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The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15, in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development.

ObjectiveIn growth plate chondrocytes, loss of Dicer, a microRNA (miRNA)-processing enzyme, causes defects in proliferation and differentiation, leading to a lethal skeletal dysplasia. However roles of miRNAs in articular chondrocytes have not been defined in vivo. To investigate the role of miRNAs in articular chondrocytes and to explore the possibility of generating a novel mouse osteoarthritis (OA) model caused by intrinsic cellular dysfunction, we ablated Drosha, another essential enzyme for miRNA biogenesis, exclusively in articular chondrocytes of postnatal mice.DesignFirst, to confirm that the essential role of miRNAs in skeletal development, we ablated the miRNA biogenesis pathway by deleting Drosha or DGCR8 in growth plate chondrocytes. Next, to investigate the role of miRNAs in articular cartilage, we deleted Drosha using Prg4-CreER(T) transgenic mice expressing a tamoxifen-activated Cre recombinase (CreER(T)) exclusively in articular chondrocytes. Tamoxifen was injected at postnatal days, 7, 14, 21, and 28 to ablate Drosha.ResultsDeletion of Drosha or DGCR8 in growth plate chondrocytes caused a lethal skeletal defect similar to that of Dicer deletion, confirming the essential role of miRNAs in normal skeletogenesis. Early postnatal Drosha deletion in articular chondrocytes significantly increased cell death and decreased Safranin-O staining. Mild OA-like changes, including surface erosion and cleft formation, were found in male mice at 6 months of age; however such changes in females were not observed even at 9 months of age.ConclusionsEarly postnatal Drosha deficiency induces articular chondrocyte death and can cause a mild OA-like pathology.

The Extracellular RNA (exRNA) Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amount

The embryonic stem cell (ESC)-enriched miR-294/302 family and the somatic cell-enriched let-7 family stabilizes the self-renewing and differentiated cell fates, respectively. The mechanisms underlying these processes remain unknown. Here we show that among many pathways regulated by miR-294/302, the combinatorial suppression of epithelial-mesenchymal transition (EMT) and apoptotic pathways is sufficient in maintaining the self-renewal of ESCs. The silencing of ESC self-renewal by let-7 was accompanied by the upregulation of several EMT regulators and the induction of apoptosis. The ectopic activation of either EMT or apoptotic program is sufficient in silencing ESC self-renewal. However, only combined but not separate suppression of the two programs inhibited the silencing of ESC self-renewal by let-7 and several other differentiation-inducing miRNAs. These findings demonstrate that combined repression of the EMT and apoptotic pathways by miR-294/302 imposes a synergistic barrier to the silencing of ESC self-renewal, supporting a model whereby miRNAs regulate complicated cellular processes through synergistic repression of multiple targets or pathways. [ABSTRACT FROM AUTHOR]

Abstract Background Neuronal phenotypes associated with hemizygosity of individual genes within the 22q11.2 deletion syndrome locus hold potential towards understanding the pathogenesis of schizophrenia and autism. Included among these genes is Dgcr8, which encodes an RNA-binding protein required for microRNA biogenesis. Dgcr8 haploinsufficient mice (Dgcr8+/-) have reduced expression of microRNAs in brain and display cognitive deficits, but how microRNA deficiency affects the development and function of neurons in the cerebral cortex is not fully understood. Results In this study, we show that Dgcr8+/- mice display reduced expression of a subset of microRNAs in the prefrontal cortex, a deficit that emerges over postnatal development. Layer V pyramidal neurons in the medial prefrontal cortex of Dgcr8+/- mice have altered electrical properties, decreased complexity of basal dendrites, and reduced excitatory synaptic transmission. Conclusions These findings demonstrate that precise microRNA expression is critical for the postnatal development of prefrontal cortical circuitry. Similar defects in neuronal maturation resulting from microRNA deficiency could represent endophenotypes of certain neuropsychiatric diseases of developmental onset.

