Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.     

Complementary expression of transmembrane ephrins and their receptors in the mouse spinal cord: a possible role in constraining the orientation of longitudinally projecting axons

In the developing spinal cord, axons project in both the transverse plane, perpendicular to the floor plate, and in the longitudinal plane, parallel to the floor plate. For many axons, the floor plate is a source of long- and short-range guidance cues that govern growth along both dimensions. We show here that B-class transmembrane ephrins and their receptors are reciprocally expressed on floor plate cells and longitudinally projecting axons in the mouse spinal cord. During the period of commissural axon pathfinding, B-class ephrin protein is expressed at the lateral floor plate boundaries, at the interface between the floor plate and the ventral funiculus. In contrast, B-class Eph receptors are expressed on decussated commissural axon segments projecting within the ventral funiculus, and on ipsilaterally projecting axons constituting the lateral funiculus. Soluble forms of all three B-class ephrins bind to, and induce the collapse of, commissural growth cones in vitro. The collapse-inducing activity associated with B-class ephrins is likely to be mediated by EphB1. Taken together, these data support a possible role for repulsive B-class Eph receptor/ligand interactions in constraining the orientation of longitudinal axon projections at the ventral midline.     

The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance

Developing axons are guided to their targets by attractive and repulsive guidance cues. In the embryonic spinal cord, the floor plate chemoattractant Netrin-1 is required to guide commissural neuron axons to the midline. However, genetic evidence suggests that other chemoattractant(s) are also involved. We show that the morphogen Sonic hedgehog (Shh) can mimic the additional chemoattractant activity of the floor plate in vitro and can act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smoothened (Smo) or conditional inactivation of Smo in commissural neurons indicate that Smo activity is important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provide evidence that Shh, acting via Smo, is a midline-derived chemoattractant for commissural axons and show that a morphogen can also act as an axonal chemoattractant.     

Sonic hedgehog is indirectly required for intraretinal axon pathfinding by regulating chemokine expression in the optic stalk

Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.     

Commissural axon pathfinding on the contralateral side of the floor plate: a role for B-class ephrins in specifying the dorsoventral position of longitudinally projecting commissural axons.

In both invertebrate and lower vertebrate species, decussated commissural axons travel away from the midline and assume positions within distinct longitudinal tracts. We demonstrate that in the developing chick and mouse spinal cord, most dorsally situated commissural neuron populations extend axons across the ventral midline and through the ventral white matter along an arcuate trajectory on the contralateral side of the floor plate. Within the dorsal (chick) and intermediate (mouse) marginal zone, commissural axons turn at a conserved boundary of transmembrane ephrin expression, adjacent to which they form a discrete ascending fiber tract. In vitro perturbation of endogenous EphB-ephrinB interactions results in the failure of commissural axons to turn at the appropriate dorsoventral position on the contralateral side of the spinal cord; consequently, axons inappropriately invade more dorsal regions of B-class ephrin expression in the dorsal spinal cord. Taken together, these observations suggest that B-class ephrins act locally during a late phase of commissural axon pathfinding to specify the dorsoventral position at which decussated commissural axons turn into the longitudinal axis.     

Distinct subpopulations of sensory afferents require F11 or axonin-1 for growth to their target layers within the spinal cord of the chick.

Dorsal root ganglion neurons project axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality. Using an in vivo approach, we demonstrate an involvement of the two immunoglobulin superfamily cell adhesion molecules axonin-1/TAG-1 and F11/F3/contactin in subpopulation-specific sensory axon guidance. Proprioceptive neurons, which establish connections with motoneurons in the ventral horn, depend on F11 interactions. Nociceptive fibers, which target to layers in the dorsal horn, require axonin-1 for pathfinding. In vitro NgCAM and NrCAM were shown to bind to both axonin-1 and F11. However, despite this fact and despite their ubiquitous expression in the spinal cord, NgCAM and NrCAM are selective binding partners for axonin-1 and F11 in sensory axon guidance. Whereas nociceptive pathfinding depends on NgCAM and axonin-1, proprioceptive fibers require NrCAM and F11.     

PKCγ免疫反应定位小鼠皮质脊髓束的实验研究

目的探讨显示皮质脊髓束在成年小鼠脑和脊髓中定位分布的简便有效方法。方法运用蛋白激酶Cγ（PKCγ）免疫组织化学染色法,观察成年ICR小鼠脑和脊髓中皮质脊髓束的定位和分布情况。结果PKCγ免疫阳性产物分布于大脑运动皮层第V层锥体细胞胞体和轴突中,锥体细胞的阳性纤维经内囊、中脑大脑脚底、脑桥基底部、下行至延髓锥体中。在延髓下段,PKCγ阳性纤维经锥体交叉后进入对侧脊髓灰质后联合背侧,形成背侧皮质脊髓束,在脊髓白质的后索腹侧深层下行,至骶髓3-4节段以下逐渐消失。在整个脊髓前索和外侧索中未见有PKCγ阳性纤维。结论PKCγ特异地表达于脊髓后索皮质脊髓束中,提示PKCγ免疫组织化学法是一种显示和观察皮质脊髓束精确定位的有效方法。     

Mouse Dispatched homolog1 is required for long-range,but not juxtacrine,Hh signaling

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, we have identified recessive ENU-induced mutations in six genes that prevent normal specification of ventral cell types in the spinal cord. We positionally cloned the genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. Our data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh.     

