Insight into Nephrocan Function in Mouse Endoderm Patterning

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5–11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn−/− mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

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Glycosylation of CaV3.2 Channels Contributes to the Hyperalgesia in Peripheral Neuropathy of Type 1 Diabetes

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2020.605312 ◽

2020 ◽

Vol 14 ◽

Author(s):

Sonja Lj. Joksimovic ◽

J. Grayson Evans ◽

William E. McIntire ◽

Peihan Orestes ◽

Paula Q. Barrett ◽

...

Keyword(s):

Type 1 Diabetes ◽

Adult Female ◽

Molecular Mechanisms ◽

Sprague Dawley ◽

Knock Out ◽

Peripheral Diabetic Neuropathy ◽

Novel Treatments

Our previous studies implicated glycosylation of the CaV3.2 isoform of T-type Ca2+ channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant CaV3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our in vitro study we used whole-cell recordings of current-voltage relationships to confirm that CaV3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced in vivo by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and CaV3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the CaV3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native CaV3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in CaV3.2 channels in vivo directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.

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Pluripotency and Epigenetic Factors in Mouse Embryonic Stem Cell Fate Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.00266-15 ◽

2015 ◽

Vol 35 (16) ◽

pp. 2716-2728 ◽

Cited By ~ 57

Author(s):

Lluis Morey ◽

Alexandra Santanach ◽

Luciano Di Croce

Keyword(s):

Cell Fate ◽

Cancer Progression ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Basic Research ◽

Cell Types ◽

Pluripotency Factors ◽

Perfect System

Embryonic stem cells (ESCs) are characterized by their ability to self-renew and to differentiate into all cell types of a given organism. Understanding the molecular mechanisms that govern the ESC state is of great interest not only for basic research—for instance, ESCs represent a perfect system to study cellular differentiationin vitro—but also for their potential implications in human health, as these mechanisms are likewise involved in cancer progression and could be exploited in regenerative medicine. In this minireview, we focus on the latest insights into the molecular mechanisms mediated by the pluripotency factors as well as their roles during differentiation. We also discuss recent advances in understanding the function of the epigenetic regulators, Polycomb and MLL complexes, in ESC biology.

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Expression Trapping: Identification of Novel Genes Expressed in Hematopoietic and Endothelial Lineages by Gene Trapping in ES Cells

Blood ◽

10.1182/blood.v92.12.4622 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4622-4631 ◽

Cited By ~ 45

Author(s):

William L. Stanford ◽

Georgina Caruana ◽

Katherine A. Vallis ◽

Maneesha Inamdar ◽

Michihiro Hidaka ◽

...

Keyword(s):

Large Scale ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Gene Trap ◽

Novel Genes ◽

Specific Expression ◽

Blood Island

Abstract We have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZreporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressedlacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.

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Expression Trapping: Identification of Novel Genes Expressed in Hematopoietic and Endothelial Lineages by Gene Trapping in ES Cells

Blood ◽

10.1182/blood.v92.12.4622.424k23_4622_4631 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4622-4631 ◽

Cited By ~ 4

Author(s):

William L. Stanford ◽

Georgina Caruana ◽

Katherine A. Vallis ◽

Maneesha Inamdar ◽

Michihiro Hidaka ◽

...

Keyword(s):

Large Scale ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Gene Trap ◽

Novel Genes ◽

Specific Expression ◽

Blood Island

We have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZreporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressedlacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.

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Fezf2 transient expression via modRNA with concurrent SIRT1 inhibition enhances differentiation of cortical subcerebral / corticospinal neuron identity from mES cells

10.1101/2020.08.13.242230 ◽

2020 ◽

Author(s):

Cameron Sadegh ◽

Wataru Ebina ◽

Anthony C. Arvanites ◽

Lance S. Davidow ◽

Lee L. Rubin ◽

...

