DEV027920 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. CAPS-GFP retains the normal function of CAPS. (A,B) Schematics illustrating ectopic synapse formation by MN12s. The distal end of ISNb that contains axons of MN12s normally forms synaptic endings exclusively on M12 (A). When the distal end of ISNb forms endings on M13, they are considered ectopic synapses (B). (C) In control larvae (24B-GAL4/+), ectopic synapses were rarely seen (n=109 segments). Expression of intact caps (n=64) or caps-GFP (n=93) in all muscles by 24B-GAL4 induces ectopic synapses. **P=5.4×10⁻⁴, ***P=4.5×10⁻⁸; Fisher’s test.

• Supplemental Figure S2 -

  Fig. S2. CAPS-GFP does not accumulate at the tips of motoneuronal filopodia. (A) CAPS-GFP expressed in motoneurons (using elav-GAL4) at 14:00 hours AEL. The neuronal filopodia indicated by arrowheads are shown at higher magnification in the inset. No strong accumulation of CAPS-GFP is seen at the tips of the filopodia (asterisks). (B) CAPS-GFP expressed in motoneurons at 15:00 hours AEL. No specific accumulation is seen at the axon terminal. Scale bar: 10 µm; 6.6 µm in inset.

• Supplemental Figure S3 -

  Fig. S3. In vivo time-lapse images of interaction between growth cones and myopodia. This time-lapse sequence shows the morphological change of growth cones (magenta) and myopodia (green) as a nascent synaptic terminal forms. Growth cones gradually transform into a bifurcated presynaptic terminal (arrows) while associating with myopodia. Myopodia transform into a lamellipodia-like structure (arrowheads). Confocal images of a dechorionated whole embryo expressing mYFP in motoneurons and mCFP in M12. Time elapsed from the first image (14:00 hours) is shown in minutes. Scale bar: 5 µm.

• Supplemental Figure S4 -

  Fig. S4. caps and caps, trn mutations do not affect axon outgrowth of MN12s. (A) Growth cones and M12 visualized by mGFP. The distance between the center of ISNb terminal growth cones and the center-line of M12 at 13:00 hours (arrow) was measured. Scale bar: 10 µm. (B) Quantification of the distance between growth cones and muscle at 13:00 hours. Mean=15.2 µm in caps caps^C28fs/ Df(3L)Ly; n=18 and 16.9 µm in caps trn caps^C28fs, trn^delta17; n=19 compared with 15.6 µm in the control (n=15) (P>0.05, two-tailed t-test).

• Supplemental Figure S5 -

  Fig. S5. Muscle size is normal in caps mutants. (A,B) Confocal images of M12 expressing mGFP in control embryos at 13:00 hours (A) and 15:00 hours (B). Scale bar: 10 µm. (C,D) Quantification of muscle size at 13:00 hours (C) and 15:00 hours (D). The length of muscle indicated by the arrows in A and B was measured in control and caps mutant caps^C28fs/ Df(3L)Ly embryos. Muscle size is normal at both 13:00 hours 34.5 µm in control, n=28; 35.4 µm in caps mutant, n=32) and 15:00 hours (40.7 µm in control, n=16; 39.6 µm in caps mutant, n=16). P>0.05, two-tailed t-test.

• Movie 1 -

  Movie 1. Interaction between myopodia and motoneuronal growth cones during early phase of synaptogenesis. Time-lapse imaging with 2-minute intervals from 13:00 hours to 14:20. Play speed, four frames per second.