Segmental characterization of Beat-Va _M and Beat-Va _L neurons. (A) VNC at wL3 with genetic mCD8::GFP labeling of Beat-Va neurons (green) and anti-Even-Skipped staining (magenta) to label the segments of the ventral nerve cord. Segments A3–A8 are denoted by tracing the Even-Skipped staining and then superimposed onto Beat-Va neurons to define segmental positions. Scale bar is 20 µm. (B) VNC at 6 h APF with genetic GFP labeling of Beat-Va neurons (green) and anti-Engrailed staining (magenta) to label the segments of the VNC, showing the segmental positions persist into metamorphosis. Scale bars are 20 µm. (C) Using segmental information and MCFO we can render single cells in Imaris and overlay positional information. Boxed areas are Beat-Va_L and Beat-Va_M termini and are displayed in high magnification (scale bars are 10 µm). CNS is outlined and boundary between VNC and brain lobes is marked with a dashed line. (D) Z-projection of ventral nerve cords with Beat-Va neurons labeled genetically with mCD8::GFP (green) and then subjected to TUNEL staining (magenta) to detect cells undergoing apoptotic cell death at 3 h APF and 4 h APF. Boxed areas containing TUNEL-positive lateral cells are magnified and segmental position is noted. Scale bar is 5 µm.