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Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins.

Hardwick LJ, Philpott A - Neural Dev (2015)

Bottom Line: We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells.Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity.Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK. ljah2@cam.ac.uk.

ABSTRACT

Background: Basic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 (also known as Xath3), the Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade.

Results: Using the developing Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation.

Conclusions: Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

No MeSH data available.


Both WT and 6T/S-A NeuroD4 generate predominantly sensory neurons (a-e). Embryos were unilaterally injected at the two-cell stage with 100 pg of either WT or 6T/S-A NeuroD4 mRNA and at stage 18, embryos were assayed by qRT-PCR for expression of pan neuronal gene neural-β-tubulin, sensory neuron marker xHox11L2, motor neuron marker xHb9 or interneuron marker xVsx1 (a, b). Data is shown on two separate graphs due to differences in scale [n = 3]. Whole embryo ISH was also conducted to compare the pattern of expression of neural-β-tubulin and xHox11L2 (c) [n = 30–38]. Views in c are all dorso-ventral with injected side to the right; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***
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Fig6: Both WT and 6T/S-A NeuroD4 generate predominantly sensory neurons (a-e). Embryos were unilaterally injected at the two-cell stage with 100 pg of either WT or 6T/S-A NeuroD4 mRNA and at stage 18, embryos were assayed by qRT-PCR for expression of pan neuronal gene neural-β-tubulin, sensory neuron marker xHox11L2, motor neuron marker xHb9 or interneuron marker xVsx1 (a, b). Data is shown on two separate graphs due to differences in scale [n = 3]. Whole embryo ISH was also conducted to compare the pattern of expression of neural-β-tubulin and xHox11L2 (c) [n = 30–38]. Views in c are all dorso-ventral with injected side to the right; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***

Mentions: The bilateral stripes of Xenopus primary neurons give rise to sensory neurons, interneurons and motor neurons (lateral to medial), which can be distinguished by expression of xHox11L2 [37], xVsx1 [38], and xHb9 respectively [39]. All three of these marker genes are endogenously expressed at stage 18, enabling their relative expression to be determined alongside neural-β-tubulin (Fig. 6). Initial analysis of expression of subtype markers was conducted by qRT-PCR, following over-expression of WT or 6T/S-A NeuroD4 mRNA (Fig. 6a-b). The relative increase in neural-β-tubulin expression (four fold for WT and 20 fold for 6T/S-A NeuroD4) is paralleled by the increase in sensory neuron marker xHox11L2 (five-fold for WT and 17 fold for 6T/S-A NeuroD4). In contrast, no significant differences are seen in motor neuron marker xHb9, and in both WT and 6T/S-A NeuroD4 injected embryos there is a reduction in interneuron marker xVsx1. Perron and colleagues also observed a reduction of interneuron marker Pax2 following over-expression of WT NeuroD4 in non-neural ectoderm [23], so this may suggest that interneuron cell fate is being diverted towards sensory cell fate and additionally, NeuroD4-induced ectopic neurons are adopting a sensory identity. We confirmed that 6T/S-A NeuroD4 induces sensory neurons like WT NeuroD4 by in situ hybridisation (Fig. 6c). Ectopic neurons induced by over-expression of WT xNgn2 and WT NeuroD4 have previously been described as Rohon-Beard sensory neurons [23, 40] and in another vertebrate model, chick homologue NeuroM is expressed in the region of the developing sensory neurons in the dorsal root ganglia [21]. This may therefore be the default path of neuronal identity induced by this Neurogenin2-NeuroD4 transcription factor cascade, and SP/TP phosphorylation does not obviously influence neuronal subtype identity. The lack of other neuronal subtypes may be due to a lack of necessary cofactors or extrinsic signals that are required for alternative cell fates. Taken together, these results indicate that phospho-mutant NeuroD4 has considerably enhanced neurogenic activity but phospho-status has no significant effect on the subtype of neurons generated.Fig. 6


Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins.

Hardwick LJ, Philpott A - Neural Dev (2015)

Both WT and 6T/S-A NeuroD4 generate predominantly sensory neurons (a-e). Embryos were unilaterally injected at the two-cell stage with 100 pg of either WT or 6T/S-A NeuroD4 mRNA and at stage 18, embryos were assayed by qRT-PCR for expression of pan neuronal gene neural-β-tubulin, sensory neuron marker xHox11L2, motor neuron marker xHb9 or interneuron marker xVsx1 (a, b). Data is shown on two separate graphs due to differences in scale [n = 3]. Whole embryo ISH was also conducted to compare the pattern of expression of neural-β-tubulin and xHox11L2 (c) [n = 30–38]. Views in c are all dorso-ventral with injected side to the right; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494719&req=5

Fig6: Both WT and 6T/S-A NeuroD4 generate predominantly sensory neurons (a-e). Embryos were unilaterally injected at the two-cell stage with 100 pg of either WT or 6T/S-A NeuroD4 mRNA and at stage 18, embryos were assayed by qRT-PCR for expression of pan neuronal gene neural-β-tubulin, sensory neuron marker xHox11L2, motor neuron marker xHb9 or interneuron marker xVsx1 (a, b). Data is shown on two separate graphs due to differences in scale [n = 3]. Whole embryo ISH was also conducted to compare the pattern of expression of neural-β-tubulin and xHox11L2 (c) [n = 30–38]. Views in c are all dorso-ventral with injected side to the right; (p < 0.05) = *; (p < 0.025) = **; (p < 0.0125) = ***
Mentions: The bilateral stripes of Xenopus primary neurons give rise to sensory neurons, interneurons and motor neurons (lateral to medial), which can be distinguished by expression of xHox11L2 [37], xVsx1 [38], and xHb9 respectively [39]. All three of these marker genes are endogenously expressed at stage 18, enabling their relative expression to be determined alongside neural-β-tubulin (Fig. 6). Initial analysis of expression of subtype markers was conducted by qRT-PCR, following over-expression of WT or 6T/S-A NeuroD4 mRNA (Fig. 6a-b). The relative increase in neural-β-tubulin expression (four fold for WT and 20 fold for 6T/S-A NeuroD4) is paralleled by the increase in sensory neuron marker xHox11L2 (five-fold for WT and 17 fold for 6T/S-A NeuroD4). In contrast, no significant differences are seen in motor neuron marker xHb9, and in both WT and 6T/S-A NeuroD4 injected embryos there is a reduction in interneuron marker xVsx1. Perron and colleagues also observed a reduction of interneuron marker Pax2 following over-expression of WT NeuroD4 in non-neural ectoderm [23], so this may suggest that interneuron cell fate is being diverted towards sensory cell fate and additionally, NeuroD4-induced ectopic neurons are adopting a sensory identity. We confirmed that 6T/S-A NeuroD4 induces sensory neurons like WT NeuroD4 by in situ hybridisation (Fig. 6c). Ectopic neurons induced by over-expression of WT xNgn2 and WT NeuroD4 have previously been described as Rohon-Beard sensory neurons [23, 40] and in another vertebrate model, chick homologue NeuroM is expressed in the region of the developing sensory neurons in the dorsal root ganglia [21]. This may therefore be the default path of neuronal identity induced by this Neurogenin2-NeuroD4 transcription factor cascade, and SP/TP phosphorylation does not obviously influence neuronal subtype identity. The lack of other neuronal subtypes may be due to a lack of necessary cofactors or extrinsic signals that are required for alternative cell fates. Taken together, these results indicate that phospho-mutant NeuroD4 has considerably enhanced neurogenic activity but phospho-status has no significant effect on the subtype of neurons generated.Fig. 6

Bottom Line: We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells.Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity.Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK. ljah2@cam.ac.uk.

ABSTRACT

Background: Basic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 (also known as Xath3), the Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade.

Results: Using the developing Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation.

Conclusions: Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

No MeSH data available.