The orphan GPCR, Gpr161, regulates the retinoic acid and canonical Wnt pathways during neurulation

highlights
• Modvl5MOLF is a genetic locus that rescues lethality and NTDs in Gpr161vl/vl.
• Bioinformatics identified Cdx1 as a QTG that contributes to the Modvl5 locus.
• Shh signaling and Gpr161 ciliary localization are not severely affected in Gpr161vl/vl.
• RA and canonical Wnt signaling are downstream to Gpr161 in regulating neurulation.
• Modvl5MOLF bypasses Gpr161vl by restoring the activity of RA and Wnt signaling.

The vacuolated lens (vl) mouse mutation arose on the C3H/HeSnJ background and results in lethality, neural tube defects (NTDs) and cataracts. The vl phenotypes are due to a deletion/frameshift mutation in the orphan GPCR, Gpr161. A recent study using a null allele demonstrated that Gpr161 functions in primary cilia and represses the Shh pathway. We show the hypomorphic Gpr161vl allele does not severely affect the Shh pathway. To identify additional pathways regulated by Gpr161 during neurulation, we took advantage of naturally occurring genetic variation in the mouse. Previously Gpr161vl-C3H was crossed to different inbred backgrounds including MOLF/EiJ and the Gpr161vl mutant phenotypes were rescued. Five modifiers were mapped (Modvl: Modifier of vl) including Modvl5MOLF. In this study we demonstrate the Modvl5MOLF congenic rescues the Gpr161vl-associated lethality and NTDs but not cataracts. Bioinformatics determined the transcription factor, Cdx1, is the only annotated gene within the Modvl5 95% CI co-expressed with Gpr161 during neurulation and not expressed in the eye. Using Cdx1 as an entry point, we identified the retinoid acid (RA) and canonical Wnt pathways as downstream targets of Gpr161. QRT-PCR, ISH and IHC determined that expression of RA and Wnt genes are down-regulated in Gpr161vl/vl but rescued by the Modvl5MOLF congenic during neurulation. Intraperitoneal RA injection restores expression of canonical Wnt markers and rescues Gpr161vl/vl NTDs. These results establish the RA and canonical Wnt as pathways downstream of Gpr161 during neurulation, and suggest that Modvl5MOLF bypasses the Gpr161vl mutation by restoring the activity of these pathways.