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R W Oppenheim

Publications and source records attributed to R W Oppenheim.

At least 19 recordsLinked to original sources
PubMed1999

Close spatial-temporal relationship between islet-1-expressing cells and growing primary afferent axons in the dorsal spinal cord of chick embryo.

The relationship between the appearance of Islet-1-expressing cells and the longitudinal growth of primary afferent axons (PAAs) in the dorsal spinal cord of chick embryos was examined. Islet-1-expressing cells first appeared in the dorsal spinal cord at embryonic days (E) 3-3.5. These immunoreactive cells were aligned in a longitudinal column in close proximity to longitudinally elongating PAAs in the presumptive dorsal funiculus. By E8, when many PAAs invade the spinal gray matter, Islet-1-expressing cells had disappeared in the dorsal spinal cord. Following the dorsoventral rotation of the spinal cord in ovo before the invasion of PAAs, a close topographical relationship between Islet-1-expressing cells and PAAs was maintained. These results suggest that Islet-1-expressing cells may play a role in the longitudinal growth of PAAs in the dorsal funiculus.

Animals↗
PubMed1998

Mechanisms of insulin-like growth factor regulation of programmed cell death of developing avian motoneurons.

During development of the avian neuromuscular system, lumbar spinal motoneurons (MNs) innervate their muscle targets in the hindlimb coincident with the onset and progression of MN programmed cell death (PCD). Paralysis (activity blockade) of embryos during this period rescues large numbers of MNs from PCD. Because activity blockade also results in enhanced axonal branching and increased numbers of neuromuscular synapses, it has been postulated that following activity blockade, increased numbers of MNs can gain access to muscle-derived trophic agents that prevent PCD. An assumption of the access hypothesis of MN PCD is the presence of an activity-dependent, muscle-derived sprouting or branching agent. Several previous studies of sprouting in the rodent neuromuscular system indicate that insulin-like growth factors (IGFs) are candidates for such a sprouting factor. Accordingly, in the present study we have begun to test whether the IGFs may play a similar role in the developing avian neuromuscular system. Evidence in support of this idea includes the following: (a) IGFs promote MN survival in vivo but not in vitro; (b) neutralizing antibodies against IGFs reduce MN survival in vivo; (c) both in vitro and in vivo, IGFs increase neurite growth, branching, and synapse formation; (d) activity blockade increases the expression of IGF-1 and IGF-2 mRNA in skeletal muscles in vivo; (e) in vivo treatment of paralyzed embryos with IGF binding proteins (IGF-BPs) that interfere with the actions of endogenous IGFs reduce MN survival, axon branching, and synapse formation; (f) treatment of control embryos in vivo with IGF-BPs also reduces synapse formation; and (g) treatment with IGF-1 prior to the major period of cell death (i.e., on embryonic day 6) increases subsequent synapse formation and MN survival and potentiates the survival-promoting actions of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) administered during the subsequent 4- to 5-day period of PCD. Collectively, these data provide new evidence consistent with the role of the IGFs as activity-dependent, muscle-derived agents that play a role in regulating MN survival in the avian embryo.

Animals↗
PubMed1998

Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurons.

Biochemical and molecular mechanisms of neuronal cell death are currently an area of intense research. It is well documented that the lumbar spinal motoneurons of the chick embryo undergo a period of naturally occurring programmed cell death (PCD) requiring new gene expression and activation of caspases. To identify genes that exhibit changed expression levels in dying motoneurons, we used a PCR-based subtractive hybridization protocol to identify messages uniquely expressed in motoneurons deprived of trophic support as compared with their healthy counterparts. We report that one upregulated message in developing motoneurons undergoing cell death is the mRNA for amyloid precursor protein (APP). Increased levels of APP and beta-amyloid protein are also detected within dying motoneurons. The predicted peptide sequence of APP indicates two potential cleavage sites for caspase-3 (CPP-32), a caspase activated in dying motoneurons. When peptide inhibitors of caspase-3 are administered to motoneurons destined to undergo PCD, decreased levels of APP protein and greatly reduced beta-amyloid production are observed. Furthermore, we show that APP is cleaved by caspase-3. Our results suggest that differential gene expression results in increased levels of APP, providing a potential substrate for one of the cell death-activated caspases that may ultimately cause the demise of the cell. These results, combined with information on the toxic role of APP and its proteolytic by-product beta-amyloid, in the neurodegenerative disease Alzheimer's, suggest that events of developmental PCD may be reactivated in early stages of pathological neurodegeneration.

