Bougerol M , Auradé F , Lambert FM , Le Ray D , Combes D , Thoby-Brisson M , Relaix F , Pollet N , Tostivint H .

	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).

Abe, Reporter mouse lines for fluorescence imaging. 2013, Pubmed

Abe, Reporter mouse lines for fluorescence imaging. 2013, Pubmed
Ampatzis, Pattern of innervation and recruitment of different classes of motoneurons in adult zebrafish. 2013, Pubmed
Babin, Zebrafish models of human motor neuron diseases: advantages and limitations. 2014, Pubmed
Becker, Zebrafish as a genomics model for human neurological and polygenic disorders. 2012, Pubmed
Blagoev, Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. 2004, Pubmed
Copeland, Recombineering: a powerful new tool for mouse functional genomics. 2001, Pubmed
Coulouarn, Cloning, sequence analysis and tissue distribution of the mouse and rat urotensin II precursors. 1999, Pubmed
Coulouarn, Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. 1998, Pubmed
Djenoune, Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates. 2014, Pubmed
Douglas, From 'gills to pills': urotensin-II as a regulator of mammalian cardiorenal function. 2004, Pubmed
Dubessy, Characterization of urotensin II, distribution of urotensin II, urotensin II-related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction. 2008, Pubmed
Dun, Urotensin II-immunoreactivity in the brainstem and spinal cord of the rat. 2001, Pubmed
Farfsing, Gene knockdown studies revealed CCDC50 as a candidate gene in mantle cell lymphoma and chronic lymphocytic leukemia. 2009, Pubmed
Fish, Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus. 2012, Pubmed , Xenbase
Flanagan-Steet, Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations. 2005, Pubmed
Forehand, Spinal cord development in anuran larvae: I. Primary and secondary neurons. 1982, Pubmed
Gabriel, Principles governing recruitment of motoneurons during swimming in zebrafish. 2011, Pubmed
Grillner, Measured motion: searching for simplicity in spinal locomotor networks. 2009, Pubmed
HUGHES, Cell degeneration in the larval ventral horn of Xenopus laevis (Daudin). 1961, Pubmed , Xenbase
Hartenstein, Early pattern of neuronal differentiation in the Xenopus embryonic brainstem and spinal cord. 1993, Pubmed , Xenbase
Hellsten, The genome of the Western clawed frog Xenopus tropicalis. 2010, Pubmed , Xenbase
Higashijima, Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer. 2000, Pubmed
Howe, The zebrafish reference genome sequence and its relationship to the human genome. 2013, Pubmed
Hwang, Efficient genome editing in zebrafish using a CRISPR-Cas system. 2013, Pubmed
Kelly, Recombineered Xenopus tropicalis BAC expresses a GFP reporter under the control of Arx transcriptional regulatory elements in transgenic Xenopus laevis embryos. 2005, Pubmed , Xenbase
Konno, Urotensin II receptor (UTR) exists in hyaline chondrocytes: a study of peripheral distribution of UTR in the African clawed frog, Xenopus laevis. 2013, Pubmed , Xenbase
Korzh, Zebrafish primary neurons initiate expression of the LIM homeodomain protein Isl-1 at the end of gastrulation. 1993, Pubmed
Kratchmarova, Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. 2005, Pubmed
Lee, Olig2 and Ngn2 function in opposition to modulate gene expression in motor neuron progenitor cells. 2005, Pubmed
Lee, A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. 2001, Pubmed
Long, Remote control of gene expression. 2007, Pubmed
López, Choline acetyltransferase immunoreactivity in the developing brain of Xenopus laevis. 2002, Pubmed , Xenbase
Marsh-Armstrong, Thyroid hormone controls the development of connections between the spinal cord and limbs during Xenopus laevis metamorphosis. 2004, Pubmed , Xenbase
McLean, A topographic map of recruitment in spinal cord. 2007, Pubmed
Nakayama, Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. 2013, Pubmed , Xenbase
Park, Spatial and temporal regulation of ventral spinal cord precursor specification by Hedgehog signaling. 2004, Pubmed
Parmentier, Comparative distribution of the mRNAs encoding urotensin I and urotensin II in zebrafish. 2008, Pubmed
Pelletier, Role of androgens in the regulation of urotensin II precursor mRNA expression in the rat brainstem and spinal cord. 2002, Pubmed
Pelletier, Androgenic down-regulation of urotensin II precursor, urotensin II-related peptide precursor and androgen receptor mRNA in the mouse spinal cord. 2005, Pubmed
Pratt, Modeling human neurodevelopmental disorders in the Xenopus tadpole: from mechanisms to therapeutic targets. 2013, Pubmed , Xenbase
Prestige, The control of cell number in the lumbar ventral horns during the development of Xenopus laevis tadpoles. 1967, Pubmed , Xenbase
Roberts, Motoneurons of the axial swimming muscles in hatchling Xenopus tadpoles: features, distribution, and central synapses. 1999, Pubmed , Xenbase
Schedl, A method for the generation of YAC transgenic mice by pronuclear microinjection. 1993, Pubmed
Schindelin, Fiji: an open-source platform for biological-image analysis. 2012, Pubmed
Schlosser, Thyroid hormone promotes neurogenesis in the Xenopus spinal cord. 2002, Pubmed , Xenbase
Schmitt, Engineering Xenopus embryos for phenotypic drug discovery screening. 2014, Pubmed , Xenbase
Seredick, Zebrafish Mnx proteins specify one motoneuron subtype and suppress acquisition of interneuron characteristics. 2012, Pubmed
Shin, Neural cell fate analysis in zebrafish using olig2 BAC transgenics. 2003, Pubmed
Sive, Inducing Ovulation in Xenopus laevis. 2007, Pubmed , Xenbase
Straka, Rhombomeric organization of brainstem motor neurons in larval frogs. 1998, Pubmed
Sugo, Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain. 2003, Pubmed
Suster, Transposon-mediated BAC transgenesis in zebrafish. 2011, Pubmed
Tostivint, Impact of gene/genome duplications on the evolution of the urotensin II and somatostatin families. 2013, Pubmed
Tostivint, Molecular evolution of GPCRs: Somatostatin/urotensin II receptors. 2014, Pubmed
Tsien, The green fluorescent protein. 1998, Pubmed
Vaudry, International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, urotensin II-related peptide, and their receptor: from structure to function. 2015, Pubmed
Vaudry, Urotensin II, from fish to human. 2010, Pubmed
Ymlahi-Ouazzani, Reduced levels of survival motor neuron protein leads to aberrant motoneuron growth in a Xenopus model of muscular atrophy. 2010, Pubmed , Xenbase
Zhang, An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. 1996, Pubmed
Zhang, Development of a spinal locomotor rheostat. 2011, Pubmed , Xenbase
de Chaumont, Icy: an open bioimage informatics platform for extended reproducible research. 2012, Pubmed
van Mier, The development of the dendritic organization of primary and secondary motoneurons in the spinal cord of Xenopus laevis. An HRP study. 1985, Pubmed , Xenbase
van Mier, The development of serotonergic raphespinal projections in Xenopus laevis. 1986, Pubmed , Xenbase