Axon Outgrowth Defects Research Articles

Axonal Growth and Guidance Defects inFrizzled3Knock-Out Mice: A Comparison of Diffusion Tensor Magnetic Resonance Imaging, Neurofilament Staining, and Genetically Directed Cell Labeling

Previous work has identified axonal outgrowth and/or guidance defects in the brain and spinal cord of prenatal Frizzled3 (Fz3)(-/-) mice. To systematically explore the axonal defects in Fz3(-/-) mice and to compare techniques for the global assessment of axon tracts in the developing mouse, we have analyzed wild-type and Fz3(-/-) brains using (1) diffusion tensor magnetic resonance imaging (muDTI), (2) neurofilament staining, and (3) two genetically directed neuronal labeling methods. Confirming and extending the previous work of Wang et al. (2002), we find that the following structures/tracts are absent or greatly reduced in the Fz3(-/-) brain: the anterior commissure, cerebral peduncle (corticospinal tract), corpus callosum, fornix, internal capsule (thalamocortical and corticothalamic tracts), stria medullaris, stria terminalis, and hippocampal commissure. An aberrant U-shaped fiber bundle immediately caudal to the optic tract connects the left and right sides of the Fz3(-/-) thalamus and likely represents a default pathway for thalamic axons that failed to enter the internal capsule. At embryonic day 18, labeling of cortical pyramidal cells with a yellow fluorescent protein reporter reveals widespread fragmentation of axons with no apparent loss of pyramidal cell bodies. Fragmentation likely represents one stage in the process that normally eliminates stalled or mistargeted axons. This work demonstrates the usefulness of muDTI and genetically directed neuronal labeling for the analysis of nervous system defects in the mouse.

Regulatory machinery of UNC‐33 Ce‐CRMP localization in neurites during neuronal development in Caenorhabditis elegans

In Caenorhabditis elegans, unc-33 encodes an orthologue of the vertebrate collapsin response mediator protein (CRMP) family. We previously reported that CRMP-2 accumulated in the distal part of the growing axon of vertebrate neurons and played critical roles in axon elongation. unc-33 mutants show axonal outgrowth defects in several neurons. It has been reported that UNC-33 accumulates in neurites, whereas a missense mutation causes the mislocalization of UNC-33 from neurites to cell body, which suggests that the localization of UNC-33 in neurites is important for axonal outgrowth. However, it is unclear how UNC-33 accumulates in neurites and regulates neuronal development. In this study, to understand the regulatory mechanisms of localization of UNC-33 in neurites, we screened for the mutants that were involved in the localization of UNC-33, and identified three mutants: unc-14 (RUN domain protein), unc-51 (ULK kinase) and unc-116 (kinesin heavy chain). UNC-14 is known to associate with UNC-51. UNC-116 forms a complex with KLC-2 as Kinesin-1, a microtubule-dependent motor complex. We found that UNC-33 interacted with UNC-14 and KLC-2 in vivo. These results suggest that the UNC-14/UNC-51 complex and Kinesin-1 are involved in the localization of UNC-33 in neurites.

Novel Caenorhabditis elegans unc‐119 axon outgrowth defects correlate with behavioral phenotypes that are partially rescued by nonneural unc‐119

UNC-119 function is necessary for the correct development of the Caenorhabditis elegans nervous system. Worms mutant for unc-119 exhibit nervous system structural defects, including supernumerary axon branches, defasciculated nerve fibers, and choice point errors. Axons of both mechanosensory (ALM) and chemo- sensory (ASI) neurons have elongation defects within the nerve ring. Expressing unc-119 cDNA in mechanosensory neurons rescues the elongation defect of ALM axons, but expression in ASI neurons does not rescue ASI axon elongation defects. Neither gross movement nor dauer larva formation defects are rescued in either case. However, expressing a construct including introns under the control of the same promoters results in substantial rescue of phenotypic defects. In these cases reporter expression expands to tissues outside those specified by the promoter, notably into head muscles. Surprisingly, expressing an unc-119 cDNA construct under the control of a muscle-specific promoter fully rescues the dauer formation defect and substantially rescues movement. Thus, although UNC-119 normally acts in a cell-autonomous fashion, the cell-nonautonomous rescue of neural function suggests that it either acts at the cell surface or that it can be transported into the cell from the extracellular environment and play its normal role.

