Permissive corridor and diffusible gradients direct medial ganglionic eminence cell migration to the neocortex

Abstract

Young neurons born in the medial ganglionic eminence (MGE) migrate a long distance dorsally, giving rise to several types of interneurons in neocortex. The mechanisms that facilitate selective dorsal dispersion of MGE cells while restricting their movement ventrally into neighboring regions are not known. Using microtransplantation into fetal brain slices and onto dissociated substrate cells on floating filters (spot assay), we demonstrate that ventral forebrain regions neighboring the MGE are nonpermissive for MGE cell migration, whereas the dorsal regions leading to the neocortex are increasingly permissive. Spot assay experiments using filters with different pore sizes indicate that the permissive factors are not diffusible. We also show that MGE cells respond to chemoattractive and inhibitory factors diffusing from the neocortex and ventromedial forebrain, respectively. We propose that the final extent and regional specificity of MGE cell dispersion is largely dictated by contact guidance through a selectively permissive environment, flanked by nonpermissive tissues. In addition, we propose that chemotactic guidance cues superimposed over the permissive corridor facilitate efficient dorsal migration of MGE cells.

Figure 1

Different regions of the developing brain are differentially permissive for MGE cell migration. (A) PKH26-labeled MGE cells (black) were transplanted into neocortex in brain slice cultures (arrow). Forty-eight hours later, many MGE cells had dispersed throughout the neocortex. Note that only a few cells crossed the boundary back to the LGE (broken line). (B) MGE cells grafted into the hypothalamus remained at the graft site (arrow), unable to penetrate into the host brain tissue. (C) Diagram of the spot assay. Dissociated cells (gray) were spotted on polycarbonate membrane floating on the surface of culture medium in a petri dish, and a reaggregate of labeled MGE cells (black) was placed in the center of the spot. Cells were cultured for 48 h. (D) Labeled MGE cells (black) readily disperse through neocortical cells. (E) MGE cells do not migrate into a spot of hypothalamic cells. (F) Quantification of MGE cell migration through spots of different brain regions (number of cells per spot ± SD). (G) Migration of MGE cells through spots containing mixed neocortical and hypothalamic cells. The percentage indicates the amount of neocortical cells in a particular spot (number of cells per spot ± SD). NCx, neocortex; HT, hypothalamus; mMGE, mantle zone of the MGE; mLGE, mantle zone of the LGE. Scale bars = 400 μm.

Figure 2

The permissive nature of the neocortex is not mediated by diffusible molecules. (A) Diagram of the flipped spot assay. Dissociated cells were spotted on floating polycarbonate membranes and cultured for 7 days. Membranes were flipped upside down, and reaggregate of MGE cells was placed on the opposite side to the cultured spot of tissue. Cells were cultured for an additional 48 h. (B) PKH26-labeled MGE cells (black) do not migrate when placed opposite to hypothalamic tissue cultured on membranes with 0.8-μm pores. (C) Labeled MGE cells (black) disperse when placed opposite to neocortical tissue on membranes with 0.8-μm pores, which allow cell–cell contact. (D) Higher magnification of cells migrating opposite neocortical spot on 0.8-μm membrane. (E) MGE cells do not migrate when placed opposite to neocortical tissue on 0.1-μm membranes, which do not allow cell–cell contact. NCx, neocortex; HT, hypothalamus.

Figure 3

Diffusible molecules influence MGE cell migration. (A) Coculture of MGE reaggregate with hypothalamic explant. More MGE cells migrate distally than proximally to the explant. (B) Coculture of MGE reaggregate with neocortical explant. More MGE cells migrate proximally than distally. (C) Quantification of the difference in the number of cells found in the distal and proximal quadrant (percentage difference ± SD). (D) Quantification of the total number of MGE cells migrating out of the explant (number of migrating cells ± SD). (E) In situ hybridization reveals strong expression of Slit1 in the hypothalamic region (arrow) next to the MGE. Slit1 is also weakly expressed in cortical plate (arrowhead), the target area of MGE cell migration. (F) In situ hybridization reveals strong expression of Slit2 in the hypothalamic region (arrow). (G) Mixing of neocortical cells with HEK293 cells expressing Slit1 or Slit2 does not result in suppression of MGE cell migration in the spot assay (the inhibitory activity of Slit-expressing cells was confirmed in coculture assays) (number of cells per spot ± SD). NCx, neocortex; HT, hypothalamus.

Figure 4

Model of the guidance of MGE cells to the neocortex. MGE is ventromedially surrounded with a nonpermissive tissue (dark gray). In contrast, neocortex is the most permissive tissue in the central nervous system for MGE cell migration (light gray). Inhibitory factors are secreted from the ventromedial forebrain (including Slit1 and Slit2), which suppress MGE cell migration in that direction. Neocortex secretes a factor(s) that seems to act as a chemoattractant for migrating MGE cells. NCx, neocortex; HT, hypothalamus.