Axon Outgrowth Defects Research Articles

Vincristine and bortezomib cause axon outgrowth and behavioral defects in larval zebrafish

Peripheral neuropathy is a common side effect of a number of pharmaceutical compounds, including several chemotherapy drugs. Among these are vincristine sulfate, a mitotic inhibitor used to treat a variety of leukemias, lymphomas, and other cancers, and bortezomib, a 26S proteasome inhibitor used primarily to treat relapsed multiple myeloma and mantle cell lymphoma. To gain insight into the mechanisms by which these compounds act, we tested their effects in zebrafish. Vincristine or bortezomib given during late embryonic development caused significant defects at both behavioral and cellular levels. Intriguingly, the effects of the two drugs appear to be distinct. Vincristine causes uncoordinated swimming behavior, which is coupled with a reduction in the density of sensory innervation and overall size of motor axon arbors. Bortezomib, in contrast, increases the duration and amplitude of muscle contractions associated with escape swimming, which is coupled with a preferential reduction in fine processes and branches of sensory and motor axons. These results demonstrate that zebrafish is a convenient in vivo assay system for screening potential pharmaceutical compounds for neurotoxic side effects, and they provide an important step toward understanding how vincristine and bortezomib cause peripheral neuropathy.

The POU transcription factor UNC‐86 controls the timing and ventral guidance of Caenorhabditis elegans axon growth

The in vivo mechanisms that coordinate the timing of axon growth and guidance are not well understood. In the Caenorhabditis elegans hermaphrodite specific neurons (HSNs), the lin-4 microRNA controls the stage of axon initiation independent of the UNC-40 and SAX-3 ventral guidance receptors. lin-4 loss-of-function mutants exhibit marked delays in axon outgrowth, while lin-4 overexpression leads to precocious growth in the L3 larval stage. Here, we show that loss of the POU transcription factor UNC-86 not only results in penetrant ventral axon growth defects in in the HSNs, but also causes processes to extend in the L1, three stages earlier than wild-type. This temporal shift is not dependent on UNC-40 or SAX-3, and does not require the presence of lin-4. We propose that unc-86(lf) HSN axons are misguided due to the temporal decoupling of axon initiation and ventral guidance responses.

ENU-3 is a novel motor axon outgrowth and guidance protein in C. elegans

During the development of the nervous system, the migration of many cells and axons is guided by extracellular molecules. These molecules bind to receptors at the tips of the growth cones of migrating axons and trigger intracellular signaling to steer the axons along the correct trajectories. We have identified a novel mutant, enu-3 (enhancer of Unc), that enhances the motor neuron axon outgrowth defects observed in strains of Caenorhabditis elegans that lack either the UNC-5 receptor or its ligand UNC-6/Netrin. Specifically, the double-mutant strains have enhanced axonal outgrowth defects mainly in DB4, DB5 and DB6 motor neurons. enu-3 single mutants have weak motor neuron axon migration defects. Both outgrowth defects of double mutants and axon migration defects of enu-3 mutants were rescued by expression of the H04D03.1 gene product. ENU-3/H04D03.1 encodes a novel predicted putative trans-membrane protein of 204 amino acids. It is a member of a family of highly homologous proteins of previously unknown function in the C. elegans genome. ENU-3 is expressed in the PVT interneuron and is weakly expressed in many cell bodies along the ventral cord, including those of the DA and DB motor neurons. We conclude that ENU-3 is a novel C. elegans protein that affects both motor axon outgrowth and guidance.

Forced Notch Signaling Inhibits Commissural Axon Outgrowth in the Developing Chick Central Nerve System

BackgroundA collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.Methodology/Principal FindingsIn this study, the neural tubes of HH10-11 chick embryos were in ovo electroporated with various Notch transgenes of activating or inhibiting Notch signaling, and then their effects on commissural axon outgrowth across the floor plate midline in the chick developing central nerve system were investigated. Our results showed that forced expression of Notch intracellular domain, constitutively active form of RBPJ, or full-length Hes1 in the rostral hindbrain, diencephalon and spinal cord at stage HH10-11 significantly inhibited commissural axon outgrowth. On the other hand, inhibition of Notch signaling by ectopically expressing a dominant-negative form of RBPJ promoted commissural axonal growth along the circumferential axis. Further results revealed that these Notch signaling-mediated axon outgrowth defects may be not due to the alteration of axon guidance since commissural axon marker TAG1 was present in the axons in floor plate midline, and also not result from the changes in cell fate determination of commissural neurons since the expression of postmitotic neuron marker Tuj1 and specific commissural markers TAG1 and Pax7 was unchanged.Conclusions/SignificanceWe first used an in vivo system to provide evidence that forced Notch signaling negatively regulates commissural axon outgrowth.