Abstract Background The regulation of gene expression is complex and occurs at many levels, including transcriptional and post-transcriptional, in metazoans. Transcriptional regulation is mainly determined by sequence elements within the promoter regions of genes while sequence elements within the 3' untranslated regions of mRNAs play important roles in post-transcriptional regulation such as mRNA stability and translation efficiency. Identifying cis-regulatory elements, or motifs, in multicellular eukaryotes is more difficult compared to unicellular eukaryotes due to the larger intergenic sequence space and the increased complexity in regulation. Experimental techniques for discovering functional elements are often time consuming and not easily applied on a genome level. Consequently, computational methods are advantageous for genome-wide cis-regulatory motif detection. To decrease the search space in metazoans, many algorithms use cross-species alignment, although studies have demonstrated that a large portion of the binding sites for the same trans-acting factor do not reside in alignable regions. Therefore, a computational algorithm should account for both conserved and nonconserved cis-regulatory elements in metazoans. Results We present CompMoby (Comparative MobyDick), software developed to identify cis-regulatory binding sites at both the transcriptional and post-transcriptional levels in metazoans without prior knowledge of the trans-acting factors. The CompMoby algorithm was previously shown to identify cis-regulatory binding sites in upstream regions of genes co-regulated in embryonic stem cells. In this paper, we extend the software to identify putative cis-regulatory motifs in 3' UTR sequences and verify our results using experimentally validated data sets in mouse and human. We also detail the implementation of CompMoby into a user-friendly tool that includes a web interface to a streamlined analysis. Our software allows detection of motifs in the following three categories: one, those that are alignable and conserved; two, those that are conserved but not alignable; three, those that are species specific. One of the output files from CompMoby gives the user the option to decide what category of cis-regulatory element to experimentally pursue based on their biological problem. Using experimentally validated biological datasets, we demonstrate that CompMoby is successful in detecting cis-regulatory target sites of known and novel trans-acting factors at the transcriptional and post-transcriptional levels. Conclusion CompMoby is a powerful software tool for systematic de novo discovery of evolutionarily conserved and nonconserved cis-regulatory sequences involved in transcriptional or post-transcriptional regulation in metazoans. This software is freely available to users at http://genome.ucsf.edu/compmoby/.

The miR-290 and miR-302 clusters of microRNAs are highly expressed in naïve and primed pluripotent stem cells, respectively. Ectopic expression of the embryonic stem cell-specific cell cycle regulating (ESCC) family of microRNAs arising from these two clusters dramatically enhances the reprogramming of both mouse and human somatic cells to induced pluripotency. Here, we used genetic knockouts to dissect the requirement for the miR-290 and miR-302 clusters during the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs) with retrovirally introduced Oct4, Sox2, and Klf4. Knockout of either cluster alone did not negatively impact the efficiency of reprogramming. Resulting cells appeared identical to their embryonic stem cell microRNA cluster knockout counterparts. In contrast, the combined loss of both clusters blocked the formation of iPSCs. While rare double knockout clones could be isolated, they showed a dramatically reduced proliferation rate, a persistent inability to fully silence the exogenously introduced pluripotency factors, and a transcriptome distinct from individual miR-290 or miR-302 mutant ESC and iPSCs. Taken together, our data show that miR-290 and miR-302 are essential yet interchangeable in reprogramming to the induced pluripotent state., Impact Statement: The process by which somatic cell reprogramming yields induced pluripotent stem cells (iPSCs) is incompletely understood. MicroRNAs from the miR-290 and miR-302 clusters have been shown to greatly increase reprogramming efficiency, but their requirement in the process has not been studied. Here, we examine this requirement by genetically removing the miRNA clusters in somatic cells. We discover that somatic cells lacking either, but not both, of these miRNA clusters can form iPSC cells. This work thus provides new important insight into mechanisms underlying reprogramming to pluripotency.

