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.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Embryonic Development/genetics , Microcephaly/genetics , Quantitative Trait Loci/genetics , Receptors, G-Protein-Coupled/genetics , Alleles , Animals , CRISPR-Cas Systems/genetics , Chromosome Mapping , Cilia/genetics , Embryo, Mammalian , Genotype , Humans , Mice , Mice, Inbred C57BL , Microcephaly/physiopathology , Prosencephalon/growth & development , Prosencephalon/metabolism , Spina Bifida Cystica/genetics , Spina Bifida Cystica/physiopathology , Synthetic Lethal Mutations/geneticsABSTRACT
Primary cilia are nearly ubiquitous, cellular projections that function to transduce molecular signals during development. Loss of functional primary cilia has a particularly profound effect on the developing craniofacial complex, causing several anomalies including craniosynostosis, micrognathia, midfacial dysplasia, cleft lip/palate and oral/dental defects. Development of the craniofacial complex is an intricate process that requires interactions between several different tissues including neural crest cells, neuroectoderm and surface ectoderm. To understand the tissue-specific requirements for primary cilia during craniofacial development we conditionally deleted three separate intraflagellar transport genes, Kif3a, Ift88 and Ttc21b with three distinct drivers, Wnt1-Cre, Crect and AP2-Cre which drive recombination in neural crest, surface ectoderm alone, and neural crest, surface ectoderm and neuroectoderm, respectively. We found that tissue-specific conditional loss of ciliary genes with different functions produces profoundly different facial phenotypes. Furthermore, analysis of basic cellular behaviors in these mutants suggests that loss of primary cilia in a distinct tissue has unique effects on development of adjacent tissues. Together, these data suggest specific spatiotemporal roles for intraflagellar transport genes and the primary cilium during craniofacial development.
Subject(s)
Craniofacial Abnormalities/genetics , Face/embryology , Gene Expression Regulation, Developmental , Skull/embryology , Adaptor Proteins, Signal Transducing/genetics , Animals , Cilia/genetics , Face/abnormalities , Female , Gene Deletion , Kinesins/genetics , Male , Mice , Neural Crest/embryology , Neural Crest/metabolism , Neural Plate/embryology , Neural Plate/metabolism , Skull/abnormalities , Skull/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Primary cilia are organelles extended from virtually all cells and are required for the proper regulation of a number of canonical developmental pathways. The role in cortical development of proteins important for ciliary form and function is a relatively understudied area. Here we have taken a genetic approach to define the role in forebrain development of three intraflagellar transport proteins known to be important for primary cilia function. We have genetically ablated Kif3a, Ift88, and Ttc21b in a series of specific spatiotemporal domains. The resulting phenotypes allow us to draw several conclusions. First, we conclude that the Ttc21b cortical phenotype is not due to the activity of Ttc21b within the brain itself. Secondly, some of the most striking phenotypes are from ablations in the neural crest cells and the adjacent surface ectoderm indicating that cilia transduce critical tissue-tissue interactions in the developing embryonic head. Finally, we note striking differences in phenotypes from ablations only one embryonic day apart, indicating very discrete spatiotemporal requirements for these three genes in cortical development.
