Robo1 modulates proliferation and neurogenesis in the developing neocortex.

The elaborate cytoarchitecture of the mammalian neocortex requires the timely production of its constituent pyramidal neurons and interneurons and their disposition in appropriate layers. Numerous chemotropic factors present in the forebrain throughout cortical development play important roles in the orchestration of these events. The Roundabout (Robo) family of receptors and their ligands, the Slit proteins, are expressed in the developing forebrain, and are known to play important roles in the generation and migration of cortical interneurons. However, few studies have investigated their function(s) in the development of pyramidal cells. Here, we observed expression of Robo1 and Slit genes (Slit1, Slit2) in cells lining the telencephalic ventricles, and found significant increases in progenitor cells (basal and apical) at embryonic day (E)12.5 and E14.5 in the developing cortex of Robo1(-/-), Slit1(-/-), and Slit1(-/-)/Slit2(-/-), but not in mice lacking the other Robo or Slit genes. Using layer-specific markers, we found that both early- and late-born pyramidal neuron populations were significantly increased in the cortices of Robo1(-/-) mice at the end of corticogenesis (E18.5). The excess number of cortical pyramidal neurons generated prenatally appears to die in early postnatal life. The observed increase in pyramidal neurons was due to prolonged proliferative activity of their progenitors and not due to changes in cell cycle events. This finding, confirmed by in utero electroporation with Robo1 short hairpin RNA (shRNA) or control constructs into progenitors along the ventricular zone as well as in dissociated cortical cell cultures, points to a novel role for Robo1 in regulating the proliferation and generation of pyramidal neurons.

Robo1 regulates the migration and laminar distribution of upper-layer pyramidal neurons of the cerebral cortex.

Laminar organization is a key feature of the mammalian cerebral cortex, but the mechanisms by which final positioning and "inside-out" distribution of neurons are determined remain largely unknown. Here, we demonstrate that Robo1, a member of the family of Roundabout receptors, regulates the correct positioning of layers II/III pyramidal neurons in the neocortex. Specifically, we used RNA interference in mice to suppress the expression of Robo1 in a subset of layers II/III neurons, and observed the positions of these cells at distinct developmental stages. In contrast to control neurons that migrated toward the pial surface by P1, Robo1-suppressed neurons exhibited a delay in entering the cortical plate at respective stages. Unexpectedly, after the first postnatal week, these neurons were predominantly located in the upper part of layers II/III, in contrast to control cells that were distributed throughout these layers. Sequential electroporation studies revealed that Robo1-suppressed cells failed to establish the characteristic inside-out neuronal distribution and, instead, they accumulated beneath the marginal zone regardless of their birthdate. These results demonstrate that Robo receptors play a crucial role in neocortical lamination and particularly in the positioning of layers II/III pyramidal neurons.

Beyond Axon Guidance: Roles of Slit-Robo Signaling in Neocortical Formation.

The formation of the neocortex relies on intracellular and extracellular signaling molecules that are involved in the sequential steps of corticogenesis, ranging from the proliferation and differentiation of neural progenitor cells to the migration and dendrite formation of neocortical neurons. Abnormalities in these steps lead to disruption of the cortical structure and circuit, and underly various neurodevelopmental diseases, including dyslexia and autism spectrum disorder (ASD). In this review, we focus on the axon guidance signaling Slit-Robo, and address the multifaceted roles of Slit-Robo signaling in neocortical development. Recent studies have clarified the roles of Slit-Robo signaling not only in axon guidance but also in progenitor cell proliferation and migration, and the maturation of neocortical neurons. We further discuss the etiology of neurodevelopmental diseases, which are caused by defects in Slit-Robo signaling during neocortical formation.

Expression profiles of Insulin-like growth factor binding protein-like 1 in the developing mouse forebrain.

Insulin-like growth factor binding protein-like 1 (IGFBPL-1) has a domain structure that resembles one in the insulin-like growth factor binding proteins (IGFBPs), which is a domain known to be bound by insulin-like growth factors (IGFs) with high affinity and to thereby affect cell growth and metabolism. However, the distribution and functions of IGFBPL-1 in the brain are not known. Thus, to clarify IGFBPL-1 expression profiles during development, we have investigated the expression patterns of IGFBPL-1 mRNA in developing mouse brains using in situ hybridization, focusing especially on the forebrain. On embryonic day (E)12, IGFBPL-1mRNA expressions were localized in the preplate of the neopallium, dorsal thalamus, the differentiating field of the epithalamus, and the hippocampus. On E14, strong expression was recognized in the subventricular zone (SVZ) and intermediate zone (IMZ) in the neopallium, hippocampus, the differentiating field of the epithalamus, and the dorsal thalamus. E14 expressions were generally the strongest among all the stages examined and signals decreased thereafter. At E16 and E18, IGFBPL-1mRNA was strongly detected in the hippocampus and the SVZ of the neopallium. In postnatal stages, mRNA was found only in the granule cell layer of the dentate gyrus (DG). Notably, at P20, expression was restricted to the deep fraction of the granule cell layer, the location of late-born DG neurons.