Amino Acid Sequence↗
PubMed1998

Characterization of spinal motoneuron degeneration following different types of peripheral nerve injury in neonatal and adult mice.

Experimental lesions have been used widely to induce motoneuron (MN) degeneration as a model to test the ability of different trophic molecules to prevent lesion-induced alterations. However, the morphological mechanisms of spinal MN death following different types of lesions is not clear at the present time. In this study, we have characterized the morphological characteristics of MN cell death by examining DNA fragmentation and the ultrastructural and light microscopic morphological features of MNs following different types of spinal nerve injury (i.e., axotomy and avulsion) in the developing and adult mouse. In neonatal mice, axotomy induced cell death as well as the atrophy of MNs that survived the injury. DNA fragmentation could be detected by using the terminal deoxynucleotidyl transferase (TUNEL) method during the cell death process following neonatal axotomy, whereas TUNEL labeling was not observed following either neonatal or adult avulsion. However, with the exception of TUNEL labeling, the morphological characteristics of MN death following neonatal axotomy and avulsion were similar, and both resembled most closely the form of programmed cell death termed cytoplasmic or type 3B, which exhibits similarities as well as differences with currently accepted definitions of apoptosis. By contrast, adult avulsion resulted in a type of degeneration that resembled necrosis more closely. However, even there, the morphology was mixed, showing characteristics of both apoptosis and necrosis. These results indicate that the mode of MN degeneration is complex and is related to developmental age and type of lesion.

Animals↗
PubMed1998

CEP-1347/KT7515 prevents motor neuronal programmed cell death and injury-induced dedifferentiation in vivo.

CEP-1347, also known as KT7515, a derivative of a natural product indolocarbazole, inhibited motor neuronal death in vitro, inhibited activation of the stress-activated kinase JNK1 (c-jun NH terminal kinase) in cultured spinal motor neurons, but had no effect on the mitogen-activated protein kinase ERK1 in these cells. Results reported here profile the functional activity of CEP-1347/KT7515 in vivo in models of motor neuronal death or dedifferentiation. Application of CEP-1347/KT7515 to the chorioallantoic membrane of embryonic chicks rescued 40% of the lumbar motor neurons that normally die during the developmental period assessed. Peripheral administration of low doses (0.5 and 1 mg/kg daily) of CEP-1347/KT7515 reduced death of motor neurons of the spinal nucleus of the bulbocavernosus in postnatal female rats, with efficacy comparable to testosterone. Strikingly, daily administration of CEP-1347/KT7515 during the 4-day postnatal window of motor neuronal death resulted in persistent long-term motor neuronal survival in adult animals that received no additional CEP-1347/KT7515. In a model of adult motor neuronal dedifferentiation following axotomy, local application of CEP-1347/KT7515 to the transected hypoglossal nerve substantially reduced the loss of choline acetyl transferase immunoreactivity observed 7 days postaxotomy compared to untreated animals. Results from these experiments demonstrate that a small organic molecule that inhibits a signaling pathway associated with stress and injury also reduces neuronal death and degeneration in vivo.

Animals↗
PubMed1998

Muscle-specific cell ablation conditional upon Cre-mediated DNA recombination in transgenic mice leads to massive spinal and cranial motoneuron loss.

