Autonomous and nonautonomous functions for Hox/Pbx in branchiomotor neuron development

The vertebrate branchiomotor neurons are organized in a pattern that corresponds with the segments, or rhombomeres, of the developing hindbrain and have identities and behaviors associated with their position along the anterior/posterior axis. These neurons undergo characteristic migrations in the hindbrain and project from stereotyped exit points. We show that lazarus/pbx4, which encodes an essential Hox DNA-binding partner in zebrafish, is required for facial (VIIth cranial nerve) motor neuron migration and for axon pathfinding of trigeminal (Vth cranial nerve) motor axons. We show that lzr/pbx4 is required for Hox paralog group 1 and 2 function, suggesting that Pbx interacts with these proteins. Consistent with this, lzr/pbx4 interacts genetically with hoxb1a to control facial motor neuron migration. Using genetic mosaic analysis, we show that lzr/pbx4 and hoxb1a are primarily required cell-autonomously within the facial motor neurons; however, analysis of a subtle non-cell-autonomous effect indicates that facial motor neuron migration is promoted by interactions amongst the migrating neurons. At the same time, lzr/pbx4 is required non-cell-autonomously to control the pathfinding of trigeminal motor axons. Thus, Pbx/Hox can function both cell-autonomously and non-cell-autonomously to direct different aspects of hindbrain motor neuron behavior.     

Programmed death of peripheral pioneer neurons in the grasshopper embryo

The Ti1 pioneer neurons arise at the distal tip of the metathoracic leg in the grasshopper embryo, and are the first neurons in the limb bud to extend axons to the central nervous system (C. M. Bate (1976) Nature (London) 260, 54-56; H. Keshishian (1980) Dev. Biol. 80, 388-397). By providing a neural pathway along which growth cones of later arising neurons migrate, these pioneer axons establish the route of one of the major nerve trunks in the leg (Keshishian, 1980; R. K. Ho and C. S. Goodman (1982) Nature (London) 297, 404-406; D. Bentley and H. Keshishian (1982) Science 218, 1082-1088). Here, we demonstrate that at the 55-59% stage of development, the two Ti1 pioneer neurons undergo programmed death. The role which these pioneers serve in establishing a nerve route appears to be their only function, and may be important for the normal development of the peripheral nervous system. The Ti1 pioneers provide an example of a previously hypothesized class (J. W. Truman (1984) Annu. Rev. Neurosci. 7, 171-188) of programmed neuron death: obsolete neurons whose function was developmental rather than behavioral.     

Specification of distinct motor neuron identities by the singular activities of individual Hox genes.

Hox genes have been implicated in specifying positional values along the anteroposterior axis of the caudal central nervous system, but their nested and overlapping expression has complicated the understanding of how they confer specific neural identity. We have employed a direct gain-of-function approach using retroviral vectors to misexpress Hoxa2 and Hoxb1 outside of the normal Hox expression domains, thereby avoiding complications resulting from possible interactions with endogenous Hox genes. Misexpression of either Hoxa2 or Hoxb1 in the anteriormost hindbrain (rhombomere1, r1) leads to the generation of motor neurons in this territory, even though it is normally devoid of this cell type. These ectopic neurons have the specific identity of branchiomotor neurons and, in the case of Hoxb1-induced cells, their axons leave the hindbrain either by fasciculating with the resident cranial motor axons at isthmic (trochlear) or r2 (trigeminal) levels of the axis or via novel ectopic exit points in r1. Next, we have attempted to identify the precise branchiomotor subtypes that are generated after misexpression and our results suggest that the ectopic motor neurons generated following Hoxa2 misexpression are trigeminal-like, while those generated following Hoxb1 misexpression are facial-like. Our data demonstrate, therefore, that at least to a certain extent and for certain cell types, the singular activities of individual Hox genes (compared to a combinatorial mode of action, for example) are sufficient to impose on neuronal precursor cells the competence to generate distinctly specified cell types. Moreover, as these particular motor neuron subtypes are normally generated in the most anterior domains of Hoxa2 and Hoxb1 expression, respectively, our data support the idea that the main site of individual Hox gene action is in the anteriormost subdomain of their expression, consistent with the phenomenon of posterior dominance.     

