The function of leak and kuzbanian during growth cone and cell migration

Axonal growth cones require an evolutionary conserved repulsive guidance system to ensure proper crossing of the CNS midline. In Drosophila, the Slit protein is a repulsive signal secreted by the midline glial cells. It binds to the Roundabout receptors, which are expressed on CNS axons in the longitudinal tracts but not in the commissural tracts. Here we present an analysis of the genes leak and kuzbanian and show that both genes are involved in the repulsive guidance system operating at the CNS midline. Mutations in leak, which encodes the Roundabout-2 Slit receptor, were first recovered by Nüsslein-Volhard and co-workers based on defects in the larval cuticle. Analysis of the head phenotype suggests that slit may be able to act as an attractive guidance cue while directing the movements of the dorsal ectodermal cell sheath. kuzbanian also regulates midline crossing of CNS axons. It encodes a metalloprotease of the ADAM family and genetically interacts with slit. Expression of a dominant negative Kuzbanian protein in the CNS midline cells results in an abnormal midline crossing of axons and prevents the clearance of the Roundabout receptor from commissural axons. Our analyses support a model in which Kuzbanian mediates the proteolytic activation of the Slit/Roundabout receptor complex.     

Slit is the midline repellent for the robo receptor in Drosophila

Previous studies suggested that Roundabout (Robo) is a repulsive guidance receptor on growth cones that binds to an unknown midline ligand. Here we present genetic evidence that Slit is the midline Robo ligand; a companion paper presents biochemical evidence that Slit binds Robo. Slit is a large extracellular matrix protein expressed by midline glia. In slit mutants, growth cones enter the midline but never leave it; they abnormally continue to express high levels of Robo while at the midline. slit and robo display dosage-sensitive genetic interactions, indicating that they function in the same pathway. slit is also required for migration of muscle precursors away from the midline. Slit appears to function as a short-range repellent controlling axon crossing of the midline and as a long-range chemorepellent controlling mesoderm migration away from the midline.     

Short-range and long-range guidance by slit and its Robo receptors. Robo and Robo2 play distinct roles in midline guidance

Previous studies showed that Roundabout (Robo) in Drosophila is a repulsive axon guidance receptor that binds to Slit, a repellent secreted by midline glia. In robo mutants, growth cones cross and recross the midline, while, in slit mutants, growth cones enter the midline but fail to leave it. This difference suggests that Slit must have more than one receptor controlling midline guidance. In the absence of Robo, some other Slit receptor ensures that growth cones do not stay at the midline, even though they cross and recross it. Here we show that the Drosophila genome encodes three Robo receptors and that Robo and Robo2 have distinct functions, which together control repulsive axon guidance at the midline. The robo,robo2 double mutant is largely identical to slit.     

Frizzled-3a and slit2 genetically interact to modulate midline axon crossing in the telencephalon

The anterior commissure forms the first axon connections between the two sides of the embryonic telencephalon. We investigated the role of the transmembrane receptor Frizzled-3a in the development of this commissure using zebrafish as an experimental model. Knock down of Frizzled-3a resulted in complete loss of the anterior commissure. This defect was accompanied by a loss of the glial bridge, expansion of the slit2 expression domain and perturbation of the midline telencephalic-diencephalic boundary. Blocking Slit2 activity following knock down of Frizzled-3a effectively rescued the anterior commissure defect which suggested that Frizzled-3a was indirectly controlling the growth of axons across the rostral midline. We have shown here that Frizzled-3a is essential for normal development of the commissural plate and that loss-of-function causes Slit2-dependent defects in axon midline crossing in the embryonic vertebrate forebrain. These data supports a model whereby Wnt signaling through Frizzled-3a attenuates expression of Slit2 in the rostral midline of the forebrain. The absence of Slit2 facilitates the formation of a midline bridge of glial cells which is used as a substrate for commissural axons. In the absence of this platform of glia, commissural axons fail to cross the rostral midline of the forebrain.     

Role of Slit proteins in the vertebrate brain.

