Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone

Abstract

Neuroblasts produced in the ventricular zone of the neocortex migrate radially and form the cortical plate, settling in an inside-out order. It is also well known that the tangential cell migration is not negligible in the embryonic neocortex. To have a better understanding of the tangential cell migration in the cortex, we disturbed the migration by making a cut in the neocortex, and we labeled the migrating cells with 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in vivo and in vitro. We also determined the birth dates of the cells. Disturbance of tangential cell migration caused an accumulation and disappearance of microtubule-associated protein 2 immunoreactive (MAP2-IR) cells on the ventral and dorsal side of the cut, respectively, which indicated that most of the MAP2-IR cells in the intermediate zone (IZ) were migrating toward the dorsal cortex. The DiI injection study in vivo confirmed the tendency of the direction of cell migration and suggested the origin of the cells to be in the lateral ganglionic eminence (LGE). DiI injection into the LGE in vitro confirmed that the LGE cells cross the corticostriatal boundary and enter the IZ of the neocortex. The migrating cells acquired multipolar shape in the IZ of the dorsal cortex and seemed to reside there. A 5-bromo-deoxyuridine incorporation study revealed that the migrating MAP2-IR cells in the IZ were early-generated neurons. We concluded that the majority of tangentially migrating cells were generated in the LGE and identified as a distinct population that was assumed not to have joined the cortical plate.

Fig. 1.

MAP2 and GABA immunohistochemistry in the E17 rat embryonic neocortex. The left side of all of these panels is arranged to face the dorsomedial direction. Most immunoreactive cells in the lower IZ had long processes directed dorsomedially. The processes were not branched in the temporal cortex but had several branches in the dorsal cortex. MAP2-IR cells in the IZ were stained most strongly and were most obvious even with low magnification. GABA immunohistochemistry equally stained three layers (the marginal zone, the subplate, and the IZ). A, MAP2 immunohistochemistry on the embryonic neocortex. B, GABA immunohistochemistry. C, MAP2-IR cells in the IZ of the temporal cortex. D, GABA-IR cells in the IZ of the temporal cortex. E, GABA-IR cells in the IZ of the dorsal cortex. Arrows in A, C, D, andE indicate the MAP2-IR or GABA-IR long processes.CP, Cortical plate; IZ, intermediate zone; MZ, marginal zone; SP, subplate;VZ, ventricular zone. Scale bar: A, 100 μm, also applies to B; C, 10 μm, also applies to D and E.

Fig. 2.

Cell migration examined after an obstacle to migration at E15 or E16 was made and detected with MAP2 immunohistochemistry at E16 or E17. MAP2-IR cells in the IZ were lost on the dorsal side of the horizontal cut (the obstacle), which was made in the middle level of the neocortex. They had accumulated on the ventral side of the horizontal cut. A, MAP2 immunohistochemistry on the frontal section of the E15 embryonic brain. An arrow (b) indicates the level where a horizontal cut was made. B, High magnification photograph of area indicated with an arrow(b) in A. Arrowsindicate the MAP2-IR cells in the IZ. C, MAP2 immunohistochemistry on the frontal section of the E16 brain with a horizontal cut in the neocortex. An asterisk indicates the point of the horizontal cut made in the neocortex.Arrows (d, e, andf) indicate the points where the photographs inD–F were taken, respectively. D, MAP2-IR cells in the neocortex of the hemisphere contralateral to the horizontal cut. Many MAP2-IR cells (arrow) were observed in the IZ. E, MAP2 immunohistochemistry on the dorsal side of the cut. The MAP2-IR cells (arrow) were reduced drastically in the IZ. F, MAP2 immunohistochemistry on the ventral side of the cut. MAP2-IR cells (arrow) were accumulated in the IZ. Arrowheads atcorners in D–F indicate the direction in which the dorsal cortex locates. Scale bars: A, 100 μm; B, 50 μm; C, 500 μm; shown inD for D, E, F, 50 μm.

Fig. 3.