Keyword(s):

Stem Cell ◽

Cortical Neurons ◽

Embryonic Stem ◽

Induced Pluripotent Stem Cell ◽

Sirtuin 1 ◽

Projection Neurons ◽

Specific Expression ◽

Specific Differentiation

AbstractDuring late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

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SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc- SMPDL3B

10.1101/2021.02.24.432815 ◽

2021 ◽

Author(s):

Wei Fan ◽

Shuang Tang ◽

Xiaojuan Fan ◽

Yi Fang ◽

Xiaojiang Xu ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Neural Differentiation ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Sphingolipid Metabolism

AbstractSphingolipids are important structural components of cell membranes and prominent signaling molecules controlling cell growth, differentiation, and apoptosis. Sphingolipids are particularly abundant in the brain, and defects in sphingolipid degradation are associated with several human neurodegenerative diseases. However, molecular mechanisms governing sphingolipid metabolism remain unclear. Here we report that sphingolipid degradation is under transcriptional control of SIRT1, a highly conserved mammalian NAD+-dependent protein deacetylase, in mouse embryonic stem cells (mESCs). Deletion of SIRT1 results in accumulation of sphingomyelin in mESCs, primarily due to reduction of SMPDL3B, a GPI-anchored plasma membrane bound sphingomyelin phosphodiesterase. Mechanistically, SIRT1 regulates transcription of Smpdl3b through c-Myc. Functionally, SIRT1 deficiency-induced accumulation of sphingomyelin increases membrane fluidity and impairs neural differentiation in vitro and in vivo. Our findings discover a key regulatory mechanism for sphingolipid homeostasis and neural differentiation, further imply that pharmacological manipulation of SIRT1-mediated sphingomyelin degradation might be beneficial for treatment of human neurological diseases.

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Abstract 78: Pnmt+ "Primer" Cells Give Rise to Cardiac Myocytes and Neurons in the Mammalian Heart: Development of New Experimental Tools for Cardiac Regenerative Medicine Applications

Circulation Research ◽

10.1161/res.111.suppl_1.a78 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Namita M Varudkar ◽

Jixiang Xia ◽

Ibrahim Abukenda ◽

Karl Pfeifer ◽

Steven Ebert

Keyword(s):

Stem Cell ◽

Regenerative Medicine ◽

Neural Crest ◽

Heart Development ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Stem Cell Differentiation ◽

Left Ventricular

Phenylethanolamine n-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine to epinephrine, and thus serves as a marker for adrenergic cells. We employed a combination of immunofluorescent histochemical staining and genetic fate-mapping strategies to show that two separate Pnmt+ cell populations contribute to heart development. Intrinsic cardiac adrenergic (ICA) cells originate from the primary heart field, and contribute to pacemaking, conduction, and working (contractile) myocardium. A second population of cardiac Pnmt+ cells is derived from migrating neural crest. These neural crest adrenergic (NCA) cells appear to contribute to cardiac neurons. By adulthood, most of the Pnmt+ cells show a distinctively left-sided orientation in the heart, with nearly 90% of them being found in the left atrium and ventricle. Surprisingly large swaths of ventricular muscle are derived from Pnmt+ primer cells. Since this region of the heart is highly vulnerable to coronary artery disease and often sustains varying degrees of damage following myocardial infarction, we hypothesize that directed stem cell differentiation into Pnmt+ primer cells could serve as a valuable resource for repair and/or regeneration of left ventricular myocardium for heart disease patients. To test this hypothesis, we have generated stable recombinant mouse embryonic stem cell (mESC) lines that express various fluorescent marker proteins under the control of the endogenous Pnmt gene regulatory network. These cells can be rapidly expanded in culture, sorted, and used for transplantation studies in animal models to determine their therapeutic effectiveness. The cells can be induced along cardiogenic or neurogenic pathways in vitro, and the resulting Pnmt+ cells from each population can then be collected and tested in vivo. To achieve this goal, we have knocked-in a nuclear-localized enhanced green fluorescent protein into the Pnmt locus to create Pnmt-nEGFP recombinant mESCs and mice. We show that nEGFP expression is specifically expressed in Pnmt+ cells in vitro and in vivo. This strategy allows us to identify and isolate Pnmt+ cells to evaluate their effectiveness for cardiac regenerative medicine applications. .

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Polypyrimidine tract-binding protein blocks miRNA-124 biogenesis to enforce its neuronal-specific expression in the mouse

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809609115 ◽

2018 ◽

Vol 115 (47) ◽

pp. E11061-E11070 ◽

Cited By ~ 6

Author(s):

Kyu-Hyeon Yeom ◽

Simon Mitchell ◽

Anthony J. Linares ◽

Sika Zheng ◽

Chia-Ho Lin ◽

...

Keyword(s):

Neuronal Differentiation ◽

Rna Binding ◽

Binding Protein ◽

Embryonic Stem ◽

Specific Expression ◽

Stem Loop ◽

Polypyrimidine Tract Binding Protein ◽

Precursor Rna

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem–loop to inhibit mature miR-124 expression in vivo and DROSHA cleavage of pri-miR-124-1 in vitro. This function for PTBP1 in repressing miR-124 biogenesis defines an additional regulatory loop in the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often found between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct derepression of miR-124, in addition to indirect effects previously described.

