Cripto Is Targeted by miR-1a-3p in a Mouse Model of Heart Development.

During cardiac differentiation, numerous factors contribute to the development of the heart. Understanding the molecular mechanisms underlying cardiac development will help combat cardiovascular disorders, among the leading causes of morbidity and mortality worldwide. Among the main mechanisms, we indeed find Cripto. Cripto is found in both the syncytiotrophoblast of ampullary pregnancies and the inner cell mass along the primitive streak as the second epithelial-mesenchymal transformation event occurs to form the mesoderm and the developing myocardium. At the same time, it is now known that cardiac signaling pathways are intimately intertwined with the expression of myomiRNAs, including miR-1. This miR-1 is one of the muscle-specific miRs; aberrant expression of miR-1 plays an essential role in cardiac diseases. Given this scenario, our study aimed to evaluate the inverse correlation between Cripto and miR-1 during heart development. We used in vitro models of the heart, represented by embryoid bodies (EBs) and embryonic carcinoma cell lines derived from an embryo-derived teratocarcinoma in mice (P19 cells), respectively. First, through a luciferase assay, we demonstrated that Cripto is a target of miR-1. Following this result, we observed that as the days of differentiation increased, the Cripto gene expression decreased, while the level of miR-1 increased; furthermore, after silencing miR-1 in P19 cells, there was an increase in Cripto expression. Moreover, inducing damage with a cobra cardiotoxin (CTX) in post-differentiation cells, we noted a decreased miR-1 expression and increased Cripto. Finally, in mouse cardiac biopsies, we observed by monitoring gene expression the distribution of Cripto and miR-1 in the right and left ventricles. These results allowed us to detect an inverse correlation between miR-1 and Cripto that could represent a new pharmacological target for identifying new therapies.

Stabilization of cell-cell adhesions prevents symmetry breaking and locks in pluripotency in 3D gastruloids.

3D embryonic stem cell (ESC) aggregates self-organize into embryo-like structures named gastruloids that recapitulate the axial organization of post-implantation embryos. Crucial in this process is the symmetry-breaking event that leads to the emergence of asymmetry and spatially ordered structures from homogeneous cell aggregates. Here, we show that budesonide, a glucocorticoid drug widely used to treat asthma, prevents ESC aggregates to break symmetry. Mechanistically, the effect of budesonide is glucocorticoid receptor independent. RNA sequencing and lineage fate analysis reveal that budesonide counteracts exit from pluripotency and modifies the expression of a large set of genes associated with cell migration, A-P axis formation, and WNT signaling. This correlates with reduced phenotypic and molecular cell heterogeneity, persistence of E-CADHERIN at the cell-cell interface, and cell aggregate compaction. Our findings reveal that cell-cell adhesion properties control symmetry breaking and cell fate transition in 3D gastruloids and suggest a potential adverse effect of budesonide on embryo development.

Capturing Transitional Pluripotency through Proline Metabolism.

In this paper, we summarize the current knowledge of the role of proline metabolism in the control of the identity of Embryonic Stem Cells (ESCs). An imbalance in proline metabolism shifts mouse ESCs toward a stable naïve-to-primed intermediate state of pluripotency. Proline-induced cells (PiCs), also named primitive ectoderm-like cells (EPLs), are phenotypically metastable, a trait linked to a rapid and reversible relocalization of E-cadherin from the plasma membrane to intracellular membrane compartments. The ESC-to-PiC transition relies on the activation of Erk and Tgfß/Activin signaling pathways and is associated with extensive remodeling of the transcriptome, metabolome and epigenome. PiCs maintain several properties of naïve pluripotency (teratoma formation, blastocyst colonization and 3D gastruloid development) and acquire a few traits of primed cells (flat-shaped colony morphology, aerobic glycolysis metabolism and competence for primordial germ cell fate). Overall, the molecular and phenotypic features of PiCs resemble those of an early-primed state of pluripotency, providing a robust model to study the role of metabolic perturbations in pluripotency and cell fate decisions.

The Multifaceted Roles of Proline in Cell Behavior.

Herein, we review the multifaceted roles of proline in cell biology. This peculiar cyclic imino acid is: (i) A main precursor of extracellular collagens (the most abundant human proteins), antimicrobial peptides (involved in innate immunity), salivary proteins (astringency, teeth health) and cornifins (skin permeability); (ii) an energy source for pathogenic bacteria, protozoan parasites, and metastatic cancer cells, which engage in extracellular-protein degradation to invade their host; (iii) an antistress molecule (an osmolyte and chemical chaperone) helpful against various potential harms (UV radiation, drought/salinity, heavy metals, reactive oxygen species); (iv) a neural metabotoxin associated with schizophrenia; (v) a modulator of cell signaling pathways such as the amino acid stress response and extracellular signal-related kinase pathway; (vi) an epigenetic modifier able to promote DNA and histone hypermethylation; (vii) an inducer of proliferation of stem and tumor cells; and (viii) a modulator of cell morphology and migration/invasiveness. We highlight how proline metabolism impacts beneficial tissue regeneration, but also contributes to the progression of devastating pathologies such as fibrosis and metastatic cancer.

