine transcriptomic and immunohistochemical study of three eutherians, armadillo (Dasypus novemcinctus), hyrax (Procavia capensis), and rabbit (Oryctolagus cuniculus); and one marsupial, opossum (Monodelphis domestica). Our results suggest that in the eutherian lineage, the ancestral inflammatory response was domesticated by suppressing one of its modules detrimental to pregnancy, namely, neutrophil recruitment by cytokine IL17A. Further, we propose that this suppression was mediated by decidual stromal cells, a novel cell type in eutherian mammals. We tested a prediction of this model in vitro and showed that decidual stromal cells can suppress the production of IL17A from helper T cells. Together, these results provide a mechanistic understanding of early stages in the evolution of eutherian pregnancy.

Author Correction: Evolution of placental invasion and cancer metastasis are causally linked.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evolution of placental invasion and cancer metastasis are causally linked.

Among mammals, placental invasion is correlated with vulnerability to malignancy. Animals with more invasive placentation (for example, humans) are more vulnerable to malignancy. To explain this correlation, we propose the hypothesis of 'Evolved Levels of Invasibility' proposing that the evolution of invasibility of stromal tissue affects both placental and cancer invasion. We provide evidence for this using an in vitro model. We find that bovine endometrial and skin fibroblasts are more resistant to invasion than are their human counterparts. Gene expression profiling identified genes with high expression in human but not in bovine fibroblasts. Knocking down a subset of them in human fibroblasts leads to stronger resistance to cancer cell invasion. Identifying the evolutionary determinants of stromal invasibility can provide important insights to develop rational antimetastatic therapeutics.

Endometrial recognition of pregnancy occurs in the grey short-tailed opossum ( Monodelphis domestica).

In human pregnancy, recognition of an embryo within the uterus is essential to support the fetus through gestation. In most marsupials, such as the opossums, pregnancy is shorter than the oestrous cycle and the steroid hormone profile during pregnancy and oestrous cycle are indistinguishable. For these reasons, it was assumed that recognition of pregnancy, as a trait, evolved in the eutherian (placental) stem lineage and independently in wallabies and kangaroos. To investigate whether uterine recognition of pregnancy occurs in species with pregnancy shorter than the oestrous cycle, we examined reproduction in the short-tailed opossum ( Monodelphis domestica), a marsupial with a plesiomorphic mode of pregnancy. We examined the morphological and gene expression changes in the uterus of females in the non-pregnant oestrous cycle and compared these to pregnancy. We found that the presence of an embryo did not alter some aspects of uterine development but increased glandular activity. Transcriptionally, we saw big differences between the uterus of pregnant and cycling animals. These differences included an upregulation of genes involved in transport, inflammation and metabolic-activity in response to the presence of a fetus. Furthermore, transcriptional differences reflected protein level differences in transporter abundance. Our results suggest that while the uterus exhibits programmed changes after ovulation, its transcriptional landscape during pregnancy responds to the presence of a fetus and upregulates genes that may be essential for fetal support. These results are consistent with endometrial recognition of pregnancy occurring in the opossum. While the effects on maternal physiology appear to differ, recognition of pregnancy has now been observed in eutherian mammals, as well as, Australian and American marsupials.

The mammalian decidual cell evolved from a cellular stress response.

Among animal species, cell types vary greatly in terms of number and kind. The number of cell types found within an organism differs considerably between species, and cell type diversity is a significant contributor to differences in organismal structure and function. These observations suggest that cell type origination is a significant source of evolutionary novelty. The molecular mechanisms that result in the evolution of novel cell types, however, are poorly understood. Here, we show that a novel cell type of eutherians mammals, the decidual stromal cell (DSC), evolved by rewiring an ancestral cellular stress response. We isolated the precursor cell type of DSCs, endometrial stromal fibroblasts (ESFs), from the opossum Monodelphis domestica. We show that, in opossum ESFs, the majority of decidual core regulatory genes respond to decidualizing signals but do not regulate decidual effector genes. Rather, in opossum ESFs, decidual transcription factors function in apoptotic and oxidative stress response. We propose that rewiring of cellular stress responses was an important mechanism for the evolution of the eutherian decidual cell type.

SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells.

Recurrent mutations in the splicing factor SRSF2 are associated with poor clinical outcomes in myelodysplastic syndromes (MDS). Their high frequency suggests these mutations drive oncogenesis, yet the molecular explanation for this process is unclear. SRSF2 mutations could directly affect pre-mRNA splicing of a vital gene product; alternatively, a whole network of gene products could be affected. Here we determine how SRSF2 mutations globally affect RNA binding and splicing in vivo using HITS-CLIP. Remarkably, the majority of differential binding events do not translate into alternative splicing of exons with SRSF2P95H binding sites. Alternative splice alterations appear to be dominated by indirect effects. Importantly, SRSF2P95H targets are enriched in RNA processing and splicing genes, including several members of the hnRNP and SR families of proteins, suggesting a "splicing-cascade" phenotype wherein mutation of a single splicing factor leads to widespread modifications in multiple RNA processing and splicing proteins. We show that splice alteration of HNRNPA2B1, a splicing factor differentially bound and spliced by SRSF2P95H, impairs hematopoietic differentiation in vivo. Our data suggests a model whereby the recurrent mutations in splicing factors set off a cascade of gene regulatory events that together affect hematopoiesis and drive cancer.

