Straightening of the body axis is a major morphogenetic event that produces the typical head-to-tail shape of the vertebrate embryo. Defects in axial straightening can lead to debilitating disorders such as idiopathic scoliosis, characterized by three-dimensional curvatures of the spine1. Although abnormal cerebrospinal fluid (CSF) flow has been implicated in the development of idiopathic scoliosis2, the molecular mechanisms operating downstream of CSF flow remain obscure. Here we show that, in zebrafish embryos, cilia-driven CSF flow transports adrenergic signals that induce urotensin neuropeptides in CSF-contacting neurons along the spinal cord. Urotensins activate their receptor on slow-twitch muscle fibers of the dorsal somite; the contraction of these fibers likely results in straightening of the body axis. Consistent with this, mutation of the urotensin receptor resulted in severe scoliosis in adult zebrafish, closely mimicking the human disorder. These findings suggest that disruption of urotensin signaling by impaired CSF flow could be a critical etiological factor underlying the pathology of idiopathic scoliosis.
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We thank G. Burns, J. Malicki, C. Duan, J. Zhou, X. Tan and L. Lu for reagents, constructs and zebrafish strains. We also thank H. Li for help with the assembly of TALEN constructs, Y. Li for transcriptome data analysis, and G. Ou for comments on earlier versions of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31422051 and 81301718), the AoShan Talents Program Supported by Qingdao National Laboratory for Marine Science and Technology (2015ASTP-ES13), and the Shandong Provincial Natural Science Foundation (JQ201506) to C.Z., and the Agency for Science, Technology and Research of Singapore (A*STAR) to S.R.
The authors declare no competing interests.
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Integrated supplementary information
a,b,b′, External phenotypes of wild-type zebrafish and mutants resulting from two different zmynd10 mutations (del7 and del10) at 72 h.p.f. c,c′,c′′, Ventral views of heart looping phenotypes in control and zmynd10 mutants. Control embryos display normal heart looping (c), while heart looping is frequently inverted (c′) or absent (c′′) in zmynd10 mutants, implying defective cilia motility in the left–right organizer. a, atrium; v, ventricle. d, Protein and genomic structures of the zmynd10 gene. The blue bar indicates the MYND-type zinc-finger domain. The bottom panel shows sequences of the zmynd10 gene in wild-type and mutant alleles. Red color shows TALEN spacer sequence. e, Lateral views of the otic vesicle showing two otoliths in wild-type embryos (e) and multiple otoliths in zmynd10 mutants (e′), implying defective motility of cilia in the otic vesicle. f, Histological analysis showing a distended pronephric tubule lumen in zmynd10 mutants (f′, asterisk; implying defective motility of pronephric duct cilia), compared with wild-type control embryos (f). Scale bars: a,b, 500 µm; e,f, 50 µm.
Supplementary Fig. 2 Specific requirement of motile cilia in the spinal canal for body-axis straightening.
a,b, External phenotypes of kif3b mutant larvae at 72 h.p.f., injected with GFP or kif17 mRNA. c, Statistical analysis of the number of beating motile cilia per a.u. in the spinal canal of kif3b mutant larvae. ***P < 0.001, Student’s t test. d–f, Confocal images showing cilia in the pronephric duct of wild-type and kif3b mutants, visualized with anti-acetylated-tubulin antibody. Arrows indicate the bundle of motile cilia formed by multiciliated cells (d), which were substantially shorter in kif3b mutants injected with either GFP (e) or kif17 (f) mRNA. Asterisks indicate neuronal axons, also labeled by anti-acetylated-tubulin antibodies. Scale bar, 10 µm.
a,b, Whole-mount in situ hybridization showing urp1 expression in wild-type (a) and foxj1a mutant (b) embryos. c, Expression of urp1 in the smo mutant. d, Expression of urp1 in a wild-type embryo treated with cyclopamine (cyc). e,f, Expression of pkd2l1 in wild-type (e) and smo mutant (f) embryos. g, Percentage of embryos with ventral body-curvature defects in control and urp1-overexpressing embryos in different mutant backgrounds; ctr, control. h,i, Compared with control wild-type siblings (h), overexpression of urp1 mRNA resulted in dorsal curvature in wild-type embryos (i). Scale bars, a–f, 20 µm; h,i, 500 µm.
a, Phenotypes of wild-type and zmynd10 (z10) mutants injected with UII-related peptides. b–i, Statistical analysis of the rescue efficiency of body-curvature defects in zmynd10 mutants (b–g) or wild-type embryos (h,i), by different UII-related peptides. mt, zmynd10 mutant. The numbers of embryos analyzed are shown at the bottom. Statistical analysis was performed by comparing the angles before and after injection. ***P < 0.001, Student’s t-test. Scale bar, 500 µm.
