Drp1-mediated mitochondrial dynamics and survival of developing chick motoneurons during the period of normal programmed cell death.

Mitochondrial morphology is dynamically remodeled by fusion and fission in neurons, and this process is implicated in nervous system development and pathology. However, the mechanism by which mitochondrial dynamics influence neuronal development is less clear. In this study, we found that the length of mitochondria is progressively reduced during normal development of chick embryo motoneurons (MNs), a process partly controlled by a fission-promoting protein, dynamin-related protein 1 (Drp1). Suppression of Drp1 activity by gene electroporation of dominant-negative mutant Drp1 in a subset of developing MNs increased mitochondrial length in vivo, and a greater proportion of Drp1-suppressed MNs underwent programmed cell death (PCD). By contrast, the survival of nontransfected MNs in proximity to the transfected MNs was significantly increased, suggesting that the suppression of Drp1 confers disadvantage during the competition for limited survival signals. Because we also monitored perturbation of neurite outgrowth and mitochondrial membrane depolarization following Drp1 suppression, we suggest that impairments of ATP production and axonal growth may be downstream factors that influence the competition of MNs for survival. Collectively, these results indicate that mitochondrial dynamics are required for normal axonal development and competition-dependent MN PCD.

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms.

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.

Neurovascular unit pathology is observed very early in disease progression in the mutant SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by motor neuron degeneration that causes neuromuscular denervation, resulting in muscle weakness and atrophy. Work over the decades using ALS mouse models has revealed that while initial pathology may occur within motor neurons, disease pathology is cell non-autologous. Impairment of the blood-spinal cord barrier (BSCB) occurs before motor neuron frank degeneration; however, precisely when the early pathogenesis of the neurovascular units occurs is not fully understood. Here we examine changes in morphology of neurovascular units, associated gene and protein expression in the lumbar spinal cord of SOD1G93A, and wild-type mice and correlate results with previous reports of early pathological events. Using RNA-sequencing and immunolabeling, we also show that both the neurovascular units and the vasculature of the SOD1G93A lumbar spinal cord present important modifications throughout the disease. Genes relevant for the neurovascular unit and immune cells were differentially expressed in the SOD1G93A ventral lumbar spinal cord compared to wild-type. A reduction in capillary density and tight junction (TJ) with overt BSCB breakdown was observed in the SOD1G93A lumbar spinal cord and ultrastructural observation revealed intact TJ. Additionally, thickened basement membrane, increased pericytes, and string vessels were observed. These alterations in neurovascular units and the vasculature are observed prior to reports of initial neuromuscular junction denervation. The identification of early pathogenesis may be critical to develop diagnostic tests and development of novel treatment strategies that target these early events.

Evidence for the spontaneous production but massive programmed cell death of new neurons in the subcallosal zone of the postnatal mouse brain.

In the last 10 years, many studies have reported that neural stem/progenitor cells spontaneously produce new neurons in a subset of adult brain regions, including the hippocampus, olfactory bulb (OB), cerebral cortex, substantia nigra, hypothalamus, white matter and amygdala in several mammalian species. Although adult neurogenesis in the hippocampus and OB has been clearly documented, its occurrence in other brain regions is controversial. In the present study, we identified a marked accumulation of new neurons in the subcallosal zone (SCZ) of Bax-knockout mice in which programmed cell death (PCD) of adult-generated hippocampal and OB neurons has been shown to be completely prevented. By contrast, in the SCZ of wild-type (WT) mice, only a few immature (but no mature) newly generated neurons were observed, suggesting that virtually all postnatally generated immature neurons in the SCZ were eliminated by Bax-dependent PCD. Treatment of 2-month-old WT mice with a caspase inhibitor, or with the neurotrophic factor brain-derived neurotrophic factor, promoted the survival of adult-generated neurons, suggesting that it is the absence of sufficient neurotrophic signaling in WT SCZ that triggers the Bax-dependent, apoptotic PCD of newly generated SCZ neurons. Furthermore, following focal traumatic brain injury to the posterior brain, SCZ neurogenesis in WT mice was increased, and a subset of these newly generated neurons migrated toward the injury site. These data indicate that the adult SCZ maintains a neurogenic potential that could contribute to recovery in the brain in response to the injury-induced upregulation of neurotrophic signaling.