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SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc- SMPDL3B

eLife ◽

10.7554/elife.67452 ◽

2021 ◽

Vol 10 ◽

Author(s):

Wei Fan ◽

Shuang Tang ◽

Xiaojuan Fan ◽

Yi Fang ◽

Xiaojiang Xu ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Neural Differentiation ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Sphingolipid Metabolism

Sphingolipids are important structural components of cell membranes and prominent signaling molecules controlling cell growth, differentiation, and apoptosis. Sphingolipids are particularly abundant in the brain, and defects in sphingolipid degradation are associated with several human neurodegenerative diseases. However, molecular mechanisms governing sphingolipid metabolism remain unclear. Here we report that sphingolipid degradation is under transcriptional control of SIRT1, a highly conserved mammalian NAD+-dependent protein deacetylase, in mouse embryonic stem cells (mESCs). Deletion of SIRT1 results in accumulation of sphingomyelin in mESCs, primarily due to reduction of SMPDL3B, a GPI-anchored plasma membrane bound sphingomyelin phosphodiesterase. Mechanistically, SIRT1 regulates transcription of Smpdl3b through c-Myc. Functionally, SIRT1 deficiency-induced accumulation of sphingomyelin increases membrane fluidity and impairs neural differentiation in vitro and in vivo. Our findings discover a key regulatory mechanism for sphingolipid homeostasis and neural differentiation, further imply that pharmacological manipulation of SIRT1-mediated sphingomyelin degradation might be beneficial for treatment of human neurological diseases.

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Transcriptome Analysis of Dnmt3l Knock-Out Mice Derived Multipotent Mesenchymal Stem/Stromal Cells During Osteogenic Differentiation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.615098 ◽

2021 ◽

Vol 9 ◽

Author(s):

Chih-Yi Yang ◽

Rita Jui-Hsien Lu ◽

Ming-Kang Lee ◽

Felix Shih-Hsian Hsiao ◽

Ya-Ping Yen ◽

...

Keyword(s):

Stromal Cells ◽

Dna Methyltransferase ◽

Bone Development ◽

Es Cells ◽

Embryonic Stem ◽

Differentiation Potential ◽

Knock Out ◽

Cell Based Therapy

Multipotent mesenchymal stem/stromal cells (MSCs) exhibit great potential for cell-based therapy. Proper epigenomic signatures in MSCs are important for the maintenance and the subsequent differentiation potential. The DNA methyltransferase 3-like (DNMT3L) that was mainly expressed in the embryonic stem (ES) cells and the developing germ cells plays an important role in shaping the epigenetic landscape. Here, we report the reduced colony forming ability and impaired in vitro osteogenesis in Dnmt3l-knockout-mice-derived MSCs (Dnmt3l KO MSCs). By comparing the transcriptome between undifferentiated Dnmt3l KO MSCs and the MSCs from the wild-type littermates, some of the differentially regulated genes (DEGs) were found to be associated with bone-morphology-related phenotypes. On the third day of osteogenic induction, differentiating Dnmt3l KO MSCs were enriched for genes associated with nucleosome structure, peptide binding and extracellular matrix modulation. Differentially expressed transposable elements in many subfamilies reflected the change of corresponding regional epigenomic signatures. Interestingly, DNMT3L protein is not expressed in cultured MSCs. Therefore, the observed defects in Dnmt3l KO MSCs are unlikely a direct effect from missing DNMT3L in this cell type; instead, we hypothesized them as an outcome of the pre-deposited epigenetic signatures from the DNMT3L-expressing progenitors. We observed that 24 out of the 107 upregulated DEGs in Dnmt3l KO MSCs were hypermethylated in their gene bodies of DNMT3L knock-down ES cells. Among these 24 genes, some were associated with skeletal development or homeostasis. However, we did not observe reduced bone development, or reduced bone density through aging in vivo. The stronger phenotype in vitro suggested the involvement of potential spreading and amplification of the pre-deposited epigenetic defects over passages, and the contribution of oxidative stress during in vitro culture. We demonstrated that transient deficiency of epigenetic co-factor in ES cells or progenitor cells caused compromised property in differentiating cells much later. In order to facilitate safer practice in cell-based therapy, we suggest more in-depth examination shall be implemented for cells before transplantation, even on the epigenetic level, to avoid long-term risk afterward.

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