Guillemot lab Neural Stem Cell Biology Laboratory
: Specification of cellular identities in the brain
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We study transcription factors that specify the neuronal or glial fates of multipotent stem cells in the embryonic and adult brain. We characterise the mechanisms that underlie their activity, including the downstream transcriptional networks that induce and stabilise cellular identities and the epigenetic mechanisms that determine the temporal competence of progenitors to adopt particular fates.
Figure 1: Scheme showing that progenitors at different stages of cortical development generate neurons destined to different layers of the cortex. This is thought to be due to sequential changes in their competence to generate neurons of different identities.
In the developing cerebral cortex, progenitors generate sequentially different subtypes of neurons that occupy progressively more superficial layers of the cortex. The competence of progenitors to produce neurons of a particular laminar identity becomes progressively restricted during embryonic development.
In the unpublished study listed below, we have identified a novel function of the proneural transcription factor Neurog2 in specification of a particular subtype of neurons in the layer 6 of the mouse cortex, cortico-thalamic neurons.
Figure 2: Experiment of in utero electroporation of Neurog2, a determinant of layer 6 cortico-thalamic neurons, demonstrating that progenitors electroporated at embryonic day 13.5 (marked by GFP) have lost the competence to respond to Neurog2 and produce cortico-thalamic neurons (marked by Zfpm2). However, silencing of Eed, a component of Polycomb Repressor Complex 2, restores the competence of Neurog2-expressing embryonic day 13.5 progenitors to differentiate into Zfpm2+ layer 6 cortico-thalamic neurons.
Overexpression and silencing experiments by in utero electroporation in the embryonic cortex have shown that Neurog2 is required to specify cortico-thalamic neurons (projecting to the thalamus) and to suppress the alternative layer 6 cortico-cortical identity (neurons projecting to the contralateral cortical hemisphere).
We have identified several transcription factors that are directly regulated by Neurog2 and form a regulatory network that establishes the cortico-thalamic identity.
Layer 6 neurons are generated during a brief time window (at embryonic day 12.5 in the mouse). While forced expression of Neurog2 by in utero electroporation of cortical progenitors at embryonic day 12.5 generates extra cortico-thalamic neurons, the same experiment performed at embryonic day 13.5 has no such effect, demonstrating that progenitors have then lost the competence to respond to Neurog2 and differentiate into layer 6 neurons.
Chromatin immunoprecipitation in day 13.5 electroporated progenitors has shown that Neurog2 retains the capacity to bind to the regulatory elements of its target genes but that an histone modification that marks compact chromatin has accumulated at Neurog2 binding sites.
Inactivation of components of the Polycomb Repressive Complex, which is involved in chromatin compaction and transcriptional silencing, in embryonic day 13.5 progenitors restores the capacity of ectopic Neurog2 to induce the formation of cortico-thalamic neurons, demonstrating that the Polycomb Repressive Complex is in part responsible for the loss of competence of day 13.5 progenitors to generate layer 6 neurons.
Interestingly, if the same experiment is performed at embryonic day 14.5, no cortico-thalamic neuron is generated demonstrating that an additional mechanism, which is independent of the Polycomb Repressive Complex and remains unidentified, restricts progenitor competence at this stage.