Genetic Associations with Gestational Duration and Spontaneous Preterm Birth.

BACKGROUND: Despite evidence that genetic factors contribute to the duration of gestation and the risk of preterm birth, robust associations with genetic variants have not been identified. We used large data sets that included the gestational duration to determine possible genetic associations. METHODS: We performed a genomewide association study in a discovery set of samples obtained from 43,568 women of European ancestry using gestational duration as a continuous trait and term or preterm (<37 weeks) birth as a dichotomous outcome. We used samples from three Nordic data sets (involving a total of 8643 women) to test for replication of genomic loci that had significant genomewide association (P<5.0×10-8) or an association with suggestive significance (P<1.0×10-6) in the discovery set. RESULTS: In the discovery and replication data sets, four loci (EBF1, EEFSEC, AGTR2, and WNT4) were significantly associated with gestational duration. Functional analysis showed that an implicated variant in WNT4 alters the binding of the estrogen receptor. The association between variants in ADCY5 and RAP2C and gestational duration had suggestive significance in the discovery set and significant evidence of association in the replication sets; these variants also showed genomewide significance in a joint analysis. Common variants in EBF1, EEFSEC, and AGTR2 showed association with preterm birth with genomewide significance. An analysis of mother-infant dyads suggested that these variants act at the level of the maternal genome. CONCLUSIONS: In this genomewide association study, we found that variants at the EBF1, EEFSEC, AGTR2, WNT4, ADCY5, and RAP2C loci were associated with gestational duration and variants at the EBF1, EEFSEC, and AGTR2 loci with preterm birth. Previously established roles of these genes in uterine development, maternal nutrition, and vascular control support their mechanistic involvement. (Funded by the March of Dimes and others.).

Embryo implantation evolved from an ancestral inflammatory attachment reaction.

The molecular changes that support implantation in eutherian mammals are necessary to establish pregnancy. In marsupials, pregnancy is relatively short, and although a placenta does form, it is present for only a few days before parturition. However, morphological changes in the uterus of marsupials at term mimic those that occur during implantation in humans and mice. We investigated the molecular similarity between term pregnancy in the marsupials and implantation in eutherian mammals using the gray short-tailed opossum (Monodelphis domestica) as a model. Transcriptomic analysis shows that term pregnancy in the opossum is characterized by an inflammatory response consistent with implantation in humans and mice. This immune response is temporally correlated with the loss of the eggshell, and we used immunohistochemistry to report that this reaction occurs at the materno-fetal interface. We demonstrate that key markers of implantation, including Heparin binding EGF-like growth factor and Mucin 1, exhibit expression and localization profiles consistent with the pattern observed during implantation in eutherian mammals. Finally, we show that there are transcriptome-wide similarities between the opossum attachment reaction and implantation in rabbits and humans. Our data suggest that the implantation reaction that occurs in eutherians is derived from an attachment reaction in the ancestral therian mammal which, in the opossum, leads directly to parturition. Finally, we argue that the ability to shift from an inflammatory attachment reaction to a noninflammatory period of pregnancy was a key innovation in eutherian mammals that allowed an extended period of intimate placentation.

Evolution of Gene Expression in the Uterine Cervix related to Steroid Signaling: Conserved features in the regulation of cervical ripening.

The uterine cervix is the boundary structure between the uterus and the vagina and is key for the maintenance of pregnancy and timing of parturition. Here we report on a comparative transcriptomic study of the cervix of four placental mammals, mouse, guinea pig, rabbit and armadillo, and one marsupial, opossum. Our aim is to investigate the evolution of cervical gene expression as related to putative mechanisms for functional progesterone withdrawal. Our findings are: 1) The patterns of gene expression in eutherian (placental) mammals are consistent with the notion that an increase in the E/P4 signaling ratio is critical for cervical ripening. How the increased E/P4 ratio is achieved, however, is variable between species. 2) None of the genes related to steroid signaling, that are modulated in eutherian species, change expression during opossum gestation. 3) A tendency for decreased expression of progesterone receptor co-activators (NCOA1, -2 and -3, and CREBBP) towards term is a shared derived feature of eutherians. This suggests that parturition is associated with broad scale histone de-acetylation. Western-blotting on mouse cervix confirmed large scale histone de-acetylation in labor. This finding may have important implications for the control of premature cervical ripening and prevention of preterm birth in humans.