Abnormal development of the neuromuscular junction in Nedd4-deficient mice.

Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. Here we show that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5-E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms.

The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes.

Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.

Misplacement of Purkinje cells during postnatal development in Bax knock-out mice: a novel role for programmed cell death in the nervous system?

During early postnatal development, the orchestrated regulation of proliferation, migration and the survival versus elimination of neurons is essential for histogenesis of the cerebellum. For instance, Purkinje cells (PCs) promote the proliferation and migration of external granule cells (EGCs), whereas EGCs in turn play a role in the migration of PCs. Considering that a substantial number of neurons undergo programmed cell death (PCD) during cerebellar development, it seems likely that neuronal loss could have a significant role in the histogenesis of the cerebellum. To address this question, we examined postnatal development of the cerebellum in Bax-knock-out (KO) mice in which the PCD of PC has been reported to be selectively reduced or eliminated, whereas EGCs are unaffected. We confirmed the absence of PC PCD as well as the normal PCD of EGCs in Bax-KO mice. We also observed a subpopulation of PCs that were misplaced in the inner granule cell layer of Bax-KO mice on postnatal day 5 (P5) to P10 and that, by the end of the major period of cerebellar histogenesis (P14), lose expression of the PC marker calbindin. These results suggest that the removal of ectopically located neurons may be a previously unrecognized function of developmental PCD.

Developing postmitotic mammalian neurons in vivo lacking Apaf-1 undergo programmed cell death by a caspase-independent, nonapoptotic pathway involving autophagy.

Previous studies have shown that caspases and Apaf-1 are required for the normal programmed cell death (PCD) in vivo of immature postmitotic neurons and mitotically active neuronal precursor cells. In contrast, caspase activity is not necessary for the normal PCD of more mature postmitotic neurons that are establishing synaptic connections. Although normally these cells use caspases for PCD, in the absence of caspase activity these neurons undergo a distinct nonapoptotic type of degeneration. We examined the survival of these more mature postmitotic neuronal populations in mice in which Apaf-1 has been genetically deleted and find that they exhibit quantitatively normal PCD of developing postmitotic neurons. We next characterized the morphological mode of PCD in these mice and show that the neurons degenerate by a caspase-independent, nonapoptotic pathway that involves autophagy. However, autophagy does not appear to be involved in the normal PCD of postmitotic neurons in which caspases and Apaf-1 are present and functional because quantitatively normal neuronal PCD occurred in the absence of a key gene required for autophagy (ATG7). Finally, we examined the possible role of another caspase-independent type of neuronal PCD involving the apoptosis-inducing factor (AIF). Mice deficient in AIF also exhibit quantitatively normal PCD of postmitotic neurons after caspase inhibition. Together, these data indicate that, when key components of the type 1 apoptotic pathway (i.e., caspases and Apaf-1) are perturbed in vivo, developing postmitotic neurons nonetheless undergo quantitatively normal PCD by a caspase-independent pathway involving autophagy and not requiring AIF.

The maintenance of specific aspects of neuronal function and behavior is dependent on programmed cell death of adult-generated neurons in the dentate gyrus.