Poliovirus receptor-related 2 (PVRL2, also known as nectin-2 or CD112) is believed to act as an immune checkpoint protein in cancer; however, most insight into its role is inferred from studies on its known receptor, poliovirus receptor (PVR)-related immunoglobulin domain protein (PVRIG, also known as CD112R). Here, we study PVRL2 itself. PVRL2 levels were found to be high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer showed a dramatic reduction in tumor growth that was immune dependent. This effect was even greater than that seen with deletion of PD-L1. PVRL2 was shown to function by suppressing CD8+ T and natural killer cells in the tumor microenvironment. The loss of PVRL2 suppressed tumor growth even in the absence of PVRIG. In contrast, PVRIG loss showed no additive effect in the absence of PVRL2. T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade combined with PVRL2 deletion resulted in a near complete block in tumor growth. This effect was not recapitulated by the combined deletion of PVRL2 with its paralog, PVR, which is the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the antitumor immune response with functions beyond that of its known receptor PVRIG. Moreover, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy., (©2024 The Authors; Published by the American Association for Cancer Research.)

Coincident transcription and DNA replication causes replication stress and genome instability. Rapidly dividing mouse pluripotent stem cells are highly transcriptionally active and experience elevated replication stress, yet paradoxically maintain genome integrity. Here, we study FOXD3, a transcriptional repressor enriched in pluripotent stem cells, and show that its repression of transcription upon S phase entry is critical to minimizing replication stress and preserving genome integrity. Acutely deleting Foxd3 leads to immediate replication stress, G2/M phase arrest, genome instability and p53-dependent apoptosis. FOXD3 binds near highly transcribed genes during S phase entry, and its loss increases the expression of these genes. Transient inhibition of RNA polymerase II in S phase reduces observed replication stress and cell cycle defects. Loss of FOXD3-interacting histone deacetylases induces replication stress, while transient inhibition of histone acetylation opposes it. These results show how a transcriptional repressor can play a central role in maintaining genome integrity through the transient inhibition of transcription during S phase, enabling faithful DNA replication., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Enhancers are essential in defining cell fates through the control of cell-type-specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers including the monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27., Results: Here we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early differentiation of mouse embryonic stem cells. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1 but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. However, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of these chromatin events from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers., Conclusions: Collectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription., (© 2023. The Author(s).)

The human placenta contains two specialized regions: the villous chorion where gases and nutrients are exchanged between maternal and fetal blood, and the smooth chorion (SC) which surrounds more than 70% of the developing fetus but whose cellular composition and function is poorly understood. Here, we use single cell RNA-sequencing to compare the cell types and molecular programs between these two regions in the second trimester human placenta. Each region consists of progenitor cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs) with similar gene expression programs. While CTBs in the villous chorion differentiate into syncytiotrophoblasts, they take an alternative trajectory in the SC producing a previously unknown CTB population which we term SC-specific CTBs (SC-CTBs). Marked by expression of region-specific cytokeratins, the SC-CTBs form a stratified epithelium above a basal layer of progenitor CTBs. They express epidermal and metabolic transcriptional programs consistent with a primary role in defense against physical stress and pathogens. Additionally, we show that SC-CTBs closely associate with EVTs and secrete factors that inhibit the migration of the EVTs. This restriction of EVT migration is in striking contrast to the villous region where EVTs migrate away from the chorion and invade deeply into the decidua. Together, these findings greatly expand our understanding of CTB differentiation in these distinct regions of the human placenta. This knowledge has broad implications for studies of the development, functions, and diseases of the human placenta., Competing Interests: BM, YZ, MK, RB No competing interests declared, SF is a consultant for Novo Nordisk. All other authors declare no competing interests, (© 2022, Marsh et al.)

Pluripotent embryonic stem cells have a unique cell cycle structure with a suppressed G1/S restriction point and little differential expression across the cell cycle phases. Here, we evaluate the link between G1/S restriction point activation, phasic gene expression, and cellular differentiation. Expression analysis reveals a gain in phasic gene expression across lineages between embryonic days E7.5 and E9.5. Genetic manipulation of the G1/S restriction point regulators miR-302 and P27 respectively accelerates or delays the onset of phasic gene expression in mouse embryos. Loss of miR-302-mediated p21 or p27 suppression expedites embryonic stem cell differentiation, while a constitutive Cyclin E mutant blocks it. Together, these findings uncover a causal relationship between emergence of the G1/S restriction point with a gain in phasic gene expression and cellular differentiation., (© 2022. The Author(s).)