We describe here a binary transgenic system based on Cre-mediated DNA recombination for genetic cell ablation in mice that enabled us to obtain skeletal muscle-deficient embryos by mating two phenotypically normal transgenic lines. In those embryos, skeletal muscles are eliminated as a consequence of the expression of the gene encoding the diphtheria toxin A fragment. Cell ablation occurs gradually beginning approximately on embryonic day (E) 12.5, and by E18-5 almost all skeletal muscle is absent. Analysis of the consequences of muscle cell ablation revealed that almost all spinal motoneurons are lost by E18.5, providing strong evidence that survival of spinal motoneurons during embryogenesis is dependent on signals from their target tissue, skeletal muscle, and that trophic signals produced by nonmuscle sources are sufficient to support survival of no more than 10% of embryonic spinal motoneurons in the absence of muscle-derived signals. There was also substantial loss of cranial (hypoglossal and facial) motoneurons in the muscle-deficient embryos, thus indicating that cranial motoneuron survival is also dependent on trophic signals produced by their target tissue. Although spinal motoneurons are a major target of spinal interneurons, the loss of motoneurons did not affect interneuron survival. Muscle-deficient embryos had a cleft palate and abnormalities of the lower jaw, raising the possibility that they might serve as a mouse model for the human disorder, Robin sequence. The data reported here demonstrate the utility of a binary transgenic system for obtaining mouse embryos in which a specific cell population has been ablated, so that its role in embryonic development can be studied.

Animals↗
PubMed1998

Peripheral target regulation of the development and survival of spinal sensory and motor neurons in the chick embryo.

Unilateral limb-bud removal (LBR) before the outgrowth of sensory or motor neurons to the leg of chick embryos was used to examine the role of limb (target)-derived signals in the development and survival of lumbar motoneurons and sensory neurons in the dorsal root ganglia (DRG). After LBR, motor and sensory neurons underwent normal initial histological differentiation, and cell growth in both populations was unaffected. Before their death, target-deprived motoneurons also expressed a cell-specific marker, the homeodomain protein islet-1. Proliferation of sensory and motor precursor cells was also unaffected by LBR, and the migration of neural crest cells to the DRG and of motoneurons into the ventral horn occurred normally. During the normal period of programmed cell death (PCD), increased numbers of both sensory and motor neurons degenerated after LBR. However, whereas motoneuron loss increased by 40-50% (90% total), only approximately 25% more sensory neurons degenerated after LBR. A significant number of the surviving sensory neurons projected to aberrant targets in the tail after LBR, and many of these were lost after ablation of both the limb and tail. Treatment with neurotrophic factors (or muscle extract) rescued sensory and motor neurons from cell death after LBR without affecting precursor proliferation of either population. Activity blockade with curare failed to rescue motoneurons after LBR, and combined treatment with curare plus muscle extract was no more effective than muscle extract alone. Treatment with the antioxidant N-acetylcysteine rescued motoneurons from normal cell death but not after LBR. Two specific inhibitors of the interleukin beta1 converting enzyme (ICE) family of cysteine proteases also failed to prevent motoneuron death after LBR. Taken together these data provide definitive evidence that the loss of spinal neurons after LBR cannot be attributed to altered proliferation, migration, or differentiation. Rather, in the absence of limb-derived trophic signals, the affected neurons fail to survive and undergo PCD. Although normal cell death and cell death after target deprivation share many features in common, the intracellular pathways of cell death in the two may be distinct.

Acetylcysteine↗
PubMed1998

A novel strategy for introducing exogenous bcl-2 into neuronal cells: the Cre/loxP system-mediated activation of bcl-2 for preventing programmed cell death using recombinant adenoviruses.

