Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo (Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons (up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.     

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.     

Transient Neuronal Populations Are Required to Guide Callosal Axons: A Role for Semaphorin 3C

The corpus callosum (CC) is the main pathway responsible for interhemispheric communication. CC agenesis is associated with numerous human pathologies, suggesting that a range of developmental defects can result in abnormalities in this structure. Midline glial cells are known to play a role in CC development, but we here show that two transient populations of midline neurons also make major contributions to the formation of this commissure. We report that these two neuronal populations enter the CC midline prior to the arrival of callosal pioneer axons. Using a combination of mutant analysis and in vitro assays, we demonstrate that CC neurons are necessary for normal callosal axon navigation. They exert an attractive influence on callosal axons, in part via Semaphorin 3C and its receptor Neuropilin-1. By revealing a novel and essential role for these neuronal populations in the pathfinding of a major cerebral commissure, our study brings new perspectives to pathophysiological mechanisms altering CC formation.     

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.     

Short-range and long-range guidance by Slit and its Robo receptors: a combinatorial code of Robo receptors controls lateral position

Slit is secreted by midline glia in Drosophila and functions as a short-range repellent to control midline crossing. Although most Slit stays near the midline, some diffuses laterally, functioning as a long-range chemorepellent. Here we show that a combinatorial code of Robo receptors controls lateral position in the CNS by responding to this presumptive Slit gradient. Medial axons express only Robo, intermediate axons express Robo3 and Robo, while lateral axons express Robo2, Robo3, and Robo. Removal of robo2 or robo3 causes lateral axons to extend medially; ectopic expression of Robo2 or Robo3 on medial axons drives them laterally. Precise topography of longitudinal pathways appears to be controlled by a combination of long-range guidance (the Robo code determining region) and short-range guidance (discrete local cues determining specific location within a region).     

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.     

White matter abnormalities in fetal alcohol spectrum disorders: Focus on axon growth and guidance

Fetal Alcohol Spectrum Disorders (FASDs) describe a range of deficits, affecting physical, mental, cognitive, and behavioral function, arising from prenatal alcohol exposure. FASD causes widespread white matter abnormalities, with significant alterations of tracts in the cerebral cortex, cerebellum, and hippocampus. These brain regions present with white-matter volume reductions, particularly at the midline. Neural pathways herein are guided primarily by three guidance cue families: Semaphorin/Neuropilin, Netrin/DCC, and Slit/Robo. These guidance cue/receptor pairs attract and repulse axons and ensure that they reach the proper target to make functional connections. In several cases, these signals cooperate with each other and/or additional molecular partners. Effects of alcohol on guidance cue mechanisms and their associated effectors include inhibition of growth cone response to repellant cues as well as changes in gene expression. Relevant to the corpus callosum, specifically, developmental alcohol exposure alters GABAergic and glutamatergic cell populations and glial cells that serve as guidepost cells for callosal axons. In many cases, deficits seen in FASD mirror aberrancies in guidance cue/receptor signaling. We present evidence for the need for further study on how prenatal alcohol exposure affects the formation of neural connections which may underlie disrupted functional connectivity in FASD.     

Slit2-Mediated chemorepulsion and collapse of developing forebrain axons

Diffusible chemorepellents play a major role in guiding developing axons toward 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) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.     

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.     

Heparan sulfate proteoglycan specificity during axon pathway formation in the Drosophila embryo

Axon guidance is influenced by the presence of heparan sulfate (HS) proteoglycans (HSPGs) on the surface of axons and growth cones (Hu, [2001]: Nat Neurosci 4:695-701; Irie et al. [2002]: Development 129:61-70; Inatani et al. [2003]: Science 302:1044-1046; Johnson et al. [2004]: Curr Biol 14:499-504; Steigemann et al. [2004]: Curr Biol 14:225-230). Multiple HSPGs, including Syndecans, Glypicans and Perlecans, carry the same carbohydrate polymer backbones, raising the question of how these molecules display functional specificity during nervous system development. Here we use the Drosophila central nervous system (CNS) as a model to compare the impact of eliminating Syndecan (Sdc) and/or the Glypican Dally-like (Dlp). We show that Dlp and Sdc share a role in promoting accurate patterns of axon fasciculation in the lateral longitudinal neuropil; however, unlike mutations in sdc, which disrupt the ability of the secreted repellent Slit to prevent inappropriate passage of axons across the midline, mutations in dlp show neither midline defects nor genetic interactions with Slit and its Roundabout (Robo) receptors at the midline. Dlp mutants do show genetic interactions with Slit and Robo in lateral fascicle formation. In addition, simultaneous loss of Dlp and Sdc demonstrates an important role for Dlp in midline repulsion, reminiscent of the functional overlap between Robo receptors. A comparison of HSPG distribution reveals a pattern that leaves midline proximal axons with relatively little Dlp. Finally, the loss of Dlp alters Slit distribution distal but not proximal to the midline, suggesting that distinct yet overlapping pattern of HSPG expression provides a spatial system that regulates axon guidance decisions.     

Surrounded by Slit--how forebrain commissural axons can be led astray.

In Drosophila, Slit acts as a barrier preventing roundabout expressing axons from entering the midline and sorting contralaterally from ipsilaterally projecting axons. Hutson and Chien, Plump et al., and Bagri et al. (all in this issue of Neuron) use Slit knockout mice and zebrafish astray/Robo2 mutants to show that in vertebrates, Robo/Slit function to channel axons into specific pathways and determine where decussation points occur. Ipsilaterally and contralaterally projected axons are equally affected.     

