Genes that control ray sensory neuron axon development in the Caenorhabditis elegans male

We have studied how a set of male-specific sensory neurons in Caenorhabditis elegans establish axonal connections during postembryonic development. In the adult male, 9 bilateral pairs of ray sensory neurons innervate an acellular fan that serves as a presumptive tactile and olfactory organ during copulation. We visualized ray axon commissures with a ray neuron-specific reporter gene and studied both known and new mutations that affect the establishment of connections to the pre-anal ganglion. We found that the UNC-6/netrin-UNC-40/DCC pathway provides the primary dorsoventral guidance cue to ray axon growth cones. Some axon growth cones also respond to an anteroposterior cue, following a segmented pathway, and most or all also have a tendency to fasciculate. Two newly identified genes, rax-1 and rax-4, are highly specific to the ray neurons and appear to be required for ray axon growth cones to respond to the dorsoventral cue. Among other genes we identified, rax-2 and rax-3 affect anteroposterior signaling or fate specification and rax-5 and rax-6 affect ray identities. We identified a mutation in sax-2 and show that the sax-2/Furry and sax-1/Tricornered pathway affects ectopic neurite outgrowth and establishment of normal axon synapses. Finally, we identified mutations in genes for muscle proteins that affect axon pathways by distorting the conformation of the body wall. Thus ray axon pathfinding relies on a variety of general and more ray neuron-specific genes and provides a potentially fruitful system for further studies of how migrating axon growth cones locate their targets. This system is applicable to the study of mechanisms underlying topographic mapping of sensory neurons into target circuitry where the next stage of information processing is carried out.     

Neuronal development: sorting out motor neurons

In the developing spinal cord, motor neurons become segregated into important functional units termed motor pools. Now it has been discovered that repertoires of cadherin surface molecules play key roles in motor pool sorting.     

Neuronal development: putting motor neurons in their place

In the developing spinal cord, motor neurons occupy discrete columns with different identities and axon projections. This organisation has now been shown to depend crucially on sequential phases of expression of Hox-c proteins, generated in response to fibroblast growth factor signals.     

A gene trap approach to identify genes that control development

One methodology called gene trap represents a versatile strategy by which murine genes that control developmental events can be captured and identified with corresponding mutants produced at the same time. Gene trap methodology has been developed and several genes and their mutants have been analyzed, but almost all of the genes reported are those already known or murine homologs of other species. In this study, the efficiency of the gene trap methodology was improved and a novel mutant mouse strain named jumonji established which displayed an intriguing defect. Homozygous fetal mice died in utero and a significant proportion of the homozygotes showed abnormal groove formation on the neural plate and a defect in neural tube closure with a mixed genetic background of 129/Ola and BALB/c. The trapped gene believed to be responsible for these phenotypes encodes a novel nuclear protein. The results reveal that the gene trap approach can identify unknown interesting genes in murine development. The gene trap strategy, however, has several problems, the greatest of which is the difficulty in prescreening embryonic stem (ES) cells for interesting trapped genes. Recent studies are solving this problem and show that the prescreening of ES cells for genes with several characteristics is possible.     

Molecular mechanisms regulating motor neuron development and degeneration

Motor neurons are a well-defined, although heterogeneous group of cells responsible for transmitting information from the central nervous system to the locomotor system. Spinal motor neurons are specified by soluble factors produced by structures adjacent to the primordial spinal cord, signaling through homeodomain proteins. Axonal pathfinding is regulated by cell-surface receptors that interact with extracellular lignads and once synaptic connections have formed, the survival of the somatic motor neuron is dependent on the provision of target-derived growth factors, although nontarget-derived factors, produced by either astrocytes or Schwann cells, are also potentially implicated. Somatic motor neuron degeneration leads to profound disability, and multiple pathogenetic mechanisms including aberrant growth factor signaling, abnormal neurofilament accumulation, excitotoxicity, and autoimmunity have been postulated to be responsible. Even when specific deficits have been identified, for example, mutations of the superoxide dismutase-1 gene in familial amyotrophic sclerosis and polyglutamine expansion of the androgen receptor in spinal and bulbar muscular atrophy, the mechanisms by which somatic motor neuronal degeneration occurs remain unclear. In order to treat motor system degeneration effectively, we will need to understand these mechanisms more thoroughly.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by gamma motor neurons may explain the increase of motor axons counted proximally to the lesion.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.     

