The vacuolated lens (vl) mouse mutant arose spontaneously on the C3H/HeSn background and exhibits neural tube defects (NTDs), congenital cataract and occasionally a white belly spot. We previously reported: i) the vl phenotypes are due to a mutation in an orphan G protein coupled receptor (GPCR), Gpr161; ii) the penetrance of the vl NTD and cataract phenotypes are affected by genetic background, allowing three unlinked QTL to be mapped (modifiers of vacuolated lens, Modvl1-3); and iii) phenotype based bioinformatics followed by genetic and molecular analysis identified a lens-specific transcription factor that contributes to the cataract modifying effect of Modvl3. We now extend this analysis in three ways. One, using the Gpr161 mutation to unequivocally identify mutant adults and embryos, we determined ~50% of vl/vl NTD affected embryos die during development. Two, the MOLF/Ei genetic background suppresses this embryonic lethality but increases the incidence of the adult belly spot phenotype. Additional QTL analysis was performed and two novel modifiers were mapped (Modvl4-LOD 4.4; Modvl5-LOD 5.0). Three, phenotype based bioinformatics identified candidate genes for these modifiers including two GPCRs that cause NTD or skin/pigmentation defects (Modvl4: Frizzled homolog 6; Modvl5: Melanocortin 5 receptor). Because GPCRs form oligomeric complexes, these genes were re-sequenced and non-synonymous coding variants were identified. Bioinformatics and protein modeling suggest these variants may be functional. Our studies further establish vl as a multigenic mouse model for NTDs and identify additional QTL that interact with Gpr161 to regulate neurulation.