Filopodial dynamics and growth cone stabilization in Drosophila visual circuit development
- University of Texas Southwestern Medical Center, United States
- Freie Universität Berlin, Germany
- Charite Universitätsmedizin Berlin, Germany
- Vlaams Instituut voor Biotechnologie, Belgium
- University of Leuven School of Medicine, Belgium
Figures
Figure 1 with 2 supplements
Development of Drosophila pupal brains in an imaging chamber.
(a) Timeline of photoreceptor circuit formation during brain development and the periods accessible by live imaging. (b) Ex vivo imaging chamber, top (left) and side (right) views (see Figure …
Figure 1—figure supplement 1
Culture imaging chamber.
(a) Step-by-step construction of the imaging chamber. (i) Spacers are placed on the Sylgard layer in a triangle formation. (ii) A drop of diluted dialyzed agarose is pipetted onto the Sylgard. (iii) …
Figure 1—figure supplement 2
Brain development in imaging chamber compared to liquid media.
Changes in brain morphology during development ex vivo in chamber vs. ex vivo in liquid media (free floating) vs. in vivo; from brains dissected at P + 24% (a-d), P + 50% (f-i) and cultured for …
Figure 2 with 2 supplements
Effects of culture conditions and laser scanning on the optic lobe development ex vivo.
Two-photon imaging of the medulla was performed with brains cultured at P + 22% for 20 hr (a) and P + 41% for 19 hr (d) all photoreceptors express CD4-tdGFP. For each experiment one image stack was …
Figure 2—figure supplement 1
Lamina rotation is incomplete ex vivo.
Two-photon imaging of the medulla was performed with brains cultured at P + 22% for 20 hr, all photoreceptors express CD4-tdGFP. Continuously scanned ex vivo culture, unscanned control optic lobe …
Figure 2—figure supplement 2
Effects of 20-Hydroxyecdysone (20-HE) and type of microscope on imaging in the culture chamber.
(a-h) 20-HE is required for early but detrimental to late pupal development in the optic lobe. (a-d) All photoreceptors were labeled with CD4-tdGFP. Cultures were set-up at P + 22% (a), with (b) or …
Figure 3 with 1 supplement
Different filopodial signatures accompany separate circuit formation steps.
Slow (30 min interval) time-lapse imaging of pupal brains dissected at P + 20% (a), P + 40% (b) and P + 55% (c) in comparison with in vivo fixed controls at the same stages. The same growth cones …
Figure 3—figure supplement 1
Filopodial dynamics are restricted to the growth cone and axon shaft inside the medulla neuropil.
(a) representative R7 terminal structures inside the medulla neuropil (grey background) reveal the transition of a more classical growth cone to a branched axonal structure. (b) 3D visualization of …
Figure 4 with 2 supplements
Distinct classes of transient and stable filopodia underlie different developmental events.
Fast (1 min interval) time-lapse imaging was performed at multiple points of three ex vivo experiments. (a) Three time points are shown; during the first-stage (P + 28%) and second-stage (P + 50%) …
Figure 4—figure supplement 1
Fast filopodial dynamics throughout pupal development.
Dynamics data from all 6 growth cones (2 independent growth cones for each time point) that were used in Figure 4. The heat maps on blue background show individual filopodia as verticals lines. The …
Figure 4—figure supplement 2
Filopodial dynamics as a function of lifetime.
(a-i) For the same growth cones depicted in Figure 4, every filopodia observed in a 1 hr period were binned into different lifetime classes: <1 min, 2–3 min, 4–7 min, 8–15 min, 16–31 min, 32–59 min …
Figure 5 with 1 supplement
R7 growth cones do not actively extend in the medulla.
(a) R7 may reach its final target layer through active extension or passive displacement and intercalation. (b) Live imaging starting at P + 30%. All photoreceptors were labeled with myr-mRFP and R7 …
Figure 5—figure supplement 1
Single growth cone tracking demonstrates R7 terminals remain passive throughout layer formation without a stationary landmark.
