Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions

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Abstract

Wnt3 proteins are lipidated and glycosylated, secreted signaling molecules that play an important role in zebrafish neural patterning and brain development. However, the transport mechanism of lipid-modified Wnts through the hydrophilic extracellular environment for long-range action remains unresolved. Here, we determine how Wnt3 accomplishes long-range distribution in the zebrafish brain. First, we characterize the Wnt3-producing source and Wnt3-receiving target regions. Subsequently, we analyze Wnt3 mobility at different length scales by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. We demonstrate that Wnt3 spreads extracellularly and interacts with heparan sulfate proteoglycans (HSPG). We then determine the binding affinity of Wnt3 to its receptor, Frizzled1 (Fzd1), using fluorescence cross-correlation spectroscopy, and show that the co-receptor, low-density lipoprotein receptor-related protein 5 (Lrp5), is required for Wnt3-Fzd1 interaction. Our results are consistent with the extracellular distribution of Wnt3 by a diffusive mechanism that is modified by tissue morphology, interactions with HSPG and Lrp5-mediated receptor binding, to regulate zebrafish brain development.

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All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 4, 5, 6 and Table 1. Videos 1 and 2 represent the raw file used to reconstruct Figures 2,3 and Videos 3,4 respectively.

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Sapthaswaran Veerapathiran

Biological Sciences, National University of Singapore, Singapore, Singapore

Competing interests
The authors declare that no competing interests exist.
Cathleen Teh

Biological Sciences, National University of Singapore, Singapore, Singapore

Competing interests
The authors declare that no competing interests exist.
Shiwen Zhu

Biological Sciences, National University of Singapore, Singapore, Singapore

Competing interests
The authors declare that no competing interests exist.
Indira Kartigayen

Biological Sciences, National University of Singapore, Singapore, Singapore

Competing interests
The authors declare that no competing interests exist.
Vladimir Korzh

Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland

Competing interests
The authors declare that no competing interests exist.
Paul T Matsudaira

Biological Sciences, National University of Singapore, Singapore, Singapore

Competing interests
The authors declare that no competing interests exist.
Thorsten Wohland

Biological Sciences and Chemistry, National University of Singapore, Singapore, Singapore

For correspondence
twohland@nus.edu.sg

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-0148-4321

Funding

Ministry of Education - Singapore (MOE2016-T3-1-005)

Thorsten Wohland

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: The study was performed in strict accordance with Institutional Animal Care and Use Committee (IACUC) protocol of Biological Resource Center (BRC), A*STAR, Singapore (IACUC #161105) and the National University of Singapore (IACUC# BR18-1023).

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.