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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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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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 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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DOI: https://doi.org/10.1038/nchembio.2124
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