Live imaging starting at P + 30%. All photoreceptors were labeled with myr-mRFP and R7 cells were sparsely labeled with CD4-tdGFP using GMR-FLP through MARCM. R7 terminal (red arrow) can be followed …
Figure 6 with 1 supplement
N-Cadherin is required for the stabilization but not the layer specific targeting of R7 growth cones.
All photoreceptors were labeled with myr-mRFP. CadN405 R7 cells were generated with MARCM, using GMR-FLP and positively labeled with CD4-tdGFP. (a) Live imaging started at P + 24% shows a mutant R7 …
Figure 6—figure supplement 1
CadN mutant R7 axons may retract completely from the medulla.
All photoreceptors were labeled with myr-mRFP. CadN 405 R7 cells were generated with MARCM, using GMR-FLP and positively labeled with CD4-tdGFP. Live imaging starting at P + 35% demonstrates an R7 …
Figure 7
N-Cadherin is required for fast filopodial dynamics.
CadN405 R7 cells were generated with MARCM, using GMR-FLP and positively labeled with CD4-tdGFP. Fast (1 min interval) time-lapse imaging was performed at P + 28%. (a), The average numbers of …
Videos
Video 1
Ex vivo imaging of Drosophila brain development in culture.
All photoreceptors are labeled with CD4-tdGFP. Two live imaging sessions (30 min intervals) starting at P + 24% (19 hr) and at P + 40% (18 hr) are shown. Four developmental processes (i) lamina …
Video 2
Long-term ex vivo imaging of R7 photoreceptor growth cone filopodial dynamics.
All photoreceptors are labeled with myr-tdTomato and R7 photoreceptors are sparsely labeled with CD4-tdGFP using GMR-FLP. Three live imaging sessions (30 min intervals) starting at P + 22% (21 hr), …
Video 3
Ex vivo imaging of fast filopodial dynamics-1.
All photoreceptors are labeled with myr-mRFP and R7 photoreceptors are sparsely labeled with CD4-tdGFP using GMR-FLP. Live imaging started P + 28% and continued for 20 hr. We used an alternating …
Video 4
Ex vivo imaging of fast filopodial dynamics-2.
All photoreceptors are labeled with myr-mRFP and R7 photoreceptors are sparsely labeled with CD4-tdGFP using GMR-FLP. Two live imaging sessions starting at P + 40% (22 hr) and P + 52% (9 hr) are …
Video 5
Second stage layer targeting of R7 and R8.
Two live imaging experiments are shown. (1) All photoreceptors are labeled with myr-mRFP and R7 photoreceptors are sparsely labeled with CD4-tdGFP using GMR-FLP. Imaging started at P + 30% and …
Video 6
N-Cadherin functions in growth cone stabilization.
All photoreceptors are labeled with myr-mRFP and approximately 10% of R7 photoreceptors were made mutant for CadN and labeled with CD4-tdGFP using GMR-FLP through MARCM. Three live imaging sessions …
Video 7
Loss of N-Cadherin leads to reduced filopodial dynamics.
Representative wild type and cadN mutant R7 growth cones are shown at P + 28%. Extraction of individual filopodia reveals reduced dynamics over the same time period (1 hr with 1 min time lapse) as …
Additional files
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Source code 1
The MATLAB function for analysis of the filopodial dynamics.
This function takes as input the Excel files including the length and orientation data for all filopodia segmented across 60 time points within a 1 hour period. "TrackID"s are also required to identify the same filopodia across different time points. The function then calculates for each TrackID the number of extension and retraction events it experienced during that filopodium's lifetime, as well as the mean and standard deviation for extension, retraction and combined speeds. The user is asked for an input (in μm) defining the amount of extension/retraction length (default=0.3 μm) that will be considered insignificant, i.e. the filopodium will be assumed static for that 1 minute step. The function then outputs the number of extension and retraction events above that threshold ("filtered"), as well as speeds calculated from only these (above-threshold) events. The function also calculates the mean and standard deviation of a filopodium's length (μm) during its lifetime. These parameters are written in a new Excel file. Finally, the function creates the heat-maps used (and explained) in Figure 4.
- https://doi.org/10.7554/eLife.10721.026
