The Role of Apoptotic Signaling in Axon Guidance.

Navigating growth cones are exposed to multiple signals simultaneously and have to integrate competing cues into a coherent navigational response. Integration of guidance cues is traditionally thought to occur at the level of cytoskeletal dynamics. Drosophila studies indicate that cells exhibit a low level of continuous caspase protease activation, and that axon guidance cues can activate or suppress caspase activity. We base a model for axon guidance on these observations. By analogy with other systems in which caspase signaling has non-apoptotic functions, we propose that caspase signaling can either reinforce repulsion or negate attraction in response to external guidance cues by cleaving cytoskeletal proteins. Over the course of an entire trajectory, incorrectly navigating axons may pass the threshold for apoptosis and be eliminated, whereas axons making correct decisions will survive. These observations would also explain why neurotrophic factors can act as axon guidance cues and why axon guidance systems such as Slit/Robo signaling may act as tumor suppressors in cancer.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.

Motor axons are guided to exit points in the spinal cord by Slit and Netrin signals.

In the spinal cord, motor axons project out the neural tube at specific exit points, then bundle together to project toward target muscles. The molecular signals that guide motor axons to and out of their exit points remain undefined. Since motor axons and their exit points are located near the floor plate, guidance signals produced by the floor plate and adjacent ventral tissues could influence motor axons as they project toward and out of exit points. The secreted Slit proteins are major floor plate repellents, and motor neurons express two Slit receptors, Robo1 and Robo2. Using mutant mouse embryos at early stages of motor axon exit, we found that motor exit points shifted ventrally in Robo1/2 or Slit1/2 double mutants. Along with the ventral shift, mutant axons had abnormal trajectories both within the neural tube toward the exit point, and after exit into the periphery. In contrast, the absence of the major ventral attractant, Netrin-1, or its receptor, DCC caused motor exit points to shift dorsally. Netrin-1 attraction on spinal motor axons was demonstrated by in vitro explant assays, showing that Netrin-1 increased outgrowth and attracted cultured spinal motor axons. The opposing effects of Slit/Robo and Netrin-1/DCC signals were tested genetically by combining Netrin-1 and Robo1/2 mutations. The location of exit points in the combined mutants was significantly recovered to their normal position compared to Netrin-1 or Robo1/2 mutants. Together, these results suggest that the proper position of motor exit points is determined by a "push-pull" mechanism, pulled ventrally by Netrin-1/DCC attraction and pushed dorsally by Slit/Robo repulsion.

Contralateral migration of oculomotor neurons is regulated by Slit/Robo signaling.

BACKGROUND: Oculomotor neurons develop initially like typical motor neurons, projecting axons out of the ventral midbrain to their ipsilateral targets, the extraocular muscles. However, in all vertebrates, after the oculomotor nerve (nIII) has reached the extraocular muscle primordia, the cell bodies that innervate the superior rectus migrate to join the contralateral nucleus. This motor neuron migration represents a unique strategy to form a contralateral motor projection. Whether migration is guided by diffusible cues remains unknown. METHODS: We examined the role of Slit chemorepellent signals in contralateral oculomotor migration by analyzing mutant mouse embryos. RESULTS: We found that the ventral midbrain expresses high levels of both Slit1 and 2, and that oculomotor neurons express the repellent Slit receptors Robo1 and Robo2. Therefore, Slit signals are in a position to influence the migration of oculomotor neurons. In Slit 1/2 or Robo1/2 double mutant embryos, motor neuron cell bodies migrated into the ventral midbrain on E10.5, three days prior to normal migration. These early migrating neurons had leading projections into and across the floor plate. In contrast to the double mutants, embryos which were mutant for single Slit or Robo genes did not have premature migration or outgrowth on E10.5, demonstrating a cooperative requirement of Slit1 and 2, as well as Robo1 and 2. To test how Slit/Robo midline repulsion is modulated, we found that the normal migration did not require the receptors Robo3 and CXCR4, or the chemoattractant, Netrin 1. The signal to initiate contralateral migration is likely autonomous to the midbrain because oculomotor neurons migrate in embryos that lack either nerve outgrowth or extraocular muscles, or in cultured midbrains that lacked peripheral tissue. CONCLUSION: Overall, our results demonstrate that a migratory subset of motor neurons respond to floor plate-derived Slit repulsion to properly control the timing of contralateral migration.

Netrin1-DCC-Mediated Attraction Guides Post-Crossing Commissural Axons in the Hindbrain.

