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Tbx16 regulates hox gene activation in mesodermal progenitor cells

Nature Chemical Biology volume 12, pages 694–701 (2016)Cite this article

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Abstract

The transcription factor T-box 16 (Tbx16, or Spadetail) is an essential regulator of paraxial mesoderm development in zebrafish (Danio rerio). Mesodermal progenitor cells (MPCs) fail to differentiate into trunk somites in tbx16 mutants and instead accumulate within the tailbud in an immature state. However, the mechanisms by which Tbx16 controls mesoderm patterning have remained enigmatic. We describe here the use of photoactivatable morpholino oligonucleotides to determine the Tbx16 transcriptome in MPCs. We identified 124 Tbx16-regulated genes that were expressed in zebrafish gastrulae, including several developmental signaling proteins and regulators of gastrulation, myogenesis and somitogenesis. Unexpectedly, we observed that a loss of Tbx16 function precociously activated posterior hox genes in MPCs, and overexpression of a single posterior hox gene was sufficient to disrupt MPC migration. Our studies support a model in which Tbx16 regulates the timing of collinear hox gene activation to coordinate the anterior–posterior fates and positions of paraxial MPCs.

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Figure 1: Optochemical regulation of Tbx16-dependent paraxial mesoderm development.
Figure 2: Optochemical analysis of the Tbx16-dependent transcriptome in trunk somite progenitors.
Figure 3: Tbx16 regulates hox gene activation during gastrulation.
Figure 4: Non-cell-autonomous signals propagate posterior hox gene activation in Tbx16-deficient embryos.
Figure 5: hoxa13b expression is sufficient to posteriorize paraxial MPCs.
Figure 6: A model for Tbx16-dependent paraxial mesoderm patterning.

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References

  1. Scaal, M. Early development of the vertebral column. Semin. Cell Dev. Biol. 49, 83–91 (2016).

    Article  PubMed  Google Scholar 

  2. Eckalbar, W.L., Fisher, R.E., Rawls, A. & Kusumi, K. Scoliosis and segmentation defects of the vertebrae. Wiley Interdiscip. Rev. Dev. Biol. 1, 401–423 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Hubaud, A. & Pourquié, O. Signalling dynamics in vertebrate segmentation. Nat. Rev. Mol. Cell Biol. 15, 709–721 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Turnpenny, P.D. et al. Novel mutations in DLL3, a somitogenesis gene encoding a ligand for the Notch signaling pathway, cause a consistent pattern of abnormal vertebral segmentation in spondylocostal dysostosis. J. Med. Genet. 40, 333–339 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Whittock, N.V. et al. Mutated MESP2 causes spondylocostal dysostosis in humans. Am. J. Hum. Genet. 74, 1249–1254 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sparrow, D.B. et al. Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype. Am. J. Hum. Genet. 78, 28–37 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Sparrow, D.B., Guillén-Navarro, E., Fatkin, D. & Dunwoodie, S.L. Mutation of Hairy-and-Enhancer-of-Split-7 in humans causes spondylocostal dysostosis. Hum. Mol. Genet. 17, 3761–3766 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Holley, S.A. Anterior-posterior differences in vertebrate segments: specification of trunk and tail somites in the zebrafish blastula. Genes Dev. 20, 1831–1837 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Mallo, M., Vinagre, T. & Carapuço, M. The road to the vertebral formula. Int. J. Dev. Biol. 53, 1469–1481 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Kimmel, C.B., Kane, D.A., Walker, C., Warga, R.M. & Rothman, M.B. A mutation that changes cell movement and cell fate in the zebrafish embryo. Nature 337, 358–362 (1989).

    Article  CAS  PubMed  Google Scholar 

  11. Ho, R.K. & Kane, D.A. Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors. Nature 348, 728–730 (1990).

    Article  CAS  PubMed  Google Scholar 

  12. Griffin, K.J., Amacher, S.L., Kimmel, C.B. & Kimelman, D. Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes. Development 125, 3379–3388 (1998).