Single-cell transcriptomics of the human placenta: inferring the cell communication network of the maternal-fetal interface.

Organismal function is, to a great extent, determined by interactions among their fundamental building blocks, the cells. In this work, we studied the cell-cell interactome of fetal placental trophoblast cells and maternal endometrial stromal cells, using single-cell transcriptomics. The placental interface mediates the interaction between two semiallogenic individuals, the mother and the fetus, and is thus the epitome of cell interactions. To study these, we inferred the cell-cell interactome by assessing the gene expression of receptor-ligand pairs across cell types. We find a highly cell-type-specific expression of G-protein-coupled receptors, implying that ligand-receptor profiles could be a reliable tool for cell type identification. Furthermore, we find that uterine decidual cells represent a cell-cell interaction hub with a large number of potential incoming and outgoing signals. Decidual cells differentiate from their precursors, the endometrial stromal fibroblasts, during uterine preparation for pregnancy. We show that decidualization (even in vitro) enhances the ability to communicate with the fetus, as most of the receptors and ligands up-regulated during decidualization have their counterpart expressed in trophoblast cells. Among the signals transmitted, growth factors and immune signals dominate, and suggest a delicate balance of enhancing and suppressive signals. Finally, this study provides a rich resource of gene expression profiles of term intravillous and extravillous trophoblasts, including the transcriptome of the multinucleated syncytiotrophoblast.

Cis-Regulatory Evolution of Forkhead Box O1 (FOXO1), a Terminal Selector Gene for Decidual Stromal Cell Identity.

Studies in human and mouse have shown that decidual stromal cells (DSC), which develop in the innermost lining of uterus, mediate placentation by regulating maternal immune response against the fetus and the extent of fetal invasion. Investigating when and how DSC evolved is thus a key step to reconstructing the evolutionary history of mammalian pregnancy. We present molecular evidence placing the origin of DSC in the stem lineage of eutherians (extant placental mammals). The transcription factor forkhead box O1 (FOXO1) is a part of the core regulatory transcription factor complex (CoRC) that establishes the cell type identity of DSC. Decidualization, the process through which DSC differentiate from endometrial stromal fibroblasts, requires transcriptional upregulation of FOXO1 Contrary to other examples in mammals where gene recruitment is caused by the origin of an alternative promoter, FOXO1 is transcribed from the same promoter in DSC as in endometrial stromal fibroblasts. Comparing the activities of FOXO1 promoters from human, mouse, manatee (Afrotheria), and opossum (marsupial) revealed that FOXO1 promoter evolved responsiveness to decidualization signals in the stem lineage of eutherians. This eutherian vs. marsupial pattern of promoter activity was not observed in some other cell types expressing FOXO1, suggesting that this cis-regulatory evolution occurred specifically in the context of the origin of DSC. Sequence comparison revealed eutherian-specifically conserved nucleotides that contribute to the eutherian promoter activity. We conclude that the cis-regulatory activity of a terminal selector gene for decidual stromal cell identity evolved in the stem lineage of eutherians supporting a model where decidual cells are a eutherian innovation.

The Transcriptomic Evolution of Mammalian Pregnancy: Gene Expression Innovations in Endometrial Stromal Fibroblasts.

The endometrial stromal fibroblast (ESF) is a cell type present in the uterine lining of therian mammals. In the stem lineage of eutherian mammals, ESF acquired the ability to differentiate into decidual cells in order to allow embryo implantation. We call the latter cell type "neo-ESF" in contrast to "paleo-ESF" which is homologous to eutherian ESF but is not able to decidualize. In this study, we compare the transcriptomes of ESF from six therian species: Opossum (Monodelphis domestica; paleo-ESF), mink, rat, rabbit, human (all neo-ESF), and cow (secondarily nondecidualizing neo-ESF). We find evidence for strong stabilizing selection on transcriptome composition suggesting that the expression of approximately 5,600 genes is maintained by natural selection. The evolution of neo-ESF from paleo-ESF involved the following gene expression changes: Loss of expression of genes related to inflammation and immune response, lower expression of genes opposing tissue invasion, increased markers for proliferation as well as the recruitment of FOXM1, a key gene transiently expressed during decidualization. Signaling pathways also evolve rapidly and continue to evolve within eutherian lineages. In the bovine lineage, where invasiveness and decidualization were secondarily lost, we see a re-expression of genes found in opossum, most prominently WISP2, and a loss of gene expression related to angiogenesis. The data from this and previous studies support a scenario, where the proinflammatory paleo-ESF was reprogrammed to express anti-inflammatory genes in response to the inflammatory stimulus coming from the implanting conceptus and thus paving the way for extended, trans-cyclic gestation.