XB-ART-48993Development June 1, 2014; 141 (11): 2216-24.
The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro.
Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. Here we demonstrate the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, we see that this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro.
PubMed ID: 24821983
Article link: Development
Species referenced: Xenopus laevis
Genes referenced: ascl1 bcl2l11 cdk2 cdknx map2 mdfic myt1 neurod1 neurog2 notch1 rai1 tub
Morpholinos: cdknx MO1
Article Images: [+] show captions
|Fig. 1. Ascl1 function is inhibited by SP-directed phosphorylation. (A) Wild-type and S-A Ascl1 translated in vitro in the presence of 35S-methionine and incubated in buffer (XB), Xenopus interphase or mitotic egg extracts with or without phosphatase, separated by SDS-PAGE. (B) Xenopus embryos injected unilaterally with 100 pg GFP, wild-type Ascl1 or S-A Ascl1, detecting β-III tubulin at stage 19 by in situ hybridisation. (C) The percentage of embryos with no difference (0), moderate increase (1) or substantial increase (2) of β-III tubulin expression comparing injected and uninjected sides (n≥57); ***P≤0.005. (D) qPCR analysis of Delta, Myt1 and β-III tubulin expression in stage 19 Xenopus embryos overexpressing 50 pg Ascl1 or S-A Ascl1 (mean±s.e.m.; *P≤0.05). I, interphase; M, mitotic; WT, wild type.|
|Fig. 2. Phosphomutant Ascl1 confers resistance to cell cycle inhibition of neurogenesis. (A) Xenopus embryos were injected unilaterally in one of two cells, injected side to right, with either 100 pg of GFP or wild-type/S-A Ascl1 mRNA, and 500 pg of CyclinA/Cdk2 (A/2) mRNA, as indicated, and subject to in situ hybridisation for for β-III tubulin at stage 19. (B) Graphical representation of percentage of injected embryos (as indicated) displaying no difference (0), moderate increase (1), substantial increase (2), moderate decrease (−1) and complete loss (−2) of neurons on the injected site compared with the uninjected side (n≥44), ***P≤0.005.|
|Fig. 3. Phosphomutant Ascl1 does not affect proliferation in vivo and induces differentiation independently of Xic1 activity. (A) Embryos were injected in one cell of a two-cell embryo with 100 pg of either GFP or Ascl1/S-A Ascl1 mRNA, and stained for pH3, a marker of mitotic activity, at stage 19. A comparison of the number of pH3+ cells in a fixed area on the injected and uninjected sides was undertaken. Data is presented as mean normalised to wild-type Ascl1±s.e.m. from two independent experiments (n≥32); P≤0.05. (B) Embryos were injected in one cell at the two-cell stage (right side) with 100 pg of either GFP or Ascl1/S-A Ascl1 mRNA, along with 45 pg of Xic mRNA or 20 ng Xic1 morpholino or control morpholino, as indicated, and subject to in situ hybridisation for βIII tubulin at stage 19. (C) Graphical representation of percentage of injected embryos (as indicated) displaying no difference (0), moderate increase (1), substantial increase (2), moderate decrease (−1) and complete loss (−2) of neurons at the injected site compared with the uninjected side (n≥43), ***P≤0.005.|
|Fig. 4. Phosphomutant Ascl1 confers resistance to lateral inhibition. (A) Xenopus embryos were injected in one cell at the two-cell stage, injected side to the right, with either 100 pg of GFP or wild-type/S-A Ascl1 mRNA, and 1 ng of NICD mRNA, as indicated, and stained for β-III tubulin at stage 19. (B) Graphical representation of percentage of injected embryos displaying no difference (0), moderate increase (1), substantial increase (2), moderate decrease (−1) and complete loss (−2) of neurons at the injected site compared with the uninjected side (n≥52) ***P≤0.005.|
|Fig. 5. Phosphomutant Ascl1 promotes neuronal maturity of transdifferentiated neurons. (A) HFL1-derived transdifferentiated neurons 21 days after transduction with wild-type or S-A ASCL1 in BAM or BAM+NEUROD protocols, as indicated, DNA (blue), Tuj1 (green). (B) Sixty randomly selected 10× visual fields were counted to determine the percentage of Hoechst-positive cells that are also Tuj1 positive. (C-G) Forty-nine or more neurons were measured to determine quantitative measurements of morphological maturation. B-G are presented as mean±s.e.m. for a duplicate experiment; *P≤0.05; ***P≤0.005. (H) Representative traces of action potentials in response to step current injections 42 days post-induction (−4 to +26 pA for wild-type BAM, −200 to +500 pA for wild-type BAMN, −4 to +14 pA for S-A BAM, −4 to +14 pA for S-A BAMN). (I) Representative traces of inward sodium and outward potassium currents in response to step depolarisations from a holding potential of −80 to +40 mM in voltage clamp mode. Scale bars: 200 µm. WT, wild type.|
|Fig. 6. Phosphomutant proneural proteins promote neuronal maturity of transdifferentiated neuronal cells in combination with SMs. (A) Immunofluorescence illustrating representative morphologies of transdifferentiated neurons stained for βIII-tub (red), MAP2 (green) expression and DNA (DAPI, blue) of HFL1 cultures under SM conditions at 21 days after transduction with either wild-type ASCL1 and wild-type Ngn2 or S-A ASCL1 and S-A Ngn2. (B) Quantitative measurements of morphological maturation comparing transdifferentiated neurons derived after either wild-type ASCL1 and wild-type Ngn2 or S-A ASCL1 and S-A Ngn2 transductions under SM conditions. (C,D) Whole-cell electrophysiological properties of wild-type ASCL1 and wild-type Ngn2 or S-A ASCL1 and S-A Ngn2 transdifferentiated neurons, as labeled at 42 days after transduction. (C) Action potential firing in response to 10 pA steps of current injection from −20 pA to +50 pA. (D) Inward sodium currents and outward potassium currents triggered in response to voltage steps from −80 mV to +50 mV (+30, 40, 50 mV shown) from a holding potential of −70 mV. Scale bars: 200 µm.|
Philpott, Multi-site phospho-regulation of proneural transcription factors controls proliferation versus differentiation in development and reprogramming. 2016, Pubmed, Xenbase