Crustacean cardioactive peptide (CCAP)-expressing neurons undergo programmed cell death (PCD) within 24 hours after adult eclosion. A subset of the doomed CCAP neurons in the ventral nerve cord also expressed the neuropeptide bursicon and thus are referred to as bursCCAP neurons. In this study, we undertook comprehensive genetic and transgenic analyses to dissect the PCD mechanisms of bursCCAP neurons. Expression of a versatile caspase inhibitor, p35, blocked PCD of bursCCAP neurons, suggesting caspase-dependent apoptosis. Further genetic analyses showed that Dronc/Dark and Drice are key caspases, but they are not sufficient to carry out the PCD fully. We did not find a role for other known caspases, Strica, Dredd, Damm, or Decay. Of interest, Dcp-1 is required not for the death of bursCCAP neurons per se but for the removal of neural projections. DIAP1 is an important survival factor that inhibits premature death of bursCCAP neurons. We found that grim functions as a principal death inducer, whereas other death genes, hid, reaper, and sickle, show no endogenous function. Taken together with other studies, our work supports the role of grim as a major death inducer particularly for the removal of obsolete larval neurons during CNS metamorphosis. Results from the ectopic expression of the mutant grim lacking either N-terminal IBM or internal GH3 domain indicated that both domains are necessary to induce CCAP cell death.

SummaryIn Drosophila melanogaster, combinatorial activities of four death genes, head involution defective (hid), reaper (rpr), grim, and sickle (skl), have been known to play crucial roles in the developmentally regulated programmed cell death (PCD) of various tissues. However, different expression patterns of the death genes also suggest distinct functions played by each. During early metamorphosis, a great number of larval neurons unfit for adult life style are removed by PCD. Among them are eight pairs of corazonin-expressing larval peptidergic neurons in the ventral nerve cord (vCrz). To reveal death genes responsible for the PCD of vCrz neurons, we examined extant and recently available mutations as well as RNA interference that disrupt functions of single or multiple death genes. We found grim as a chief proapoptotic gene and skl and rpr as minor ones. The function of grim is also required for PCD of the mitotic sibling cells of the vCrz neuronal precursors (EW3-sib) during embryonic neurogenesis. An intergenic region between grim and rpr, which, it has been suggested, may enhance expression of three death genes in embryonic neuroblasts, appears to play a role for the vCrz PCD, but not for the EW3-sib cell death. The death of vCrz neurons and EW3-sib is triggered by ecdysone and the Notch signaling pathway, respectively, suggesting distinct regulatory mechanisms of grim expression in a cell- and developmental stage-specific manner.

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