Antagonistic roles for Ultrabithorax and Antennapedia in regulating segment-specific apoptosis of differentiated motoneurons in the Drosophila embryonic central nervous system

The generation of morphological diversity among segmental units of the nervous system is crucial for correct matching of neurons with their targets and for formation of functional neuromuscular networks. However, the mechanisms leading to segment diversity remain largely unknown. We report here that the Hox genes Ultrabithorax (Ubx) and Antennapedia (Antp) regulate segment-specific survival of differentiated motoneurons in the ventral nerve cord of Drosophila embryos. We show that Ubx is required to activate segment-specific apoptosis in these cells, and that their survival depends on Antp. Expression of the Ubx protein is strongly upregulated in the motoneurons shortly before they undergo apoptosis, and our results indicate that this late upregulation is required to activate reaper-dependent cell death. We further demonstrate that Ubx executes this role by counteracting the function of Antp in promoting cell survival. Thus, two Hox genes contribute to segment patterning and diversity in the embryonic CNS by carrying out opposing roles in the survival of specific differentiated motoneurons.     

Segment-specific generation of Drosophila Capability neuropeptide neurons by multi-faceted Hox cues

In the Drosophila ventral nerve cord, the three pairs of Capability neuropeptide-expressing Va neurons are exclusively found in the second, third and fourth abdominal segments (A2–A4). To address the underlying mechanisms behind such segment-specific cell specification, we followed the developmental specification of these neurons. We find that Va neurons are initially generated in all ventral nerve cord segments and progress along a common differentiation path. However, their terminal differentiation only manifests itself in A2–A4, due to two distinct mechanisms: segment-specific programmed cell death (PCD) in posterior segments, and differentiation to an alternative identity in segments anterior to A2. Genetic analyses reveal that the Hox homeotic genes are involved in the segment-specific appearance of Va neurons. In posterior segments, the Hox gene Abdominal-B exerts a pro-apoptotic role on Va neurons, which involves the function of several RHG genes. Strikingly, this role of Abd-B is completely opposite to its role in the segment-specific apoptosis of other classes of neuropeptide neurons, the dMP2 and MP1 neurons, where Abd-B acts in an anti-apoptotic manner. In segments A2–A4 we find that abdominal A is important for the terminal differentiation of Va cell fate. In the A1 segment, Ultrabithorax acts to specify an alternate Va neuron fate. In contrast, in thoracic segments, Antennapedia suppresses the Va cell fate. Thus, Hox genes act in a multi-faceted manner to control the segment-specific appearance of the Va neuropeptide neurons in the ventral nerve cord.     

The prostate apoptosis response-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. Here, we report higher Par-4 levels in lumbar spinal cord samples from patients with amyotrophic lateral sclerosis (ALS) than in lumbar spinal cord samples from neurologically normal patients. We also compared the levels of Par-4 in lumbar spinal cord samples from wild-type and transgenic mice expressing the human Cu/Zn-superoxide dismutase gene with a familial ALS mutation. Relative to control samples, higher Par-4 levels were observed in lumbar spinal cord samples prepared from the transgenic mice at a time when they had hind-limb paralysis. Immunohistochemical analyses of human and mouse lumbar spinal cord sections revealed that Par-4 is localized to motor neurons in the ventral horn region. In culture studies, exposure of primary mouse spinal cord motor neurons or NSC-19 motor neuron cells to oxidative insults resulted in a rapid and large increase in Par-4 levels that preceded apoptosis. Pretreatment of the motor neuron cells with a Par-4 antisense oligonucleotide prevented oxidative stress-induced apoptosis and reversed oxidative stress-induced mitochondrial dysfunction that preceded apoptosis. Collectively, these data suggest a role for Par-4 in models of motor neuron injury relevant to ALS.     