Commissural axons grow along precise trajectories that are guided by several cues secreted from the ventral midline. After initial attraction to the floor plate using Netrin1 activation of its main attractive receptor, DCC (deleted in colorectal cancer), axons cross the ventral midline, and many turn to grow longitudinally on the contralateral side. After crossing the midline, axons are thought to lose their responsiveness to Netrin1 and become sensitive to midline Slit-Robo repulsion. We aimed to address the in vivo significance of Netrin1 in guiding post-crossing axon trajectories in mouse embryos. Surprisingly, in contrast to the spinal cord, Netrin1 and DCC mutants had abundant commissural axons crossing in the hindbrain. In Netrin1 and DCC mutants, many post-crossing axons made normal turns to grow longitudinally, but projected abnormally at angles away from the midline. In addition, exposure of cultured hindbrain explants to ectopic Netrin1 caused attractive deflection of post-crossing axons. Thus, Netrin1-DCC signaling is not required to attract pre-crossing axons toward the hindbrain floor plate, but is active in post-crossing guidance. Also in contrast with spinal cord, analysis of hindbrain post-crossing axons in Robo1/2 mutant embryos showed that Slit-Robo repulsive signaling was not required for post-crossing trajectories. Our findings show that Netrin1-DCC attractive signaling, but not Slit-Robo repulsive signaling, remains active in hindbrain post-crossing commissural axons to guide longitudinal trajectories, suggesting surprising regional diversity in commissural axon guidance mechanisms. SIGNIFICANCE STATEMENT: The left and right sides of the brainstem and spinal cord are connected primarily by axon fibers that grow across the ventral midline, and then away on the other side to their targets. Based on spinal cord, axons are initially attracted by diffusible attractive protein signals to approach and cross the midline, and then are thought to switch to repulsive cues to grow away on the opposite side. Our results in the hindbrain show that the major midline attractant, Netrin1, is not required for midline crossing. However, the post-crossing axons depend on Netrin1 attraction to set their proper trajectories on the other side. Overall, these findings suggest that commissural axons use distinct mechanisms to navigate in different CNS regions.

Motor neuron cell bodies are actively positioned by Slit/Robo repulsion and Netrin/DCC attraction.

Motor neurons differentiate from a ventral column of progenitors and settle in static clusters, the motor nuclei, next to the floor plate. Within these cell clusters, motor neurons receive afferent input and project their axons out to muscle targets. The molecular mechanisms that position motor neurons in the neural tube remain poorly understood. The floor plate produces several types of guidance cues with well-known roles in attracting and repelling axons, including the Slit family of chemorepellents via their Robo receptors, and Netrin1 via its DCC attractive receptor. In the present study we found that Islet1(+) motor neuron cell bodies invaded the floor plate of Robo1/2 double mutant mouse embryos or Slit1/2/3 triple mutants. Misplaced neurons were born in their normal progenitor column, but then migrated tangentially into the ventral midline. Robo1 and 2 receptor expression in motor neurons was confirmed by reporter gene staining and anti-Robo antibody labeling. Mis-positioned motor neurons projected their axons longitudinally within the floor plate, and failed to reach their normal exit points. To test for potential counteracting ventral attractive signals, we examined Netrin-1 and DCC mutants, and found that motor neurons shifted dorsally in the hindbrain and spinal cord, suggesting that Netrin-1/DCC signaling normally attracts motor neurons closer to the floor plate. Our results show that motor neurons are actively migrating cells, and are normally trapped in a static position by Slit/Robo repulsion and Netrin-1/DCC attraction.

Pioneer midbrain longitudinal axons navigate using a balance of Netrin attraction and Slit repulsion.

BACKGROUND: Longitudinal axons grow parallel to the embryonic midline to connect distant regions of the central nervous system. Previous studies suggested that repulsive midline signals guide pioneer longitudinal axons by blocking their entry into the floor plate; however, the role of midline attractants, and whether attractant signals may cooperate with repulsive signals, remains unclear. In this study we investigated the navigation of a set of pioneer longitudinal axons, the medial longitudinal fasciculus, in mouse embryos mutant for the Netrin/Deleted in Colorectal Cancer (DCC) attractants, and for Slit repellents, as well as the responses of explanted longitudinal axons in vitro. RESULTS: In mutants for Netrin1 chemoattractant or DCC receptor signaling, longitudinal axons shifted away from the ventral midline, suggesting that Netrin1/DCC signals act attractively to pull axons ventrally. Analysis of mutants in the three Slit genes, including Slit1/2/3 triple mutants, suggest that concurrent repulsive Slit/Robo signals push pioneer axons away from the ventral midline. Combinations of mutations between the Netrin and Slit guidance systems provided genetic evidence that the attractive and repulsive signals balance against each other. This balance is demonstrated in vitro using explant culture, finding that the cues can act directly on longitudinal axons. The explants also reveal an unexpected synergy of Netrin1 and Slit2 that promotes outgrowth. CONCLUSIONS: These results support a mechanism in which longitudinal trajectories are positioned by a push-pull balance between opposing Netrin and Slit signals. Our evidence suggests that longitudinal axons respond directly and simultaneously to both attractants and repellents, and that the combined signals constrain axons to grow longitudinally.