Fig. S1

Immobilization of Embryos, Processing of Longitudinal Functional Imaging Data, and Identification and Characterization of KA Neurons in the Developing Spinal Cord, Related to Figure 1 and STAR Methods (A) Comparison of immobilization efficiency for embryos injected at the 1-cell stage with 50 ng/μl mRNA of alpha-bungarotoxin (αBTX) with zebrafish membrane-tethering sequences (blue), 50 ng/μl mRNA αBTX with mouse membrane-tethering sequences (red), and for uninjected siblings (orange) at 20, 24, 36 and 48 hpf. Membrane-tethered αBTX effectively immobilizes over 80% of the injected embryos until 24 hpf. Efficiency is shown as the percentage of embryos that do not react to dechorionation at 20 hpf or touch response at 24, 36 and 48 hpf. (B) Comparison of parameters characterizing the activity patterns underlying real and fictive coiling behavior in non-immobilized embryos and in embryos injected with 50 ng/μl mRNA of membrane-tethered alpha-bungarotoxin at 22-24 hpf. No significant difference (Wilcoxon rank-sum test, p > 0.1) was observed in behavior frequency (left panel), ratio of left versus right patterns (middle panel) or left-right alternation index (right panel). The alternation index is defined as the number of consecutive pairs of patterned events occurring on opposite sides of the spinal cord, divided by the total number of events minus one, over a 10 min recording time period. (C) Flowchart of the semi-automated cell tracking pipeline for functional image data (see STAR Methods for details). First, raw images are pre-processed and subdivided into groups of 200 time points. For each group, a single image stack is computed by maximum-intensity projection along the time axis. Second, the resulting stacks are input into an automatic tracking algorithm, which comprises initialization, update and merge detection/split steps. In parallel, active neurons are identified and selected based on the last 20 min of the time-lapse recording. Subsequently, tracks of the selected active neurons are validated by manual curation. Finally, the locations of the tracked cells are interpolated across the full time series and the calcium signal for each cell is extracted from the images as a function of time to calculate ΔF/F. Procedures in black boxes are performed by automated algorithms, steps in orange boxes are interactively integrated with a manual curation workflow using the Fiji plugins MTrackJ and MaMuT. (D) Identification of morphology and types of neurons involved in patterned activity. Active neurons involved in patterned activity on the left (green) and right (magenta) side of the spinal cord were identified and their morphologies visualized by voxel-based independent component analysis (ICA) of volumetric functional image data of a pan-neuronally expressed cytosolic calcium indicator (elavl3:GCaMP6f). The ICA results are furthermore superimposed with the cell-type specific marker mnx1:TagRFP-T (blue) that labels MNs and VeLD INs. The morphologies of the corresponding neuron populations are visualized here using maximum-intensity projections showing dorsal and lateral views of the spinal cord (left), together with enlarged views of the regions highlighted by white boxes on the right. White arrows in the dorsal-view image indicate the commissural projections of mnx- INs, and the white arrow in the lateral-view image indicates the location of an mnx- neuron cell body located in the dorsal spinal cord. Scale bars, 50 μm (left and middle) and 20 μm (right). (E) Dorsal-view images of an embryo from a cross of the transgenic lines Tg(elavl3:H2B-GCaMP6f) and Tg(Gal4s1020t)Tg(UASmCherry) at different depths along the dorso-ventral axis. The pan-neuronal expression pattern of elavl3:H2BGCaMP6f (green) covers all cells, including KA neurons (example indicated by white arrow), labeled by the transgenic line Tg(Gal4s1020t)Tg(UASmCherry) (magenta). Scale bar, 20 μm. (F) Identification of KA neurons and representative MNsduring longitudinal functional imaging of elavl3:H2B-GCaMP6f. A dorsal view of the spinal cord is shown as a maximum-intensity projection of the image volume at 22 hpf, with four KA neurons and four MNs highlighted by green circles. Scale bar, 50 μm. (G) Calcium traces of the four KA neurons and four MNs highlighted in panel (F) at 22 hpf. Notably, the activity of KA neurons is not synchronized or in phase with the rhythmic motor patterns. (H) Activity classification of the four KA neurons and four MNs highlighted in panel (F) during spinal circuit development. KA neurons become active later than the earliest active MNs.