RGMa-mediated repulsion of newborn interneurons is suppressed by Netrin-1.

<p>(A) The extent of interneuron migration was calculated as the ratio of the distal to proximal area (guidance ratio) occupied by migrating cells. (B–E) Anti-βIII-tubulin immunolabeling (red) revealed the extent of interneuron migration out of E14.5 MGE VZ explants placed adjacent to agarose blocks containing control HEK293 cells (B), cells producing Netrin-1 (C), RGMa (D) or RGMa + Netrin-1 (E). Note the significant increase in neuron density on the distal side of the explant in the presence of RGMa (D). (E) Quantification of the guidance ratio for newborn interneuron migration out of the MGE explants in response to guidance cues. Dotted lines indicate the body of the explant. Control, n = 10; Netrin-1, n = 8; RGMa, n = 12; RGMa + Netrin-1, n = 7. *p<0.5, **p<0.01.</p

<i>Neo</i> is expressed on newborn cortical interneurons and the maturing calbindin and parvalbumin subpopulations.

<p>(A) 2 day cultures from the E14.5 MGE were immunolabeled with anti-Neo (H-175, green) and anti-βIII-tubulin (red; merge, yellow), a marker for newborn neurons. (B,C,D) MGE cells were isolated at E14.5, differentiated for 4 days and then colabeled with (B) anti-Neo (MAB1079, red) and anti-GAD65/67, a GABAergic interneuron marker (green; merge, yellow), (C) anti-Neo (MAB1079, red) and anti-calbindin (green; merge, yellow), or (D) anti-Neo (H-175, green) and anti-parvalbumin (red; merge, yellow).</p

RGMa does not influence neurogenic divisions or interneuronal fate.

<p>MGE cells were isolated at E14.5 and differentiated for 4 days in recombinant RGMa. (A) No significant difference was observed in the percentage of neurons generated in the presence of 200 or 400 ng/ml RGMa or in the absence of RGMa. (B) Addition of the RGMa inhibitory peptide (Pep2, 10 µM) or scrambled control peptide (ScPep, 10 µM) had no significant effect on the number of neurons generated in the presence or absence of RGMa. Number of neurons counted per condition >520.</p

<i>RGMa</i> and <i>Neo</i> are expressed by radial progenitors in the VZ of the ganglionic eminences.

<p>(A) Coronal sections of E14.5 embryos show that RGMa was restricted to the VZ and SVZ in the ganglionic eminences. (B) Low levels of Neo were detected on radial progenitors in the LGE and MGE VZ (arrows). Newborn neurons within the migratory corridor were also Neo-positive (arrowhead). Neurons in the cortical plate (cp) and striatum (st) expressed high levels of <i>Neo</i>. No immunoreactivity was seen with an isotype-matched IgG control antibody (inset). Colabeling of E14.5 MGE VZ cells cultured for 2 days with antibodies to Neo (H-175, green), the radial progenitor marker, GLAST (C, red; merge, yellow), the cell cycle marker, Ki67 (D, red; merge, yellow), or the M-phase marker, phospho-vimentin55 (E, red; merge, yellow). GE, ganglionic eminence. Scale bars: A, 670 µm; B, 1.00 mm.</p