Diffusible chemorepellents play a major role in guiding developing axons towards their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptors and their secreted ligand Slits. Three distinct slit genes (slit1, slit2 and slit3) and three distinct robo genes (robo1, robo2 and rig-1) have been cloned in mammals. In collagen gel co-cultures, Slit1 and Slit2 can repel and collapse olfactory axons. However, there is also some positive effect associated with Slits, as Slit2 stimulates the formation of axon collateral branches by NGF-responsive neurons of the dorsal root ganglia (DRG). Slit2 is a large ECM glycoproteins of about 200 kD, which is proteolytically processed into 140 kD N-terminal and 55-60 kD C-terminal fragments. Slit2 cleavage fragments appear to have different cell association characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. This suggested that the different fragments might have different functional activities in vivo. We have begun to explore these questions by engineering mutant and truncated versions of hSlit2 representing the two cleavage fragments, N- and C-, and the uncleavable molecule and examining the activities of these mutants in binding and functional assays. We found that an axon's response to Slit2 is not absolute, but rather is reflective of the context in which the protein is encountered.     

Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain

Three major axon pathways cross the midline of the vertebrate forebrain early in embryonic development: the postoptic commissure (POC), the anterior commissure (AC) and the optic nerve. We show that a small population of Gfap+ astroglia spans the midline of the zebrafish forebrain in the position of, and prior to, commissural and retinal axon crossing. These glial ;bridges' form in regions devoid of the guidance molecules slit2 and slit3, although a subset of these glial cells express slit1a. We show that Hh signaling is required for commissure formation, glial bridge formation, and the restricted expression of the guidance molecules slit1a, slit2, slit3 and sema3d, but that Hh does not appear to play a direct role in commissural and retinal axon guidance. Reducing Slit2 and/or Slit3 function expanded the glial bridges and caused defasciculation of the POC, consistent with a ;channeling' role for these repellent molecules. By contrast, reducing Slit1a function led to reduced midline axon crossing, suggesting a distinct role for Slit1a in midline axon guidance. Blocking Slit2 and Slit3, but not Slit1a, function in the Hh pathway mutant yot (gli2DR) dramatically rescued POC axon crossing and glial bridge formation at the midline, indicating that expanded Slit2 and Slit3 repellent function is largely responsible for the lack of midline crossing in these mutants. This analysis shows that Hh signaling helps to pattern the expression of Slit guidance molecules that then help to regulate glial cell position and axon guidance across the midline of the forebrain.     

Lateral neuron--glia interactions steer the response of axons to the Robo code

Glia are required for axon pathfinding along longitudinal trajectories, but it is unknown how this relates to the molecular paradigm of axon guidance across the midline. Most interneuron axons in bilateral organisms cross the midline only once. Preventing them from recrossing the midline requires the expression of Robo receptors on the axons. These sense the repulsive signal Slit, which is produced by the midline. The lateral positioning of longitudinal axons depends on the response to Slit by the combination of Robo receptors expressed by the axons, on selective fasciculation, and on longitudinal (lateral) glia. Here, we analyse how longitudinal glia influence reading of the 'Robo code' by axons. We show that whereas loss of robo1 alone only affects the most medial axons, loss of both glial cells missing (gcm) and robo1 causes a severe midline collapse of longitudinal axons, similar to that caused by the loss of multiple Robo receptors. Furthermore, whereas ectopic expression of robo2 is sufficient to displace the medial MP2 axons along a more lateral trajectory, this does not occur in gcm-robo1 double-mutant embryos, where axons either do not extend at all or they misroute exiting the CNS. Hence, lateral neuron-glia interactions steer the response of axons to the Robo code.     

