MiceTime-pregnant ICR wild-type mice were purchased from SLC (Shizuoka, Japan). Mice expressing green fluorescent protein (GFP; green mice) were provided by Okabe et al. (Okabe et al., 1997). Xt J /Xt J mutant embryos, which have deletions in the Gli3 gene, were generated as described previously (Tomioka et al., 2000). When the Xt J mutation was introduced into green mice, green mice were crossed with Xt J /+ mice and the resulting Xt J /+ mice expressing GFP were intercrossed to obtain Xt J /Xt J green mouse embryos. Heterozygous netrin 1 mutant mice (Serafini et al., 1996) and Dcc mutant mice (Fazeli et al., 1997) were crossed to obtain homozygous embryos. The day on which a vaginal plug was found was designated embryonic day 0.5 (E0.5). All experimental protocols were approved by the Animal Committee of National Institute of Genetics.
Organotypic culture of telencephalon stripsEach telencephalic hemisphere of the E10.5 mouse embryo was dissected along the dorsoventral axis into a strip ~1.5 mm wide, including the neocortex, the presumptive LOT area and the lateral and medial ganglionic eminences (GE) (see Fig. 1A). In the strip, the presumptive LOT area was located around the middle along the dorsoventral axis. In a standard culture, the strip was labeled by injection of dextran tetramethylrhodamine In the developing nervous system, functional neural networks are constructed with intricate coordination of neuronal migrations and axonal projections. We have previously reported a ventral tangential migration of a special type of cortical neurons, lot cells, in the mouse embryo. These neurons originate from the ventricular zone of the entire neocortex, tangentially migrate in the surface layer of the neocortex into the ventral direction, align in the future pathway of the lateral olfactory tract (LOT) and eventually guide the projection of LOT axons. In this study, we developed an organotypic culture system to investigate the regulation of this cell migration in the developing telencephalon. Our data show that the neocortex contains the signals that direct lot cells ventrally, that the ganglionic eminence excludes lot cells by repelling the migration and that lot cells are attracted to netrin 1, an axon guidance factor. Furthermore, we demonstrate that mutations in the genes encoding netrin 1 and its functional receptor Dcc lead to inappropriate distribution of lot cells and subsequent partial disruption of LOT projection. These results suggest that netrin 1 regulates the migration of lot cells and LOT projections, possibly by ensuring the correct distribution of these guidepost neurons. Molecular Probes, Eugene, OR) or 1,1-dioctadecyl-3,3,3Ј3Ј-tetrametylindocarbacyanine perchlorate (DiI; Molecular Probes) into a small area of the dorsal neocortex, unfolded on a collagen-coated membrane filter (Transwell-COL inserts #3492, Corning, Acton, MA) and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Sigma-Aldrich, St Louis, MO) containing 10% fetal bovine serum (Cansera, Rexdale, ONT, Canada) and 5% hor...