A considerable number of new neurons are generated daily in the dentate gyrus (DG) of the adult hippocampus, but only a subset of these survive, as many adult-generated neurons undergo programmed cell death (PCD). However, the significance of PCD in the adult brain for the functionality of DG circuits is not known. Here, we examined the electrophysiological and behavioral characteristics of Bax-knockout (Bax-KO) mice in which PCD of post-mitotic neurons is prevented. The continuous increase in DG cell numbers in Bax-KO mice resulted in the readjustment of afferent and efferent synaptic connections, represented by age-dependent reductions in the dendritic arborization of DG neurons and in the synaptic contact ratio of mossy fibers with CA3 dendritic spines. These neuroanatomical changes were associated with reductions in synaptic transmission and reduced performance in a contextual fear memory task in 6-month-old Bax-KO mice. These results suggest that the elimination of excess DG neurons via Bax-dependent PCD in the adult brain is required for the normal organization and function of the hippocampus.

Adult Hippocampal Neurogenesis in Mammals (and Humans): The Death of a Central Dogma in Neuroscience and its Replacement by a New Dogma.

The review is a critical appraisal of the history and present status of the phenomenon of adult hippocampal neurogenesis (AHN) in the mammalian and human brain. Previous as well as most current studies of AHN have focused on highly inbred domestic mice and rats that are examined in rigorously controlled laboratory environments using psychology-based behavioral tests. However, this approach cannot reveal the adaptive significance of AHN, a key unresolved question in the field. After the publication of several thousand articles in the field over the last 20 years, little progress has been made in our understanding of the biological utility of AHN in the real world. To accomplish this will require comparative studies employing a greater diversity of species and species-specific behaviors that are investigated in a more naturalistic, evolutionary context. Although efforts along these lines are on the rise, they remain "voices in the wilderness." This review is an attempt to hasten and increase those efforts.

The sympathetic nervous system regulates skeletal muscle motor innervation and acetylcholine receptor stability.

AIM: Symptoms of autonomic failure are frequently the presentation of advanced age and neurodegenerative diseases that impair adaptation to common physiologic stressors. The aim of this work was to examine the interaction between the sympathetic and motor nervous system, the involvement of the sympathetic nervous system (SNS) in neuromuscular junction (NMJ) presynaptic motor function, the stability of postsynaptic molecular organization, and the skeletal muscle composition and function. METHODS: Since muscle weakness is a symptom of diseases characterized by autonomic dysfunction, we studied the impact of regional sympathetic ablation on muscle motor innervation by using transcriptome analysis, retrograde tracing of the sympathetic outflow to the skeletal muscle, confocal and electron microscopy, NMJ transmission by electrophysiological methods, protein analysis, and state of the art microsurgical techniques, in C57BL6, MuRF1KO and Thy-1 mice. RESULTS: We found that the SNS regulates motor nerve synaptic vesicle release, skeletal muscle transcriptome, muscle force generated by motor nerve activity, axonal neurofilament phosphorylation, myelin thickness, and myofibre subtype composition and CSA. The SNS also modulates the levels of postsynaptic membrane acetylcholine receptor by regulating the Gαi2 -Hdac4-Myogenin-MuRF1pathway, which is prevented by the overexpression of the guanine nucleotide-binding protein Gαi2 (Q205L), a constitutively active mutant G protein subunit. CONCLUSION: The SNS regulates NMJ transmission, maintains optimal Gαi2 expression, and prevents any increase in Hdac4, myogenin, MuRF1, and miR-206. SNS ablation leads to upregulation of MuRF1, muscle atrophy, and downregulation of postsynaptic AChR. Our findings are relevant to clinical conditions characterized by progressive decline of sympathetic innervation, such as neurodegenerative diseases and aging.

The kiss of death.

The programmed cell death (PCD) of neurons is generally thought to be cell autonomous and not to require a death signal from other cells. A recent study by Marín-Teva et al., in this issue of Neuron, brings this theory into question and suggests that neighboring microglia actively participate in the PCD of Purkinje cells in the cerebellum.

Astrocyte and muscle-derived secreted factors differentially regulate motoneuron survival.