We have established a novel strategy for introducing exogenous Bcl-2 into neuronal cells that is mediated by Cre/loxP recombination using recombinant adenoviral vectors. An on/off-switching cassette for Bcl-2 (CALNLbcl-2) was designed to express Bcl-2 by recombinase Cre-mediated excisional deletion of a spacer DNA flanked by a pair of loxP sites. Exogenous Bcl-2 was clearly induced in PC12 cell lines carrying CALNLbcl-2 after infection with recombinant adenovirus producing recombinase Cre (AxCANCre). Dual infection with both AxCANCre and a recombinant adenovirus bearing CALNLbcl-2 showed efficient delivery of exogenous Bcl-2 into a hybrid motoneuronal cell line and primary chicken spinal motoneurons. The delivery of foreign Bcl-2 promoted survival of motoneurons in medium either containing or lacking trophic support. Thus, this strategy for delivery of exogenous Bcl-2 will be useful for studying neuronal death as well as for introducing foreign genes into postmitotic neurons under the control of recombinase Cre.

Adenoviridae↗
PubMed1998

Involvement of specific caspases in motoneuron cell death in vivo and in vitro following trophic factor deprivation.

The caspases have been shown to be key components of programmed cell death (PCD) in various cell types, including neurons. Caspase-3 (CPP32) is the predominant caspase that appears to be involved in cell death in several systems. In embryonic motoneuron cultures, caspase-3 activity increases beginning at 20 h following deprivation of trophic support, as determined by the cleavage of its specific substrates. Inhibition of caspase-3 by peptide inhibitors prevents the PCD of motoneurons following trophic factor deprivation in vitro, as well as in vivo. We also investigated the cleavage of poly(ADP-ribose) polymerase (PARP) in motoneurons after trophic factor withdrawal. No PARP cleavage was detected in either viable or dying cells. These data suggest that some components of the cell death machinery such as the involvement of caspases may be conserved in different cell types undergoing PCD, whereas the activation and specific substrates of the caspases may differ from one cell type to another.

Animals↗
PubMed1997

Cloning and expression of the cDNA encoding rat caspase-2.

We have isolated cDNA clones for rat caspase-2 (also called Nedd2/Ich-1), that encodes a protein similar to interleukin-1beta-converting enzyme (ICE) and the product of the nematode Caenorhabditis elegans cell death gene ced-3 both of which play an important role in programmed cell death (PCD). The rat caspase-2 cDNA clones have an open reading frame (ORF) of 452 amino acids (aa). The predicted aa sequence of rat caspase-2 is highly similar to that of mouse Nedd2 (97.3%) and human Ich-1L (91.3%). The aa sequence QACRG containing the active Cys residue, that is necessary for the proteolytic activity of ICE/Ced-3 (caspase) family of proteases, is also conserved in rat caspase-2. Rat caspase-2 also has several Asp residues in the amino and carboxyl cleavage regions similar to other caspase family proteins. We have developed PC12 cells carrying an on/off switching cassette of caspase-2 (named PC-Nd cells), which contains the neo gene flanked by a pair of loxP sites, the Cre-specific recognition sequence of 34 nucleotides (nt), that lies between the promoter and the caspase-2 cDNA. This expression cassette was designed to express the neo gene initially and to turn on the expression of caspase-2 by site-specific recombinase Cre-mediated excisional deletion of the neo gene. After infection with Cre-producing recombinant adenovirus (re-Ad), the expression of caspase-2 was highly induced in PC-Nd cells and presumptive actively processed fragments of caspase-2 were also observed. This gene activation strategy of caspase-2 will be useful for the study of the biological effects of caspase family proteins in PCD.

Amino Acid Sequence↗
PubMed1997

Effects of excitatory amino acids on neuromuscular development in the chick embryo.

