Lis1 is necessary for normal non-radial migration of inhibitory interneurons

Abstract

Type I lissencephaly is a central nervous system (CNS) malformation characterized by mental retardation and epilepsy. These clinical features suggest a deficit in inhibitory neurons may, in part, underlie the pathogenesis of this disorder. Mutations in, or deletions of, LIS1 are the most commonly recognized genetic anomaly associated with type I lissencephaly. The pathogenesis of type I lissencephaly is believed to be a defect in radial neuronal migration, a process requiring LIS1. In contrast the inhibitory neurons migrate non-radially from the basal forebrain to the neocortex and hippocampus. Given that Lis1 is expressed in all neurons, we hypothesized that Lis1 also functions in non-radial migrating inhibitory neurons. To test this hypothesis we used a combination of in vivo and in vitro studies with Lis1 mutant mice and found non-radial cell migration is also affected. Our data indicate Lis1 is required for normal non-radial neural migration and that the Lis1 requirement is primarily cell autonomous, although a small cell non-autonomous effect could not be excluded. These data indicate inhibitory neuron migration is slowed but not absent, similar to that found for radial cell migration. We propose that the defect in non-radial cell migration is likely to contribute to the clinical phenotype observed in individuals with a LIS1 mutation.

Figure 1

Inhibitory interneurons migrate a shorter distance in E14.5 Lis1 +/− animals. Immunohistochemistry for calretinin (a, b, d, e) and GABA (c and f) on coronal sections of the telencephalon in E14.5 wild-type (a, b, c) and Lis1 +/− (d, e, f) embryos. GABA (c)-labeled and calretinin (a)-labeled neurons have migrated farther in wild-type embryos when compared to the mutant mice (f for GABA, d for calretinin) (see Figure 2 for quantitation). At higher power, taken from boxed areas in a and d, the leading edge cells with morphologies and orientations of non-radially migration interneurons can be seen in the dorso-lateral cortex of the wild-type (b), but only in medio-lateral cortex of the Lis1 +/− animals, indicating that interneurons have not migrated as far in the mutant animals at similar points in development.

Figure 2

Total distance and percentage of distance from GE to dorsum of cortex migrated in Lis1 +/− and wild-type E14.5 embryos. The averaged distance migrated by all calretinin-positive cells past the cortico-striatal notch is significantly greater in wild-type animals by E14.5 (A). When only the leading GABAergic cells are examined, the differences between mutant and wild-type animals is greater in magnitude, as are the total distances traveled (B). Comparing the percentage of the distance from the cortico-striatal notch to the dorsum of the cortex in both cases reveals that Lis1 +/− animals also traverse a significantly smaller portion of the developing brain than do wild-type age-matched controls (C and D)

Figure 3

Migration of DiI-labeled inhibitory interneurons is impaired in Lis1 +/− E14.5 embryonic slice culture. Representative examples of E14.5 embryonic brain slice cultures with DiI crystal implants in wild-type (a) and Lis1 +/− animals (b). Arrows in (a) and (b) indicate approximate average distance migrated dorsally from the DiI crystal edge by all labeled (arrowhead) or the leading 25 (arrow) cells (for quantification, see Figure 5). Cells labeled with DiI displaying characteristic morphologies of non-radially migrating cells (c) also label with antibody against a marker of inhibitory interneurons, calretinin (d, and merged image in e).

Figure 4

Average distance migrated by cells from Lis1 +/− mice in E14.5 slice cultures is significantly less than by wild-type cells. The average distance migrated by all DiI-labeled cells dorsally from the DiI crystal edge is statistically less in Lis1 +/− mice than in wild-type animals, at E14.5 (A). Similarly, when comparing the average distance traveled by the leading 25 cells, Lis1 +/− animals exhibit a migratory defect relative to wild-type animals (B)

Figure 5

Distribution of distance migrated by mutant and wild-type cells in slice culture. Cells from both wild-type (white bars) and Lis1 +/− (black bars) eminences are placed into bins by distances of 100 μm migrated from the DiI crystal edge. The distributions of both populations are similarly shaped and approximately normal. There is no statistical difference in the number of cells at distances between 300 and 500 μm, inclusive. At all farther distances there is a statistically greater number of wild-type than mutant cells, and at the farthest distances only wild-type cells are found (beyond 2 mm past the DiI crystal). Bins less than 300 μm cannot be considered as the halo from the DiI crystal often made identification of individual cells impossible. Comparison within bins were made by two-tailed Student’s t-test; *, denotes P < 0.01

Figure 6

Schematic representation of E14.5 transplant migration assay. Brain slices were obtained as for standard slice culture. Before DiI crystal implantation, the neocortex was excised from the ventral telencephalon along dotted line, and re-apposed to either a homo- or heterogenic ventral telencephalon (source of migratory, GE-derived cells) (blue and tan denote different genotypes, they can be interchanged such that if blue represents mutant and thus the source of GE cells in the image the figure would show mutant cells migrating on wild-type substrate. The reverse genotype, ie, tan being mutant, would give wild-type cells migrating on mutant substrate. For those slices where blue is re-apposed with blue or tan with tan, the resulting section would mutant with mutant or wild-type with wild-type giving all possible combinations; see Figure 7). DiI crystals (red sphere) were then implanted, and slices were cultured and assayed as cell migration (far right).

Figure 7

Migratory defect in Lis1 +/− animals is both cell autonomous and non-autonomous. Representative examples of E14.5 embryonic brain slice transplant cultures with DiI crystal implants in cultures with either wild-type GEs (a and b) or Lis1 +/− GEs (c and d) apposed to either wild-type (a and c) or Lis1 +/− cortices (b and d). Arrows in (a–d) indicate approximate average distance migrated dorsally from the DiI crystal edge by all labeled (arrowhead) or the leading 25 (arrow) cells (for quantification, see Figure 8). (GE, ganglionic eminence; cortex, cortical substrate, see Figure 6)

Figure 8

Quantification of cell autonomous and non-autonomous migratory defect in Lis1 +/− mice in E14.5 slice cultures. Migratory cells derived from wild-type GEs migrated farther than those from Lis1 GEs, regardless of the cortical migratory substrate’s genotype, as assessed by the averaged distance migrated dorsally by all (A) or the leading 25 (B) DiI-labeled cells. This indicates a cell autonomous defect in migratory ability in the Lis1 +/− animals. When comparing how well cells migrated on wild-type or mutant cortex, a cell non-autonomous effect is evidenced by the significantly greater distance traveled by the leading 25 wild-type cells in wild-type rather than Lis1 +/− cortex (B).