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. 2011 Apr 20;31(16):6174-87.
doi: 10.1523/JNEUROSCI.5464-10.2011.

Robo1 Regulates Semaphorin Signaling to Guide the Migration of Cortical Interneurons Through the Ventral Forebrain

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Free PMC article

Robo1 Regulates Semaphorin Signaling to Guide the Migration of Cortical Interneurons Through the Ventral Forebrain

Luis R Hernández-Miranda et al. J Neurosci. .
Free PMC article

Abstract

Cortical interneurons, generated predominantly in the medial ganglionic eminence, migrate around and avoid the developing striatum in the subpallium en route to the cortex. This is attributable to the chemorepulsive cues of class 3 semaphorins expressed in the striatal mantle and acting through neuropilin (Nrp1 and Nrp2) receptors expressed in these cells. Cortical interneurons also express Robo receptors, and we show here that in mice lacking Robo1, but not Robo2, these cells migrate aberrantly through the striatum. In vitro experiments demonstrated that interneurons lacking Robo1 function are significantly less responsive to the effects of semaphorins. Failure to respond to semaphorin appears to be attributable to a reduction in Nrp1 and PlexinA1 receptors within these cells. Biochemical studies further demonstrated that Robo1 binds directly to Nrp1, but not to semaphorins, and this interaction is mediated by a region contained within its first two Ig domains. Thus, we show for the first time that Robo1 interacts with Nrp1 to modulate semaphorin signaling in the developing forebrain and direct the migration of interneurons through the subpallium and into the cortex.

