Abnormal Axon Research Articles

Abnormal axon guidance signals and reduced interhemispheric connection via anterior commissure in neonates of marmoset ASD model

In autism spectrum disorder (ASD), disrupted functional and structural connectivity in the social brain has been suggested as the core biological mechanism underlying the social recognition deficits of this neurodevelopmental disorder. In this study, we aimed to identify genetic and neurostructural abnormalities at birth in a non-human primate model of ASD, the common marmoset with maternal exposure to valproic acid (VPA), which has been reported to display social recognition deficit in adulthood. Using a comprehensive gene expression analysis, we found that 20 genes were significantly downregulated in VPA-exposed neonates. Of these, Frizzled3 (FZD3) and PIK3CA were identified in an axon guidance signaling pathway. FZD3 is essential for the normal development of the anterior commissure (AC) and corpus callosum (CC); hence, we performed diffusion tensor magnetic resonance imaging with a 7-Tesla scanner to measure the midsagittal sizes of these structures. We found that the AC size in VPA-exposed neonates was significantly smaller than that in age-matched controls, while the CC size did not differ. These results suggest that downregulation of the genes related to axon guidance and decreased AC size in neonatal primates may be linked to social brain dysfunctions that can happen later in life.

Asxl1 ablation in mouse embryonic stem cells impairs neural differentiation without affecting self-renewal

Additional sex comb-like1 (Asxl1) is known as a chromatin modulator that plays dual functions in transcriptional regulation depending on the cell type. Recent studies using Asxl1 knockout mice revealed that Asxl1 is important for the proliferation and differentiation of hematopoietic progenitor cells, and the development of organs. Although we previously reported Asxl1 as a Sox2 target gene, its function in embryonic stem cells (ESCs) remains largely unknown. For this purpose, we isolated ESCs from the blastocyst inner cell mass of Asxl1−/− mice. Asxl1 deficiency in ESCs exhibited no effect on cell proliferation, expression of core pluripotent transcription factors, or alkaline phosphatase activity, suggesting dispensability of Asxl1 for self-renewal of ESCs. By contrast, the differentiation of Asxl1−/− ESCs was significantly affected as shown by size reductions of embryoid bodies accompanied with apoptosis, aberrant expression of differentiation genes, downregulation of bivalent neurogenesis genes, and abnormal axon formation in neurons. Overall, our findings indicated that Asxl1 played a critical role in regulating genes associated with neural differentiation without affecting self-renewal of mouse ESCs.

Exposure to ZnO nanoparticles alters neuronal and vascular development in zebrafish: Acute and transgenerational effects mitigated with dissolved organic matter

Exposure to ZnO-nanoparticles (NPs) in embryonic zebrafish reduces hatching rates which can be mitigated with dissolved organic material (DOM). Although hatching rate can be a reliable indicator of toxicity and DOM mitigation potential, a fish that has been exposed to ZnO-NPs or any other toxicant may also exhibit other abnormal phenotypes not readily detected by the unaided eye. In this study, we moved beyond hatching rate analysis to investigate the consequences of ZnO-NPs exposure on the nervous and vascular systems in developing zebrafish. Zebrafish exposed to ZnO-NPs (1–100 ppm) exhibited an array of cellular phenotypes including: abnormal secondary motoneuron (SMN) axonal projections, abnormal dorsal root ganglion development and abnormal blood vessel development. Dissolved Zn (<10 kDa) exposure also caused abnormal SMN axonal projections, but to a lesser extent than ZnO-NPs. The ZnO-NPs-induced abnormal phenotypes were reversed in embryos concurrently exposed with various types of DOM. In these acute mitigation exposure experiments, humic acid and carbohydrate, along with natural organic matter obtained from the Suwannee River in Georgia and Milwaukee River in Wisconsin, were the best mitigators of ZnO-NPs-induced motoneuron toxicity at 96 h post fertilization. Further experiments were performed to determine if the ZnO-NPs-induced, abnormal axonal phenotypes and the DOM mitigated axonal phenotypes could persist across generations. Abnormal SMN axon phenotypes caused by ZnO-NPs-exposure were detected in F1 and F2 generations. These are fish that have not been directly exposed to ZnO-NPs. Fish mitigated with DOM during the acute exposure (F0 generation) had a reduction in abnormal motoneuron axon errors in larvae of subsequent generations. Therefore, ZnO-NPs exposure results in neurotoxicity in developing zebrafish which can persist from one generation to the next. Mitigation with DOM can reverse the abnormal phenotypes in an acute embryonic exposure context, as well as across generations, resulting in healthy fish.