Are Presynaptic Proteins Predisposed to Self-Assemble?

Tight control of synapse formation ensures that neurons connect to appropriate targets. In this issue of Neuron, Klassen et al. identify ARL-8 GTPase as a regulator of presynaptic assembly. Without ARL-8, presynaptic material aggregates en route to its destination, suggesting that ARL-8 acts like a dispersant to prevent premature synaptic assembly in the axon.

Retrograde BMP Signaling Controls Synaptic Growth at the NMJ by Regulating Trio Expression in Motor Neurons

Retrograde signaling is essential for coordinating the growth of synaptic structures; however, it is not clear how it can lead to modulation of cytoskeletal dynamics and structural changes at presynaptic terminals. We show that loss of retrograde bone morphogenic protein (BMP) signaling at the Drosophila larval neuromuscular junction (NMJ) leads to a significant reduction in levels of Rac GEF Trio and a diminution of transcription at the trio locus. We further find that Trio is required in motor neurons for normal structural growth. Finally, we show that transgenic expression of Trio in motor neurons can partially restore NMJ defects in larvae mutant for BMP signaling. Based on our findings, we propose a model in which a retrograde BMP signal from the muscle modulates GTPase activity through transcriptional regulation of Rac GEF trio, thereby regulating the homeostasis of synaptic growth at the NMJ.

Motor Neuron Synapse and Axon Defects in a C. elegans Alpha-Tubulin Mutant

Regulation of microtubule dynamics underlies many fundamental cellular mechanisms including cell division, cell motility, and transport. In neurons, microtubules play key roles in cell migration, axon outgrowth, control of axon and synapse growth, and the regulated transport of vesicles and structural components of synapses. Loss of synapse and axon integrity and disruption of axon transport characterize many neurodegenerative diseases. Recently, mutations that specifically alter the assembly or stability of microtubules have been found to directly cause neurodevelopmental defects or neurodegeneration in vertebrates. We report here the characterization of a missense mutation in the C-terminal domain of C. elegans alpha-tubulin, tba-1(ju89), that disrupts motor neuron synapse and axon development. Mutant ju89 animals exhibit reduction in the number and size of neuromuscular synapses, altered locomotion, and defects in axon extension. Although null mutations of tba-1 show a nearly wild-type pattern, similar axon outgrowth defects were observed in animals lacking the beta-tubulin TBB-2. Genetic analysis reveals that tba-1(ju89) affects synapse development independent of its role in axon outgrowth. tba-1(ju89) is an altered function allele that most likely perturbs interactions between TBA-1 and specific microtubule-associated proteins that control microtubule dynamics and transport of components needed for synapse and axon growth.

The Guanine Exchange FactorvavControls Axon Growth and Guidance duringDrosophilaDevelopment

The Vav proteins are guanine exchange factors (GEFs) that trigger the activation of the Rho GTPases in general and the Rac family in particular. While the role of the mammalian vav genes has been extensively studied in the hematopoietic system and the immune response, there is little information regarding the role of vav outside of these systems. Here, we report that the single Drosophila vav homolog is ubiquitously expressed during development, although it is enriched along the embryonic ventral midline and in the larval eye discs and brain. We have analyzed the role that vav plays during development by generating Drosophila null mutant alleles. Our results indicate that vav is required during embryogenesis to prevent longitudinal axons from crossing the midline. Later on, during larval development, vav is required within the axons to regulate photoreceptor axon targeting to the optic lobe. Finally, we demonstrate that adult vav mutant escapers, which exhibit coordination problems, display axon growth defects in the ellipsoid body, a brain area associated with locomotion control. In addition, we show that vav interacts with other GEFs known to act downstream of guidance receptors. Thus, we propose that vav acts in coordination with other GEFs to regulate axon growth and guidance during development by linking guidance signals to the cytoskeleton via the modulation of Rac activity.

