The role of Robo3 in the development of cortical interneurons

Abstract

A number of studies in recent years have shown that members of the Roundabout (Robo) receptor family, Robo1 and Robo2, play significant roles in the formation of axonal tracks in the developing forebrain and in the migration and morphological differentiation of cortical interneurons. Here, we investigated the expression and function of Robo3 in the developing cortex. We found that this receptor is strongly expressed in the preplate layer and cortical hem of the early cortex where it colocalizes with markers of Cajal-Retzius cells and interneurons. Analysis of Robo3 mutant mice at early (embryonic day [E] 13.5) and late (E18.5) stages of corticogenesis revealed no significant change in the number of interneurons, but a change in their morphology at E13.5. However, preliminary analysis on a small number of mice that lacked all 3 Robo receptors indicated a marked reduction in the number of cortical interneurons, but only a limited effect on their morphology. These observations and the results of other recent studies suggest a complex interplay between the 3 Robo receptors in regulating the number, migration and morphological differentiation of cortical interneurons.

Figure 1.

Expression of Robo3 protein at rostral (A, E, I), middle (B, F, J), and caudal (C, G, K) levels of the embryonic mouse forebrain during preplate stages of development (E11.5–13.5). (D, H) Higher magnification images taken through middle levels of the cortex at E11.5 (D) and E12.5 (H). Scale bar in (C), (G), and (K) is 200 μm and applies to panels of equivalent ages; scale bar in H is 200 μm and also applies to (D) (Cx, cortex; CH cortical hem).

Figure 2.

(A–C) Localisation of Robo- and reelin proteins in coronal sections through the developing mouse cortex at E13.5. Robo1 (A), Robo2 (B), and Robo3 (C) proteins (shown in green) colocalize with reelin (red) in some cells in the CH and MZ of the neocortex (yellow). Reelin labeling is predominantly present in the more superficial aspect of the MZ, whereas Robo expression is prevalent in the lower part of this layer, where some colocalization of the 2 proteins is evident (yellow). (A′–C′) Higher magnification images of the CH, in (A–C), respectively, illustrate colocalization in a number of cells (yellow) some of which are pointed with arrows. (D–F) Robo3 (red) colocalizes with GAD67 GFP+ (green) in the MGE and in a stream of cells extending toward the corticostriatal boundary in coronal sections through the forebrain at E10.5. (G–I) A number of dissociated GAD67 GFP+ MGE neurons (green) express Robo3 (red) (shown in yellow) (I). Scale bar in A is 100 μm and corresponds to A–C; in D is 100 μm and corresponds to D–F; in G is 30 μm and corresponds to G–I; scale bar in A′ is 75 μm and corresponds to A′–C′ (CH, cortical hem; LV, lateral ventricle).

Figure 3.

(A, B) Photomicrographs of coronal sections through the caudal cortex of Robo3^+/+ and Robo3^−/− mice stained for calbindin. (C–F) Analysis of the number and distribution of calbindin-labeled cells in all layers of the cortex of Robo3^+/− and Robo3^−/− mice at E13.5 (C, D) and E18.5 (E, F). Counts were made in rostral-middle (C, E) and caudal (D, F) regions of the cortex. Scale bar in (A, B) is 150 μm (*P < 0.01) (SVZ, subventricular zone; IZ, intermediate zone; SP, subplate; CP, cortical plate).