Radial glial dependent and independent dynamics of interneuronal migration in the developing cerebral cortex

Abstract

Interneurons originating from the ganglionic eminence migrate tangentially into the developing cerebral wall as they navigate to their distinct positions in the cerebral cortex. Compromised connectivity and differentiation of interneurons are thought to be an underlying cause in the emergence of neurodevelopmental disorders such as schizophrenia. Previously, it was suggested that tangential migration of interneurons occurs in a radial glia independent manner. Here, using simultaneous imaging of genetically defined populations of interneurons and radial glia, we demonstrate that dynamic interactions with radial glia can potentially influence the trajectory of interneuronal migration and thus the positioning of interneurons in cerebral cortex. Furthermore, there is extensive local interneuronal migration in tangential direction opposite to that of pallial orientation (i.e., in a medial to lateral direction from cortex to ganglionic eminence) all across the cerebral wall. This counter migration of interneurons may be essential to locally position interneurons once they invade the developing cerebral wall from the ganglionic eminence. Together, these observations suggest that interactions with radial glial scaffold and localized migration within the expanding cerebral wall may play essential roles in the guidance and placement of interneurons in the developing cerebral cortex.

Figure 1. Analysis of interneuron-radial glial interactions in the embryonic cerebral cortex.

(A) Interneurons express GFP in a coronal slice of E16 cortex from Dlx5/6-cre-IRES-EGFP (Dlx5/6-CIE) mice. Radial glial scaffold is labeled with RC2 antibodies (blue). (B–D) Higher magnification view of the outlined region (B, radial glial scaffold; C, GFP⁺ interneurons, D, merge) illustrates the potential for interneuron-radial glial interactions as the neurons invade the cerebral wall. (E) Assay for live imaging of radial glia-interneuronal interactions. Dlx5/6-CIE E16 cerebral cortices were electroporated with BLBP-DsRed2 to label multiple radial glia. Time-lapse imaging of slices from these electroporated cortices were used to evaluate interneuron [arrowhead]-radial glial [arrow] interactions. GE, ganglionic eminence; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Scale bar: A, 1250 µm; B–D, 350 µm; E, 80 µm.

Figure 2. Radial glia modulates the patterns of interneuronal migration.

(A) An interneuron [arrowhead] migrating in the marginal zone area turn towards a radial glial endfeet [red]. (B) An interneuron [arrowhead] ascending towards the cortical plate uses radial glial strand [red] as a migratory guide, before detaching from it. (C) Contact with a radial glial cell soma [red] in the ventricular zone alters the trajectory of an interneuron [arrowhead] from tangential to sharply radial. Time elapsed since the beginning of observations is indicated in minutes. (Also, see the AVI movie files provided in the supporting information). Scale bar: A, C, 100 µm; B, 80 µm.

Figure 3. Changes in interneuronal migration following interactions with radial glia.

(A–C) Changes in interneuronal migratory behavior before and after contact with radial glial endfeet (A), process (B), and cell soma (C) were monitored in real time. Interneurons displayed one of the following four types of behavior when they come in contact with a radial glial cell: (1) turn towards the radial glial cell, (2) migrate pass the radial glia without pausing, (3) turn back and migrate in opposite direction after contacting radial glia, or (4) remain attached to the radial glial cell following contact. The extent of these four types of interactions were measured per radial glial endfeet, process, or cell soma during the period of observation. Distinct radial glial cells can alter the migratory behavior of subsets of interneurons in the developing cerebral wall. Number of cells/group>1000. Data shown are mean±SEM.

Figure 4. Quantification of the direction of interneuronal migration in the embryonic cortex.

(A–C) Neurons migrating in four distinct directions (towards the pial surface, towards the ventricular surface, tangentially towards cortex [subpallium→ pallium], tangentially towards ganglionic eminence [pallium→ subpallium]) within the cortical plate (A), intermediate zone (B), and ventricular zone (C) were measured. Surprisingly, significant numbers of interneurons across the cerebral wall migrate in cortex→ ganglionic eminence direction. Number of cells/group>1500. Data shown are mean±SEM.

Figure 5. Distinct migratory dynamics of tangentially migrating interneurons.

Interneurons migrating tangentially in subpallium → pallium orientation extend branched leading processes (arrowhead, A) with elaborate growth cones (arrowhead, B) trailed by pre-somal (asterisk, A, B) swellings. (C, D) The leading processes of interneurons migrating in the opposite direction [pallium→ subpallium] tend not to branch and their tips are often club-like and small (arrowhead, C, D). Pre-somal swellings are evident in these neurons (asterisk, C, D). These panels are freeze frame images of actively migrating neurons. Scale bar: 20 µm.

Figure 6. Migratory dynamics of distinct subsets of interneurons.

(A, B) Interneurons in embryonic day 16 cerebral cortex were labeled with anti-calbindin or calretinin antibodies. (C) The migratory orientation of labeled interneurons as indicated by the direction of their leading processes was quantified across the cerebral wall. Majority of calretinin⁺ cells were oriented radially, towards pial surface direction, whereas a higher percentage of calbindin⁺ interneurons were oriented tangentially towards the cortex. Distinct subsets of interneurons may therefore undergo distinct patterns of migration within the cerebral wall. CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Data shown are mean±SEM (n = 10); asterisk, significant when compared with controls at p<0.01 (Student's t test). Scale bar: 75 µm.

Figure 7. In vivo two-photon microscopy of interneurons in living embryos.

(A) Living embryos (E15) attached to the mother were immobilized on an optical stage. Two-photon imaging of GFP positive interneurons in the superficial surface of parietal cortical area (B) indicates multidirectional orientation of migrating interneurons. (C) Time lapse imaging of these neurons illustrate multidirectional movement of these neurons as they navigate to their appropriate locations within the developing cortex. This multidirectional movement of interneurons suggest that once interneurons invade cortex from the ganglionic eminence, local cues may influence the allocation of interneurons into distinct cortical areas. Arrows indicate neurons moving in different orientations. Time elapsed is indicated in minutes. M, medial; L, lateral; R, rostral; C, caudal. Scale bar: B, 30 µm; C, 40 µm.

Figure 8. Interneuron-radial glial interactions in the developing cerebral cortex.

Interneurons (green) migrating into the cerebral wall from the ganglionic eminence(GE) interact with radial glia (red) and can exhibit changes in direction of migration after contacting radial glia. Interneurons can use radial glia as a scaffold upon which to migrate as they ascend to the cortical plate (CP) or descend in the direction of the ventricular zone (VZ). Particular orientation and morphological dynamics of migration may be associated with particular subsets of interneurons. Once the interneurons invade the cortex from the ganglionic eminence, differential interactions between interneurons and radial glial scaffold and localized multidirectional migration of interneurons influenced by local guidance cues may facilitate interneuronal positioning within distinct domains of the developing cerebral cortex. Potential differences among interneurons and radial glia are indicated by shades of green and red, respectively. Putative local guidance cues are indicated by a gradient of pink. Blue arrows indicate direction of migration.