Our results reveal a mechanism by which early activity and genetic factors interact to drive differentiation. The data support a model in which Ca spike activity prior to synapse formation modulates the genetic pathway for specification of neurotransmitter phenotype (Supplementary Fig. 8a
). It is currently unclear whether Ca spike activity functions cell-autonomously. Ca spikes could cause phenotypic changes within the spiking cell or they could initiate a signaling cascade to neighbouring cells via diffusible factors. However, the downstream signaling cascade in dorsal neurons initiated by Ca activity is cell-autonomous -- it involves phosphorylation of cJun that regulates tlx3
transcription through a CRE site in its promoter (Supplementary Fig. 8b
). cJun appears to function as a repressor of tlx3
transcription. Although cJun is often described as an activator, it functions as a repressor in some cases, operating by recruiting co-repressors to the promoter or sequestering other activation factors 40
The conservation of the variant CRE site in Xenopus
, mouse and human tlx3
suggests that activity-dependent regulation of tlx3
is present in mammals, as well as in X. tropicalis
. However, there appear to be differences in the specification of glutamatergic and GABAergic neurons in Xenopus
and mammals. In mice, tlx3
functions to antagonize lbx1
: in the absence of tlx3
neurons default to a GABAergic fate under the control of lbx1
, but when lbx1
is also knocked out, the glutamatergic fate is restored 24
appear to be largely non-overlapping in Rohon-Beard cells of Xenopus tropicalis
, so this mechanism is not conserved. Another transcription factor may fulfill the same role as lbx1
may act alone in Xenopus
. Although there is strong conservation in the promoter sequence of tlx3
across species, other species-specific sites regulating activity-dependence are not excluded.
Strikingly, a strong cell-non-autonomous effect is also observed in both tlx3 loss-of-function and cJun gain-of-function embryos: the increase in GABA-IR cells is greater than the loss of VGluT1-IR cells and there are additional ventral GABA-IR neurons in the tlx3 morphants where tlx3 is not expressed. This result raises the possibility that other mechanisms exist to maintain circuit homeostasis in the developing spinal cord, perhaps by altering neurotransmitter specification in downstream neurons or generating compensatory synaptic connections.
Many other transcription factors are involved in neurotransmitter specification, but their relation to activity-dependent processes is unclear. However, several examples of activity-dependent neurotransmitter regulation involve transcription. Changes in Ca spike activity change the number of neurons expressing tyrosine hydroxylase transcripts in the postembryonic brain 27
. Synaptic activation leads to appearance of GAD67 and vesicular GABA transporter transcripts in adult glutamatergic granule cells and GABAA
receptor-mediated responses to granule cell stimulation 41
. These results suggest that the interplay between activity and genetic pathways in neurotransmitter specification is extensive. Indeed, the difference between the changes in VGluT1-IR and GABA-IR that we observe when manipulating activity is likely caused by the involvement of other transcription factors, which may or may not be differentially regulated by activity. Future work will determine whether the mechanisms underlying activity-dependent specification of other neurotransmitters share common elements.
This integration of activity and genetic pathways may have important implications for development of the nervous system. The Ca activity-dependent switch from excitatory glutamatergic to inhibitory GABAergic phenotype through regulation of a binary selection gene could reverse the polarity of a developing neuronal circuit. Our findings raise the intriguing possibility that activity-dependent regulation occurs at other fundamental choice points during development. Activity-responsive genes may identify developmental switches where extrinsic factors impinge on intrinsic pathways.
Activity plays an important role in many aspects of neuronal development. The functions and mechanisms of synaptic activity have been extensively studied. For example, this form of activity regulates the number and strength of synapses formed 42,43
and cell survival 44
. Less is known about the function of earlier forms of activity, although it has been identified throughout the nervous system in many organisms. Early activity regulates proliferation of neuronal precursors and neuronal migration as well as neuronal differentiation 2
. Ca transients occur at many times and in many places during development, but the mechanisms by which their patterns regulate differentiation have not been fully elucidated. It is unclear how Ca transients generated at frequencies of 1–20 hr −1
initiate the signaling cascade regulating neurotransmitter phenotype, although bursts of these Ca transients stimulate generation of cAMP transients 45
. Coding of transcription by the frequency of Ca transients has been reported in T lymphocytes 46
and in basophilic leukaemia cells 47
. Stimulation of CaM kinase II, NFAT and OCT/OAP transcription factor activity depends on relatively high frequencies of Ca transients 46–48
. In contrast, activation of the NF-kB transcription factor by Ca oscillations occurs at low frequencies 46
that are within the range of those generated by developing Xenopus
spinal neurons. It will be interesting to assess the role of cJun and CRE in other organisms and to identify signaling components linking Ca spikes to changes in cJun activity. N-terminal Jun kinases regulate cJun activity via phosphorylation of S63/S73 and T91/T93 (39), making them attractive candidates.