Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants
John Snedeker1,2, William J Gibbons1, David F Paulding1
1Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
Abstract:
The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.
Insights
Genetic background significantly impacts Ttc21b-related microcephaly. Researchers identified Gpr63 as a modifier gene, validating its role in embryonic development and ciliopathies.
Area of Science:
- Genetics
- Developmental Biology
- Cell Biology
Background:
- The primary cilium is vital for embryonic development, acting as a signaling center.
- Ttc21b mutations cause organogenesis defects, including microcephaly, with severity influenced by genetic background.
- FVB/NJ mice exhibit smaller forebrains compared to C57BL/6J mice when Ttc21b is mutated.
Purpose of the Study:
- To identify genetic modifiers influencing Ttc21b-associated microcephaly.
- To investigate the role of Gpr63 as a potential modifier gene.
- To validate Gpr63's function in primary cilia and its interaction with Ttc21b.
Main Methods:
- Quantitative Trait Locus (QTL) analysis to map modifier genes.
- Congenic strain construction for QTL validation.
- CRISPR-Cas9 genome editing to create specific Gpr63 alleles in mice.
- In vitro ciliary localization assays for Gpr63.
Main Results:
- Two major QTLs, Moaq1 and Moaq2, were identified on chromosomes 4 and 6, respectively.
- Gpr63 was identified as a candidate gene within the Moaq1 locus, with a strain-specific missense mutation.
- Engineered Gpr63 alleles altered ciliary localization and genetically interacted with Ttc21b, causing spina bifida aperta and increased embryonic lethality.
Conclusions:
- Gpr63 is a key modifier of Ttc21b-associated neural phenotypes.
- Gpr63 functions within the primary cilium and plays a role in embryonic development.
- Gpr63 is validated as a quantitative trait gene (QTG) within the Moaq1 locus.