Reproductive immunology has moved on from the classical Medawar question of 60 years ago " why doesn't the mother reject the fetus? ". Looking beyond fetal-maternal tolerance, modern reproductive immunology focuses on how the maternal immune system supports fetal growth. Maternal uterine natural killer (uNK) cells, in partnership with fetal trophoblast cells, regulate physiological vascular changes in the uterus of pregnant women and mice. These vascular changes are necessary to build the placenta and sustain fetal growth. NK cell functions in the uterus and elsewhere, including anti-viral and anti-tumour immunity mediated mostly by blood NK cells, are modulated by NK cell education, a quantifiable process that determines cellular activation thresholds. This process relies largely on interactions between self-MHC class I molecules and inhibitory NK cell receptors. By getting to know self, the maternal immune system sets up uNK cells to participate to tissue homeostasis in the womb. Placentation can be viewed as a form of natural transplantation unique in vertebrates and this raises the question of how uNK cell education or missing-self recognition affect their function and, ultimately fetal growth. Here, using combinations of MHC-sufficient and -deficient mice, we show that uNK cell education is linked to maternal and not fetal MHC, so that MHC-deficient dams produce more growth-restricted fetuses, even when the fetuses themselves express self-MHC. We also show that, while peripheral NK cells reject bone marrow cells according to the established rules of missing-self recognition, uNK cells educated by maternal MHC do not reject fetuses that miss self-MHC and these fetuses grow to their full potential. While these results are not directly applicable to clinical research, they show that NK education by maternal MHC-I is required for optimal fetal growth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Depierreux, Kieckbusch, Shreeve, Hawkes, Marsh, Blelloch, Sharkey and Colucci.)

Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan-binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived small extracellular vesicles (EVs), leading to the robust quantification of 953 cell and EV surface annotated proteins. We identified a newly recognized subset of EV-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer EVs. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types., Competing Interests: LK, SE, JY, JB, JG, RB, JW No competing interests declared, (© 2022, Kirkemo et al.)

Hundreds of microRNAs (miRNAs) are expressed in distinct spatial and temporal patterns during embryonic and postnatal mouse development. The loss of all miRNAs through the deletion of critical miRNA biogenesis factors results in early lethality. The function of each miRNA stems from their cumulative negative regulation of multiple mRNA targets expressed in a particular cell type. During development, miRNAs often coordinate the timing and direction of cell fate transitions. In adults, miRNAs frequently contribute to organismal fitness through homeostatic roles in physiology. Here, we review how the recent dissection of miRNA-knockout phenotypes in mice as well as advances related to their targets, dosage, and interactions have collectively informed our understanding of the roles of miRNAs in mammalian development and adaptive responses.

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., Competing Interests: BM, RB No competing interests declared, (© 2020, Marsh and Blelloch.)

Profilin2 (PFN2) is a target of the embryonic stem cell (ESC)-enriched miR-290 family of microRNAs (miRNAs) and an actin/dynamin-binding protein implicated in endocytosis. Here we show that the miR-290-PFN2 pathway regulates many aspects of ESC biology. In the absence of miRNAs, PFN2 is up-regulated in ESCs, with a resulting decrease in endocytosis. Reintroduction of miR-290, knockout of Pfn2 , or disruption of the PFN2-dynamin interaction domain in miRNA-deficient cells reverses the endocytosis defect. The reduced endocytosis is associated with impaired extracellular signal-regulated kinase (ERK) signaling, delayed ESC cell cycle progression, and repressed ESC differentiation. Mutagenesis of the single canonical conserved 3' UTR miR-290-binding site of Pfn2 or overexpression of the Pfn2 open reading frame alone in otherwise wild-type cells largely recapitulates these phenotypes. Taken together, these findings define an axis of posttranscriptional control, endocytosis, and signal transduction that is important for ESC proliferation and differentiation., Competing Interests: The authors declare no competing interest.