Mutations in Drosophila sec15 Reveal a Function in Neuronal Targeting for a Subset of Exocyst Components

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. We have isolated an exocyst component, sec15, in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. Our data suggest a role of Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions.

Brn-3a Transcription Factor Blocks p53-mediated Activation of Proapoptotic Target Genes Noxa and Bax in Vitro and in Vivo to Determine Cell Fate

The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a-/- embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a-/- embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.

Coordinated regulation of gene expression by Brn3a in developing sensory ganglia.

Mice lacking the POU-domain transcription factor Brn3a exhibit marked defects in sensory axon growth and abnormal sensory apoptosis. We have determined the regulatory targets of Brn3a in the developing trigeminal ganglion using microarray analysis of Brn3a mutant mice. These results show that Brn3 mediates the coordinated expression of neurotransmitter systems, ion channels, structural components of axons and inter- and intracellular signaling systems. Loss of Brn3a also results in the ectopic expression of transcription factors normally detected in earlier developmental stages and in other areas of the nervous system. Target gene expression is normal in heterozygous mice, consistent with prior work showing that autoregulation by Brn3a results in gene dosage compensation. Detailed examination of the expression of several of these downstream genes reveals that the regulatory role of Brn3a in the trigeminal ganglion appears to be conserved in more posterior sensory ganglia but not in the CNS neurons that express this factor.

A genetic screen for neurite outgrowth mutants in Caenorhabditis elegans reveals a new function for the F-box ubiquitin ligase component LIN-23.

Axon pathfinding and target recognition are highly dynamic and tightly regulated cellular processes. One of the mechanisms involved in regulating protein activity levels during axonal and synaptic development is protein ubiquitination. We describe here the isolation of several Caenorhabditis elegans mutants, termed eno (ectopic/erratic neurite outgrowth) mutants, that display defects in axon outgrowth of specific neuron classes. One retrieved mutant is characterized by abnormal termination of axon outgrowth in a subset of several distinct neuron classes, including ventral nerve cord motor neurons, head motor neurons, and mechanosensory neurons. This mutant is allelic to lin-23, which codes for an F-box-containing component of an SCF E3 ubiquitin ligase complex that was previously shown to negatively regulate postembryonic cell divisions. We demonstrate that LIN-23 is a broadly expressed cytoplasmically localized protein that is required autonomously in neurons to affect axon outgrowth. Our newly isolated allele of lin-23, a point mutation in the C-terminal tail of the protein, displays axonal outgrowth defects similar to those observed in null alleles of this gene, but does not display defects in cell cycle regulation. We have thus defined separable activities of LIN-23 in two distinct processes, cell cycle control and axon patterning. We propose that LIN-23 targets distinct substrates for ubiquitination within each process.

PAK4 kinase is essential for embryonic viability and for proper neuronal development.

The serine/threonine kinase PAK4 is a target for the Rho GTPase Cdc42 and has been shown to regulate cell morphology and cytoskeletal organization in mammalian cells. To examine the physiological and developmental functions of PAK4, we have disrupted the PAK4 gene in mice. The absence of PAK4 led to lethality by embryonic day 11.5, a result most likely due to a defect in the fetal heart. Striking abnormalities were also evident in the nervous systems of PAK4-deficient embryos. These embryos had dramatic defects in neuronal development and axonal outgrowth. In particular, spinal cord motor neurons and interneurons failed to differentiate and migrate to their proper positions. This is probably related to the role for PAK4 in the regulation of cytoskeletal organization and cell and/or extracellular matrix adhesion. PAK4-null embryos also had defects in proper folding of the caudal portion of the neural tube, suggesting an important role for PAK4 in neural tube development.

Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by a loss of α motoneurons in the spinal cord. SMA is caused by low levels of the ubiquitously expressed survival motor neuron (Smn) protein. As it is unclear how low levels of Smn specifically affect motoneurons, we have modeled SMA in zebrafish, a vertebrate model organism with well-characterized motoneuron development. Using antisense morpholinos to reduce Smn levels throughout the entire embryo, we found motor axon–specific pathfinding defects. Reduction of Smn in individual motoneurons revealed that smn is acting cell autonomously. These results show for the first time, in vivo, that Smn functions in motor axon development and suggest that these early developmental defects may lead to subsequent motoneuron loss.

Isolation of Rho GTPase effector pathways during axon development

The Rho GTPases Rac1 and Cdc42 have been implicated in the regulation of axon outgrowth and guidance. However, the downstream effector pathways through which these GTPases exert their effects on axon development are not well characterized. Here, we report that axon outgrowth defects within specific subsets of motoneurons expressing constitutively active Drosophila Rac1 largely persist even with the addition of an effector-loop mutation to Rac1 that disrupts its ability to bind to p21-activated kinase (Pak) and other Cdc42/Rac1 interactive-binding (CRIB)-motif effector proteins. While hyperactivation of Pak itself does not lead to axon outgrowth defects as when Rac1 is constitutively activated, live analysis reveals that it can alter filopodial activity within specific subsets of neurons similar to constitutive activation of Cdc42. Moreover, we show that the axon guidance defects induced by constitutive activation of Cdc42 persist even in the absence of Pak activity. Our results suggest that (1) Rac1 controls axon outgrowth through downstream effector pathways distinct from Pak, (2) Cdc42 controls axon guidance through both Pak and other CRIB effectors, and (3) Pak's primary contribution to in vivo axon development is to regulate filopodial dynamics that influence growth cone guidance.

Neurotrophins and Netrins Require Calcineurin/NFAT Signaling to Stimulate Outgrowth of Embryonic Axons

Axon outgrowth is the first step in the formation of neuronal connections, but the pathways that regulate axon extension are still poorly understood. We find that mice deficient in calcineurin-NFAT signaling have dramatic defects in axonal outgrowth, yet have little or no defect in neuronal differentiation or survival. In vitro, sensory and commissural neurons lacking calcineurin function or NFATc2, c3, and c4 are unable to respond to neurotrophins or netrin-1 with efficient axonal outgrowth. Neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons. These data indicate that the ability of these embryonic axons to respond to growth factors with rapid outgrowth requires activation of calcineurin/NFAT signaling by these factors. The precise parsing of signals for elongation turning and survival could allow independent control of these processes during development.

Neurons Are Dying for CREB

Two reports demonstrate the importance of signaling to the adenosine 3′-5′ monophosphate (cAMP) response-element-binding protein (CREB) for neuronal survival. Lonze et al. reported enhanced apoptosis and neuronal degeneration, as well as defects in axon growth in mouse embryos deficient for the Creb gene. Hardingham et al. discovered that in hippocampal neurons in culture two pathways can regulate CREB activity and that the extrasynaptic pathway that inhibits CREB activity can result in apoptosis. Analysis of the Creb −/− mice showed that by embryonic day 17.5 there was increased apoptosis and loss of peripheral neurons of the trigeminal ganglia, dorsal root ganglia (DRG), and superior cervical ganglia. At earlier times, neuron number and morphology of the DRG was similar to wild-type mouse DRG, which suggests that CREB is not required for the initial differentiation of sensory neurons, but is required for neuronal survival. Hardingham et al. monitored the phosphorylation of CREB and the expression of CRE-containing genes in response to activation of synaptic and extrasynaptic N -methyl-D-aspartate type of glutamate receptors. They found that activation of synaptic glutamate receptors (by evoked action potentials) stimulated CREB phosphorylation and CRE-dependent gene expression. However, activation of extrasynaptic receptors (by bath application of glutamate under conditions in which the synaptic receptors were inhibited) promoted CREB dephosphorylation and promoted apoptosis of the cells. Thus, the compartmentalized activation of glutamate receptor signaling can produce two opposing signals: one to activate CREB and to promote cell survival and one to inhibit CREB activity and to promote apoptosis. The implications of this dual signaling and the potential differences in these pathways during development are discussed by Riccio and Ginty. B. E. Lonze, A. Riccio, S. Cohen, D. D. Ginty, Apoptosis, axonal growth defects, and degeneration of peripheral neurons in mice lacking CREB. Neuron 34 , 371-385 (2002). [Online Journal] G. E. Hardingham, Y. Fukunaga, H. Bading, Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nature Neurosci. 5 , 405-414 (2002). [Online Journal] A. Riccio, D. D. Ginty, What a privilege to reside at the synapse: NMDA receptor signaling to CREB. Nature Neurosci. 5 , 389-390 (2002). [Online Journal]

