Wild-type Axons Research Articles

Local glycolysis supports injury-induced axonal regeneration.

Successful axonal regeneration following injury requires the effective allocation of energy. How axons withstand the initial disruption in mitochondrial energy production caused by the injury and subsequently initiate regrowth is poorly understood. Transcriptomic data showed increased expression of glycolytic genes after optic nerve crush in retinal ganglion cells with the co-deletion of Pten and Socs3. Using retinal cultures in a multicompartment microfluidic device, we observed increased regrowth and enhanced mitochondrial trafficking in the axons of Pten and Socs3 co-deleted neurons. While wild-type axons relied on mitochondrial metabolism, after injury, in the absence of Pten and Socs3, energy production was supported by local glycolysis. Specific inhibition of lactate production hindered injury survival and the initiation of regrowth while slowing down glycolysis upstream impaired regrowth initiation, axonal elongation, and energy production. Together, these observations reveal that glycolytic ATP, combined with sustained mitochondrial transport, is essential for injury-induced axonal regrowth, providing new insights into the metabolic underpinnings of axonal regeneration.

Boosting peripheral BDNF rescues impaired in vivo axonal transport in CMT2D mice.

Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterizing Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor tropomyosin receptor kinase receptor B (TrkB), underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice displayed early and persistent disturbances in axonal transport of neurotrophin-containing signaling endosomes in vivo. We discovered that brain-derived neurotrophic factor (BDNF)/TrkB impairments correlated with transport disruption and overall CMT2D neuropathology and that inhibition of this pathway at the nerve-muscle interface perturbed endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restored physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.

Regulation of degenerative spheroids after injury

Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1−/− and DR6−/−, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1−/− and DR6−/−, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.

The ubiquitin ligase PHR promotes directional regrowth of spinal zebrafish axons

To reconnect with their synaptic targets, severed axons need to regrow robustly and directionally along the pre-lesional trajectory. While mechanisms directing axonal regrowth are poorly understood, several proteins direct developmental axon outgrowth, including the ubiquitin ligase PHR (Mycbp2). Invertebrate PHR also limits regrowth of injured axons, whereas its role in vertebrate axonal regrowth remains elusive. Here we took advantage of the high regrowth capacity of spinal zebrafish axons and observed robust and directional regrowth following laser transection of spinal Mauthner axons. We found that PHR directs regrowing axons along the pre-lesional trajectory and across the transection site. At the transection site, initial regrowth of wild-type axons was multidirectional. Over time, misdirected sprouts were corrected in a PHR-dependent manner. Ablation of cyfip2, known to promote F-actin-polymerization and pharmacological inhibition of JNK reduced misdirected regrowth of PHR-deficient axons, suggesting that PHR controls directional Mauthner axonal regrowth through cyfip2- and JNK-dependent pathways.

Axonal Degeneration in Retinal Ganglion Cells Is Associated with a Membrane Polarity-Sensitive Redox Process.

Axonal degeneration is a pathophysiological mechanism common to several neurodegenerative diseases. The slow Wallerian degeneration (WldS) mutation, which results in reduced axonal degeneration in the central and peripheral nervous systems, has provided insight into a redox-dependent mechanism by which axons undergo self-destruction. We studied early molecular events in axonal degeneration with single-axon laser axotomy and time-lapse imaging, monitoring the initial changes in transected axons of purified retinal ganglion cells (RGCs) from wild-type and WldS rat retinas using a polarity-sensitive annexin-based biosensor (annexin B12-Cys101,Cys260-N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine). Transected axons demonstrated a rapid and progressive change in membrane phospholipid polarity, manifested as phosphatidylserine externalization, which was significantly delayed and propagated more slowly in axotomized WldS RGCs compared with wild-type axons. Delivery of bis(3-propionic acid methyl ester)phenylphosphine borane complex, a cell-permeable intracellular disulfide-reducing drug, slowed the onset and velocity of phosphatidylserine externalization in wild-type axons significantly, replicating the WldS phenotype, whereas extracellular redox modulation reversed the WldS phenotype. These findings are consistent with an intra-axonal redox mechanism for axonal degeneration associated with the initiation and propagation of phosphatidylserine externalization after axotomy.SIGNIFICANCE STATEMENT Axonal degeneration is a neuronal process independent of somal apoptosis, the propagation of which is unclear. We combined single-cell laser axotomy with time-lapse imaging to study the dynamics of phosphatidylserine externalization immediately after axonal injury in purified retinal ganglion cells. The extension of phosphatidylserine externalization was slowed and delayed in Wallerian degeneration slow (WldS) axons and this phenotype could be reproduced by intra-axonal disulfide reduction in wild-type axons and reversed by extra-axonal reduction in WldS axons. These results are consistent with a redox mechanism for propagation of membrane polarity asymmetry in axonal degeneration.

