P19 Embryonal Carcinoma Research Articles

Cell quality control mechanisms maintain stemness and differentiation potential of P19 embryonic carcinoma cells

ABSTRACTGiven the relatively long life of stem cells (SCs), efficient mechanisms of quality control to balance cell survival and resistance to external and internal stress are required. Our objective was to test the relevance of cell quality control mechanisms for SCs maintenance, differentiation and resistance to cell death. We compared cell quality control in P19 stem cells (P19SCs) before and after differentiation (P19dCs). Differentiation of P19SCs resulted in alterations in parameters involved in cell survival and protein homeostasis, including the redox system, cardiolipin and lipid profiles, unfolded protein response, ubiquitin-proteasome and lysosomal systems, and signaling pathways controlling cell growth. In addition, P19SCs pluripotency was correlated with stronger antioxidant protection, modulation of apoptosis, and activation of macroautophagy, which all contributed to preserve SCs quality by increasing the threshold for cell death activation. Furthermore, our findings identify critical roles for the PI3K-AKT-MTOR pathway, as well as autophagic flux and apoptosis regulation in the maintenance of P19SCs pluripotency and differentiation potential.Abbreviations: 3-MA: 3-methyladenine; AKT/protein kinase B: thymoma viral proto-oncogene; AKT1: thymoma viral proto-oncogene 1; ATG: AuTophaGy-related; ATF6: activating transcription factor 6; BAX: BCL2-associated X protein; BBC3/PUMA: BCL2 binding component 3; BCL2: B cell leukemia/lymphoma 2; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CL: cardiolipin; CTSB: cathepsin B; CTSD: cathepsin D; DDIT3/CHOP: DNA-damage inducible transcript 3; DNM1L/DRP1: dynamin 1-like; DRAM1: DNA-damage regulated autophagy modulator 1; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2, subunit alpha; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; ESCs: embryonic stem cells; KRT8/TROMA-1: cytokeratin 8; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NANOG: Nanog homeobox; NAO: 10-N-nonyl acridine orange; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; OPA1: OPA1, mitochondrial dynamin like GTPase; P19dCs: P19 differentiated cells; P19SCs: P19 stem cells; POU5F1/OCT4: POU domain, class 5, transcription factor 1; PtdIns3K: phosphatidylinositol 3-kinase; RA: retinoic acid; ROS: reactive oxygen species; RPS6KB1/p70S6K: ribosomal protein S6 kinase, polypeptide 1; SCs: stem cells; SOD: superoxide dismutase; SHC1-1/p66SHC: src homology 2 domain-containing transforming protein C1, 66 kDa isoform; SOX2: SRY (sex determining region Y)-box 2; SQSTM1/p62: sequestosome 1; SPTAN1/αII-spectrin: spectrin alpha, non-erythrocytic 1; TOMM20: translocase of outer mitochondrial membrane 20; TRP53/p53: transformation related protein 53; TUBB3/betaIII-tubulin: tubulin, beta 3 class III; UPR: unfolded protein response; UPS: ubiquitin-proteasome system

E2f1 regulates the induction of promyelocytic leukemia zinc finger transcription in neuronal differentiation of pluripotent P19 embryonal carcinoma cells

During brain development, the expression of promyelocytic leukemia zinc finger (Plzf) in neural stem cells is precisely controlled to maintain the balance between neural stem cell self-renewal and differentiation. However, the mechanism underlying transcriptional regulation of Plzf in neural stem cell is still unclear. Herein, using P19 embryonal carcinoma cells as a model, we observed that Plzf expression was induced in the P19-derived embryonic bodies, which enrich neural stem-like cell populations, as demonstrated by the expression of neural stem cell markers, Nestin and Sox2. We then characterized the Plzf promoter and identified two E2f1 binding sites (−755/-751 and −53/-49, the transcription start site was designated as +1) are important for the activation of Plzf promoter. Finally, we found that the induction of Plzf in the neural stem-like cells derived from pluripotent P19 cells is decrease by E2f1 knockdown. Taken together, we conclude that E2f1 is an important transcription factor that regulates Plzf transcription and may involve in maintaining the self-renewal ability of neural stem cells.