Apoptosis, Axonal Growth Defects, and Degeneration of Peripheral Neurons in Mice Lacking CREB

CRE-binding protein (CREB) belongs to a family of transcription factors that mediates stimulus-dependent gene expression in neuronal and non-neuronal cells. Here we show that CREB is phosphorylated on its transcriptional regulatory site, Ser-133, in vivo in a neurotrophin-dependent manner. In mice harboring a null mutation in the Creb gene, sensory neurons exhibit excess apoptosis and degeneration, and display impaired axonal growth and projections. Interestingly, excess apoptosis is not observed in the central nervous system. CREB is required within sensory and sympathetic neurons for survival and axon extension since both of these neurotrophin-dependent processes are compromised in cultured neurons from CREB null mice. Thus, during their period of neurotrophin dependency, peripheral neurons require CREB-mediated gene expression for both survival and growth in vivo.

P190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation.

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. Here we identify the potent Rho regulatory protein, p190 RhoGAP (GTPase-activating protein), as the principal Src substrate detected in the developing and mature nervous system. We also find that mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and we find that p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, our results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase.

Defects in Sensory Axon Growth Precede Neuronal Death in Brn3a-Deficient Mice

Brn3a/Brn-3.0 is a POU-domain transcription factor expressed in primary sensory neurons of the cranial and dorsal root ganglia and in specific neurons in the caudal CNS. Mice lacking Brn3a undergo extensive sensory neural death late in gestation and die at birth. To further examine Brn3a expression and the abnormalities that accompany its absence, we constructed a transgene containing 11 kb of Brn3a upstream regulatory sequence linked to a LacZ reporter. Here we show that these regulatory sequences direct transgene expression specifically to Brn3a peripheral sensory neurons of the cranial and dorsal root ganglia. Furthermore, expression of the 11 kb/LacZ reporter in the sensory neurons of the mesencephalic trigeminal, but not other Brn3a midbrain neurons, demonstrates that cell-specific transgene expression is targeted to a functional class of neurons rather than to an anatomical region. We then interbred the 11 kb/LacZ reporter strain with mice carrying a null mutant allele of Brn3a to generate 11 kb/LacZ, Brn3a knock-out mice. beta-Galactosidase expression in these mice reveals significant axonal growth defects, including excessive and premature branching of the major divisions of the trigeminal nerve and a failure to correctly innervate whisker follicles, all of which precede sensory neural death in these mice. These defects in Brn3a(-/-) mice resemble strongly those seen in mice lacking the mediators of sensory pathfinding semaphorin 3A and neuropilin-1. Here we show, however, that sensory neurons are able to express neuropilin-1 in the absence of Brn3a.

Ciboulot Regulates Actin Assembly during Drosophila Brain Metamorphosis

A dynamic actin cytoskeleton is essential for the remodeling of cell shape during development, but the specific roles of many actin partners remain unclear. Here we characterize a novel actin binding protein, Ciboulot (Cib), which plays a major role in axonal growth during Drosophila brain metamorphosis. Loss of Cib function leads to axonal growth defects in the central brain, while overexpression of the gene during development leads to overgrown projections. The Cib protein displays strong sequence similarity to β-thymosins but has biochemical properties like profilin: the Cib-actin complex participates in actin filament assembly exclusively at the barbed end, and Cib enhances actin-based motility in vitro. Genetic experiments show that Cib and the Drosophila profilin protein Chickadee (Chic) cooperate in central brain metamorphosis.