The Sonic Hedgehog signaling pathway regulates inferior alveolar nerve regeneration

Activation of Shh signaling is known to be observed following injury of the peripheral nerves such as the sciatic nerve. However, the precise role of Shh signaling during peripheral nerve regeneration is not fully understood. The inferior alveolar nerve (IAN) is most commonly injured during oral surgery. Unlike the sciatic nerve, the IAN is isolated from other craniofacial tissues, as it resides in a long bony canal within the mandible. The IAN is thus an excellent experimental model for investigating peripheral nerve regeneration. In this study, the role of Shh signaling in peripheral nerve regeneration was investigated using the mouse IAN transection model. During regeneration, Shh signaling was activated within the entire distal region of the IAN and proximal stumps. Inhibition of Shh signaling by cyclopamine application at the transection site led to abnormal axon growth in random directions, a reduced number of macrophages, and an increase in myelin debris within the distal region. Shh signaling is thus involved in peripheral nerve regeneration via the regulation of myelin degradation.

3-2-01. Decreased motor axonal potassium currents in peripheral nerve hyperexcitability syndrome with negativity for anti-VGKC antibody and positivity for anti-CRMP5 antibody

Peripheral nerve hyperexcitability (PNH) syndromes are caused by spontaneous discharges originating from motor axons. Antibodies against voltage-gated potassium channels (VGKC) are detected in some patients with PNH syndrome, but the cause in the remaining patients has not yet been clarified. The index patient was a 46-year-old female with recurred thymoma and myasthenia gravis. Six month after the start of chemotherapy, she developed myokymia, fasciculations and muscle cramps in both her lower limbs. A nerve conduction studies revealed mild axonal polyneuropathy and a motor F-wave examination showed marked after-discharges. Needle electromyography showed fasciculation potentials and myokymic discharges. From these results, the patient was diagnosed with PNH syndrome. Nerve excitability study showed extremely greater supernormality in the recovery cycle and greater changes in depolarizing threshold electrotonus than control, suggesting decreased slow potassium currents in the motor axons. Serological tests revealed the absence of anti-VGKC antibody and the presence of anti-collapsin response mediator protein 5 (CRMP5) antibody. A recent study reported that CRMP5 plays an important role in axon–Schwann cell cooperation during development and nerve regeneration. The coexistence of nerve regeneration and anti-CRMP5 antibody may cause abnormal axon–Schwann cell interaction, which would result in slow potassium channel dysfunction and associated symptoms such as myokymia.

Regulation of mitochondria-dynactin interaction and mitochondrial retrograde transport in axons.

Mitochondrial transport in axons is critical for neural circuit health and function. While several proteins have been found that modulate bidirectional mitochondrial motility, factors that regulate unidirectional mitochondrial transport have been harder to identify. In a genetic screen, we found a zebrafish strain in which mitochondria fail to attach to the dynein retrograde motor. This strain carries a loss-of-function mutation in actr10, a member of the dynein-associated complex dynactin. The abnormal axon morphology and mitochondrial retrograde transport defects observed in actr10 mutants are distinct from dynein and dynactin mutant axonal phenotypes. In addition, Actr10 lacking the dynactin binding domain maintains its ability to bind mitochondria, arguing for a role for Actr10 in dynactin-mitochondria interaction. Finally, genetic interaction studies implicated Drp1 as a partner in Actr10-dependent mitochondrial retrograde transport. Together, this work identifies Actr10 as a factor necessary for dynactin-mitochondria interaction, enhancing our understanding of how mitochondria properly localize in axons.

Physiological Exploration of the Long Term Evolutionary Selection against Expression of N-Glycolylneuraminic Acid in the Brain

All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specific Cmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gc-preferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens.

A Local Inflammatory Renin-Angiotensin System Drives Sensory Axon Sprouting in Provoked Vestibulodynia.

