Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions
- National University of Singapore, Singapore
- 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
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
-
- 1,949
- views
-
- 244
- downloads
-
- 16
- 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)
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
Further reading
-
- Developmental Biology
Cells called alveolar myofibroblasts, which have a central role in the development of the lung after birth, receive an orchestrated input from a range of different signaling pathways.
-
- Developmental Biology
Premature infants with bronchopulmonary dysplasia (BPD) have impaired alveolar gas exchange due to alveolar simplification and dysmorphic pulmonary vasculature. Advances in clinical care have improved survival for infants with BPD, but the overall incidence of BPD remains unchanged because we lack specific therapies to prevent this disease. Recent work has suggested a role for increased transforming growth factor-beta (TGFβ) signaling and myofibroblast populations in BPD pathogenesis, but the functional significance of each remains unclear. Here, we utilize multiple murine models of alveolar simplification and comparative single-cell RNA sequencing to identify shared mechanisms that could contribute to BPD pathogenesis. Single-cell RNA sequencing reveals a profound loss of myofibroblasts in two models of BPD and identifies gene expression signatures of increased TGFβ signaling, cell cycle arrest, and impaired proliferation in myofibroblasts. Using pharmacologic and genetic approaches, we find no evidence that increased TGFβ signaling in the lung mesenchyme contributes to alveolar simplification. In contrast, this is likely a failed compensatory response, since none of our approaches to inhibit TGFβ signaling protect mice from alveolar simplification due to hyperoxia while several make simplification worse. In contrast, we find that impaired myofibroblast proliferation is a central feature in several murine models of BPD, and we show that inhibiting myofibroblast proliferation is sufficient to cause pathologic alveolar simplification. Our results underscore the importance of impaired myofibroblast proliferation as a central feature of alveolar simplification and suggest that efforts to reverse this process could have therapeutic value in BPD.