DNA topoisomerase IIbeta and neural development.

DNA topoisomerase IIbeta is shown to have an unsuspected and critical role in neural development. Neurogenesis was normal in IIbeta mutant mice, but motor axons failed to contact skeletal muscles, and sensory axons failed to enter the spinal cord. Despite an absence of innervation, clusters of acetylcholine receptors were concentrated in the central region of skeletal muscles, thereby revealing patterning mechanisms that are autonomous to skeletal muscle. The defects in motor axon growth in IIbeta mutant mice resulted in a breathing impairment and death of the pups shortly after birth.

New ways to look at axons inCaenorhabditis elegans

In the nematode Caenorhabditis elegans, a well-established model organism for the analysis of nervous system development and function, nerve processes can be labelled in the intact animal with markers based on the "Green Fluorescent Protein" (GFP). The generation of GFP variants with improved brightness and modified emission spectra potentiated the use of this marker for in vivo labelling of subcellular structures. This made it possible to label different groups of neurons and their axons in the same animal with GFP variants of different spectral characteristics. Here I show with double labelling experiments that spatial relationships of axons in small axon bundles can now be resolved at the light microscopic level. In the future this will largely circumvent the need for time-consuming electron microscopic reconstructions to detect local defects in axon outgrowth. Furthermore, I demonstrate that neuronal processes can now be traced even in the head ganglia, an area of the nervous system that was previously almost inaccessible for analysis due to the compact arrangement of cell bodies and axons.

VP16-activation of the C. elegans neural specification transcription factor UNC-86 suppresses mutations in downstream genes and causes defects in neural migration and axon outgrowth.

The POU homeobox gene unc-86 specifies many neuroblast and neural fates in the developing C. elegans nervous system. Genes regulated by unc-86 are mostly unknown. Here we describe a genetic strategy for the identification of downstream pathways regulated by unc-86. We activate UNC-86 transcription activity by inserting the VP16 activation domain into an unc-86 genomic clone that bears all regulatory sequences necessary for normal expression in C. elegans. unc-86/VP16 complements unc-86 mutations in the specification of neuroblast and neural cell fates, but displays novel genetic activities: it can suppress non-null mutations in the downstream genes mec-3 and mec-7 that are necessary for mechanosensory neuron differentiation and function. These data suggest that UNC-86/VP16 increases the expression of mec-3 and mec-7 to compensate for the decreased activities of mutant MEC-3 or MEC-7 proteins. The suppression of mutations in downstream genes by an activated upstream transcription factor should be a general strategy for the identification of genes in transcriptional cascades. unc-86/VP16 also causes neural migration and pathfinding defects and novel behavioral defects. Thus, increased or unregulated expression of genes downstream of unc-86 can confer novel neural phenotypes suggestive of roles for unc-86-regulated genes in neural pathfinding and function. Genetic suppression of these unc-86/VP16 phenotypes may identify the unc-86 downstream genes that mediate these events in neurogenesis.

A Putative Cyclic Nucleotide–Gated Channel Is Required for Sensory Development and Function in C. elegans

In vertebrate visual and olfactory systems, a cyclic nucleotide–gated channel couples receptor activation to electrical activity of the sensory neurons. The Caenorhabditis elegans tax-2 gene is required for some forms of olfaction, for chemosensation of salts, and for thermosensation. We show here that tax-2 encodes a predicted subunit of a cyclic nucleotide–gated channel that is expressed in olfactory, gustatory, and thermosensory neurons, implicating this channel in multiple sensory modalities. Some sensory neurons display axon outgrowth defects in tax-2 mutants. Thus, the channel has an unexpected role in sensory neuron development in addition to its role in sensation. Consistent with this proposed dual function, a Tax-2::GFP fusion protein is present both in sensory cilia and in sensory axons.