Netrin-1 is an evolutionarily conserved secreted extracellular matrix protein discovered using genetic and biochemical screens for its role in axon guidance at the central nervous system (CNS) midline1,2. Netrin-1 is expressed by cells localized at CNS midline, such as the floor plate in vertebrate embryos1,3. Growth cone turning assays and 3D gel diffusion assays showed that netrin-1 can attract commissural axons2,4–6. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in absence of netrin-13,7–9. Together these data support a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here, we selectively ablated netrin-1 expression in floor plate cells using a Netrin-1 conditional mouse line. We found that hindbrain and spinal cord commissural axons develop normally in absence of floor plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone with the potential to release it at the pial surface where it binds to commissural axons. Importantly, netrin-1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Netrin-1 knockout mice. These results show that the classical textbook view that attraction of commissural axons is mediated by a gradient of floor plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesion.

All vertebrate brains develop following a common Bauplan defined by anteroposterior (AP) and dorsoventral (DV) subdivisions, characterized by largely conserved differential expression of gene markers. However, it is still unclear how this Bauplan originated during evolution. We studied the relative expression of 48 genes with key roles in vertebrate neural patterning in a representative amphioxus embryonic stage. Unlike nonchordates, amphioxus develops its central nervous system (CNS) from a neural plate that is homologous to that of vertebrates, allowing direct topological comparisons. The resulting genoarchitectonic model revealed that the amphioxus incipient neural tube is unexpectedly complex, consisting of several AP and DV molecular partitions. Strikingly, comparison with vertebrates indicates that the vertebrate thalamus, pretectum, and midbrain domains jointly correspond to a single amphioxus region, which we termed Di-Mesencephalic primordium (DiMes). This suggests that these domains have a common developmental and evolutionary origin, as supported by functional experiments manipulating secondary organizers in zebrafish and mice.

Hindbrain rhombomeres in general are differentially specified molecularly by unique combinations of Hox genes with other developmental genes. Rhombomere 1 displays special features, including absence of Hox gene expression. It lies within the hindbrain range of the Engrailed genes (En1, En2), controlled by the isthmic organizer via diffusion of FGF8. It is limited rostrally by the isthmus territory, and caudally by rhombomere 2. It is double the normal size of any other rhombomere. Its dorsal part generates the cerebellar hemispheres and its ventral part gives rise to several populations, such as some raphe nuclei, the interpeduncular nucleus, the rhabdoid nucleus, anterior, dorsal, ventral and posterodorsal tegmental nuclei, the cholinergic pedunculopontine and laterodorsal tegmental nuclei, rostral parts of the hindbrain reticular formation, the locus coeruleus, and part of the lateral lemniscal and paralemniscal nuclei, among other formations. Some of these populations migrate tangentially before reaching their final positions. The morphogen Sonic Hedgehog (Shh) is normally released from the local floor plate and underlying notochord. In the present report we explore, first, whether Shh is required in the specification of these r1 populations, and, second, its possible role in the guidance of tangentially migrating neurons that approach the midline. Our results indicate that when Shh function is altered selectively in a conditional mutant mouse strain, most populations normally generated in the medial basal plate of r1 are completely absent. Moreover, the relocation of some neurons that normally originate in the alar plate and migrate tangentially into the medial basal plate is variously altered. In contrast, neurons that migrate radially (or first tangentially and then radially) into the lateral basal plate were not significantly affected.

