Figure 1. Pomca Neurons Target Spinal (Pre-)Motor Circuits in Zebrafish Larvae (A) Schematic illustration of the imaged region. (B–D) Immunohistochemistry of pomca:EGFPras crossed to vsx2:Gal4FF; UAS-E1b:nfsb-mCherry transgenic larvae of 10–11 mm standard length (SL). (B) shows lateral view of whole-mount spinal cord preparation; top panel shows maximum intensity projection; for boxed region, single-plane images are shown below (see also Video S1 for z stack). (C) and (D) show transverse sections of the spinal cord. (C) Pomca projections are in proximity (arrow) or more distant (arrowhead) to V2a neurons. (D) Motoneurons backfilled with rhodamine-dextran in proximity to Pomca projections. (E) Dorso-ventral topology of Pomca projections in transverse sections of the spinal cord at SL 10. Position of ascending neurons adapted from Pedroni and Ampatzis [34]. (F) Dorso-ventral quantification of Pomca and V2a contacts. (G–I) Colorimetric in situ hybridization for mc4r, vsx2, and gfp (in mnx1:GFP transgenic animal) on spinal transverse sections at 9–10 mm SL. (J) Lateral view of whole-mount spinal cord at 9 mm SL stained with colorimetric in situ hybridization against mc4r. Motoneurons without signal (arrowhead) and with signal (arrow) are magnified in the right panel. (K) mc4r colorimetric in situ hybridization signal overlapped with fluorescence of V2a neurons of vsx2:GFP transgenic animal. Right panels show magnified views of exemplary neurons of indicated categories from different sections. (L) Quantification of co-localization in images as in (K) and the defined regions of interest (ROI, right panel). N = 3 animals, n = 9 spinal cord sections. cc, central canal; ma, Mauthner axons; D, dorsal; P, posterior; M, medial; L, lateral; n.s., not significant. Error bars show 95% confidence intervals. Scale bars, 25 μm.
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