    CAS  PubMed  Google Scholar 

  13. Ho, R.K. Cell movements and cell fate during zebrafish gastrulation. Dev. Suppl. 1992, 65–73 (1992).

    Google Scholar 

  14. Yamamoto, A. et al. Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm. Development 125, 3389–3397 (1998).

    CAS  PubMed  Google Scholar 

  15. Griffin, K.J. & Kimelman, D. One-Eyed Pinhead and Spadetail are essential for heart and somite formation. Nat. Cell Biol. 4, 821–825 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Fior, R. et al. The differentiation and movement of presomitic mesoderm progenitor cells are controlled by Mesogenin 1. Development 139, 4656–4665 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. O'Neill, K. & Thorpe, C. BMP signaling and spadetail regulate exit of muscle precursors from the zebrafish tailbud. Dev. Biol. 375, 117–127 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Szeto, D.P. & Kimelman, D. The regulation of mesodermal progenitor cell commitment to somitogenesis subdivides the zebrafish body musculature into distinct domains. Genes Dev. 20, 1923–1932 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Garnett, A.T. et al. Identification of direct T-box target genes in the developing zebrafish mesoderm. Development 136, 749–760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mueller, R.L., Huang, C. & Ho, R.K. Spatio-temporal regulation of Wnt and retinoic acid signaling by tbx16/spadetail during zebrafish mesoderm differentiation. BMC Genomics 11, 492 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Shestopalov, I.A., Sinha, S. & Chen, J.K. Light-controlled gene silencing in zebrafish embryos. Nat. Chem. Biol. 3, 650–651 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Yamazoe, S., Shestopalov, I.A., Provost, E., Leach, S.D. & Chen, J.K. Cyclic caged morpholinos: conformationally gated probes of embryonic gene function. Angew. Chem. Int. Edn Engl. 51, 6908–6911 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  24. Shestopalov, I.A., Pitt, C.L. & Chen, J.K. Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development. Nat. Chem. Biol. 8, 270–276 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Myers, D.C., Sepich, D.S. & Solnica-Krezel, L. Bmp activity gradient regulates convergent extension during zebrafish gastrulation. Dev. Biol. 243, 81–98 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Yamazoe, S., Liu, Q., McQuade, L.E., Deiters, A. & Chen, J.K. Sequential gene silencing using wavelength-selective caged morpholino oligonucleotides. Angew. Chem. Int. Edn Engl. 53, 10114–10118 (2014).

    Article  CAS  Google Scholar 

  27. Payumo, A.Y., Walker, W.J., McQuade, L.E., Yamazoe, S. & Chen, J.K. Optochemical dissection of T-box gene-dependent medial floor plate development. ACS Chem. Biol. 10, 1466–1475 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Weinberg, E.S. et al. Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development 122, 271–280 (1996).

    CAS  PubMed  Google Scholar 

  29. Kimmel, C.B., Warga, R.M. & Schilling, T.F. Origin and organization of the zebrafish fate map. Development 108, 581–594 (1990).

    CAS  PubMed  Google Scholar 

  30. Warga, R.M. & Nüsslein-Volhard, C. Origin and development of the zebrafish endoderm. Development 126, 827–838 (1999).

    CAS  PubMed  Google Scholar 

  31. Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651–12656 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bouldin, C.M. et al. Wnt signaling and tbx16 form a bistable switch to commit bipotential progenitors to mesoderm. Development 142, 2499–2507 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Amores, A. et al. Zebrafish hox clusters and vertebrate genome evolution. Science 282, 1711–1714 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Montavon, T. & Soshnikova, N. Hox gene regulation and timing in embryogenesis. Semin. Cell Dev. Biol. 34, 76–84 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Yu, P.B. et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4, 33–41 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Wells, S., Nornes, S. & Lardelli, M. Transgenic zebrafish recapitulating tbx16 gene early developmental expression. PLoS One 6, e21559 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kim, J.H. et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, e18556 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Martin, B.L. & Kimelman, D. Regulation of canonical Wnt signaling by Brachyury is essential for posterior mesoderm formation. Dev. Cell 15, 121–133 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Martin, B.L. & Kimelman, D. Canonical Wnt signaling dynamically controls multiple stem cell fate decisions during vertebrate body formation. Dev. Cell 22, 223–232 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Yabe, T. & Takada, S. Mesogenin causes embryonic mesoderm progenitors to differentiate during development of zebrafish tail somites. Dev. Biol. 370, 213–222 (2012).

    Article  CAS  PubMed  Google Scholar 

  41. Neave, B., Holder, N. & Patient, R. A graded response to BMP-4 spatially coordinates patterning of the mesoderm and ectoderm in the zebrafish. Mech. Dev. 62, 183–195 (1997).

    Article  CAS  PubMed  Google Scholar 

  42. Row, R.H. & Kimelman, D. Bmp inhibition is necessary for post-gastrulation patterning and morphogenesis of the zebrafish tailbud. Dev. Biol. 329, 55–63 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Harvey, S.A., Tümpel, S., Dubrulle, J., Schier, A.F. & Smith, J.C. no tail integrates two modes of mesoderm induction. Development 137, 1127–1135 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Martin, B.L. & Kimelman, D. Brachyury establishes the embryonic mesodermal progenitor niche. Genes Dev. 24, 2778–2783 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Iimura, T. & Pourquié, O. Collinear activation of Hoxb genes during gastrulation is linked to mesoderm cell ingression. Nature 442, 568–571 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Denans, N., Iimura, T. & Pourquié, O. Hox genes control vertebrate body elongation by collinear Wnt repression. eLife 4, e04379 (2015).

    Article  CAS  PubMed Central  Google Scholar 

  47. Manning, A.J. & Kimelman, D. Tbx16 and Msgn1 are required to establish directional cell migration of zebrafish mesodermal progenitors. Dev. Biol. 406, 172–185 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lengerke, C. et al. BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway. Cell Stem Cell 2, 72–82 (2008).

    Article  CAS  PubMed  Google Scholar 

  49. Warga, R.M., Mueller, R.L., Ho, R.K. & Kane, D.A. Zebrafish Tbx16 regulates intermediate mesoderm cell fate by attenuating Fgf activity. Dev. Biol. 383, 75–89 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Westerfield, M. The Zebrafish Book (University of Oregon Press, 1995).