This study provides evidence that local inflammation leads to angiotensin II formation, which acts on the AT2 to induce nociceptor axon sprouting in vulvodynia. Preventing inflammation and blocking AT2 therefore present potential pharmacological strategies for reducing vestibular pain.

Two novel variants in CNTNAP1 in two siblings presenting with congenital hypotonia and hypomyelinating neuropathy.

Homozygous frameshift variants in CNTNAP1 have recently been reported in patients with arthrogryposis and abnormal axon myelination. In two brothers with severe congenital hypotonia and foot deformities, we identified compound heterozygous variants in CNTNAP1, reporting the first causative missense variant, p.(Cys323Arg). Motor nerve conductions were markedly decreased. Nerve microscopical lesions confirmed a severe hypomyelinating process and showed loss of attachment sites of the myelin loops on the axons, which could be a characteristic of Caspr loss-of-function. We discuss the pathophysiology of the myelination process and we propose to consider this disorder as a congenital hypomyelinating neuropathy.

Residual neurogenesis in chronically epileptic hippocampus of mice

Abnormal hippocampal neurogenesis after acute seizures has been well addressed. However, whether newly generated cells continued to be disturbed even they were born in the chronic stage after pilocarpine-induce status epilepticus has remained elusive. Labeling dividing progenitors and their progeny with retroviral vector expressing green fluorescent protein or proliferation marker 5-bromo-2′-deoxyuridine at 3 months post pilocarpine-induced status epilepticus in mice, a spot of newly born neurons exhibiting hilar ectopic location (4.57±2.3%), aberrant basal dendrites (8.09±1.5%) and abnormal axon sprouting into inner molecular layer of dentate gyrus was identified when examined 6 weeks later. No significant difference on the amount of mossy fiber sprouting was found when cohorts of newborn cells were eliminated by methylazoxymethanol acetate injection initiated at 3 months after SE, suggesting that adult generated neurons in the chronically epileptic hippocampus don’t contribute a lot to the mossy fiber sprouting. These results indicated that the aberrant neurogenesis in the chronically epileptic hippocampus occurs only in a small population of newly generated granule cells.

Presynaptic protein Synaptotagmin1 regulates the neuronal polarity and axon differentiation in cultured hippocampal neurons.

BackgroundHippocampal neurons in the brain polarize to form multiple dendrites and one long axon. The formation of central synapses remains poorly understood. Although several of the intracellular proteins involved in the clustering of central neurotransmitter receptors and ion channels have been identified, the signals involved in pre- and postsynaptic differentiation remain elusive. Synaptotagmin1 is an abundant and important presynaptic vesicle protein that binds Ca2+ (J Biol Chem 277:7629–7632, 2002) in regulation of synaptic vesicle exocytosis at the synapse. Synapse consists of the formation of synaptic connections and requires precise coordination of Synaptotagmin1. It was reported Synaptotagmin1 plays an important roles in the formation of axonal filopodia and branches in chicken forebrain neurons (Dev Neurobiol 73:27–44, 2013). To determine if Synaptotagmin1 could have a role in formation of axon in hippocampal neurons, we investigated the effects of Synaptotagmin1 overexpression and knockdown using the shRNA on the growth and branching of the axons of primary hippocampal neurons. We showed that overexpression of Synaptotagmin1 leads to abnormal multiple axon formation in cultured rat hippocampal neurons.ResultsWe first examined the effects of Synaptotagmin1 on the numbers of axon and dendrites. We found that the overexpression of Synaptotagmin1 led to the formation of multiple axons and induced an increase in the number of endogenous postsynaptic protein Homer1c clusters in cultured hippocampal neurons. Endogenous initial segment of axon was detected with anti-sodium channel (anti-NaCh) antibody and with anti-Tau1 (J Neurosci 24: 4605–4613, 2004). The endogenous initial segment of axon was stained with anti-NaCh antibodies and with anti-Tau1 antibodies. Then the numbers of prominence dyed positive were counted as axon. We attempted to specifically knockdown the endogenous Synaptotagmin1 with small hairpin RNAs (shRNAs). To further dissect the functions of endogenous Synaptotagmin1 in neuronal polarity, we used the shRNA of Synaptotagmin1 that specifically blocks the existence of endogenous Synaptotagmin1. When the shRNA of Synaptotagmin1 was introduced to the cells, the number of axons and dendrites did not change.ConclusionsThese results indicate that the accumulation of Synaptotagmin1 may play an important role in axon/dendrite differentiation.

