Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

cJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification

Nature Neuroscience volume 13, pages 944–950 (2010)Cite this article

Subjects

Abstract

Neuronal differentiation is accomplished through cascades of intrinsic genetic factors initiated in neuronal progenitors by external gradients of morphogens. Activity has been thought to be important only late in development, but recent evidence suggests that activity also regulates early neuronal differentiation. Activity in post-mitotic neurons before synapse formation can regulate phenotypic specification, including neurotransmitter choice, but the mechanisms are not clear. We identified a mechanism that links endogenous calcium spike activity with an intrinsic genetic pathway to specify neurotransmitter choice in neurons in the dorsal embryonic spinal cord of Xenopus tropicalis. Early activity modulated transcription of the GABAergic/glutamatergic selection gene tlx3 through a variant cAMP response element (CRE) in its promoter. The cJun transcription factor bound to this CRE site, modulated transcription and regulated neurotransmitter phenotype via its transactivation domain. Calcium signaled through cJun N-terminal phosphorylation, which integrated activity-dependent and intrinsic neurotransmitter specification. This mechanism provides a basis for early activity to regulate genetic pathways at critical decision points, switching the phenotype of developing neurons.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Activity-dependent specification of GABA and glutamate in embryonic spinal neurons requires tlx3.
Figure 2: A variant CRE binding site is required for activity-dependent transcriptional regulation.
Figure 3: cJun interacts with the CRE site.
Figure 4: cJun regulates transcription and specification of GABA and glutamate.
Figure 5: Ca2+ spike activity regulates phosphorylation of cJun.
Figure 6: cJun signaling integrates genetic and activity-dependent neurotransmitter specification.

Similar content being viewed by others

References

  1. Goodman, C.S. & Shatz, C.J. Developmental mechanisms that generate precise patterns of neuronal connectivity. Cell 72, 77–98 (1993).

    Article  Google Scholar 

  2. Spitzer, N.C. Electrical activity in early neuronal development. Nature 444, 707–712 (2006).

    Article  CAS  Google Scholar 

  3. Weissman, T.A., Riquelme, P.A., Ivic, L., Flint, A.C. & Kriegstein, A.R. Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex. Neuron 43, 647–661 (2004).

    Article  CAS  Google Scholar 

  4. Komuro, H. & Rakic, P.K. Intracellular Ca2+ fluctuations modulate the rate of neuronal migration. Neuron 17, 275–285 (1996).

    Article  CAS  Google Scholar 

  5. Hanson, M.G. & Landmesser, L.T. Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules. Neuron 43, 687–701 (2004).

    Article  CAS  Google Scholar 

  6. Hanson, M.G. & Landmesser, L.T. Increasing the frequency of spontaneous rhythmic activity disrupts pool-specific axon fasciculation and pathfinding of embryonic spinal motoneurons. J. Neurosci. 26, 12769–12780 (2006).

    Article  CAS  Google Scholar 

  7. Buffelli, M. et al. Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition. Nature 424, 430–434 (2003).

    Article  CAS  Google Scholar 

  8. Yano, S., Tokumitsu, H. & Soderling, T.R. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature 396, 584–587 (1998).

    Article  CAS  Google Scholar 

  9. Borodinsky, L.N. et al. Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. Nature 429, 523–530 (2004).

    Article  CAS  Google Scholar 

  10. Lin, Y. et al. Activity-dependent regulation of inhibitory synapse development by Npas4. Nature 455, 1198–1204 (2008).

    Article  CAS  Google Scholar 

  11. Flavell, S.W. & Greenberg, M.E. Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annu. Rev. Neurosci. 31, 563–590 (2008).

    Article  CAS  Google Scholar 

  12. Yamada, T., Pfaff, S.L., Edlund, T. & Jessell, T.M. Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate. Cell 73, 673–686 (1993).

    Article  CAS  Google Scholar 

  13. Liem, K.F.J., Tremml, G., Roelink, H. & Jessell, T.M. Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82, 969–979 (1995).

    Article  CAS  Google Scholar 

  14. Thor, S., Andersson, S.G., Tomlinson, A. & Thomas, J.B. A LIM-homeodomain combinatorial code for motor-neuron pathway selection. Nature 397, 76–80 (1999).

    Article  CAS  Google Scholar 

  15. Jessell, T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).

    Article  CAS  Google Scholar 

  16. Baumgardt, M., Miguel-Aliaga, I., Karlsson, D., Ekman, H. & Thor, S. Specification of neuronal identities by feedforward combinatorial coding. PLoS Biol. 5, e37 (2007).

