Mode and tempo of tangential cell migration in the cerebellar external granular layer

Abstract

After their final mitosis, cerebellar granule cells remain in the external granular layer (EGL) for 20-48 hr before initiating their radial migration across the molecular layer (ML), but the significance of this latent period is not well understood. In the present study, we used a confocal microscope to examine morphogenetic changes and behavior of postmitotic granule cells restricted to the EGL in slice preparations of the postnatal mouse cerebellum. We found that, coincident with the extension of two uneven horizontal processes oriented parallel to the longitudinal axis of the folium, postmitotic granule cells start to migrate tangentially in the direction of the larger process. Interestingly, their morphology and the speed of cell movement change systematically with their position within the EGL. The rate of tangential cell movement is fastest (approximately 14.8 micrometer/hr) in the middle of the EGL, when cells have two short horizontal processes. As granule cells elongate their somata and extend longer horizontal processes at the bottom of the EGL, they move at a reduced rate (approximately 12.6 micrometer/hr). At the interface of the EGL and ML where cells migrate tangentially at the slowest rate (approximately 4.1 micrometer/hr), their somata round and then begin to extend couples of the descending processes into the ML. After the stationary period, granule cells abruptly extend a single vertical process and initiate the transition from tangential to radial migration, reshaping their rounded somata into a vertically elongated spindle. These observations suggest that tangential migration of granule cells within the EGL may provide the developmental mechanisms for their appropriate allocation across parasagittal compartments of the expanding cerebellar cortex.

Fig. 1.

A, Time course of translocation of postmitotic granule cells in the EGL. P10 mice were injected with BrdU intraperitoneally and killed 2 hr (a), 1 d (b), and 2 d (c) later. Two hours after injection, BrdU-labeled cells were localized at the top level of the EGL. One day after injection, the BrdU-labeled cells occupied the entire EGL. Two days after injection, approximately half of BrdU-labeled cells left the EGL and translocated to the ML, PCL, and IGL. B, Morphology of granule cell precursors and postmitotic granule cells in the EGL of P10 mouse cerebellum. The granule cells in the EGL at the pyramis were visualized 2 hr after DiI staining. The granule cell precursor (a) had a round soma without any long processes. Postmitotic granule cells (b, c) had spindle-shaped cell bodies oriented parallel to the longitudinal axis of the folium with two horizontal processes. On the EGL–ML border, the postmitotic granule cell (d) had a rounded cell body with vertically oriented short processes penetrating the ML. Scale bars, 10 μm. PC, Purkinje cell.

Fig. 2.

Tangential migration of a granule cell in the middle level of the EGL. A, During the observation period, the granule cell soma (white asterisks) gradually moved toward the left side of the cerebellar hemisphere at a rate of 18.8 μm/hr. The distal portion of the leading process had large motile lamellipodia (small white arrowheads). Time interval (in minutes) is indicated on the top right of each photograph. Scale bar, 10 μm. The total distance traversed by the granule cell soma (B), the direction and distance traveled by the soma during each 10 min of the testing period (C), the length/width ratio of the soma (D), and the leading process length (E) were plotted as a function of elapsed time. In C, positive values represent forward cell movement, and negative values represent backward cell movement.

Fig. 3.

Postmitotic granule cell extending its voluminous leading process without active cell movement. A, In the middle level of the EGL, the cell body of a granule cell (white asterisks) remained stationary for >3 hr, while its voluminous leading process (white arrows) extended continuously. Time interval (in minutes) is indicated on the top leftof each photograph. Scale bar, 10 μm. The total distance traversed by the granule cell soma (B), the direction and distance traveled by the soma during each 5 min of the testing period (C), the leading process length (D), and changes in the leading process length during each 5 min of the testing period (E) were plotted as a function of elapsed time.

Fig. 4.

Tangential movement of a granule cell in the bottom level of the EGL. A, During the observation period, the spindle-shaped soma of the granule cell having two long horizontal processes (white arrowheads) migrated toward the left side of the hemisphere. Time interval (in minutes) is indicated on the top right of each photograph. Scale bar, 10 μm. The total distance traversed by the granule cell soma (B), the direction and distance traveled by the soma during each 10 min of the testing period (C), and the length/width ratio of the soma (D) were plotted as a function of elapsed time.

Fig. 5.

A reverse in the direction of tangential movement of a granule cell in the bottom level of the EGL. A, The horizontally oriented cell body (white asterisks) of the granule cell initially moved toward the left hemisphere at a rate of 14.6 μm/hr, and 80 min later, changed its direction of movement and started to migrate toward the right hemisphere at a rate of 17.9 μm/hr. Black circles represent a reference point. Time interval (in minutes) is indicated on the bottom rightof each photograph. Scale bar, 10 μm. The total distance traversed by the granule cell soma (B), the direction and distance traveled by the soma during each 10 min of the testing period (C), and the length/width ratio of the soma (D) were plotted as a function of elapsed time. In B and C, positive values represent cell movement toward the left hemisphere, and negative values represent cell movement toward the right hemisphere.