Coexistence of Charcot Marie Tooth disease type 1A and diabetes in Taiwan: A clinicopathological study

BackgroundCharcot Marie Tooth disease type 1A (CMT1A) is the most commonly inherited demyelinating polyneuropathy with variable phenotypes, affected by several comorbidities, especially diabetes mellitus (DM). Previous studies showed that DM exacerbates the clinical manifestations of CMT1A. Patients and methodsWe retrospectively evaluated patients with CMT1A in our hospital, and identified three groups among 12 cases, which comprised four patients with CMT1A, four with CMT1A+DM, and four with DM. We reviewed the CMT neuropathy score (CMTNS), electrophysiological data, and histomorphological parameters of the sural nerve, including fiber density, myelin thickness, axon diameter, g-ratio, regenerative clusters, and regeneration ratio. ResultsThe CMTNS was significantly higher in patients with CMT1A+DM (21.5±2.52) than in those with CMT1A only (10.8±4.4; p=0.03). Pathological findings in patients with CMT1A+DM included a significant decrease of myelinated fiber density (p=0.02) and reduction in the regenerative ratio (p=0.01), indicating severe degeneration with impaired regeneration. In non-parametric analyses, DM was found to play a more important role than CMT1A in influencing nerve degeneration and regeneration. ConclusionsIn patients with CMT1A, DM exacerbated clinical and pathological manifestations including increased loss of myelinated fibers, abnormal axon–myelin interaction, and impaired nerve regeneration.

Activating the translational repressor 4E-BP or reducing S6K-GSK3β activity prevents accelerated axon growth induced by hyperactive mTOR in vivo.

Abnormal axonal connectivity and hyperactive mTOR complex 1 (mTORC1) are shared features of several neurological disorders. Hyperactive mTORC1 alters axon length and polarity of hippocampal neurons in vitro, but the impact of hyperactive mTORC1 on axon growth in vivo and the mechanisms underlying those effects remain unclear. Using in utero electroporation during corticogenesis, we show that increasing mTORC1 activity accelerates axon growth without multiple axon formation. This was prevented by counteracting mTORC1 signaling through p70S6Ks (S6K1/2) or eukaryotic initiation factor 4E-binding protein (4E-BP1/2), which both regulate translation. In addition to regulating translational targets, S6K1 indirectly signals through GSK3β, a regulator of axogenesis. Although blocking GSK3β activity did not alter axon growth under physiological conditions in vivo, blocking it using a dominant-negative mutant or lithium chloride prevented mTORC1-induced accelerated axon growth. These data reveal the contribution of translational and non-translational downstream effectors such as GSK3β to abnormal axon growth in neurodevelopmental mTORopathies and open new therapeutic options for restoring long-range connectivity.

Excitability properties of motor axons in adults with cerebral palsy

Cerebral palsy (CP) is a permanent disorder caused by a lesion to the developing brain that significantly impairs motor function. The neurophysiological mechanisms underlying motor impairment are not well understood. Specifically, few have addressed whether motoneuron or peripheral axon properties are altered in CP, even though disruption of descending inputs to the spinal cord may cause them to change. In the present study, we have compared nerve excitability properties in seven adults with CP and fourteen healthy controls using threshold tracking techniques by stimulating the median nerve at the wrist and recording the compound muscle action potential over the abductor pollicis brevis. The excitability properties in the CP subjects were found to be abnormal. Early and late depolarizing and hyperpolarizing threshold electrotonus was significantly larger (i.e., fanning out), and resting current–threshold (I/V) slope was smaller, in CP compared to control. In addition resting threshold and rheobase tended to be larger in CP. According to a modeling analysis of the data, an increase in leakage current under or through the myelin sheath, i.e., the Barrett–Barrett conductance, combined with a slight hyperpolarization of the resting membrane potential, best explained the group differences in excitability properties. There was a trend for those with greater impairment in gross motor function to have more abnormal axon properties. The findings indicate plasticity of motor axon properties far removed from the site of the lesion. We suspect that this plasticity is caused by disruption of descending inputs to the motoneurons at an early age around the time of their injury.

