cJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification

Journal name:
Nature Neuroscience
Volume:
13,
Pages:
944–950
Year published:
DOI:
doi:10.1038/nn.2582
Received
Accepted
Published online

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.

At a glance

Figures

  1. Activity-dependent specification of GABA and glutamate in embryonic spinal neurons requires tlx3.
    Figure 1: Activity-dependent specification of GABA and glutamate in embryonic spinal neurons requires tlx3.

    (a) GABA specification was suppressed by overexpression of potassium channels (Kir) together with a control morpholino (MOctl) and enhanced by reducing expression of tlx3 by splice-blocking morpholino injection (MOtlx3). Combining Kir overexpression and MOtlx3 injection (Kir + MOtlx3) yielded the MOtlx3 phenotype. GABA and vesicular glutamate transporter immunoreactivities (GABA-IR and vGlut1-IR) for stage 41 larvae are shown on the left and quantified per 100 μm of spinal cord on the right. Panels show central regions of the spinal cord (dashed outlines in insets) enlarged to make cell bodies clear. Insets show the entire spinal cord, including cell bodies and lateral axon tracts. Cells included in the counts are marked with arrows; other profiles were counted in adjacent sections. (b) Glutamate specification was enhanced by Kir overexpression and reduced by MOtlx3 injection. Combining Kir overexpression and MOtlx3 injection produced the MOtlx3 phenotype. (c) GABA specification was enhanced by overexpression of sodium channels (Nav) and suppressed by overexpression of tlx3. Combining the two perturbations generated the tlx3 overexpression phenotype. (d) Glutamate specification was suppressed by overexpression of sodium channels and enhanced by overexpression of tlx3. Combining the two perturbations yielded the tlx3 overexpression phenotype. Scale bar represents 100 μm for insets and 25 μm for magnified panels. Data are mean ± s.e.m. (n ≥ 7 larvae, *P < 0.05).

  2. A variant CRE binding site is required for activity-dependent transcriptional regulation.
    Figure 2: A variant CRE binding site is required for activity-dependent transcriptional regulation.

    (a) A control construct in which 384 bp of the tlx3 promoter drives firefly luciferase reported an increase in relative luminescence when co-injected with Kir and a decrease in relative luminescence when co-injected with Nav. WT, wild type. (b) Mutating the AP1 site (red) had no effect on luciferase expression. (c) Mutating the CRE site (red) eliminated the activity-dependence of luciferase expression. Data are mean ± s.e.m. (n ≥ 3 clutches, *P < 0.05).

  3. cJun interacts with the CRE site.
    Figure 3: cJun interacts with the CRE site.

    (a,b) Electrophoretic mobility shift assays showed a shift in CRE probe migration in response to cJun (a) and ATF2 (b) that was eliminated by competition with excess unlabeled probe (comp), but not by an unlabeled probe with a mutant CRE (compmut) or a nonspecific probe (compNS). Specific bands are indicated by arrows and nonspecific bands by asterisks; free probe is indicated by arrowheads. (c) Constitutively overexpressing a dominant-negative cJun or ATF2 construct lacking the transactivation domain (cJunTAM, ATF2TAM) increased the activity of the wild-type tlx3 luciferase reporter, but only the increase caused by cJunTAM required an intact CRE. Data are mean ± s.e.m. (n ≥ 3 clutches,*P < 0.05; NS, not significant). (d) RT-PCR performed on isolated spinal cords revealed that cJun transcripts were expressed at stages 22, 25 and 28. W, stage 28 whole embryos as positive control; −RT: stage 28 whole embryos without reverse transcriptase in the reaction. (e) cJun immunoreactivity in the spinal cord of a stage 25 embryo. Scale bar represents 25 μm.

  4. cJun regulates transcription and specification of GABA and glutamate.
    Figure 4: cJun regulates transcription and specification of GABA and glutamate.

    (a,b) Inducing overexpressed wild-type cJun at stage 24 caused a reduction in activity of the wild-type tlx3 luciferase reporter, whereas inducing the dominant-negative cJun produced the opposite result. Mutating the CRE site abolished these effects. (c,d) Inducing overexpressed wild-type cJun at stage 24 caused an increase in the number of GABA-immunoreactive neurons and a decrease in the number of vGlut1-immunoreactive neurons, whereas inducing the overexpressed dominant-negative cJun resulted in a decreased incidence of GABA-immunoreactive neurons and an increase in the number of vGlut1-immunoreactive neurons. Overexpression of either construct without induction had no effect. Immunostaining for GABA and vGlut1 immunoreactivity was performed on stage 41 larvae and data are presented as in Figure 1. Scale bar represents 100 μm for insets and 25 μm for magnified panels. Data are mean ± s.e.m. (n ≥ 3 clutches for a,b; n ≥ 15 larvae for c,d; *P < 0.05).

  5. Ca2+ spike activity regulates phosphorylation of cJun.
    Figure 5: Ca2+ spike activity regulates phosphorylation of cJun.

    (a) The number of cells expressing cJun was not changed by Ca2+ activity manipulations. (b,c) Ca2+ spike suppression caused a decrease in the number of cells expressing cJun that was phosphorylated at residues S73 (p-cJun (S73)-IR) and T91 (p-cJun (T91)-IR), whereas Ca2+ spike enhancement caused an increase in the number of cells expressing cJun phosphorylated at S73 and T91. Kir or Nav was injected into one cell of two-cell embryos to suppress or enhance Ca2+ spike activity, respectively. Immunostaining was performed on stage 28 embryos. Scale bar represents 25 μm. Data are mean ± s.e.m. (n ≥ 15 embryos, *P < 0.05).

  6. cJun signaling integrates genetic and activity-dependent neurotransmitter specification.
    Figure 6: cJun signaling integrates genetic and activity-dependent neurotransmitter specification.

    (a) Inducing an overexpressed phosphorylation mutant of cJun (cJunmut; S63A, S73A, T91A and T93A) at stage 24 caused an increase in the activity of the wild-type tlx3 luciferase reporter. Mutating the CRE site abolished this effect. (b,c) Inducing the overexpressed phosphorylation mutant of cJun at stage 24 caused a decrease in the number of GABA-immunoreactive neurons and an increase in the number of vGlut1-immunoreactive neurons. Overexpression without induction had no effect. Immunostaining for GABA and vGlut1 immunoreactivity was performed on stage 41 larvae and data are presented as in Figure 1. Scale bar represents 100 μm for insets and 25 μm for magnified panels. Data are mean ± s.e.m. (n ≥ 3 clutches for a; n ≥ 15 larvae for b,c; *P < 0.05).

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Author information

  1. These authors contributed equally to this work.

    • Kurt W Marek &
    • Lisa M Kurtz

Affiliations

  1. Neurobiology Section, Division of Biological Sciences and Center for Neural Circuits, 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

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.

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The authors declare no competing financial interests.

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