Differentiation of human embryonic stem cells into pancreatic β cells holds great promise for the treatment of diabetes. Recent advances have led to the production of glucose-responsive insulin-secreting cells in vitro, but resulting cells remain less mature than their adult primary β cell counterparts. The barrier(s) to in vitro β cell maturation are unclear. Here, we evaluated a potential role for microRNAs. MicroRNA profiling showed high expression of let-7 family microRNAs in vivo, but not in in vitro differentiated β cells. Reduced levels of let-7 in vitro were associated with increased levels of the RNA binding protein LIN28B, a negative regulator of let-7 biogenesis. Ablation of LIN28B during human embryonic stem cell (hESC) differentiation toward β cells led to a more mature glucose-stimulated insulin secretion profile and the suppression of juvenile-specific genes. However, let-7 overexpression had little effect. These results uncover LIN28B as a modulator of β cell maturation in vitro., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Pluripotent stem cells give rise to all cells of the adult organism, making them an invaluable tool in regenerative medicine. In response to differentiation cues, they can activate markedly distinct lineage-specific gene networks while turning off or rewiring pluripotency networks. Recent innovations in chromatin and nuclear structure analyses combined with classical genetics have led to novel insights into the transcriptional and epigenetic mechanisms underlying these networks. Here, we review these findings in relation to their impact on the maintenance of and exit from pluripotency and highlight the many factors that drive these processes, including histone modifying enzymes, DNA methylation and demethylation, nucleosome remodeling complexes and transcription factor-mediated enhancer switching., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The enhancer landscape of pluripotent stem cells undergoes extensive reorganization during early mammalian development. The functions and mechanisms behind such reorganization, however, are unclear. Here, we show that the transcription factor GRHL2 is necessary and sufficient to activate an epithelial subset of enhancers as naive embryonic stem cells (ESCs) transition into formative epiblast-like cells (EpiLCs). Surprisingly, many GRHL2 target genes do not change in expression during the ESC-EpiLC transition. Instead, enhancers regulating these genes in ESCs diminish in activity in EpiLCs while GRHL2-dependent alternative enhancers become activated to maintain transcription. GRHL2 therefore assumes control over a subset of the naive network via enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency. These data evoke a model where the naive pluripotency network becomes partitioned into smaller, independent networks regulated by EpiLC-specific transcription factors, thereby priming cells for lineage specification., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Translation and mRNA degradation are intimately connected, yet the mechanisms that link them are not fully understood. Here, we studied these mechanisms in embryonic stem cells (ESCs). Transcripts showed a wide range of stabilities, which correlated with their relative translation levels and that did not change during early ESC differentiation. The protein DHH1 links translation to mRNA stability in yeast; however, loss of the mammalian homolog, DDX6, in ESCs did not disrupt the correlation across transcripts. Instead, the loss of DDX6 led to upregulated translation of microRNA targets, without concurrent changes in mRNA stability. The Ddx6 knockout cells were phenotypically and molecularly similar to cells lacking all microRNAs ( Dgcr8 knockout ESCs). These data show that the loss of DDX6 can separate the two canonical functions of microRNAs: translational repression and transcript destabilization. Furthermore, these data uncover a central role for translational repression independent of transcript destabilization in defining the downstream consequences of microRNA loss., Competing Interests: JF, TH, RB No competing interests declared, (© 2018, Freimer et al.)