Argonaute2 Suppresses Drosophila Fragile X Expression Preventing Neurogenesis and Oogenesis Defects

Fragile X Syndrome is caused by the silencing of the Fragile X Mental Retardation gene (FMR1). Regulating dosage of FMR1 levels is critical for proper development and function of the nervous system and germ line, but the pathways responsible for maintaining normal expression levels are less clearly defined. Loss of Drosophila Fragile X protein (dFMR1) causes several behavioral and developmental defects in the fly, many of which are analogous to those seen in Fragile X patients. Over-expression of dFMR1 also causes specific neuronal and behavioral abnormalities. We have found that Argonaute2 (Ago2), the core component of the small interfering RNA (siRNA) pathway, regulates dfmr1 expression. Previously, the relationship between dFMR1 and Ago2 was defined by their physical interaction and co-regulation of downstream targets. We have found that Ago2 and dFMR1 are also connected through a regulatory relationship. Ago2 mediated repression of dFMR1 prevents axon growth and branching defects of the Drosophila neuromuscular junction (NMJ). Consequently, the neurogenesis defects in larvae mutant for both dfmr1 and Ago2 mirror those in dfmr1 null mutants. The Ago2 null phenotype at the NMJ is rescued in animals carrying an Ago2 genomic rescue construct. However, animals carrying a mutant Ago2 allele that produces Ago2 with significantly reduced endoribonuclease catalytic activity are normal with respect to the NMJ phenotypes examined. dFMR1 regulation by Ago2 is also observed in the germ line causing a multiple oocyte in a single egg chamber mutant phenotype. We have identified Ago2 as a regulator of dfmr1 expression and have clarified an important developmental role for Ago2 in the nervous system and germ line that requires dfmr1 function.

Caenorhabditits elegans LRK-1 and PINK-1 Act Antagonistically in Stress Response and Neurite Outgrowth

Mutations in two genes encoding the putative kinases LRRK2 and PINK1 have been associated with inherited variants of Parkinson disease. The physiological role of both proteins is not known at present, but studies in model organisms have linked their mutants to distinct aspects of mitochondrial dysfunction, increased vulnerability to oxidative and endoplasmic reticulum stress, and intracellular protein sorting. Here, we show that a mutation in the Caenorhabditits elegans homologue of the PTEN-induced kinase pink-1 gene resulted in reduced mitochondrial cristae length and increased paraquat sensitivity of the nematode. Moreover, the mutants also displayed defects in axonal outgrowth of a pair of canal-associated neurons. We demonstrate that in the absence of lrk-1, the C. elegans homologue of human LRRK2, all phenotypic aspects of pink-1 loss-of-function mutants were suppressed. Conversely, the hypersensitivity of lrk-1 mutant animals to the endoplasmic reticulum stressor tunicamycin was reduced in a pink-1 mutant background. These results provide the first evidence of an antagonistic role of PINK-1 and LRK-1. Due to the similarity of the C. elegans proteins to human LRRK2 and PINK1, we suggest a common role of both factors in cellular functions including stress response and regulation of neurite outgrowth. This study might help to link pink-1/PINK1 and lrk-1/LRRK2 function to the pathological processes resulting from Parkinson disease-related mutants in both genes, the first manifestations of which are cytoskeletal defects in affected neurons.

