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

  1. Sapthaswaran Veerapathiran
  2. Cathleen Teh
  3. Shiwen Zhu
  4. Indira Kartigayen
  5. Vladimir Korzh
  6. Paul T Matsudaira
  7. Thorsten Wohland  Is a corresponding author
  1. National University of Singapore, Singapore
  2. International Institute of Molecular and Cell Biology in Warsaw, Poland

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.

Data availability

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.

Article and author information

Author details

  1. Sapthaswaran Veerapathiran

    Biological Sciences, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  2. Cathleen Teh

    Biological Sciences, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  3. Shiwen Zhu

    Biological Sciences, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  4. Indira Kartigayen

    Biological Sciences, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  5. 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.
  6. Paul T Matsudaira

    Biological Sciences, National University of Singapore, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.
  7. 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.
    ORCID icon "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

© 2020, Veerapathiran et al.

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.

Metrics

  • 2,024
    views
  • 249
    downloads
  • 19
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Sapthaswaran Veerapathiran
  2. Cathleen Teh
  3. Shiwen Zhu
  4. Indira Kartigayen
  5. Vladimir Korzh
  6. Paul T Matsudaira
  7. Thorsten Wohland
(2020)
Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions
eLife 9:e59489.
https://doi.org/10.7554/eLife.59489

Share this article

https://doi.org/10.7554/eLife.59489

Further reading

    1. Cancer Biology
    2. Developmental Biology
    Sara Jaber, Eliana Eldawra ... Franck Toledo
    Research Article

    Missense ‘hotspot’ mutations localized in six p53 codons account for 20% of TP53 mutations in human cancers. Hotspot p53 mutants have lost the tumor suppressive functions of the wildtype protein, but whether and how they may gain additional functions promoting tumorigenesis remain controversial. Here, we generated Trp53Y217C, a mouse model of the human hotspot mutant TP53Y220C. DNA damage responses were lost in Trp53Y217C/Y217C (Trp53YC/YC) cells, and Trp53YC/YC fibroblasts exhibited increased chromosome instability compared to Trp53-/- cells. Furthermore, Trp53YC/YC male mice died earlier than Trp53-/- males, with more aggressive thymic lymphomas. This correlated with an increased expression of inflammation-related genes in Trp53YC/YC thymic cells compared to Trp53-/- cells. Surprisingly, we recovered only one Trp53YC/YC female for 22 Trp53YC/YC males at weaning, a skewed distribution explained by a high frequency of Trp53YC/YC female embryos with exencephaly and the death of most Trp53YC/YC female neonates. Strikingly, however, when we treated pregnant females with the anti-inflammatory drug supformin (LCC-12), we observed a fivefold increase in the proportion of viable Trp53YC/YC weaned females in their progeny. Together, these data suggest that the p53Y217C mutation not only abrogates wildtype p53 functions but also promotes inflammation, with oncogenic effects in males and teratogenic effects in females.

    1. Developmental Biology
    Mengjie Li, Aiguo Tian, Jin Jiang
    Research Advance

    Stem cell self-renewal often relies on asymmetric fate determination governed by niche signals and/or cell-intrinsic factors but how these regulatory mechanisms cooperate to promote asymmetric fate decision remains poorly understood. In adult Drosophila midgut, asymmetric Notch (N) signaling inhibits intestinal stem cell (ISC) self-renewal by promoting ISC differentiation into enteroblast (EB). We have previously shown that epithelium-derived Bone Morphogenetic Protein (BMP) promotes ISC self-renewal by antagonizing N pathway activity (Tian and Jiang, 2014). Here, we show that loss of BMP signaling results in ectopic N pathway activity even when the N ligand Delta (Dl) is depleted, and that the N inhibitor Numb acts in parallel with BMP signaling to ensure a robust ISC self-renewal program. Although Numb is asymmetrically segregated in about 80% of dividing ISCs, its activity is largely dispensable for ISC fate determination under normal homeostasis. However, Numb becomes crucial for ISC self-renewal when BMP signaling is compromised. Whereas neither Mad RNA interference nor its hypomorphic mutation led to ISC loss, inactivation of Numb in these backgrounds resulted in stem cell loss due to precocious ISC-to-EB differentiation. Furthermore, we find that numb mutations resulted in stem cell loss during midgut regeneration in response to epithelial damage that causes fluctuation in BMP pathway activity, suggesting that the asymmetrical segregation of Numb into the future ISC may provide a fail-save mechanism for ISC self-renewal by offsetting BMP pathway fluctuation, which is important for ISC maintenance in regenerative guts.