DEV042135 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Opa1 is a mitochondrial protein and its knockdown affects mitochondrial morphology of S2 cells. (A) Western blot analysis of Opa1 in S2 cells. cyto: cytoplasmic fraction; mito: mitochondrion-enriched fraction. Individual proteins were detected by the antibodies listed on the right. α-tubulin and CV-α, a component of mitochondrial F₁F₀ ATP synthase, are loading controls for cytoplasmic and mitochondrial proteins, respectively. Long and short forms of Opa1 are indicated by the arrowheads. The 708 bp fragment encoding the Opa1 C-terminus was used as a template to produce dsRNA. (B-D) Light micrographs (B) and ultrastructure of mitochondria (C,D) in opa1 knockdown S2 cells. In the light micrographs, mitochondria were visualized by using Mitotracker Orange. Scale bars: 10 µm in B; 200 nm in C,D.

• Supplemental Figure S2 -

  Fig. S2. prel expression pattern in da neurons. (A-F) For immunofluorescence analysis of da neurons, larvae were dissected and stained according to standard protocols with the following antibodies: rabbit anti-GFP antibody A11122 (Invitrogen) and mouse 22C10 (Developmental Studies Hybridoma Bank). Anti-Milton antibody 2A108 (Strowers et al., 2002) was a gift from T. Schwarz (Harvard Medical School). One specimen was NP0738, in which a P-element had been inserted into the 5′UTR (Fig. 3A); and another was prel-Gal4, which had a 2 kb region upstream of the coding sequence fused to Gal4. In both crosses, the Gal4-driven GFP expression was detected strongly in class IV and only weakly in the other classes in dissected larva. We could not detect endogenous Prel in larval da neurons immunostained with our antibody (Fig. 1A). Therefore, whether the Gal4 expression represents relative Prel levels in different da subclasses or not awaits further study. (A-C) Gal4^NP0738 /UAS-mCD8::GFP was doubly labeled for the pan-sensory neuron marker 22C10 (A; magenta in C) and for GFP (B; green in C). (D-F) prel-GAL4 (Gal4^prel/UAS-mCD8::GFP) was double labeled for 22C10 (D; magenta in F) and for GFP (E; green in F). (G-I) A pan-da marker line (Gal4^109(2)80 UAS-mCD8::GFP) was doubly labeled for GFP (G; green in 'I') and for Milton (H; magenta in I). (C,F,I) Merged images. Individual da neurons were identified and labeled as follows: C (class IV ddaC), D (class I ddaD), E (class I ddaE), and F (class III ddaF). Scale bars: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. prel loss-of-function causes breakage of dendritic branches more in the proximal area than in the distal one. (A,B) Dendritic arbors of representative class IV ddaC neurons of the prel mutant clones. (A′ ,B′ ) High-magnification images of the yellow boxes in A and B, respectively. The branches of the prel mutant neurons had broken and become detached from the dendritic arbors (arrows in A′,B′). Scale bars: 100 µm in A,B and 50 µm in A′,B′. (C) Composite plot that shows the distribution of breakage points of branches in the arbors of nine prel mutant neurons. Each symbol shows the location of the proximal end of a detached branch of a mutant neuron. Each concentric circle with increasing radius of 100 µm indicates the distance from cell body (cross). The most distal ends of the arbors of the wild-type neurons are distributed within the magenta annulus, whereas the blue annulus indicates a zone of the most distal ends of the mutant arbor. Twenty-two out of the 55 breakage points were found in the proximal area of the arbor (distance from the soma: 0-100 µm).

• Supplemental Figure S4 -

  Fig. S4. The prel phenotype is not modified by expression of caspase inhibitor and active caspase signal is not detected in prel mutant class IV ddaC neurons. (A-D) To study whether prel loss-of-function led to apoptosis, we blocked the caspase pathway in the prel mutant class IV neurons at the third instar larval stage. The dendritic phenotype was not modified either by expression of a dominant-negative form of the fly initiator caspase Dronc or p35 (A,B), and there was no significant rescue of the phenotype to normal (C,D). (E-G) We also investigated whether prel mutant class IV neurons could be labeled with an anti-cleaved caspase-3 Asp175 antibody (Cell Signaling); however, signals were not detected in the cell bodies, dendrites or axons. (A,B) Dendritic arbors of prel mutant ddaC clones that expressed either a dominant-negative form of DRONC (A) or p35 (B). Clone genotypes were hs-FLP Gal4^elavC155 UAS-mCD8::GFP; prel FRTG13/prel FRTG13; UAS-dronc DN/+ (A) and hs-FLP Gal4^elavC155 UAS-mCD8::GFP; prel FRTG13/prel FRTG13; UAS-p35/+ (B). Scale bars: 100 µm. (C,D) Quantification of total length of dendritic branches (C) and the number of terminal branches (D) of wild-type ddaC clones (WT), prel mutant clones (prel), the prel mutant clones expressing dominant-negative form of Dronc (prel + UAS-dronc DN), and the prel mutant clones expressing p35 (prel + UAS-p35). Data are presented as means ± s.d. ***P<0.001 (ANOVA with Tukey's HSD post hoc analysis). (E-G) prel mutant clone that was stained for two markers: clone marker GFP (green in E andG) and an antibody against cleaved caspase-3 (F; magenta in E and G). Genotype was hs-FLP Gal4^elavC155 UAS-mCD8::GFP; prel FRTG13/prel FRTG13. Scale bars: 100 µm in E, 20 µm in F and G.

