Figure 1.
Neurons labeled in the cerebellar cortex by the electroporation of pCAGGS-mCherry.
(A) A Purkinje cell. The cell extends an axon (arrows) towards the WM and axon collaterals (arrowheads) ending in the Purkinje cell layer. (B) Two Golgi cells in the IGL extending their dendrites into the ML and ramified axons into the IGL. (C) A Lugaro cell in the IGL, bipolar in shape, extending dendrites in parallel with the Purkinje cell layer. (D) A UBC in the IGL. (E1) An mCherry-labeled cell in the IGL shows the morphology of Golgi cells. Maximum projection images for a series of confocal images. (E2,E3) Double-labeling immunohistochemistry for neurogranin (blue) and mCherry (orange) confirmed the Golgi cell identity. Confocal plane images. ML, molecular layer; IGL, internal granular layer. Scale bars: A, 20 µm; B–E1, 10 µm.
Table 1.
Summary of cells in the cerebellar cortex labeled at each stage of electroporation.
Figure 2.
Labeled cells in the cerebellar cortex by IUE of transposon plasmids.
(A) Low magnification view of a sagittal section of a Tol2-GFP electroporated cerebellum. (B) A basket cell extending its axon branches towards the Purkinje cell layer (lower left). (C) A stellate cell in the upper ML extending ramified processes. Black dots in the insets in B and C show the location of the respective cell bodies. (D) A granule cell with radiating dendrites (arrow) and an axon extending towards the ML (arrowheads). (E,F) Numerous parallel fibers occupy the ML. F is a high-magnification view of the boxed region in E. (G) A UBC in the IGL. (H) A Bergmann glia. (I) An astrocyte in the IGL. (J) An oligodendrocyte in the WM. (K) A large-diameter DN neuron (arrowhead). (L) A small-diameter DN neuron. (M1, N1, O1, P1) Maximum projection images for each series of confocal images of each cell. (M2–M4, N2–N4, O2–O4, P2–P4) Confocal plane images. (M1–M4) A Tol2-GFP labeled cell in the upper part of the ML shows the morphology of stellate cells. Double-labeling immunohistochemistry for parvalbumin (magenta) and GFP (green) confirmed the stellate cell identity. (N1–N4) A Tol2-GFP labeled cell in the lower part of the ML shows the morphology of basket cells. Double-labeling immunohistochemistry for parvalbumin (magenta) and GFP (green) confirmed the basket cell identity. (O1–O4) A Tol2-GFP labeled cell in the IGL shows the morphology of astrocytes. Double-labeling immunohistochemistry for GFAP (magenta) and GFP (green) confirmed the astrocyte identity. (P1–P4) A Tol2-GFP labeled cell in the white matter shows the morphology of oligodendrocytes. Double-labeling immunohistochemistry for MBP (magenta) and GFP (green) confirmed the oligodendrocyte identity. Scale bars: 10 µm ML, molecular layer; PCL, Purkinje cell layer; IGL, internal granular layer. Scale bars: A, 1 mm; B, C, E, F, K, L, M1, N1, O1, and P1, 10 µm; in G, 10 µm for D and G; in J, 10 µm for H, I, and J.
Figure 3.
Electroporation of Cre recombinase into Ai9 reporter mice caused labeling of all cerebellar cell types.
Electroporation was performed at E10.5–E12.5, and electroporated animals were observed at P21–P25 (see also Table 1). As shown in A, numerous cells were labeled in the cerebellum and included Purkinje cells (B), Golgi/Lugaro cells (C), stellate cells (D) and basket cells (E), granule cells (F) and UBCs (G), all of which were readily identified by their morphology and laminar positions. Glial cells, including Bergmann glia, were also labeled (data not shown). These findings support the hypothesis that the labeling of all cerebellar cell types by Tol2 transposon-mediated gene transfer was due to the integration of the GFP gene into the genome of the progenitors and the corresponding progeny. In cases in which many granule cells were labeled in the internal granular layer, it was difficult to identify Golgi/Lugaro cells based on morphological features only. In such cases, identification was facilitated by immunohistochemistry for neurogranin. Scale bar: A, 100 µm; B–G, 10 µm.
Table 2.
Summary of cells in the DN labeled at each stage of electroporation.
Figure 4.
Labeled cells at the site of electroporation.
(A,B) Labeled cells in the cerebellar primordium (cp). Electroporation at E10.5. (C) Magnified view of the boxed region (c) in B. (D) Schematic illustrating of dorsal view of the midbrain-hindbrain showing the plane of sections (blue). (E,F) Labeled cells attached to the ventricular surface directly (E) or by extending an apical process (F). (G,H) A cell with an apical endfoot and a radial process reaching the pial surface in the uRL region (G) and a cell with a radial glia-like morphology in the VZ (H). Electroporation at E12.5. (I) A radial glia-like cells in the VZ with a basal process (arrowheads) and apical process that reach the pial and ventricular surface, respectively. Electroporation at E14.5. (J) A mixture of pCAGGS-mCherry, pCAGGS-Cre, and pCALNL5-EGFP were electroporated and observed 12 h after electroporation. Parasagittal sections. cp, cerebellar primordium; VZ, ventricular zone. Scale bars: in B, 500 µm for A,B; C, 50 µm; E–J, 10 µm.
Figure 5.
Emigration of neurons from the cerebellar primordium.
UBCs labeled in the dorsal cochlear nucleus of the brainstem. (A) The fluorescent image is a higher magnified view of the dotted-boxed region in the inset, which is a Nissl-stained consecutive section. (B) A high magnified view of the boxed region (white solid line) in A. The inset is a magnified view of a UBC (arrow). Parasagittal sections. Cb, cerebellum; Hb, hindbrain. Scale bars: A, B, 100 µm; in the inset of A, 1 mm; in the inset of B, 10 µm.
Figure 6.
Schematic summarizing the present results.
Progenitors having a radial glia-like morphology are present both in the VZ and uRL. Green bars signify that progenitors are self-renewing at indicated stages. Orange bars show stages at which progenitors are generating differentiated cells. GC/LC, Golgi cell and Lugaro cell; PC, Purkinje cell; SC/BC, stellate and basket cell; BG, Bergmann glia; As, astrocyte; Ol, oligodendrocyte; sDN, small-diameter deep nuclei neuron; lDN, large-diameter deep nuclei neuron; grC, granule cell.