Pleiotropic effects of spastin on neurite growth depending on expression levels

Hereditary spastic paraplegia (HSP) is characterized by weakness and spasticity of the lower limbs, owing to degeneration of corticospinal axons. The most common form is due to heterozygous mutations in the SPG4 gene, encoding spastin, a microtubule (MT)-severing protein. Here, we show that neurite growth in immortalized and primary neurons responds in pleiotropic ways to changes in spastin levels. Spastin depletion alters the development of primary hippocampal neurons leading to abnormal neuron morphology, dystrophic neurites, and axonal growth defects. By live imaging with End-Binding Protein 3-Fluorescent Green Protein (EB3-GFP), a MT plus-end tracking protein, we ascertained that the assembly rate of MTs is reduced when spastin is down-regulated. Spastin over-expression at high levels strongly suppresses neurite maintenance, while slight spastin up-regulation using an endogenous promoter enhances neurite branching and elongation. Spastin severing activity is exerted preferentially on stable acetylated and detyrosinated MTs. We further show that SPG4 nonsense or splice site mutations found in hereditary spastic paraplegia patients result in reduced spastin levels, supporting haploinsufficiency as the molecular cause of the disease. Our study reveals that SPG4 is a dosage-sensitive gene, and broadens the understanding of the role of spastin in neurite growth and MT dynamics.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Oxygen deprived (The zebrafish embryo as a dynamic model of anoxia tolerance by Bryce A. Mendelsohn, Bethany L. Kassebaum, and Jonathan D. Gitlin, Dev Dyn 237:1780–1788; and Coordination of development and metabolism in the pre-midblastula transition zebrafish embryo by Bryce A. Mendelsohn and Jonathan D. Gitlin, Dev Dyn 237:1789–1798) It is hard to grow when you cannot breath—consequently, zebrafish embryos postpone development in an anoxic environment. Depending on the embryonic stage and the metabolic state, development proceeds when oxygen becomes available. The authors characterized this intriguing tolerance phenomenon in back-to-back studies. One question addressed is, how might anoxia trigger arrest? Mendelsohn, Kassebaum, and Gitilin tested if energy homeostasis was involved. The 24-hour-old embryos were incubated in normal oxygen with chemicals such as cyanide that block oxidative phosphorylation—causing a shift from aerobic to anaerobic energy production. This shift, leads to developmental arrest similar to that seen under anoxic conditions. What mechanism is involved in sensing the change in energy metabolism? One contender is the energy sensing AMP activated protein kinase (AMPK) pathway. Consistent with this supposition, the authors show, using phosphorylation assays, that AMPK and downstream factors are activated in embryos exposed to anoxia or inhibitors of oxidative phosphorylation. In their second study, Mendelsohn and Gitilin show that younger embryos also stall development upon chemical induced anoxia. Developmental arrest was initiated before the mid blastula transition, when maternal components rule, revealing that in this case zygotic transcription is not required. Maternal AMPK is activated by anoxia, suggesting a conservation of response between young and old embryos. The AMPK pathway regulates essential processes including translation, cell cycle control, and glycolysis that are required for development to proceed making the pathway a good candidate for the link from energy sensing to anoxia tolerance. Sympathetic development (Loss of the Prader-Willi Syndrome protein necdin causes defective migration, axonal outgrowth, and survival of embryonic sympathetic neurons by Alysa A. Tennese, Christopher B. Gee, and Rachel Wevrick, Dev Dyn 237:1935–1943) Prader-Willi syndrome is a neurodevelopmental disorder with multiple symptoms including low muscle tone, learning disability, excessive hunger leading to obesity, and difficulty swallowing. The disease is caused by deletion of several imprinted genes on chromosome 15. One, Ndn, encodes necdin—a protein previously shown in mice to be required for normal development of sensory and central neurons. Tennese, Gee, and Wevrick now show that Ndn knock out mice also have developmental defects in a subset of sympathetic neurons, primarily in the superior cervical ganglia (SCG), which innervate salivary, parotid, submandibular glands, and nasal mucosa. Phox2B and tyrosine hydroxylase mark sympathetic neurons, and were used to follow SCG during proliferation and migration. They show that a subset of SCG neurons failed to migrate to their final rostral position between vertebral levels C1 and C4. Moreover, SCG neurons were reduced in size and number, which corresponds to an observed increase in cell death. Finally, defects in SCG axon outgrowth and neuronal morphology likely contribute to an observed reduction in innervation of target tissues. Some Prader-Willi patients have difficulty swallowing due to decreased saliva production, suggesting an analogous disruption could be the cause. Exit the pool (Effect of canonical Wnt inhibition in the neurogenic cortex, hippocampus, and premigratory dentate gyrus progenitor pool by Nina Solberg, Ondrej Machon, and Stefan Krauss, Dev Dyn 237:1799–1811) Canonical Wnt signaling, through β-catenin, is required for hippocampal and dentate gyrus (DG) development. But its precise role in the development of these tissues is not clear. Just before formation of DG progenitor cells, Solberg, Machon, and Krauss, decreased Wnt signaling in mice by driving expression of the inhibitor, Dickkopf1 (Dkk1) in the developing cortex and hippocampus. They found, similar to conditional mutants in Wnt3A and β-catenin, that there was a diminution of the hippocampus and a severe reduction in granule cells of the DG. Furthermore, they determined that reduced cell number in the DG was caused by an increase in cell cycle length and a decrease in proliferation of the progenitor pool. Importantly, adults lack two DG structures, the internal blade and part of the external blade. This finding shows that there was no compensation as development proceeded, suggesting the progenitor pool was depleted during embryogenesis.

Plastin 3 is a protective modifier of autosomal recessive spinal muscular atrophy.

Homozygous deletion of the survival motor neuron 1 gene (SMN1) causes spinal muscular atrophy (SMA), the most frequent genetic cause of early childhood lethality. In rare instances, however, individuals are asymptomatic despite carrying the same SMN1 mutations as their affected siblings, thereby suggesting the influence of modifier genes. We discovered that unaffected SMN1-deleted females exhibit significantly higher expression of plastin 3 (PLS3) than their SMA-affected counterparts. We demonstrated that PLS3 is important for axonogenesis through increasing the F-actin level. Overexpression of PLS3 rescued the axon length and outgrowth defects associated with SMN down-regulation in motor neurons of SMA mouse embryos and in zebrafish. Our study suggests that defects in axonogenesis are the major cause of SMA, thereby opening new therapeutic options for SMA and similar neuromuscular diseases.