SGTb regulates a surface localization of a guidance receptor BOC to promote neurite outgrowth

Neuritogenesis is a critical event for neuronal differentiation and neuronal circuitry formation during neuronal development and regeneration. Our previous study revealed a critical role of a guidance receptor BOC in a neuronal differentiation and neurite outgrowth. However, regulatory mechanisms for BOC signaling pathway remain largely unexplored. In the current study, we have identified Small glutamine-rich tetratricopeptide repeat (TPR)-containing b (SGTb) as a BOC interacting protein through yeast two-hybrid screening. Like BOC, SGTb is highly expressed in brain and P19 embryonal carcinoma (EC) cells differentiated into neuronal cells. BOC and SGTb proteins co-precipitate in mouse brain and differentiated P19 EC cells. Furthermore, BOC and SGTb co-localize in neurites and especially are concentrated at the tip of neurites in various neuronal cells. SGTb depletion attenuates neuronal differentiation of P19 cells through reduction of the surface level of BOC. Additionally, SGTb depletion causes BOC localization at neurite tip, coinciding with decreased p-JNK levels critical for actin cytoskeleton remodeling. The overexpression of SGTb or BOC restores JNK activation in BOC or SGTb-depleted cells, respectively. Finally, SGTb elevates the level of surface-resident BOC in BOC-depleted cells, restoring JNK activation. Taken together, our data suggest that SGTb interacts with BOC and regulates its surface level and consequent JNK activation, thereby promoting neuronal differentiation and neurite outgrowth.

Dynamic spatiotemporal patterns of alternative splicing of an F-actin scaffold protein, afadin, during murine development

An F-actin scaffold protein, afadin, comprises two splice variants called l-afadin (a long isoform) and s-afadin (a short isoform). It is known that in adult tissues, l-afadin is ubiquitously expressed while s-afadin is restrictedly expressed in brain. In cultured cortical neurons, l-afadin potentiates axonal branching whereas s-afadin blocks axonal branching by functioning as a naturally occurring dominant-negative isoform that forms a heterodimer with l-afadin. However, the temporal and spatial expression pattern of s-afadin during development or across multiple tissues and organs has not been fully understood. In this study, using Western blotting and RT-qPCR techniques and the fluorescent splicing reporters, we examined the detailed expression patterns of l- and s-afadin isoforms across various tissues and cell types, including rat organs at developmental stages, primary cultured neuronal and non-neuronal cells prepared from the developing rat brain, and in neurons in vitro generated from P19 embryonal carcinoma (EC) cells. Both mRNA and protein of s-afadin were abundantly expressed in various regions of rat neuronal tissues, and their expression dynamically changed during development in vivo. The expression of s-afadin was also detected in primary rat cortical neurons, but not in astrocytes or fibroblasts, and the neuronal expression increased during neuronal maturation in vitro. The dynamic alternative splicing event of afadin during development was successfully visualized with the newly developed fluorescent splicing reporter plasmids at a single cell level. Moreover, s-afadin was undetectable in undifferentiated EC cells, and the expression was induced upon differentiation of the cells into neurons. These data suggest that spatiotemporal and dynamic alternative splicing produces differential expression patterns of afadin isoforms and that alternative splicing of afadin is controlled by the neuron-specific regulator(s) whose activity is triggered and dynamically altered during neuronal differentiation and maturation.