During development, motoneurons (MNs) undergo a highly stereotyped, temporally and spatially defined period of programmed cell death (PCD), the result of which is the loss of 40-50% of the original neuronal population. Those MNs that survive are thought to reflect the successful acquisition of limiting amounts of trophic factors from the target. In contrast, maturation of MNs limits the need for target-derived trophic factors, because axotomy of these neurons in adulthood results in minimal neuronal loss. It is unclear whether MNs lose their need for trophic factors altogether or whether, instead, they come to rely on other cell types for nourishment. Astrocytes are known to supply trophic factors to a variety of neuronal populations and thus may nourish MNs in the absence of target-derived factors. We investigated the survival-promoting activities of muscle- and astrocyte-derived secreted factors and found that astrocyte-conditioned media (ACM) was able to save substantially more motoneurons in vitro than muscle-conditioned media (MCM). Our results indicate that both ACM and MCM are significant sources of MN trophic support in vitro and in ovo, but only ACM can rescue MNs after unilateral limb bud removal. Furthermore, we provide evidence suggesting that MCM facilitates the death of a subpopulation of MNs in a p75(NTR) - and caspase-dependent manner; however, maturation in ACM results in MN trophic independence and reduced vulnerability to this negative, pro-apoptotic influence from the target.

Exogenous delivery of heat shock protein 70 increases lifespan in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder that results in the progressive loss of motoneurons (MNs) in the CNS. Several survival and death mechanisms of MNs have been characterized and it has been determined that MNs do not appear to mount a complete stress response, as determined by the lack of heat shock protein 70 (Hsp70) upregulation after several stress paradigms. Hsp70 has been shown to confer neuroprotection and the insufficient availability of Hsp70 may contribute to MNs' susceptibility to death in ALS mice. In this study, recombinant human Hsp70 (rhHsp70) was intraperitoneally injected three times weekly, beginning at postnatal day 50 until endstage, to G93A mutant SOD1 (G93A SOD1) mice. The administration of rhHsp70 was effective at increasing lifespan, delaying symptom onset, preserving motor function and prolonging MN survival. Interestingly, injected rhHsp70 localized to skeletal muscle and was not readily detected in the CNS. Treatment with rhHsp70 also resulted in an increased number of innervated neuromuscular junctions compared with control tissue. Together these results suggest rhHsp70 may delay disease progression in the G93A SOD1 mouse via a yet to be identified peripheral mechanism.

Impaired migration in the rostral migratory stream but spared olfactory function after the elimination of programmed cell death in Bax knock-out mice.

Rats and mice exhibit neurogenesis of olfactory bulb (OB) interneurons throughout adulthood. To homeostatically maintain stable neuron numbers, it is necessary to continuously remove a subset of OB neurons by programmed cell death (PCD). Here we demonstrate that Bax is critical for the elimination of OB neurons by showing that Bax-KO mice exhibit greatly reduced PCD in the OB. Despite the reduction of PCD, however, proliferation of progenitors and the size of the OB were virtually unaffected in Bax-knock-out (KO) mice. However, reducing PCD by Bax deletion affected the migration of a subset of adult-produced neurons by the disruption of glial tube formation as well as by premature detachment of neuroblasts from the migratory chain. Rescued cells aberrantly remained in the subventricular zone (SVZ)-rostral migratory stream (RMS), in which they differentiated into calretinin+ or GABA-expressing interneurons. Because of the migratory deficit, OB cell homeostasis involving new cell entry and PCD (neuronal turnover) was virtually absent in adult Bax-KO mice. Despite this, Bax-KO mice exhibited normal olfactory behaviors such as odor discrimination and olfactory memory which are thought to be influenced by adult neurogenesis. These results demonstrate that PCD is involved in the regulation of RMS migration and differentiation after OB neurogenesis, but that animals maintain normal olfactory function in the absence of PCD.

Neuromuscular development in the absence of programmed cell death: phenotypic alteration of motoneurons and muscle.

The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal death in vivo to ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from alpha- to gamma-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic gene Bax. However, there was an increase in intrafusal muscle fibers (spindles) in Bax knock-out versus both wild-type and MyoGDNF mice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.