Pioneer growth cone behavior at a differentiating limb segment boundary in the grasshopper embryo

The first neurons to extend axons through embryonic grasshopper limbs are a pair of sibling pioneer neurons. After migrating proximally along the limb axis, the pioneer growth cones normally make an abrupt ventral turn. In some cases (less than 20%) this turn is directly toward the proximo-ventrally located Cx1 guidepost neurons. However, in the majority of cases (greater than 80%) the pioneer growth cones make a more acute ventral turn along a single circumferential line which lies distal to the Cx1 neurons. Growth cones from other afferent neurons orient along the same line. Growth cones can extend along this line around more than half of the circumference of the limb and can grow in either direction along it. The circumferential line appears to be the prospective trochanter-coxa segment boundary. Afferent axons on the segment boundary leave it and contact the proximo-ventrally located Cx1 neurons. The site at which pioneer growth cones leave the boundary is variable and appears to be the point from which filopodial contact with Cx1 cells is first established. In addition to the trochanter-coxa segment boundary, the pioneer growth cones and axons also respond to the tibia-femur and femur-trochanter segment boundaries. The role of segment boundaries as barriers to growth cone movement and the effect of such barriers on the timing and placement of differentiation of pioneer neurons are discussed.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis.

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by approximately 30%. The death of motor neurons was confirmed using the terminal transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl-modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord.     

Formation of the transverse nerve in moth embryos. II. Stereotyped growth by the axons of identified neuroendocrine neurons

We are interested in the cellular mechanisms that guide neuroendocrine axons to their neurohaemal target regions and that regulate the extent and positioning of their terminal arbor. The neurohaemal organ we have studied is the segmentally repeated transverse nerve of the moth Manduca. In the mature animal, two motor neurons and a heterogeneous set of identified neuroendocrine neurons project to this nerve; the latter release hormonal peptides from along its length. In the preceding report, we demonstrated that during embryogenesis, the position, trajectory and extent of the transverse nerve are anticipated by two sets of nonneuronal cells, the strap and the bridge. In this paper we show that four identified neuroendocrine neurons (L1 and B1-3), like the identified motor neurons before them, elaborate growth cones that use this preexisting scaffolding as a substrate for axonal elongation. Moreover, growth cone navigation by these neuroendocrine neurons is as precise and invariant as that displayed by the motor neurons. One feature that differentiates the behavior of the developing neuroendocrine cells from that of the motor neurons is a stereotyped interaction that the L1 and B1-3 axons undergo with an identified syncytial cell that lies in close proximity to the strap. Each neuroendocrine neuron specifically adheres to the syncytium by extending numerous filopodia, and an occasional large lamellopodium, over its surface. These contacts are maintained by the neuroendocrine axons after their growth cones have left the vicinity of the syncytium and proceeded into the strap/bridge complex. Adhesion to the syncytium is transient and specific to the neuroendocrine neurons: although motor neuron axons are present at this same time and place, they display no affinity for the syncytium. This distinction correlates with the fact that the neuroendocrine neurons go on to elaborate arbor within the confines of the transverse nerve, while the motor neurons do not. We suggest that the syncytium may act as a "fictive target" for these neurons to aid in the differentiation of features that are specific to their cellular phenotype.     

Expression of the SM-20 Gene Promotes Death in Nerve Growth Factor-Dependent Sympathetic Neurons

Sympathetic neurons undergo apoptosis when deprived of nerve growth factor (NGF). Inhibitors of RNA or protein synthesis block this death, suggesting that gene expression is important for apoptosis in this system. We have identified SM-20 as a new gene that increases in expression in sympathetic neurons after NGF withdrawal. Expression of SM-20 also increases during neuronal death caused by cytosine arabinoside or the phosphatidylinositol 3-kinase inhibitor LY294002. In addition, SM-20 protein synthesis is elevated in NGF-deprived neurons compared with neurons maintained with NGF. Importantly, expression of SM-20 in sympathetic neurons causes cell death in the presence of NGF. These results suggest that SM-20 may function to regulate cell death in neurons.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by ∼30%. The death of motor neurons was confirmed using the terminal transferase‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl‐modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 185–201, 1999     

Injury-induced spinal motor neuron apoptosis is preceded by DNA single-strand breaks and is p53- and Bax-dependent.

