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.
Vertebrate development requires communication among cells in order to define the body axis (front/back, head/tail, or left/right). Secreted factors such as Wnts play key roles in a vast array of cellular processes, including patterning of the body axis. One arm of the Wnt-signaling network, the non-canonical 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 GAPs and are uniquely situated to control the amplitude of a Wnt signal. Here, we combine cellular, molecular, and genetic analyses with high resolution calcium imaging to identify a role for 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. Analysis of endogenous calcium release dynamics in developing zebrafish revealed that both rgs3 and wnt5b are required for appropriate frequency and amplitude of calcium release. Our results provide new evidence that a member of the RGS protein family is essential for modulating the non-canonical Wnt network to assure normal tissue patterning during development.