Alejevski F , Leemans M , Gaillard AL , Leistenschneider D , de Flori C , Bougerol M , Le Mével S , Herrel A , Fini JB , Pézeron G , Tostivint H .

Xla Wt + uts2rl3 CRISPR (Fig. 11 a)

	Figure 1. Primary structure of frog Urp2 precursors. (a) Nucleotide sequence of the X. tropicalis urp2 cDNA and deduced amino acid sequence. (b) Alignment of the aa sequences of X. tropicalis and X. laevis prepro-Urp2. An asterisk denotes conserved residues. Precursor sizes are indicated. Xtr, X. tropicalis; Xla, X. laevis. (c) Comparison of Urp2 and Urp1 primary sequences in Xenopus and zebrafish. Initiation (ATG) and stop codons are boxed. Signal peptides are in grey. Putative bioactive peptides are highlighted in red. Cleavage sites are in bold. The sequences of the two X. laevis Urp2 cDNAs have been deposited in the GenBank database under the accession number MZ054702 and MZ054703, respectively.
	Figure 2. Phylogenetic tree of vertebrate UII-Urp precursor sequences. Phylogenetic analysis of 47 vertebrate prepro UII-Urp amino acid sequences was performed using the NJ distance-based method, with 1000 bootstrap replicates. The number shown at each branch node indicates in percentage the bootstrap value. Sequence references and alignment are given in the electronic supplementary material, figures S1 and S10, respectively
	Figure 3. Synteny of genes in the urp2 locus in five selected osteichthyan species: human (H. sapiens), chicken (G. gallus), western clawed frog (X. tropicalis), spotted gar (L. oculatus) and zebrafish (D. rerio). Genes are represented by block arrows. The position of the genes (in megabases, Mb) is displayed below each box, according to the Ensembl database. The detailed chromosomal locations of genes displayed in this map are included in the electronic supplementary material, table S3.
	Figure 4. urp2-L and urp2-S gene expression during X. laevis development. Relative mRNA expression levels were measured by quantitative RT-PCR and represented as ratios to the mRNA levels of the housekeeping genes sub1-L and slc35b1-L
	Figure 5. The tissue expression of urp2-L and urp2-L genes in juvenile X. laevis frog. Relative mRNA expression levels were measured by quantitative RT-PCR and represented as ratios to the mRNA level of the housekeeping gene rps13. Values are estimated for samples from six frogs.
	Figure 6. Localization of urp2-L mRNA in X. laevis embryo revealed by in situ hybridization. Lateral view of embryos (stage NF29-30) hybridized with antisense (a) and sense (b) urp2-L probes. Staining of cells contacting the central canal is indicated by arrowheads. (a’), detail of (a) in the transverse section. cc, central canal; nc, notochord. Scale bars: 450 µm.
	Figure 7. Localization of urp2-L mRNA in the brain and spinal cord of juvenile X. laevis frog revealed by in situ hybridization. Transverse sections (40 µm) of juvenile frog hindbrain (a) and spinal cord (b) hybridized with antisense urp2-L probe. (c,d) Sagittal sections (40 µm) of juvenile frog spinal cord hybridized with antisense urp2-L (c) and pkd2l1 (d) probes. cc, central canal; IV, fourth ventricle. (e) Schematic sagittal section depicting the distribution of urp2 mRNA hybridization signals (red dots). The level of the coronal sections in (a) and (b) is indicated by arrows. Ce, cerebellum; dh, dorsal horn of the spinal cord; Di, diencephalon; Is, isthmic nucleus; MO, medulla oblongata; Ob, olfactive bulbs; Rs, nucleus reticularis superior; SC, spinal cord; Tel, telencephalon; TO, tectum opticum; vh, ventral horn of the spinal cord. Anatomical structures are designated according to [37]. Scale bars: 150 µm (a,b), 50 µm (c,d).