Zfp57 inactivation illustrates the role of ICR methylation in imprinted gene expression during neural differentiation of mouse ESCs.

ZFP57 is required to maintain the germline-marked differential methylation at imprinting control regions (ICRs) in mouse embryonic stem cells (ESCs). Although DNA methylation has a key role in genomic imprinting, several imprinted genes are controlled by different mechanisms, and a comprehensive study of the relationship between DMR methylation and imprinted gene expression is lacking. To address the latter issue, we differentiated wild-type and Zfp57-/- hybrid mouse ESCs into neural precursor cells (NPCs) and evaluated allelic expression of imprinted genes. In mutant NPCs, we observed a reduction of allelic bias of all the 32 genes that were imprinted in wild-type cells, demonstrating that ZFP57-dependent methylation is required for maintaining or acquiring imprinted gene expression during differentiation. Analysis of expression levels showed that imprinted genes expressed from the non-methylated chromosome were generally up-regulated, and those expressed from the methylated chromosome were down-regulated in mutant cells. However, expression levels of several imprinted genes acquiring biallelic expression were not affected, suggesting the existence of compensatory mechanisms that control their RNA level. Since neural differentiation was partially impaired in Zfp57-mutant cells, this study also indicates that imprinted genes and/or non-imprinted ZFP57-target genes are required for proper neurogenesis in cultured ESCs.

Long Non-coding RNA T-UCstem1 Controls Progenitor Proliferation and Neurogenesis in the Postnatal Mouse Olfactory Bulb through Interaction with miR-9.

Neural stem cell populations generate a wide spectrum of neuronal and glial cell types in a highly ordered fashion. MicroRNAs are essential regulators of this process. T-UCstem1 is a long non-coding RNA containing an ultraconserved element, and in vitro analyses in pluripotent stem cells provided evidence that it regulates the balance between proliferation and differentiation. Here we investigate the in vivo function of T-UCstem1. We show that T-UCstem1 is expressed in the forebrain neurogenic lineage that generates interneurons for the postnatal olfactory bulb. Gain of function in neural stem cells increased progenitor proliferation at the expense of neuron production, whereas knockdown had the opposite effect. This regulatory function is mediated by its interaction with miR-9-3p and miR-9-5p. Based thereon, we propose a mechanistic model for the role of T-UCstem1 in the dynamic regulation of neural progenitor proliferation during neurogenesis.

Interplay between DNA and RNA Modifications: A Constantly Evolving Process.

The epigenome refers to the entirety of DNA methylations, histone modifications, nucleosome occupancy, and coding and non-coding RNAs (and their modifications) in different cell types [...].

Long non-coding RNA in stem cell pluripotency and lineage commitment: functions and evolutionary conservation.

LncRNAs have recently emerged as new and fundamental transcriptional and post-transcriptional regulators acting at multiple levels of gene expression. Indeed, lncRNAs participate in a wide variety of stem cell and developmental processes, acting in cis and/or in trans in the nuclear and/or in the cytoplasmic compartments, and generating an intricate network of interactions with RNAs, enhancers, and chromatin-modifier complexes. Given the versatility of these molecules to operate in different subcellular compartments, via different modes of action and with different target specificity, the interest in this research field is rapidly growing. Here, we review recent progress in defining the functional role of lncRNAs in stem cell biology with a specific focus on the underlying mechanisms. We also discuss recent findings on a new family of evolutionary conserved lncRNAs transcribed from ultraconserved elements, which show perfect conservation between human, mouse, and rat genomes, and that are emerging as new player in this complex scenario.

LncRNAs and PRC2: Coupled Partners in Embryonic Stem Cells.

The power of embryonic stem cells (ESCs) lies in their ability to self-renew and differentiate. Behind these two unique capabilities is a fine-tuned molecular network that shapes the genetic, epigenetic, and epitranscriptomic ESC plasticity. Although RNA has been shown to be functionally important in only a small minority of long non-coding RNA genes, a growing body of evidence has highlighted the pivotal and intricate role of lncRNAs in chromatin remodeling. Due to their multifaceted nature, lncRNAs interact with DNA, RNA, and proteins, and are emerging as new modulators of extensive gene expression programs through their participation in ESC-specific regulatory circuitries. Here, we review the tight cooperation between lncRNAs and Polycomb repressive complex 2 (PRC2), which is intimately involved in determining and maintaining the ESC epigenetic landscape. The lncRNA-PRC2 partnership is fundamental in securing the fully pluripotent state of ESCs, which must be primed to differentiate properly. We also reflect on the advantages brought to this field of research by the advent of single-cell analysis.

