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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.

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