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First published online 3 May 2006
doi: 10.1242/dev.02376

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Programmed cell death mechanisms of identifiable peptidergic neurons in Drosophila melanogaster

Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.

View larger version (77K): [in a new window]   Fig. 1. Crz expression in the CNS during metamorphosis. (A,B) In situ hybridization of Crz mRNA. (A) Third-instar larval CNS, showing three pairs of DL neuron in the brain (Br) and eight pairs of vCrz neurons (brackets). (B) Pupal CNS at 36 hours APF. The number of neurons in the brain (arrowhead) is found to be six to eight per lobe, whereas none of vCrz neurons are detectable. (C,D) Crz-IR neurons in third-instar larval CNS (C) and pupal CNS at 12-24 hours APF (D). Consistent with the in situ hybridization results, no Crz-IR neurons are detectable in the VNC of the pupal CNS (arrow). At least five specimens were processed for each panel. (E) Colocalization (center) of Crz-gal4-induced GFP expression (left) in Crz-IR neurons (right). Scale bars: 100 µm.

View larger version (103K): [in a new window]   Fig. 2. Rescue of vCrz-cell death by transgenic expression of p35. (A) Control 1-day old pupa (UAS-lacZ/+; Crz-gal4/+). (B) One-day-old pupa expressing p35 in Crz neurons (UAS-lacZ/+; Crz-gal4, UAS-p35/+). Persistent vCrz neuronal somata and ascending projections are designated by arrows and arrowheads, respectively. (C-F) Progressive removal of lacZ-marked vCrz neurons (UAS-lacZ/+; Crz-gal4/+) during metamorphosis. White prepupae were aged at 25°C for durations designated in each panel, and then processed for X-gal staining. (C) White prepupa (n=5). (D) 2 hours APF (n=6). (E) 3 hours APF (n=4). Arrow points to discontinued projection. (F) 6 hours APF (n=9). Faint dots (arrow) most probably come from residual ß-galactosidase activity rather than from live cells. Scale bar: 100 µm.

View larger version (59K): [in a new window]   Fig. 3. DNA fragmentation in vCrz neurons. Wild-type prepupal CNSs at 2 hours APF (A) or 3 hours APF (B) were subjected to TUNEL assay (right), followed by Crz immunohistochemistry (left). These images were merged to show superimposition of the two signals (center). TUNEL-positive vCrz neurons are indicated by arrows and TUNEL signals in non-vCrz cells by arrowheads. Scale bar: 10 µm.

View larger version (100K): [in a new window]   Fig. 4. Inhibition of vCrz-cell death by targeted expression of EcR-B1 dominant-negative forms. (A-C) CNSs were dissected at 12 hours APF and processed for X-gal staining. (A) Control (UAS-lacZ/+; Crz-gal4/+). An arrowhead indicates the staining from residual ß-galactosidase activity. (B) UAS-lacZ/+; Crz-gal4/UAS-EcR-B1F645A (n=4). (C) UAS-lacZ/+; Crz-gal4/UAS-EcR-B1W650A (n=4). Live vCrz neurons are shown within the brackets. (D) (Left) A confocal image showing nuclear EcR-B1-IRy (green) in a vCrz neuron (orange, arrow) at white prepupal stage of wild-type. (Right) An independent specimen showing two vCrz neurons containing EcR-B1-IRy (arrows). (E-H) Crz-IRy in various EcR mutants at 12-17 hours APF. (E) Wild type. (F) EcR-A mutant (EcRM554fs/EcR112). (G) EcR-B mutant (EcR31/EcR99) after completion of apolysis. Surviving vCrz neurons are shown in the brackets (see also Fig. 5A). (H) EcR-B1 mutant (EcR31/EcRQ50st). Scale bars: in A, 50 µm for A-C,E-H; in D, 10 µm for D.

View larger version (40K): [in a new window]   Fig. 5. Rescue of EcR-B mutant phenotype by transgenic expression of EcR-B1 or EcR-B2-isoform. (A) EcR-B mutant control (EcR31/EcR99). (B) EcR31/EcR99; UAS-EcR-B1/Crz-gal4. (C) EcR31/EcR99; UAS-EcR-B2/Crz-gal4. The histograms indicate percentages of the CNSs showing no cell death (open bar), partial (gray bar), or complete death (black bar). As described in the text, the CNSs were grouped into `E' (Early), `I' (Intermediate), or `L' (Late), depending on their developmental status. Numbers of tissues examined in each category are noted on the right of each bar (N), and mean numbers of vCrz neurons (± s.d.) are in parentheses. The CNSs belonging to the `E' group were not found in the EcR-B2 rescue group. Representative images taken from the `I' group are shown in the left. Scale bar: 100 µm.

View larger version (104K): [in a new window]   Fig. 6. The role of rpr in vCr2 PCD. (A-D) Crz-IRy in (A) H99/TM6B, (B) XR38/TM6B, (C) rpr-null mutant (XR38/H99) at 1216 hours APF, and (D) hid loss-of-function mutant (X14/hid05014) at 8 hours APF. Note that 14 vCrz neurons are still detectable in the rpr mutant (brackets in C). (E,F) Rpr in situ hybridization of the CNSs dissected from (E) third-instar larva (Crz-gal4, UAS-p35/UAS-rpr) and (F) wild-type prepupa at 1.5 hours APF. The hybridization signals are indicated by arrowheads or by a circle. (G) Double labeling of wild-type CNS with rpr in situ hybridization probe (top) and anti-Crz (middle) at 1.5 hours APF. Merge of the two images (bottom) shows co-localization of rpr mRNA in a vCrz neuron (arrow). Expression of rpr in non-vCrz neurons is indicated by arrowheads. (H) rpr promoter activity in vCrz neurons at 1 hour APF. Crz-IRy (top), rpr-gal4-driven GFP expression (middle) and merge of the two (bottom). A vCrz neuron positive for both GFP and Crz-IRy appears in yellow. (I) Rpr-gal4-mediated p35 expression (rpr-gal4/UAS-p35) suppresses the death of 10 vCrz neurons (brackets) at 7-9 hours APF. Two different specimens are shown here. Scale bars: in D, 50 µm for A-D,I; in E, 50 µm for E,F; 10 µm in G,H.

View larger version (92K): [in a new window]   Fig. 7. Lack of role for diap1 in the vCrz PCD. (A) Control (UAS-mCD8-GFP/+;;Crz-gal4/+). (B) Targeted expression of the diap1RNAi in Crz neurons (UAS-mCD8-GFP/+; symUAS-diap1RNAi/+; Crz-gal4/+) (cf. Huh et al., 2004). Intact GFP signals (vCrz) at 1 hour APF suggest that knockdown of endogenous diap1 does not cause premature cell death. (C) Effect of diap1RNAi on premature death of the salivary glands. The symUAS-diap1RNAi or y w (control) was crossed to the 34B-gal4; UAS-GFP, and then white prepupal progeny were examined for GFP. In the control, intense GFP signals are seen in a pair of the salivary glands (left, arrow), whereas the signals are significantly weakened by diap1RNAi (right, arrow). (D) Crz-IRy in homozygous thSL at 6.5 hours APF. (E) X-gal histochemistry of UAS-lacZ/+; Crz-gal4/UAS-diap1 at 6 hours APF. Both genetic and transgenic diap1 gain of function do not prevent vCrz death. (F) Rescue of the salivary gland degeneration by diap1. In a control pupa (34B-gal4/+; UAS-GFP/+, n=5) at 18 hours APF, faint GFP signals (left, arrow) reflect the PCD in this tissue. By contrast, strong GFP signals (right, arrow) are maintained by diap1 expression (34B-gal4/+; UAS-GFP/UAS-diap1, n=8). (G,H) Delayed PCD of vCrz neurons (G) in a dronc-null mutant (dronc51/droncI24) or (H) in a homozygous dark-null mutant (darkCD4). Arrows indicate Crz-IR neurons that still survive at stages indicated in each panel. Scale bars: in A, 100 µm for A,B,D,E; in G, 50 µm for G,H.