The outermost region of the developing cortical plate is crucial for both the switch of the radial migration mode and the Dab1-dependent "inside-out" lamination in the neocortex - PubMed (original) (raw)

Figure 1.

Figure 1.

Time-lapse imaging of the terminal translocation. A, Time-lapse images of the terminal translocation of neurons were obtained from a cortical slice prepared at E17.5 after electroporation of GFP expression vectors at E14.5. Note that the cell soma (arrow) tended to stop near the top of the CP (2:30–3:00). After this pause, the soma alone moved rapidly toward the top of the CP, with a shortening of the leading process (3:00, arrowheads). Scale bar, 20 μm. The time indicates the time (hours and minutes) elapsed since the start of the observation period. B, Tracking graph of the terminal translocation. The somal movement of cell 1 (shown in A) was tracked using NIH ImageJ software. Note that the speed of the somal movement changed at 3:00 (arrow). The migration distance after this time point until the end of migration was 54 μm. Representative graphs of the terminal translocation for cell 2 and cell 3 are also shown. C, Average terminal translocation distance. The distances from the point at which the migratory speed changed until the end of migration were plotted. The average ± SEM distance was 50.9 ± 2.8 μm (n = 40, 5 independent slices obtained from different embryos).

Figure 2.

Figure 2.

The outermost region of the developing CP was composed of immature neurons. A–A″, Nissl staining of the wild-type developing cortex. A, At E18.5. Note that the staining pattern of the outermost region of the CP differed from that of the other parts of the CP. A higher magnification of the square is shown in A′. Note the accumulation of the darkly stained neurons around the superficial part of the CP (white arrows). Accumulation of these cells was also identified at E16.5 but not at E15.5 (A″, white arrows). VZ, Ventricular zone. Scale bars: A, 200 μm; A′, A″, 50 μm. B, NeuN staining at P0.5; the outermost part of the CP showed weak or almost negative staining for NeuN. The arrows indicate the NeuN-negative zone. Scale bars, 100 μm. C, Developmental course of the NeuN-negative zone (white arrows). At E15.5, the subplate cells (SP) were NeuN positive (green), whereas the other cells in the CP were NeuN negative. Magenta is the nuclear staining. At E16.5, some cells in the CP became NeuN positive. From E17.5 to P1.5, the thicknesses of the NeuN-negative zone at the outermost part of the CP remained almost the same. Thereafter, the thickness gradually decreased, and, at P4.5 the NeuN-negative zone entirely disappeared. Note that the cells in the deep part of the CP showed weak or negative staining for NeuN until approximately P1.5. Scale bar, 100 μm. D, Nissl staining of the reeler cortex at P0. Note that no obvious accumulation of darkly stained neurons was visible. Scale bar, 200 μm. E, NeuN staining of the reeler cortex at P0. An admixture of NeuN-positive cells and NeuN-negative cells was observed in the deep part of the mutant cortex. Most of the cells at the superficial part of the reeler cortex were NeuN negative, corresponding to the deep part of the wild-type CP. Scale bars, 100 μm.

Figure 3.

Figure 3.

