Knocking out a negative regulator of Hedgehog signaling blocks differentiation of cells into neurons

Hedgehog (Hh) signaling, one of many different protein signaling pathways found in mammals, is vital in many stage of neural development. A major negative regulator of Hh signaling is a protein known as Suppressor of Fused (SUFU), which acts to sequester the full length Gli transcription factors, proteins that can turn genes on and off, in the cytoplasm or facilitates its conversion to a repressive form. The P19 embryonal carcinoma cell line is a model of hind-brain neuronal differentiation and the involvement of Hh signaling, in particular the role of SUFU in this process has yet to be explored. We hypothesize that SUFU is required for P19 neuronal differentiation to occur, and that altering the expression of this protein through CRISPR/Cas9 knockout will result in a loss of differentiation potential. Throughout normal, retinoic acid (RA) induced, differentiation of P19 cells the abundance of SUFU mRNA levels show modest changes, although protein abundance does not change. Knocking out Sufu blocked neuronal differentiation, without changing the stem-ness of the cells or their ability to grow normally. During RA-induced neuron formation, Hh signaling is inhibited in the later stages of the differentiation process, as seen by increased Gli repressor levels over those days. It is likely, then, that knocking out the negative regulator SUFU does not facilitate the conversion of Gli proteins from their full-length active state to their smaller repressive state in those final stages of neuron formation. Together these results show the dynamic nature of Hh signaling during neuron development and showcase the critical role of SUFU in regulating this developmental event

NOX1 and NOX4 are required for the differentiation of mouse F9 cells into extraembryonic endoderm

Mouse F9 cells differentiate to primitive endoderm (PrE) when treated with retinoic acid (RA). Differentiation is accompanied by increased reactive oxygen species (ROS) levels, and while treating F9 cells with antioxidants attenuates differentiation, H2O2 treatment alone is sufficient to induce PrE. We identified the NADPH oxidase (NOX) complexes as candidates for the source of this endogenous ROS, and within this gene family, and over the course of differentiation, Nox1 and Nox 4 show the greatest upregulation induced by RA. Gata6, encoding a master regulator of extraembryonic endoderm is also up-regulated by RA and we provide evidence that NOX1 and NOX4 protein levels increase in F9 cells overexpressing Gata6. Pan-NOX and NOX1-specific inhibitors significantly reduced the ability of RA to induce PrE, and this was recapitulated using a genetic approach to knockdown Nox1 and/or Nox4 transcripts. Interestingly, overexpressing either gene in untreated F9 cells did not induce differentiation, even though each elevated ROS levels. Thus, the data suggests that ROS produced during PrE differentiation is dependent in part on increased NOX1 and NOX4 levels, which is under the control of GATA6. Furthermore, these results suggest that the combined activity of multiple NOX proteins is necessary for the differentiation of F9 cells to primitive endoderm

O-GlcNAcylation and Regulation of Galectin-3 in Extraembryonic Endoderm Differentiation

The regulation of proteins through the addition and removal of O-linked β-N-acetylglucosamine (O-GlcNAc) plays a role in many signaling events, specifically in stem cell pluripo-tency and the regulation of differentiation. However, these post-translational modifications have not been explored in extraembryonic endoderm (XEN) differentiation. Of the plethora of proteins regulated through O-GlcNAc, we explored galectin-3 as a candidate protein known to have various intracellular and extracellular functions. Based on other studies, we predicted a reduction in global O-GlcNAcylation levels and a distinct galectin expression profile in XEN cells relative to embryonic stem (ES) cells. By conducting dot blot analysis, XEN cells had decreased levels of global O-GlcNAc than ES cells, which reflected a disbalance in the expression of genes encoding O-GlcNAc cycle enzymes. Immunoassays (Western blot and ELISA) revealed that although XEN cells (low O-GlcNAc) had lower concentrations of both intracellular and extracellular galectin-3 than ES cells (high O-GlcNAc), the relative secretion of galectin-3 was significantly increased by XEN cells. Inducing ES cells toward XEN in the presence of an O-GlcNAcase inhibitor was not sufficient to inhibit XEN differentiation. However, global O-GlcNAcylation was found to decrease in differentiated cells and the extracellular localization of galectin-3 accompanies these changes. Inhibiting global O-GlcNAcylation status does not, however, impact pluripotency and the ability of ES cells to differentiate to the XEN lineage

NOX1 and NOX4 are required for the differentiation of mouse F9 cells into extraembryonic endoderm

Mouse F9 cells differentiate to primitive endoderm (PrE) when treated with retinoic acid (RA). Differentiation is accompanied by increased reactive oxygen species (ROS) levels, and while treating F9 cells with antioxidants attenuates differentiation, H2O2 treatment alone is sufficient to induce PrE. We identified the NADPH oxidase (NOX) complexes as candidates for the source of this endogenous ROS, and within this gene family, and over the course of differentiation, Nox1 and Nox4 show the greatest upregulation induced by RA. Gata6, encoding a master regulator of extraembryonic endoderm is also up-regulated by RA and we provide evidence that NOX1 and NOX4 protein levels increase in F9 cells overexpressing Gata6. Pan-NOX and NOX1-specific inhibitors significantly reduced the ability of RA to induce PrE, and this was recapitulated using a genetic approach to knockdown Nox1 and/or Nox4 transcripts. Interestingly, overexpressing either gene in untreated F9 cells did not induce differentiation, even though each elevated ROS levels. Thus, the data suggests that ROS produced during PrE differentiation is dependent in part on increased NOX1 and NOX4 levels, which is under the control of GATA6. Furthermore, these results suggest that the combined activity of multiple NOX proteins is necessary for the differentiation of F9 cells to primitive endoderm

<i>Nox</i> overexpression activates canonical Wnt signaling, but does not promote differentiation.

<p><b>(A)</b> Dual-luciferase assay of TCF activity in F9 cells transfected with <i>EV</i> and treated with DMSO or RA, or transfected with pcDNA3.1-<i>Nox1</i> or pcDNA3.1-<i>Nox4</i>. <b>(B)</b> <i>Dab2</i> expression in F9 cells transfected with <i>EV</i> and treated with DMSO or RA, or transfected with pcDNA3.1-<i>Nox1</i>. <b>(C)</b> <i>Dab2</i> expression in F9 cells transfected with <i>EV</i> and treated with DMSO or RA, or transfected with pcDNA3.1-<i>Nox4</i>. <b>(D)</b> Immunoblot analysis for DAB2, TROMA1, and OCT4 in F9 cells transfected with <i>EV</i> and treated with DMSO or RA, or transfected with pcDNA3.1-<i>Nox1</i>, pcDNA3.1-<i>Nox4</i> or both vectors. β-actin was used as a loading control. A total of 3 independent experiments were analyzed and results are presented as mean ± SEM. Letters denote groups of significance (<i>P</i> < 0.05) tested by a One-Way ANOVA followed by a Tukey’s test.</p

Chemical inhibition of NOX proteins attenuates RA-mediated differentiation of F9 cells.

<p><b>(A)</b> MTT cell viability assay of F9 cells treated with DMSO, RA, VAS2870 or ML171. <b>(B)</b> F9 cells were treated with DMSO, RA, VAS2870 and RA, or ML171 and RA and assayed for ROS production using Amplex Red. <b>(C)</b> <i>Dab2</i> expression of F9 cells treated with DMSO, RA, VAS2870, or VAS2870 and RA. <b>(D)</b> Immunoblot analysis for DAB2, TROMA1, and OCT4 in F9 cells treated with DMSO, RA, VAS2870, or VAS2870 and RA. <b>(E)</b> <i>Dab2</i> expression of F9 cells treated with DMSO, RA, ML171, or ML171 and RA. <b>(F)</b> Immunoblot analysis for DAB2, TROMA,1 and OCT4 in F9 cells treated with DMSO, RA, ML171, or ML171 and RA. β-actin was used as a loading control. A total of 4 independent experiments were analyzed and results are presented as mean ± SEM. Letters denote groups of significance (<i>P</i> < 0.05) tested by a One-Way ANOVA followed by a Tukey’s test.</p

<i>Nox1</i> and <i>Nox4</i> knockdown reduces ROS production.

<p>Total RNA was collected from F9 cells transfected with scrambled (scr) si-<i>RNA</i>, si-<i>Nox1</i> and/or si-<i>Nox4</i> siRNA and cultured 4 days with RA treatment. <b>(A)</b> Expression of <i>Nox1</i> and <b>(B)</b> NOX1 protein levels following transfection with <i>scr</i> or si<i>-Nox1</i> and RA treatment. <b>(C)</b> Expression of <i>Nox4</i> and <b>(D)</b> NOX4 protein levels following transfection with <i>scr</i> or si<i>-Nox1</i> and RA treatment. <b>(E)</b> ROS production detected using Amplex Red of F9 cells transfected with <i>scr</i>, si<i>-Nox1</i>, or si<i>-Nox4</i> and treated with RA. A total of 3 independent experiments were analyzed and results are presented as mean ± SEM. * denotes significance (<i>P</i> < 0.05) tested by a Student’s t-Test, whereas letters denote groups of significance (<i>P</i> < 0.05) tested by a One-Way ANOVA followed by a Tukey’s test.</p

O-GlcNAcylation and Regulation of Galectin-3 in Extraembryonic Endoderm Differentiation

The regulation of proteins through the addition and removal of O-linked &beta;-N-acetylglucosamine (O-GlcNAc) plays a role in many signaling events, specifically in stem cell pluripotency and the regulation of differentiation. However, these post-translational modifications have not been explored in extraembryonic endoderm (XEN) differentiation. Of the plethora of proteins regulated through O-GlcNAc, we explored galectin-3 as a candidate protein known to have various intracellular and extracellular functions. Based on other studies, we predicted a reduction in global O-GlcNAcylation levels and a distinct galectin expression profile in XEN cells relative to embryonic stem (ES) cells. By conducting dot blot analysis, XEN cells had decreased levels of global O-GlcNAc than ES cells, which reflected a disbalance in the expression of genes encoding O-GlcNAc cycle enzymes. Immunoassays (Western blot and ELISA) revealed that although XEN cells (low O-GlcNAc) had lower concentrations of both intracellular and extracellular galectin-3 than ES cells (high O-GlcNAc), the relative secretion of galectin-3 was significantly increased by XEN cells. Inducing ES cells toward XEN in the presence of an O-GlcNAcase inhibitor was not sufficient to inhibit XEN differentiation. However, global O-GlcNAcylation was found to decrease in differentiated cells and the extracellular localization of galectin-3 accompanies these changes. Inhibiting global O-GlcNAcylation status does not, however, impact pluripotency and the ability of ES cells to differentiate to the XEN lineage

<i>Nox1</i> and <i>Nox4</i> knockdown attenuates RA-induced differentiation.

<p>RNA was collected from F9 cells transfected with scrambled (<i>scr</i>) si-<i>RNA</i>, si-<i>Nox1</i> and/or si-<i>Nox4</i> siRNA and cultured 4 days with RA treatment. <b>(A)</b> <i>Dab2</i> expression of F9 cells transfected with <i>scr</i>, si<i>-Nox1</i>, or si<i>-Nox4</i> and treated with RA. <b>(B)</b> Immunoblot analysis for DAB2, TROMA1, and OCT4 in F9 cells transfected with <i>scr</i>, si<i>-Nox1</i>, or si<i>-Nox4</i> and treated with RA. <b>(C)</b> Densitometric analysis for DAB2, TROMA1, and <b>(D)</b> OCT4 in F9 cells transfected with <i>scr</i>, si<i>-Nox1</i>, or si<i>-Nox4</i> and treated with RA. β-actin was used as a loading control. A total of 3 independent experiments were analyzed and results are presented as mean ± SEM. Letters and symbols denote groups of significance (<i>P</i> < 0.05) tested by a One-Way ANOVA followed by a Tukey’s test.</p

RA increases NOX1 and NOX4 levels and ROS production.

<p>Total RNA and protein was harvested from F9 cells treated with either DMSO or RA for 4 days. <b>(A)</b> <i>Nox1</i> and <b>(B)</b> <i>Nox4</i> expression following DMSO or RA treatment. (<b>C</b>) NOX1 and NOX4 protein levels following DMSO and RA treatment. <b>(D)</b> Protein levels of NADPH oxidase complex accessory subunits NOXA1, NOXO1, and p22Phox following DMSO and RA treatment. <b>(E)</b> <i>Rac1</i> expression following DMSO or RA treatment. <b>(F)</b> ROS detection using CM-H₂DCFDA or <b>(G)</b> Amplex Red in F9 cells treated with either DMSO or RA. A total of 3–5 independent experiments were analyzed and results presented as mean ± SEM. * denotes significance (<i>P</i> < 0.05) tested by a Student’s t-Test.</p