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	Fig 2. uts2b mRNA is restricted to the ventral spinal cord and hindbrainin X. laevis tadpoles.Distribution of the uts2b mRNA revealed by in situ hybridization with uts2b antisense probe on whole tadpoles (A–D and H), dissected and opened CNS (E), or CNS sections (F, G) at stages 24 (A), 27 (B), 33/34 (C), 37/38 (D) 51 (E), and 55 (F, G). H. Stage 37/38 tadpole hybridized with uts2b sense probe used as negative control. A–D and H. Lateral views of tadpoles with dorsal side up and rostral to the right. E. Dorsal view of whole-mount preparation of hindbrain-spinal cord (CNS). Note that in this preparation, the more ventral structures are closer to the midline. F and G. Coronal sections at the level of the spinal cord (F) and the posterior part of brainstem (G) with dorsal up. Arrowheads point to uts2b mRNA detection in stage 24 tadpole and in F and G coronal sections. White arrows designate the region of nuclei of the trigeminal (V) nerve; asterisks denote the region of nuclei of the vagus/hypoglossal/accessory (IX–XI) nerves. r, rhombomere; V4, fourth ventricule.
	Fig 3. Spinal uts2b+ cells are cholinergic neurons in X. laevis tadpoles.Confocal images of combined fluorescent in situ hybridization of uts2b mRNA (A1, B1) and ChAT immunolabeling (A2, B2) in a whole-mount dissected and opened spinal cord preparation of stage 50 wild type tadpole. A3, B3. Merged image obtained when uts2b and ChAT stainings were superimposed. Dorsal view with rostral up. Note that in this preparation, the more ventral structures are close to the midline (dashed line). The boxed region in A3 is shown at higher magnification in B1–B3.
	Fig 4. Spinal uts2b+ cells of X. laevis tadpoles project to tail myotomes.Confocal image of combined fluorescent in situ hybridization of uts2b mRNA (A1, B1) and retrograde labeling of spinal axial (A2) and appendicular (B2) motoneurons in stage 55 tadpole whole-mount spinal cord preparations. A3, B3. Merged images of uts2b staining and retrograde labeling. All images display dorsal view of hemi-cords, with the rostral side up (dashed-line on the left indicates the midline). Axial motoneurons (Ax MN) were labeled with rhodamine dextran dye (RDA) injected into tail myotomes while appendicular motoneurons (Ap MN) were labeled from posterior leg muscles with Alexa Dextran 647 dye (A.D. 647; see upper scheme on the left panel). The drawing on the left panel illustrates the localization of Ax MN and developing Ap MN in larval spinal cord at stage 55. Yellow arrowheads indicate double stained cells. Red arrowheads indicate uts2b—retrograde labeled cells. sp.sgt 7–10, spinal segments 7 to 10.
	Fig 5. Most of the fluorescence visible in transgenic uts2b-GFP X. laevis tadpoles occurs in cells located in the spinal cord and in motor axon projections.A. GFP fluorescence imaging of a representative transgenic tadpole at stage 58. The tail is seen in lateral view while the head is seen in dorsal view. B. GFP expression at the level of a dissected and opened whole-mount CNS of a stage 50 transgenic tadpole. The CNS was optically exposed by removing the dorsal part of the tail and the top of the head. Note that GFP+ cells are restricted to the spinal cord. Dorsal view, rostral to the right. C. Detail of A showing GFP+ motor axon projections (arrowheads) extending towards axial musculature. White dashed vertical lines indicate myotome boundaries. SC, spinal cord. OT, optic tectum.
	Fig 6. The uts2b expression pattern is partially reproduced by GFP in transgenic uts2b-GFP X. laevis tadpoles.Confocal images of combined immuno-labeling of GFP+ neurons (A1, B1) and fluorescent in situ hybridization of uts2b mRNA (A2, B2) in a whole-mount spinal cord preparation of stage 50 uts2b-GFP tadpole. A3, B3. Merged image obtained when GFP and uts2b stainings were superimposed. Dorsal view with rostral up. Only the hemi-cord is shown. The dashed-line on the left side represents the midline. Note that in this preparation, the more ventral structures are closer to the midline. The boxed region in A3 is shown at higher magnification in B1–B3. Yellow arrowheads denote cells that express both GFP and uts2b. Red arrowheads designate uts2b+/GFP− cells and green arrowheads designate uts2b−/GFP+ cells.
	Fig 7. Spinal GFP+ cells of transgenic uts2b-GFP X. laevis tadpoles mainly express ChAT.Confocal images of combined immuno-labeling of ChAT+ (A1) and GFP+ (A2) neurons in a whole-mount spinal cord preparation of stage 50 transgenic tadpole. A3. Merged image obtained when ChAT and GFP stainings were superimposed. Dorsal view with rostral up. Only the hemi-cord is shown. The dashed-line on the right side represents the midline. Note that in this preparation, the more ventral structures are closer to the midline. Confocal images of combined immuno-labeling of ChAT+ (B1–C1) and GFP+ (B2–C2) neurons in lumbar spinal cord (B) and brainstem (C) cross-sections of stage 60 uts2b-GFP tadpoles. B3, C3. Merged images obtained when ChAT and GFP stainings were superimposed. The cross-section in C1–3 originates from the caudal hindbrain (rhombomeres 7–8) where IX-XI motor nuclei are located. Yellow arrowheads designate GFP+/ChAT+ cells; green arrowheads designate GFP+/ChAT− cells; White arrowheads designate GFP−/ChAT+ cells. D, dorsal; M, medial; V4, 4th ventricule.
