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
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Goodman, C.S. & Shatz, C.J. Developmental mechanisms that generate precise patterns of neuronal connectivity. Cell 72, 77–98 (1993).
Spitzer, N.C. Electrical activity in early neuronal development. Nature 444, 707–712 (2006).
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).
Komuro, H. & Rakic, P.K. Intracellular Ca2+ fluctuations modulate the rate of neuronal migration. Neuron 17, 275–285 (1996).
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).
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).
Buffelli, M. et al. Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition. Nature 424, 430–434 (2003).
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).
Borodinsky, L.N. et al. Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. Nature 429, 523–530 (2004).
Lin, Y. et al. Activity-dependent regulation of inhibitory synapse development by Npas4. Nature 455, 1198–1204 (2008).
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).
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).
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).
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).
Jessell, T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).
Baumgardt, M., Miguel-Aliaga, I., Karlsson, D., Ekman, H. & Thor, S. Specification of neuronal identities by feedforward combinatorial coding. PLoS Biol. 5, e37 (2007).
Ma, Q. Transcriptional regulation of neuronal phenotype in mammals. J. Physiol. (Lond.) 575, 379–387 (2006).
Tanabe, Y., William, C.M. & Jessell, T.M. Specification of motor neuron identity by the MNR2 homeodomain protein. Cell 95, 67–80 (1998).
Pierani, A. et al. Control of interneuron fate in the developing spinal cord by the progenitor homeodomain protein Dbx1. Neuron 29, 367–384 (2001).
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).
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).
Mizuguchi, R. et al. Ascl1 and Gsh1/2 control inhibitory and excitatory cell fate in spinal sensory interneurons. Nat. Neurosci. 9, 770–778 (2006).
Cheng, L. et al. Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates. Nat. Neurosci. 7, 510–517 (2004).
Cheng, L. et al. Lbx1 and Tlx3 are opposing switches in determining GABAergic versus glutamatergic transmitter phenotypes. Nat. Neurosci. 8, 1510–1515 (2005).
Liu, X. et al. Regulation of cholinergic phenotype in developing neurons. J. Neurophysiol. 99, 2443–2455 (2008).
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).
Dulcis, D. & Spitzer, N.C. Illumination controls differentiation of dopamine neurons regulating behavior. Nature 456, 195–201 (2008).
Gu, X. & Spitzer, N.C. Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients. Nature 375, 784–787 (1995).
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).
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).
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).
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).
Ovcharenko, I., Stubbs, L. & Loots, G.G. Interpreting mammalian evolution using Fugu genome comparisons. Genomics 84, 890–895 (2004).
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).
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).
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).
Peng, Y. et al. Neural inhibition by c-Jun as a synergizing factor in bone morphogenetic protein 4 signaling. Neuroscience 109, 657–664 (2002).
Picard, D. Posttranslational regulation of proteins by fusions to steroid-binding domains. Methods Enzymol. 327, 385–401 (2000).
Raivich, G. c-Jun expression, activation and function in neural cell death, inflammation and repair. J. Neurochem. 107, 898–906 (2008).
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).
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).
Flavell, S.W. et al. Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number. Science 311, 1008–1012 (2006).
Lin, Y. et al. Activity-dependent regulation of inhibitory synapse development by Npas4. Nature 455, 1198–1204 (2008).
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).
Gorbunova, Y.V. & Spitzer, N.C. Dynamic interactions of cyclic AMP transients and spontaneous Ca2+ spikes. Nature 418, 93–96 (2002).
Dolmetsch, R.E., Xu, K. & Lewis, R.S. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392, 933–936 (1998).
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).
De Koninck, P. & Schulman, H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. Science 279, 227–230 (1998).
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).
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).
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
Authors and Affiliations
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
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
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nn.2582
This article is cited by
-
Transcriptomic analysis reveals distinct adaptive molecular mechanism in the hippocampal CA3 from rats susceptible or not-susceptible to hyperthermia-induced seizures
Scientific Reports (2023)
-
A Spacetime Odyssey of Neural Progenitors to Generate Neuronal Diversity
Neuroscience Bulletin (2023)
-
Exercise enhances motor skill learning by neurotransmitter switching in the adult midbrain
Nature Communications (2020)
-
Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons
Cell Death & Disease (2013)
-
A neurochemical map of the developing amphioxus nervous system
BMC Neuroscience (2012)