Movement disorder and neuromuscular change in zebrafish embryos after exposure to caffeine

Though caffeine is broadly distributed in many plants and foods, little is known about the teratogenic effects of caffeine during early embryonic development. Here, we used zebrafish as a model to test toxicity and teratogenicity since they have transparent eggs, making the organogenesis of zebrafish embryos easier to observe. When the exposure doses of caffeine were less than 150 ppm (17.5, 35, 50, 100 and 150 ppm), the zebrafish embryos exhibited no significant differences in survival rates after comparison with vehicle-control (0 ppm) group. As the exposure dosages increased, the survival rates decreased. No embryos survived after treatment with 300 ppm caffeine or higher dosages. The most evident change in embryos treated with caffeine was a shorter body length (vehicle-control: 3.26 ± 0.01 mm, n = 49; vs 150 ppm of caffeine: 2.67 ± 0.03 mm, n = 50). In addition, caffeine-treated embryos exhibited significantly reduced tactile sensitivity frequencies of touch-induced movement (vehicle-control: 9.93 ± 0.77 vs 17.5–150 ppm caffeine: 5.37 ± 0.52–0.10 ± 0.06). Subtle changes are easily observed by staining with specific monoclonal antibodies F59, Znp1 and Zn5 to detect morphological changes in muscle fibers, primary motor axons and secondary motor axon projections, respectively. Our data show that the treatment of caffeine leads to misalignment of muscle fibers and motor neuron defects, especially secondary motor neuron axonal growth defects.

The SMN binding protein gemin2 is not involved in motor axon outgrowth

A paramount question in spinal muscular atrophy (SMA) research is why reduced levels of SMN, a ubiquitously expressed protein, leads to a motoneuron-specific disease. It has been hypothesized that SMN may have a dual function: a role in snRNP assembly and a novel function that affects axons. We have previously shown that decreasing Smn levels in zebrafish causes defects in motor axon outgrowth. To determine whether decreasing other components of the snRNP complex would also cause motor axon defects, we knocked down Gemin2, a SMN binding protein involved in snRNP assembly. Moderate knockdown of Gemin2 yields a large percentage of morphologically abnormal embryos with shortened trunks and overall delayed development. Examination of motor axons revealed that only embryos with abnormal body morphology had aberrant motor axons indicating that the motor axon defects are secondary to the overall body defects observed in these embryos. To directly test this, we knocked down Gemin2 specifically in motoneurons using two separate approaches and found that motor axons developed normally. Furthermore, wild-type neurons transplanted into morphologically abnormal gemin2 morphants had aberrant motor axons indicating that the motor axon defects observed when Gemin2 is decreased are secondary to the defects in body morphology. These data show that reduction of Gemin2, unlike reduction of SMN, in zebrafish embryos does not directly cause motor axon outgrowth defects. Since Gemin2 and SMN both function in snRNP biogenesis yet only SMN knockdown causes motor axon defects, these data are consistent with an additional role for SMN that is snRNP independent.

HSPC300 and its role in neuronal connectivity

BackgroundThe WAVE/SCAR complex, consisting of CYFIP (PIR121 or Sra1), Kette (Nap1), Abi, SCAR (WAVE) and HSPC300, is known to regulate the actin nucleating Arp2/3 complex in a Rac1-dependent manner. While in vitro and in vivo studies have demonstrated that CYFIP, Kette, Abi and SCAR work as subunits of the complex, the role of the small protein HSPC300 remains unclear.ResultsIn the present study, we identify the HSPC300 gene and characterize its interaction with the WAVE/SCAR complex in the Drosophila animal model. On the basis of several lines of evidence, we demonstrate that HSPC300 is an indispensable component of the complex controlling axonal and neuromuscular junction (NMJ) growth. First, the Drosophila HSPC300 expression profile resembles that of other members of the WAVE/SCAR complex. Second, HSPC300 mutation, as well as mutations in the other complex subunits, results in identical axonal and NMJ growth defects. Third, like with other complex subunits, defects in NMJ architecture are rescued by presynaptic expression of the respective wild-type gene. Fourth, HSPC300 genetically interacts with another subunit of the WAVE/SCAR complex. Fifth, HSPC300 physically associates with CYFIP and SCAR.ConclusionPresent data provide the first evidence for HSPC300 playing a role in nervous system development and demonstrate in vivo that this small protein works in the context of the WAVE/SCAR complex.

