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Tissue clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue images have been limited to counting sparse cell populations in stereotypical mice. Here we introduce NuMorph, a group of image analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after tissue clearing and imaging by a conventional light-sheet microscope. We applied NuMorph to investigate two distinct mouse models: aTopoisomerase 1(Top1) conditional knockout model with severe neurodegenerative deficits and aNeurofibromin 1(Nf1) conditional knockout model with a more subtle brain overgrowth phenotype. In each case, we identified differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of 3D whole brain imaging approaches and the tools are widely applicable for studying 3D structural deficits of the brain at cellular resolution in animal models of neuropsychiatric disorders.

Many developmental syndromes have been linked to genetic mutations that cause abnormal ERK/MAPK activity; however, the neuropathological effects of hyperactive signaling are not fully understood. Here, we examined whether hyperactivation of MEK1 modifies the development of GABAergic cortical interneurons (CINs), a heterogeneous population of inhibitory neurons necessary for cortical function. We show that GABAergic-neuron specific MEK1 hyperactivation in vivo leads to increased cleaved caspase-3 labeling in a subpopulation of immature neurons in the embryonic subpallial mantle zone. Adult mutants displayed a significant loss of parvalbumin (PV), but not somatostatin, expressing CINs and a reduction in perisomatic inhibitory synapses on excitatory neurons. Surviving mutant PV-CINs maintained a typical fast-spiking phenotype but showed signs of decreased intrinsic excitability that coincided with an increased risk of seizure-like phenotypes. In contrast to other mouse models of PV-CIN loss, we discovered a robust increase in the accumulation of perineuronal nets, an extracellular structure thought to restrict plasticity. Indeed, we found that mutants exhibited a significant impairment in the acquisition of behavioral response inhibition capacity. Overall, our data suggest PV-CIN development is particularly sensitive to hyperactive MEK1 signaling, which may underlie certain neurological deficits frequently observed in ERK/MAPK-linked syndromes.

SUMMARY Tissue-clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue images have been limited to counting sparse cell populations in stereotypical mice. Here, we introduce NuMorph, a group of analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after clearing and imaging by light-sheet microscopy. We apply NuMorph to investigate two distinct mouse models: a Topoisomerase 1 (Top1) model with severe neurodegenerative deficits and a Neurofibromin 1 (Nf1) model with a more subtle brain overgrowth phenotype. In each case, we identify differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of whole-brain imaging approaches, and the tools are widely applicable for studying brain structure phenotypes at cellular resolution., Graphical abstract, In brief Krupa et al. develop an image analysis toolbox called NuMorph that accurately quantifies all nuclei within the mouse cortex after tissue clearing and light-sheet imaging. They implement NuMorph to characterize region- and cell-type-specific deficits present in two distinct mouse models, demonstrating the advantages of a whole-brain imaging approach.

During development of the vertebrate CNS, the basic helix-loop-helix (bHLH) transcription factor Olig2 sustains replication competence of progenitor cells that give rise to neurons and oligodendrocytes. A pathological counterpart of this developmental function is seen in human glioma, wherein Olig2 is required for maintenance of stem-like cells that drive tumor growth. The mitogenic/gliomagenic functions of Olig2 are regulated by phosphorylation of a triple serine motif (S10, S13, and S14) in the amino terminus. Here, we identify a set of three serine/threonine protein kinases (glycogen synthase kinase 3α/β [GSK3α/β], casein kinase 2 [CK2], and cyclin-dependent kinases 1/2 [CDK1/2]) that are, collectively, both necessary and sufficient to phosphorylate the triple serine motif. We show that phosphorylation of the motif itself serves as a template to prime phosphorylation of additional serines and creates a highly charged "acid blob" in the amino terminus of Olig2. Finally, we show that small molecule inhibitors of this forward-feeding phosphorylation cascade have potential as glioma therapeutics.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function., Author summary The RASopathies are a large and complex family of syndromes caused by mutations in the RAS/MAPK signaling cascade with no known cure. Individuals with these syndromes often present with heart defects, craniofacial differences, and neurological abnormalities, such as developmental delay, cognitive changes, epilepsy, and an increased risk of autism. However, there is wide variation in the extent of intellectual ability between individuals. It is currently unclear how different RASopathy mutations affect brain development. Here, we describe the cellular and behavioral consequences of a mutation in a gene called Raf1 that is associated with a common RASopathy, Noonan Syndrome. We find that mice harboring a mutation in Raf1 show moderate increases in the number of two subsets of glial cells, which is also observed in a number of other RASopathy brain samples. Surprisingly, we found that Raf1 mutant mice show improved performance in several learning and memory tasks. Our work highlights potential mutation-specific changes in RASopathy brain function and helps set the framework for future personalized therapeutic approaches.