The mechanisms of injury-induced apoptosis of neurons within the spinal cord are not understood. We used a model of peripheral nerve-spinal cord injury in the rat and mouse to induce motor neuron degeneration. In this animal model, unilateral avulsion of the sciatic nerve causes apoptosis of motor neurons. We tested the hypothesis that p53 and Bax regulate this neuronal apoptosis, and that DNA damage is an early upstream signal. Adult mice and rats received unilateral avulsions causing lumbar motor neurons to achieve endstage apoptosis at 7-14 days postlesion. This motor neuron apoptosis is blocked in bax(-/-) and p53(-/-) mice. Single-cell gel electrophoresis (comet assay), immunocytochemistry, and quantitative immunogold electron microscopy were used to measure molecular changes in motor neurons during the progression of apoptosis. Injured motor neurons accumulate single-strand breaks in DNA by 5 days. p53 accumulates in nuclei of motor neurons destined to undergo apoptosis. p53 is functionally activated by 4-5 days postlesion, as revealed by immunodetection of phosphorylated p53. Preapoptotically, Bax translocates to mitochondria, cytochrome c accumulates in the cytoplasm, and caspase-3 is activated. These results demonstrate that motor neuron apoptosis in the adult spinal cord is controlled by upstream mechanisms involving DNA damage and activation of p53 and downstream mechanisms involving upregulated Bax and cytochrome c and their translocation, accumulation of mitochondria, and activation of caspase-3. We conclude that adult motor neuron death after nerve avulsion is DNA damage-induced, p53- and Bax-dependent apoptosis.     

Axon guidance in the inner ear

Statoacoustic ganglion (SAG) neurons send their peripheral processes to navigate into the inner ear sensory organs where they will ultimately become post-synaptic to mature hair cells. During early ear development, neuroblasts delaminate from a restricted region of the ventral otocyst and migrate to form the SAG. The pathfinding mechanisms employed by the processes of SAG neurons as they search for their targets in the periphery are the topic of this review. Multiple lines of evidence exist to support the hypothesis that a combination of cues are working to guide otic axons to their target sensory organs. Some pioneer neurites may retrace their neuronal migratory pathway back to the periphery, yet additional guidance mechanisms likely complement this process. The presence of chemoattractants in the ear is supported by in vitro data showing that the otic epithelium exerts both trophic and tropic effects on the statoacoustic ganglion. The innervation of ectopic hair cells, generated after gene misexpression experiments, is further evidence for chemoattractant involvement in the pathfinding of SAG axons. While the source(s) of chemoattractants in the ear remains unknown, candidate molecules, including neurotrophins, appear to attract otic axons during specific time points in their development. Data also suggest that classical axon repellents such as Semaphorins, Eph/ephrins and Slit/Robos may be involved in the pathfinding of otic axons. Morphogens have recently been implicated in guiding axonal trajectories in many other systems and therefore a role for these molecules in otic axon guidance must also be explored.     

Suppression of death receptor signaling in cerebellar Purkinje neurons protects neighboring granule neurons from apoptosis via an insulin-like growth factor I-dependent mechanism

Neuronal apoptosis contributes to the progression of neurodegenerative disease. Primary cerebellar granule neurons are an established in vitro model for investigating neuronal death. After removal of serum and depolarizing potassium, granule neurons undergo apoptosis via a mechanism that requires intrinsic (mitochondrial) death signals; however, the role of extrinsic (death receptor-mediated) signals is presently unclear. Here, we investigate involvement of death receptor signaling in granule neuron apoptosis by expressing adenoviral, AU1-tagged, dominant-negative Fas-associated death domain (Ad-AU1-deltaFADD). Ad-AU1-deltaFADD decreased apoptosis of granule neurons from 65 +/- 5 to 27 +/- 2% (n = 7, p < 0.01). Unexpectedly, immunocytochemical staining for AU1 revealed that <5% of granule neurons expressed deltaFADD. In contrast, deltaFADD was expressed in >95% of calbindin-positive Purkinje neurons ( approximately 2% of the cerebellar culture). Granule neurons in proximity to deltaFADD-expressing Purkinje cells demonstrated markedly increased survival. Both granule and Purkinje neurons expressed insulin-like growth factor-I (IGF-I) receptors, and deltaFADD-mediated survival of granule neurons was inhibited by an IGF-I receptor blocking antibody. These results demonstrate that the selective suppression of death receptor signaling in Purkinje neurons is sufficient to rescue neighboring granule neurons that depend on Purkinje cell-derived IGF-I. Thus, the extrinsic death pathway has a profound but indirect effect on the survival of cerebellar granule neurons.