Cortical neurons gradually attain a post-mitotic state

Abstract

Once generated, neurons are thought to permanently exit the cell cycle and become irreversibly differentiated. However, neither the precise point at which this post-mitotic state is attained nor the extent of its irreversibility is clearly defined. Here we report that newly born neurons from the upper layers of the mouse cortex, despite initiating axon and dendrite elongation, continue to drive gene expression from the neural progenitor tubulin α1 promoter (Tα1p). These observations suggest an ambiguous post-mitotic neuronal state. Whole transcriptome analysis of sorted upper cortical neurons further revealed that neurons continue to express genes related to cell cycle progression long after mitotic exit until at least post-natal day 3 (P3). These genes are however down-regulated thereafter, associated with a concomitant up-regulation of tumor suppressors at P5. Interestingly, newly born neurons located in the cortical plate (CP) at embryonic day 18-19 (E18-E19) and P3 challenged with calcium influx are found in S/G2/M phases of the cell cycle, and still able to undergo division at E18-E19 but not at P3. At P5 however, calcium influx becomes neurotoxic and leads instead to neuronal loss. Our data delineate an unexpected flexibility of cell cycle control in early born neurons, and describe how neurons transit to a post-mitotic state.

Figure 1

Neurons in the CP and layer II initiated gradually neuronal morphological differentiation with the formation of axon and dendrites. (A) At E18, E15-in utero electroporated cells in the CP are scattered migrating to their final positions and have already initiated axon extension (arrows at the inset). (B) At E19, migration is reduced in transfected cells which are arranged as a layer within the upper CP with single axon (arrows at the inset). (C, D) At P3, transfected cells develop basal dendrites (dendrites, black arrowheads; axon, arrows) and at P5 their apical dendrite, or initially leading process, is remodeled and sculpted to a more complex structure (apical dendrite, white arrowheads; basal dendrites, black arrowheads; axon, arrows). Scale bar: 200 μm (left panels from A, B) and 10 μm (inset from A-D).

Figure 2

Cells in the CP and layer II that drive gene expression from the neuronal promoter pNeuroD and the neurogenic intermediate neuronal progenitor promoter Tα1p. (A) Cells in the CP co-express pNeuroD-GFP/mCherry, and are not immunoreactive for Ki67 (white arrowheads, inset from the left panel), a marker of cycling cells. (B) Quantification of experiment from A (P < 0.0001 by one-way ANOVA, post hoc Dunnett test ^***P < 0.001; values are mean ± SEM) (C) At E19 and P3, Tα1p-mCherry is expressed in Venus transfected cells from the CP and layer II, respectively. (D) Quantification of mCherry/Venus-transfected cells in the CP and layer II at E19 and P3, respectively (^***P = 0.0002 by t-test). Scale bar: 200 μm (A) and 20 μm (inset from A, C).

Figure 3

Transcriptional landscape of cortical layer II neurons at P3 and P5 suggests a not well-defined post-mitotic identity at P3 compared to P5. (A, B) Scatter plots showing genes up-regulated at P3 and P5 that are represented in proliferation of cells, and neurotransmission bio-functions. (C, D) Summary of selected categories of over-represented GO terms at P3 and P5 as calculated by DAVID. The GO categories are ranked by their associated −log(P-value). Horizontal bar (threshold) indicates P < 0.1.

Figure 4

Calcium influx induces faster nuclear mAG-hGem expression in neurons from the CP compared to neurons from layer II. (A) mCherry/mAG-hGem-transfected cells located in the CP at E19 express the S/G₂/M marker mAG-hGem (white arrowheads) upon ionomycin treatment (5 μM). Left panels: cortical slice used for time-lapse at E19 (in utero electroporated at E15). Right panels: Time-lapse sequence of CP-neurons upon ionomycin treatment (inset from left panel). (B) mCherry/mAG-hGem-transfected cells located in layer II at P3 express the S/G₂/M marker mAG-hGem (white arrowheads) upon ionomycin treatment (5 μM). Left panels: cortical slice used for time-lapse at P3 (in utero electroporated at E15). Right panels: Time-lapse sequence of layer II-neurons upon ionomycin treatment (inset from left panel). (C) 24 h ionomycin treatment induced similarly nuclear mAG-hGem expression in mCherry-electroporated neurons from the CP at E18-E19 and layer II at P3 (^****P < 0.0001 by one-way ANOVA, post hoc Dunnett test; ^***P < 0.001, ^**P < 0.01; values are mean ± SEM). (D) Nuclear mAG-hGem expression in neurons from the CP at E18-E19 is faster compared to layer II neurons at P3 (one-way ANOVA, post hoc Tukey test; E18 vs E19, not significant; E18 vs P3, ^***P < 0.001; E19 vs P3, ^**P < 0.01; values are mean ± SEM). Scale bar: 150 μm (left panels) and 100 μm (right panel).

Figure 5

Calcium influx induces mAG-hGem expression but not cell division in developing neurons from layer II at P3. Neuron with apical dendrite and axon (arrowhead and arrow respectively, in the time-lapse sequence) in layer II from P3 coronal brain slice (left panels, in utero electroporated at E15 with mCherry and mAG-hGem plasmids) express the S/G₂/M marker mAG-hGem (white arrowheads, insets from time-lapse sequence) upon ionomycin treatment (5 μM). Scale bar: 50 μm (left panel) and 10 μm (time-lapse sequence).

Figure 6

Calcium influx induces mAG-hGem expression and cell division in developing neurons from the CP. Neuron with a leading process and trailing process or axon (arrowhead and arrow respectively, in the time-lapse sequence) in the CP from E18 coronal brain slice (left panels, in utero electroporated at E15 with mCherry and mAG-hGem plasmids) express the S/G₂/M marker mAG-hGem (white arrowheads, insets from time-lapse sequence) and divide upon ionomycin treatment (5 μM). Scale bar: 50 μm (left panel) and 10 μm (time-lapse sequence).

Figure 7

Calcium influx in developing neurons induces a limited proliferative potential. (A) Diagram depicting the steps followed during the experiments. (B) Control neurons in the CP are negative for BrdU and display a radial organization. Slices treated with ionomycin (5 μM) and washout for 2-3 days, contain mCherry-transfected cells in the CP positive for BrdU without a radial organization (white arrowheads, inset from the left panel). (C) Slices treated with ionomycin (5 μM) and washout for 2-3 days contain mCherry-transfected cells in the CP, positive for BrdU and NeuN (white arrowheads, inset from the left panel) that lost radial dendritic organization. (D) Quantification of mCherry-transfected cells in the CP positive for BrdU (^***P = 0.0003 by t-test; values are mean ± SEM). (E) Quantification of mCherry-transfected cells in the CP positive for NeuN (P = 0.0814 by t-test; values are mean ± SEM). Scale bar: 10 μm.