Slit-Robo signals regulate pioneer axon pathfinding of the tract of the postoptic commissure in the mammalian forebrain.

During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5-E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.

The Slit receptor Robo1 is predominantly expressed via the Dutt1 alternative promoter in pioneer neurons in the embryonic mouse brain and spinal cord.

Robo1 is a member of the Roundabout (Robo) family of receptors for the Slit axon guidance cues. In mice (and humans), the Robo1 locus has alternative promoters producing two transcript isoforms, Robo1 and Dutt1. These isoforms have unique 5' termini, predicted to encode distinct N-terminal amino acids, but share the rest of their 3' exons. To determine the spatial expression of the Robo1 and Dutt1 isoforms, we generated isoform-specific RNA probes, and carried out in situ hybridization on E10.5 mouse embryos, the stage in early neuron differentiation when many major axon pathways are established. The two isoforms had distinct expression patterns that partially overlapped. Dutt1 was the predominant isoform, with widespread expression in regions of post-mitotic neurons and neuroepithelial cells. The Robo1 isoform had a distinct expression pattern restricted to subsets of neurons, many of which were Dutt1-negative. Dutt1 was the main isoform expressed in spinal cord commissural neurons. For both probes, the main hybridization signal was limited to two spots in the nuclei of individual cells. This study shows distinct expression patterns for the Dutt1 and Robo1 alternative promoters in the embryonic nervous system.

Longitudinal axons are guided by Slit/Robo signals from the floor plate.

Longitudinal axons grow long distances along precise pathways to connect major CNS regions. However, during embryonic development, it remains largely undefined how the first longitudinal axons choose specific positions and grow along them. Here, we review recent evidence identifying a critical role for Slit/Robo signals to guide pioneer longitudinal axons in the embryonic brain stem. These studies indicate that Slit/Robo signals from the floor plate have dual functions: to repel longitudinal axons away from the ventral midline, and also to maintain straight longitudinal growth. These dual functions likely cooperate with other guidance cues to establish the major longitudinal tracts in the brain.

Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals.

Longitudinal axons transmit all signals between the brain and spinal cord. Their axon tracts through the brain stem are established by a simple set of pioneer axons with precise trajectories parallel to the floor plate. To identify longitudinal guidance mechanisms in vivo, the overall role of floor plate tissue and the specific roles of Slit/Robo signals were tested. Ectopic induction or genetic deletion of the floor plate diverted longitudinal axons into abnormal trajectories. The expression patterns of the diffusible cues of the Slit family were altered in the floor plate experiments, suggesting their involvement in longitudinal guidance. Genetic tests of Slit1 and Slit2, and the Slit receptors Robo1 and Robo2 were carried out in mutant mice. Slit1;Slit2 double mutants had severe longitudinal errors, particularly for ventral axons, including midline crossing and wandering longitudinal trajectories. Robo1 and Robo2 were largely genetically redundant, and neither appeared to specify specific tract positions. However, combined Robo1 and Robo2 mutations strongly disrupted each pioneer tract. Thus, pioneer axons depend on long-range floor plate cues, with Slit/Robo signaling required for precise longitudinal trajectories.