Cell migration observed in vivo by injecting DiI into the E16 embryonic brain and examined at E18.A, A frontal section of an embryonic brain through an injection site at the corticostriatal sulcus. Many migrating cells were observed in the IZ of the cortex. At the same time many retrogradely labeled cells were observed in the subplate and the cortical plate.B, A confocal microphotograph of migrating cells in the IZ of the temporal cortex. A white arrow indicates the dorsal direction. C, A frontal section of an embryonic brain through an injection site in the dorsal cortex. D, Migrating cells in C with higher magnification.E, A confocal microphotograph of migrating cells in the IZ of the dorsal cortex. F, A frontal section of an embryonic brain through an injection site in the LGE. G, Migrating cells at the corticostriatal sulcus. CP, Cortical plate; HIP, hippocampus; IZ, intermediate zone; LGE, lateral ganglionic eminence;MGE, medial ganglionic eminence; SP, subplate. Dorsal direction is the top of all panels exceptB. Arrows indicate the migrating cells.Large arrowheads indicate the corticostriatal sulcus.Small arrowheads indicate migrating cells crossing the corticostriatal boundary. Scale bars: A, 500 μm, also applies to F; B, D, 50 μm;E (shown in B), 50 μm; C, G, 100 μm.

Fig. 4.

Schematic diagrams to summarize the direction and number of migrating cells observed in vivo after DiI injection into the embryonic brain. Asterisks indicate the injection sites. Arrows indicate the direction of cell migration. Size of arrows and thenumber nearby stand for the number of cells migrating in each direction. A, DiI crystal was injected into the medial wall of the telencephalic vesicle. Ten migrating cells were observed outside of the injection site. All of them were directed ventrally toward the corticoseptal boundary. Regarding the rostrocaudal distribution, the migrating cells were found in five serial sections obtained from around the level of the injection site (within 500 μm width). B, DiI crystal was injected into the dorsal wall of the telencephalic vesicle (dorsal cortex). Nineteen migrating cells were observed. Eighteen cells were directed ventrally in the medial wall of the telencephalic vesicle. Only one cell was found migrating laterally. The cells were distributed in six serial sections obtained from around the level of the injection site. C, DiI was injected into the corticostriatal sulcus. More than 500 migrating cells were observed in 12 serial frontal sections (1.2 mm) obtained more from the rostral side of the injection site. Most of them were directed dorsally along the IZ. Several migrating cells were also observed in the temporal cortex. D, DiI was injected into the ventricular zone between the MGE and LGE, and in total >500 migrating cells were also observed in this case. The labeled migrating cells from the LGE were distributed more widely in the rostrocaudal direction but tilted toward the rostral direction (in 17 serial frontal sections obtained more rostrally to the injection site). Most of them were directed dorsally. Some of them (<50) were directed toward the temporal cortical surface with radial migration.

Fig. 5.

Top. Cell migration observed in vitro by injecting DiI into the LGE of the E16 embryonic brain and cultured for 24 hr (Case 1 in Table 2). A, A cultured rostral half of the embryonic brain. B, A frontal section through the LGE and the neocortex of the cultured brain. Arrows (c andd) indicate the migrating cells in the IZ and the internal capsule shown in C and D, respectively. An arrowhead indicates the corticostriatal sulcus. C, A migrating cell in the IZ with higher magnification. D, A migrating cell in the internal capsule with higher magnification. LGE, Lateral ganglionic eminence; MGE, medial ganglionic eminence. Scale bars: A, 1 mm; B, 500 μm;C, 50 μm, and also applies to D.

Fig. 7.

The time of origin of MAP2-IR cells in the IZ of the E17 embryo brain. BrdU was injected at E12, E13, E14, E15, or E16. The number of BrdU and MAP2 double-labeled cells contained in one side of the IZ in one frontal section was obtained from 21 embryo brains. These numbers were used to make the histogram of birth dates of the MAP2-IR cells in the IZ of the E17 neocortex. The most numerous double-labeled cell somata were found after BrdU injection at E14 (24.1 ± 1.75; mean ± SD; n = 5).