To investigate the presumptive role of excitatory amino acids (EAAs) in the regulation of normally occurring motoneuron (MN) death, chick embryos were treated with the glutamate receptor antagonists dizocilpine maleate and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium. Both failed to alter the number of surviving MNs at the end of the critical period of programmed cell death. However, treatment with 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, a competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, was able to rescue a significant number of MNs from death. Treatment with several EAA agonists induced extensive excitotoxic lesions in the spinal cord. MN degeneration induced by excitotoxins exhibited changes characteristic of necrosis rather than apoptosis. However, when either 0.5 or 1 mg of NMDA was applied acutely on embryonic day (E) 7, about 50% of treated embryos failed to exhibit NMDA-induced excitoxicity but rather showed a clear reduction in the number of pyknotic MNs. This apparent neuroprotective effect of NMDA was also observed in a subset of embryos chronically treated with NMDA, in which an excessive number of MNs was detected when examined on E9. Surprisingly, in the same experiment other embryos showed either normal or highly reduced MN numbers. Embryos with motoneuronal depletion induced by NMDA also showed a delayed impairment of later neuromuscular development with the appearance of degenerative changes in surviving MNs and apoptosis of skeletal muscle cells. Because some of the alterations reported here are similar to those described in MN diseases, our experimental model may be useful for gaining insights into the mechanisms that control both developmentally regulated and pathological MN death.

Animals↗
PubMed1997

Neurotrophin-3 promotes the differentiation of muscle spindle afferents in the absence of peripheral targets.

The neurons of the dorsal root ganglia (DRG) that supply muscle spindles require target-derived factors for survival. One necessary factor for these neurons is neurotrophin-3 (NT3). To determine whether NT3 can promote the survival of these neurons in the absence of other target-derived factors, we analyzed the effects of exogenous NT3 after early limb bud deletion in the chick. In control embryos, limb bud deletion eliminated approximately 90% of the trkC-positive (trkC+) neurons in lumbar DRG on the deleted side. In addition, the deletion led to a dramatic loss of collateral sensory projections to motoneurons. Exogenous NT3 restored a normal population of trkC+ neurons in lumbar DRG on the deleted side and increased the number of trkC+ neurons in DRG with normal targets (contralateral lumbar and thoracic). The effect was highly selective; NT3 increased the number of trkC+ neurons without significantly changing the number of either trkA+ or trkB+ neurons. The effect of NT3 was attributable to the rescue of DRG neurons from cell death, because exogenous NT3 reduced the number of pyknotic nuclei without significantly altering proliferation. Analysis of spinal projections showed further that many of the trkC+ neurons rescued by NT3 projected to the ventral spinal cord. These neurons thus had central projections characteristic of muscle spindle afferents. Together, our results indicate that NT3 signaling is both necessary and sufficient for the development of the proprioceptive phenotype, even in the absence of other signals from limb muscle.

Animals↗
PubMed1997

Neuromuscular development in the avian paralytic mutant crooked neck dwarf (cn/cn): further evidence for the role of neuromuscular activity in motoneuron survival.

Neuromuscular transmission and muscle activity during early stages of embryonic development are known to influence the differentiation and survival of motoneurons and to affect interactions with their muscle targets. We have examined neuromuscular development in an avian genetic mutant, crooked neck dwarf (cn/cn), in which a major phenotype is the chronic absence of the spontaneous, neurally mediated movements (motility) that are characteristic of avian and other vertebrate embryos and fetuses. The primary genetic defect in cn/cn embryos responsible for the absence of motility appears to be the lack of excitation-contraction coupling. Although motility in mutant embryos is absent from the onset of activity on embryonic days (E) 3-4, muscle differentiation appears histologically normal up to about E8. After E8, however, previously separate muscles fuse or coalesce secondarily, and myotubes exhibit a progressive series of histological and ultrastructural degenerative changes, including disarrayed myofibrils, dilated sarcoplasmic vesicles, nuclear membrane blebbing, mitochondrial swelling, nuclear inclusions, and absence of junctional end feet. Mutant muscle cells do not develop beyond the myotube stage, and by E18-E20 most muscles have almost completely degenerated. Prior to their breakdown and degeneration, mutant muscles are innervated and synaptic contacts are established. In fact, quantitative analysis indicates that, prior to the onset of muscle degeneration, mutant muscles are hyperinnervated. There is increased branching of motoneuron axons and an increased number of synaptic contacts in the mutant muscle on E8. Naturally occurring cell death of limb-innervating motoneurons is also significantly reduced in cn/cn embryos. Mutant embryos have 30-40% more motoneurons in the brachial and lumbar spinal cord by the end of the normal period of cell death. Electrophysiological recordings (electromyographic and direct records form muscle nerves) failed to detect any differences in the activity of control vs. mutant embryos despite the absence of muscular contractile activity in the mutant embryos. The alpha-ryanodine receptor that is genetically abnormal in homozygote cn/cn embryos is not normally expressed in the spinal cord. Taken together, these data argue against the possibility that the mutant phenotype described here is caused by the perturbation of a central nervous system (CNS)-expressed alpha-ryanodine receptor. The hyperinnervation of skeletal muscle and the reduction of motoneuron death that are observed in cn/cn embryos also occur in genetically paralyzed mouse embryos and in pharmacologically paralyzed avian and rat embryos. Because a primary common feature in all three of these models is the absence of muscle activity, it seems likely that the peripheral excitation of muscle by motoneurons during normal development is a major factor in regulating retrograde muscle-derived (or muscle-associated) signals that control motoneuron differentiation and survival.

