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, 9 (1), 5874

Role of Per3, a Circadian Clock Gene, in Embryonic Development of Mouse Cerebral Cortex

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Role of Per3, a Circadian Clock Gene, in Embryonic Development of Mouse Cerebral Cortex

Mariko Noda et al. Sci Rep.

Abstract

Per3 is one of the primary components of circadian clock system. While circadian dysregulation is known to be involved in the pathogenesis of several neuropsychiatric diseases. It remains largely unknown whether they participate in embryonic brain development. Here, we examined the role of clock gene Per3 in the development of mouse cerebral cortex. In situ hybridization analysis revealed that Per3 is expressed in the developing mouse cortex. Acute knockdown of Per3 with in utero electroporation caused abnormal positioning of cortical neurons, which was rescued by RNAi-resistant Per3. Per3-deficient cells showed abnormal migration phenotypes, impaired axon extension and dendritic arbor formation. Taken together, Per3 was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Characterization of RNAi vectors for Per3. (a) pCAG-Myc-Per1, –Per2 or –Per3 was co-transfected into COS7 cells with control pSuper vector, pSuper-mPer3#1 and #2 in various combinations. After 48 h, cells were harvested and subjected to western blotting (20 µg protein per lane) with anti-Myc. Anti-Sept11 was used for a loading control. Molecular weight markers are shown at the right. Relative levels of Myc-Per proteins were calculated with ImageJ software based on densitometry and normalized against Sept11. Error bars indicate SD (n = 3). The data shown are representatives of 3 independent experiments. **P 0.01 and ***P < 0.001 (control vs RNAi) by one-way ANOVA < with Fisher’s LSD post hoc test. (b) Characterization of RNAi-resistant Per3, Per3-R. pCAG-Myc-Per3 or –Per3-R was co-transfected into COS7 cells with pSuper-control or pSuper-mPer3#1. Analyses were done as in (a). (c) Coronal brain sections prepared from E16.5, P2 and P10 mice were examined by in situ hybridization with mPer3 riboprobe. Note that only weak signal was detected with the sense probe at E16.5. Scale bars, 100 μm.
Figure 2
Figure 2
Effects of Per3-knockdown on neuronal migration during corticogenesis. (a) pCAG-EGFP was co-electroporated with control pSuper (i), pSuper-mPer3#1 (ii) or #2 (iii) into cerebral cortices at E14.5. For the rescue experiments, pCAG-EGFP was co-electroporated with pSuper-mPer3#1 together with pCAG-Myc-mPer3-R (iv). Coronal sections were prepared at P2. Nuclei were stained with DAPI (blue). Dotted lines represent the pial and ventricular surfaces. Scale bars in (a,d), 100 µm. (b) Quantification of the distribution of GFP-positive neurons in distinct regions of cerebral cortex for each condition shown in (a). Error bars indicate SD; control (n = 5), pSuper-mPer3#1 (n = 5), pSuper-mPer3#2 (n = 3), pSuper-mPer3#1 + Per3-R (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 (vs. control) by one-way ANOVA with Fisher’s LSD post hoc test. (c) Representative images of Per3-deficient neurons migrating in the lower CP. Scale bar, 10 µm. (d) Migration defects of Per3-deficient cortical neurons at P10. Coronal sections were immunostained with anti-RORβ as a layer IV marker. Dotted lines represent the pial surface. Analyses were done as in (a). Note that vascular structure was nonspecifically stained. (e) Quantification of the distribution of GFP-positive neurons in distinct regions in (d). Error bars indicate SD; control (n = 9), pSuper-mPer3#1 (n = 8), pSuper-mPer3#1 + Per3-R (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 (vs. control) by Fisher’s LSD.
Figure 3
Figure 3
Time-lapse imaging of migration of Per3-deficient neurons. Analyses were repeated 3 times for each case, and the migration pattern was observed for 20 cells in each imaging. Representative results were shown in (a–e). (a) Tracing of control and Per3-deficient cells in the IZ-CP boundary. E14.5 cortices were co-electroporated with pCAG-EGFP together with control pSuper or pSuper-mPer3#1, followed by coronal section slice preparation at E16.5 and time-lapse imaging for 20 h. Scale bars in (b–e), 20 µm. (b,c) Migratory tracks of control (b) and Per3-deficient neurons (c) in the IZ/CP boundary. (d,e) Migratory tracks of control (d) and the deficient neurons (e) in the lower-middle CP. (f) Calculation of migration velocity of control and the deficient cells in the middle-upper CP. More than 10 cells were analyzed in each experiment (n = 3). Error bars indicate SD. ***P < 0.0001 by Students t-test.
Figure 4
Figure 4
Effects of Per3-knockdown on axon growth. (a) Cortical neurons isolated at E14.5 were co-transfected with pCAG-EGFP together with control pSuper, pSuper-mPer3#1 or pSuper-mPer3#1+pCAG-Myc-Per3-R. At day 3 in vitro, cells were fixed and stained for GFP. Scale bar, 10 µm. (b) Quantitative analysis of axon growth in (a). Axon was recognized as the longest neurite. Axonal length was quantified using ImageJ software. Error bars indicate SD (n = 3). ***P < 0.0001 and ###P < 0.0001 by Fisher’s LSD. (c) pCAG-Turbo-RFP was co-electroporated in utero with control pSuper, pSuper-mPer3#1 or pSuper-mPer3#1+pCAG-Myc-Per3-R into cerebral cortices at E14.5. Coronal sections were prepared at P2 and stained for RFP. Scale bar, 500 µm. (d) Quantitative analyses of axon growth in (c). The ratio of the intensity of RFP-positive axon in the area (green) of electroporated ipsilateral cortex to that in the area (magenta) of contralateral cortex in (c) was calculated with ImageJ software. Error bars indicate SD (n = 3). ***P < 0.0001 and ###P < 0.0001 by Fisher’s LSD. (e) Estimation of axon growth at P10. Representative images of the terminal arbors of axons expressing RFP with control pSuper, pSuper-Per3#1 or pSuper-Per3#1+Per3-R were shown. The experiments were repeated 3 times. Densitometric analyses of RFP fluorescence intensity were shown as blue (control), red (pSuper-mPer3#1) and green (pSuper-mPer3#1+Per3-R) lines. Shadows represent SD (control, n = 3; pSuper-mPer3#1, n = 3; pSuper-mPer3#1+Per3-R, n = 3). Scale bar, 200 µm.
Figure 5
Figure 5
Effects of Per3-knockdown on dendritic growth. (a) pCAG-loxP-GFP was co-electroporated for sparse expression with pCAG-M-Cre together with pSuper-control, pSuper-mPer3#1 or pSuper-mPer3#1+pCAG-Myc-Per3-R into cerebral cortices at E14.5. Analyses were carried out with cortical slices at P10. Representative average Z-stack projection images of GFP fluorescence of cortical neurons in the upper CP were shown. Scale bar, 50 µm. (bd) One section from each brain was analyzed for pSuper-control (n = 5), pSuper-Per3#1 (n = 7) and pSuper-mPer3#1 + Per3-R (n = 5). (b) Branch point number of dendrites was analyzed by Sholl test. Error bars indicate SD (n = 3). Branching point number (c) or total length (d) was calculated for apical as well as basal dendrites. *P < 0.05, $P < 0.05, **P < 0.01 and ***P < 0.001 (*control vs pSuper-Per3#1, $control vs pSuper-Per3#1+Per3-R) by Fisher’s LSD.

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