a, Quantification of the number of beating motile cilia per a.u. in the spinal canal of control embryos and urp1 and uts2ra morphants. NS, not significant; P = 0.42 for urp1 and P = 0.84 for uts2ra. b, Diagram showing the structure of the urp1 gene and sequences of urp1 alleles in wild-type and mutant embryos. The underlining shows the conserved amino acids of the cyclic region of wild-type Urp1; one of the cysteines is mutated in the mutants. c, Percentage of embryos with body curvature defects in wild-type or mutant urp1 mRNA overexpressing embryos, which were collected from cross between two zmynd10 heterozygotes d, Top, reverse transcription (RT)–PCR amplification of the urp2 transcript in wild-type and urp2 SP (Splicing-blocking Morpholino) morphants at 24 h.p.f. Note the decreased level of mature urp2 transcript in the morphants. Bottom, genomic structure of the urp2 gene. The position of the SP morpholino oligonucleotide and primers used for RT–PCR are shown. e, Percentage of embryos with a curly body axis in control, urp1 and urp2 morphants at different combinations as indicated. f, Percentage of embryos with body-curvature defects in wild-type or urp1 mutants injected with urp2 SP morpholino oligonucleotide. In panels c, e and f, the numbers of embryos analyzed are shown on top of each bar.
a–d, Phenotypes of kif3b (a,b) and ovl (c,d) mutants treated with DMSO or epinephrine (epi). e,f, Epinephrine treatment induced dorsal curvature in wild-type embryos. g, Statistical analysis of the angles of body curvature of kif3b (a,b) and ovl (c,d) mutants treated with DMSO or epinephrine. ***P < 0.001, Student’s t-test. h, Relative travel distance of fluorescent beads in wild-type or zmynd10 (z10) mutants after DMSO or epinephrine treatment. NS, not significant with two-way ANOVA test. Scale bar, 500 µm.
Statistical analysis of the angles of body curvature in zmynd10 mutants treated with different catecholamines at different concentrations. The numbers of embryos analyzed are shown at the bottom.
a, Phylogenetic tree of urotensin II receptors in different vertebrates. Zebrafish contains four UII receptor homologs: Uts2r, Si:dkey-27n14.1 (Uts2ra), Si:dkey-33c3.1 and Si:ch73-379f7.4. The analysis was conducted using the neighbor-joining method in MEGA7 software with 1,000 bootstrap replicates. Dre, zebrafish; Gga, chicken; Hsa, human; Mmu, mouse; Xtr, Xenopus; Tru, Fugu. b, qPCR data showing relative expression of the four UII-receptor homologs in zebrafish embryos at 28 h.p.f. c,d, Expression of GFP driven by the 5.3-kb uts2ra promoter in the dorsal muscle cells of a zebrafish embryo at 28 h.p.f. The dotted line in d marks the midline of the notochord. e–h, Whole-mount in situ hybridization showing the expression of uts2ra and eng2a in DMSO- or cyclopamine-treated embryos. The eng2a gene is a key marker of muscle-cell types induced by Shh signaling. Arrows in e indicate expression of uts2ra in the dorsal somites. i, Statistical analysis of the angles of body curvature in control or uts2ra morphants treated with DMSO or epinephrine. cMO, control MO. ***P < 0.001, Student’s t-test. NS, not significant, P = 0.24. Scale bar, 50 µm.
a,b, Phenotypes of α-BTX-treated wild-type embryos before (a) and 5 min after (b) Urp1 peptide injection. c,d, Statistical analysis of the angles of body-axis curvature of α-BTX-treated (c) or Fas-II-treated (d) wild-type embryos before and 5 min after Urp1 peptide injection. ***P < 0.001, Student’s t-test.
a, Diagram of the uts2ra gene and sequences of uts2ra alleles in wild-type (wt) and mutant (mt) embryos. Underlined nucleotides show premature stop codons in the mutant. b, qPCR results showing relative expression of UII-receptor genes in uts2ra mutants. c, Percentage of embryos with a curled body axis in uts2r morphants. Note that the mutant embryos were collected from crosses between uts2ra heterozygous males and homozygous females. The number (n) of embryos is shown at the top of each bar. d,e, Confocal images showing the results of staining 22-d.p.f. juvenile wild-type and uts2ra mutant embryos with F59 antibody. In panel e, the asterisk indicates weak fluorescence signals resulting from the presence of pigment cells.
Supplementary Figures 1–10 and Supplementary Tables 2–5
List of compounds used in the screen
Time-lapse movie of motile cilia in the spinal canal of a 30 h.p.f. wild-type embryo carrying the Tg(bactin2::Arl13b-gfp) transgene. Images were captured at 65 frames per second (f.p.s.) for the duration of 200 frames, which is the same for Supplementary Videos 2 and 5
Time-lapse movie of motile cilia in the spinal canal of a 30 h.p.f. zmynd10 mutant embryo carrying the Tg(bactin2::Arl13b-gfp) transgene
Time-lapse movie showing the body-axis changes of a wild-type embryo injected with the Urp1 neuropeptide. Images were captured every 30 s after injection. The arrow points to the injection site. ‘s’ indicates seconds
Time-lapse movie showing the body-axis changes of a zmynd10 mutant embryo injected with the Urp1 neuropeptide. Images were captured every 30 s after injection. The arrow points to the injection site. ‘s’ indicates seconds
Time-lapse movie of motile cilia in the spinal canal of a 30 h.p.f. urp1 morphant carrying the Tg(bactin2::Arl13b-gfp) transgene
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Zhang, X., Jia, S., Chen, Z. et al. Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis. Nat Genet 50, 1666–1673 (2018). https://doi.org/10.1038/s41588-018-0260-3
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