Abstract
Wnt3 proteins are lipidated and glycosylated 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.
Highlights
Wnt proteins represent a family of secreted signaling glycoproteins having multiple functions in embryonic development such as specification of the vertebrate axis, embryonic induction, maintenance of cell potency, cell fate determination, cell migration, cell division, and apoptosis, to name a few (Clevers and Nusse, 2012; Hikasa and Sokol, 2013; Logan and Nusse, 2004; Moon et al, 2002)
Since the 4 kb wnt3 promoter contains most of the regulatory elements (Teh et al, 2015), Tg(À4.0wnt3:PMTmApple) is a faithful reporter of Wnt3 expression, which marks the plasma membrane of the Wnt3-producing cells
The localization of PMTmApple is restricted to its source cells, as it remains tethered to the inner leaflet of the plasma membrane
Summary
Wnt proteins represent a family of secreted signaling glycoproteins having multiple functions in embryonic development such as specification of the vertebrate axis, embryonic induction, maintenance of cell potency, cell fate determination, cell migration, cell division, and apoptosis, to name a few (Clevers and Nusse, 2012; Hikasa and Sokol, 2013; Logan and Nusse, 2004; Moon et al, 2002). Wnts are hydrophobic and water insoluble due to their post-translational lipidation in the endoplasmic reticulum (ER) (Mikels and Nusse, 2006). Porcupine (Porc), an O-acyltransferase localized on the membrane of the ER, catalyzes the acylation of Wnts and provides Wnts hydrophobic characteristics (Herr and Basler, 2012). The acylation facilitates the interaction of Wnts with Wntless, a transmembrane protein that shuttles Wnts to the plasma membrane (Galli et al, 2007). They are secreted and transported to Wnt-receiving cells. The acylation of Wnts is a critical step for their trafficking, secretion, and activity (Coudreuse and Korswagen, 2007)
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