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.
Highlights
Primary cilia are microtubule-based organelles known to play essential roles in proper development and function of a number of organ systems including the central nervous system (CNS) [1,2,3,4]
Mice homozygous for a null allele of Ttc21b have a spectrum of ciliopathy phenotypes, including microcephaly
Further work has shown that the severity of the microcephaly significantly depends on the genetic background of the mouse model
Summary
Primary cilia are microtubule-based organelles known to play essential roles in proper development and function of a number of organ systems including the central nervous system (CNS) [1,2,3,4]. Ciliopathies are a class of human diseases caused by pathogenic variants in genes encoding proteins responsible for the proper form and function of cilia [5,6,7]. The frequent presentation of cognitive impairment in ciliopathy patients, in addition to severely compromised brain development in a number of ciliary mutant mouse models, clearly displays the importance of primary cilia in CNS health and development [8, 9]. IFT-A proteins have been shown to play a role in the trafficking of select GPCRs in and out of the cilium [13,14,15]
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