Molecular interaction between projection neuron precursors and invading interneurons via stromal-derived factor 1 (CXCL12)/CXCR4 signaling in the cortical subventricular zone/intermediate zone

Abstract

Most cortical interneurons are generated in the subpallial ganglionic eminences and migrate tangentially to their final destinations in the neocortex. Within the cortex, interneurons follow mainly stereotype routes in the subventricular zone/intermediate zone (SVZ/IZ) and in the marginal zone. It has been suggested that interactions between invading interneurons and locally generated projection neurons are implicated in the temporal and spatial regulation of the invasion process. However, so far experimental evidence for such interactions is lacking. We show here that the chemokine stromal-derived factor 1 (SDF-1; CXCL12) is expressed in the main invasion route for cortical interneurons in the SVZ/IZ. Most SDF-1-positive cells are proliferating and express the homeodomain transcription factors Cux1 and Cux2. Using MASH-1 mutant mice in concert with the interneuron marker DLX, we exclude that interneurons themselves produce the chemokine in an autocrine manner. We conclude that the SDF-1-expressing cell population represents the precursors of projection neurons during their transition and amplification in the SVZ/IZ. Using mice lacking the SDF-1 receptor CXCR4 or Pax6, we demonstrate that SDF-1 expression in the cortical SVZ/IZ is essential for recognition of this pathway by interneurons. These results represent the first evidence for a molecular interaction between precursors of projection neurons and invading interneurons during corticogenesis.

Figure 1.

Expression of SDF-1 and Cux2 in the developing forebrain. ISH for SDF-1 (a, c, e, g) and Cux2 (b, d, f, h) on frontal serial forebrain vibratome sections at E14.5 (a, b) and cryostat sections at E13.5 (c, d), E15.5 (e, f), and E18.5 (g, h). a, c, At E13.5 and E14.5, SDF-1 expression is confined to the meninges and a cell population deep within the pallium. b, d, Cux2 labels the SVZ/IZ, interneurons in the MZ, and the first cohort of upper-layer neurons. Cux2 staining extends into the ventral forebrain. The deep SDF-1 expression domain (a, c) is mainly overlapping with the Cux2 domain, demarcating the SVZ/IZ (b, d). e, g, At E15.5 (e) and E18.5 (g), SDF-1 expression is strong in the meninges but is confined to the deep aspect in the SVZ/IZ (arrowhead). f, h, At the same time, Cux2 expression is found in upper-layer neurons of the developing CP. Furthermore, cells in the SVZ/IZ and radially oriented cells traversing the already formed deep layers express the transcription factor. Scale bars: a, b, e–h, 200 μm; c, d, 100 μm. Me, Meninges; AA, amygdala anlage; MGE, medial GE.

Figure 2.

Identification of SDF-1-expressing cells in the SVZ/IZ. a, b, Double FISH for SDF-1 (green) and Cux2 (red) at E13.5 in the pallium. b, High-magnification view of the SVZ/IZ. Most cells in the deep aspect of the SVZ/IZ are colabeled (green and red precipitates in the same cell); single-labeled cells for SDF-1 (arrowhead) and for Cux2 (arrow) can be identified. c, Quantification of single- and double-labeled cell populations in the SVZ/IZ. d–g, Combined ISH for SDF-1 (blue) with immunohistochemistry (brown staining) for Cux1 (d, e), PCNA (f), and DLX (g) at E15.5. d, Strong Cux1 immunoreactivity is found in upper-layer neurons of the developing CP, whereas weaker staining is observed in precursors in the SVZ/IZ. e, Higher magnification shows that SDF-1-expressing cells in the SVZ/IZ exhibit nuclear immunoreactivity for Cux1. f, The majority of SDF-1-positive cells coexpress the mitotic cell marker PCNA. g, SDF-1-positive cells never express the interneuron marker DLX. h–k, ISH for SDF1 (h, j) and Dlx5 (i, k) at E15.5 in wild types (h, i) and Mash1 mutants (j, k). In the wild type, SDF-1 (h) and Dlx5 (i) are both expressed in the SVZ/IZ. In the Mash1 mutant brain, Dlx5 (k) in the SVZ/IZ is absent, indicating the lack of interneurons. However, SDF-1 expression (j) in mutants is apparently unaltered. Scale bars: a, d, h–k, 100 μm; b, e, f, 10 μm. UL, Upper-layer neurons; DL, deep-layer neurons.

Figure 3.

SDF-1 expression in the SVZ/IZ is necessary for correct interneuron migration. a–d, ISH for SDF-1 (a, b) and for the interneuron marker Lhx6 (c, d) in the wild type (a, c) and in the Pax6 mutant (b, d) at E14.5. In Pax6 mutants, SDF1 mRNA expression is absent in the SVZ/IZ but remains unchanged in the meninges. At the same time, Lhx6-positive interneurons disappear from the SVZ/IZ (d), whereas larger amounts of these cells appear in the MZ pathway. Scale bar, 100 μm. Me, Meninges.

Figure 4.

Altered interneuron migration in the pallium of CXCR4 mutant mice. a, ISH for CXCR4 at E15.5. CXCR4 is strongly expressed in cells in the MZ and in the SVZ/IZ. b, Double FISH for Lhx6 (red) and CXCR4 (green) in the SVZ/IZ. Lhx6-positive interneurons coexpress CXCR4 (arrows). c–f, ISH for Lhx6 in WT (c, e) and CXCR4 mutant (d, f) mice at E14.5 (c, d) and E18.5 (e, f). g, Quantitative analysis of the distribution of Lhx6-expressing neurons in the pallium of WT and CXCR4 mutant E14.5 forebrain. At E14.5, invading interneurons expressing Lhx6 in the WT cortex (c) are concentrated in the SVZ/IZ and the MZ. At E18.5, interneurons are relatively evenly distributed over the entire CP in the wild type (e), while in the mutants (f) they accumulate in intermediate positions. Quantitative analysis showed that in CXCR4 mutants (d), both SVZ/IZ and MZ contain significantly less interneurons, whereas intermediate routes are preferentially used (*p > 0.05, significant differences between bins in wild type and mutant). Scale bars: a, c–f, 100 μm; b, 10 μm. UL, Upper-layer neurons; DL, deep-layer neurons.