Stroke Literature Synopses: Basic Science

The mitochondrion is a membrane-bounded cell organelle, which plays critical roles in cellular survival by supplying energy or mediating intracellular signaling pathway. Mitochondrial dysfunction has been implicated in many CNS disorders including stroke. Three recent studies elucidate novel mechanisms for how mitochondria are involved in neuronal damage under diseased conditions. Li et al. (A TIGAR-regulated metabolic pathway is critical for protection of brain ischemia. The Journal of Neuroscience. 2014;34:7458-7471) demonstrated that TP53-induced glycolysis and apoptosis regulator (TIGAR) protects neurons against ischemic stress. TIGAR has been reported to block glycolysis by reducing the levels of Fru-2,6-P2 and therefore increase the flow of pentose phosphate pathway. The western blots and immunostaining analyses found that TIGAR was highly expressed in brain neurons and was rapidly upregulated in response to ischemia/reperfusion insults. In addition, both in animal and cellular stroke models, ischemia/reperfusion or oxygen-glucose-deprivation (OGD)/reoxygenation stress increased mitochondrial localization of TIGAR. To explore the roles of TIGAR in cerebral ischemic injury, the authors injected lentiviral vectors encoding TIGAR or sh-TIGAR into mouse lateral ventricle and striatum, and then subjected the mice to stroke insults. Compared to control-lentiviral-vector-injected mice, overexpression of TIGAR reduced the infarct size, neurological deficits, and brain edema, whereas TIGAR knockdown aggravated the damage. Similarly, in their neuronal cell culture system, overexpression of TIGAR increased the cell viability under OGD/reoxygenation conditions, whereas knockdown of TIGAR decreased the cell viability. Furthermore, overexpression of TIGAR in neuron cultures prevented the decreases in mitochondrial transmembrane potential caused by OGD/reoxygenation insults. Therefore, the TIGAR-regulated metabolic pathway may be a new therapeutic target for stroke. Mitochondria maintain intra-cellular homeostasis, and as needed, they mediate pro-apoptotic signaling by releasing apoptosis-inducing factor (AIF), which plays crucial roles in caspase-independent neuronal death. Doti et al. (Inhibition of the AIF/CypA complex protects against intrinsic death pathways induced by oxidative stress. Cell Death and Disease. 2014;5:e993) showed that the AIF amino-acid residues 370-394 mediates the protein complex formation of AIF with cytosolic cyclophilin A (CypA). AIF is a mitochondrial flavoprotein, and after release from the mitochondria, it binds to CypA to initiate the translocation to the nucleus, where the AIF/CypA complex generates a lethal DNA-degrading complex. The authors designed AIF peptide AIF(370-394), which targets the AIF-binding site on CypA. To evaluate the neuroprotective potency of AIF/CypA complex formation inhibition, the AIF peptide was applied to HT-22 neuronal cell lines, wherein glutamate-induced oxidative stress causes AIF-dependent cell death. Firstly, the authors confirmed that CypA knockdown by CypA-siRNA gene silencing blocked glutamate-induced nuclear translocation of AIF and protected HT-22 neuronal cells against glutamate-induced oxidative stress. Similarly, AIF(370-394)-transfected HT-22 exhibited less glutamate-induced nuclear translocation of AIF and more resistance to glutamate-induced oxidative stress. Furthermore, the decrease of mitochondrial membrane potential by glutamate treatment was significantly attenuated in the HT-22 cells transfected with AIF(370-394). Hence, drugs or peptides that inhibit AIF/CypA complex formation may act as a promising neuroprotectant against ischemic stress. Axonal degeneration is one of the major hallmarks of neuronal damage in stroke. Ohno et al. (Mitochondrial immobilization mediated by syntaphilin facilitates survival of demyelinated axons. Proceedings of the National Academy of Sciences. 2014;111:9953-9958) defined the roles of mitochondrial volume and distribution in axonal degeneration following CNS demyelination. Most axonal mitochondria do not appear to translocate and are located at their stationary sites on microtubules, and syntaphilin tethers/immobilizes axonal mitochondria to microtubles at the stationary sites. This study compared mitochondrial function in wild-type axons with the one in syntaphilin-deficient axons. The volume of mitochondrial stationary sites was increased in demyelinated CNS axons in wild-type white matters. In contrast, although the size of mitochondrial stationary sites was similar in wild-type and syntaphilin-deficient myelinated axons, demyelinated syntaphilin-deficient axons exhibited similar size of mitochondrial stationary sites as myelinated syntaphilin-deficient axons. Correspondingly, compared to wild-type mice, larger axonal pathology was observed in the demyelinated corpus callosum of syntaphilin-deficient mice. Failure to increase mitochondrial volume in the syntaphilin-deficient demyelinated axons may reduce ATP production and increase axonal Na+, leading to augmented axonal degeneration. In fact, a Na+ channel blocker flecainide was beneficial for the survival of demyelinated syntaphilin-deficient axons. Taken together, therapeutic stabilization of mitochondria-microtuble docking interactions would prevent or reduce degeneration of demyelinated axons. Clinically effective neuroprotectants in the stroke field are not yet developed. However, the ideas described above propose novel therapeutic approaches in targeting mitochondria to protect neurons against ischemic insults. Future studies are warranted to pursue this direction in preclinical and clinical studies.

