Abstract
Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.
Highlights
The Wnt signaling network is classified into b-catenindependent and b-catenin-independent pathways [1,2,3]. b-catenin-dependent Wnt signaling acts through the stabilization of bcatenin and subsequent transcriptional activation of b-catenin targets [4], whereas b-catenin-independent Wnt signaling influences cell polarity. b-catenin-independent Wnt signaling has been shown to lead to calcium (Ca2+) release as well as activation of Rac, Rho and other cytoskeletal components in vertebrates [5,6]
We combine cellular, molecular, and genetic analyses with high resolution calcium imaging to identify a role for Regulator of G protein Signaling (RGS) modulation of Wnt-mediated calcium release dynamics and developmental patterning events
We find that loss of rgs3 gene function produced body patterning defects like those observed with loss of wnt5b gene function
Summary
The Wnt signaling network is classified into b-catenindependent and b-catenin-independent pathways [1,2,3]. b-catenin-dependent Wnt signaling acts through the stabilization of bcatenin and subsequent transcriptional activation of b-catenin targets [4], whereas b-catenin-independent Wnt signaling influences cell polarity (known as Planar Cell Polarity or PCP in Drosophila). b-catenin-independent Wnt signaling has been shown to lead to calcium (Ca2+) release as well as activation of Rac, Rho and other cytoskeletal components in vertebrates [5,6]. The Wnt signaling network is classified into b-catenindependent and b-catenin-independent pathways [1,2,3]. B-catenin-independent Wnt signaling has been shown to lead to calcium (Ca2+) release as well as activation of Rac, Rho and other cytoskeletal components in vertebrates [5,6]. Wnt is enriched in the anterior and mutants show anterior extension and eye fusion defects, while Wnt5b is enriched in the posterior and mutants show altered cell movements during gastrulation, often resulting in convergence extension and somite defects [9,10,11]. Zebrafish embryos demonstrate Ca2+ release dynamics during epiboly, gastrulation, convergent extension and organogenesis [12,13,14,15,16,17,18,19,20,21]. The zebrafish wnt5b genetic mutant (pipetail) shows reduced Ca2+ release [24] and the ventralized maternal effect mutant hecate shows ectopic Ca2+
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