Animals↗
PubMed1997

Target contact regulates expression of synaptotagmin genes in spinal motor neurons in vivo.

During neuromuscular development, neuronal contact with peripheral targets is associated with an increase in synaptic vesicle protein (SVP) gene expression, suggesting that target contact and upregulation of SVP genes are causally related. To test this idea, we analyzed the developmental expression pattern of synaptotagmin (syt) mRNAs in the chick lateral motor column (LMC) using in situ hybridization. Syt I mRNA in the LMC is upregulated from Embryonic Day 4.5 (E4.5) to E5.5, coincident with the time these neurons begin to make contact with their muscle targets. In contrast, levels of mRNA for neurofilament do not change during this time. Extirpation of the limb bud prior to motor axon outgrowth eliminates the increase in syt I mRNA ipsilaterally. Later in development, there is a switch in syt isoform abundance in the LMC, with syt II mRNA being upregulated between E15 and E20 and syt I mRNA being downregulated. Our results suggest that contact with targets upregulates syt I gene expression during neuromuscular synapse formation in vivo, and that a later stage of synaptic maturation involves changes in SVP isoform abundance.

Animals↗
PubMed1996

Altered development of spinal cord in the mouse mutant (Patch) lacking the PDGF receptor alpha-subunit gene.

The platelet-derived growth factor receptor alpha subunit (PDGFR alpha) is expressed by glial precursors, glial cells, and some peripheral neurons during normal rodent development. Its ligands are expressed ubiquitously in neurons, including sensory and motor neurons. Thus, neuronally secreted PDGF-A may play a paracrine role in the development of both glial cells and peripheral neurons. The Patch (Ph) mutation, which is a deletion of the PDGFR alpha, is a homozygous embryonic lethal mutation in the mouse. Previously, several developmental abnormalities, including deficiencies in connective tissues in many organs, aberrant neural crest cell migration, and defects in non-neuronal derivatives of crest cells, have been shown to be associated with the Patch mutation. However, whether and the extent to which motor and sensory neurons are affected by the mutation are not known. Here, we have examined the survival and/or morphological differentiation of spinal motor and sensory (dorsal root ganglion) neurons during the period of naturally occurring cell death, i.e., between E14 and E18, in control and Ph/Ph mice. The results show a 65-70% decrease in motor and sensory neuron numbers in Ph/Ph mice, compared to controls, at all stages examined. Furthermore, motoneurons in Ph/Ph mice were significantly smaller than those in controls. Because of the bidirectional nature of neuron-glial cell interactions, these results suggest that PDGFR alpha plays an important role in glial cell development and, thus, indirectly in neuronal cell development or, alternatively, that PDGF and the PDGFR alpha are directly involved in peripheral neuron survival and development by an autocrine/paracrine mechanism.

Animals↗
PubMed1996

Schwann cell apoptosis during normal development and after axonal degeneration induced by neurotoxins in the chick embryo.