WldS and PGC-1α Regulate Mitochondrial Transport and Oxidation State after Axonal Injury

Mitochondria carry out many of the processes implicated in maintaining axon health or causing axon degeneration, including ATP and reactive oxygen species (ROS) generation, as well as calcium buffering and protease activation. Defects in mitochondrial function and transport are common in axon degeneration, but how changes in specific mitochondrial properties relate to degeneration is not well understood. Using cutaneous sensory neurons of living larval zebrafish as a model, we examined the role of mitochondria in axon degeneration by monitoring mitochondrial morphology, transport, and redox state before and after laser axotomy. Mitochondrial transport terminated locally after injury in wild-type axons, an effect that was moderately attenuated by expressing the axon-protective fusion protein Wallerian degeneration slow (WldS). However, mitochondrial transport arrest eventually occurred in WldS-protected axons, indicating that later in the lag phase, mitochondrial transport is not required for axon protection. By contrast, the redox-sensitive biosensor roGFP2 was rapidly oxidized in the mitochondrial matrix after injury, and WldS expression prevented this effect, suggesting that stabilization of ROS production may mediate axon protection. Overexpression of PGC-1α, a transcriptional coactivator with roles in both mitochondrial biogenesis and ROS detoxification, dramatically increased mitochondrial density, attenuated roGFP2 oxidation, and delayed Wallerian degeneration. Collectively, these results indicate that mitochondrial oxidation state is a more reliable indicator of axon vulnerability to degeneration than mitochondrial motility.

When Schwann Cells Conspire with Mitochondria, Neighboring Axons Are under Attack by Glia-Derived Neurotoxic Lipids

In this issue of Neuron, Viader etal. (2013) report on the production of toxic lipids by Schwann cells deficient in mitochondrial respiration, which are capable of destroying neighboring axons. This may have implications for understanding the neurobiology and treatment of mitochondrial neuropathies.

Doublecortin (Dcx) Family Proteins Regulate Filamentous Actin Structure in Developing Neurons

Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule-binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semiquantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, widespread differences are found in actin-associated proteins as compared with wild-type axons. Decreases in actin-binding proteins α-actinin-1 and α-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued by full-length Dcx and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C-terminal S/P-rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through α-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin-like kinase 1 deficiency is consistent with actin defect, as these axons are selectively deficient in axon guidance, but not elongation.

Intra-axonal calcium changes after axotomy in wild-type and slow Wallerian degeneration axons

Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt–Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (WldS) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4–24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from WldS rats. The step gradient of calcium distribution in WldS is absent in around 20% of the intact nerves beneath SLCs but 4–24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between WldS and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in WldS neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that WldS neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.

A DLK And JNK Dependent Axon Self- Destruction Program Promotes Wallerian Degeneration

A DLK And JNK Dependent Axon Self- Destruction Program Promotes Wallerian Degeneration

Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?

Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs). NFs are type IV intermediate filaments (IFs) that can be composed of four subunits, neurofilament heavy (NF-H), neurofilament medium (NF-M), neurofilament light (NF-L), and α-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP) repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations.

WldS prevents axon degeneration through increased mitochondrial flux and enhanced mitochondrial Ca2+ buffering.