Epilepsy in Multigene Tubulin Family Mutations

Mutations in α- and β-tubulin genes are responsible for a large spectrum of brain malformations secondary to abnormal neuronal migration, organization, differentiation, and axon guidance and maintenance. Motor and language developmental disorders, cognitive impairment and epilepsy are the main clinical associated symptoms. Frequency and severity of these disorders are largely related with the involvement of specific tubulin genes and their functions. The present study summarizes all the published data on tubulin family gene mutations and the associated clinical phenotype in order to define epilepsy recurrence and its characteristics. Mutations disrupting the stability of microtubules, mainly mechanisms involving neuronal migration and organization, may play an important role in epileptogenicity. Moreover, since mutations in the α- and β- tubulin genes are responsible for a large spectrum of cortical and brain malformations, they do not show a high specificity and correlation with an epileptic phenotype unlike other MCDs-related genes. Usually seizures start early (in the first year of life) and they are more often generalized (infantile spasms, tonic, tonic-clonic) as expression of a diffuse cortical maldevelopment. Mutations in the TUBA1A gene, causing various degrees of agyria-pachigyria spectrum, classical lissencephaly or polymicrogyria, represent among tubulin genes, the major responsible of epilepsy (about 50% of the cases). Disorders of cortical development as polymicrogyria, various types of gyral disorganization and schizencephaly were also described in cases carrying TUBB2B gene mutations showing epilepsy in the 40%. As far as mutations in the TUBB4A gene are concerned, despite the brain phenotype is represented by a form of leukoencephalopathy characterized by hypomielination with atrophy of the basal ganglia and cerebellum and not by MCDs, epilepsy is present in about 40% of the mutated subjects. On the contrary, mutations in the TUBB3 gene cause epilepsy only in 15% of cases, while seizures have never been described in association with TUBB gene.

Label-free quantitative proteomic profiling identifies disruption of ubiquitin homeostasis as a key driver of Schwann cell defects in spinal muscular atrophy.

Low levels of survival of motor neuron (SMN) protein cause the neuromuscular disease spinal muscular atrophy (SMA), characterized by degeneration of lower motor neurons and atrophy of skeletal muscle. Recent work demonstrated that low levels of SMN also trigger pathological changes in Schwann cells, leading to abnormal axon myelination and disrupted deposition of extracellular matrix proteins in peripheral nerve. However, the molecular pathways linking SMN depletion to intrinsic defects in Schwann cells remained unclear. Label-free proteomics analysis of Schwann cells isolated from SMA mouse peripheral nerve revealed widespread changes to the Schwann cell proteome, including disruption to growth/proliferation, cell death/survival, and molecular transport pathways. Functional clustering analyses revealed significant disruption to a number of proteins contributing to ubiquitination pathways, including reduced levels of ubiquitin-like modifier activating enzyme 1 (Uba1). Pharmacological suppression of Uba1 in Schwann cells was sufficient to reproduce the defective myelination phenotype seen in SMA. These findings demonstrate an important role for SMN protein and ubiquitin-dependent pathways in maintaining Schwann cell homeostasis and provide significant additional experimental evidence supporting a key role for ubiquitin pathways and, Uba1 in particular, in driving SMA pathogenesis across a broad range of cells and tissues.

DsRNA binding protein PACT/RAX in gene silencing, development and diseases.

PACT (Protein kinase, interferon-inducible double stranded RNA dependent activator) and its murine ortholog RAX (PKR-associated protein X) were originally identified as a protein activator for the dsRNA-dependent, interferon-inducible protein kinase (PKR). Endogenous PACT/RAX activates PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. PACT/RAX heterodimerized with PKR and activated it with its third motif in the absence of dsRNA. The activation of PKR leads to enhanced eIF2α phosphorylation followed by apoptosis or inhibition of growth. Besides the role of activating PKR, PACT is associated with a ~500 kDa complex that contains Dicer, hAgo2, and TRBP (TAR RNA binding protein) and it associates with Dicer to facilitate the production of small interfering RNA. PACT/RAX plays an important role in diverse physiological and pathological processes. Pact-/- mice exhibit notable developmental abnormalities including microtia, with craniofacial ear, and hearing defects. Pact-/- mice had smaller body sizes and fertility defects, both of which were caused by defective pituitary functions. It was found that dRAX disrupted fly embryos homozygous, displayed highly abnormal commissural axon structure of the central nervous system, and 70% of the flies homozygous for the mutant allele died prior to adulthood. Using high density SNP genotyping arrays, it was found that a mutation in PRKRA (the PACT/RAX gene) is the causative genetic mutation in DYT16, a novel autosomal recessive dystonia-parkinsonism syndrome in Brazilian patients.

