The G protein-coupled receptor Gpr161 regulates forelimb formation, limb patterning and skeletal morphogenesis in a primary cilium-dependent manner

Abstract

The role of basal suppression of the sonic hedgehog (Shh) pathway and its interaction with Indian hedgehog (Ihh) signaling during limb/skeletal morphogenesis is not well understood. The orphan G protein-coupled receptor Gpr161 localizes to primary cilia and functions as a negative regulator of Shh signaling by promoting Gli transcriptional repressor versus activator formation. Here, we show that forelimb buds are not formed in knockout mouse embryos despite establishment of prospective limb fields. Limb-specific deletion of resulted in prematurely expanded Shh signaling and ectopic Shh-dependent patterning defects resulting in polysyndactyly. In addition, endochondral bone formation in forearms, including formation of both trabecular bone and bone collar was prevented. Endochondral bone formation defects resulted from accumulation of proliferating round/periarticular-like chondrocytes, lack of differentiation into columnar chondrocytes, and corresponding absence of Ihh signaling. deficiency in craniofacial mesenchyme also prevented intramembranous bone formationin calvarium. Defects in limb patterning, endochondral and intramembranous skeletal morphogenesis were suppressed in the absence of cilia. Overall, Gpr161 promotes forelimb formation, regulates limb patterning, prevents periarticular chondrocyte proliferation and drives osteoblastogenesis in intramembranous bones in a cilium-dependent manner.

BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone

“…S1J). The decreased threshold of detection when 9 pulse-labeling with mSA647 ensures a more faithful visualization of exit kinetics than when exit 10 is monitored by immunostaining ( Fig. 1B and F).…”

“…7E, Movie S5). No co-movement was observed 9 between the faint BBSome trains and SSTR3 in the absence of Arl6 ( Fig. 7E and S3D).…”

“…We note that PKA 7 inhibition was not sufficient to trigger retrieval of GPR161 or SSTR3 in the absence of receptor 8 stimulation ( Fig. 4C-D), suggesting the existence of mechanisms that act non-redundantly with 9 the Gα i -PKA-BBSome axis (e.g. β -arrestin 2).…”

“…GPR161 and the 9 prototypical ciliary GPCR somatostatin receptor 3 (SSTR3) both undergo retrieval from the (Fig. 1A).…”

“…GPCRs were tagged on the FlpIn locus, agonist-dependent exit of SSTR3 from cilia was undetectable. Molecular counting 7 of GFP and comparison of immunofluorescence intensities revealed that pEF1α-driven 8 expression resulted in SSTR3 levels that were an order of magnitude greater than in neurons 9 ( Fig. 1C, …”