The status of MAPK cascades contributes to the induction and activation of Gata4 and Nkx2.5 during the stepwise process of cardiac differentiation

Cardiac differentiation in vitro is a complex, stepwise process that is rigidly governed by a subset of transcription factors and signaling cascades. In this study, we investigated the cooperation of cardiac-specific transcription factors Gata4 and Nkx2.5, as well as mitogen-activated protein kinase (MAPK) cascades. P19 embryonic carcinoma cells were induced into spontaneously beating cardiomyocytes utilizing a two-step protocol that comprised an early stage and a late stage of differentiation. During early-stage differentiation in suspension culture, P19 cells aggregated to form embryoid bodies (EBs), and the Gata4 and Nkx2.5 genes were induced. However, Gata4 expressed at the early stage of differentiation was incapable of activating downstream gene expression, as it was localized in the cytoplasm and prone to degradation. After EBs were plated for late-stage differentiation in adherent culture, the MAPK cascades were highly activated and contributed to the activation of Gata4 and Nkx2.5. Specifically, we revealed that p38 signaling participated in regulating the localization and stabilization of Gata4 and Nkx2.5. Additionally, the JNK cascade regulated late-stage cardiac differentiation; JNK kinase reduced Gata4 stabilization and conversely alleviated Nkx2.5 degradation by direct interaction and phosphorylation of Nkx2.5. Finally, we found that the C-terminal domain of Nkx2.5 was required for its stabilization under conditions of oxidative stress and JNK activation. Overall, our results indicated that the induction and activation of Gata4 and Nkx2.5 during early- and late-stage cardiac differentiation was closely associated with the function of the MAPK signaling cascades.

Aryl hydrocarbon receptor mediates the cardiac developmental toxicity of EOM from PM2.5 in P19 embryonic carcinoma cells

Ambient fine particulate matter (PM2.5) has been found to be associated with congenital heart defects, but the molecular mechanisms remain to be elucidated. Our previous study revealed that extractable organic matter (EOM) from PM2.5 exerted cardiac developmental toxicity in zebrafish embryos. The aim of the current study is to explore the effects of EOM on cardiac differentiation of P19 mouse embryonic carcinoma stem cells. We found that EOM at 10 μg/ml (a non-cytotoxic dose level) significantly reduced the proportion of cardiac muscle troponin (cTnT) positive cells and the percentage of spontaneously beating embryoid bodies, indicating a severe inhibition of cardiac differentiation. Immunofluorescence and qPCR data demonstrated that EOM increased the expression levels of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1A1 and diminished the expression level of β-catenin. Furthermore, EOM treatment significantly upregulated cell proliferation rate and elevated the percentage of γH2A.X positive cells without affecting apoptosis. It is worth noting that the EOM-induced changes in gene expression, cellular proliferation and DNA double strain breaks were attenuated by the AhR antagonist CH223191. In conclusion, our data indicate that AhR mediates the inhibitory effects of EOM (from PM2.5) on the cardiac differentiation of P19 cells.

The combined effects of three-dimensional cell culture and natural tissue extract on neural differentiation of P19 embryonal carcinoma stem cells.

Tissue engineering, as a novel transplantation therapy, aims to create biomaterial scaffolds resembling the extracellular matrix in order to regenerate the damaged tissues. Adding bioactive factors to the scaffold would improve cell-tissue interactions. In this study, the effect of chitosan polyvinyl alcohol nanofibres containing carbon nanotube scaffold with or without active bioglass (BG+ /BG- ), in combination with neonatal rat brain extract on cell viability, proliferation, and neural differentiation of P19 embryonic carcinoma stem cells was investigated. To induce differentiation, the cells were cultured in α-MEM supplemented with neonatal rat brain extract on the scaffolds. The expression of undifferentiated stem cell markers as well as neuroepithelial and neural-specific markers was evaluated and confirmed by real-time Reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence procedures. Finally, the three-dimensional (3D) cultured cells were implanted into the damaged neural tubes of chick embryos, and their fates were followed in ovo. Based on the histological and immunofluorescence observations, the transplanted cells were able to survive, migrate, and penetrate into the host embryonic tissues. Gene network analysis suggested the possible involvement of neurotransmitters as a downstream target of synaptophysin and tyrosine hydroxylase. Overall, the results of this study indicated that combining the effects of 3D cell culture and natural brain tissue extract can accelerate the differentiation of P19 embryonic carcinoma cells into neuronal phenotype cells.

