Fig. 4.

Immunofluorescence microscopy images of dumbbell-shaped nuclei in Lmnb1^CS/CS neurons (where the nuclear lamina is separated from the bulk of the chromatin). Cortical NPCs were isolated from E13.5 Lmnb1^+/+ and Lmnb1^CS/CS embryos and cultured in serum-containing medium to generate neurospheres. The neurospheres were then cultured in differentiation medium to promote neuronal differentiation and migration. (A) Low-magnification images of cells stained with antibodies against LAP2β (green) and lamin B1 (red). DNA was stained with DAPI (blue). Images of neurons migrating near the edge of the neurospheres (at the bottom of the images) are shown. The nuclei of Lmnb1^+/+ neurons were mostly round, and LAP2β and lamin B1 were located mainly at the nuclear rim. In contrast, many migrating Lmnb1^CS/CS neurons had dumbbell-shaped nuclei (outlined in white) surrounded by LAP2β. [The localization of LAP2β around both ends of the nucleus was clear in higher-magnification images (Fig. 5A).] In those cells, the nonfarnesylated lamin B1 was found at one end of the dumbbell, but the bulk of the chromatin was located in the other end of the dumbbell and lacked a coat of lamin B1 (naked chromatin). (Scale bars, 20 µm.) (B) Higher-magnification images of migrating Lmnb1^CS/CS neurons stained with antibodies against pericentrin (green) and lamin B1 (red). DNA was visualized with DAPI (white). In the dumbbell-shaped Lmnb1^CS/CS nuclei, lamin B1 was always found closer to the leading edge of the cells, as judged by pericentrin staining (arrows); naked chromatin was found in the trailing edge (arrowheads). The length of the thin strand of DNA connecting the two ends of dumbbell-shaped nuclei was variable but was occasionally longer than 70 µm. (Scale bars, 10 µm.)