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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.

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.

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.

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.

Determinations of dorsal root ganglion (DRG) neuron loss in nerve growth factor (NGF) and neurotrophin-3 (NT-3) null mutant mice have supported the concept that neurons can switch neurotrophin dependence by revealing that many neurons must require both of these factors acting either sequentially or simultaneously during development. The situation is complex, however, in thatNT-3(−/−)mutant mice show far greater neuron loss than mice deficient in the NT-3 receptor TrkC, suggesting that NT-3 may support many DRG neurons via actions on the NGF receptor TrkA. To assess the possibility of ligand-receptor cross-talk as a developmental mechanism, we have compared the onset of survival dependence of lumbar DRG neurons on NT-3, TrkC, NGF, and TrkA signaling in mice deficient in these molecules as a result of gene targeting. At embryonic day 11.5 (E11.5), virtually all lumbar DRG cells express TrkC mRNA and many require NT-3 and TrkC signaling for survival. In contrast, although many lumbar DRG cells also express TrkA at E11.5, there is little survival dependence on TrkA signaling. By E13.5, most lumbar DRG cells have downregulated TrkC mRNA. The onset of survival dependence on NGF and TrkA-signaling is concurrent and of equal magnitude at E13.5, demonstrating that NT-3 alone does not support DRG neurons via TrkA, nor can NT-3 compensate for the loss of NGF. We conclude that many murine DRG cells require NT-3 for survival before exhibiting NGF dependence and that NT-3 activation of TrkA is unimportant to these early NT-3 survival-promoting actions. We suggest that the discrepancy in cell loss betweenNT-3(−/−)andtrkC(−/−)mutants is attributable to the ability of NT-3 to support DRG neurons via TrkA in the artificial situation where TrkC is absent.

Recent advances in defining neurotrophin signaling mediators have provided insights into the signal transduction mechanisms that underlie axon growth. Evidence is accumulating that major Trk effectors regulate the morphological development of embryonic peripheral neurons. Less is known about signaling related to the robust axon extension that follows peripheral axotomy of adult neurons. Regenerative axon growth can be mimicked in vitro by a "conditioning" lesion performed 2 weeks before culture (Smith and Skene, 1997). Previous work has implicated both neurotrophins and cytokines in this response. Because signal transduction mediators of both of these families of growth factors are well characterized, we have compared the role of neurotrophin and cytokine signaling in developmental versus regenerative sensory axon growth. Chemical inhibitors were administrated to embryonic and axotomized sensory neurons in vitro to block the activation of Erk kinase (MEK)-extracellular signal-regulated kinase (ERK), phosphatidylinositol-3 kinase (PI3-K), and janus kinase (JAK) signaling. As expected, both MEK and PI3-K inhibition blocked axon growth from both naive and NGF-stimulated embryonic day 13 sensory neurons, whereas inhibition of JAK phosphorylation had no effect. In contrast, neither MEK nor PI3-K inhibitors blocked elongation of adult sensory neurons after a conditioning lesion. However, the addition of a JAK2 inhibitor prevented the regenerative axon response. Consistent with these pharmacological results, the percentage of neurons showing intense nuclear signal transducers and activators of transcription 3 phosphorylation after a conditioning lesion was markedly increased compared with controls. These observations demonstrate that the signaling mediators that underlie regenerative axon growth are distinct from those used during development and suggest that cytokine signaling may be critical to peripheral nervous system regeneration.

The enormous range of animal size raises a fundamental problem: How do larger animals maintain adequate control of peripheral structures that are many times more massive and extensive than the homologous structures in smaller animals? To explore this question, we have determined neuronal number, the number of axons that innervate each neuron (convergence) and the number of neurons innervated by each axon (divergence), in a peripheral sympathetic pathway of several mammals (mouse, hamster, rat, guinea pig, and rabbit). The average adult weights of these species vary over approximately a 65-fold range. However, the number of superior cervical ganglion cells increases by only a factor of 4 between the smallest of these animals (mice; about 25 gm) and the largest (rabbits; about 1700 gm); the number of spinal preganglionic neurons that innervate the ganglion increases by only a factor of 2. Thus, the number of nerve cells in the sympathetic system does not increase in proportion to animal size. On the other hand, our results indicate that there are systematic differences across these species in the number of axons that innervate each ganglion cell and in the number of ganglion cells innervated by each axon. We suggest that modulation of convergence and divergence in sympathetic ganglia allows this part of the nervous system to effectively activate homologous peripheral targets over a wide range of animal size.