Expression pattern of Ci-GlyR. (A-E) Whole-mount in situ hybridization of the Ci-GlyR antisense probe during larval development. The signal in the nervous system is marked with red arrowheads, and that in muscle cells is with open black arrowheads. eTB, early tailbud stage; mTB, mid-tailbud stage; lTB, late tailbud stage; hL, hatched larva stage. Scale bar on E = 100 μm for A-E. (F-G). Pseudo-color images of fluorescently detected Ci-GlyR and Ci-ChAT expression. (F) In early tailbud (eTB), Ci-GlyR (green, yellow arrowheads) and Ci-ChAT (magenta) expression can be detected in the cytoplasm of single neuronal cells (determined by labeling the cell nuclei (blue) with SYTOX Green), although it should be noted that the expression patterns do not overlap until the mid-tailbud stage (though nuclear staining show that the signals are in the same cell). Scale bar = 10 μm. Beside the scale bar, the location of the depicted region is outlined with an orange rectangle. (G) Ci-GlyR and Ci-ChAT transcripts are co-localized at the mid tailbud stage (mTB). The dashed line indicates the midline of the nerve cord. Scale bar = 10 μm.