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium.

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES+ neural progenitors (~ 46%), TUBB3+ immature neurons (~ 6%), MAP2+ mature neurons (~ 2%), and GFAP+ astrocytes (~ 50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.

P19 Cells as a Model for Studying the Circadian Clock in Stem Cells before and after Cell Differentiation.

In mammals, circadian rhythmicity is sustained via a transcriptional/translational feedback loop referred to as the canonical molecular circadian clock. Circadian rhythm is absent in undifferentiated embryonic stem cells; it begins only after differentiation. We used pluripotent P19 embryonal carcinoma stem cells to check the biological clock before and after differentiation into neurons using retinoic acid. We show that the central clock genes ARNTL (Bmal), Per2 and Per3, and the peripheral clock genes Rev-erb-α and ROR-α, oscillate before and after differentiation, as does the expression of the neuronal differentiation markers Hes5, β-3-tubulin (Tubb3) and Stra13, but not Neurod1. Furthermore, the known clock-modulating compounds ERK, EGFR, Pi3K, p38, DNA methylation and Sirtiun inhibitors, in addition to Rev-erb-α ligands, modulate the expression of central and peripheral clock genes. Interestingly Sirtinol, Sirt1 and Sirt2 inhibitors had the greatest significant effect on the expression of clock genes, and increased Hes5 and Tubb3 expression during neuronal differentiation. Our findings reveal a new frontier of circadian clock research in stem cells: contrary to what has been published previously, we have shown the clock to be functional and to oscillate, even in undifferentiated stem cells. Modulating the expression of clock genes using small molecules could affect stem cell differentiation.

Dural effects of oxidative stress on cardiomyogenesis via Gata4 transcription and protein ubiquitination

Oxidative stress generates reactive oxygen species (ROS) that can promote or inhibit cardiac differentiation of stem cells dependent on the intensity of stimuli as well as cellular context in redox and differentiation status. In the current study, we confirmed that suitable intensity of hydrogen peroxide at the formation stage of embryoid bodies (EBs) effectively favored the formation of spontaneously beating cardiomyocytes from P19 embryonal carcinoma cells. Mechanistic studies implicated that extrinsic ROS enhanced the Caspase-mediated degradation of Oct4 and Nanog, two factors that governing pluripotent property. Further experiments suggested that a cohort of Nanog together with histone deacetylase 4 (Hdac4) played a critical role in establishing and maintaining the silent transcriptional status of Gata4 and Nkx2.5 in undifferentiated cells. Thus, an impulse of hydrogen peroxide depleted Nanog and Hdac4 via a caspase-dependent manner to ameliorate the repression on Gata4 and Nkx2.5 promoters, thereby generating a persistent activation on cardiac differentiation program. Meanwhile, we found that excessive ROS-activated JNK cascade to facilitate the ubiquitination and subsequent degradation of Gata4 protein. Overall, our results indicate that suitable ROS promotes the activation of Gata4 in transcription, while excessive ROS targets Gata4 protein for proteasome-dependent degradation. Gata4 is an important modulator balancing the promoting and inhibitory effects of oxidative stress on differentiation program of cardiomyogenesis.

Retinoic Acid Signaling in P19 Stem Cell Differentiation.