Hyperactive glial cells contribute to axonal pathologies in the spinal cord of Npc1 mutant mice

Niemann-Pick disease type C1 (NPC1) is a neurodegenerative disease with various progressive pathological features, for example, neuronal loss, dysmyelination, abnormal axon swelling, and gliosis, in the brain. Pathological activation of p38-mitogen-activated protein kinase (MAPK) results in hyperphosphorylation of tau protein, which contributes to the development of neurodegenerative diseases. In this study, axonal varicosities or spheroids and presynaptic aggregates in the spinal cord of the Npc1 mutant mice were found from postnatal day (P) 35 onwards, as indicated by the increased hyperphosphorylated neurofilament and synaptophysin immunoreactivity as well as the findings from electron microscopy. However, activities of astrocytes and microglia in the Npc1 mutant spinal cord were progressively increased earlier from P10 onwards, accompanied by increased expression of interleukin-1β and apolipoprotein E, as well as up-regulated p38-MAPK activity and enhanced phosphorylated tau protein, but not cyclin-dependent kinase 5/p35 complex and glycogen synthase kinase-3β. Taken together, our data suggest that the axonal pathologies in the Npc1 mutant spinal cord are strongly correlated with the increase of activated glial cells, which produce IL-1β and ApoE, resulting in the activation of p38-MAPK signaling pathway and enhanced phosphorylated tau protein.

Ultra-structural analysis of the brain in aDrosophilamodel of Alzheimer's disease using FIB/SEM microscopy

Alzheimer's disease (AD), one of the most prevalent neurodegenerative brain diseases, has been extensively researched for years. However, its synaptic structure, which is a basis for understanding neurodegenerative disorders, has not yet been understood clearly. Defining the structures of neurons and their synaptic connections is the significant goal of brain research. To study synaptic connectivity, three-dimensional (3D) reconstructions of the nervous system are very helpful. In this study, the 3D structure of brain synapses in the Drosophila melanogaster Swedish amyloid precursor protein (APP) mutant, which is characterized by early onset AD, was analyzed using focused ion beam/scanning electron microscopy (FIB/SEM). This technique is one of the most useful for 3D reconstruction, as the process of obtaining serial images is fully automated and thus avoids the problems inherent in hand-operated ultrathin serial sectioning. The 3D images of normal and AD brains reported in this study reveal characteristic features of AD such as appearance of autophagy, abnormal axon formation and increased mitochondrial size. This 3D analysis reveals structural change as a basis for understanding neurodegenerative disorder.

A Forward Genetic Screen in Mice Identifies Mutants with Abnormal Cortical Patterning

Formation of a 6-layered cortical plate and axon tract patterning are key features of cerebral cortex development. Abnormalities of these processes may be the underlying cause for a range of functional disabilities seen in human neurodevelopmental disorders. To identify mouse mutants with defects in cortical lamination or corticofugal axon guidance, N-ethyl-N-nitrosourea (ENU) mutagenesis was performed using mice expressing LacZ reporter genes in layers II/III and V of the cortex (Rgs4-lacZ) or in corticofugal axons (TAG1-tau-lacZ). Four lines with abnormal cortical lamination have been identified. One of these was a splice site mutation in reelin (Reln) that results in a premature stop codon and the truncation of the C-terminal region (CTR) domain of reelin. Interestingly, this novel allele of Reln did not display cerebellar malformation or ataxia, and this is the first report of a Reln mutant without a cerebellar defect. Four lines with abnormal cortical axon development were also identified, one of which was found by whole-genome resequencing to carry a mutation in Lrp2. These findings demonstrated that the application of ENU mutagenesis to mice carrying transgenic reporters marking cortical anatomy is a sensitive and specific method to identify mutations that disrupt patterning of the developing brain.