Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death

Additional file 1:

Dendritic pruning and migrating hemocytes. Class IV ddaC and hemocytes were imaged at 5-minute intervals with a 1.5 μm Z-step between 4 h 50 minutes APF and 6 h APF. Dendritic branches became detached from the cell body and eventually disappeared. The hemocytes were migrating by generating prominent lamellipodia. Genotype: UAS-EGFP; ppk-EGFP/pxn-Gal4.

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Additional file 2:

Identification of da neurons in live whole-mount pupae. (A) Time-lapse recordings of A4 tergite of a whole-mount pupa of genotype Gal4^109(2)80 UAS-mCD8::GFP/Gal4^109(2)80 UAS-mCD8::GFP. By 27 h APF, dendrites had degenerated, and four cells (arrows at 27 h APF) were aligned along the dorsal-ventral axis. Those four cells were identified as indicated. Scale bar: 50 μm. (B-H) We also observed whole-mount pupae of the subset markers and addressed whether the specificity of the marker expression persisted in pupae or not. (B-E) The class IV marker ppk-EGFP [13] primarily labeled ddaC in the tergite and v'ada in the pleura (not shown) as it did in larvae. Tergite of a whole-mount 60 h APF pupa of genotype Gal4^109(2)80 UAS-mmRFP/Gal4^109(2)80 UAS- mmRFP; ppk-GFP/ppk-EGFP. The channel signal of RFP (B), GFP (C), the merge of these (D), and tracings (E) are shown. (F-H) Example of other markers for 'subsets' of larval da neuons. A3 or A4 tergites of whole-mount 60 h APF pupae of genotype ppk-Gal4 UAS-mCD8::GFP/ppk-Gal4 UAS-mCD8::GFP (F), which labeled all da neurons, Gal4⁴⁷⁷ UAS-mCD8::GFP/Gal4⁴⁷⁷ UAS-mCD8::GFP (G), which labeled both da and es neurons, and UAS-mCD8::GFP/UAS-mCD8::GFP; C161/TM3 (H). In (H), ddaC was weakly labeled and difficult to see in this sample. Expression profiles of various Gal4 lines in the adult are summarized in Table 2. Scale bars: 50 μm.

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Additional file 3:

ddaD and ldaA/ldaA-like in adults. (A) Tracings of dendritic arbors of ddaD clones in A3/A4 (top) and A5/A6 (bottom). Each dendritic branch of ddaD was sometimes difficult to resolve. (B) Gr66aGal4 visualizes dendritic arbors of ldaA or ldaA-like at single-cell resolution. Pleura of A2 to A6 of UAS-RedStinger/+; Gr66aGal4 UAS-mCD8::GFP/+ was imaged through the channel of GFP (green) and DsRed (magenta; overlap with green, white). In each hemisegment, the nucleus of a single neuron (ldaA or lda-like) was labeled (inset; compare with that in Figure 5M). Arrows point to cell bodies. sp: spiracle. Scale bars: 50 μm (A); 100 μm (B).

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Additional file 4:

Segment-dependent variability in the composition of da neurons in the tergite. (A) A fillet preparation of a pharate adult female was stained with anti-HRP antibody. This image is a high-power view of the boxed region in Figure 2E. Yellow brackets indicate dendritic arbors of ddaE in A2 to A4; ddaE-like cells that formed such bushy dendritic arbors were not found in A5 or A6 (n = 9). Yellow arrowheads indicate cell bodies of representative es neurons, and blue arrows dendrites of dbds. Genotype: Gal4^109(2)80 UAS-mCD8::GFP/Gal4^109(2)80 UAS-mCD8::GFP. Scale bar: 50 μm. (B-F) Another fillet preparation of the same genotype as in (A), which was doubly stained with anti-mCD8 antibody (B, C-F) and antibody against a pan-neuronal nuclear protein, Elav (C-F). Boxed regions of A2 to A5 in (B) are highlighted in (C-F). (C-F) Three panels of each hemisegment show mCD8 staining (left; green in the right panel), Elav staining (middle; magenta or white in the right panel), and a merged image (right panel). Yellow arrows point to nuclei of da neurons, and blue arrows to those of dbd. Other smaller Elav-positive nuclei are those of es neurons. Typically, the number of the neuronal nuclei of da in the tergite was two in A5 and A6 (F), in comparison to three in A3 and A4 (D, E). Together with the results shown in (A) and Figure 8A, these results strongly suggest that ddaE was absent in A5 and A6 in the pharate adult. The A2 tergite had two, not three, da neurons (C). Our observation using ppk-EGFP (a marker of ddaC; Additional file 2C) and C161 Gal4 (a marker of ddaE; Figure 8A) showed that A2 had ddaC and ddaE, but not ddaD. (G) Collectively, the composition of da neurons in each segment at the pharate stage is summarized. Plus and minus signs represent the presence and absence of each neuronal type, respectively, and a plus sign with an asterisk means cell death within 1 week after eclosion. Scale bars: 100 μm (B); 50 μm (C-F).

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Additional file 5:

Degeneration of ddaE in A5. Time-lapse recording of tergites of A4 and A5 (right). Images were taken every 10 minutes with a 2 μm Z-step between 14 h APF and 62 h APF. A branch of ddaE in A5 was degraded after it started regeneration. Towards the end of the movie, the dorsal internal oblique muscle 1 (DIOM1) is seen at the top (dorsal) in both A4 and A5, whereas DIOM3 was present at a lateral position in only A4. We performed time-lapse recordings of pupae of this genotype a total of four times. Genotype: UAS-mCD8::GFP/UAS-mCD8::GFP; C161 UAS-mCD8::GFP/TM3 Ser Sb.

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