Reelin, Rap1 and N-cadherin orient the migration of multipolar neurons in the developing neocortex

Abstract

Projection neurons migrate from the ventricular zone to the neocortical plate during the development of the mouse brain. Their overall movement is radial, but they become multipolar and move nonradially in the intermediate zone. Here we show that Reelin, the Rap1 GTPase and N-cadherin (NCad) are important for multipolar neurons to polarize their migration toward the cortical plate. Inhibition and rescue experiments indicated that Reelin regulates migration through Rap1 and Akt, and that the Rap1-regulated GTPases RalA, RalB, Rac1 and Cdc42 are also involved. We found that Rap1 regulated the plasma membrane localization of NCad and NCad rescued radial polarization when Rap1 was inhibited. However, inhibition of Rap1 or NCad had little effect on glia-dependent locomotion. We propose a multistep mechanism in which Reelin activates Rap1, Rap1 upregulates NCad, and NCad is needed to orient cell migration.

Figure 1. Rap activity is required cell-autonomously for neuron entry into the upper intermediatezone and cortical plate

(a–c) Fetal brains were electroporated in utero at embryonic day 14.5 (E14.5) with plasmids expressing the indicated proteins along with GFP or ChFP. Rap1GAP was expressed in progenitors and post-mitotic neurons under the CAG promoter; Rap1A^17Nand Rap1A^63E were expressed in post-mitotic neurons using the NeuroD promoter. (a) Positions of transfected cells at E17.5. The cerebral wall was subdivided into RMZ (CP and upper IZ), MMZ (middle and lower IZ and sVZ) and VZ (GFP, green; DAPI, blue). (b) Percentage of electroporated cells in each region. (c) Sequential electroporation with GFP, followed, 10 min later, by Rap1GAP and ChFP, shows that Rap role in migration is cell autonomous. Scale bars, 100 μm. RMZ, radial morphology zone; MMZ, multipolar morphology zone; VZ, ventricular zone; CP, cortical plate; IZ, intermediate zone; sVZ, subventricular zone. Error bars, s.e.m. **, P < 0.01; ***, P < 0.001; NS, non significant.

Figure 2

Rap is required to orient multipolar cells but not for migration of bipolar neurons. The indicated plasmids were electroporated in utero at E14.5 along with plasmids expressing GFP or ChFP. (a) Computer-based reconstruction of shapes of GFP-positive neurons in E16.5 cortices at the transition between MMZ and RMZ. (b) Golgi staining (green, arrowheads) of MMZ neurons (magenta). The percentage of cells with Golgi facing the CP was calculated. (c) Tracks of migration paths followed by control and Rap-inhibited multipolar cells in the upper part of the MMZ. Positions of cell centroids in successive frames (circles) are linked by lines. The start position is marked by an arrow. (d) Migration of control and Rap inhibited GFP-positive multipolar (Control: N = 28 cells in 3 movies; Rap1GAP: N = 21 in 3 movies) and bipolar (Control: N = 121 cells in 6 movies; Rap1GAP: N = 51 in 4 movies) neurons in cortical slices prepared at E16.5 from brains electroporated at E14.5. Migration of bipolar GFP-positive cortical neurons in lattice culture (Control: N = 22 cells; Rap1GAP: N = 14). Mean + s.e.m. of average migration speed.. Scale bars, 50 μm. **, P < 0.01; NS, non significant.

Figure 3. Rap regulates adhesion to NCad and membrane NCad levels

(a) HA antibody staining (green) of MMZ neurons that had been electroporated in utero with HA-tagged NCad^WT, ChFP and vector (control) or Rap1GAP. (b) Mean ± s.e.m. of HA-NCad fluorescence intensity profiles across the bodies of N = 9 control and N = 9 Rap1GAP-expressing cells. (c) Immunofluorescence of the endogenous NCad in dissociated cortical neurons electroporated with GFP expressing plasmid alone or along with the Rap1GAP expressing plasmid. (d–e) E15.5 dissociated cortical neurons were electroporated with the indicated plasmids and incubated overnight on dishes coated with NCad-Fc or PDL. (d) GFP-expressing cells were counted before and after washes to determine the percentage of attached cells. (e) The morphology of GFP-expressing attached cells was observed after a further one day of differentiation. Scale bars, 20 μm.

Figure 4. NCad is required to orient multipolar cells, under control of Rap1

(a–b) Electroporation of a dominant-negative NCad^DN in progenitor cells (a) or post-mitotic cells (b) at E14.5 and labeled for the indicated proteins 3 days later. (c) Percentage of electroporated cells in each region (mean + s.e.m). NCad^DNAAA is an inactive point mutant of NCad^DN. (d) Computer-based reconstruction of shapes of GFP-positive neurons in E16.5 cortices at the transition between MMZ and RMZ. (e) Percentage of cells with Golgi facing the CP and fraction of multipolar neurons migrating towards the CP in time-lapse videomicroscopy. (f) Tracks of migrating NCad^DN-expressing multipolar cells in the upper part of the MMZ. Positions of cell centroids in successive frames (circles) are linked by lines. The start position is marked by an arrow. (g) Migration of GFP-positive neurons in cortical slices prepared at E16.5 from brains electroporated at E14.5. Cadherin-inhibited multipolar (Control: N = 28 cells in 3 movies; NCad^DN: N = 27 in 3 movies) and bipolar (Control: N = 121 cells in 6 movies; NCad^DN: N = 65 cells in 4 movies) neurons migrate at a normal speed. Migration of GFP-positive neurons in lattice culture (N =16 cells). Mean + s.e.m. of average migration speed. (h) Wildtype NCad^WT, expressed from the NeuroD promoter, partly rescues the migration defect of Rap1GAP-expressing cells. (i) Percentage of electroporated cells in each region (mean + s.e.m.). Scale bars represent 20 μm (d) and 100 μm (a, b, h). **, P < 0.01; ***, P < 0.001; NS, non significant.

Figure 5. Reelin regulates Rap and entry into the radial migration zone

(a) Immunostaining for NCad in the cerebral wall of wildtype and Reeler mutant embryos at E16.5. (b) Quantification of the NCad staining for 2 brains in each genotype. (c) Expression of dominant-negative VLDLRΔC interferes with neuron entry to the RMZ without affecting radial glia morphology. Left: red, ChFP; blue: DAPI. Right: Nestin. (d) Dominant-negative VLDLRΔC interferes with Reelin-dependent Dab1 degradation in the MMZ. In utero electroporation with ChFP and vector or VLDLRΔC plasmids, stained for ChFP (red) and Dab1 protein (green).(e) Rap1.GTP loading. Neurons were electroporated with HA-Rap1A^WT and GFP-RalGDSRBD along with Rap1GAP or VLDLRΔC. Five days after electroporation, neurons were treated with control (lane 1) or Reelin containing-supernatants (lanes 2–4) for 20 min prior to lysis. Lysates were analyzed directly or after pull-down with GFP-RalGDS-RBD to purify Rap1.GTP. Samples were immunoblotted with HA antibodies. The graph shows the Rap.GTP levels from 2 experiments. Similar results were obtained in two additional experiments with stimulation 3 days after electroporation. (f) Rap1 and Akt partly rescue inhibition by VLDLRΔC. (g) Percentage of electroporated cells in each region (mean + s.e.m.). (h) HA antibody staining (green) of MMZ neurons that had been electroporated in utero with HA-tagged NCad, ChFP and VLDLRΔC. Scale bars, 100 μm (a, c, f), 50 μm (d) and 20 μm (h). *, P < 0.05, **, P < 0.01; ***, P < 0.001; NS, non significant.