Fig. 6.

Slowdown of tangential movement of a granule cell with longer horizontal processes. A, During the period of observation, the cell with longer horizontal processes moved toward the right side of the hemisphere at a reduced rate of 9.1 μm/hr. Interestingly, the cell did not protrude filopodia and lamellipodia from its cell body and horizontal processes. Time interval (in minutes) is indicated on the top right of each photograph. Scale bars, 10 μm. B, A photograph representing the long horizontal process of tangentially migrating granule cell shown inA. The total distance traversed by the granule cell soma (C), the direction and distance traveled by the soma during each 10 min of the testing period (D), and the length/width ratio of the soma (E) were plotted as a function of elapsed time.

Fig. 7.

Slowly migrating granule cell with descending processes near the EGL–ML border. A, The highly motile processes having lamellipodia-like structure (open arrows) and filopodia (white arrows), extended from the cell body. During the process extension, the horizontally oriented cell body (asterisks) exhibited the tangential movement toward the right hemisphere of the cerebellum at a significantly reduced rate of 3.8 μm/hr. Time interval (in minutes) is indicated on the bottom left of each photograph. Scale bar, 10 μm. The total distance traversed by the granule cell soma (B), the direction and distance traveled by the soma during each 5 min of the testing period (C), and the length/width ratio of the soma (D) were plotted as a function of elapsed time.

Fig. 8.

Initiation of vertical migration and formation of a T-shaped axon. A, At the beginning, a granule cell (asterisks) located near the EGL–ML border (dotted line), had vertically oriented soma with two horizontal and one vertical process. The cell remained stationary for the first 30 min before its soma quickly moved radially toward the bottom of the ML. As a result, the granule cell developed a trailing process (small arrow) and a T-shaped axon. Time interval (in minutes) is indicated on the bottom right of each photograph. Open arrows represent rapid extension of the horizontal process of other granule cells. Scale bar, 10 μm. The total distance traversed by the granule cell soma (B), the direction and distance traveled by the soma during each 10 min of the testing period (C), and the length/width ratio of the soma (D) were plotted as a function of elapsed time.

Fig. 9.

Extrusion of one side of the parallel fibers during initiation of vertical migration of a granule cell.A, During the first 30 min, the granule cell (asterisks) located near the EGL–ML border (dotted line), had a single horizontal process extending toward the left side. Forty minutes later, the cell developed a small horizontal process (open arrow) at the rear part of its soma toward the right side. As a result of downward movement of the soma, the cell developed a T-shaped axon branch. Time interval (in minutes) is indicated on the bottom right of each photograph. Scale bar, 10 μm. The total distance traversed by the granule cell soma (B), the direction and distance traveled by the soma during each 5 min of the testing period (C), and the length/width ratio of the soma (D) were plotted as a function of elapsed time.

Fig. 10.

A–E, Histograms of soma length (A), soma width (B), leading process length (C), movement rate(D), and movement direction (E) of granule cells in the EGL. To categorize the morphology and behavior of the postmitotic granule cells within the EGL, we divided the width of the EGL of the P10 mouse into five levels:levels I, II (proliferating cell layer; within 12 μm of the pial surface); level III(postmitotic cell layer; within 18 μm of the pial surface);level IV (postmitotic cell layer; within 24 μm of the pial surface); and level V (postmitotic cell layer; over 24 μm from the pial surface). Each column represents the average values obtained from 80 migrating granule cells in level III, 75 cells in level IV, and 64 cells in level V. *p < 0.05 and **p < 0.01 indicate statistical significance.

Fig. 11.

A, Shift of the geography of developing cerebellar cortex over time. At P10, the granule cell precursors proliferate within ∼19–21 hr of the cell cycle within levels I and II of the EGL. Approximately 21 hr after their final cell division, tangentially migrating granule cells change the orientation of their somata from horizontal to vertical and start to leave the EGL. As a result, their horizontal processes become parallel fibers, and the EGL–ML border moves up toward the pial surface (as seen at P11). Therefore, the granule cell, which initially migrates tangentially at level III of the EGL at P10, will be located at the level V after 24 hr (at P11), without undergoing downward movement. B, Schematic drawing illustrating the hypothesis that granule cells may be specified to settle in a particular A-P compartment and have to travel tangentially over relatively long distances in the middle and the bottom of the EGL to reach a target compartment. Granule cells that underwent final cell division in the same area at the top level of the EGL may settle in different compartments in the IGL. For example, green granule cells may be instructed to enter a green compartment, whereas red granule cells may be instructed to enter ared compartment. Furthermore, postmitotic granule cells located in the left or right side of the hemisphere tend to migrate toward the midline region, whereas the cells located in the midline region do not exhibit a preference in the movement direction between the left and right side.