Integration of Shallow Gradients of Shh and Netrin-1 Guides Commissural Axons

During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.     

Axon guidance and somites

The segmental arrangement of spinal nerves in higher vertebrate embryos provides a simple system in which to study the factors that influence axon pathfinding. Developing motor and sensory axons are intimately associated with surrounding tissues that direct axon guidance. We argue that two distinct guidance mechanisms, viz. contact repulsion and chemorepulsion, act simultaneously to prescribe spinal axon trajectories by ’surround-repulsion’. Motor and sensory axons grow freely within the anterior half of each mesodermal somite, because they are excluded from posterior half-somites by contact repulsion. By contrast, the dorsoventral trajectory that bipolar sensory axons of the dorsal root ganglia follow is governed by diffusible repellents originating from the notochord medially and dermamyotome laterally. Even though spinal nerve development appears to be a simple system for elucidating axon guidance mechanisms, many distinct candidate guidance molecules have been implicated and their relative contributions remain to be evaluated. Received: 28 May 1997 / Accepted: 27 June 1997     

Macroautophagy supports Sonic Hedgehog signaling by promoting Patched1 degradation

Autophagy is a highly conservative self-digestion process to maintain intracellular homeostasis and to ensure the survival of cells under stress. Activation of Sonic Hedgehog (Shh) signaling depends on the normal endocytic degradation of pathway receptor Patched1 (Ptch1). It is unclear whether autophagy participates in the receptor endocytosis and modulates Shh signaling transduction. Here we found that blocking macroautophagy attenuates Shh signaling due to the failed transport of Smoothened (Smo) into primary cilia. At the upstream of Smo, Ptch1 was poly-ubiquitinated through K63-conjugated ubiquitin chains. Macroautophagy participates Shh-induced degradation of poly-ubiquitinated Ptch1, contributing to the activation of Shh signaling.     

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.     

Netrin-1 signaling for sensory axons

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a ‘waiting period’ of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.     

Orienting axon growth: spinal nerve segmentation and surround-repulsion

The study of spinal nerve trajectories in higher vertebrate embryos has revealed an inherent polarity within somites along the antero-posterior axis, and provides a simple system in which to study the factors that influence axon pathfinding. We argue that the orientation of spinal axons is determined by the simultaneous operation of two distinct guidance mechanisms, contact repulsion and chemorepulsion. Motor and sensory axons traverse the anterior half of each somite because they are excluded by contact repulsion from the posterior half-somite, and the molecular nature of several candidate contact repellents is reviewed. In contrast, we find that the dorsoventral trajectory of primary sensory axons is oriented by diffusible repellents originating from the notochord medially and dermamyotome laterally. In this system, therefore, repulsion by surrounding tissues ('surround-repulsion') is the main force directing axon growth in three dimensions.     

Type III neuregulin 1 regulates pathfinding of sensory axons in the developing spinal cord and periphery

Sensory axons must develop appropriate connections with both central and peripheral targets. Whereas the peripheral cues have provided a classic model for neuron survival and guidance, less is known about the central cues or the coordination of central and peripheral connectivity. Here we find that type III Nrg1, in addition to its known effect on neuron survival, regulates axon pathfinding. In type III Nrg1(-/-) mice, death of TrkA(+) nociceptive/thermoreceptive neurons was increased, and could be rescued by Bax elimination. In the Bax and type III Nrg1 double mutants, axon pathfinding abnormalities were seen for TrkA(+) neurons both in cutaneous peripheral targets and in spinal cord central targets. Axon guidance phenotypes in the spinal cord included penetration of axons into ventral regions from which they would normally be repelled by Sema3A. Accordingly, sensory neurons from type III Nrg1(-/-) mice were unresponsive to the repellent effects of Sema3A in vitro, which might account, at least in part, for the central projection phenotype, and demonstrates an effect of type III Nrg1 on guidance cue responsiveness in neurons. Moreover, stimulation of type III Nrg1 back-signaling in cultured sensory neurons was found to regulate axonal levels of the Sema3A receptor neuropilin 1. These results reveal a molecular mechanism whereby type III Nrg1 signaling can regulate the responsiveness of neurons to a guidance cue, and show that type III Nrg1 is required for normal sensory neuron survival and axon pathfinding in both central and peripheral targets.