• Supplemental Figure S5 -

  Fig. S5. In class I-III neurons, neither prel loss-of-function nor its overexpression causes abnormal dendritic phenotypes. (A-P) Mitochondria were distributed in cell bodies, dendrites, and axons of class I neurons as they were in the class IV ones, and mitochondria in cell bodies of class I neurons also took on the tubular structure (A,B,E,F). Loss of prel function or its overexpression caused fragmentation of mitochondria in class I neurons as they did in class IV (insets of C and G). Nevertheless, both dendrites and axons of the prel mutant or prel-overexpressing class I neurons still possessed a number of mito::GFP signals, and their densities were not affected (C,G,K,L). Furthermore, neither loss of prel function nor its overexpression caused malformation of the comb-like shape of class I dendritic arbors (compare B with D, and F with H) and caused no detectable quantitative changes in dendrite morphogenesis (M,N). In addition, we observed that class II neurons extended dendritic branches normally (data not shown). Class III neurons produced spikes as the control neurons did (compare I with J) and showed no detectable quantitative changes in dendrite morphogenesis (O,P). (A-H) Distributions of mitochondria in class I ddaE neurons. Mitochondria were visualized with mito::GFP (A,C,E,G; green in B,D,F,H) and neuronal plasma membranes were labeled with myr::mRFP (magenta in B,D,F,H). Insets in A,C,E, and G are high-magnification images of cell bodies. (A-D) Clones of the wild-type (A,B) and prel (C,D). (E-H) Control ddaE (E, F) and ddaE overexpressing Prel::3HA (G,H). Tracings of axons (blue) and dendritic arbors (magenta) of representative prel mutant ddaE (D) and ddaE overexpressing Prel::3HA (H) are shown on the right of the merged images. (I,J) Dendritic arbors of representative class III ddaA neurons of the wild-type (I) and prel (J). Genotypes were hs-FLP Gal4^elavC155; FRTG13/FRTG13; UAS-myr::mRFP UAS-mito::GFP (A, B), hs-FLP Gal4^elavC155; prel FRTG13/prel FRTG13 UAS-myr::mRFP UAS-mito::GFP (C, D), Gal4^2-21 UAS-myr::mRFP UAS-mito::GFP/+ (E, F), Gal4^2-21 UAS-myr::mRFP UAS-mito::GFP/Gal4^ppk UAS- Prel::3HA (G, H), hs-FLP Gal4^elavC155 UAS-mCD8::GFP; FRTG13/FRTG13 (I), and hs-FLP Gal4^elavC155 UAS-mCD8::GFP; prel FRTG13/prel FRTG13 (J). Scale bars: 50 µm in A-J and 10 µm in insets of A,C,E,G. The yellow asterisk indicates an auto-fluorescent signal of the trachea. (K,L) Mitochondrial signals in both dendrites (K) and axons (L) of clones of the wild-type (WT) and those of prel (prel) were quantified as described in the legend of Fig. 3J-M. (M-P) Quantification of total length of dendritic branches (M,O) and the number of terminal branches (N,P) of wild-type clones (WT), and prel mutant clones (prel). (M, N) Class I ddaD and ddaE neurons. (O,P) Class III ddaA neurons. Quantification of the length and the number included those of spikes that are unique to the class III neuron. Data are presented as means ± s.d. Each value as compared with control neurons by Student's t-test.

• Supplemental Figure S6 -

  Fig. S6. Neither Buffy expression nor co-expression of Buffy and Drob-1 affects the dendritic pattern of class IV ddaC neurons. (A-F) Dendritic arbors and distribution of mitochondria of a wild-type ddaC that overexpressed buffy (A,B), those of a ddaC neuron that co-overexpressed drob-1 and buffy (C,D), and those of a ddaC neuron that co-overexpressed prel and buffy (E,F) are shown. Mitochondria were visualized with mito::GFP (A,C,E; green in B,D,F) and neuronal plasma membranes were labeled with myr::mRFP (magenta in B,D,F). The yellow arrows indicate ddaC neurons. Genotype was Gal4^ppk UAS-myr::mRFP UAS-mito::GFP/UAS-Buffy (A,B), Gal4^ppk UAS-myr::mRFP UAS-mito::GFP/UAS-Buffy; UAS-Drob-1/+ (C,D) or Gal4^ppk UAS-myr::mRFP UAS-mito::GFP/UAS-Buffy UAS-prel (E,F). Scale bars: 100 µm.

• Supplemental Figure S7 -

  Fig. S7. Most terminal branches of the wild-type clones grow over the time period observed. Time-lapse recordings of the wild-type class IV ddaC clones. Boxed regions in the left images are enlarged on the right. The arrows indicate elaborated terminal branches. The genotype was hs-FLP Gal4^elavC155 UAS-mCD8::GFP; FRTG13/FRTG13. Scale bars: 100 µm.

• Supplemental Figure S8 -

  Fig. S8. Terminal branches of the prel mutant neurons tend to be retracted over the observed time period. Time-lapse recordings of prel mutant class IV clones. Boxed regions in the left images are enlarged on the right. The arrows indicate eliminated terminal branches. The genotype was hs-FLP Gal4 ^elavC155 UAS-mCD8::GFP; prel FRTG13/prel FRTG13. Scale bars: 100 µm