Verslag – Rapport – Bericht – Report - GSKE - FMRE

candidate-based approach with a proteomic screen to compare the cortical acetylome (proteome of
acetylated proteins) of WT and cKO Elp3 mice. One of the candidate proteins is a connexin. We found
that this connexin is enriched in the developing cerebral cortex and is massively acetylated. Western
blot analyses performed on cortical extracts from Elp3 conditional knockout (Elp3lox Foxg1:Cre) E12
mouse embryos showed a dramatic reduction of the level of acetylation of the connexin. In addition,
co-immunoprecipitation assays with cortical tissue extracts demonstrated an interaction between the
connexin and Elp1, the scaffold subunit of the Elongator complex. Thus, this connexin is a strong candidate
for acetylation by Elongator. These results have been confirmed in several mouse and human cell lines.
In addition, we found that HDAC6 is a KDAC responsible for the deacetylation of this connexin. We are
currently assessing the putative role of connexin acetylation in corticogenesis, focussing on the control
of the interkinetic nuclear movement (INM). We performed BrdU injection followed by immunohistology to
study INM kinetics. BrdU injections were performed in pregnant mice to label cycling cortical progenitors
in S or G2 phases in E14.5 Elp3 cKO or WT embryos followed by sacrifice after several timings (1h or 4h
for assessing S-phase or G2 phase, respectively). Our preliminary result suggests that INM is affected
upon depletion of Elp3. This biological event has previously been linked to connexin expression but not
its acetylation. We are currently identifying the lysine residues of Connexin 43 targeted by Elp3 with mass
spectrometry on N2A cells, that expressed or not Elp3. We will perform mutations to important lysine
residues by arginines. Plasmids coding for various lysine mutant forms will be engineered and delivered
together with Cre-expressing vectors (to remove the endogenous Elp3) by in utero electroporation into
cortical progenitors from cKO Elp3 embryos. A second aspect is the control of cortical progenitors
specification and their ability to generate projection neurons. The overall phenotype of Elp3 cKO cortex is
a reduced cortical plate thickness arising as a consequence of the major depletion postmitotic projections
neurons. We discovered that Tbr2 is significantly impaired in Elp3 cKO cortices as compared to control.
These cells are required for the production of most projection neurons dedicated to all cortical layers.
Our preliminary results suggest that Elongator may control the proliferation and or specification of Tbr2
intermediate cortical progenitors whose reduction would impact on the total neuronal output and could
thus explain the cortical phenotype of Elp3 cKO embryos. We are now investigating if and how Elongator
controls the expression of Tbr2 in the developing cortex.
To investigate the role(s) of Elongator in tangential migration of cortical interneurons, we used an Elp3
flox; Dlx5,6 Cre-GFP mouse line (Elp3 cKO) newly generated in our laboratory. Real-time experiments
on Elp3 cKO or WT MGE explants were performed on interneurons to analyse their migration as well as
their cell shape modifications. After 24 hours of culture, control interneurons that have migrated out of
MGE explants exhibited a polarized morphology with branched leading process. Our preliminary data
indicated that interneurons that lack Elp3 expression had a significant decrease of migration velocity as
well as a reduced frequency and amplitude of nuclear translocations. In addition, only 40% of Elp3 cKO
migrating interneurons displayed a swelling. Real time imaging also indicated that the formation and
division of growth cones (that underlie the production of new branches) were both, less frequent and
less stable in Elp3 cKO interneurons, as compared to control experiments. Some of these results were
confirmed in situ, cultured brain slices. Furthermore, immunolabeling of E12.5 embryo sections showed
that the loss of Elp3 expression resulted in abnormal cellular shape and in a significant reduction of the
number of GABAergic interneurons that entered into the cortex. This observation suggests that the
conditional removal of Elp3 resulted in a tangential migration delay and supports the time-lapse results
on MGE explants. Collectively, our data describe a novel role for Elp3 in the control of nucleokinesis
kinetics, branching dynamics of interneurons, growth cone splitting and stability of newly formed
neurites. To determine how Elp3 controls these parameters, we will combine time–lapse recording
of MGE explants from Elp3 cKO GABAergic interneurons with rescue experiments. For this, we will
Verslag – Rapport – Report 2012 63