Retinoic acid (RA), especially all-trans retinoic acid is the most potent natural metabolite of vitamin A. RA is involved in a variety of biological functions including embryogenesis, cell differentiation and apoptosis. RA acts through its nuclear receptors to induce transcription of specific target genes. Mouse P19 embryonic carcinoma (EC) stem cells (ES) are one of the most studied in vitro systems for RA-induced differentiation. P19 ES cells can differentiate to endodermal-like, mesodermal-like, and neuronal-like phenotypes in response to specific morphogens including RA and dimethyl sulfoxide (DMSO). At low concentrations, RA directs P19 ES cells to differentiate into cells displaying an endodermal phenotype, whereas at higher concentrations it induces differentiation to neuroectoderm. In the past, many RA---regulated genes have been discovered in EC and ES cells and efforts are ongoing to elucidate the exact mechanisms of RA-induced ES cell differentiation and apoptosis. In the RA-triggered differentiation process of the P19 ES cells, several proteins belonging to different families participate, some being obligatory while others, dispensable. Revealing the mechanisms behind RA-induced effects on ES cells has a bearing on understanding how cells proliferate, differentiate and undergo apoptosis that can provide greater insight into cancer biology and therapy. In addition to summarizing the reports on gene/protein targets of RA in stem cells, the signaling pathways driven by some of the specific class of proteins in the presence or absence of RA in P19 ES cell differentiation, especially to an endodermal phenotype, are the focus of this review.

Extracellular Signal-regulated Kinases (ERKs) Phosphorylate Lin28a Protein to Modulate P19 Cell Proliferation and Differentiation

Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA (miRNA) and is implicated in cell proliferation and pluripotency control. However, little is known about how Lin28a function is modulated at the post-translational level and thereby responds to major signaling pathways. Here we show that Lin28a is directly phosphorylated by ERK1/2 kinases at Ser-200. By editing lin28a gene with the CRISPR/Cas9-based method, we generated P19 mouse embryonic carcinoma stem cells expressing Lin28a-S200A (phospho-deficient) and Lin28a-S200D (phospho-mimetic) mutants, respectively, to study the functional impact of Ser-200 phosphorylation. Lin28a-S200D-expressing cells, but not Lin28a-S200A-expressing or control P19 embryonic carcinoma cells, displayed impaired inhibition of let-7 miRNA and resulted in decreased cyclin D1, whereas Lin28a-S200A knock-in cells expressed less let-7 miRNA, proliferated faster, and exhibited differentiation defect upon retinoic acid induction. Therefore our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process.

Cytotoxicity Evaluation of Methanol Extracts of Some Medicinal Plants on P19 Embryonal Carcinoma Cells

Ionic Liquid-based Microwave-Assisted Extraction (Il-MAE) of Oxyresveratrol from Morus alba RootsMazaya Fadhila, Abdul Mun’im, Mahdi Jufri

Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells

Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation.

A Sonic hedgehog coreceptor, BOC regulates neuronal differentiation and neurite outgrowth via interaction with ABL and JNK activation

Neurite outgrowth is a critical step for neurogenesis and remodeling synaptic circuitry during neuronal development and regeneration. An immunoglobulin superfamily member, BOC functions as Sonic hedgehog (Shh) coreceptor in canonical and noncanonical Shh signaling in neuronal development and axon outgrowth/guidance. However signaling mechanisms responsible for BOC action during these processes remain unknown. In our previous studies, a multiprotein complex containing BOC and a closely related protein CDO promotes myogenic differentiation through activation of multiple signaling pathways, including non-receptor tyrosine kinase ABL. Given that ABL and Jun. N-terminal kinase (JNK) are implicated in actin cytoskeletal dynamics required for neurogenesis, we investigated the relationship between BOC, ABL and JNK during neuronal differentiation. Here, we demonstrate that BOC and ABL are induced in P19 embryonal carcinoma (EC) cells and cortical neural progenitor cells (NPCs) during neuronal differentiation. BOC-depleted EC cells or Boc−/− NPCs exhibit impaired neuronal differentiation with shorter neurite formation. BOC interacts with ABL through its putative SH2 binding domain and seems to be phosphorylated in an ABL activity-dependent manner. Unlike wildtype BOC, ABL-binding defective BOC mutants exhibit impaired JNK activation and neuronal differentiation. Finally, Shh treatment enhances JNK activation which is diminished by BOC depletion. These data suggest that BOC interacts with ABL and activates JNK thereby promoting neuronal differentiation and neurite outgrowth.

Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells.

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is a commonly abused recreational drug that causes neurotoxic effects in both humans and animals. The mechanism behind MDMA-induced neurotoxicity is suggested to be species-dependent and needs to be further investigated on the cellular level. In this study, the effects of MDMA in neuronally differentiated P19 mouse embryonal carcinoma cells have been examined. MDMA produces a concentration-, time- and temperature-dependent toxicity in differentiated P19 neurons, as measured by intracellular MTT reduction and extracellular LDH activity assays. The P19-derived neurons express both the serotonin reuptake transporter (SERT), that is functionally active, and the serotonin metabolizing enzyme monoamine oxidase A (MAO-A). The involvement of these proteins in the MDMA-induced toxicity was investigated by a pharmacological approach. The MAO inhibitors clorgyline and deprenyl, and the SERT inhibitor fluoxetine, per se or in combination, were not able to mimic the toxic effects of MDMA in the P19-derived neurons or block the MDMA-induced cell toxicity. Oxidative stress has been implicated in MDMA-induced neurotoxicity, but pre-treatment with the antioxidants α-tocopherol or N-acetylcysteine did not reveal any protective effects in the P19 neurons. Involvement of mitochondria in the MDMA-induced cytotoxicity was also examined, but MDMA did not alter the mitochondrial membrane potential (ΔΨm) in the P19 neurons. We conclude that MDMA produce a concentration-, time- and temperature-dependent neurotoxicity and our results suggest that the mechanism behind MDMA-induced toxicity in mouse-derived neurons do not involve the serotonergic system, oxidative stress or mitochondrial dysfunction.

Disruption of Retinol (Vitamin A) Signaling by Phthalate Esters: SAR and Mechanism Studies.

A spectrum of reproductive system anomalies (cryptorchidism, hypospadias, dysgenesis of Wolffian duct-derived tissues and prostate, and reduced sperm production) in male rats exposed in utero to phthalate esters (PEs) are thought to be caused by PE inhibition of fetal testosterone production. Recently, dibutyl and dipentyl phthalate (DBuP, DPnP) were shown to disrupt the retinol signaling pathway (RSP) in mouse pluripotent P19 embryonal carcinoma cells in vitro. The RSP regulates the synthesis and cellular levels of retinoic acid (RA), the active metabolite of retinol (vitamin A). In this new study, a total of 26 di- and mono-esters were screened to identify additional phthalate structures that disrupt the RSP and explore their mechanisms of action. The most potent PEs, those causing > 50% inhibition, contained aryl and cycloalkane groups or C4-C6 alkyl ester chains and were the same PEs reported to cause malformations in utero. They shared similar lipid solubility; logP values were between 4 and 6 and, except for PEs with butyl and phenyl groups, were stable for prolonged periods in culture. Mono- and cognate di-esters varied in ability to disrupt the RSP; e.g., DEHP was inactive but its monoester was active while DBuP was active yet its monoester was inactive. DBuP and dibenzyl phthalate both disrupted the synthesis of RA from retinol but not the ability of RA to activate gene transcription. Both PEs also disrupted the RSP in C3H10T1/2 multipotent mesenchymal stem cells. Based on this in vitro study showing that some PEs disrupt retinol signaling and previous in vivo studies that vitamin A/RA deficiency and PEs both cause strikingly similar anomalies in the male rat reproductive system, we propose that PE-mediated inhibition of testosterone and RA synthesis in utero are both causes of malformations in male rat offspring.

BRG1 interacts with GLI2 and binds Mef2c gene in a hedgehog signalling dependent manner during in vitro cardiomyogenesis.