D-Aspartate treatment attenuates myelin damage and stimulates myelin repair.

Glutamate signaling may orchestrate oligodendrocyte precursor cell (OPC) development and myelin regeneration through the activation of glutamate receptors at OPC-neuron synapses. D-Aspartate is a D-amino acid exerting modulatory actions at glutamatergic synapses. Chronic administration of D-Aspartate has been proposed as therapeutic treatment in diseases related to myelin dysfunction and NMDA receptors hypofunction, including schizophrenia and cognitive deficits. Here, we show, by using an in vivo remyelination model, that administration of D-Aspartate during remyelination improved motor coordination, accelerated myelin recovery, and significantly increased the number of small-diameter myelinated axons. Chronically administered during demyelination, D-Aspartate also attenuated myelin loss and inflammation. Interestingly, D-Aspartate exposure stimulated OPC maturation and accelerated developmental myelination in organotypic cerebellar slices. D-Aspartate promoting effects on OPC maturation involved the activation of glutamate transporters, AMPA and NMDA receptors, and the Na+/Ca2+ exchanger NCX3. While blocking NMDA or NCX3 significantly prevented D-Aspartate-induced [Ca2+]i oscillations, blocking AMPA and glutamate transporters prevented both the initial and oscillatory [Ca2+]i response as well as D-Aspartate-induced inward currents in OPC Our findings reveal that D-Aspartate treatment may represent a novel strategy for promoting myelin recovery.

An Ultraconserved Element Containing lncRNA Preserves Transcriptional Dynamics and Maintains ESC Self-Renewal.

Ultraconserved elements (UCEs) show the peculiar feature to retain extended perfect sequence identity among human, mouse, and rat genomes. Most of them are transcribed and represent a new family of long non-coding RNAs (lncRNAs), the transcribed UCEs (T-UCEs). Despite their involvement in human cancer, the physiological role of T-UCEs is still unknown. Here, we identify a lncRNA containing the uc.170+, named T-UCstem1, and provide in vitro and in vivo evidence that it plays essential roles in embryonic stem cells (ESCs) by modulating cytoplasmic miRNA levels and preserving transcriptional dynamics. Specifically, while T-UCstem1::miR-9 cytoplasmic interplay regulates ESC proliferation by reducing miR-9 levels, nuclear T-UCstem1 maintains ESC self-renewal and transcriptional identity by stabilizing polycomb repressive complex 2 on bivalent domains. Altogether, our findings provide unprecedented evidence that T-UCEs regulate physiological cellular functions and point to an essential role of T-UCstem1 in preserving ESC identity.

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum.

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 ß signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.

Tracking the evolution of epialleles during neural differentiation and brain development: D-Aspartate oxidase as a model gene.

We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.

Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/ß-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.

ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells.

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.

c-Myc modulation: a key role in melanoma drug response.

Understanding molecular mechanisms involved in melanoma resistance to drugs is a big challenge. Experimental evidences suggested a correlation between mutational status in B-RAF and melanoma cell susceptibility to drugs, such as paclitaxel, doxorubicin and temozolomide, which generate an accumulation of hydrogen peroxide (H2O2) in the cells. We investigated the survival phenotype and the protein level of c-myc, a B-RAF target molecule, in melanoma cells, carrying a different mutational status in B-RAF, upon paclitaxel, doxorubicin and H2O2 treatment. For the first time, we reported c-myc modulation is critical for melanoma drug response. It appeared drug-specific and post-transcriptionally driven through PP2A; in correlation, cell pre-treatment with okadaic acid (OA), a specific PP2A inhibitor, as well as PP2A silencing of melanoma cells, was able to increase melanoma cell drug-sensitivity and c-myc protein level. This is relevant for designing efficacious therapeutic strategies in melanoma.

Ran signaling in melanoma: implications for the development of alternative therapeutic strategies.