Mouse oocyte maturation, fertilization, and reprogramming occur in the absence of transcription, and thus, changes in mRNA levels and translation rate are regulated through post-transcriptional mechanisms [1]. Surprisingly, microRNA function, which is a major form of post-transcriptional regulation, is absent during this critical period of mammalian development [2, 3]. Here, we investigated the mechanisms underlying the global suppression of microRNA activity. In both mouse and frogs, microRNA function was active in growing oocytes but then absent during oocyte maturation. RNA sequencing (RNA-seq) of mouse oocytes uncovered that the microRNA effector protein AGO2 is predominantly expressed as an alternative isoform that encodes a truncated protein lacking all of the known essential domains. Full-length Ago2 as well as the related Argonautes (Ago1, Ago3, and Ago4) were lowly expressed in maturing mouse oocytes. Reintroduction of full-length AGO2 together with an exogenous microRNA in either mouse or frog oocytes restored translational repression of a target reporter. However, levels of endogenous transcripts remained unchanged. Consistent with a lack of microRNA activity, analysis of transcripts with alternative polyadenylation sites showed increased stability of transcripts with a longer 3' UTR during oocyte maturation. Redundant mechanisms protecting endogenous transcripts and the conserved loss of microRNA activity suggest a strong selection for suppressing microRNA function in vertebrate oocytes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The vertebrate-specific ESCC microRNA family arises from two genetic loci in mammals: miR-290/miR-371 and miR-302. The miR-302 locus is found broadly among vertebrates, whereas the miR-290/miR-371 locus is unique to eutheria, suggesting a role in placental development. Here, we evaluate that role. A knock-in reporter for the mouse miR-290 cluster is expressed throughout the embryo until gastrulation, when it becomes specifically expressed in extraembryonic tissues and the germline. In the placenta, expression is limited to the trophoblast lineage, where it remains highly expressed until birth. Deletion of the miR-290 cluster gene ( Mirc5 ) results in reduced trophoblast progenitor cell proliferation and a reduced DNA content in endoreduplicating trophoblast giant cells. The resulting placenta is reduced in size. In addition, the vascular labyrinth is disorganized, with thickening of the maternal-fetal blood barrier and an associated reduction in diffusion. Multiple mRNA targets of the miR-290 cluster microRNAs are upregulated. These data uncover a crucial function for the miR-290 cluster in the regulation of a network of genes required for placental development, suggesting a central role for these microRNAs in the evolution of placental mammals., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

While years of investigation have elucidated many aspects of embryonic stem cell (ESC) regulation, the contributions of post-transcriptional and translational mechanisms to the pluripotency network remain largely unexplored. In particular, little is known in ESCs about the function of RNA binding proteins (RBPs), the protein agents of post-transcriptional regulation. We performed an unbiased RNAi screen of RBPs in an ESC differentiation assay and identified two related genes, NF45 (Ilf2) and NF90/NF110 (Ilf3) , whose knockdown promoted differentiation to an epiblast-like state. Characterization of NF45 KO, NF90 + NF110 KO, and NF110 KO ESCs showed that loss of NF45 or NF90 + NF110 impaired ESC proliferation and led to dysregulated differentiation down embryonic lineages. Additionally, we found that NF45 and NF90/NF110 physically interact and influence the expression of each other at different levels of regulation. Globally across the transcriptome, NF45 KO ESCs and NF90 + NF110 KO ESCs show similar expression changes. Moreover, NF90 + NF110 RNA immunoprecipitation (RIP)-seq in ESCs suggested that NF90/NF110 directly regulate proliferation, differentiation, and RNA-processing genes. Our data support a model in which NF45, NF90, and NF110 operate in feedback loops that enable them, through both overlapping and independent targets, to help balance the push and pull of pluripotency and differentiation cues., (© 2017 Ye et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Micropatterned cocultures are a useful experimental tool for the study of cell-cell interactions. Patterning methods often rely on sequential seeding of different cell types or removal of a barrier separating two populations, but it is difficult to pattern sharp interfaces between pure populations with low cross-contamination when using these approaches. Patterning by the use of reconfigurable substrates can overcome these limitations, but such methods can be costly and challenging to employ in a typical biology laboratory. Here, we describe a low-cost and simple-to-use reconfigurable substrate comprised of a transparent elastic material that is partially cut to form a slit that opens when the device is stretched. The slit seals back up when released, allowing two initially separate, adherent cell populations to be brought together to form a contact interface. Fluorescent imaging of patterned cocultures demonstrates the early establishment of a sharp cellular interface. As a proof of principle, we demonstrate the use of this device to study competition at the interface of two stem cell populations.