Stat5 constitutive activation rescues defects in spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is a motor neuron degeneration disorder for which there is currently no effective treatment. Here, we report three compounds (sodium vanadate, trichostatin A and aclarubicin) that effectively enhance SMN2 expression by inducing Stat5 activation in SMA-like mouse embryonic fibroblasts and human SMN2-transfected NSC34 cells. We found that Stat5 activation enhanced SMN2 promoter activity with increase in both full-length and deletion exon 7 SMN transcripts in SMN2-NSC34 cells. Knockdown of Stat5 expression disrupted the effects of sodium vanadate on SMN2 activation but did not influence SMN2 splicing, suggesting that Stat5 signaling is involved in SMN2 transcriptional regulation. In addition, constitutive activation of Stat5 mutant (Stat5A1*6) profoundly increased the number of nuclear gems in SMA-patient lymphocytes and reduced SMA-like motor neuron axon outgrowth defects. These results demonstrate that Stat5 signaling could be a possible pharmacological target for treating SMA.

Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks

The Drosophila element Mos1 is a class II transposon, which moves by a 'cut-and-paste' mechanism and can be experimentally mobilized in the Caenorhabditis elegans germ line. Here, we triggered the excision of identified Mos1 insertions to create chromosomal breaks at given sites and further manipulate the broken loci. Double-strand break (DSB) repair could be achieved by gene conversion using a transgene containing sequences homologous to the broken chromosomal region as a repair template. Consequently, mutations engineered in the transgene could be copied to a specific locus at high frequency. This pathway was further characterized to develop an efficient tool--called MosTIC--to manipulate the C. elegans genome. Analysis of DSB repair during MosTIC experiments demonstrated that DSBs could also be sealed by end-joining in the germ line, independently from the evolutionarily conserved Ku80 and ligase IV factors. In conjunction with a publicly available Mos1 insertion library currently being generated, MosTIC will provide a general tool to customize the C. elegans genome.

The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo

Hereditary spastic paraplegia (HSP) is a collection of neurological disorders characterized by developmental failure or degeneration of motor axons in the corticospinal tract and progressive lower limb spasticity. SPG4 mutations are the most common cause of autosomal dominant HSP and Spastin (the SPG4 gene product) is a microtubule severing protein that shares homology with katanin, the microtubule severing activity of which promotes axon growth in cultured neurons. Given the sequence and functional similarity between spastin and katanin, we hypothesized that spastin promotes the dynamic disassembly and remodelling of microtubules required for robust, properly directed motor axon outgrowth. To investigate this hypothesis, we cloned the zebrafish spg4 orthologue and used morpholino antisense oligonucleotides directed against the translation start site and the intron 7-8 splice donor site to knock down spastin function in the developing zebrafish embryo. Reduced spg4 function caused dramatic defects in motor axon outgrowth without affecting the events driving the initial specification of motor neurones. Other neuronal subtypes also exhibited a requirement for spg4 function, since spg4 knock down caused both widespread defects in neuronal connectivity and extensive CNS-specific apoptosis. Our results reveal a critical requirement for spastin to promote axonal outgrowth during embryonic development, and they validate the zebrafish embryo as a novel model system to dissect the pathogenetic mechanisms underlying HSP. Taken together with other recent studies, our findings suggest that axon outgrowth defects may be a common feature of childhood SPG3A and SPG4 cases.

Regulation of FGF10 by POU transcription factor Brn3a in the developing trigeminal ganglion

The POU-domain transcription factor Brn3a is expressed in specific neurons of the caudal CNS and peripheral sensory nervous system. The sensory neurons of mice lacking Brn3a exhibit marked defects in axon growth and extensive apoptosis in late gestation. Here we show that expression of the developmental regulator FGF10 is approximately 35-fold increased in the developing trigeminal ganglia of Brn3a-null mice. In order to determine whether FGF10 regulates other changes in gene expression observed in Brn3a knock-out ganglia, we have used a sensory-specific enhancer to over-express FGF10 in transgenic mice. Microarray analysis of trigeminal ganglia from individual transgenic founders effectively excludes the cell-autonomous activity of FGF10 as a mechanism for mediating the downstream effects of the loss of Brn3a, probably because developing sensory neurons lack the appropriate type of FGF receptor.