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Programmed cell death of an identified motoneuron in vitro: temporal requirements for steroid exposure and protein
11254 Institute of Neuroscience, University of Oregon, Eugene 97403-1254, USA.
Abstract:
Ecdysteroid hormones trigger the programmed cell death (PCD) of a segmental subset of accessory planta retractor (APR) motoneurons at pupation in the moth, Manduca sexta. APRs from abdominal segment four [APR (4)s] survive through the pupal stage, whereas homologous APR(6)s die 24-48 h after pupal ecdysis (PE) (the shedding of the larval cuticle), in response to the prepupal peak of ecdysteroids. Following retrograde labeling with the vital fluorescent dye, DiI, the morphology of APR(4)s and APR(6)s in vivo was examined at PE and 24-48 h later. During this period, APR(4) somata remained large and ovoid while APR(6)s somata became shrunken and rounded. Similar phenotypes were observed when DiI-labeled APRs were cultured at PE and examined 24 h to 1 week later. During initial shrinkage and rounding of APR(6)s, the plasma membrane remained intact but DNA condensation occurred and mitochondrial activity was lost. The requirements for ecdysteroids and new protein synthesis for APR(6) death were tested by culturing cells with ecdysteroids and cycloheximide (CHX). When cultured at PE, the death of APR(6)s was independent of further exposure to ecdysteroids and could not be blocked by CHX. In contrast, APR(6)s cultured 24 h earlier required additional exposure to ecdysteroids to die and their death was inhibited by CHX. Thus, the final 24 h of larval life represents an important transition period in the commitment of APR(6)s to undergo PCD, and is of interest for pursuing underlying mechanisms of steroid-induced PCD.
Insights
Ecdysteroid hormones induce programmed cell death (PCD) in specific moth neurons. APR(6) neurons commit to PCD late in larval development, independent of further steroid exposure.
Area of Science:
- Developmental biology
- Neuroscience
- Endocrinology
Background:
- Ecdysteroid hormones regulate insect development and metamorphosis.
- Specific motoneurons undergo programmed cell death (PCD) during insect development.
- Accessory planta retractor (APR) motoneurons in Manduca sexta exhibit differential survival during pupation.
Purpose of the Study:
- To investigate the cellular and molecular mechanisms underlying ecdysteroid-induced PCD in APR motoneurons.
- To determine the critical timing of developmental commitment to PCD in APR(6) neurons.
- To elucidate the role of ecdysteroids and protein synthesis in APR(6) neuron death.
Main Methods:
- Retrograde labeling with DiI to visualize motoneuron morphology in vivo.
- In vitro culture of APR neurons with and without ecdysteroids and cycloheximide (CHX).
- Microscopic examination of cell morphology, plasma membrane integrity, DNA condensation, and mitochondrial activity.
Main Results:
- APR(4) motoneurons survive pupation, while APR(6) motoneurons undergo PCD 24-48 hours after pupal ecdysis.
- APR(6) neurons exhibit somatic shrinkage, DNA condensation, and loss of mitochondrial activity during PCD.
- APR(6) neurons cultured at pupal ecdysis die independently of further ecdysteroids and CHX, but those cultured earlier require ecdysteroids and are sensitive to CHX.
Conclusions:
- The final 24 hours of larval life are crucial for APR(6) neuron commitment to PCD.
- Steroid-induced PCD involves a critical transition period in late larval development.
- Further research is needed to uncover the molecular pathways of steroid-induced PCD in these neurons.