	Figure 8. utr gene expression during X. laevis development. Relative mRNA expression levels were measured by quantitative RT-PCR and represented as ratios to the mRNA levels of the housekeeping genes sub1-L and slc35b1-L
	Figure 9. Localization of urotensin II receptor (utr) mRNAs in X. laevis embryo revealed by in situ hybridization. Lateral view of embryos (stages NF29-30) hybridized with antisense utr1-L (a), utr4-L (b), utr3-L (c) and utr5-L (d) antisense RNA probes. (a0 ) and (b0 ) are details of (a) and (b), in dorsal view and transverse sections, showing the staining in notochord (nc, filled arrowheads) and somites (so, empty arrowheads), respectively. Scale bars: 450 µm.
	Figure 10. The phenotypic effect of utr4 knock-out. Still images showing the curved body phenotype of utr4 crispant (a) and control (b) tadpoles. (c) Statistical analysis of the angle of body curvature of utr4 crispant tadpoles compared to control tadpoles at stages NF46 to NF52. n = 41 controls and 29 utr4 crispants, ****p < 0.0001.
	Figure 11. Figure illustrating the tracking of a sequence of an utr4 crispant (a) and a control (b) tadpole. Noticeable is the erratic trajectory of the utr4 crispant (in red) characterized by long-axis rotations visible as marked deviations from the straight-line path (illustrated by the arrows for part of the trajectory) in contrast with the rectilinear displacement of the control tadpole.
	uts2r (urotensin 2 receptor) gene expression in Xenopus laevis embryo, assayed through in situ hybridization, NF stage 29-30, lateral view, anterior left, dorsal up.
	uts2rl3 (urotensin-2 receptor-like 3) gene expression in Xenopus laevis embryo, assayed through in situ hybridization, NF stage 29-30, lateral view, anterior left, dorsal up.
	Supplementary Figure S9. (a) Phylogenetic tree of X. tropicalis and X. laevis Utrs constructed with the NJ method. Bootstrap test was performed with 1000 replications.
	Supplementary Figure S9 (b) [Utr1] Alignment of the aa sequences of X. tropicalis and X. laevis Utrs. Transmembrane domains are highlighted in red. Pair-wise comparison of each Utr subtype are indicated. Values represent percentage of amino acid identity.
	Supplementary Figure S9 (b) [Utr3] Alignment of the aa sequences of X. tropicalis and X. laevis Utrs. Transmembrane domains are highlighted in red. Pair-wise comparison of each Utr subtype are indicated. Values represent percentage of amino acid identity.
	Supplementary Figure S9 (b) [Utr4] Alignment of the aa sequences of X. tropicalis and X. laevis Utrs. Transmembrane domains are highlighted in red. Pair-wise comparison of each Utr subtype are indicated. Values represent percentage of amino acid identity.
	Supplementary Figure S9 (b) [Utr5] Alignment of the aa sequences of X. tropicalis and X. laevis Utrs. Transmembrane domains are highlighted in red. Pair-wise comparison of each Utr subtype are indicated. Values represent percentage of amino acid identity.

Almagro Armenteros, SignalP 5.0 improves signal peptide predictions using deep neural networks. 2019, Pubmed

Almagro Armenteros, SignalP 5.0 improves signal peptide predictions using deep neural networks. 2019, Pubmed
Ames, Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. 1999, Pubmed
Bandín, Immunohistochemical analysis of Pax6 and Pax7 expression in the CNS of adult Xenopus laevis. 2014, Pubmed , Xenbase
Blitz, Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system. 2013, Pubmed , Xenbase
Boswell, Understanding Idiopathic Scoliosis: A New Zebrafish School of Thought. 2017, Pubmed
Bougerol, Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the urotensin II-related peptide (ust2b) gene as a driver. 2015, Pubmed , Xenbase
Cantaut-Belarif, Adrenergic activation modulates the signal from the Reissner fiber to cerebrospinal fluid-contacting neurons during development. 2020, Pubmed
Cantaut-Belarif, The Reissner Fiber in the Cerebrospinal Fluid Controls Morphogenesis of the Body Axis. 2018, Pubmed
Cheng, Adolescent idiopathic scoliosis. 2015, Pubmed
Cui, Characterization of four urotensin II receptors (UTS2Rs) in chickens. 2021, Pubmed
Dai, Novel Mutations in UTS2R are Associated with Adolescent Idiopathic Scoliosis in the Chinese Population. 2021, Pubmed
