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Motoneurons deprived of trophic support in vitro require new gene expression to undergo programmed cell death
C E Milligan1, R W Oppenheim, L M Schwartz
1Department of Biology, University of Massachusetts, Amherst.
Abstract:
During normal development, large numbers of neurons die by programmed cell death. This phenomena has been extensively studied in the lateral motor column of chick embryos, where approximately 50% of the motoneurons that are initially produced, subsequently die due in part to competition for a limited supply of target-derived trophic support. Inhibitors of RNA and protein synthesis block this cell loss in vivo, indicating a requirement for new gene expression (Oppenheim et al., 1990). Prior to their commitment to death, motoneurons can be isolated as a relatively pure population from chick spinal cord for in vitro study. Cells plated with muscle extract, a potent source of target-derived trophic support, survive, and have large, phase-bright cell bodies and extensive neurite outgrowth. In contrast, motoneurons cultured in the absence of muscle extract die within 48 h. This death can be blocked by the RNA synthesis inhibitor actinomycin D, at the time when the cells become committed to die, suggesting that new gene expression is required for cell death. DNA fragmentation and nuclear condensation indicate that some of these cells die by apoptosis. Therefore, it appears that many aspects of motoneuron development observed in vivo can be reconstituted in vitro. These cultures can be used as a model system for studying neuronal death and may contribute to an understanding of the molecular mechanisms that mediate programmed cell death during neuronal development.
Insights
During chick embryo development, about 50% of motor neurons undergo programmed cell death due to limited trophic support. New gene expression is required for this neuronal cell death, as shown by RNA synthesis inhibitors blocking the process.
Area of Science:
- Neuroscience
- Developmental Biology
- Cell Biology
Background:
- Programmed cell death is a critical process during normal development.
- In chick embryos, ~50% of developing motor neurons in the lateral motor column undergo cell death.
- This cell death is partly driven by competition for target-derived trophic factors.
Purpose of the Study:
- To investigate the molecular mechanisms underlying programmed cell death in developing motor neurons.
- To establish an in vitro model for studying neuronal development and cell death.
- To determine the role of new gene expression in motor neuron death.
Main Methods:
- Isolation of pure populations of chick spinal cord motoneurons.
- In vitro culture of motoneurons with and without muscle extract (trophic support).
- Treatment with RNA and protein synthesis inhibitors (e.g., actinomycin D).
- Assessment of cell survival, morphology, neurite outgrowth, DNA fragmentation, and nuclear condensation.
Main Results:
- Motoneurons cultured without muscle extract exhibited significant cell death within 48 hours.
- Inhibition of RNA synthesis (actinomycin D) blocked cell death when cells were committed to die.
- Motoneurons cultured with muscle extract survived and showed robust neurite outgrowth.
- Evidence of apoptosis, including DNA fragmentation and nuclear condensation, was observed in dying cells.
Conclusions:
- In vitro cultures of chick motoneurons effectively model in vivo developmental cell death.
- New gene expression is essential for the execution of programmed cell death in developing motor neurons.
- This model system facilitates the study of molecular mechanisms governing neuronal development and apoptosis.