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.Key words: Slit, Robo, longitudinal axon, hindbrain, axon guidance     

Roundabout gene family functions during sensory axon guidance in the drosophila embryo are mediated by both Slit-dependent and Slit-independent mechanisms

roundabout (robo) family genes play key roles in axon guidance in a wide variety of animals. We have investigated the roles of the robo family members, robo, robo2, and robo3, in the guidance of sensory axons in the Drosophila embryo. In robo(-/-), slit(-/-), and robo(-/+) slit(-/+) mutants, lateral cluster sensory neurons misproject to cells and axons in the nearby ventral' (v') cluster. These phenotypes, together with the normal expression pattern of Slit and Robo, suggest that Slit ligand secreted from the epidermis interacts with Robo receptors on lateral cluster sensory growth cones to limit their exploration of nearby attractive substrates. The most common sensory axon phenotype seen in robo2(-/-) mutants was misprojection of dorsal cluster sensory axons away from their normal growth substrate, the transverse connective of the trachea. slit appears to play no role in this aspect of sensory axon growth. Robo2 is expressed, not on the dorsal sensory axons, but on the transverse connective. These results suggest a novel, non-cell-autonomous mechanism for axon guidance by robo family genes: Robo2 expressed on the trachea acts as an attractant for the dorsal sensory growth cones.     

Conserved roles for Slit and Robo proteins in midline commissural axon guidance

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.     

Early posterior/ventral fate specification in the vertebrate embryo

Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation.     

Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain.

We report that Slit proteins, a family of secreted chemorepellents, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 and, even more so, mice deficient in both Slit1 and Slit2 show significant axon guidance errors in a variety of pathways, including corticofugal, callosal, and thalamocortical tracts. Analysis of multiple pathways suggests several generalizations regarding the functions of Slit proteins in the brain, which appear to contribute to (1) the maintenance of dorsal position by prevention of axonal growth into ventral regions, (2) the prevention of axonal extension toward and across the midline, and (3) the channeling of axons toward particular regions.     

Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain

The floor plate is known to be a source of repellent signals for cranial motor axons, preventing them from crossing the midline of the hindbrain. However, it is unknown which molecules mediate this effect in vivo. We show that Slit and Robo proteins are candidate motor axon guidance molecules, as Robo proteins are expressed by cranial motoneurons, and Slit proteins are expressed by the tissues that delimit motor axon trajectories, i.e. the floor plate and the rhombic lip. We present in vitro evidence showing that Slit1 and Slit2 proteins are selective inhibitors and repellents for dorsally projecting, but not for ventrally projecting, cranial motor axons. Analysis of mice deficient in Slit and Robo function shows that cranial motor axons aberrantly enter the midline, while ectopic expression of Slit1 in chick embryos leads to specific motor axon projection errors. Expression of dominant-negative Robo receptors within cranial motoneurons in chick embryos strikingly perturbs their projections, causing some motor axons to enter the midline, and preventing dorsally projecting motor axons from exiting the hindbrain. These data suggest that Slit proteins play a key role in guiding dorsally projecting cranial motoneurons and in facilitating their neural tube exit.     

Slit-roundabout signaling neutralizes netrin-Frazzled-mediated attractant cue to specify the lateral positioning of longitudinal axon pathways

An extending axon growth cone is subjected to attractant and repellent cues. It is not clear how these growth cones discriminate the two opposing forces and select their projection paths. Here, we report that in the Drosophila nerve cord the growth cones of longitudinal tracts are subjected to attraction by the Netrin-Frazzled pathway. However, the midline Slit neutralizes this pathway in a Robo-dependent manner and prevents Netrin-Frazzled-mediated attraction of longitudinal tracts. Our results suggest that the loss of a neutralizing effect on the Netrin-mediated attraction is responsible for the longitudinal tracts entering the midline in slit mutants as opposed to a loss of repulsion as is currently believed. This effect is not via a direct inhibition of Frazzled by Robo; instead, it is at a level downstream of Frazzled. Thus, the growth cones of longitudinal tracts subjected to two opposing forces are able to block one with the other and specify their correct lateral positioning along the midline.     

Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons

The olfactory bulb plays a central role in olfactory information processing through its connections with both peripheral and cortical structures. Axons projecting from the olfactory bulb to the telencephalon are guided by a repulsive activity in the septum. The molecular nature of the repellent is not known. We report here the isolation of vertebrate homologs of the Drosophila slit gene and show that Slit protein binds to the transmembrane protein Roundabout (Robo). Slit is expressed in the septum whereas Robo is expressed in the olfactory bulb. Functionally, Slit acts as a chemorepellent for olfactory bulb axons. These results establish a ligand-receptor relationship between two molecules important for neural development, suggest a role for Slit in olfactory bulb axon guidance, and reveal the existence of a new family of axon guidance molecules.     