    Article  Google Scholar 

  17. Ma, Q. Transcriptional regulation of neuronal phenotype in mammals. J. Physiol. (Lond.) 575, 379–387 (2006).

    Article  CAS  Google Scholar 

  18. Tanabe, Y., William, C.M. & Jessell, T.M. Specification of motor neuron identity by the MNR2 homeodomain protein. Cell 95, 67–80 (1998).

    Article  CAS  Google Scholar 

  19. Pierani, A. et al. Control of interneuron fate in the developing spinal cord by the progenitor homeodomain protein Dbx1. Neuron 29, 367–384 (2001).

    Article  CAS  Google Scholar 

  20. Mo, Z., Li, S., Yang, X. & Xiang, M. Role of the Barhl2 homeobox gene in the specification of glycinergic amacrine cells. Development 131, 1607–1618 (2004).

    Article  CAS  Google Scholar 

  21. Pillai, A., Mansouri, A., Behringer, R., Westphal, H. & Goulding, M. Lhx1 and Lhx5 maintain the inhibitory-neurotransmitter status of interneurons in the dorsal spinal cord. Development 134, 357–366 (2007).

    Article  CAS  Google Scholar 

  22. Mizuguchi, R. et al. Ascl1 and Gsh1/2 control inhibitory and excitatory cell fate in spinal sensory interneurons. Nat. Neurosci. 9, 770–778 (2006).

    Article  CAS  Google Scholar 

  23. Cheng, L. et al. Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates. Nat. Neurosci. 7, 510–517 (2004).

    Article  CAS  Google Scholar 

  24. Cheng, L. et al. Lbx1 and Tlx3 are opposing switches in determining GABAergic versus glutamatergic transmitter phenotypes. Nat. Neurosci. 8, 1510–1515 (2005).

    Article  CAS  Google Scholar 

  25. Liu, X. et al. Regulation of cholinergic phenotype in developing neurons. J. Neurophysiol. 99, 2443–2455 (2008).

    Article  CAS  Google Scholar 

  26. Brosenitsch, T.A. & Katz, D.M. Expression of Phox2 transcription factors and induction of the dopaminergic phenotype in primary sensory neurons. Mol. Cell. Neurosci. 20, 447–457 (2002).

    Article  CAS  Google Scholar 

  27. Dulcis, D. & Spitzer, N.C. Illumination controls differentiation of dopamine neurons regulating behavior. Nature 456, 195–201 (2008).

    Article  CAS  Google Scholar 

  28. Gu, X. & Spitzer, N.C. Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients. Nature 375, 784–787 (1995).

    Article  CAS  Google Scholar 

  29. Root, C.M., Velázquez-Ulloa, N.A., Monsalve, G.C., Minakova, E. & Spitzer, N.C. Embryonically expressed GABA and glutamate drive electrical activity regulating neurotransmitter specification. J. Neurosci. 28, 4777–4784 (2008).

    Article  CAS  Google Scholar 

  30. Chang, L.W. & Spitzer, N.C. Spontaneous calcium spike activity in embryonic spinal neurons is regulated by developmental expression of the Na+, K+-ATPase β3 subunit. J. Neurosci. 29, 7877–7885 (2009).

    Article  CAS  Google Scholar 

  31. Patterson, K.D. & Krieg, P.A. Hox11-family genes XHox11 and XHox11L2 in Xenopus: XHox11L2 expression is restricted to a subset of the primary sensory neurons. Dev. Dyn. 214, 34–43 (1999).

    Article  CAS  Google Scholar 

  32. Nakano, T., Windrem, M., Zappavigna, V. & Goldman, S.A. Identification of a conserved 125 base-pair Hb9 enhancer that specifies gene expression to spinal motor neurons. Dev. Biol. 283, 474–485 (2005).

    Article  CAS  Google Scholar 

  33. Ovcharenko, I., Stubbs, L. & Loots, G.G. Interpreting mammalian evolution using Fugu genome comparisons. Genomics 84, 890–895 (2004).

    Article  CAS  Google Scholar 

  34. Borghini, S., Vargiolu, M., Di Duca, M., Ravazzolo, R. & Ceccherini, I. Nuclear factor Y drives basal transcription of the human TLX3, a gene overexpressed in T-cell acute lymphocytic leukemia. Mol. Cancer Res. 4, 635–643 (2006).