  51. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B. & Schilling, T.F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310 (1995).

    Article  CAS  PubMed  Google Scholar 

  52. Sato, T., Takahoko, M. & Okamoto, H. HuC:Kaede, a useful tool to label neural morphologies in networks in vivo. Genesis 44, 136–142 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Mich, J.K., Payumo, A.Y., Rack, P.G. & Chen, J.K. In vivo imaging of Hedgehog pathway activation with a nuclear fluorescent reporter. PLoS One 9, e103661 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Thisse, B. & Thisse, C. High throughput expression analysis of ZF models consortium clones. ZFIN Direct Data Submission <http://zfin.org/search?q=High+throughput+expression+analysis+of+ZF+models+consortium+clones> (2005).

  55. Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. Cell Rep. 2, 666–673 (2012).

    Article  CAS  PubMed  Google Scholar 

  56. Ellett, F., Pase, L., Hayman, J.W., Andrianopoulos, A. & Lieschke, G.J. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117, e49–e56 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. Crumpton, B. Gomez and O. Herman of the Stanford Shared FACS Facility for technical assistance with flow cytometry, Z. Weng of the Stanford Sequencing Service Center for assistance with RNA library sequencing, I. Yanai and F. Wagner for assistance with sequence-read processing, S. Amacher (Ohio State University) for tbx16b104/+ zebrafish, M. Lardelli (University of Adelaide), G. Lieschke (Monash University) and H. Okamoto (RIKEN Brain Science Institute) for plasmids, and K. Mruk for helpful discussions. This work was supported by the NIH (DP1 HD075622, R01 GM087292 and R01 GM108952 to J.K.C.; P50 GM107615 to the Stanford Center for Systems Biology), an A.P. Giannini Foundation Fellowship for Medical Research (L.E.M.) and a Japan Society for the Promotion of Science Fellowship (S.Y.).

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  1. Lindsey E McQuade & Sayumi Yamazoe

    Present address: Present addresses: College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA (L.E.M.); Discovery Chemistry, Genentech Inc., South San Francisco, California, USA (S.Y.).,

Authors and Affiliations

  1. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California, USA

    Alexander Y Payumo, Lindsey E McQuade, Whitney J Walker, Sayumi Yamazoe & James K Chen

  2. Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, USA

    James K Chen

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Contributions

A.Y.P., L.E.M. and J.K.C. designed the experiments. A.Y.P., L.E.M. and W.J.W. performed the experiments. A.Y.P., L.E.M. and J.K.C. analyzed data. S.Y. synthesized reagents for cMO preparation. A.Y.P. and J.K.C. wrote the manuscript.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–16. (PDF 7256 kb)

Ventral MPCs lacking Tbx16 function do not exhibit movement defects during gastrulation.

Time-lapse imaging of embryos injected with either Kaede-NLS mRNA alone or in combination with the tbx16 cMO and spot-irradiated in the ventral margin at 6 hpf. Optically targeted cells can then be identified by the red fluorescence of photoconverted Kaede-NLS protein. Morphogenetic movements from 6 hpf to 9 hpf were imaged at a rate of 1 frame/5 minutes, and the movie is shown at a rate of 10 frames (50 minutes of development)/second. Embryo orientations: ventral view, animal pole up. Scale bar: 200 μm. The same embryos were imaged at later stages to generate Supplementary Video 2 (MOV 1117 kb)

Ventral MPCs lacking Tbx16 function exhibit movement defects during somitogenesis.

Time-lapse imaging of embryos injected with either Kaede-NLS mRNA alone or in combination with the tbx16 cMO and spot-irradiated in the ventral margin at 6 hpf. Optically targeted cells can then be identified by the red fluorescence of photoconverted Kaede- NLS protein. Morphogenetic movements from 10.5 to 17 hpf were imaged at a rate of 1 frame/5 minutes, and movie is shown a rate of 10 frames (50 minutes of development)/second. Embryo orientations: lateral view, dorsal up. Scale bar: 200 μm. The same embryos were imaged at earlier stages to generate Supplementary Video 1. (MOV 3406 kb)

MPCs overexpressing hoxa13b exhibit movement defects during somitogenesis.

Time-lapse imaging of embryos injected with either tbx16:EGFP-P2A-mCherry-NLS or tbx16:EGFP-P2A-hoxa13b-NLS constructs at the 1- to 4-cell stage. Morphogenetic movements from 10.5 to 16.25 hpf were imaged at a rate of 1 frame/5 minutes, and movie is 29 shown a rate of 10 frames (50 minutes of development)/second. Embryo orientations: lateral view, dorsal up. Scale bar: 200 μm. (MOV 3383 kb)

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Payumo, A., McQuade, L., Walker, W. et al. Tbx16 regulates hox gene activation in mesodermal progenitor cells. Nat Chem Biol 12, 694–701 (2016). https://doi.org/10.1038/nchembio.2124

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