Figures

Figure 1.
Figure 1.
Deletion of Robo1 receptor increases the number of GABAergic neurons in the cerebral cortex and striatum. A, B, Coronal sections through the brains of a Robo1+/−;GAD67GFP mouse (A) and a Robo1−/−;GAD67-GFP littermate (B) at E15.5. The areas bracketed in A and B are shown at higher magnifications in C and D, respectively, whereas boxes in A and B are shown at higher magnifications in E and F, respectively. E′, F′, The boxed areas in E and F are shown at an even higher magnification. G, Counts of GAD67-GFP+ cells show increased number throughout the rostral-caudal extent of the striatum of Robo1−/− mice at E15.5. Scale bars: A, B, 500 μm; C–F′, 150 μm. **p < 0.01. Error bars indicate SEM. Abbreviations: CP, Cortical plate; Cx, cerebral cortex; hem, cortical hem; IZ, intermediate zone; LV, lateral ventricle; MZ, marginal zone; Sp, septum; Str, striatum; SVZ, subventricular zone; VZ, ventricular zone.
Figure 2.
Figure 2.
No differences in the number of developing striatal projection neurons or striatal interneurons between Robo1+/− and Robo1−/− embryos. A, B, D, E, Coronal sections from Robo1+/− and Robo1−/− mice at E15.5 (A, B) and at E18.5 (D, E) were immunostained for FOXP2. C, F, Counts of FOXP2+ cells showed no differences throughout the rostral-caudal extent of the developing striatum of Robo1−/− and Robo1+/− littermates at E15.5 (C) and E18.5 (F). G, H, Coronal brain sections taken from Robo1+/− and Robo1−/− mice at E15.5 and processed by in situ hybridization for Nkx2.1. J, K, Similar sections were processed by in situ hybridization for Lhx8. I, L, Counts of labeled cells showed no differences for either gene between the two groups of animals. Scale bars: A, B, D, E, 100 μm; G, H, J, K, 120 μm. Abbreviation: Str, striatum. Error bars indicate SEM.
Figure 3.
Figure 3.
No differences in the number of striatal projection neurons or interneurons between Robo1+/− and Robo1−/− adult mice. Immunostaining of coronal brain sections from adult Robo1+/− and Robo1−/− mice for the striatal projection neuron markers CB (A, B) and DARP-32 (D, E), and for the interneuron markers PV (G, H), SST (J, K), and ChAT (M, N). The quantitation of immunopositive neurons for each marker is shown adjacent to the sections (C, F, I, L, O). Scale bar, 100 μm. Abbreviations: Str, Striatum; WM, white matter. Error bars indicate SEM.
Figure 4.
Figure 4.
Increased number of CB+ cells in the striatum of Robo1−/− mouse embryos. Immunostaining of coronal sections of Robo1+/− and Robo1−/− brains for CB at E15.5 (A, B), E18.5 (D, E), and P0 (G, H). The quantification of the number of CB+ cells in the striatum of Robo1−/− animals and Robo1+/− littermates at each age is shown adjacent to the sections (C, F, I, respectively). Scale bar, 100 μm. *p < 0.05; **p < 0.01. Abbreviation: Str, Striatum. Error bars indicate SEM.
Figure 5.
Figure 5.
No differences in the number of CB+ or FOXP2+ cells in the striatum of Robo2−/−, Slit1−/−;Slit2−/− mice, and control littermates. A, B, D, E, Coronal sections taken from the brains of Robo2+/+ (A, D) and Robo2−/− (B, E) mice at E15.5 were immunostained for FOXP2 (A, B) and CB (D, E). C, F, Quantitation of the number of FOXP2+ and CB+ cells in the striatum at E15.5. G, H, J, K, Coronal sections from brains of Slit+/−;Slit2+/− (G, J) and Slit1−/−;Slit2−/− (H, K) mice at E15.5 were immunostained for FOXP2 (G, H) and CB (J, K). I, L, Quantitation of the number of FOXP2+ and CB+ cells in the striatum at E15.5. Scale bar, 100 μm. **p < 0.01. Abbreviation: Str, Striatum. Error bars indicate SEM.
Figure 6.
Figure 6.
MGE-derived cells from Robo1−/− mutants do not respond to Sema3A or Sema3F. A–D, Migration of cells away from E13.5 MGE explants prepared from Robo1+/− (A, C) and Robo1−/− (B, D) littermates treated with control conditioned media (CM-myc) (A, B) or CM-Sema3A (C, D). E, F, Quantification of migration from Robo1+/− and Robo1−/− MGE explants treated with Sema3A (E) or Sema3F (F). G, H, Quantification of the number of dissociated MGE cells from Robo1+/− and Robo1−/− mice used in a Boyden's chamber assay to test their response to Sema3A (G) or Sema3F (H). Scale bar, 100 μm. **p < 0.01; ***p < 0.001. Error bars indicate SEM.
Figure 7.
Figure 7.
GN11 cells express Robo receptors and respond to class 3 semaphorins and Slit1. A, Reverse transcription-PCR analysis showed expression of Robo1–3 in GN11 cells. B, Immunohistochemistry confirmed the expression of Robo receptors in GN11 cells. C, D, Quantification of GN11 cell migration in a Boyden's chamber, containing Sema3A, Sema3F, Slit1, or control (CM-myc) conditioned media (C). D, Robo1-DN-transfected GN11 cells are less responsive to Sema3A than control (GFP)-transfected cells. Scale bar, 100 μm. ***p < 0.001. Error bars indicate SEM.
Figure 8.
Figure 8.
Downregulation of Nrp and PlexinA in MGE cells derived from Robo1−/−;GAD67-GFP mice and Robo1-DN-transfected GN11 cells. A, B, Sema3A and Sema3F do not bind to Robo1. Cell lysates from COS-7 cells, which had been transiently transfected with myc-tagged full-length Plexin, Nrp, or Robo1 and treated with Flag-tagged Sema3F in the culture medium, were immunoprecipitated with anti-myc antibody and immunoblotted with anti-Flag and anti-myc antibodies (A). B, Sema3A-AP or Sema3F-AP was added to COS-7 cells transiently transfected with full-length Nrp1, Nrp2, Robo1, or control. Sema3A binding was only observed with cells expressing Nrp1, and Sema3F binding was only observed with cells expressing Nrp2; no binding was observed to cells expressing Robo1. C, QPCR for semaphorin receptors was performed on GN11 cells transfected with Robo1-DN or control (GFP) constructs and of MGE cells derived from E15.5 Robo1+/−;GAD67-GFP and Robo1−/−;GAD67-GFP mice. D, Immunoblot analyses of MGE cells derived from E15.5 Robo1+/−;GAD67-GFP and Robo1−/−;GAD67-GFP mice, and of GN11 cells transfected with Robo1-DN cells showed reduced levels of Nrp1 and PlexinA1, but not Nrp2. Abbreviations: −RT, Without reverse transcriptase (negative control). Genes examined are listed next to the gel bands. E, QPCR for VEGF receptors was performed on MGE-derived cells from E15.5 Robo1+/−;GAD67-GFP and Robo1−/−;GAD67-GFP mice. Genes examined are listed next to the gel bands. Lack of expression of VEGF receptors was observed in MGE cells derived from both groups of animals but were present in total brain extract.
Figure 9.
Figure 9.
Robo1 interacts directly with Nrp1. A, E15.5 brain lysates were immunoprecipitated and immunoblotted with the indicated antibodies; note that Robo1 forms complexes with Robo2, Nrp1, Nrp2, and PlexinA1, but not VEGFR3. B, Cell lysates from COS-7 cells, transiently transfected with constructs for the indicated proteins or control expression vector, were immunoprecipitated with Fc-tagged Nrp1 or Robo1, and immunoblotted with anti-myc antibody. Robo1 bound homophilically to itself and heterophilically to Nrp1. C, Covasphere aggregation assay to identify Robo1 heterophilic interactions. Beads coated with the indicated Fc proteins were imaged with a 25× objective at the end of the aggregation assay. Histograms show the corresponding changes in the proportion of beads (red, green, and yellow) over time; heterophilic interactions are indicated by yellow. Robo1 appears to bind homophilically to itself, and heterophilically to Nrp1, but not Nrp2 or Sema3A. D, Cell lysates from COS-7 cells, which were transiently transfected with myc-tagged full-length Nrp1 and incubated with Robo1-Fc, Robo1Δ1,2-Fc, or Robo1Δ3,4,5-Fc in the culture medium, were immunoprecipitated with the same Fc-tagged protein and immunoblotted with anti-myc antibody. Robo1Δ1,2-Fc failed to coimmunoprecipitate Nrp1, suggesting that the neuropilin binding domain resides within the first two Ig domains of Robo1. E, Covasphere aggregation assays using Nrp-Fc (green) and Robo1-Fc, Robo1Δ1,2-Fc, and Robo1Δ3,4,5-Fc (red) confirmed these findings. Error bars indicate SEM.

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