    Article  CAS  Google Scholar 

  35. Sugiyama, C. et al. Activator protein-1 responsive to the group II metabotropic glutamate receptor subtype in association with intracellular calcium in cultured rat cortical neurons. Neurochem. Int. 51, 467–475 (2007).

    Article  CAS  Google Scholar 

  36. Heckert, L.L., Schultz, K. & Nilson, J.H. The cAMP response elements of the alpha subunit gene bind similar proteins in trophoblasts and gonadotropes but have distinct functional sequence requirements. J. Biol. Chem. 271, 31650–31656 (1996).

    Article  CAS  Google Scholar 

  37. Peng, Y. et al. Neural inhibition by c-Jun as a synergizing factor in bone morphogenetic protein 4 signaling. Neuroscience 109, 657–664 (2002).

    Article  CAS  Google Scholar 

  38. Picard, D. Posttranslational regulation of proteins by fusions to steroid-binding domains. Methods Enzymol. 327, 385–401 (2000).

    Article  CAS  Google Scholar 

  39. Raivich, G. c-Jun expression, activation and function in neural cell death, inflammation and repair. J. Neurochem. 107, 898–906 (2008).

    CAS  PubMed  Google Scholar 

  40. Ghosh, S., Wu, Y., Li, R. & Hu, Y. Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element. Oncogene 24, 2236–2246 (2005).

    Article  CAS  Google Scholar 

  41. Gómez-Lira, G., Lamas, M., Romo-Parra, H. & Gutiérrez, R. Programmed and induced phenotype of the hippocampal granule cells. J. Neurosci. 25, 6939–6946 (2005).

    Article  Google Scholar 

  42. Flavell, S.W. et al. Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number. Science 311, 1008–1012 (2006).

    Article  CAS  Google Scholar 

  43. Lin, Y. et al. Activity-dependent regulation of inhibitory synapse development by Npas4. Nature 455, 1198–1204 (2008).

    Article  CAS  Google Scholar 

  44. Hardingham, G.E., Fukunaga, Y. & Bading, H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. Neurosci. 5, 405–414 (2002).

    Article  CAS  Google Scholar 

  45. Gorbunova, Y.V. & Spitzer, N.C. Dynamic interactions of cyclic AMP transients and spontaneous Ca2+ spikes. Nature 418, 93–96 (2002).

    Article  CAS  Google Scholar 

  46. Dolmetsch, R.E., Xu, K. & Lewis, R.S. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392, 933–936 (1998).

    Article  CAS  Google Scholar 

  47. Li, W., Llopis, J., Whitney, M., Zlokarnik, G. & Tsien, R.Y. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature 392, 936–941 (1998).

    Article  CAS  Google Scholar 

  48. De Koninck, P. & Schulman, H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. Science 279, 227–230 (1998).

    Article  CAS  Google Scholar 

  49. Sive, H.L., Grainger, R.M. & Harland, R.M. Early Development of Xenopus laevis: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2000).

  50. Ovcharenko, I., Nobrega, M.A., Loots, G.G. & Stubbs, L. ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Res. 32, 280–286 (2004).

    Article  Google Scholar 

Download references

Acknowledgements

We thank S. Chung, G. Monsalve and A. de la Torre for technical assistance. We thank D. Berg, A. Ghosh and J. Gleeson for critical comments on the manuscript and I. Hsieh for technical support. This work was supported by a Damon Runyon Cancer Research Foundation Postdoctoral Fellowship to K.W.M., a US National Institutes of Health Predoctoral Fellowship (T32 AG00216) to L.M.K. and by a US National Institutes of Health grant (MH 074702) to N.C.S.

Author information

Author notes

  1. Kurt W Marek and Lisa M Kurtz: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Biological Sciences and Center for Neural Circuits, Neurobiology Section, Kavli Institute for Brain and Mind, University of California San Diego, La Jolla, California, USA

    Kurt W Marek, Lisa M Kurtz & Nicholas C Spitzer

  2. Neurosciences Graduate Program, University of California San Diego, La Jolla, California, USA

    Lisa M Kurtz

Authors
  1. Kurt W Marek
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lisa M Kurtz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicholas C Spitzer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.W.M., L.M.K. and N.C.S. designed the experiments, K.W.M. and L.M.K. performed and analyzed the experiments and K.W.M., L.M.K. and N.C.S. wrote the manuscript.

Corresponding author

Correspondence to Kurt W Marek.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1417 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Marek, K., Kurtz, L. & Spitzer, N. cJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification. Nat Neurosci 13, 944–950 (2010). https://doi.org/10.1038/nn.2582

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.2582

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing