Caenorhabditis elegans sine oculis/SIX-type homeobox genes act as homeotic switches to define neuronal subtype identities.

The classification of neurons into distinct types reveals hierarchical taxonomic relationships that reflect the extent of similarity between neuronal cell types. At the base of such taxonomies are neuronal cells that are very similar to one another but differ in a small number of reproducible and select features. How are very similar members of a neuron class that share many features instructed to diversify into distinct subclasses? We show here that the six very similar members of the Caenorhabditis elegans IL2 sensory neuron class, which are all specified by a homeobox terminal selector, unc-86/BRN3, differentiate into two subtly distinct subclasses, a dorsoventral subclass and a lateral subclass, by the toggle switch-like action of the sine oculis/SIX homeobox gene unc-39. unc-39 is expressed only in the lateral IL2 neurons, and loss of unc-39 leads to a homeotic transformation of the lateral into the dorsoventral class; conversely, ectopic unc-39 expression converts the dorsoventral subclass into the lateral subclass. Hence, a terminal selector homeobox gene controls both class- as well as subclass-specific features, while a subordinate homeobox gene determines the ability of the class-specific homeobox gene to activate subtype-specific target genes. We find a similar regulatory mechanism operating in a distinct class of six motor neurons. Our findings underscore the importance of homeobox genes in neuronal identity control and invite speculations about homeotic identity transformations as potential drivers of evolutionary novelty during cell-type evolution in the brain.

Distinct subtypes of proprioceptive dorsal root ganglion neurons regulate adaptive proprioception in mice.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice.

Efficient and rapid conversion of human astrocytes and ALS mouse model spinal cord astrocytes into motor neuron-like cells by defined small molecules.

BACKGROUND: Motor neuron degeneration or loss in the spinal cord is the characteristic phenotype of motor neuron diseases or spinal cord injuries. Being proliferative and located near neurons, astrocytes are considered ideal cell sources for regenerating neurons. METHODS: We selected and tested different combinations of the small molecules for inducing the conversion of human and mouse astrocytes into neurons. Microscopic imaging and immunocytochemistry analyses were used to characterize the morphology and phenotype of the induced neurons while RT-qPCR was utilized to analyze changes in gene expression. In addition, whole-cell patch-clamp recordings were measured to examine the electrophysiological properties of induced neurons. RESULTS: The results showed that human astrocytes could be rapidly and efficiently converted into motor neuron-like cells by treatment with defined small molecules, with a yield of over 85% motor neuron-like cells attained. The induced motor neuron-like cells expressed the pan-neuronal markers TUJ1, MAP2, NeuN, and Synapsin 1 and motor neuron markers HB9, ISL1, CHAT, and VAChT. During the conversion process, the cells did not pass through a proliferative neural progenitor cell intermediate. The induced motor neurons were functional, showing the electrophysiological properties of neurons. The same chemical cocktail could induce spinal cord astrocytes from an amyotrophic lateral sclerosis mouse model carrying a SOD1 mutation to become motor neuron-like cells that exhibited a decrease in cell survival and an increase in oxidative stress compared to that observed in wild-type MNs derived from healthy mice. Moreover, the chemical induction reduced oxidative stress in the mutant astrocytes. CONCLUSION: The results of the present study demonstrated the feasibility of chemically converting human and mouse astrocytes into motor neuron-like cells that are useful for neurodegenerative disease modeling and regenerative medicine.

Motor Unit Number Estimate and Isometric Hand Grip Strength in Military Veterans with or Without Muscular Complaints: Reference Values for Longitudinal Follow-up.

INTRODUCTION: It remains unclear if Gulf War (GW) veterans have a higher risk of developing motor neuron disorder. We intended to establish baseline neurophysiological values, including thenar motor unit number estimate (MUNE) and isometric hand grip (IHG) strength, to compare future follow-ups of deployed GW veterans with or without muscular complaints. MATERIALS AND METHODS: We evaluated 19 GW veterans with self-reported weakness, cramps, or excessive muscle fatigue (Ill-19) and compared them with 18 controls without such muscular complaints (C-18). We performed MUNE on hand thenar muscles using adapted multipoint stimulation method for Ill-19 and 15 controls (C-15). We measured IHG strength (maximum force, endurance, and fatigue level) on Ill-19 and C-18 with a hand dynamometer. We performed nerve conduction studies on all study participants to determine which subjects had mild carpal tunnel syndrome (CTS). We compared the MUNE and IHG strength measures between Ill group and controls and between those with CTS and those without CTS. RESULTS: We obtained thenar MUNE of Ill-19 (95% CI of mean: 143-215; mean age: 46 yr) and compared it with that of C-15 (95% CI of mean: 161-230; mean age: 45 yr), and 95% of CI of mean among IHG strength variables (maximum force: 324-381 Newton; endurance: 32-42 s; fatigue level: 24%-33%) compared with C-18 (maximum force: 349-408 Newton; endurance: 35-46 s; fatigue level: 21%-27%). There was no significant difference in either MUNE or IHG strength between Ill-19 group and controls. The MUNE and IHG maximum forces were significantly lower in those with CTS compared with those without CTS. As a surrogate of mild CTS, the median versus ulnar distal sensory latency on nerve conduction study was only weakly associated with MUNE, maximum force, and fatigue level, respectively. CONCLUSION: To our knowledge, no published study on MUNE reference values of military veteran population has been available. The quantifiable values of both thenar MUNE and IHG strength of military veterans serve as baselines for our longitudinal follow-up of motor neuron function of deployed troops. These reference values are also useful for other laboratories to study veterans' motor system with or without mild CTS.

Gaskell revisited: new insights into spinal autonomics necessitate a revised motor neuron nomenclature.

Several concepts developed in the nineteenth century have formed the basis of much of our neuroanatomical teaching today. Not all of these were based on solid evidence nor have withstood the test of time. Recent evidence on the evolution and development of the autonomic nervous system, combined with molecular insights into the development and diversification of motor neurons, challenges some of the ideas held for over 100 years about the organization of autonomic motor outflow. This review provides an overview of the original ideas and quality of supporting data and contrasts this with a more accurate and in depth insight provided by studies using modern techniques. Several lines of data demonstrate that branchial motor neurons are a distinct motor neuron population within the vertebrate brainstem, from which parasympathetic visceral motor neurons of the brainstem evolved. The lack of an autonomic nervous system in jawless vertebrates implies that spinal visceral motor neurons evolved out of spinal somatic motor neurons. Consistent with the evolutionary origin of brainstem parasympathetic motor neurons out of branchial motor neurons and spinal sympathetic motor neurons out of spinal motor neurons is the recent revision of the organization of the autonomic nervous system into a cranial parasympathetic and a spinal sympathetic division (e.g., there is no sacral parasympathetic division). We propose a new nomenclature that takes all of these new insights into account and avoids the conceptual misunderstandings and incorrect interpretation of limited and technically inferior data inherent in the old nomenclature.

Distinct projection targets define subpopulations of mouse brainstem vagal neurons that express the autism-associated MET receptor tyrosine kinase.

Detailed anatomical tracing and mapping of the viscerotopic organization of the vagal motor nuclei has provided insight into autonomic function in health and disease. To further define specific cellular identities, we paired information based on visceral connectivity with a cell-type specific marker of a subpopulation of neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (nAmb) that express the autism-associated MET receptor tyrosine kinase. As gastrointestinal disturbances are common in children with autism spectrum disorder (ASD), we sought to define the relationship between MET-expressing (MET+) neurons in the DMV and nAmb, and the gastrointestinal tract. Using wholemount tissue staining and clearing, or retrograde tracing in a METEGFP transgenic mouse, we identify three novel subpopulations of EGFP+ vagal brainstem neurons: (a) EGFP+ neurons in the nAmb projecting to the esophagus or laryngeal muscles, (b) EGFP+ neurons in the medial DMV projecting to the stomach, and (b) EGFP+ neurons in the lateral DMV projecting to the cecum and/or proximal colon. Expression of the MET ligand, hepatocyte growth factor (HGF), by tissues innervated by vagal motor neurons during fetal development reveal potential sites of HGF-MET interaction. Furthermore, similar cellular expression patterns of MET in the brainstem of both the mouse and nonhuman primate suggests that MET expression at these sites is evolutionarily conserved. Together, the data suggest that MET+ neurons in the brainstem vagal motor nuclei are anatomically positioned to regulate distinct portions of the gastrointestinal tract, with implications for the pathophysiology of gastrointestinal comorbidities of ASD.

A distinct functional distribution of α and γ motoneurons in the rat trigeminal motor nucleus.

Gamma-motoneurons (γMNs) play a crucial role in regulating isometric muscle contraction. The slow jaw-closing during mastication is one of the most functional isometric contractions, which is developed by the rank-order recruitment of alpha-motoneurons (αMNs) in a manner that reflects the size distribution of αMNs. In a mouse spinal motor nucleus, there are two populations of small and large MNs; the former was identified as a population of γMNs based on the positive expression of the transcription factor estrogen-related receptor 3 (Err3) and negative expression of the neuronal DNA-binding protein NeuN, and the latter as that of αMNs based on the opposite pattern of immunoreactivity. However, the differential identification of αMNs and γMNs in the trigeminal motor nucleus (TMN) remains an assumption based on the size of cell bodies that were retrogradely stained with HRP. We here examined the size distributions of αMNs and γMNs in the dorsolateral TMN (dl-TMN) by performing immunohistochemistry using anti-Err3 and anti-NeuN antibodies. The dl-TMN was identified by immunopositivity for vesicular glutamate transporter-1. Immunostaining for choline acetyltransferase and Err3/NeuN revealed that the dl-TMN is composed of 65% αMNs and 35% γMNs. The size distribution of αMNs was bimodal, while that of γMNs was almost the same as that of the population of small αMNs, suggesting the presence of αMNs as small as γMNs. Consistent with the size concept of motor units, the presence of smaller jaw-closing αMNs was coherent with the inclusion of jaw-closing muscle fibers with smaller diameters compared to limb muscle fibers.

Meis/UNC-62 isoform dependent regulation of CoupTF-II/UNC-55 and GABAergic motor neuron subtype differentiation.

Gene regulatory networks orchestrate the assembly of functionally related cells within a cellular network. Subtle differences often exist among functionally related cells within such networks. How differences are created among cells with similar functions has been difficult to determine due to the complexity of both the gene and the cellular networks. In Caenorhabditis elegans, the DD and VD motor neurons compose a cross-inhibitory, GABAergic network that coordinates dorsal and ventral muscle contractions during locomotion. The Pitx2 homologue, UNC-30, acts as a terminal selector gene to create similarities and the Coup-TFII homologue, UNC-55, is necessary for creating differences between the two motor neuron classes. What is the organizing gene regulatory network responsible for initiating the expression of UNC-55 and thus creating differences between the DD and VD motor neurons? We show that the unc-55 promoter has modules that contain Meis/UNC-62 binding sites. These sites can be subdivided into regions that are capable of activating or repressing UNC-55 expression in different motor neurons. Interestingly, different isoforms of UNC-62 are responsible for the activation and the stabilization of unc-55 transcription. Furthermore, specific isoforms of UNC-62 are required for proper synaptic patterning of the VD motor neurons. Isoform specific regulation of differentiating neurons is a relatively unexplored area of research and presents a mechanism for creating differences among functionally related cells within a network.

Targeted assessment of lower motor neuron burden is associated with survival in amyotrophic lateral sclerosis.

Estimating survival in amyotrophic lateral sclerosis (ALS) is challenging due to heterogeneity in clinical features of disease and a lack of suitable markers that predict survival. Our aim was to determine whether scoring of upper or lower motor neuron weakness is associated with survival. With this objective, 161 ALS subjects were recruited from two tertiary referral centres. Scoring of upper (UMN) and lower motor neuron (LMN) signs was performed, in addition to a brief questionnaire. Subjects were then followed until the censorship date. Univariate analysis was performed to identify variables associated with survival to either non-invasive ventilation (NIV) or death, which were then further characterized using Cox regression. Results showed that factors associated with reduced survival included older age, bulbar and respiratory involvement and shorter diagnostic delay (all p < 0.05). Whole body LMN score was strongly associated with time to NIV or death (p ≤0.001) whereas UMN scores were poorly associated with survival. In conclusion, our results suggest that, early in disease assessment and in the context of other factors (age, bulbar, respiratory status), the burden of LMN weakness provides an accurate estimate of outcome. Such a scoring system could predict prognosis, and thereby aid in selection of patients for clinical trials.

A structural and genotypic scaffold underlying temporal integration.

The accumulation and storage of information over time, temporal integration, is key to numerous behaviors. Many oculomotor tasks depend on integration of eye-velocity signals to eye-position commands, a transformation achieved by a hindbrain cell group termed the velocity-to-position neural integrator (VPNI). Although the VPNI's coding properties have been well characterized, its mechanism of function remains poorly understood because few links exist between neuronal activity, structure, and genotypic identity. To fill this gap, we used calcium imaging and single-cell electroporation during oculomotor behaviors to map VPNI neural activity in zebrafish onto a hindbrain scaffold consisting of alternating excitatory and inhibitory parasagittal stripes. Three distinct classes of VPNI cells were identified. One glutamatergic class was medially located along a stripe associated with the alx transcription factor; these cells had ipsilateral projections terminating near abducens motoneurons and collateralized extensively within the ipsilateral VPNI in a manner consistent with integration through recurrent excitation. A second glutamatergic class was more laterally located along a stripe associated with transcription factor dbx1b; these glutamatergic cells had contralateral projections collateralizing near abducens motoneurons, consistent with a role in disconjugate eye movements. A third class, immunohistochemically suggested to be GABAergic, was located primarily in the dbx1b stripe and also had contralateral projections terminating near abducens motoneurons; these cells collateralized extensively in the dendritic field of contralateral VPNI neurons, consistent with a role in coordinating activity between functionally opposing populations. This mapping between VPNI activity, structure, and genotype may provide a blueprint for understanding the mechanisms governing temporal integration.

Accumulation of glycogen in axotomized adult rat facial motoneurons.

This study biochemically determined glycogen content in the axotomized facial nucleus of adult rats up to 35 days postinsult. The amounts of glycogen in the transected facial nucleus were significantly increased at 5 days postinsult, peaked at 7 days postinsult, and declined to the control levels at 21-35 days postinsult. Immunohistochemical analysis with antiglycogen antibody revealed that the quantity of glycogen granules in the axotomized facial nucleus was greater than that in the control nucleus at 7 days postinjury. Dual staining methods with antiglycogen antibody and a motoneuron marker clarified that the glycogen was localized mainly in motoneurons. Immunoblotting and quantification analysis revealed that the ratio of inactive glycogen synthase (GS) to total GS was significantly decreased in the injured nucleus at about 1-3 days postinsult and significantly increased from 7 to 14 days postinsult, suggesting that glycogen is actively synthesized in the early period postinjury but suppressed after 7 days postinsult. The enhanced glycogen at about 5-7 days postinsult is suggested to be responsible for the decrease in inactive GS levels, and the decrease of glycogen after 7 days postinsult is considered to be caused by increased inactive GS levels and possibly the increase in active glycogen phosphorylase.

Gene co-regulation by Fezf2 selects neurotransmitter identity and connectivity of corticospinal neurons.

The neocortex contains an unparalleled diversity of neuronal subtypes, each defined by distinct traits that are developmentally acquired under the control of subtype-specific and pan-neuronal genes. The regulatory logic that orchestrates the expression of these unique combinations of genes is unknown for any class of cortical neuron. Here, we report that Fezf2 is a selector gene able to regulate the expression of gene sets that collectively define mouse corticospinal motor neurons (CSMN). We find that Fezf2 directly induces the glutamatergic identity of CSMN via activation of Vglut1 (Slc17a7) and inhibits a GABAergic fate by repressing transcription of Gad1. In addition, we identify the axon guidance receptor EphB1 as a target of Fezf2 necessary to execute the ipsilateral extension of the corticospinal tract. Our data indicate that co-regulated expression of neuron subtype-specific and pan-neuronal gene batteries by a single transcription factor is one component of the regulatory logic responsible for the establishment of CSMN identity.

Selective disruption of acetylcholine synthesis in subsets of motor neurons: a new model of late-onset motor neuron disease.

Motor neuron diseases are characterized by the selective chronic dysfunction of a subset of motor neurons and the subsequent impairment of neuromuscular function. To reproduce in the mouse these hallmarks of diseases affecting motor neurons, we generated a mouse line in which ~40% of motor neurons in the spinal cord and the brainstem become unable to sustain neuromuscular transmission. These mice were obtained by conditional knockout of the gene encoding choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine. The mutant mice are viable and spontaneously display abnormal phenotypes that worsen with age including hunched back, reduced lifespan, weight loss, as well as striking deficits in muscle strength and motor function. This slowly progressive neuromuscular dysfunction is accompanied by muscle fiber histopathological features characteristic of neurogenic diseases. Unexpectedly, most changes appeared with a 6-month delay relative to the onset of reduction in ChAT levels, suggesting that compensatory mechanisms preserve muscular function for several months and then are overwhelmed. Deterioration of mouse phenotype after ChAT gene disruption is a specific aging process reminiscent of human pathological situations, particularly among survivors of paralytic poliomyelitis. These mutant mice may represent an invaluable tool to determine the sequence of events that follow the loss of function of a motor neuron subset as the disease progresses, and to evaluate therapeutic strategies. They also offer the opportunity to explore fundamental issues of motor neuron biology.

Localization and chemical coding of the dorsal motor vagal nucleus (DMX) neurons projecting to the porcine stomach prepyloric area in the physiological state and after stomach partial resection.

The aim of our study was to localize and define immunocytochemical characteristic of the dorsal motor nucleus of the vagus (DMX) neurons projecting to the porcine stomach prepyloric region in the physiological state and after gastric partial resection. To identify the stomach-projecting perikarya, the neuronal retrograde tracer--Fast Blue (FB) was injected into the studied region of control and resection group (RES). In the RES group, on 22nd day after FB injection, the partial resection of the stomach region previously injected with FB was performed. Sections were immunostained with ChAT, pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), galanin (GAL), substance P (SP), leu-enkephalin (LENK), and cocaine- and amphetamine-regulated transcript (CART). In the DMX of control and RES group, the stomach-projecting perikarya were found in the entire extent of the nucleus bilaterally. Within control animals, 30.08 ± 1.97 % of the gastric DMX perikarya expressed PACAP, while other substances were found only in the neuronal fibers. In the RES group DMX, PACAP was found in 45.58 ± 2.2 %, VIP in 28.83 ± 3.63 %, NOS in 21.22 ± 3.32 %, and GAL in 5.67 ± 1.49 % of the FB-labeled gastric perikarya. Our data implicate PACAP, VIP, NOS, and GAL as neuronal survival promoting substances and the CART-, LENK-, SP- NOS-, and GAL-immunoreactive processes in control of the gastric vagal neurons in the pig.

Generating spinal motor neuron diversity: a long quest for neuronal identity.

Understanding how thousands of different neuronal types are generated in the CNS constitutes a major challenge for developmental neurobiologists and is a prerequisite before considering cell or gene therapies of nervous lesions or pathologies. During embryonic development, spinal motor neurons (MNs) segregate into distinct subpopulations that display specific characteristics and properties including molecular identity, migration pattern, allocation to specific motor columns, and innervation of defined target. Because of the facility to correlate these different characteristics, the diversification of spinal MNs has become the model of choice for studying the molecular and cellular mechanisms underlying the generation of multiple neuronal populations in the developing CNS. Therefore, how spinal motor neuron subpopulations are produced during development has been extensively studied during the last two decades. In this review article, we will provide a comprehensive overview of the genetic and molecular mechanisms that contribute to the diversification of spinal MNs.

Morphology and ultrastructure of medial rectus subgroup motoneurons in the macaque monkey.

There are two muscle fiber types in extraocular muscles: those receiving a single motor endplate, termed singly innervated fibers (SIFs), and those receiving multiple small terminals along their length, termed multiply innervated fibers (MIFs). In monkeys, these two fiber types receive input from different motoneuron pools: SIF motoneurons found within the extraocular motor nuclei, and MIF motoneurons found along their periphery. For the monkey medial rectus muscle, MIF motoneurons are found in the C-group, while SIF motoneurons lie in the A- and B-groups. We analyzed the somatodendritic morphology and ultrastructure of these three subgroups of macaque medial rectus motoneurons to better understand the structural determinants controlling the two muscle fiber types. The dendrites of A- and B-group motoneurons lay within the oculomotor nucleus, but those of the C-group motoneurons were located outside the nucleus, and extended into the preganglionic Edinger-Westphal nucleus. A- and B-group motoneurons were very similar ultrastructurally. In contrast, C-group motoneurons displayed significantly fewer synaptic contacts on their somata and proximal dendrites, and those contacts were smaller in size and lacked dense-cored vesicles. However, the synaptic structure of C-group distal dendrites was quite similar to that observed for A- and B-group motoneurons. Our anatomical findings suggest that C-group MIF motoneurons have different physiological properties than A- and B-group SIF motoneurons, paralleling their different muscle fiber targets. Moreover, primate C-group motoneurons have evolved a special relationship with the preganglionic Edinger-Westphal nucleus, suggesting these motoneurons play an important role in near triad convergence to support increased near work requirements.

Single low-threshold afferents innervating the skin of the human foot modulate ongoing muscle activity in the upper limbs.

We have shown for the first time that single cutaneous afferents in the foot dorsum have significant reflex coupling to motoneurons supplying muscles in the upper limb, particularly posterior deltoid and triceps brachii. These observations strengthen what we know from whole nerve stimulation, that skin on the foot and ankle can contribute to the modulation of interlimb muscles in distant innervation territories. The current work provides evidence of the mechanism behind the reflex, where one single skin afferent can evoke a reflex response, rather than a population. Nineteen of forty-one (46%) single cutaneous afferents isolated in the dorsum or plantar surface of the foot elicited a significant modulation of muscle activity in the upper limb. Identification of single afferents in this reflex indicates the strength of the connection and, ultimately, the importance of foot skin in interlimb coordination. The median response magnitude was 2.29% of background EMG, and the size of the evoked response did not significantly differ among the four mechanoreceptor classes (P > 0.1). Interestingly, although the distribution of afferents types did not differ across the foot dorsum, there was a significantly greater coupling response from receptors located on the medial aspect of the foot dorsum (P < 0.01). Furthermore, the most consistent coupling with upper limb muscles was demonstrated by type I afferents (fast and slowly adapting). This work contributes to the current literature on receptor specificity, supporting the view that individual classes of cutaneous afferents may subserve specific roles in kinesthesia, reflexes, and tactile perception.

Proprioceptive coupling within motor neurons drives C. elegans forward locomotion.

Locomotion requires coordinated motor activity throughout an animal's body. In both vertebrates and invertebrates, chains of coupled central pattern generators (CPGs) are commonly evoked to explain local rhythmic behaviors. In C. elegans, we report that proprioception within the motor circuit is responsible for propagating and coordinating rhythmic undulatory waves from head to tail during forward movement. Proprioceptive coupling between adjacent body regions transduces rhythmic movement initiated near the head into bending waves driven along the body by a chain of reflexes. Using optogenetics and calcium imaging to manipulate and monitor motor circuit activity of moving C. elegans held in microfluidic devices, we found that the B-type cholinergic motor neurons transduce the proprioceptive signal. In C. elegans, a sensorimotor feedback loop operating within a specific type of motor neuron both drives and organizes body movement.

Zebrafish Mnx proteins specify one motoneuron subtype and suppress acquisition of interneuron characteristics.

BACKGROUND: Precise matching between motoneuron subtypes and the muscles they innervate is a prerequisite for normal behavior. Motoneuron subtype identity is specified by the combination of transcription factors expressed by the cell during its differentiation. Here we investigate the roles of Mnx family transcription factors in specifying the subtypes of individually identified zebrafish primary motoneurons. RESULTS: Zebrafish has three Mnx family members. We show that each of them has a distinct and temporally dynamic expression pattern in each primary motoneuron subtype. We also show that two Mnx family members are expressed in identified VeLD interneurons derived from the same progenitor domain that generates primary motoneurons. Surprisingly, we found that Mnx proteins appear unnecessary for differentiation of VeLD interneurons or the CaP motoneuron subtype. Mnx proteins are, however, required for differentiation of the MiP motoneuron subtype. We previously showed that MiPs require two temporally-distinct phases of Islet1 expression for normal development. Here we show that in the absence of Mnx proteins, the later phase of Islet1 expression is initiated but not sustained, and MiPs become hybrids that co-express morphological and molecular features of motoneurons and V2a interneurons. Unexpectedly, these hybrid MiPs often extend CaP-like axons, and some MiPs appear to be entirely transformed to a CaP morphology. CONCLUSIONS: Our results suggest that Mnx proteins promote MiP subtype identity by suppressing both interneuron development and CaP axon pathfinding. This is, to our knowledge, the first report of transcription factors that act to distinguish CaP and MiP subtype identities. Our results also suggest that MiP motoneurons are more similar to V2 interneurons than are CaP motoneurons.

A microfluidic device to investigate axon targeting by limited numbers of purified cortical projection neuron subtypes.

While much is known about general controls over axon guidance of broad classes of projection neurons (those with long-distance axonal connections), molecular controls over specific axon targeting by distinct neuron subtypes are poorly understood. Corticospinal motor neurons (CSMN) are prototypical and clinically important cerebral cortex projection neurons; they are the brain neurons that degenerate in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases, and their injury is central to the loss of motor function in spinal cord injury. Primary culture of purified immature murine CSMN has been recently established, using either fluorescence-activated cell sorting (FACS) or immunopanning, enabling a previously unattainable level of subtype-specific investigation, but the resulting number of CSMN is quite limiting for standard approaches to study axon guidance. We developed a microfluidic system specifically designed to investigate axon targeting of limited numbers of purified CSMN and other projection neurons in culture. The system contains two chambers for culturing target tissue explants, allowing for biologically revealing axonal growth "choice" experiments. This device will be uniquely enabling for investigation of controls over axon growth and neuronal survival of many types of neurons, particularly those available only in limited numbers.