Dab1 is required for neuronal entry into the NeuN-negative zone or PCZ. A, In vitro analysis of the Dab1–KD vectors. Wild-type or mutant Dab1-expressing vectors were transfected with or without Dab1–KD vectors into Neuro2A cells. The cell lysates were analyzed by Western blotting. Dab1–KD vectors effectively suppressed the expression of wild-type Dab1 but not the expression of mutant Dab1, which was designed to be resistant to this Dab1–KD vector. B–B″, C–C″, D–D″, Effects of Dab1–KD on neuronal migration. Electroporation was performed at E14.5, and the brains were examined at E17.5, E18.5, and P0.5. The area from the outer margin of the CP to the inner margin of the IMZ was divided into 10 bins. B–B″, Control case. C–D″, The distribution of the Dab1–KD neurons was almost the same as that of the control neurons at E17.5 (C, D), but, even by E18.5 (C′, D′, black arrow) or P0.5 (C″, D″, black arrow), most of these neurons had not entered the NeuN-negative PCZ (white arrows). Note that some cells were abnormally observed in the IMZ (C′) and the third bin of the Dab1–KD experiments at E18.5 (D′, *), but most of these neurons had migrated to the CP by P0.5. n = 3 brains each. Scale bars, 100 μm. E–E″, F–F″, Dab1 is required for neuronal entry into the NeuN-negative PCZ. E–E″, Control case at P0.5. GFP-expressing cells had entered the top of the CP. Note that this zone was negative or weak for NeuN (red) (arrows). The nuclei were showed by DAPI (blue). F–F″, Dab1–KD neurons (green) were located beneath the NeuN-negative zone. Scale bars, 50 μm. G, Time-lapse images of the Dab1–KD neurons. The Dab1–KD neuron (arrow) migrated toward the surface of the CP but stopped migrating at 8:00 and did not move anymore. Note that the long leading process (arrowheads) is attached to the MZ. Scale bar, 20 μm. The time indicates the time (hours and minutes) elapsed since the start of the observation period. H, Three tracking graphs of the Dab1–KD neurons. Cell 1 is the tracking graph of the cell shown in G. The red line is the tracking graph of the pSilencer–control vector-transfected neuron. I–I′, Rescue experiments of the Dab1–KD phenotype. Electroporation was performed at E14.5, and the brains were examined at P0.5. In the rescue experiments (cotransfection of a Dab1–KD vector and a Tα1–Dab1–3MT–HA vector), many cells were successfully located in the NeuN-negative PCZ (white arrow). Note some neuronal accumulation around the middle of the CP; such accumulation was also observed when both a pSilencer–control vector and a Tα1–Dab1–3MT–HA vector were cotransfected (I′) but not observed in the Dab1–KD experiments (C″). Scale bar, 100 μm. J, Bin analysis of the rescue experiment. The area from the outer margin of the CP to the inner margin of the IMZ was divided into 10 bins. Note the accumulation of cells in the seventh to ninth bins (*). n = 5 brains for rescue experiments (black bar) and n = 3 brains for Dab1 overexpression experiments (gray bar). K–K″, Higher-magnification views of the rescue experiments around the PCZ. Note that the Dab1–KD phenotype was rescued by cotransfection of a Tα1–Dab1–3MT–HA vector and located within the NeuN-negative PCZ (arrow). Scale bar, 50 μm. L, Statistical analysis of the distances from the outer margin of the CP to the soma of the neurons. Only neurons located above the middle of the CP were counted. The distance was 26.5 ± 0.5 μm for the control neurons (n = 4 brains, >500 neurons were counted), 55.7 ± 0.7 μm for the Dab1–KD neurons (n = 4 brains, >500 neurons were counted), and 26.7 ± 1.1 μm for the Dab1–KD+Rescue neurons (n = 5 brains, >250 neurons were counted). **p < 0.01, Scheffé's F test.

Figure 4.

Figure 4.

