Interaction between opposing modes of phospho-regulation of the proneural proteins Ascl1 and Ngn2

Abstract

From the relatively simple nervous system of Drosophila to the elaborate mammalian cortex, neurogenesis requires exceptional spatial and temporal precision to co-ordinate progenitor cell proliferation and subsequent differentiation to a diverse range of neurons and glia. A limited number of transiently expressed proneural basic-helix-loop-helix (bHLH) transcription factors, for example achaete-scute-complex (as-c) and atonal (ato) in Drosophila and the vertebrate homologues Ascl1 and Neurogenin2 (Ngn2), are able to orchestrate the onset of neuronal determination, context-dependent subtype selection and even influence later aspects of neuronal migration and maturation. Within the last decade, two models have emerged to explain how the temporal activity of proneural determination factors is regulated by phosphorylation at distinct sites. One model describes how cell-cycle associated phosphorylation on multiple sites in the N and C termini of vertebrate proneural proteins limits neuronal differentiation in cycling progenitor cells. A second model describes phosphorylation on a single site in the bHLH domain of Drosophila atonal that acts as a binary switch, where phosphorylation terminates proneural activity. Here we combine activating mutations of phosphorylation sites in the N- and C- termini with an inhibitory phospho-mimetic mutation in the bHLH domain of Ascl1 and Ngn2 proteins, and test their functions in vivo using Xenopus embryos to determine which mode of phospho-regulation dominates. Enhancing activity by preventing N- and C terminal phosphorylation cannot overcome the inhibitory effect of mimicking phosphorylation of the bHLH domain. Thus we have established a hierarchy between these two modes of proneural protein control and suggest a model of temporal regulation for proneural protein activity.

Keywords: Ascl1; Neurogenin2; Proneural; Xenopus.; neurogenesis; phospho-regulation.

Figure 1.. S150D single site mutation inactivates both WT and 6S-A Ascl1.

( A) Schematic representation of WT Ascl1 and the three mutant constructs tested. The relative location of the S150D phospho-mimetic substitution within the bHLH domain is indicated (blue), along with the location of the six Serine to Alanine phospho-mutant substitutions in the N and C termini (red). ( B) Western blot analysis of stage 11 whole embryo extracts over-expressing 200pg of each construct, with tubulin as a loading control. All four constructs are expressed in embryos and the additional S150D mutation has no significant effect on WT or 6S-A Ascl1 protein migration or accumulation. ( C– E) Two cell stage embryos were unilaterally injected with 40pg of mRNA encoding each construct. At stage 18, embryos were assayed for expression of neural-β-tubulin relative to uninjected control embryos. ( C) qPCR data [N=2] with significance calculated by paired student T test; * = p< 0.05. ( D) Semi-quantitative scoring of grade of neurogenesis after ISH [N=53-73 embryos per category from two experiments]. ( E) Representative images of embryos with injected side to the right, stained with pale blue β-gal tracer. Induction of neural-β-tubulin by WT and 6S-A Ascl1 is prevented with the respective introduction of the single S150D mutation.

Figure 2.. T149D single site mutation significantly inhibits both WT and 9S-A Ngn2.

( A) Schematic representation of WT Ngn2 and the three mutant constructs tested. The relative location of the T149D phospho-mimetic substitution within the bHLH domain is indicated (blue), along with the location of the nine Serine to Alanine phospho-mutant substitutions in the N and C termini (red). ( B– D) Two cell stage embryos were unilaterally injected with 25pg of mRNA encoding each construct. At stage 18, embryos were assayed for expression of neural-β-tubulin relative to uninjected control embryos. ( B) qPCR data [N=2] with significance calculated by paired student T test; * = p< 0.05; *** = p< 0.0125. ( C) Semi-quantitative scoring of grade of neurogenesis after ISH [N=36–39 embryos per category from two experiments]. ( D) Representative images of embryos with injected side to the right, stained with pale blue β-gal tracer. Introduction of the T149D mutation prevents induction of neural-β-tubulin by WT Ngn2 and significantly inhibits 9S-A Ngn2.