We performed a comparative study between two human metastatic melanoma cell lines (A375 and 526), and melanocytes (FOM78) by gene expression profiling and pathway analysis, using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) software. Genes involved in Ran signaling were significantly over-represented (p ≤ 0.001) and up-regulated in melanoma cells. A melanoma-associated molecular pathway was identified, where Ran, Aurora Kinase A (AurkA) and TERT were up-regulated, while c-myc and PTEN were down-regulated. A consistent high Ran and AurkA gene expression was detected in about 48% and 53%, respectively, of 113 tissue samples from metastatic melanoma patients. AurkA down-regulation was observed in melanoma cells, by Ran knockdown, suggesting AurkA protein is a Ran downstream target. Furthermore, AurkA inhibition, by exposure of melanoma cells to MLN8054, a specific AurKA inhibitor, induced apoptosis in both melanoma cell lines and molecular alterations in the IPA-identified molecular pathway. These alterations differed between cell lines, with an up-regulation of c-myc protein level observed in 526 cells and a slight reduction seen in A375 cells. Moreover, Ran silencing did not affect the A375 invasive capability, while it was enhanced in 526 cells, suggesting that Ran knockdown, by AurkA down-regulation, resulted in a Ran-independent enhanced melanoma cell invasion. Finally, AurK A inhibition induced a PTEN up-regulation and its action was independent of B-RAF mutational status. These findings provide insights relevant for the development of novel therapeutic strategies as well as for a better understanding of mechanisms underlying therapy resistance in melanoma.

Reducing glypican-4 in ES cells improves recovery in a rat model of Parkinson's disease by increasing the production of dopaminergic neurons and decreasing teratoma formation.

The heparan sulfate proteoglycan Glypican 4 (Gpc4) is strongly expressed in mouse embryonic stem (ES) cells where it controls the maintenance of self-renewal by modulating Wnt/ß-catenin signaling activities. Here we show that mouse ES cells carrying a hypomorphic Gpc4 allele, in a single-step neuronal differentiation protocol, show increased differentiation into dopaminergic neurons expressing tyrosine hydroxylase (TH) and nuclear receptor related-1 protein (Nurr1) 1. In contrast to wild-type cells, these differentiating Gpc4-mutant cells expressed high levels of DOPA decarboxylase and the dopamine transporter, two markers expressed by fully mature dopaminergic neurons. Intrastriatal transplantation of Gpc4 hypomorphic cells into a 6-OHDA rat model for Parkinson's disease improved motor behavior in the cylinder test and amphetamine-induced rotations at a higher level than transplanted wild-type cells. Importantly, Gpc4 hypomorphic cell grafts, in contrast to wild-type cells, did not generate teratomas in the host brains, leading to strongly enhanced animal survival. Therefore, control of Gpc4 activity level represents a new potential strategy to reduce ES cell tumorigenic features while at the same time increasing neuronal differentiation and integration.

L-Proline induces a mesenchymal-like invasive program in embryonic stem cells by remodeling H3K9 and H3K36 methylation.

Metabolites are emerging as key mediators of crosstalk between metabolic flux, cellular signaling, and epigenetic regulation of cell fate. We found that the nonessential amino acid L-proline (L-Pro) acts as a signaling molecule that promotes the conversion of embryonic stem cells into mesenchymal-like, spindle-shaped, highly motile, invasive pluripotent stem cells. This embryonic-stem-cell-to-mesenchymal-like transition (esMT) is accompanied by a genome-wide remodeling of the H3K9 and H3K36 methylation status. Consistently, L-Pro-induced esMT is fully reversible either after L-Pro withdrawal or by addition of ascorbic acid (vitamin C), which in turn reduces H3K9 and H3K36 methylation, promoting a mesenchymal-like-to-embryonic-stem-cell transition (MesT). These findings suggest that L-Pro, which is produced by proteolytic remodeling of the extracellular matrix, may act as a microenvironmental cue to control stem cell behavior.

The G-protein-coupled receptor APJ is expressed in the second heart field and regulates Cerberus-Baf60c axis in embryonic stem cell cardiomyogenesis.

AIMS: Mammalian cardiomyogenesis occurs through a multistep process that requires a complex network of tightly regulated extracellular signals, which integrate with the genetic and epigenetic machinery to maintain, expand, and regulate the differentiation of cardiac progenitor cells. Pluripotent embryonic stem cells (ESCs) recapitulate many aspects of development, and have provided an excellent opportunity to dissect the molecular mechanisms underlying cardiomyogenesis, which is still incompletely defined. METHODS AND RESULTS: We provide new in vivo evidence that the G-protein-coupled receptor angiotensin receptor-like 1 (Apj) is expressed in the mesodermal cells of the second heart field, a population of cardiac progenitors that give rise to a major part of the definitive heart. By combining loss-and-gain of function studies in mouse ESCs, we show that Apj (i) controls the balance between proliferation and cardiovascular differentiation, (ii) regulates the Nodal/Bone Morphogenetic Protein antagonist Cerberus and the Baf60c/Smarcd3 subunit of the Brg1/Brm-associated factors (BAF) chromatin-remodelling complex. CONCLUSION: We propose a model in which Apj controls a regulatory Cerberus-Baf60c pathway in pluripotent stem cell cardiomyogenesis, and speculate that this regulatory circuit may regulate cardiac progenitor cell behaviour.