The glycosylation pathway is required for the secretion of Slit and for the maintenance of the Slit receptor Robo on axons.

Slit proteins act as repulsive axon guidance cues by activating receptors of the Roundabout (Robo) family. During early neurogenesis in Drosophila melanogaster, Slit prevents the growth cones of longitudinal tract neurons from inappropriately crossing the midline, thus restricting these cells to trajectories parallel to the midline. Slit is expressed in midline glial cells, and Robo is present in longitudinal axon tracts and growth cones. We showed that the enzyme Mummy (Mmy) controlled Slit-Robo signaling through mechanisms that affected both the ligand and the receptor. Mmy was required for the glycosylation of Slit, which was essential for Slit secretion. Mmy was also required for maintaining the abundance and spatial distribution of Robo through an indirect mechanism that was independent of Slit secretion. Moreover, secretion of Slit was required to maintain the fasciculation and position of longitudinal axon tracts, thus maintaining the hardwiring of the nervous system. Thus, Mmy is required for Slit secretion and for maintaining Robo abundance and distribution in the developing nervous system in Drosophila.

The midline protein regulates axon guidance by blocking the reiteration of neuroblast rows within the Drosophila ventral nerve cord.

Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.