title: Modulating the expression level of secreted Wnt3 influences cerebellum development in zebrafish transgenics.
abstract: 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.
authors.affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, 138673 Singapore.
authors.forename: Cathleen
authors.initials: C
authors.lastname: Teh
authors.affiliation: Department of Chemistry, National University of Singapore, 117543 Singapore Center for Bioimaging Sciences, National University of Singapore, 117557 Singapore.
authors.forename: Guangyu
authors.initials: G
authors.lastname: Sun
authors.affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, 138673 Singapore.
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Shen
authors.affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, 138673 Singapore Department of Biological Sciences, National University of Singapore, 117543 Singapore vlad@imcb.a-star.edu.sg twohland@nus.edu.sg.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Korzh
authors.affiliation: Department of Chemistry, National University of Singapore, 117543 Singapore Center for Bioimaging Sciences, National University of Singapore, 117557 Singapore Department of Biological Sciences, National University of Singapore, 117543 Singapore vlad@imcb.a-star.edu.sg twohland@nus.edu.sg.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wohland
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002531:Cerebellum / Q000254:growth & development / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D060449:Wnt Signaling Pathway; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D011518:Proto-Oncogene Proteins; D051153:Wnt Proteins; C491438:Wnt5 protein, Drosophila; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Brain ventricle; C59 Wnt inhibitor; Cerebellum; Fluorescence correlation spectroscopy (FCS); Ligand; Protein fractions