Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis

Abstract

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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Fig. 1: Body curvature correlates with impaired cilia-driven CSF flow in ciliary mutants.
Fig. 2: Urp1 functions downstream of motile cilia and CSF flow to regulate body-axis straightening.
Fig. 3: CSF epinephrine signals regulate body-axis development.
Fig. 4: Uts2ra is the receptor for the neuropeptide Urp1 in axial straightening.

Data availability

RNA-seq data have been deposited to the Sequence Read Archive (SRA) database under accession numbers SRX4719722 and SRX4719723. All of the data that support the findings of this study are available from the corresponding author upon reasonable request.

Change history

  • 12 December 2018

    In the HTML version of the article originally published, there were several errors in the posting of the supplementary videos. In particular, the links for Supplementary Videos 1–4 originally led to Supplementary Videos 2–5, respectively, and the video for Supplementary Video 1 was not posted. The errors have been corrected in the HTML version of the article.

References

  1. 1.

    Cheng, J. C. et al. Adolescent idiopathic scoliosis. Nat. Rev. Dis. Primers 1, 15030 (2015).

    Article  Google Scholar 

  2. 2.

    Grimes, D. T. et al. Zebrafish models of idiopathic scoliosis link cerebrospinal fluid flow defects to spine curvature. Science 352, 1341–1344 (2016).

    CAS  Article  Google Scholar 

  3. 3.

    Lun, M. P., Monuki, E. S. & Lehtinen, M. K. Development and functions of the choroid plexus-cerebrospinal fluid system. Nat. Rev. Neurosci. 16, 445–457 (2015).

    CAS  Article  Google Scholar 

  4. 4.

    Goetz, S. C. & Anderson, K. V. The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331–344 (2010).

    CAS  Article  Google Scholar 

  5. 5.

    Song, Z., Zhang, X., Jia, S., Yelick, P. C. & Zhao, C. Zebrafish as a model for human ciliopathies. J. Genet. Genomics 43, 107–120 (2016).

    Article  Google Scholar 

  6. 6.

    Zariwala, M. A. et al. ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6. Am. J. Hum. Genet. 93, 336–345 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Zhao, C., Omori, Y., Brodowska, K., Kovach, P. & Malicki, J. Kinesin-2 family in vertebrate ciliogenesis. Proc. Natl Acad. Sci. USA 109, 2388–2393 (2012).

    CAS  Article  Google Scholar 

  8. 8.

    Tsujikawa, M. & Malicki, J. Intraflagellar transport genes are essential for differentiation and survival of vertebrate sensory neurons. Neuron 42, 703–716 (2004).

    CAS  Article  Google Scholar 

  9. 9.

    Kramer-Zucker, A. G. et al. Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer’s vesicle is required for normal organogenesis. Development 132, 1907–1921 (2005).

    CAS  Article  Google Scholar 

  10. 10.

    Borovina, A., Superina, S., Voskas, D. & Ciruna, B. Vangl2 directs the posterior tilting and asymmetric localization of motile primary cilia. Nat. Cell Biol. 12, 407–412 (2010).

    CAS  Article  Google Scholar 

  11. 11.

    Ross, B., McKendy, K. & Giaid, A. Role of urotensin II in health and disease. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298, R1156–R1172 (2010).

    CAS  Article  Google Scholar 

  12. 12.

    Quan, F. B. et al. 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. PLoS ONE 10, e0119290 (2015).

    Article  Google Scholar 

  13. 13.

    Djenoune, L. et al. Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates. Front. Neuroanat. 8, 26 (2014).

    Article  Google Scholar 

  14. 14.

    van Rooijen, E. et al. LRRC50, a conserved ciliary protein implicated in polycystic kidney disease. J. Am. Soc. Nephrol. 19, 1128–1138 (2008).

    Article  Google Scholar 

  15. 15.

    Yu, X., Ng, C. P., Habacher, H. & Roy, S. Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nat. Genet. 40, 1445–1453 (2008).

    CAS  Article  Google Scholar 

  16. 16.

    Panizzi, J. R. et al. CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms. Nat. Genet. 44, 714–719 (2012).

    CAS  Article  Google Scholar 

  17. 17.

    van Eeden, F. J. et al. Mutations affecting somite formation and patterning in the zebrafish, Danio rerio. Development 123, 153–164 (1996).

    PubMed  Google Scholar 

  18. 18.

    Odenthal, J., van Eeden, F. J., Haffter, P., Ingham, P. W. & Nusslein-Volhard, C. Two distinct cell populations in the floor plate of the zebrafish are induced by different pathways. Dev. Biol. 219, 350–363 (2000).

    CAS  Article  Google Scholar 

  19. 19.

    Vaudry, H. et al. Urotensin II, from fish to human. Ann. N. Y. Acad. Sci. 1200, 53–66 (2010).

    CAS  Article  Google Scholar 

  20. 20.

    Rossi, A. et al. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524, 230–233 (2015).

    CAS  Article  Google Scholar 

  21. 21.

    Lehtinen, M. K. & Walsh, C. A. Neurogenesis at the brain-cerebrospinal fluid interface. Annu. Rev. Cell Dev. Biol. 27, 653–679 (2011).

    CAS  Article  Google Scholar 

  22. 22.

    Xie, L. et al. Sleep drives metabolite clearance from the adult brain. Science 342, 373–377 (2013).