Wld(S) (slow Wallerian degeneration) is a remarkable protein that can suppress Wallerian degeneration of axons and synapses, but how it exerts this effect remains unclear. Here, using Drosophila and mouse models, we identify mitochondria as a key site of action for Wld(S) neuroprotective function. Targeting the NAD(+) biosynthetic enzyme Nmnat to mitochondria was sufficient to fully phenocopy Wld(S), and Wld(S) was specifically localized to mitochondria in synaptic preparations from mouse brain. Axotomy of live wild-type axons induced a dramatic spike in axoplasmic Ca(2+) and termination of mitochondrial movement-Wld(S) potently suppressed both of these events. Surprisingly, Wld(S) also promoted increased basal mitochondrial motility in axons before injury, and genetically suppressing mitochondrial motility in vivo dramatically reduced the protective effect of Wld(S). Intriguingly, purified mitochondria from Wld(S) mice exhibited enhanced Ca(2+) buffering capacity. We propose that the enhanced Ca(2+) buffering capacity of Wld(S+) mitochondria leads to increased mitochondrial motility, suppression of axotomy-induced Ca(2+) elevation in axons, and thereby suppression of Wallerian degeneration.

Myelinated axons need a 4.1G connection

![Figure][1] In wild-type axons (top), potassium channels (white) localize along the juxtamesaxonal line (arrowhead), but this organization is lost when Schwann cells lack 4.1G (bottom). Ivanovic et al. report that a glial cytoskeletal adaptor protein organizes the membranes of Schwann

PINK1 and Parkin Target Miro for Phosphorylation and Degradation to Arrest Mitochondrial Motility

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson's disease proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin, but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome.

Neurons Help Bridge the Brain's Communication Gap

Neurons Help Bridge the Brain's Communication Gap

Research Highlights

Research Highlights

NMNAT expression in mitochondrial matrix delays Wallerian degeneration

Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt–Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (WldS) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3 mm of the lesion site by 4–24 h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from WldS rats. The step gradient of calcium distribution in WldS is absent in around 20% of the intact nerves beneath SLCs but 4–24 h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between WldS and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in WldS neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that WldS neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.

The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model

Glaucoma is a leading cause of blindness caused by progressive degeneration of retinal ganglion cells (RGCs) and their axons. The pathogenesis of glaucoma remains incompletely understood, but optic nerve (ON) axonal injury appears to be an important trigger of RGC axonal and cell body degeneration. Rat models are widely used in glaucoma research to explore pathogenic mechanisms and to test novel neuroprotective approaches. Here we investigated the mechanism of axon loss in glaucoma, studying axon degeneration in slow Wallerian degeneration (Wld(S)) rats after increasing intraocular pressure. Wld(S) delays degeneration of experimentally transected axons for several weeks, so it can provide genetic evidence for Wallerian-like degeneration in disease. As apoptosis is unaffected, Wld(S) also provides information on whether cell death results from axon degeneration or arises independently, an important question yet to be resolved in glaucoma. Having confirmed expression of Wld(S) protein, we found that Wld(S) delayed ON axonal degeneration in experimental rat glaucoma for at least 2 weeks, especially in proximal ON where wild-type axons are most severely affected. The duration of axonal protection is similar to that after ON transection and crush, suggesting that axonal degeneration in glaucoma follows a Wallerian-like mechanism. Axonal degeneration must be prevented for RGCs to remain functional, so pharmacologically mimicking and enhancing the protective mechanism of Wld(S) could offer an important route towards therapy. However, Wld(S) did not protect RGC bodies in glaucoma or after ON lesion, suggesting that combination treatments protecting both axons and cell bodies offer the best therapeutic prospects.

A Molecular Program for Contralateral Trajectory: Rig-1 Control by LIM Homeodomain Transcription Factors

Despite increasing evidence for transcriptional control of neural connectivity, how transcription factors regulate discrete steps in axon guidance remains obscure. Projection neurons in the dorsal spinal cord relay sensory signals to higher brain centers. Some projection neurons send their axons ipsilaterally, whereas others, commissural neurons, send axons contralaterally. We show that two closely related LIM homeodomain proteins, Lhx2 and Lhx9, are expressed by a set of commissural relay neurons (dI1c neurons) and are required for the dI1c axon projection. Midline crossing by dI1c axons is lost in Lhx2/9 double mutants, a defect that results from loss of expression of Rig-1 from dI1c axons. Lhx2 binds to a conserved motif in the Rig-1 gene, suggesting that Lhx2/9 regulate directly the expression of Rig-1. Our findings reveal a link between the transcriptional programs that define neuronal subtype identity and the expression of receptors that guide distinctive aspects of their trajectory.