The ERK/MAPK intracellular signaling pathway is hypothesized to be a key regulator of striatal activity via modulation of synaptic plasticity and gene transcription. However, prior investigations into striatal ERK/MAPK functions have yielded conflicting results. Further, these studies have not delineated the cell-type-specific roles of ERK/MAPK signaling due to the reliance on globally administered pharmacological ERK/MAPK inhibitors and the use of genetic models that only partially reduce total ERK/MAPK activity. Here, we generated mouse models in which ERK/MAPK signaling was completely abolished in each of the two distinct classes of medium spiny neurons (MSNs). ERK/MAPK deletion in D1R-MSNs (direct pathway) resulted in decreased locomotor behavior, reduced weight gain, and early postnatal lethality. In contrast, loss of ERK/MAPK signaling in D2R-MSNs (indirect pathway) resulted in a profound hyperlocomotor phenotype. ERK/MAPK-deficient D2R-MSNs exhibited a significant reduction in dendritic spine density, markedly suppressed electrical excitability, and suppression of activity-associated gene expression even after pharmacological stimulation. Our results demonstrate the importance of ERK/MAPK signaling in governing the motor functions of the striatal direct and indirect pathways. Our data further show a critical role for ERK in maintaining the excitability and plasticity of D2R-MSNs.SIGNIFICANCE STATEMENT Alterations in ERK/MAPK activity are associated with drug abuse, as well as neuropsychiatric and movement disorders. However, genetic evidence defining the functions of ERK/MAPK signaling in striatum-related neurophysiology and behavior is lacking. We show that loss of ERK/MAPK signaling leads to pathway-specific alterations in motor function, reduced neuronal excitability, and the inability of medium spiny neurons to regulate activity-induced gene expression. Our results underscore the potential importance of the ERK/MAPK pathway in human movement disorders.

SummaryCoordinated migration of distinct classes of neurons to appropriate positions leads to the formation of functional neuronal circuitry in the cerebral cortex. The two major classes of cortical neurons, interneurons and projection neurons, utilize distinctly different modes (radial versus tangential) and routes of migration to arrive at their final positions in the cerebral cortex. Here, we show that adenomatous polyposis coli (APC) modulates microtubule (MT) severing in interneurons to facilitate tangential mode of interneuron migration, but not the glial-guided, radial migration of projection neurons. APC regulates the stability and activity of the MT-severing protein p60-katanin in interneurons to promote the rapid remodeling of neuronal processes necessary for interneuron migration. These findings reveal how severing and restructuring of MTs facilitate distinct modes of neuronal migration necessary for laminar organization of neurons in the developing cerebral cortex.

Axons fail to regenerate after central nervous system (CNS) injury. Modulation of the PTEN/mTORC1 pathway in retinal ganglion cells (RGCs) promotes axon regeneration after optic nerve injury. Here, we report that AKT activation, downstream of Pten deletion, promotes axon regeneration and RGC survival. We further demonstrate that GSK3β plays an indispensable role in mediating AKT-induced axon regeneration. Deletion or inactivation of GSK3β promotes axon regeneration independently of the mTORC1 pathway, whereas constitutive activation of GSK3β reduces AKT-induced axon regeneration. Importantly, we have identified eIF2Bε as a novel downstream effector of GSK3β in regulating axon regeneration. Inactivation of eIF2Bε reduces both GSK3β and AKT-mediated effects on axon regeneration. Constitutive activation of eIF2Bε is sufficient to promote axon regeneration. Our results reveal a key role of the AKT-GSK3β-eIF2Bε signaling module in regulating axon regeneration in the adult mammalian CNS. DOI: http://dx.doi.org/10.7554/eLife.11903.001, eLife digest The central nervous system consists of the neurons that make up the brain, retina, and spinal cord. Neurons transmit electrical signals along a cable-like structure called an axon. However, an axon cannot regenerate itself, and so injuries that crush or sever the axons can lead to permanent damage. This happens for two reasons: neurons don’t have the same regenerative ability as other cells, and the environment in the central nervous system restricts cell growth. The optic nerve transmits visual information from the eye to the brain. Studies in mice with a damaged optic nerve show that it is possible to regenerate the axons of neurons that lack a protein known as PTEN. These studies revealed one molecular pathway by which eliminating PTEN helps to boost the regrowth of axons. Now, Guo et al. identify another independent pathway by which eliminating PTEN helps promote axon regeneration in damaged mouse optic nerves. This pathway starts with a growth-promoting enzyme called AKT, which is turned on in neurons that lack PTEN. Indeed, injecting mice with an active form of this enzyme caused the optic nerve fiber to regrow in mice whose optic nerve had been crushed. Further experiments revealed that AKT activates a pathway in which another enzyme called GSK3β acts on a protein called eIF2Bε. A future challenge is to simultaneously manipulate the different signaling pathways that have been linked to axon regrowth to investigate whether this combined approach could help repair damage to the central nervous system. DOI: http://dx.doi.org/10.7554/eLife.11903.002

Aberrant signaling through the Raf/MEK/ERK (ERK/MAPK) pathway causes pathology in a family of neurodevelopmental disorders known as 'RASopathies' and is implicated in autism pathogenesis. Here, we have determined the functions of ERK/MAPK signaling in developing neocortical excitatory neurons. Our data reveal a critical requirement for ERK/MAPK signaling in the morphological development and survival of large Ctip2+ neurons in layer 5. Loss of Map2k1/2 (Mek1/2) led to deficits in corticospinal tract formation and subsequent corticospinal neuron apoptosis. ERK/MAPK hyperactivation also led to reduced corticospinal axon elongation, but was associated with enhanced arborization. ERK/MAPK signaling was dispensable for axonal outgrowth of layer 2/3 callosal neurons. However, Map2k1/2 deletion led to reduced expression of Arc and enhanced intrinsic excitability in both layers 2/3 and 5, in addition to imbalanced synaptic excitation and inhibition. These data demonstrate selective requirements for ERK/MAPK signaling in layer 5 circuit development and general effects on cortical pyramidal neuron excitability. DOI: http://dx.doi.org/10.7554/eLife.11123.001, eLife digest In the nervous system, cells called neurons form networks that relay information in the form of electrical signals around the brain and the rest of the body. Typically, an electrical signal travels from branch-like structures at one end of the cell, through the cell body and then along a long fiber called an axon to reach junctions with another neurons. The connections between neurons start to form as the nervous system develops in the embryo, and any errors or delays in this process can cause severe neurological disorders and intellectual disabilities. For example, genetic mutations affecting a communication system within cells known as the ERK/MAPK pathway can lead to a family of syndromes called the “RASopathies”. Abnormalities in this pathway may also contribute to certain types of autism. However, it is not clear how alterations to the ERK/MAPK pathway cause these conditions. Xing et al. investigated whether ERK/MAPK signaling regulates the formation of connections between neurons and the activity of neurons in mouse brains. The experiments showed that the growth of axons that extend from an area of the brain called the cerebral cortex towards the spinal cord are particularly sensitive to changes in the level of signaling through the ERK/MAPK pathway. On the other hand, inhibiting the pathway has relatively little effect on the growth of axons within the cerebral cortex. Further experiments showed that many neurons in the cerebral cortex require the ERK/MAPK pathway to activate genes that alter neuronal activity and the strength of the connections between neurons. Xing et al.’s findings suggest that defects in the connections between the cerebral cortex and different regions of the nervous system may contribute to the symptoms observed in patients with conditions linked to alterations in ERK/MAPK activity. Future studies will focus on understanding the molecular mechanisms by which ERK/MAPK pathway influences the organization and activity of neuron circuits during the development of the nervous system. DOI: http://dx.doi.org/10.7554/eLife.11123.002

In this issue of Neuron, Napoli et al. (2012) demonstrate that elevated ERK/MAPK signaling in Schwann cells is a crucial trigger for Schwann cell dedifferentiation in vivo. Moreover, the authors show that dedifferentiated Schwann cells have the potential to coordinate much of the peripheral nerve response to injury.

Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3α and GSK3β, while a splice variant of GSK3β (GSK3β2), containing a 13 amino acid insert, is enriched in neurons. GSK3α and GSK3β deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, β-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking GSK3β, yet normal in cortex lacking GSK3α). Furthermore, the neuron-enriched GSK3β2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3β1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3β isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3β splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of GSK3β1 and GSK3β2 are similar.

Disrupted ERK1/2 ( MAPK3 / MAPK1 ) MAPK signaling has been associated with several developmental syndromes in humans; however, mutations in ERK1 or ERK2 have not been described. We demonstrate haplo-insufficient ERK2 expression in patients with a novel ≈1 Mb micro-deletion in distal 22q11.2, a region that includes ERK2 . These patients exhibit conotruncal and craniofacial anomalies that arise from perturbation of neural crest development and exhibit defects comparable to the DiGeorge syndrome spectrum. Remarkably, these defects are replicated in mice by conditional inactivation of ERK2 in the developing neural crest. Inactivation of upstream elements of the ERK cascade ( B-Raf and C-Raf, MEK1 and MEK2 ) or a downstream effector, the transcription factor serum response factor resulted in analogous developmental defects. Our findings demonstrate that mammalian neural crest development is critically dependent on a RAF/MEK/ERK/serum response factor signaling pathway and suggest that the craniofacial and cardiac outflow tract defects observed in patients with a distal 22q11.2 micro-deletion are explained by deficiencies in neural crest autonomous ERK2 signaling.

Astrocytes produce an assortment of signals that promote neuronal maturation according to a precise developmental timeline. Is this orchestrated timing and signaling altered in human neurodevelopmental disorders? To address this question, the astroglial lineage was investigated in two model systems of a developmental disorder with intellectual disability caused by mutant Harvey rat sarcoma viral oncogene homolog (HRAS) termed Costello syndrome: mutant HRAS human induced pluripotent stem cells (iPSCs) and transgenic mice. Human iPSCs derived from patients with Costello syndrome differentiated to astroglia more rapidly in vitro than those derived from wild-type cell lines with normal HRAS, exhibited hyperplasia, and also generated an abundance of extracellular matrix remodeling factors and proteoglycans. Acute treatment with a farnesyl transferase inhibitor and knockdown of the transcription factor SNAI2 reduced expression of several proteoglycans in Costello syndrome iPSC-derived astrocytes. Similarly, mice in which mutant HRAS was expressed selectively in astrocytes exhibited experience-independent increased accumulation of perineuronal net proteoglycans in cortex, as well as increased parvalbumin expression in interneurons, when compared to wild-type mice. Our data indicate that astrocytes expressing mutant HRAS dysregulate cortical maturation during development as shown by abnormal extracellular matrix remodeling and implicate excessive astrocyte-to-neuron signaling as a possible drug target for treating mental impairment and enhancing neuroplasticity.

Chondroitin sulfate proteoglycans (CSPGs) and myelin-based inhibitors are the most studied inhibitory molecules in the adult central nervous system. Unlike myelin-based inhibitors, few studies have reported ways to overcome the inhibitory effect of CSPGs. Here, by using regenerating adult dorsal root ganglion (DRG) neurons, we show that chondroitin sulfate proteoglycans inhibit axon assembly by a different mechanism from myelin-based inhibitors. Furthermore, we show that neither Rho inhibition nor cAMP elevation rescues extracellular factor-induced axon assembly inhibited by CSPGs. Instead, our data suggest that CSPGs block axon assembly by interfering with integrin signaling. Surprisingly, we find that nerve growth factor (NGF) promotes robust axon growth of regenerating DRG neurons over CSPGs. We have found that, unlike naive neurons that require simultaneous activation of neurotrophin and integrin pathways for axon assembly, either neurotrophin or integrin signaling alone is sufficient to induce axon assembly of regenerating neurons. Thus, our results suggest that the ability of NGF to overcome CSPG inhibition in regenerating neurons is probably due to the ability of regenerating neurons to assemble axons using an integrin-independent pathway. Finally, our data show that the GSK-3β-APC pathway, previously shown to mediate developing axon growth, is also necessary for axon regeneration.

GSK-3is a master kinase that controls many different aspects of nervous system development and function and has been implicated in neurodegeneration. In a Perspective, Zhou and Snider discuss emerging evidence that reveals how GSK-3regulates the morphogenesis of axons by controlling the assembly of microtubules that are needed for axonal extension.

At a certain point in development, axons in the mammalian CNS undergo a profound loss of intrinsic growth capacity, which leads to poor regeneration after injury. Overexpression of Bcl-2 prevents this loss, but the molecular basis of this effect remains unclear. Here, we report that Bcl-2 supports axonal growth by enhancing intracellular Ca(2+) signaling and activating cAMP response element binding protein (CREB) and extracellular-regulated kinase (Erk), which stimulate the regenerative response and neuritogenesis. Expression of Bcl-2 decreases endoplasmic reticulum (ER) Ca(2+) uptake and storage, and thereby leads to a larger intracellular Ca(2+) response induced by Ca(2+) influx or axotomy in Bcl-2-expressing neurons than in control neurons. Bcl-x(L), an antiapoptotic member of the Bcl-2 family that does not affect ER Ca(2+) uptake, supports neuronal survival but cannot activate CREB and Erk or promote axon regeneration. These results suggest a novel role for ER Ca(2+) in the regulation of neuronal response to injury and define a dedicated signaling event through which Bcl-2 supports CNS regeneration.

To study the role of NT3 in directing axonal projections of proprioceptive dorsal root ganglion (DRG) neurons, NT3−/− mice were crossed with mice carrying a targeted deletion of the proapoptotic gene Bax. In Bax−/−/NT3−/− mice, NT3-dependent neurons survived and expressed the proprioceptive neuronal marker parvalbumin. Initial extension and collateralization of proprioceptive axons into the spinal cord occurred normally, but proprioceptive axons extended only as far as the intermediate spinal cord. This projection defect is similar to the defect in mice lacking the ETS transcription factor ER81 (Arber et al., 2000). Few if any DRG neurons from Bax−/−/NT3−/− mice expressed ER81 protein. Expression of a NT3 transgene in muscle restored DRG ER81 expression in NT3−/− mice. Finally, addition of NT3 to DRG explant cultures resulted in induction of ER81 protein. Our data indicate that NT3 mediates the formation of proprioceptive afferent-motor neuron connections via regulation of ER81.

Neuronal morphological differentiation is regulated by numerous polypeptide growth factors (neurotrophic factors). Recently, significant progress has been achieved in clarifying the roles of neurotrophins as well as glial cell line-derived neurotrophic factor family members in peripheral axon elongation during development. Additionally, advances have been made in defining the signal transduction mechanisms employed by these factors in mediating axon morphological responses. Several studies addressed the role of neurotrophic factors in regenerative axon growth and suggest that signaling mechanisms in addition to those triggered by receptor tyrosine kinases may be required for successful peripheral nervous system regeneration. Finally, recent investigations demonstrate that neurotrophic factors can enhance axon growth after spinal cord injuries.

GSK-3 is an essential mediator of several signaling pathways that regulate cortical development. We therefore created conditional mouse mutants lacking both GSK-3α and GSK-3β in newly born cortical excitatory neurons. Gsk3-deleted neurons expressing upper layer markers exhibited striking migration failure in all areas of the cortex. Radial migration in hippocampus was similarly affected. In contrast, tangential migration was not grossly impaired after Gsk3 deletion in interneuron precursors. Gsk3-deleted neurons extended axons and developed dendritic arbors. However, the apical dendrite was frequently branched while basal dendrites exhibited abnormal orientation. GSK-3 regulation of migration in neurons was independent of Wnt/β-catenin signaling. Importantly, phosphorylation of the migration mediator, DCX, at ser327, and phosphorylation of the semaphorin signaling mediator, CRMP-2, at Thr514 were markedly decreased. Our data demonstrate that GSK-3 signaling is essential for radial migration and dendritic orientation and suggest that GSK-3 mediates these effects by phosphorylating key microtubule regulatory proteins. DOI: http://dx.doi.org/10.7554/eLife.02663.001, eLife digest In the brain, one of the most striking features of the cerebral cortex is that its neurons are organized into different layers that are specifically connected to one another and to other regions of the brain. How newly generated neurons find their appropriate layer during the development of the brain is an important question; and, in humans, when this process goes awry, it can often result in seizures and mental retardation. An enzyme called GSK-3 regulates several major signaling pathways important to brain development. The GSK-3 enzyme switches other proteins on or off by adding phosphate groups to them. Morgan-Smith et al. set out to better understand the role of GSK-3 in brain development by deleting the genes for this enzyme specifically in the cerebral cortex of mice. Mice have two genes that encode slightly different forms of the GSK-3 enzyme. Deleting both of these in different groups of neurons during brain development revealed that a major group of neurons need GSK-3 in order to migrate to the correct layer. Specifically, the movement of neurons from where they arise in the central region of the brain to the outermost layer (a process called radial migration) was disrupted when the GSK-3 genes were deleted. Morgan-Smith et al. further found that cortical neurons without GSK-3 were unable to develop the shape needed to undertake radial migration because they failed to switch from having many branches to having just two main branches. Additional experiments revealed that these abnormalities did not depend on certain signaling pathways, such as the Wnt-signaling pathway or the PI3K signaling pathway that can control GSK-3 activity. Instead, Morgan-Smith et al. found that two proteins that are normally targeted by the GSK-3 enzyme have fewer phosphate groups than normal in the cortical neurons that did not contain the enzyme: both of these proteins regulate the shape of neurons by interacting with the molecular ‘scaffolding’ within the cell. The GSK-3 enzyme was already known to modify the activities of many other proteins that affect the migration of cells. Thus, the findings of Morgan-Smith et al. suggest that this enzyme may coordinate many of the mechanisms thought to underlie this process during brain development. DOI: http://dx.doi.org/10.7554/eLife.02663.002

Defects in ependymal (E) cells, which line the ventricle and generate cerebrospinal fluid flow through ciliary beating, can cause hydrocephalus. Dishevelled genes (Dvls) are essential for Wnt signaling, and Dvl2 has been shown to localize to the rootlet of motile cilia. Using the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mouse, we show that compound genetic ablation of Dvls causes hydrocephalus. In hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mutants, E cells differentiated normally, but the intracellular and intercellular rotational alignments of ependymal motile cilia were disrupted. As a consequence, the fluid flow generated by the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) E cells was significantly slower than that observed in control mice. Dvls were also required for the proper positioning of motile cilia on the apical surface. Tamoxifen-induced conditional removal of Dvls in adult mice also resulted in defects in intracellular rotational alignment and positioning of ependymal motile cilia. These results suggest that Dvls are continuously required for E cell planar polarity and may prevent hydrocephalus.

We report a novel role for long-distance retrograde neurotrophin signaling in the establishment of synapses in the sympathetic nervous system. Target-derived NGF is both necessary and sufficient for formation of post-synaptic specializations on dendrites of sympathetic neurons. This, in turn, is prerequisite for formation of pre-synaptic specializations but not preganglionic axonal ingrowth from the spinal cord into sympathetic ganglia. We also find that NGF–TrkA signaling endosomes travel from distal axons to cell bodies and dendrites where they promote PSD clustering. Furthermore, the p75 neurotrophin receptor restricts PSD formation, suggesting an important role for antagonistic NGF–TrkA and p75 signaling pathways during retrograde control of synapse establishment. Thus, in addition to defining the appropriate number of sympathetic neurons that survive the period of developmental cell death, target-derived NGF also exerts control over the degree of connectivity between the spinal cord and sympathetic ganglia through retrograde control of synapse assembly.

Because of discrepancies in previous reports regarding the role of glial cell line-derived neurotrophic factor (GDNF) in motoneuron (MN) development and survival, we have reexamined MNs in GDNF-deficient mice and in mice exposed to increased GDNF afterin uterotreatment or in transgenic animals overexpressing GDNF under the control of the muscle-specific promoter myogenin (myo-GDNF). With the exception of oculomotor and abducens MNs, the survival of all other populations of spinal and cranial MNs were reduced in GDNF-deficient embryos and increased in myo-GDNF andin uterotreated animals. By contrast, the survival of spinal sensory neurons in the dorsal root ganglion and spinal interneurons were not affected by any of the perturbations of GDNF availability.In wild-type control embryos, all brachial and lumbar MNs appear to express the GDNF receptors c-ret and GFRα1 and the MN markers ChAT, islet-1, and islet-2, whereas only a small subset express GFRα2. GDNF-dependent MNs that are lost in GDNF-deficient animals express ret/GFRα1/islet-1, whereas many surviving GDNF-independent MNs express ret/GFRα1/GFRα2 and islet-1/islet-2. This indicates that many GDNF-independent MNs are characterized by the presence of GFRα2/islet-2. It seems likely that the GDNF-independent population represent MNs that require other GDNF family members (neurturin, persephin, artemin) for their survival. GDNF-dependent and -independent MNs may reflect subtypes with distinct synaptic targets and afferent inputs.

Semaphorin 6A (Sema6A) (previously named Semaphorin VIa) is the originally described member of the vertebrate semaphorin class 6, a group of transmembrane semaphorins homologous to the insect semaphorin class 1. Although Sema-1a (previously named semaphorin I) has been implicated in axon guidance in insects, the function of Sema6A is currently unknown. We have expressed the extracellular domain of Sema6A in mammalian cells as either a monomeric or a dimeric fusion protein and tested for potential axon guidance effects on two populations of embryonic neurons in growth cone collapse and collagen matrix chemorepulsion assays. Sema6A was observed to induce growth cone collapse of sympathetic neurons with an EC50 of ∼200 pm, although a 10-fold higher (EC50 of ∼2 nm) concentration was necessary to induce growth cone collapse of dorsal root ganglion neurons. The activity of Sema6A is likely to depend on protein dimerization or oligomerization. Although Sema6A mRNA is expressed in complex patterns during embryonic development, it is strikingly absent from sympathetic ganglia. Sema6A is, however, expressed in areas avoided by sympathetic axons and in areas innervated by sympathetics, but before their arrival. Our results demonstrate that transmembrane semaphorins, like the secreted ones, can act as repulsive axon guidance cues. Our findings are consistent with a role for Sema6A in channeling sympathetic axons into the sympathetic chains and controlling the temporal sequence of sympathetic target innervation.

Neurotrophins are powerful regulators of neuronal morphology. Several lines of evidence are consistent with the idea that characteristic axonal and dendritic morphologies throughout the nervous system may be determined by local patterns of neurotrophin and neurotrophin receptor expression. Neurotrophin receptor tryosine kinases (Trks) exist in both tyrosine-containing (TK+) and tyrosine-lacking (TK-) isoforms, both of which are expressed in many neuronal populations. However, ratios of TK+ to TK- isoforms may vary at different stages of development and may be differentially distributed to cellular compartments. To test whether these isoforms have different functions related to axon outgrowth, full-length or tyrosine kinase-lacking TrkC receptors were overexpressed in embryonic dorsal root ganglion neurons maintained in explant cultures in neurotrophin-3 (NT-3)-containing media. Neurons were transfected with plasmid DNA encoding enhanced yellow fluorescent protein (EYFP) and TrkC receptor isoforms by particle-mediated gene transfer. Control neurons possessed 3.7 +/- 1.3 primary processes and 113.8 +/- 46 branch points. About 80% of the branches were located along the distal part of the axon. Transfection with the trkC TK+ increased the number of primary processes (6.5 +/- 2.8), whereas transfection with trkC TK- reduced the formation of primary processes (3.0 +/- 1.3). Surprisingly, the distribution of branch points was shifted to the proximal region of axons in neurons transfected with trkC TK-. These observations are consistent with the idea that differential expression of Trk isoforms during development may sculpt axonal morphology.

Evidence garnered from both human autopsy studies and genetic animal models has suggested a potential role for astrocytes in the pathogenesis of amyotrophic lateral sclerosis (ALS). Currently, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) represent the only known cause of motoneuron loss in the disease, producing 21q linked familial ALS (FALS). To determine whether astrocytic dysfunction has a primary role in familial ALS, we have generated multiple lines of transgenic mice expressing G86R mutant SOD1 restricted to astrocytes. In GFAP-m SOD1 mice, astrocytes exhibit significant hypertrophy and increased GFAP reactivity as the animals mature. However, GFAP-mutant SOD1 transgenic mice develop normally and do not experience spontaneous motor deficits with increasing age. Histological examination of spinal cord in aged GFAP-mSOD1 mice reveals normal motoneuron and microglial morphology. These results indicate that 21q linked FALS is not a primary disorder of astrocytes, and that expression of mutant SOD1 restricted to astrocytes is not sufficient to cause motoneuron degenerationin vivo. Expression of mutant SOD1 in other cell types, most likely neurons, is critical for the initiation of disease.

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha1 or GFR alpha2). Neurturin-deficient (NTN-/-) mice generated by homologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons. GFR alpha2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha2/Ret receptor complexes as the critical mediators of NTN function in vivo.

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)–E13, stages of rapid axon growth toward targetsin vivo,Bax−/− sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substantially. Neurotrophin-3 (NT-3) supported striking terminal arborization of subsets ofBax−/− neurons, whereas NGF produced predominantly axon elongation in a different subset. We conclude that axon growthin vitrois neurotrophin dependent from the earliest stages of sensory neuron development. Furthermore, neurotrophins support the appearance of distinct axonal morphologies that characterize different sensory neuron subpopulations.

Over the past decade, mutations in genes encoding RAS family members, other components of an intracellular signaling cascade that RAS controls, and proteins that modify the cascade have been recognized as causes of developmental syndromes. Collectively, these syndromes are often referred to as “RASopathies.” Not surprisingly, RASopathies have numerous manifestations, including propensity to cancer, craniofacial abnormalities, cardiac defects, cutaneous abnormalities, neurodevelopmental delay, and varying degrees of cognitive dysfunction. Uncovering the causes and developing treatments for the neurodevelopmental abnormalities are a challenge because of the myriad cellular elements in the brain and the complexity of nervous system function. A recent study by Krencik et al. ( 1 ) takes a major step toward identifying the cellular pathology underlying Costello syndrome, a RASopathy that is characterized by delayed development, craniofacial and heart problems, and cognitive impairment. The latter appears to be linked to abnormal development and function of a population of nonneuronal cells (astrocytes) in the brain.

In this minireview, we have discussed new data highlighting the distinct properties of subsets of nociceptors that in turn raise questions regarding their functional roles. The concept of two major nociceptor classes—distinguished by their responses to chemical mediators and growth factors in the periphery, and by their central connectivity and pharmacology—may prove important for the understanding (and possibly the treatment) of chronic pain.The most clear-cut implication of the distinction of nociceptor classes is in the setting of inflammation. Here, increases in NGF synthesis in the periphery, the associated upregulation of putative neuromodulators such as substance P and BDNF in TrkA-expressing nociceptors, and the consequent induction of central sensitization seem critical events underpinning abnormal pain sensitivity. The profound regulatory effects of NGF on nociceptor function and the powerful analgesic effects of anti-NGF on inflammatory hyperalgesia strongly suggest that interference with this ligand–receptor interaction provides a novel therapeutic opportunity. Given the current overwhelming clinical reliance on just two classes of analgesic drugs (nonsteroidal anti-inflammatory drugs [NSAIDS] and opiates), the distinct target offered by NGF represents a genuine conceptual advance.The normal functions of the IB4-binding, GDNF-responsive class of nociceptors are, at the moment, mysterious. Some of their properties (lack of responsiveness to NGF in the periphery and less direct access to the NK1 pathway in the dorsal horn) suggest a very different role for these nociceptors. Other properties (potential responsiveness to ATP and the expression of the neuromodulator somatostatin in a subset of these cells) as yet do not imply clear functional roles. We are also still relatively ignorant of the conditions and factors regulating GDNF expression. However, a fascinating issue for the future relates to the importance of the IB4-binding neurons in chronic pain, perhaps particularly in neuropathic pain states. Clearly, GDNF or related molecules may also offer an important target in the development of novel analgesic therapies.

Overexpression of glial cell line–derived neurotrophic factor (GDNF) by muscle greatly increased the number of motor axons innervating neuromuscular junctions in neonatal mice. The extent of hyperinnervation correlated with the amount of GDNF expressed in four transgenic lines. Overexpression of GDNF by glia and overexpression of neurotrophin-3 and neurotrophin-4 in muscle did not cause hyperinnervation. Thus, increased amounts of GDNF in postsynaptic target cells can regulate the number of innervating axons.

The proapoptotic molecule BAX is required for death of sympathetic and motor neurons in the setting of trophic factor deprivation. Furthermore, adultBax−/−mice have more motor neurons than do their wild-type counterparts. These findings raise the possibility that BAX regulates naturally occurring cell death during development in many neuronal populations. To test this idea, we assessed apoptosis using TUNEL labeling in several well-studied neural systems during embryonic and early postnatal development inBax−/−mice. Remarkably, naturally occurring cell death is virtually eliminated between embryonic day 11.5 (E11.5) and postnatal day 1 (PN1) in most peripheral ganglia, in motor pools in the spinal cord, and in the trigeminal brainstem nuclear complex. Additionally, reduction, although not elimination, of cell death was noted throughout the developing cerebellum, in some layers of the retina, and in the hippocampus. Saving of cells was verified by axon counts of dorsal and ventral roots, as well as facial and optic nerves that revealed 24–35% increases in axon number. Interestingly, many of the supernumerary axons had very small cross-sectional areas, suggesting that the associated neurons are not normal. We conclude that BAX is a critical mediator of naturally occurring death of peripheral and CNS neurons during embryonic life. However, rescue from naturally occurring cell death does not imply that the neurons will develop normal functional capabilities.

To clarify the role of muscle-derived neurotrophin-3 (NT-3) in the development of sensory neurons, we generated transgenic mice selectively overexpressing NT-3 in skeletal muscles under the control of a myogenin promoter ( myo-NT-3 mice). The myo-NT-3 transgene was then bred into an NT-3 null mutant (−/−) line to generate myo-NT-3 , NT-3 −/− mice in which NT-3 was expressed in muscles, but not elsewhere. Transient overexpression of NT-3 in developing muscles increased the number of proprioceptive neurons as well as the density of both their central and peripheral projections, resulting in more Ia afferents in spinal cord and more spindles (end organs of Ia afferents) in muscles. NT-3 expression restricted to muscles was sufficient to secure the development of proprioceptive neurons and their central and peripheral projections in myo-NT-3 , NT-3 −/− mice. The loss of nonproprioceptive neurons observed in NT-3 −/− mice was not reversed by the transgene, suggesting that these neurons are regulated by NT-3 from sources other than muscle. We conclude that target-derived rather than intraganglionic NT-3 is preeminent in supporting the development of proprioceptive neurons. The level of NT-3 in developing muscles may be the principal factor determining the number of proprioceptive neurons in dorsal root ganglions and spindles in skeletal muscles of adults.

Nerve growth factor (NGF), signaling through its receptor tyrosine kinase, TrkA, is required for the survival of all small and many intermediate-sized murine dorsal root ganglion (DRG) neurons during development, accounting for 80% of the total DRG population. Surprisingly, NGF/TrkA-dependent neurons include a large population that does not express TrkA in adult mice (Silos-Santiago et al., 1995). This finding suggests two hypotheses: Neurons lacking TrkA in the adult may express TrkA during development, or they may be maintained through a paracrine mechanism by TrkA-expressing neurons. To determine whether TrkA is expressed transiently by DRG neurons that lack the receptor in adulthood, we examined the distribution of TrkA protein during development. We show here that TrkA expression is strikingly developmentally regulated. Eighty percent of DRG neurons expressed TrkA during embryogenesis and early postnatal life, whereas only 43% expressed TrkA at postnatal day (P) 21. Because the period of TrkA down-regulation corresponds with a critical period during which nociceptive phenotype can be altered by NGF (see Lewin and Mendell [1993] Trends Neurosci. 16:353-359), we examined whether NGF modulates the down-regulation of TrkA. Surprisingly, neither NGF deprivation nor augmentation altered the extent of TrkA down-regulation. Our results demonstrate a novel form of regulation of neurotrophin receptor expression that occurs late in development. All DRG neurons that require NGF for survival express TrkA during embryogenesis, and many continue to express TrkA during a postnatal period when neuronal phenotype is regulated by NGF. The subsequent down-regulation of TrkA is likely to be importantly related to functional distinctions among nociceptive neurons in maturity.

The formation of laminar-specific projections is a key event in the development of appropriate neuronal connections in many regions of the central nervous system. In order to provide a framework for defining functions of molecules related to spinal laminar targeting of dorsal root ganglion neurons in mice, we have characterized the initial trajectories of sensory axons in relation to the maturation of their target laminae in the spinal cord. We show that morphological and biochemical differentiation of distinct clusters of neurons in the dorsal region of the spinal cord precedes initial collateral branching from sensory axons. Between embryonic day (E)12.5 and E13.5, sensory axons develop swellings (“nodes”) along their entire intraspinal extent and elaborate interstitial collateral branches from these nodes. Collaterals from the different classes of sensory axons then penetrate the gray matter of the spinal cord sequentially. Each class of sensory axons projects directly to its target lamina, never branching into inappropriate laminae en route. Some cutaneous afferents traverse the entire width of the spinal cord to reach superficial laminae on the contralateral side, strictly avoiding both the ventral spinal cord and inappropriate laminae of the deep dorsal horn. The pathways taken by developing sensory afferents are compatible with the idea that cells in inappropriate laminae exert inhibitory influences on sensory axons which regulate their laminar specificity. J. Comp. Neurol. 380:215–229, 1997. © 1997 Wiley-Liss, Inc.

Laminins are heterotrimers of alpha, beta, and gamma chains. At present, five alpha, three beta, and two gamma chains have been described. The best characterized laminin (laminin 1 = alpha 1, beta 1, gamma 1) promotes neurite outgrowth from virtually all classes of developing neurons, implying that laminins may serve as axon guidance molecules in vivo. Moreover, different laminin trimers exert distinct effects on subsets of laminin-1-responsive cells, suggesting that isoform diversity may underlie some axonal choices in vivo. As a first step toward evaluating these hypotheses, we have documented the expression patterns of all 10-known laminin chains in the peripheral nervous system and spinal cord of the murine embryo. The alpha 2, alpha 4, beta 1, and gamma 1 chains are expressed in peripheral axonal pathways by embryonic day (E) 11.5, when sensory and motor axonal outgrowth is underway. Thus, laminins (but not laminin 1) may promote peripheral axonal outgrowth. By E 13.5, laminin chains are differentially expressed in the limb-bud, with prominent expression of alpha 2 and alpha 4 in muscle and of alpha 3 and alpha 5 in skin. This pattern raises the possibility that laminin isoform diversity contributes to the ability of cutaneous and muscle sensory axons to distinguish their targets. Later in development, some chains (e.g., alpha 2, alpha 4, and beta 1) are downregulated in peripheral nerve while others (e.g., gamma 1), continue to be expressed by Schwann cells into adulthood. In contrast to peripheral nerves and ganglia, laminin chains are expressed at low levels, if at all, in the developing spinal cord gray matter.

Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

We describe a novel semaphorin family member, Sema VIa, with 25–36% sequence identity at the amino acid level in the semaphorin domain to previously published mouse homologues. This novel family member shares considerable homology with the best characterized murine semaphorin, Sema III (also known as SemD), at the 5′ end but is divergent from Sema III near the 3′ end because it contains a putative transmembrane domain. Remarkably, of the known semaphorins, Sema VIa bears the greatest structural similarity to insect Sema I, although it contains a much larger intracellular domain. We propose, therefore, that Sema VIa is the prototype of a new class (class VI) of semaphorins. In order to gain insights into potential functions of Sema VIa, we have compared mRNA expression of Sema VIa to that of Sema III during development. In the nervous system, Sema VIa is expressed in strikingly localized and transient patterns that are markedly different from those of Sema III. Interestingly, Sema VIa and Sema III frequently exhibit complementary or adjacent loci of expression. We suggest that Sema VIa may be important to nervous system development via a mechanism that involves cell–cell communication.