Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex

Abstract

Two distinct modes of radial neuronal migration, locomotion and somal translocation, have been reported in the developing cerebral cortex. Although these two modes of migration have been well documented, the cortical intermediate zone contains abundant multipolar cells, and they do not resemble the cells migrating by locomotion or somal translocation. Here, we report that these multipolar cells express neuronal markers and extend multiple thin processes in various directions independently of the radial glial fibers. Time-lapse analysis of living slices revealed that the multipolar cells do not have any fixed cell polarity, and that they very dynamically extend and retract multiple processes as their cell bodies slowly move. They do not usually move straight toward the pial surface during their radial migration, but instead frequently change migration direction and rate; sometimes they even remain in almost the same position, especially when they are in the subventricular zone. Occasionally, the multipolar cells jump tangentially during their radial migration. Because the migration modality of these cells clearly differs from locomotion or somal translocation, we refer to their novel type of migration as "multipolar migration." In view of the high proportion of cells exhibiting multipolar migration, this third mode of radial migration must be an important type of migration in the developing cortex.

Figure 1.

Histological features of multipolar cells in mouse developing cerebral cortex. A, B, Coronal sections of brains transfected with EF1α-GFP/E13:E16. In both the dorsomedial cortex (A) and the lateral cortex (B), most of the GFP-expressing cells in the IZ and SVZ exhibited multipolar cell morphology, whereas cells in the CP they had a radially oriented bipolar cell morphology. The dashed lines indicate the border between VZ and SVZ, or IZ and CP. C, High-magnification views of the GFP-positive cells in the CP. D-G, In the CAG-GFP/E14.5:E16 brains, the GFP-positive cells exhibiting multipolar cell morphology in the IZ-SVZ (D, left panel; E, F; extended focus view of the confocal image in F is shown in G) expressed the neuron markers Hu (D, middle and right panels) and TuJ1 (E, F). The dashed line in D and E indicates the border between VZ and SVZ. The arrows in D and E indicate the cytoplasm of multipolar cells. H, Although some of the thin processes of the multipolar cells were apposed to the radial fibers (arrowhead), which were stained with anti-nestin, most of them extended independently from the radial fibers. EF1α-GFP/E13:E16 brains were analyzed. Scale bars: A, B, 100 μm; C-E, H, 20 μm; F, G, 10 μm.

Figure 2.

The GFP-labeled multipolar cells originated from the cortical VZ. A, Plasmid DNA was injected into the lateral ventricle on one side, and an anode was placed on the opposite side of the injected hemisphere so that the dorsomedial region was labeled selectively (CAG-GFP/E14.5:E16). The dashed lines indicate the margin of the tissue. GE, Ganglionic eminence. B, High magnification of the boxed region in A revealed that many of the GFP-expressing cells in the IZ-SVZ exhibited multipolar cell morphology, whereas the progenitor cells in the GE were not labeled (A). The dashed line indicates the border of VZ and SVZ. C, D, The GFP-positive multipolar cells (C and D, green in the right panels) were calbindin negative (C, left panel and purple in the right panel) and GABA negative (D, left panel and purple in the right panel). The calbindin-positive cells are indicated by the arrows. The EF1α-GFP/E13:E16 brains (C) and CAG-GFP/E14.5:E16 brains (D) were analyzed. E, Tbr1 immunostaining on the CAG-GFP/E12.5:E13.5 brains. High expression of Tbr1 was seen in the CP, and low expression was detected in the IZ (purple in the left and middle panels). A single confocal section of the boxed region in the left panel is shown in the middle panel. The GFP-positive (green) cells were Tbr1 positive (purple). The extended-focus view of the green channel of the middle panel revealed that the GFP-positive cells exhibited a multipolar cell morphology (right panel). Scale bars: A, E, 200 μm; B, 100 μm; C, D, 20 μm.

Figure 3.

Time-lapse observation of multipolar migration. A, B, The movement of the multipolar cells was observed on living slices prepared from EF1α-GFP/E12.5:E14 brains. A, Multipolar cells advanced toward the pial surface (top) very slowly, while extending and retracting multiple processes dynamically. B, DsRed-positive multipolar cells were colocalized frequently with DsRed-positive radial glial cells. In this specimen, three labeled multipolar cells (arrowheads and arrow) were colocalized closely with a labeled radial glial cell, the body of which (asterisk) was undergoing mitosis (M phase at t = 7.5 hr). One of the multipolar cells, indicated by the arrow, advanced toward the pial surface (dashed line) by means of the dynamic movement of its processes and passed another multipolar cell. The short horizontal bars on the right in A and B represent the initial position (t = 0 hr) of the cell observed. C, The locomotion cells within the CP were observed on the living slices taken from the CAG-GFP/E14.5:E18 brains. The migration rates of cells a-c were measured (see Results) (movie files, available at www.jneurosci.org). The position of cell c is indicated by the arrow. D, The multipolar cells near the border between IZ and CP tend to extend major leading processes toward the pial surface. Scale bars, 20 μm.

Figure 4.

Migratory course of the multipolar neurons. A, Tracing of multipolar cell movement. The slices were prepared from CAG-GFP/E12.5:E14 brains, and time-lapse observations were made in the IZ. The position of 10 individual multipolar cells was plotted at 1 hr intervals for 10 hr. The image taken 2 hr after the first time point is shown in the left panel, and the trajectory is shown in the right panel. Many of the multipolar cells occasionally remained in nearly the same positions for several hours during their migration (dotted circles). The border between the IZ and CP is indicated by a dashed line. The traced cells are indicated by the arrows. B, Tangential jump of multipolar cells. One multipolar cell (arrow) temporarily assumed locomotion cell-like morphology by extending a thick process tangentially and jumping in that direction (t = 2.5-8.5 hr). The cell then retracted the thick tangential process and extended a radially oriented thick process. Finally, the cell resumed radial multipolar migration toward the pial surface (top) (t = 10.5-14 hr). The other cell (arrowhead; t = 6.5) also jumped tangentially (t = 4.5-8.5 hr) (movie file, available at www.jneurosci.org). C, D, The cell bodies and tangential processes of the multipolar cells (green) tended to be oriented in parallel with the NF-positive axon bundles (purple). CAG-GFP/E14.5:E16 (C) or CAG-GFP/E16:E17.5 (D) brains were analyzed. Scale bars, 20 μm.