MicroRNAs act posttranscriptionally to suppress multiple target genes within a cell population. To what extent this multi-target suppression occurs in individual cells and how it impacts transcriptional heterogeneity and gene co-expression remains unknown. Here we used single-cell sequencing combined with introduction of individual microRNAs. miR-294 and let-7c were introduced into otherwise microRNA-deficient Dgcr8 knockout mouse embryonic stem cells. Both microRNAs induce suppression and correlated expression of their respective gene targets. The two microRNAs had opposing effects on transcriptional heterogeneity within the cell population, with let-7c increasing and miR-294 decreasing the heterogeneity between cells. Furthermore, let-7c promotes, whereas miR-294 suppresses, the phasing of cell cycle genes. These results show at the individual cell level how a microRNA simultaneously has impacts on its many targets and how that in turn can influence a population of cells. The findings have important implications in the understanding of how microRNAs influence the co-expression of genes and pathways, and thus ultimately cell fate.

Metabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor β signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Early development is governed by the ability of pluripotent cells to retain the full range of developmental potential and respond accurately to developmental cues. This property is achieved in large part by the temporal and contextual regulation of gene expression by enhancers. Here, we evaluated regulation of enhancer activity during differentiation of embryonic stem to epiblast cells and uncovered the forkhead transcription factor FOXD3 as a major regulator of the developmental potential of both pluripotent states. FOXD3 bound to distinct sites in the two cell types priming enhancers through a dual-functional mechanism. It recruited the SWI/SNF chromatin remodeling complex ATPase BRG1 to promote nucleosome removal while concurrently inhibiting maximal activation of the same enhancers by recruiting histone deacetylases1/2. Thus, FOXD3 prepares cognate genes for future maximal expression by establishing and simultaneously repressing enhancer activity. Through switching of target sites, FOXD3 modulates the developmental potential of pluripotent cells as they differentiate., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The intrinsic (mitochondrial) apoptotic pathway is a conserved cell death program crucial for eliminating superfluous, damaged, or incorrectly specified cells, and the multi-domain pro-death BCL-2 family proteins BAX and BAK are required for its activation. In response to internal damage or developmental signals, BAX and/or BAK permeabilize the mitochondrial outer membrane, resulting in cytochrome c release and activation of effector caspases such as Caspase-3 (Casp3). While the mitochondrial apoptotic pathway plays a critical role during late embryonic development in mammals, its role during early development remains controversial. Here, we show that Bax(-/-)Bak(-/-) murine embryonic stem cells (ESCs) display defects during the exit from pluripotency, both in culture and during teratoma formation. Specifically, we find that when ESCs are stimulated to differentiate, a subpopulation fails to do so and instead upregulates FAS in a p53-dependent manner to trigger Bax/Bak-dependent apoptosis. Blocking this apoptotic pathway prevents the removal of these poorly differentiated cells, resulting in the retention of cells that have not exited pluripotency. Taken together, our results provide further evidence for heterogeneity in the potential of ESCs to successfully differentiate and reveal a novel role for apoptosis in promoting efficient ESC differentiation by culling cells that are slow to exit pluripotency., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In human prostate cancer, the microRNA biogenesis machinery increases with prostate cancer progression. Here, we show that deletion of the Dgcr8 gene, a critical component of this complex, inhibits tumor progression in a Pten-knockout mouse model of prostate cancer. Early stages of tumor development were unaffected, but progression to advanced prostatic intraepithelial neoplasia was severely inhibited. Dgcr8 loss blocked Pten null-induced expansion of the basal-like, but not luminal, cellular compartment. Furthermore, while late-stage Pten knockout tumors exhibit decreased senescence-associated beta-galactosidase activity and increased proliferation, the simultaneous deletion of Dgcr8 blocked these changes resulting in levels similar to wild type. Sequencing of small RNAs in isolated epithelial cells uncovered numerous miRNA changes associated with PTEN loss. Consistent with a Pten-Dgcr8 association, analysis of a large cohort of human prostate tumors shows a strong correlation between Akt activation and increased Dgcr8 mRNA levels. Together, these findings uncover a critical role for microRNAs in enhancing proliferation and enabling the expansion of the basal cell compartment associated with tumor progression following Pten loss., (© 2015 The Authors.)

