Figure 2.
Evidence that pupal tissue movements exert force on c4da neuron dendrites. (A and B) Time-lapse analysis of c4da neuron dendrite severing and correlation with tissue movements during the early pupal stage. A–A‴ are stills from a representative time-lapse movie (1 frame every 5 min between 0–6.5 h APF) of a dorsal c4da neuron labeled by ppk-eGFP (see also Video 3). The colored trace shows cell body position over the analysis, with color-coded timepoints. Red arrowheads mark severed dendrites. (B) Graph depicting average movement magnitude over time from 13 time-lapse movies. Error envelope represents standard deviation, arrows over graph indicate dendrite severing events. (C–I) C4da neuron cell bodies and distal dendrite branchpoints (labeled by ppk-eGFP) as well as epidermal cell tricellular junctions (labeled by DE-Cad::GFP) were traced in time-lapse movies (e.g., Video 4) starting at 0 h APF. (C) C4da neuron at 0 h APF (start of the analysis) with cell body (red), dendrite (blue) and epidermal landmarks (yellow) indicated. (C′) The same neuron at the end of the time-lapse experiment, with the traces of the respective landmarks indicated. (D) Graph showing distances between the dendrite branchpoint or the cell body and the epidermis landmark in C–C′ to assess synchronicity of movement over time. The black curve shows dendrite length (between soma and dendrite branchpoint landmark) over time. All values were normalized to the initial timepoint. (E) Distribution of cell body/epidermis and dendrite/epidermis distances after 5 h APF normalized to the distance at t0 (N = 10 time-lapse movies). (F) The dendrite segment between cell body and dendrite landmark from time-lapse movie in C–C′. Upper panel shows the dendrite at 0 h APF, the lower panel shortly before severing (7.5 h APF). The segment is marked with a red line for length comparison. (G) In these time-lapse movies, the length of primary dendrite proximal segments (between soma and second branchpoint) was measured at the onset of the pupal stage (t0) and shortly before severing. N = 9, values are normalized to t0. (H and H′) Image showing a dendrite branch point distal to an eventual severing site at the onset of metamorphosis (H) and after severing (H′). The branching angle is indicated by red lines. (I) Graph depicting branching angles at branchpoints distal to severing sites at the start of metamorphosis (0–90 min APF) and after severing (250–515 min APF). **P < 0.005, Mann–Whitney U test (N = 14). Scale bars in A, C, and H are 20, 50, and 10 µm, respectively.

Evidence that pupal tissue movements exert force on c4da neuron dendrites. (A and B) Time-lapse analysis of c4da neuron dendrite severing and correlation with tissue movements during the early pupal stage. A–A‴ are stills from a representative time-lapse movie (1 frame every 5 min between 0–6.5 h APF) of a dorsal c4da neuron labeled by ppk-eGFP (see also Video 3). The colored trace shows cell body position over the analysis, with color-coded timepoints. Red arrowheads mark severed dendrites. (B) Graph depicting average movement magnitude over time from 13 time-lapse movies. Error envelope represents standard deviation, arrows over graph indicate dendrite severing events. (C–I) C4da neuron cell bodies and distal dendrite branchpoints (labeled by ppk-eGFP) as well as epidermal cell tricellular junctions (labeled by DE-Cad::GFP) were traced in time-lapse movies (e.g., Video 4) starting at 0 h APF. (C) C4da neuron at 0 h APF (start of the analysis) with cell body (red), dendrite (blue) and epidermal landmarks (yellow) indicated. (C′) The same neuron at the end of the time-lapse experiment, with the traces of the respective landmarks indicated. (D) Graph showing distances between the dendrite branchpoint or the cell body and the epidermis landmark in C–C′ to assess synchronicity of movement over time. The black curve shows dendrite length (between soma and dendrite branchpoint landmark) over time. All values were normalized to the initial timepoint. (E) Distribution of cell body/epidermis and dendrite/epidermis distances after 5 h APF normalized to the distance at t0 (N = 10 time-lapse movies). (F) The dendrite segment between cell body and dendrite landmark from time-lapse movie in C–C′. Upper panel shows the dendrite at 0 h APF, the lower panel shortly before severing (7.5 h APF). The segment is marked with a red line for length comparison. (G) In these time-lapse movies, the length of primary dendrite proximal segments (between soma and second branchpoint) was measured at the onset of the pupal stage (t0) and shortly before severing. N = 9, values are normalized to t0. (H and H′) Image showing a dendrite branch point distal to an eventual severing site at the onset of metamorphosis (H) and after severing (H′). The branching angle is indicated by red lines. (I) Graph depicting branching angles at branchpoints distal to severing sites at the start of metamorphosis (0–90 min APF) and after severing (250–515 min APF). **P < 0.005, Mann–Whitney U test (N = 14). Scale bars in A, C, and H are 20, 50, and 10 µm, respectively.

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