Recombinant inbreeding in mice reveals thresholds in embryonic corpus callosum development

The inbred strains BALB/cWah1 and 129P1/ReJ both show incomplete penetrance for absent corpus callosum (CC); about 14% of adult mice have no CC at all. Their F(1) hybrid offspring are normal, which proves that the strains differ at two or more loci pertinent to absent CC. Twenty-three recombinant inbred lines were bred from the F(2) cross of BALB/c and 129, and several of these expressed a novel and severe phenotype after only three or four generations of inbreeding - total absence of the CC and severe reduction of the hippocampal commissure (HC) in every adult animal. As inbreeding progressed, intermediate sizes of the CC and the HC remained quite rare. This striking phenotypic distribution in adults arose from developmental thresholds in the embryo. CC axons normally cross to the opposite hemisphere via a tissue bridge in the septal region at midline, where the HC forms before CC axons arrive. The primary defect in callosal agenesis in the BALB/c and 129 strains is severe retardation of fusion of the hemispheres in the septal region, and failure to form a CC is secondary to this defect. The putative CC axons arrive at midline at the correct time and place in all groups, but in certain genotypes, the bridge is not yet present. The relative timing of axon growth and delay of the septal bridge create a narrow critical period for forming a normal brain.     

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.     

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     

EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure

The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains. Consistent with the roles for these receptors in formation of the CC and ACp, we detect expression of these receptors in multiple brain regions associated with the formation of these forebrain structures. Taken together, our findings suggest that a combination of forward and reverse EphB1/2 receptor‐mediated signaling contribute to ACp and CC axon guidance. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 405–420, 2016     

The autophagy-inducing kinases,ULK1 and ULK2, regulate axon guidance in the developing mouse forebrain via a noncanonical pathway

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, Caenorhabditis elegans UNC-51, and Drosophila melanogaster Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking Ulk1 and Ulk2 in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in ulk1/2 double-knockout mice and central nervous system-specific (Nes-Cre) Ulk1/2-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., Atg7 or Rb1cc1 [RB1-inducible coiled-coil 1]). The brains of Ulk1/2-deficient mice did not show stem cell defects previously attributed to defective autophagy in ambra1 (autophagy/Beclin 1 regulator 1)- and Rb1cc1-deficient mice or accumulation of SQSTM1 (sequestosome 1)⁺ or ubiquitin⁺ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.     

Slit proteins are not dominant chemorepellents for olfactory tract and spinal motor axons.

Members of the Slit family are large extracellular glycoproteins that may function as chemorepellents in axon guidance and neuronal cell migration. Their actions are mediated through members of the Robo family that act as their receptors. In vertebrates, Slit causes chemorepulsion of embryonic olfactory tract, spinal motor, hippocampal and retinal ganglion cell axons. Since Slits are expressed in the septum and floor plate during the period when these tissues cause chemorepulsion of olfactory tract and spinal motor axons respectively, it has been proposed that Slits function as guidance cues. We have tested this hypothesis in collagen gel co-cultures using soluble Robo/Fc chimeras, as competitive inhibitors, to disrupt Slit interactions. We find that the addition of soluble Robo/Fc has no effect on chemorepulsion of olfactory tract and spinal motor axons when co-cultured with septum or floor plate respectively. Thus, we conclude that although Slits are expressed in the septum and floor plate, their proteins do not contribute to the major chemorepulsive activities emanating from these tissues which cause repulsion of olfactory tract and spinal motor axons.     

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.     

Wnt/calcium signaling mediates axon growth and guidance in the developing corpus callosum

It has been shown in vivo that Wnt5a gradients surround the corpus callosum and guide callosal axons after the midline (postcrossing) by Wnt5a-induced repulsion via Ryk receptors. In dissociated cortical cultures we showed that Wnt5a simultaneously promotes axon outgrowth and repulsion by calcium signaling. Here to test the role of Wnt5a/calcium signaling in a complex in vivo environment we used sensorimotor cortical slices containing the developing corpus callosum. Plasmids encoding the cytoplasmic marker DsRed and the genetically encoded calcium indicator GCaMP2 were electroporated into one cortical hemisphere. Postcrossing callosal axons grew 50% faster than pre-crossing axons and higher frequencies of calcium transients in axons and growth cones correlated well with outgrowth. Application of pharmacological inhibitors to the slices showed that signaling pathways involving calcium release through IP3 receptors and calcium entry through TRP channels regulate post-crossing axon outgrowth and guidance. Co-electroporation of Ryk siRNA and DsRed revealed that knock down of the Ryk receptor reduced outgrowth rates of postcrossing but not precrossing axons by 50% and caused axon misrouting. Guidance errors in axons with Ryk knockdown resulted from reduced calcium activity. In the corpus callosum CaMKII inhibition reduced the outgrowth rate of postcrossing (but not precrossing) axons and caused severe guidance errors which resulted from reduced CaMKII-dependent repulsion downstream of Wnt/calcium. We show for the first time that Wnt/Ryk calcium signaling mechanisms regulating axon outgrowth and repulsion in cortical cultures are also essential for the proper growth and guidance of postcrossing callosal axons which involve axon repulsion through CaMKII.     

C. elegans slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor

Robo receptors interact with ligands of the Slit family. The nematode C. elegans has one Robo receptor (SAX-3) and one Slit protein (SLT-1), which direct ventral axon guidance and guidance at the midline. In larvae, slt-1 expression in dorsal muscles repels axons to promote ventral guidance. SLT-1 acts through the SAX-3 receptor, in parallel with the ventral attractant UNC-6 (Netrin). Removing both UNC-6 and SLT-1 eliminates all ventral guidance information for some axons, revealing an underlying longitudinal guidance pathway. In the embryo, slt-1 is expressed at high levels in anterior epidermis. Embryonic expression of SLT-1 provides anterior-posterior guidance information to migrating CAN neurons. Surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3 mutants, suggesting that SAX-3 has both Slit-dependent and Slit-independent functions in development.