6 research outputs found
Induction of Olig2+ Precursors by FGF Involves BMP Signalling Blockade at the Smad Level
During normal development oligodendrocyte precursors (OPCs) are generated in the ventral spinal cord in response to Sonic hedgehog (Shh) signalling. There is also a second, late wave of oligodendrogenesis in the dorsal spinal cord independent of Shh activity. Two signalling pathways, controlled by bone morphogenetic protein and fibroblast growth factor (FGF), are active players in dorsal spinal cord specification. In particular, BMP signalling from the roof plate has a crucial role in setting up dorsal neural identity and its inhibition is sufficient to generate OPCs both in vitro and in vivo. In contrast, FGF signalling can induce OPC production from dorsal spinal cord cultures in vitro. In this study, we examined the cross-talk between mitogen-activated protein kinase (MAPK) and BMP signalling in embryonic dorsal spinal cord cultures at the SMAD1/5/8 (SMAD1) transcription factor level, the main effectors of BMP activity. We have previously shown that FGF2 treatment of neural precursor cells (NPCs) derived from rat E14 dorsal spinal cord is sufficient to generate OPCs in vitro. Utilising the same system, we now show that FGF prevents BMP-induced nuclear localisation of SMAD1-phosphorylated at the C-terminus (C-term-pSMAD1). This nuclear exclusion of C-term-pSMAD1 is dependent on MAPK activity and correlates with OLIG2 upregulation, the obligate transcription factor for oligodendrogenesis. Furthermore, inhibition of the MAPK pathway abolishes OLIG2 expression. We also show that SMAD4, which acts as a common partner for receptor-regulated Smads including SMAD1, associates with a Smad binding site in the Olig2 promoter and dissociates from it upon differentiation. Taken together, these results suggest that FGF can promote OPC generation from embryonic NPCs by counteracting BMP signalling at the Smad1 transcription factor level and that Smad-containing transcriptional complexes may be involved in direct regulation of the Olig2 promoter
Smad expression, MAPK signalling analysis and quantification of OLIG2 expressing cells in DSC neural precursor cell cultures.
<p>A. Semi-quantitative RT-PCR analysis of <i>Smad1</i>, <i>5</i>, and <i>8</i> expression in primary E14 rat dorsal spinal cord cultures. Rat E14 trunk cDNA was used as positive control. B. Western blot analysis of extracts from different DSC culture conditions using anti-phospho ERK1/2 polyclonal antibody to assess MAPK activity. Anti-ERK2 antibody was used as a loading control. C. Primary cultures of E14 dorsal neural precursor cells were treated with FGF2, BMP4 or FGF2+U0126 for 6, 24 or 72 hours and stained for OLIG2 expression. Pictographs showing representative fields for different culture conditions after 72 hours of treatment. D. Cells expressing OLIG2 were counted and represented as percent of the total cell number. Data represent mean +/β SEM. FGF2 treatment 72 h time point is statistically different from 6 and 24 h (ANOVA with Tukey HSD test, p<0.01). FGF2 treatment is also statistically different at each time point compared to other conditions (*,p<0.05; **, p<0.0001; *** p<0.0001; ANOVA with Tukey HSD).</p
FGF2 regulates sub-cellular localisation of SMAD1.
<p>A. Primary cultures of E14 dorsal neural precursor cells were treated with FGF2, BMP4 or FGF2+U0126 for 6 hours or 24 hours and labelled for C-term-pSMAD1. Representative fields from 6 hours treatment is shown. B. Cells showing an exclusive nuclear or an exclusive cytoplasmic localisation were counted and are represented as a percent of total C-term-pSMAD1 expressing cells. Data represent mean +/β SEM. FGF2 treatment at each time point is statistically different from the three other groups (ANOVA and t-Test with Bonferonni, p<0.001). C. Alignment of the linker regions of receptor-regulated Smads and identification of MAPK phosphorylation sites. Conserved phosphorylation motifs between human SMAD1 and rat SMADs are highlighted in grey; green marking the peptide L-pSMAD1 antibody is raised against. D. Primary cultures of E14 dorsal neural precursor cells were treated with FGF2, or BMP4 for 6 hours and stained for L-pSMAD1. Representative fields are shown. E. Comparison of L-pSMAD1 cytoplasmic/nuclear signal levels between FGF2 and BMP4 treated samples. Data represent mean +/β SEM (p<0.05, paired T-test).</p
SMAD4, co-partner for all Receptor-Smads, binds to <i>Olig2</i> promoter.
<p>A. Semi-quantitative RT-PCR analysis of <i>Olig2</i> expression in undifferentiated (control) and neuralised (FGF2 only and FGF2/Shh/Retinoic Acid (F/S/R)) W9.5 mES cells. mES cells neuralised in chemically defined medium (CDM) for 4 days were further differentiated for 4 days in CDM+FGF2 (d8 FGF) or CDM+F/S/R (d8 F/S/R). <i>Olig2</i> transcription is not detected in undifferentiated mES cells but is strongly induced upon 4 day differentiation in FGF2/Shh/Retinoic Acid containing medium. B. Schematic representation of putative Smad binding sites (BS) targeted for analysis in 4 kb promoter region of <i>Olig2</i>. PCR products identified by agarose gel electrophoresis after chromatin immunoprecipitation from W9.5 mES cells with Smad4 antibody. Smad4 binding was detected at BS3 only. No Smad4 binding is detected using beads only or at 3β² UTR of <i>Olig2</i>. Smad4 binding at BS3 site is lost upon differentiation. Nucleotides represented in uppercase are exact matches to previously described consensus binding sequences at respective Smad sites in the <i>Olig2</i> promoter. The positions of the binding sites are shown relative to the transcription start site.</p
Ubiquitylation on Canonical and Non-canonical Sites Targets the Transcription Factor Neurogenin for Ubiquitin-mediated Proteolysis*
Polyubiquitylation targets multiple proteins for degradation by the proteasome. Typically, the first ubiquitin is linked to lysine residues in the substrate for degradation via an isopeptide bond, although rarely ubiquitin linkage to the N-terminal residue has also been observed. We have recently shown that Neurogenin (NGN), a basic helix-loop-helix transcription factor that plays a central role in regulating neuronal differentiation, is degraded by ubiquitin-mediated proteolysis. We have taken a biochemical and mutagenesis approach to investigate sites of ubiquitylation of NGN, initially using extracts of eggs from the frog Xenopus laevis as a source of ubiquitylation and degradation components. NGN can be targeted for destruction by ubiquitylation via lysines or the N terminus. However, we see that a modified NGN, where canonical lysine ubiquitylation and N-terminally linked ubiquitylation are prevented, is nevertheless ubiquitylated and degraded by the proteasome. We show that polyubiquitin chains covalently attach to non-canonical cysteine residues in NGN, and these non-canonical linkages alone are capable of targeting NGN protein for destruction. Importantly, canonical and non-canonical ubiquitylation occurs simultaneously in the native protein and may differ in importance for driving degradation in interphase and mitosis. We conclude that native NGN is ubiquitylated on multiple canonical and non-canonical sites by cellular ubiquitin ligases, and all types of linkage can contribute to protein turnover