Graphical Abstract Highlights d Floor-plate-derived netrin1 (FP-netrin1) guides commissural axons at long range d FP-netrin1 and Shh collaborate to guide commissural axons in the ventral spinal cord d Both FP-netrin1 and ventricular zone-netrin1 contribute to commissural axon guidance d FP-netrin1 may guide via haptotaxis and/or chemotaxis In Brief Recent studies have queried the role of netrin1 from floor plate (FP-netrin1) in guiding commissural axons. Wu et al. show that, in spinal cord, FP-netrin1 is required and acts at long range to guide commissural axons, collaborating with Shh. SUMMARYAn important model for axon pathfinding is provided by guidance of embryonic commissural axons from dorsal spinal cord to ventral midline floor plate (FP). FP cells produce a chemoattractive activity, comprised largely of netrin1 (FP-netrin1) and Sonic hedgehog (Shh), that can attract the axons at a distance in vitro. netrin1 is also produced by ventricular zone (VZ) progenitors along the axons' route (VZ-netrin1). Recent studies using region-specific netrin1 deletion suggested that FP-netrin1 is dispensable and VZ-netrin1 sufficient for netrin guidance activity in vivo. We show that removing FP-netrin1 actually causes guidance defects in spinal cord consistent with long-range action (i.e., over hundreds of micrometers), and double mutant analysis supports that FP-netrin1 and Shh collaborate to attract at long range. We further provide evidence that netrin1 may guide via chemotaxis or haptotaxis. These results support the model that netrin1 signals at both short and long range to guide commissural axons in spinal cord.

Highlights d Both floor-plate-and VZ-derived netrin-1 guide spinal cord commissural axons d Commissural axons go astray to the midline in the absence of floor plate netrin-1 d Commissural axon fasciculation is modified in the absence of VZ netrin-1 d Midline crossing mechanisms differ between the spinal cord and hindbrain

The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviors and show select disease vulnerability, including in Parkinson's disease. Despite progress in identifying mDA neuron subtypes, how these neuronal subsets develop and organize into functional brain structures remains poorly understood. Here we generate and use an intersectional genetic platform, Pitx3-ITC, to dissect the mechanisms of substantia nigra (SN) development and implicate the guidance molecule Netrin-1 in the migration and positioning of mDA neuron subtypes in the SN. Unexpectedly, we show that Netrin-1, produced in the forebrain and provided to the midbrain through axon projections, instructs the migration of GABAergic neurons into the ventral SN. This migration is required to confine mDA neurons to the dorsal SN. These data demonstrate that neuron migration can be controlled by remotely produced and axon-derived secreted guidance cues, a principle that is likely to apply more generally.

Whereas sensory perception relies on specialized sensory pathways, it is unclear whether these pathways originate as modality-specific circuits. We demonstrated that somatosensory and visual circuits are not by default segregated but require the earliest retinal activity to do so. In the embryo, somatosensory and visual circuits are intermingled in the superior colliculus, leading to cortical multimodal responses to whisker pad stimulation. At birth, these circuits segregate, and responses switch to unimodal. Blocking stage I retinal waves prolongs the multimodal configuration into postnatal life, with the superior colliculus retaining a mixed somato-visual molecular identity and defects arising in the spatial organization of the visual system. Hence, the superior colliculus mediates the timely segregation of sensory modalities in an input-dependent manner, channeling specific sensory cues to their appropriate sensory pathway.

Growth differentiation factor 10 (Gdf10), also known as Bmp3b, is a member of the transforming growth factor (TGF)-ß superfamily. Gdf10 is expressed in Bergmann glial cells, which was investigated by single-cell transcriptional profiling (Koirala and Corfas, (2010) PLoS ONE 5: e9198). Here we provide a detailed characterization of Gdf10 expression from E14, the stage at which Gdf10 is expressed for the first time in the cerebellum, until P28. We detected Gdf10 expression in both germinal zones: in the ventricular zone (VZ) of the 4th ventricle as well as in the rhombic lip (RL). The VZ has been postulated to give rise to GABAergic neurons and glial cells, whereas the RL gives rise to glutamatergic neurons. Thus, it was very surprising to discover a gene that is expressed exclusively in glial cells and is not restricted to an expression in the VZ, but is also present in the RL. At postnatal stages Gdf10 was distributed equally in Bergmann glial cells of the cerebellum. Furthermore, we found Gdf10 to be regulated by Sonic hedgehog (Shh), which is secreted by Purkinje cells of the cerebellum. In the conditional Shh mutants, glial cells showed a reduced expression of Gdf10, whereas the expression of Nestin and Vimentin was unchanged. Thus, we show for the first time, that Gdf10, expressed in Bergmann glial cells, is affected by the loss of Shh as early as E18.5, suggesting a regulation of glial development by Shh.