The Extracellular RNA (exRNA) Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amounts of RNA can be detected in serum, plasma, and other bodily fluids. The exact mechanism(s) by which RNA can be secreted from cells and the mechanisms for the delivery and uptake by recipient cells remain to be determined. This review will summarize current knowledge about the biogenesis and delivery of exRNA and outline projects seeking to understand the functional impact of exRNA.

Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1(+) and subsequent PDX1(+)/NKX6.1(+) pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1(+)/NKX6.1(+) progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells., (© 2015 The Authors.)

Embryonic stem cells (ESCs) have adopted an accelerated cell-cycle program with shortened gap phases and precocious expression of cell-cycle regulatory proteins, including cyclins and cyclin-dependent kinases (CDKs). We examined the effect of CDK inhibition on the pathways regulating proliferation and survival of ESCs. We found that inhibiting cyclin-dependent kinase 1 (CDK1) leads to activation of the DNA damage response, nuclear p53 stabilization, activation of a subset of p53 target genes including NOXA, and negative regulation of the anti-apoptotic protein MCL1 in human and mouse ESCs, but not differentiated cells. We demonstrate that MCL1 is highly expressed in ESCs and loss of MCL1 leads to ESC death. Finally, we show that clinically relevant CDK1 inhibitors prevent formation of ESC-derived tumors and induce necrosis in established ESC-derived tumors. Our data demonstrate that ES cells are uniquely sensitive to CDK1 inhibition via a p53/NOXA/MCL1 pathway., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Establishment, maintenance, and exit from pluripotency require precise coordination of a cell's molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Serum microRNAs hold great promise as easily accessible and measurable biomarkers of disease. In prostate cancer, serum miRNA signatures have been associated with the presence of disease as well as correlated with previously validated risk models. However, it is unclear whether miRNAs can provide independent prognostic information beyond current risk models. Here, we focus on a group of low-risk prostate cancer patients who were eligible for active surveillance, but chose surgery. A major criteria for the low risk category is a Gleason score of 6 or lower based on pre-surgical biopsy. However, a third of these patients are upgraded to Gleason 7 on post surgical pathological analysis. Both in a discovery and a validation cohort, we find that pre-surgical serum levels of miR-19, miR-345 and miR-519c-5p can help identify these patients independent of their pre-surgical age, PSA, stage, and percent biopsy involvement. A combination of the three miRNAs increased the area under a receiver operator characteristics curve from 0.77 to 0.94 (p<0.01). Also, when combined with the CAPRA risk model the miRNA signature significantly enhanced prediction of patients with Gleason 7 disease. In-situ hybridizations of matching tumors showed miR-19 upregulation in transformed versus normal-appearing tumor epithelial, but independent of tumor grade suggesting an alternative source for the increase in serum miR-19a/b levels or the release of pre-existing intracellular miR-19a/b upon progression. Together, these data show that serum miRNAs can predict relatively small steps in tumor progression improving the capacity to predict disease risk and, therefore, potentially drive clinical decisions in prostate cancer patients. It will be important to validate these findings in a larger multi-institutional study as well as with independent methodologies.

Ectopic expression of specific factors such as Oct4, Sox2, and Klf4 (OSK) is sufficient to reprogram somatic cells into induced pluripotent stem cells (iPSCs). In this study, we examine the paths taken by cells during the reprogramming process by following the transcriptional activation of two pluripotent miRNA clusters (mir-290 and mir-302) in individual cells in vivo and in vitro with knockin reporters. During embryonic development and embryonic stem cell differentiation, all cells sequentially expressed mir-290 and mir-302. In contrast, during OSK-induced reprogramming, cells activated the miRNA loci in a stochastic, nonordered manner. However, the addition of Sall4 to the OSK cocktail led to a consistent reverse sequence of locus activation (mir-302 then mir-290) and increased reprogramming efficiency. These results demonstrate that cells can follow multiple paths during the late stages of reprogramming, and that the trajectory of any individual cell is strongly influenced by the combination of factors introduced., (Copyright © 2014 Elsevier Inc. All rights reserved.)