Dab1 is required for neuronal entry to the outermost region of the CP during all developmental stages. A–A″, Control or Dab1–KD vector was introduced at E16 and the brains were examined at P3.5. Control neurons were positioned within the NeuN-negative PCZ (A, arrow), whereas the Dab1–KD neurons could not enter this zone (A′, arrow). A″, Bin analysis. The thickness of the CP was divided into 10 bins (n = 3 brains each, >120 neurons were counted.) Scale bar, 50 μm. B–B′, C–C′, D–D″, Control or Dab1–KD vector was introduced at E13 and the brains were examined at E16.5. Many control neurons were positioned in the NeuN-negative PCZ (D, arrow), and these neurons were positive for Cux1 (B–B′) but negative for Ctip2 (C–C′). The Dab1–KD neurons, conversely, could not enter the PCZ (D′, arrow). D″, Bin analysis. The distance between the outer margin of the CP and the inner margin of the IMZ was divided into 10 bins. Note that the abnormal accumulation of the Dab1–KD neurons in the ninth bin, but the distribution pattern in the deeper parts of the cortex was similar to that in the control (n = 4 brains each, >400 neurons were counted.) Scale bars, 100 μm. E–E″, F–F′, G–G′, H, Control or Dab1–KD vector was introduced into the dorsomedial cortex at E12.5 and the brains were examined at E15.5. Control neurons were positive for Ctip2 but negative for Brn2 (E–E″), and they were positioned in the middle one-third of the CP (F). Many Dab1–KD neurons, conversely, were located at the border between the CP and the IMZ (F′, *). Arrows indicate the thickness of the CP. G–G′, Higher-magnification view of the Dab1–KD neurons located at the border between the CP and the IMZ. Note that their leading processes are attached to the MZ (arrowheads). H, Bin analysis. The cortex was divided into upper CP, middle CP, lower CP, and IMZ. Note that Dab1–KD neurons were abnormally accumulated in the lower CP (*p = 0.0495, Mann–Whitney's test, n = 3 brains each, >200 neurons were counted.) Scale bars: E-E″, F–F′, 50 μm; G–G′, 10 μm. I–I″, J–J″, K–K′, L–L′, M–M‴, N, Control or Dab1–KD vector was introduced into the lateral cortex at E12.5 and the brains were examined at E15.5. Control neurons were positioned in the outermost region of the CP (K), and many of them were double-positive for Ctip2 and Brn2 (I–I″) or Cux1 and Brn2 (J–J″). Dab1–KD neurons, conversely, were accumulated in three different areas (*, **, ***) (K′). * denotes the neuronal accumulation around the border between the upper one-third of the CP and the middle one-third of the CP. ** denotes the neuronal accumulation around the border between the CP and the IMZ. *** denotes the neuronal accumulation below the CP. Arrows indicate the thickness of the CP. L–L′, Higher-magnification view of Dab1–KD neurons located at the border between the upper CP and the middle CP (representative images at *). Note that their leading processes reach the MZ (arrowheads). M–M‴, Higher-magnification view of Dab1–KD neurons located at the border between the CP and the IMZ (representative images at **). Note that their leading processes also reach the MZ (arrowheads). Red represents Ctip2-positive cells, and blue represents Brn2-positive cells. N, Bin analysis. The cortex was divided into the upper CP, middle CP, lower CP, and IMZ (*p = 0.0209, Mann–Whitney's test; n = 4 brains each, >700 neurons were counted.) Scale bars: I–I″, J–J″, K–K′, 50 μm; L–L′, M–M‴, 10 μm. O–O′, P, The control or Dab1–KD vector was introduced into the lateral cortex at E12.5 and the brains were examined at E17.5. O, Control case. O′, Most of the Dab1–KD neurons were located in the CP, but they were settled in a slightly deeper place than the control neurons. Note the positioning of some Dab1–KD neurons at the border between the CP and the IMZ (*). Arrows indicate the thickness of the CP. P, Bin analysis. The distance between the outer margin of the CP and the inner margin of the IMZ was divided into 10 bins (n = 3 brains each, >400 neurons were counted.). Scale bar, 100 μm.

Figure 5.

Figure 5.

Sequential electroporation can visualize the process of the inside-out alignment of neurons in the wild-type cortex. A–A‴, The GFP expression vector was introduced at E14.5, and the mCherry expression vector was electroporated at E15.5. At E18.5, most of the GFP-labeled earlier-born control neurons were seen in the superficial part of the CP, whereas the mCherry-labeled later-born control neurons were mainly located in the IMZ and the deep part of the CP. Scale bar, 100 μm. A‴, Bin analysis. The distance between the outer margin of the CP and the inner margin of the IMZ was divided into 20 bins. n = 3 brains. B–B‴, At P1.5, the mCherry-labeled later-born neurons were located more superficially than the GFP-labeled earlier-born neurons in the outermost region of the CP. B‴, Bin analysis. The distance between the outer margin of the CP and the inner margin of the IMZ was divided into 20 bins. Many mCherry-labeled later-born neurons were located in the top 20th bin, whereas the GFP-labeled earlier-born neurons were seen in the 19th bin. Scale bar, 100 μm. n = 4 brains. C–C‴, At P7. Note that the mCherry-labeled later-born neurons and the GFP-labeled earlier-born neurons exhibited a highly segregated inside-out pattern. C‴, Bin analysis. The thickness of the CP (from the outer border of the layer II/III to the inner border of the layer VI) was divided into 20 bins. The mean ± SEM distances from the top of the CP were 117.4 ± 22.7 μm for the GFP-labeled neurons and 59.8 ± 7.3 μm for the mCherry-labeled neurons, respectively (Mann–Whitney's test, _p_=0.0495, n = 3 brains, >700 neurons were counted). Scale bar, 100 μm.

Figure 6.

Figure 6.