In the present work, we show that chick embryo Schwann cells die by apoptosis both during normal development and after axonal degeneration induced by neurotoxin treatment. Schwann cell apoptosis during development takes place during a period roughly coincidental with normally occurring motoneuron death. Administration of NMDA to chick embryos on embryonic day 7 induces extensive excitotoxic motoneuronal damage in the spinal cord without any apparent effects on neurons in the dorsal root ganglia (DRG). The death of Schwann cells in ventral nerve roots after NMDA treatment causes degenerative changes that display ultrastructural features of apoptosis and exhibit in situ detectable DNA fragmentation. By contrast, NMDA treatment does not increase the death of Schwann cells in dorsal nerve roots. In situ detection of DNA fragmentation in combination with the avian Schwann cell marker 1E8 antibody demonstrates that dying cells in ventral nerve roots are in the Schwann cell lineage. Administration of cycloheximide does not prevent the toxic effects of NMDA on motoneurons, but dramatically reduces the number of pyknotic Schwann cells and DNA fragmentation profiles in the ventral nerve roots. In ovo administration of various tissue extracts (muscle, brain, and spinal cord) from the chick embryo or of the motoneuron conditioned medium fails to prevent Schwann cell apoptosis in NMDA-treated embryos. Intramuscular administration of the snake toxin beta-bungarotoxin produces a massive death of both lateral motor column motoneurons and DRG neurons, resulting in a substantial increase in the number of pyknotic Schwann cells in both ventral and dorsal nerve roots. It is concluded that during development, axonal-derived trophic signals are involved in the regulation of Schwann cell survival in peripheral nerves.

Animals↗
PubMed1996

A novel type of programmed neuronal death in the cervical spinal cord of the chick embryo.

We examined the massive early cell death that occurs in the ventral horn of the cervical spinal cord of the chick embryo between embryonic days 4 and 5 (E4 and E5). Studies with immunohistochemical, in situ hybridization, and retrograde-tracing methods revealed that many dying cells express Islet proteins and Lim-3 mRNA (motoneuron markers) and send their axons to the somatic region of the embryo before cell death. Together, these data strongly suggest that the dying cells are somatic motoneurons. Cervical motoneurons die by apoptosis and can be rescued by treatment with cycloheximide and actinomycin D. Counts by motoneuron numbers between E3.5 and E10 revealed that, in addition to cell death between E4 and E5, motoneuron death also occur between E6 and E10 in the cervical cord. Studies with [3H]thymidine autoradiography and morphological techniques revealed that in the early cell-death phase (E4-E5), genesis of motoneurons, axonal elongation, and innervation of muscles is still ongoing. However, studies with [3H]thymidine autoradiography also revealed that the cells dying between E4 and E5 become postmitotic before E3.5. Increased size of peripheral targets, treatment with neuromuscular blockade, and treatment with partially purified muscle or brain extracts and defined neurotropic agents, such as NGF, BDNF, neurotrophin-3, CNTF, bFGF, PDGF, S100-beta, activin, cholinergic differentiation factor/leukemia inhibitory factor, bone morphogenetic protein-2, IGF-I, interleukin-6, and TGF-beta 1, were all ineffective in rescuing motoneurons dying between E4 and E5. By contrast, motoneurons that undergo programmed cell death at later stages (E6-E10) in the cervical cord are target-dependent and respond to activity blockade and trophic factors. Experimental approaches revealed that early cell death also occurs in a notochord-induced ectopic supernumerary motoneuron column in the cervical cord. Transplantation of the cervical neural tube to other segmental regions failed to alter the early death of motoneurons, whereas transplantation of other segments to the cervical region failed to induce early motoneuron death. These results suggest that the mechanisms that regulate motoneuron death in the cervical spinal cord between E4 and E5 are independent of interactions with targets. Rather, this novel type of cell death seems to be determined by signals that either are cell-autonomous or are derived from other cells within the cervical neural tube.

Animals↗