Djenoune, Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates. 2014, 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
Elurbe, Regulatory remodeling in the allo-tetraploid frog Xenopus laevis. 2017, Pubmed , Xenbase
Hänzi, Developmental changes in head movement kinematics during swimming in Xenopus laevis tadpoles. 2017, Pubmed , Xenbase
Konno, Identification and signaling characterization of four urotensin II receptor subtypes in the western clawed frog, Xenopus tropicalis. 2020, 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
Kumar, TimeTree: A Resource for Timelines, Timetrees, and Divergence Times. 2017, Pubmed
Larkin, Clustal W and Clustal X version 2.0. 2007, Pubmed
Liu, Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14. 1999, Pubmed
Lu, Reissner fibre-induced urotensin signalling from cerebrospinal fluid-contacting neurons prevents scoliosis of the vertebrate spine. 2020, Pubmed
Meiniel, The subcommissural organ and Reissner's fiber complex. An enigma in the central nervous system? 1996, Pubmed
Mori, Urotensin II is the endogenous ligand of a G-protein-coupled orphan receptor, SENR (GPR14). 1999, Pubmed
Muffato, Genomicus: a database and a browser to study gene synteny in modern and ancestral genomes. 2010, Pubmed
Mughal, Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis. 2018, Pubmed , Xenbase
Nakajima, Generation of albino Xenopus tropicalis using zinc-finger nucleases. 2012, Pubmed , Xenbase
Nobata, Potent cardiovascular effects of homologous urotensin II (UII)-related peptide and UII in unanesthetized eels after peripheral and central injections. 2011, Pubmed
Nothacker, Identification of the natural ligand of an orphan G-protein-coupled receptor involved in the regulation of vasoconstriction. 1999, Pubmed
Orts-Del'Immagine, Sensory Neurons Contacting the Cerebrospinal Fluid Require the Reissner Fiber to Detect Spinal Curvature In Vivo. 2020, Pubmed
Parmentier, Comparative distribution of the mRNAs encoding urotensin I and urotensin II in zebrafish. 2008, Pubmed
Parmentier, Occurrence of two distinct urotensin II-related peptides in zebrafish provides new insight into the evolutionary history of the urotensin II gene family. 2011, Pubmed
Pearson, Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes. 1980, Pubmed
Quan, Characterization of the true ortholog of the urotensin II-related peptide (URP) gene in teleosts. 2012, Pubmed
Quan, Comparative distribution and in vitro activities of the urotensin II-related peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal cerebrospinal fluid-contacting neurons. 2015, Pubmed
Rose, SCO-Spondin Defects and Neuroinflammation Are Conserved Mechanisms Driving Spinal Deformity across Genetic Models of Idiopathic Scoliosis. 2020, Pubmed
Roy, Adolescent Idiopathic Scoliosis: Fishy Tales of Crooked Spines. 2021, Pubmed
Saitou, The neighbor-joining method: a new method for reconstructing phylogenetic trees. 1987, Pubmed
Session, Genome evolution in the allotetraploid frog Xenopus laevis. 2016, Pubmed , Xenbase
Suzuki, High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos. 2013, Pubmed , Xenbase
Tamura, MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. 2013, Pubmed
Tostivint, Impact of gene/genome duplications on the evolution of the urotensin II and somatostatin families. 2013, Pubmed
Tostivint, Comparative genomics provides evidence for close evolutionary relationships between the urotensin II and somatostatin gene families. 2006, Pubmed
Tostivint, Molecular evolution of GPCRs: Somatostatin/urotensin II receptors. 2014, Pubmed
Troutwine, The Reissner Fiber Is Highly Dynamic In Vivo and Controls Morphogenesis of the Spine. 2020, 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
Wajchenberg, Adolescent idiopathic scoliosis: current concepts on neurological and muscular etiologies. 2016, Pubmed
Wlizla, Generation and Care of Xenopus laevis and Xenopus tropicalis Embryos. 2018, Pubmed , Xenbase
Yulis, Floor plate and the subcommissural organ are the source of secretory compounds of related nature: comparative immunocytochemical study. 1998, Pubmed , Xenbase
Yun, Prevertebrate Local Gene Duplication Facilitated Expansion of the Neuropeptide GPCR Superfamily. 2015, Pubmed
Zhang, Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis. 2018, Pubmed