Dab1 is required for the eventual inside-out alignment of the mature cortex. A–A‴, Control-Dab1–KD case at E18.5. The distributions of the GFP-labeled earlier-born control neurons and the mCherry-labeled later-born Dab1–KD neurons were almost the same as those in the control–control case (Fig. 5_A-A‴_). A‴, Bin analysis. The distance between the outer margin of the CP and the inner margin of the IMZ was divided into 20 bins. Scale bar, 100 μm. n = 3 brains. B–B‴, Control–Dab1–KD case at P1.5. The mCherry-labeled later-born Dab1–KD neurons were seen at almost the same position as the GFP-labeled earlier-born control neurons. B‴, Bin analysis. The distance between the outer margin of the CP and the inner margin of the IMZ was divided into 20 bins. The later-born Dab1–KD neurons were seen in the 19th bin, which was almost similar to the case of the earlier-born control neurons. Scale bar, 100 μm. n = 4 brains. C–C‴, Control–Dab1–KD case at P7. The mCherry-labeled later-born Dab1–KD neurons were located at almost the same position as the GFP-labeled earlier-born control neurons, showing disruption of the inside-out alignment pattern. C‴, Bin analysis. The thickness of the CP was divided into 20 bins. The distribution of the later-born Dab1–KD neurons was similar to that of the earlier-born control neurons. The mean ± SEM distances from the top of the CP were 132.9 ± 16.1 μm for the GFP-labeled neurons and 124.4 ± 10.3 μm for the mCherry-labeled neurons, respectively (p = 0.8273, Mann–Whitney's test, n = 3 brains, >800 neurons were counted.). Scale bar, 100 μm.

Figure 7.

Figure 7.

Dab1–KD neurons can go past the Dab1–KD predecessors until they reach the region just beneath the PCZ. A–A′, Sequential electroporation of Dab1–KD neurons was performed at E14.5 and E15.5. The brains were then examined at P7. Blue is the nuclear staining. A′, Bin analysis. The thickness of the CP was divided into 20 bins. Note that the later-born neurons were located above the earlier-born neurons (the mean ± SEM distances from the top of the CP to the soma were 236.3 ± 10.9 μm for the earlier-born neurons and 138.9 ± 6.9 μm for the later-born neurons, respectively; n = 3 brains, >700 neurons were counted; p = 0.0495, Mann–Whitney's test). Scale bar, 100 μm. B–B′, The brains were examined at P1.5. B′, Bin analysis. The distance between the outer margin of the CP and the inner margin of the IMZ was divided into 20 bins. n = 4 brains. Scale bar, 100 μm. C–C′, D–D′, E–E′, Staining for nestin (cyan), a marker for radial fibers, was unaffected in these sequential electroporation experiments at P1.5. C, C′, Control–control case. D, D′, Control–Dab1–KD case. E, E′, Dab1–KD–Dab1–KD case. Scale bar, 50 μm. F–F‴, G–G‴, H–H‴, NeuN staining at P1.5 in the series of sequential electroporation. F–F‴, In the control–control case, the GFP-labeled earlier-born neurons were seen below the NeuN-negative zone (F′), whereas the mCherry-labeled later-born neurons were seen within the NeuN-negative zone (F″). Cyan blue staining showed the NeuN-positive nuclei, and the white line is the lower border of the NeuN-negative zone (F‴). Scale bar, 100 μm. G–G‴, In the control–Dab1–KD case, mCherry-labeled Dab1–KD later-born neurons were located below the PCZ. H–H‴, In the Dab1–KD–Dab1–KD case, both earlier-born neurons and later-born neurons were located below the PCZ. I, Statistical analysis of the percentages of the mCherry-labeled later-born neurons within the PCZ. Control–control case; the percentage ± SEM of the mCherry-positive cells within the PCZ was 47.4 ± 3.5% (n = 4 brains, >600 neurons were counted). Control–Dab1–KD case; the percentage of the mCherry-positive cells within the PCZ was 14.2 ± 4.3% (n = 3 brains, >180 neurons were counted). Dab1–KD–Dab1–KD case; the percentage of the mCherry-positive cells within the PCZ was 17.6 ± 2.2% (n = 3 brains, >250 neurons were counted). **p < 0.01, ANOVA followed by the Scheffé's F test.

Figure 8.

Figure 8.

The Dab1-dependent terminal translocation forms the inside-out neuronal alignment within the PCZ. In the developing CP, there are two distinct predecessors: the immature predecessors within the PCZ and the mature predecessors below the PCZ. Terminal translocation is necessary for both neuronal entry into the PCZ and establishment of the Dab1-dependent inside-out alignment of neurons. Conversely, locomoting neurons can pass through the mature predecessors independent of Dab1 until they reach the region just beneath the PCZ. Note that the PCZ is a transient structure that is formed from the inside to the outside and that Dab1–KD neurons cannot enter the PCZ.