Complete dissociation of motor neuron death from motor dysfunction by Bax deletion in a mouse model of ALS.

The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.

Synaptic dysfunction in disease and following injury in the developing and adult nervous system: caveats in the choice of therapeutic intervention.

A cardinal feature of most developmental and adult onset neurodegenerative diseases is the death of specific populations of neurons. Largely as a result of the progress made in elucidating the cellular and molecular mechanisms underlying the neuronal death that occurs during development, approaches ameliorating them often focus on the manipulation of neuronal death pathways. Recent evidence derived from the study of animal models of various neuropathological conditions, however, has revealed that damage to axons and synapses long precedes the activation of death pathways. We recently extended these findings to the most commonly studied animal model of familial amyotrophic lateral sclerosis (fALS). Inhibiting the cell death pathway by deletion of the pro-apoptotic gene Bax completely rescued spinal MNs yet failed to prevent disease in fALS transgenic mice. However, we observed distinct abnormalities within presynaptic terminals of spinal MNs at the neuromuscular junction (NMJ), as well as profound denervation. These results suggest that therapies aimed at preserving the synapse rather than the soma may be more effective at treating these neuropathologies.

Survival and death of mature avian motoneurons in organotypic slice culture: trophic requirements for survival and different types of degeneration.

We have developed an organotypic culture technique that uses slices of chick embryo spinal cord, in which trophic requirements for long-term survival of mature motoneurons (MNs) were studied. Slices were obtained from E16 chick embryos and maintained for up to 28 days in vitro (DIV) in a basal medium. Under these conditions, most MNs died. To promote MN survival, 14 different trophic factors were assayed. Among these 14, glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor were the most effective. GDNF was able to promote MN survival for at least 28 DIV. K(+) depolarization or caspase inhibition prevented MN death but also induced degenerative-like changes in rescued MNs. Agents that elevate cAMP levels promoted the survival of a proportion of MNs for at least 7 DIV. Examination of dying MNs revealed that, in addition to cells exhibiting a caspase-3-dependent apoptotic pattern, some MNs died by a caspase-3-independent mechanism and displayed autophagic vacuoles, an extremely convoluted nucleus, and a close association with microglia. This organotypic spinal cord slice culture may provide a convenient model for testing conditions that promote survival of mature-like MNs that are affected in late-onset MN disease such as amyotrophic lateral sclerosis.

Phosphorylation of c-Jun in avian and mammalian motoneurons in vivo during programmed cell death: an early reversible event in the apoptotic cascade.

c-Jun is a transcription factor that is involved in various cellular events, including apoptotic cell death. For example, phosphorylation of c-Jun is one of the earliest biochemical changes detected in dying sympathetic neurons after NGF deprivation in vitro. However, currently, it is not known whether a similar molecular event is involved in the developmental programmed cell death (PCD) of neurons in vivo. We observed that only a subpopulation of motoneurons (MNs) exhibit c-Jun phosphorylation during the PCD period in chick [embryonic day 5 (E5)-E12] and mouse (E13-E18) embryos. Experimental perturbation of MN survival-promoting signals by limb bud removal (reduced signals) or by activity blockade (increased signals) in the chick embryo demonstrated that the presence of those signals is negatively correlated with the number of c-Jun-phosphorylated MNs. This suggests that insufficient survival signals (e.g., neurotrophic factors) may induce c-Jun phosphorylation of MNs in vivo. Consistent with the idea that c-Jun phosphorylation is a reversible event during normal PCD of MNs, we found that c-Jun phosphorylation was transiently observed in a subpopulation of mouse MNs rescued from PCD by deletion of the proapoptotic gene Bax. Inhibition of c-Jun signaling significantly reduced MN death in chick embryo, indicating that activation of c-Jun signaling is necessary for the PCD of MNs. Together, c-Jun phosphorylation appears to be required for the initiation of an early and reversible event in the intracellular PCD cascade in vivo after loss of survival-promoting signals such as neurotrophic factors.