BackgroundThe Hedgehog (HH) signalling pathway regulates cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (mES) cell model. To further assess the transcriptional role of HH signalling during cardiomyogenesis in stem cells, we studied the effects of overexpressing GLI2, a primary transducer of the HH signalling pathway, in mES cells.ResultsStable GLI2 overexpression resulted in an enhancement of cardiac progenitor-enriched genes, Mef2c, Nkx2-5, and Tbx5 during mES cell differentiation. In contrast, pharmacological blockade of the HH pathway in mES cells resulted in lower expression of these genes. Mass spectrometric analysis identified the chromatin remodelling factor BRG1 as a protein which co-immunoprecipitates with GLI2 in differentiating mES cells. We then determined that BRG1 is recruited to a GLI2-specific Mef2c gene element in a HH signalling-dependent manner during cardiomyogenesis in P19 EC cells, a mES cell model.ConclusionsThus, we propose a mechanism where HH/GLI2 regulates the expression of Mef2c by recruiting BRG1 to the Mef2c gene, most probably via chromatin remodelling, to ultimately regulate in vitro cardiomyogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0127-8) contains supplementary material, which is available to authorized users.

Dynamic expression of the mouse orthologue of the human amyotropic lateral sclerosis associated gene C9orf72 during central nervous system development and neuronal differentiation.

The hexanucleotide repeat in the first intron of the C9orf72 gene is the most significant cause of amyotropic lateral sclerosis as well as some forms of fronto-temporal dementia. The C9orf72 protein has been previously reported to be expressed in post-mortem human brain as well as in late embryonic and some postnatal stages in mice. Herein, we present a detailed study of the distribution of C9orf72 protein in the embryonic, postnatal and adult mouse brain, spinal cord as well as during the differentiation of P19 embryonal carcinoma cells to neurons including motor neurons. We show that the expression levels of the C9orf72 transcripts in the developing and adult mouse brain as well as in differentiating neurons, are dynamic. Besides the strong expression in the cerebellum and motor cortex reported previously, we show for the first time that C9orf72 is expressed strongly in the olfactory bulb and also in the hippocampus. Our immunostaining data also reveal a hitherto unreported switch in the cellular distribution of C9orf72 from a predominantly cytoplasmic to a nucleo-cytoplasmic distribution during corticogenesis. This switch in distribution was also observed during differentiation of the pluripotent embryonal carcinoma P19 cell line to mature neurons. Our findings have implications for interpreting the pathophysiology caused by the repeat expansions in C9orf72 in mouse models.

Abstract 203: A Shh Coreceptor Cdo is Required for Efficient Cardiomyogenesis of Pluripotent Stem Cells

Sonic hedgehog (Shh) signaling plays an important role for early heart development, such as heart looping and cardiomyogenesis of pluripotent stem cells. A multifunctional receptor Cdo functions as a Shh coreceptor together with Boc and Gas1 to activate Shh signaling and these coreceptors seem to play compensatory roles in early heart development. Thus in this study, we examined the role of Cdo in cardiomyogenesis by utilizing an in vitro differentiation of pluripotent stem cells. Here we show that Cdo is required for efficient cardiomyogenesis of pluripotent stem cells by activation of Shh signaling. Cdo is induced concurrently with Shh signaling activation upon induction of cardiomyogenesis of P19 embryonal carcinoma (EC) cells. Cdo -depleted P19 EC and Cdo –/– mouse embryonic stem (ES) cells display decreased expression of key cardiac regulators, including Gata4, Nkx2.5 and Mef2c and this decrease coincides with reduced Shh signaling activities. Furthermore Cdo deficiency causes a stark reduction in formation of mature contractile cardiomyocytes. This defect in cardiomyogenesis is overcome by reactivation of Shh signaling at the early specification stage of cardiomyogenesis. The Shh agonist treatment restores differentiation capacities of Cdo -deficient ES cells into contractile cardiomyocytes by recovering both the expression of early cardiac regulators and structural genes such as cardiac troponin T and Connexin 43. Therefore Cdo is required for efficient cardiomyogenesis of pluripotent stem cells and an excellent target to improve the differentiation potential of stem cells for generation of transplantable cells to treat cardiomyopathies.