    CAS  Article  Google Scholar 

  23. 23.

    Lleo, A. et al. Cerebrospinal fluid biomarkers in trials for Alzheimer and Parkinson diseases. Nat. Rev. Neurol. 11, 41–55 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Ames, R. S. et al. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 401, 282–286 (1999).

    CAS  Article  Google Scholar 

  25. 25.

    Wu, X., Shen, W., Zhang, B. & Meng, A. The genetic program of oocytes can be modified in vivo in the zebrafish ovary. J. Mol. Cell Biol. https://doi.org/10.1093/jmcb/mjy044 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Jackson, H. E. & Ingham, P. W. Control of muscle fibre-type diversity during embryonic development: the zebrafish paradigm. Mech. Dev. 130, 447–457 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Freund, J. B., Goetz, J. G., Hill, K. L. & Vermot, J. Fluid flows and forces in development: functions, features and biophysical principles. Development 139, 1229–1245 (2012).

    CAS  Article  Google Scholar 

  28. 28.

    Baik, J. S. et al. Scoliosis in patients with Parkinson’s disease. J. Clin. Neurol. 5, 91–94 (2009).

    Article  Google Scholar 

  29. 29.

    Zeman, R. J., Zhang, Y. & Etlinger, J. D. Clenbuterol, a beta2-adrenoceptor agonist, reduces scoliosis due to partial transection of rat spinal cord. Am. J. Physiol. 272, E712–E715 (1997).

    CAS  PubMed  Google Scholar 

  30. 30.

    Cantaut-Belarif, Y., Sternberg, J. R., Thouvenin, O., Wyart, C. & Bardet, P. L. The Reissner fiber in the cerebrospinal fluid controls morphogenesis of the body axis. Curr. Biol. 28, 2479–2486 (2018).

    CAS  Article  Google Scholar 

  31. 31.

    Caprile, T., Hein, S., Rodriguez, S., Montecinos, H. & Rodriguez, E. Reissner fiber binds and transports away monoamines present in the cerebrospinal fluid. Brain Res. Mol. Brain Res. 110, 177–192 (2003).

    CAS  Article  Google Scholar 

  32. 32.

    Barresi, M. J., Stickney, H. L. & Devoto, S. H. The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity. Development 127, 2189–2199 (2000).

    CAS  PubMed  Google Scholar 

  33. 33.

    Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    CAS  Article  Google Scholar 

  34. 34.

    Chang, J. T. & Sive, H. Manual drainage of the zebrafish embryonic brain ventricles. J. Vis. Exp. 70, e4243 (2012).

    Google Scholar 

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Acknowledgements

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.

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Affiliations

Authors

Contributions

C.Z. designed the project. X.Z. and S.J. performed most of the experiments, including the screening of zmynd10, urp1 and uts2ra mutants, analysis of body curvature, injection of urotensin II related peptides, screening of the GPCR compound library, and CSF drainage. Z.C. generated the uts2ra BAC construct and performed expression analysis. Z.C. and X.W. performed the OMIS experiments. Y.L.C. generated foxj1a mutants and performed urp expression analysis in ciliary and smo mutants, uts2ra BAC expression in smo mutants, and epinephrine treatment of foxj1a mutants. H.X. performed mutant screening and immunohistochemistry analysis. D.F. conducted qPCR and whole-mount in situ hybridization experiments. D.Z.S. and X.Z. performed fluorescent-dye tracing analysis. C.Z. and S.R. analyzed the data and wrote the manuscript. All authors read and endorsed the final version of the manuscript.

Corresponding author

Correspondence to Chengtian Zhao.

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Integrated supplementary information

Supplementary Fig. 1 Phenotypes of zmynd10 mutants.

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. df, 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.

Supplementary Fig. 3 Expression of urp1 is downregulated in ciliary mutants.

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, af, 20 µm; h,i, 500 µm.

Supplementary Fig. 4 UII-related peptides rescue body curvature in zmynd10 mutants.

a, Phenotypes of wild-type and zmynd10 (z10) mutants injected with UII-related peptides. bi, Statistical analysis of the rescue efficiency of body-curvature defects in zmynd10 mutants (bg) 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.

Supplementary Fig. 5 Redundant functions of Urp1 and Urp2 during body-axis straightening.

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.

Supplementary Fig. 6 Epinephrine treatment rescues body-curvature defects in ciliary mutants.

ad, 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.

Supplementary Fig. 7 Rescue of body curvature in zmynd10 mutants using different catecholamines.

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.

Supplementary Fig. 8 Uts2ra is the major Urp1 receptor expressed in the dorsal somite cells.

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. eh, 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.

Supplementary Fig. 9 Inhibition of neuromuscular junction activity does not affect Urp signaling.

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.

Supplementary Fig. 10 Phenotypes of uts2ra mutants.

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 information

41588_2018_260_MOESM4_ESM.mp4

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

41588_2018_260_MOESM5_ESM.mp4

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

41588_2018_260_MOESM6_ESM.mp4

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

41588_2018_260_MOESM7_ESM.mp4

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

41588_2018_260_MOESM8_ESM.mp4

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

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 2–5

Reporting Summary

Supplementary Table 1

List of compounds used in the screen

Supplementary Video 1

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

Supplementary Video 2

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

Supplementary Video 3

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

Supplementary Video 4

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

Supplementary Video 5

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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