GSK-3 signaling in developing cortical neurons is essential for radial migration and dendritic orientation
- University of North Carolina, United States
Figures
Figure 1 with 1 supplement
GSK-3 signaling is essential for proper lamination of the developing cortex.
(A–A') Cux-1 staining (red) in coronal sections from control and Gsk3:Neurod6 mice at E19.5. Cux-1 neurons are strikingly mislocalized in Gsk3:Neurod6 mutants (orange arrows) including a small …
Figure 1—figure supplement 1
Migration defect apparent by E16 after Gsk3 deletion in cortical excitatory neurons.
Coronal cryostat sections at E16. Layer 6 TBR1 neurons populate appropriate layers in control and Gsk3:Neurod6 mutants. Cux-1 (red) expressing neurons, marker for upper layer 2/3 neurons, are …
Figure 2 with 1 supplement
Migration defects in Gsk3-deleted mice are persistent.
(A) P0:Cux 1 staining (red) in coronal sections of Gsk3loxp:Neurod6 mutants and littermate heterozygote controls. Cux1-expressing neurons are localized to layer2/3 in controls (denoted by yellow …
Figure 2—figure supplement 1
Gsk3 overexpression enhances radial migration.
(A) E19.5 coronal sections showing normal migration after in utero electroporation of Neurod1-Cre and lox-STOP-lox-Ai9 (red neurons) and (B) Coronal sections showing abnormal neuronal migration …
Figure 3 with 1 supplement
GSK-3 signaling is dispensable for tangential migration, but required for radial hippocampal migration.
(A–A') E19.5 coronal sections showing EYFP-expressing interneurons in heterozygous control and Gsk3:Dlx5/6 mutants crossed with the Ai3 reporter line. Gsk3-deleted interneurons (green) enter the …
Figure 3—figure supplement 1
No apparent migration defect in Gsk3:Dlx5/6 mice.
P0 quantification of Ai3-positive neurons in control and Gsk3-deleted interneurons using 8 bin analysis spanning white matter to the dorsal stream. p-values reaching significance are shown in …
Figure 4 with 1 supplement
GSK-3 deletion delays the multipolar to bipolar transition.
(A–A') Representative E19 coronal sections after in utero electroporation at E14.5 with Neurod1-Cre and Z/EG plasmids. Electroporated cells were visualized with anti-EGFP (green), and nuclei were …
Figure 4—figure supplement 1
Gsk3-deleted neurons polarize and are highly dynamic.
(A) Stage progression analysis of dissociated control and Gsk3-deleted neurons. Stage 1 immature neurons display lamellapodial and filopodial protrusions. Stage 2 neurons have transitioned to form …
Figure 5 with 1 supplement
GSK-3 signaling is required for proper dendrite orientation.
(A) Gsk3 deleted neurons at P15 shown after in utero electroporation at E14.5 with Neurod1-Cre and Z/EG plasmids. Multiple neurons with obvious abnormalities in dendritic orientation were observed …
Figure 5—figure supplement 1
Quantification of dendritic branching at P15 in control and Gsk3-deleted neurons.
(A) Representitive images of control and Gsk3-deleted neurons used for Sholl analysis with specific processes pseudocolored for identification. (B) Apical dendrite Sholl analysis of Gsk3-deleted …
Figure 6 with 1 supplement
Lamination in other signaling mutants.
(A) P0 representative coronal sections of control (heterozygous for floxed allele) and Ctnnb1Ex3:Neurod6 mutants stained for Cux1 (red) and Hoechst (blue). (n = 5) Scale bar = 100 μm. (B) E18 …
Figure 6—figure supplement 1
Quantification of lamination in other signaling mutants.
(A–D) P0 quantification of Cux1 expressing neurons using 8 Bin quantification in conditional mutants and control shown in Figure 6. (A) Ctnnb1Ex3:Neurod6, (B) Stk11:Neurod6, (C) Cdc42:Neurod6, (D) Pt…
Figure 7
Phosphorylation status of GSK-3 substrates.
(A–A') Western blots of P0 cortical lysates from Gsk3loxp:Neurod6 mutants and wild-type controls performed in triplicate. Levels of GSK-3 proteins and phospho-target proteins are shown. Strong …
Videos
Video 1
Live cell imaging of radially migrating neurons in a cortical slice preparation.
Control neurons electroporated at E15.5 migrate towards the pial surface after 3 days ex vivo. Neurons are imaged using time-lapse microscopy with images taken every 45 min for a 20-hr session.
Video 2
Gsk3-deleted neurons fail to migrate in cortical slice preparation.
Gsk3a−/−Gsk3bloxp/loxp mice electroporated at E15.5 with Neurod1-cre and Z/EG have an elongated multipolar stage and do not migrate towards the pial surface. Images were taken every 45 min over a …
