Cathleen Teh1,*, Guangyu Sun2,3,*, Hongyuan Shen1, Vladimir Korzh1,4,‡ and Thorsten Wohland2,3,,4,‡
1 Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive Singapore 138673
2 Department of Chemistry, National University of Singapore, 117543 Singapore.
3 Center for Bioimaging Sciences, National University of Singapore, 117557 Singapore.
4 Department of Biological Sciences, National University of Singapore, 117543 Singapore.
* These authors contributed equally to this work.
‡ Authors for correspondence (firstname.lastname@example.org; email@example.com)
Published in Development on September 24th 2015.
The boundaries of brain regions are associated with the tissue-specific secretion of ligands from different signaling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. Using light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics, we demonstrated that Wnt3 regulates cerebellum development during embryogenesis using zebrafish wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP. The results suggest a state of dynamic equilibrium of Wnt3EGFP mobility in polarized neuroepithelial-like progenitors in the dorsal midline and cerebellar progenitors on the lateral side. Wnt3EGFP is secreted from the cerebellum as shown by measurements of its mobility in the ventricular cavity. The importance of Wnt secretion in brain patterning was validated with the Porcn inhibitor Wnt-C59 (C59), which, when applied early, reduced membrane-bound and secreted fractions of Wnt3EGFP and led to a malformed brain characterized by the absence of epithalamus, optic tectum and cerebellum. Likewise, interference with Wnt secretion later on during cerebellar development negatively impacted cerebellar growth and patterning. Our work, supported by quantitative analysis of protein dynamics in vivo, highlights the importance of membrane-localized and secreted Wnt3 during cerebellum development.
Figure legend: Exposure of Tg(wnt3:Wnt3EGFP)F3 larvae to Wnt inhibitor C59 decreased the segmental volume of cerebellum in Tg(memKR15-16). (A-D) In vivo images of 3 dpf double transgenic larvae: control Tg(wnt3:EGFP)F1/Tg(memKR15-16) (A), Tg(wnt3:Wnt3EGFP)F3/Tg(memKR15-16) (B-D). All Tg(wnt3:wnt3EGFP)F3 double transgenic larvae were exposed to either 1%DMSO (B), 5 μM C59 (C) or 50 μM IWR-1 (D) at 36 hpf-stage for 36 h before assaying expression of fluorescent reporter at 72 hpf. High-magnification view of representative EGFP/Wnt3EGFP-expressing cells from different treatment groups (insets). KillerRed expression in the MHB and cerebellum of Tg(memKR15-16) is in red; EGFP or Wnt3EGFP expression in wnt3 transgenics is in green; and yellow represents a 3D view of the computed volume. (E) Comparison of the resultant KillerRed-positive brain segment in Tg(memKR15-16). A significant difference was observed between segmental volumes in 1% DMSO-treated Tg(wnt3:Wnt3EGFP)F3 vs C59-treated Tg(wnt3:Wnt3EGFP)F3 (unpaired t-test, P=0.0001).
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