Administration of Hsp70 in vivo inhibits motor and sensory neuron degeneration

The induction of heat shock proteins (Hsps) serves not only as a marker for cellular stress but also as a promoter of cell survival, which is especially important in the nervous system. We examined the regulation of the constitutive and stress-induced 70-kD Hsps (Hsc70 and Hsp70, respectively) after sciatic nerve (SN) axotomy in the neonatal mouse. Additionally, the prevention of axotomy-induced SN cell death by administration of several preparations of exogenous Hsc70 and Hsp70 was tested. Immunohistochemistry and Western blot analyses showed that endogenous levels of Hsc70 and Hsp70 did not increase significantly in lumbar motor neurons or dorsal root ganglion sensory neurons up to 24 hours after axotomy. When a variety of Hsc70 and Hsp70 preparations at doses ranging from 5 to 75 microg were applied to the SN stump after axotomy, the survival of both motor and sensory neurons was significantly improved. Thus, it appears that motor and sensory neurons in the neonatal mouse do not initiate a typical Hsp70 response after traumatic injury and that administration of exogenous Hsc/Hsp70 can remedy that deficit and reduce the subsequent loss of neurons by apoptosis.     

Neuronal death induced by endogenous extracellular ATP in retinal cholinergic neuron density control

The precise assembly of neuronal circuits requires that the correct number of pre- and postsynaptic neurons form synaptic connections. Neuronal cell number is thus tightly controlled by cell death during development. Investigating the regulation of cell number in the retina we found an ATP gated mechanism of neuronal death control. By degrading endogenous extracellular ATP or blocking the P2X(7) ATP receptors we found that endogenous extracellular ATP triggers the death of retinal cholinergic neurons during normal development. ATP-induced death eliminates cholinergic cells too close to one another, thereby controlling the total number, the local density and the regular spacing of these neurons.     

Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.     

Disruption of peripheral target contact influences the development of identified central dendritic branches in a leech motor neuron in vivo

Retrograde signaling from target tissues has been shown to influence many aspects of neuronal development in a number of developmental systems. In these experiments using embryonic leeches (Hirudo medicinalis), we examined how depriving a neuron of contact with its peripheral target affects the development of the cell's central arborization. We focused our attention on the motor neuron cell 3, which normally stimulates dorsal longitudinal muscle fibers to contract. At different locations in the periphery and in embryos of several different stages, we cut the nerve containing the growing axon of cell 3. This surgery led to dramatic overgrowth of cell 3's central dendritic branches, which normally accept synaptic contacts from other neurons, including the inhibitory motor neuron cell 1. When cell 3's peripheral axon was cut relatively early in development, its overgrown central branches eventually retracted. However, cells that were disrupted later in development retained their overextended branches into adulthood. In addition, if the axon was cut close to the ganglion early in development, depriving the cell of contact with any dorsal tissues, the central branches failed to retract and were instead retained into adulthood. Unlike cell 3, the central branches of cell 1, which has the same peripheral target muscles as cell 3, remained unchanged following all axotomy protocols. These results suggest that in at least some neurons contact with peripheral targets can influence development of the central processes that normally mediate synaptic contacts.     

Metals in motor neuron diseases

Degenerative processes within the nervous system are common features in disease entities such as dementia of Alzheimer type (DAT), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease with unknown etiology; widespread muscle wasting and respiratory failure lead to death within a few years. Denervation can be detected with electromyography and axonal deterioration monitored by motor unit number estimates. Several suggestions about the cause of ALS have emerged but no solid theory has yet precipitated. Lead or mercury exposure has been suggested. Exposure data alone cannot support this connection. Alterations in metal kinetics may underlie the deterioration of motor function observed in patients with ALS. In this review the role of metals in motor neuron disease is discussed. Both classic studies on exposure and recent understanding of metal binding proteins are considered. Aspects of peak exposure and excretion are merged toward an understanding of metal dynamics in ALS. An overview of chemical and electrophysiological investigations is given in the context of neurodegeneration.