Local inhibition guides the trajectory of early longitudinal tracts in the developing chick brain

During development of the chick central nervous system, the trajectories of the descending medial and lateral longitudinal fascicles (MLF and LLF) are pioneered by axons originating from the interstitial nucleus of Cajal (INC) and the mesencephalic trigeminal nucleus (MTN), respectively. Both tracts cross rhombomere 1 at two specific locations in the basal plate. In this study, we have investigated the molecular properties of these crossing points and find that they are permissive regions situated in an otherwise inhibitory boundary region. We show that the dorsal part of rhombomere 1 is inhibitory for the growth of both MTN and INC axons. Ventrally, MLF and LLF axons are repelled from the midline by Slit proteins. Our results reveal the existence of a new repulsive/inhibitory mechanism for axons in the alar plate in addition to the ventral repulsion by Slit proteins. This suggests a model where MLF and LLF axons are channeled longitudinally within the neural tube by both dorsal and ventral constraints.     

The Drosophila ARF6-GEF Schizo controls commissure formation by regulating Slit

The CNS of bilateral symmetric organisms is characterized by intensive contralateral axonal connections. Genetic screens in Drosophila have identified only a few genes required for guiding commissural growth cones toward and across the midline. Two evolutionarily conserved signaling molecules, Netrin and Slit, are expressed in the CNS midline cells. Netrin acts primarily as an attractive signaling cue, whereas Slit mediates repulsive functions. Here, we describe a detailed analysis of the Drosophila gene schizo, which is required for commissure formation. schizo leads to a commissural phenotype reminiscent of netrin mutant embryos. Double-mutant analyses indicate that Netrin and Schizo act independently. The schizo mutant phenotype can be suppressed by either expressing netrin in the CNS midline cells or by a reduction of the slit gene dose, indicating that the balance of attractive and repulsive signaling is impaired in schizo mutants. Overexpression of the schizo RNA in the CNS midline using the GAL4/UAS system leads to a slit phenocopy, suggesting that schizo primarily antagonizes Slit signaling. This is further supported by cell type-specific rescue experiments. The schizo gene generates at least two proteins containing a conserved Sec7 and a pleckstrin homology domain (PH) characteristic for guanine nucleotide exchange factors (GEF) acting on ARF GTPases, which are known to regulate endocytosis. In support of the notion that schizo regulates Slit expression via endocytosis, we found that block of endocytosis leads to a schizo-like phenotype. We thus propose that the balance of the two signaling cues Netrin and Slit can be regulated, controlling membrane dynamics.     

Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.     

Patched regulation of axon guidance is by specifying neural identity in the<Emphasis Type="Italic"> Drosophila</Emphasis> nerve cord

Within an axon bundle, one or two are pioneering axons and the rest are follower axons. Pioneering axons are projected first and the follower axons are projected later but follow a pioneering axon(s) pathway. It is not clear whether the pioneering axons have a guidance role for follower axons. In this paper, we have investigated the role of Patched (Ptc) in regulating the guidance of medial tract, one of the longitudinal tracts in the nerve cord. In patched mutants the medial longitudinal tract fails to fasciculate on its own side along the nerve cord, instead it abnormally crosses the midline and fasciculates with the contralateral tract. Interestingly, the medial tracts cross the midline ignoring the axon-repellant Slit on the midline and Roundabout on growth cones. The medial tract is pioneered by neurons pCC and vMP2. Our results show that guidance defects of this tract are due to loss and mis-specification of vMP2, which results in the projection from pCC to either stall or project outward near the location of vMP2. Thus, both pioneering neurons are necessary for the proper guidance of pioneering and follower axons. We also show that the loss of Ptc activity in the neuroectoderm prior to the formation of S1 and S2 neuroblasts causes the majority of axon guidance defects. These results provide insight into how mis-specification and loss of neurons can non-autonomously contribute to defects in axon pathfinding.     

Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos

Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Here, evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons. Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues.