Lateral Motor Column Research Articles

The histone demethylase Kdm6b regulates subtype diversification of mouse spinal motor neurons during development

How a single neuronal population diversifies into subtypes with distinct synaptic targets is a fundamental topic in neuroscience whose underlying mechanisms are unclear. Here, we show that the histone H3-lysine 27 demethylase Kdm6b regulates the diversification of motor neurons to distinct subtypes innervating different muscle targets during spinal cord development. In mouse embryonic motor neurons, Kdm6b promotes the medial motor column (MMC) and hypaxial motor column (HMC) fates while inhibiting the lateral motor column (LMC) and preganglionic motor column (PGC) identities. Our single-cell RNA-sequencing analyses reveal the heterogeneity of PGC, LMC, and MMC motor neurons. Further, our single-cell RNA-sequencing data, combined with mouse model studies, demonstrates that Kdm6b acquires cell fate specificity together with the transcription factor complex Isl1-Lhx3. Our study provides mechanistic insight into the gene regulatory network regulating neuronal cell-type diversification and defines a regulatory role of Kdm6b in the generation of motor neuron subtypes in the mouse spinal cord.

TDP-43 is essential for Eph receptor-class-specific spinal motor axon trajectory into the limb

During the neural circuit formation, neuronal growth cones must be guided precisely to their neuronal or muscle targets, which can be achieved by the activation of membrane-bound guidance receptors at the periphery. However, the mechanisms that regulate the temporal availability of these receptors remain largely unknown. TAR DNA binding protein-43 (TDP-43) has been proposed to bind with the mRNAs of guidance receptors, thus prompting us to investigate its role in axon guidance of the spinal lateral motor column (LMC) neurons into the limb mesenchyme. We first identified the TDP-43 expression in the LMC neurons at the stage of axons growth into the limb using in situ mRNA hybridization. The loss and gain of TDP-43 function in chick LMC neurons redirected their axon trajectory with opposite effects. In mice, a spinal motor neuron-specific TDP-43 deletion led to the misrouting of LMC axons. Further, ectopic TDP-43 expression increased EphB protein levels in LMC neurons, suggesting that TDP-43 mediates LMC pathfinding by regulating EphB expression. Finally, TDP-43 levels influenced the growth preference of LMC neurites against ephrin-B, but not Netrin-1 and Semaphorin ligands. Our results demonstrate that TDP-43 is essential for the ephrinB:EphB signaling-mediated axon trajectory selection of LMC subtypes into the limb.

Vav2 is required for Netrin-1 receptor-class-specific spinal motor axon guidance.

Proper guidance of neuronal axons to their targets is required to assemble neural circuits during the development of the nervous system. However, the mechanism by which the guidance of axonal growth cones is regulated by specific intermediaries activated by receptor signaling pathways to mediate cytoskeleton dynamics is unclear. Vav protein members have been proposed to mediate this process, prompting us to investigate their role in the limb selection of the axon trajectory of spinal lateral motor column (LMC) neurons. We found Vav2 and Vav3 expression in LMC neurons when motor axons grew into the limb. Vav2, but not Vav3, loss-of-function perturbed LMC pathfinding, while Vav2 gain-of-function exhibited the opposite effects, demonstrating that Vav2 plays an important role in motor axon growth. Vav2 knockdown also attenuated the redirectional phenotype of LMC axons induced by Dcc, but not EphA4, in vivo and lateral LMC neurite growth preference to Netrin-1 in vitro. This study showed that Vav2 knockdown and ectopic nonphosphorylable Vav2 mutant expression abolished the Src-induced stronger growth preference of lateral LMC neurites to Netrin-1, suggesting that Vav2 is downstream of Src in this context. Vav2 is essential for Netrin-1-regulated LMC motor axon pathfinding through Src interaction.

Paxillin Is Required for Proper Spinal Motor Axon Growth into the Limb.

To assemble the functional circuits of the nervous system, the neuronal axonal growth cones must be precisely guided to their proper targets, which can be achieved through cell-surface guidance receptor activation by ligand binding in the periphery. We investigated the function of paxillin, a focal adhesion protein, as an essential growth cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show paxillin expression in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Paxillin loss-of-function and gain-of-function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of paxillin in motor axon guidance. In addition, a neuron-specific paxillin deletion in mice led to LMC axon trajectory selection errors. We also show that knocking down paxillin attenuates the growth preference of LMC neurites against ephrins in vitro, and erythropoietin-producing human hepatocellular (Eph)-mediated retargeting of LMC axons in vivo, suggesting paxillin involvement in Eph-mediated LMC motor axon guidance. Finally, both paxillin knockdown and ectopic expression of a nonphosphorylable paxillin mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating paxillin as a Src target in Eph signal relay in this context. In summary, our findings demonstrate that paxillin is required for motor axon guidance and suggest its essential role in the ephrin-Eph signaling pathway resulting in motor axon trajectory selection.SIGNIFICANCE STATEMENT During the development of neural circuits, precise connections need to be established among neurons or between neurons and their muscle targets. A protein family found in neurons, Eph, is essential at different stages of neural circuit formation, including nerve outgrowth and pathfinding, and is proposed to mediate the onset and progression of several neurodegenerative diseases, such as Alzheimer's disease. To investigate how Ephs relay their signals to mediate nerve growth, we investigated the function of a molecule called paxillin and found it important for the development of spinal nerve growth toward their muscle targets, suggesting its role as an effector of Eph signals. Our work could thus provide new information on how neuromuscular connectivity is properly established during embryonic development.

A Fiber Alginate Co-culture Platform for the Differentiation of mESC and Modeling of the Neural Tube.

Alginate hydrogels are a commonly used substrate for in vitro 3D cell culture. These naturally derived biomaterials are highly tunable, biocompatible, and can be designed to mimic the elastic modulus of the adult brain at 1% w/v solution. Recent studies show that the molecular weight of the alginate can affect cell viability and differentiation. The relationship between the molecular weight, viscosity and ratio of G:M monomers of alginate hydrogels is complex, and the balance between these factors must be carefully considered when deciding on a suitable alginate hydrogel for stem cell research. This study investigates the formation of embryoid bodies (EB) from mouse embryonic stem cells, using low molecular weight (LMW) and high molecular weight (HMW) alginates. The cells are differentiated using a retinoic acid-based protocol, and the resulting aggregates are sectioned and stained for the presence of stem cells and the three germ layers (endoderm, mesoderm, and ectoderm). The results highlight that aggregates within LMW and HMW alginate are true EBs, as demonstrated by positive staining for markers of the three germ layers. Using tubular alginate scaffolds, formed with an adapted gradient maker protocol, we also propose a novel 3D platform for the patterned differentiation of mESCs, based on gradients of retinoic acid produced in situ by lateral motor column (LMC) motor neurons. The end product of our platform will be of great interest as it can be further developed into a powerful model of neural tube development.

Development of fin‐innervating motor neurons after peripheral target removal in medaka fish

Peripheral targets regulate the development and survival of the nerve centers that serve them, because the elimination of the target normally results in massive death of the developing neurons that innervate it. This widely accepted theory appears to be well supported by developing limbs and their innervation in tetrapods, but it is unclear whether this concept applies to primitive vertebrates that have paired appendages. In this study, we examined the development of spinal motor neurons following pectoral fin bud removal (FBR) in medaka fish. After FBR, motor axons initially extended to the plexus region in a morphologically normal pattern. During the period of fin innervation, motor axons in the FBR-medaka failed to form the normal brachial plexus and elongated ventrally toward the abdominal region. In the ventral horn that would normally innervate the pectoral fin, however, neurons did not undergo cell death following FBR. There were no differences in the numbers of axons in the ventral roots between the FBR and control sides. Motor neuron markers, RALDH2 and FOXP1, that are expressed in limb-innervating motor neurons in the lateral motor column in tetrapods, were also expressed in the ventral horns of both the control and FBR sides in medaka fish. These results suggest that, although both tetrapod and medaka motor neurons share the same molecular characteristics for innervating paired appendages, the fates of neurons differ following the removal of their peripheral target. Therefore, the relationship between the peripheral target and its nerve center may be altered among vertebrates.

The Geometry of Limb Motor Innervation is Controlled by the Dorsal–Ventral Compartment Boundary in the Chick Limbless Mutant

Precise control of limb muscles, and ultimately of limb movement, requires accurate motor innervation. Motor innervation of the vertebrate limb is established by sequential selection of trajectories at successive decision points. Motor axons of the lateral motor column (LMC) segregate at the base of the limb into two groups that execute a choice between dorsal and ventral tissue: medial LMC axons innervate the ventral limb, whereas lateral LMC axons innervate the dorsal limb. We investigated how LMC axons are targeted to the limb using the chick mutant limbless (ll), which has a dorsal transformation of the ventral limb mesenchyme. In ll the spatial pattern of motor projections to the limb is abnormal while their targeting is normal. While extensive, the dorsal transformation of the ll ventral limb mesenchyme is incomplete whereas the generation, specification and targeting of spinal motor neurons are apparently unaffected. Thus, the dorsal–ventral motor axon segregation is an active choice that is independent of the ratio between dorsal and ventral tissue but dependent on the presence of both tissues. Therefore, the fidelity of the motor projections to the limb depends on the presence of both dorsal and ventral compartments, while the geometry of motor projections is controlled by the position of limb dorsal–ventral compartment boundary.

Filtering the good from the bad: A focus on kidney development and disease to celebrate John Bertram's long‐standing career in anatomy and renal research

Filtering the good from the bad: A focus on kidney development and disease to celebrate John Bertram's long‐standing career in anatomy and renal research

Characterization of Human iPSC-derived Spinal Motor Neurons by Single-cell RNA Sequencing

Human induced pluripotent stem cells (iPSCs) offer the opportunity to generate specific cell types from healthy and diseased individuals, allowing the study of mechanisms of early human development, modelling a variety of human diseases, and facilitating the development of new therapeutics. Human iPSC-based applications are often limited by the variability among iPSC lines originating from a single donor, as well as the heterogeneity among specific cell types that can be derived from iPSCs. The ability to deeply phenotype different iPSC-derived cell types is therefore of primary importance to the successful and informative application of this technology. Here we describe a combination of motor neuron (MN) derivation and single-cell RNA sequencing approaches to generate and characterize specific MN subtypes obtained from human iPSCs. Our studies provide evidence for rapid and robust generation of MN progenitor cells that can give rise to a heterogenous population of MNs. Approximately 58% of human iPSC-derived MNs display molecular characteristics of lateral motor column MNs, with a number of molecularly distinct subpopulations present within this MN group. Roughly 19% of induced MNs resemble hypaxial motor column MNs, while ∼6% of induced MNs have features of median motor column MNs. The present study has the potential to improve our understanding of iPSC-derived MN subtype function and dysfunction, possibly leading to improved iPSC-based applications for the study of human MN biology and disease.

Ephrin-A5 potentiates netrin-1 axon guidance by enhancing Neogenin availability

Axonal growth cones are guided by molecular cues in the extracellular environment. The mechanisms of combinatorial integration of guidance signals at the growth cone cell membrane are still being unravelled. Limb-innervating axons of vertebrate spinal lateral motor column (LMC) neurons are attracted to netrin-1 via its receptor, Neogenin, and are repelled from ephrin-A5 through its receptor EphA4. The presence of both cues elicits synergistic guidance of LMC axons, but the mechanism of this effect remains unknown. Using fluorescence immunohistochemistry, we show that ephrin-A5 increases LMC growth cone Neogenin protein levels and netrin-1 binding. This effect is enhanced by overexpressing EphA4 and is inhibited by blocking ephrin-A5-EphA4 binding. These effects have a functional consequence on LMC growth cone responses since bath addition of ephrin-A5 increases the responsiveness of LMC axons to netrin-1. Surprisingly, the overexpression of EphA4 lacking its cytoplasmic tail, also enhances Neogenin levels at the growth cone and potentiates LMC axon preference for growth on netrin-1. Since netrins and ephrins participate in a wide variety of biological processes, the enhancement of netrin-1 signalling by ephrins may have broad implications.

Critical roles of ARHGAP36 as a signal transduction mediator of Shh pathway in lateral motor columnar specification.

During spinal cord development, Sonic hedgehog (Shh), secreted from the floor plate, plays an important role in the production of motor neurons by patterning the ventral neural tube, which establishes MN progenitor identity. It remains unknown, however, if Shh signaling plays a role in generating columnar diversity of MNs that connect distinct target muscles. Here, we report that Shh, expressed in MNs, is essential for the formation of lateral motor column (LMC) neurons in vertebrate spinal cord. This novel activity of Shh is mediated by its downstream effector ARHGAP36, whose expression is directly induced by the MN-specific transcription factor complex Isl1-Lhx3. Furthermore, we found that AKT stimulates the Shh activity to induce LMC MNs through the stabilization of ARHGAP36 proteins. Taken together, our data reveal that Shh, secreted from MNs, plays a crucial role in generating MN diversity via a regulatory axis of Shh-AKT-ARHGAP36 in the developing mouse spinal cord.

Mir-17∼92 Confers Motor Neuron Subtype Differential Resistance to ALS-Associated Degeneration.

Progressive degeneration of motor neurons (MNs) is the hallmark of amyotrophic lateral sclerosis (ALS). Limb-innervating lateral motor column MNs (LMC-MNs) seem to be particularly vulnerable and are among the first MNs affected in ALS. Here, we report association of this differential susceptibility with reduced expression of the mir-17∼92 cluster in LMC-MNs prior to disease onset. Reduced mir-17∼92 is accompanied by elevated nuclear PTEN in spinal MNs of presymptomatic SOD1G93A mice. Selective dysregulation of the mir-17∼92/nuclear PTEN axis in degenerating SOD1G93A LMC-MNs was confirmed in a double-transgenic embryonic stem cell system and recapitulated in human SOD1+/L144F-induced pluripotent stem cell (iPSC)-derived MNs. We further show that overexpression of mir-17∼92 significantly rescues human SOD1+/L144F MNs, and intrathecal delivery of adeno-associated virus (AAV)9-mir-17∼92 improves motor deficits and survival in SOD1G93A mice. Thus, mir-17∼92 may have value as a prognostic marker of MN degeneration and is a candidate therapeutic target in SOD1-linked ALS. VIDEO ABSTRACT.

Nck2 is essential for limb trajectory selection by spinal motor axons

The development of a functioning nervous system requires precise assembly of neuronal connections, which can be achieved by the guidance of axonal growth cones to their proper targets. How axons are guided by signals transmitted to the cytoskeleton through cell surface-expressed guidance receptors remains unclear. We investigated the function of Nck2 adaptor protein as an essential guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory into the limb. Nck2 mRNA and protein are preferentially expressed in the medial subgroups of chick LMC neurons during axon trajectory into the limb. Nck2 loss- and gain-of-function in LMC neurons using in ovo electroporation perturb LMC axon trajectory selection demonstrating an essential role of Nck2 in motor axon guidance. We also showed that Nck2 knockdown and overexpression perturb the growth preference of LMC neurites against ephrins in vitro and Eph-mediated redirection of LMC axons in vivo. Finally, the significant changes of LMC neurite growth preference against ephrins in the context of Nck2 and α2-chimaerin loss- and gain-of-function implicated Nck2 function to modulate α2-chimaerin activity. Here, we showed that Nck2 is required for Eph-mediated axon trajectory selection from spinal motor neurons through possible interaction with α2-chimaerin. Developmental Dynamics 247:1043-1056, 2018. © 2018 Wiley Periodicals, Inc.

All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS.

SummaryHuman induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, “Optopatch,” pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

Influence of Brain Stem on Axial and Hindlimb Spinal Locomotor Rhythm Generating Circuits of the Neonatal Mouse.

The trunk plays a pivotal role in limbed locomotion. Yet, little is known about how the brain stem controls trunk activity during walking. In this study, we assessed the spatiotemporal activity patterns of axial and hindlimb motoneurons (MNs) during drug-induced fictive locomotor-like activity (LLA) in an isolated brain stem-spinal cord preparation of the neonatal mouse. We also evaluated the extent to which these activity patterns are affected by removal of brain stem. Recordings were made in the segments T7, L2, and L5 using calcium imaging from individual axial MNs in the medial motor column (MMC) and hindlimb MNs in lateral motor column (LMC). The MN activities were analyzed during both the rhythmic and the tonic components of LLA, the tonic component being used as a readout of generalized increase in excitability in spinal locomotor networks. The most salient effect of brain stem removal was an increase in locomotor rhythm frequency and a concomitant reduction in burst durations in both MMC and LMC MNs. The lack of effect on the tonic component of LLA indicated specificity of action during the rhythmic component. Cooling-induced silencing of the brain stem reproduced the increase in rhythm frequency and accompanying decrease in burst durations in L2 MMC and LMC, suggesting a dependency on brain stem neuron activity. The work supports the idea that the brain stem locomotor circuits are operational already at birth and further suggests an important role in modulating trunk activity. The brain stem may influence the axial and hindlimb spinal locomotor rhythm generating circuits by extending their range of operation. This may represent a critical step of locomotor development when learning how to walk in different conditions and environments is a major endeavor.

The developmental and genetic basis of 'clubfoot' in the peroneal muscular atrophy mutant mouse.

Genetic factors underlying the human limb abnormality congenital talipes equinovarus ('clubfoot') remain incompletely understood. The spontaneous autosomal recessive mouse 'peroneal muscular atrophy' mutant (PMA) is a faithful morphological model of human clubfoot. In PMA mice, the dorsal (peroneal) branches of the sciatic nerves are absent. In this study, the primary developmental defect was identified as a reduced growth of sciatic nerve lateral motor column (LMC) neurons leading to failure to project to dorsal (peroneal) lower limb muscle blocks. The pma mutation was mapped and a candidate gene encoding LIM-domain kinase 1 (Limk1) identified, which is upregulated in mutant lateral LMC motor neurons. Genetic and molecular analyses showed that the mutation acts in the EphA4-Limk1-Cfl1/cofilin-actin pathway to modulate growth cone extension/collapse. In the chicken, both experimental upregulation of Limk1 by electroporation and pharmacological inhibition of actin turnover led to defects in hindlimb spinal motor neuron growth and pathfinding, and mimicked the clubfoot phenotype. The data support a neuromuscular aetiology for clubfoot and provide a mechanistic framework to understand clubfoot in humans.

Ephexin1 Is Required for Eph-Mediated Limb Trajectory of Spinal Motor Axons.

The precise assembly of a functional nervous system relies on the guided migration of axonal growth cones, which is made possible by signals transmitted to the cytoskeleton by cell surface-expressed guidance receptors. We investigated the function of ephexin1, a Rho guanine nucleotide exchange factor, as an essential growth-cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show that ephexin1 is expressed in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Ephexin1 loss of function and gain of function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of ephexin1 in motor axon guidance. In addition, ephexin1 loss in mice of either sex led to LMC axon trajectory selection errors. We also show that ephexin1 knockdown attenuates the growth preference of LMC neurites against ephrins in vitro and Eph receptor-mediated retargeting of LMC axons in vivo, suggesting that ephexin1 is required in Eph-mediated LMC motor axon guidance. Finally, both ephexin1 knockdown and ectopic expression of nonphosphorylatable ephexin1 mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating ephexin1 as an Src target in Eph signal relay in this context. In summary, our findings demonstrate that ephexin1 is essential for motor axon guidance and suggest an important role in relaying ephrin:Eph signals that mediate motor axon trajectory selection.SIGNIFICANCE STATEMENT The proper development of functioning neural circuits requires precise nerve connections among neurons or between neurons and their muscle targets. The Eph tyrosine kinase receptors expressed in neurons are important in many contexts during neural-circuit formation, such as axon outgrowth, axon guidance, and synaptic formation, and have been suggested to be involved in neurodegenerative disorders, including amyotrophic lateral sclerosis and Alzheimer's disease. To dissect the mechanism of Eph signal relay, we studied ephexin1 gain of function and loss of function and found ephexin1 essential for the development of limb nerves toward their muscle targets, concluding that it functions as an intermediary to relay Eph signaling in this context. Our work could thus shed new light on the molecular mechanisms controlling neuromuscular connectivity during embryonic development.

Glial cell line-derived neurotrophic factor as a treatment after spinal cord injury.

Glial cell line-derived neurotrophic factor as a treatment after spinal cord injury.

Columnar-Intrinsic Cues Shape Premotor Input Specificity in Locomotor Circuits

Control of movement relies on the ability of circuits within the spinal cord to establish connections with specific subtypes of motor neuron (MN). Although the pattern of output from locomotor networks can be influenced by MN position and identity, whether MNs exert an instructive role in shaping synaptic specificity within the spinal cord is unclear. We show that Hox transcription-factor-dependent programs in MNs are essential in establishing the central pattern of connectivity within the ventral spinal cord. Transformation of axially projecting MNs to a limb-level lateral motor column (LMC) fate, through mutation of the Hoxc9 gene, causes the central afferents of limb proprioceptive sensory neurons to targetMNs connected to functionally inappropriate muscles. MN columnar identity also determines the pattern and distribution of inputs from multiple classes of premotor interneurons, indicating that MNs broadly influence circuit connectivity. These findings indicate that MN-intrinsic programs contribute to the initial architecture of locomotor circuits.

Motor neurons with limb-innervating character in the cervical spinal cord are sculpted by apoptosis based on the Hox code in chick embryo.

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1+ MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1+ MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1+ MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1+ MNs committed to LMC neurons, depending on the Hox expression pattern.