Electron microscopy volumes and reconstruction

Four electron microscopy volumes were used in this study. They comprise a whole or partial central nervous system of first instar Drosophila larvae. Two of these are control volumes which have been previously reported (Ohyama et al., 2015): a whole-central nervous system (CNS) volume (A1 segment, Control 1) and a 1.5-segment long volume (A2/A3 segment, Control 2). Some neurons from the control volumes were previously reconstructed by members and collaborators of the Cardona lab (Janelia Research Campus, HHMI). Control 2 volume had a gap in sections that prevented the complete reconstruction of Griddle, Drunken, and Ladder interneurons, but allowed complete reconstruction of Basin dendrites. The mechano > FraRobo and mechano > TNT EM volumes were acquired for this study using the same preparation and imaging protocols reported for the control volumes (Ohyama et al., 2015). These volumes include a 1.5-segment fraction of the central nervous system (A1/A2 segment) of 1^st instar larvae. The genotypes for these volumes are: 1) w;; iav-GAL4/UAS-FraRobo 2) w; UAS-TNT/+; iav-GAL4/+. They have an image resolution of 3.8 nm by 3.8 nm by 40 nm in x, y and z, respectively. The neurons of interest were reconstructed using CATMAID (Saalfeld et al., 2009) to obtain the skeletonized structure and connectivity of the cells of interest. The neuronal reconstruction process has been detailed previously (Ohyama et al., 2015; Schneider-Mizell et al., 2016).