	Fig 8. Spinal GFP+ axon motor projections of transgenic uts2b-GFP X. laevis tadpoles co-localize with α-bungarotoxin, a marker of postsynaptic neuromuscular junctions.Combined fluorescence of GFP (A) and immuno-labeling of α-bungarotoxin (B) at the level of spinal axon motor projections in a whole-mount stage 57 transgenic tadpole. C. Merged image obtained when GFP fluorescence and α-bungarotoxin staining were superimposed. Postsynaptic nicotinic acetylcholine receptors labeled by α-bungarotoxin and GFP+ motor neuron axons overlap (arrowheads). Lateral views, rostral to the left.
	Fig 9. Spinal GFP+ cells of transgenic uts2b-GFP X. laevis tadpoles project to tail myotomes.Confocal image of combined immuno-labeling of spinal GFP+ neurons (A1) and retrograde labeling of spinal motoneurons (A2) in a stage 50 uts2b-GFP tadpole whole-mount spinal cord preparation. A3. Merged image obtained when GFP staining and retrograde labeling were superimposed. Dorsal view with rostral up. Only the hemi-cord is shown. The dashed-line on the right side represents the midline. Axial motoneurons (Ax MN) innervating myotomes were labeled from tail muscles with rhodamine (RDA) dextran dye whereas appendicular motoneurons (Ap MN) innervating limbs were labeled from hindlimb buds with alexa dextran 647 dye (A.D. 647; see scheme on the left panel). The drawing at the top left panel illustrates the medio-lateral localization of Ax MN and developing Ap MN in larval spinal cord at stage 50. Yellow arrowheads indicate double stained cells. Cross-section confocal image of combined immuno-labeling of spinal GFP+ neurons (B1, C1) and retrograde labeling of dorsal thoracic motoneurons (dTh MN, B2) and Ap MN, (C2) in stage 60 metamorphosing uts2b-GFP tadpole. B3, C3. Merged images obtained GFP staining and retrograde labeling were superimposed. dTh MN were labeled from dorsalis trunci muscles with RDA whereas Ap MN were labeled from groups of extensor and flexor of the hindlimb with A.D. 647 (see scheme on the left side). The cross-section drawing at the bottom left panel illustrates the dorso-ventral localization of dTh MN and Ap MN in the future adult spinal cord at stage 60. sp.sgt 7–10, spinal segments 7 to 10, Vr, ventral root. D, dorsal; L, lateral.
	Fig 10. GFP+ cells exhibit a typical pattern of motoneuronal electrical activity during spontaneous fictive swimming episodes.Visualization of spinal GFP+ neurons at x40 in fluorescence condition (A1) and infrared condition (A2) in a whole-mount isolated in vitro preparation of brainstem-spinal cord with dorsal-side opened. Patch-clamp intracellular recordings (bottom traces) of spinal GFP+ neurons (Intra.) either with a persisting (B1), a non-persisting firing pattern (B2), or a slow-delayed firing pattern (B3) during spontaneous fictive swimming episodes (f. sw.). The upper trace in B1–B3 represents the extracellular fictive swimming activity recorded simultaneously in a ventral motor root (Vr). DIC, differential interference contrast. The white arrowhead points to the GFP+ neuron targeted for patch clamp recording.
	uts2b (urotensin 2B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	uts2b (urotensin 2B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior left, dorsal up.
	uts2b (urotensin 2B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
	uts2b (urotensin 2B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 38, lateral view, anterior left, dorsal up.
	Fig 1. Strategy for constructing the recombinant uts2b BAC containing uts2b-EGFP expression cassette.The X. tropicalis uts2b BAC clone, ALNOAAA14YB24, contains approximately 164 kbp genomic DNA. The uts2b gene is approximately 8.2 kbp long as represented by a red arrow, and it comprises 5 exons. The targeting vector was designed to contain two uts2b genomic DNAs on its either side (denoted 5’- and 3’-arms), that allows a specific homologous recombination between the targeting vector and the uts2b BAC DNA at the genomic region surrounding the first exon. Homology arms for the first exon of uts2b were ligated to both ends of an EGFP.FRT-Promotor Bacterial Minimal.kanamycin-FRT.2pA expression cassette. Through the first homologous recombination, the expression cassette was inserted in place of the first uts2b exon in the uts2b BAC clone. Then, the kanamycin resistance cassette was selectively eliminated by expressing Flipase, leading to the uts2b-EGFP BAC clone containing only the EGFP cDNA expression cassette under control of the uts2b promoter. Finally, note that the ccdc50 gene, located close to the uts2b locus, was deleted from the uts2b BAC to avoid possible artifacts due to its overexpression (not shown).

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