Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants

Autoři: John Snedeker aff001;  William J. Gibbons, Jr. aff001;  David F. Paulding aff001;  Zakia Abdelhamed aff001;  Daniel R. Prows aff001;  Rolf W. Stottmann aff001
Působiště autorů: Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America aff001;  Neuroscience Graduate Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America aff002;  Department of Anatomy and Embryology, Faculty of Medicine (Girl’s Section), Al-Azhar University, Cairo, Egypt aff003;  Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America aff004;  Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America aff005;  Shriner’s Hospital for Children - Cincinnati, Cincinnati, Ohio, United States of America aff006
Vyšlo v časopise: Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants. PLoS Genet 15(11): e32767. doi:10.1371/journal.pgen.1008467
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008467


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.

Klíčová slova:

Alleles – Cilia – Embryos – Mammalian genomics – Molecular genetics – Phenotypes – Quantitative trait loci – Sequence motif analysis


1. Guemez-Gamboa A, Coufal NG, Gleeson JG. Primary cilia in the developing and mature brain. Neuron. 2014;82(3):511–21. Epub 2014/05/09. doi: 10.1016/j.neuron.2014.04.024 24811376.

2. Valente EM, Rosti RO, Gibbs E, Gleeson JG. Primary cilia in neurodevelopmental disorders. Nat Rev Neurol. 2014;10(1):27–36. Epub 2013/12/04. doi: 10.1038/nrneurol.2013.247 24296655.

3. Goetz SC, Anderson KV. The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet. 2010;11(5):331–44. Epub 2010/04/17. doi: 10.1038/nrg2774 20395968.

4. Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med. 2011;364(16):1533–43. Epub 2011/04/22. doi: 10.1056/NEJMra1010172 21506742.

5. Wheway G, Mitchison HM. Opportunities and Challenges for Molecular Understanding of Ciliopathies-The 100,000 Genomes Project. Front Genet. 2019;10:127. Epub 2019/03/28. doi: 10.3389/fgene.2019.00127 30915099.

6. Wang L, Dynlacht BD. The regulation of cilium assembly and disassembly in development and disease. Development. 2018;145(18). Epub 2018/09/19. doi: 10.1242/dev.151407 30224385.

7. Badano JL, Mitsuma N, Beales PL, Katsanis N. The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet. 2006;7:125–48. Epub 2006/05/26. doi: 10.1146/annurev.genom.7.080505.115610 16722803.

8. Han YG, Alvarez-Buylla A. Role of primary cilia in brain development and cancer. Curr Opin Neurobiol. 2010;20(1):58–67. Epub 2010/01/19. doi: 10.1016/j.conb.2009.12.002 20080044.

9. Reiter JF, Leroux MR. Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol. 2017;18(9):533–47. Epub 2017/07/13. doi: 10.1038/nrm.2017.60 28698599.

10. Wheway G, Nazlamova L, Hancock JT. Signaling through the Primary Cilium. Front Cell Dev Biol. 2018;6:8. Epub 2018/02/24. doi: 10.3389/fcell.2018.00008 29473038.

11. Schou KB, Pedersen LB, Christensen ST. Ins and outs of GPCR signaling in primary cilia. EMBO Rep. 2015;16(9):1099–113. Epub 2015/08/25. doi: 10.15252/embr.201540530 26297609.

12. Prevo B, Scholey JM, Peterman EJG. Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery. FEBS J. 2017;284(18):2905–31. Epub 2017/03/28. doi: 10.1111/febs.14068 28342295.

13. Fu W, Wang L, Kim S, Li J, Dynlacht BD. Role for the IFT-A Complex in Selective Transport to the Primary Cilium. Cell Rep. 2016;17(6):1505–17. Epub 2016/11/03. doi: 10.1016/j.celrep.2016.10.018 27806291.

14. Hirano T, Katoh Y, Nakayama K. Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein-coupled receptors. Mol Biol Cell. 2017;28(3):429–39. Epub 2016/12/10. doi: 10.1091/mbc.E16-11-0813 27932497.

15. Mukhopadhyay S, Wen X, Chih B, Nelson CD, Lane WS, Scales SJ, et al. TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia. Genes Dev. 2010;24(19):2180–93. Epub 2010/10/05. doi: 10.1101/gad.1966210 20889716.

16. Tran PV, Haycraft CJ, Besschetnova TY, Turbe-Doan A, Stottmann RW, Herron BJ, et al. THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia. Nat Genet. 2008;40(4):403–10. Epub 2008/03/11. doi: 10.1038/ng.105 18327258.

17. Huangfu D, Anderson KV. Cilia and Hedgehog responsiveness in the mouse. Proc Natl Acad Sci U S A. 2005;102(32):11325–30. Epub 2005/08/03. doi: 10.1073/pnas.0505328102 16061793.

18. Hui CC, Angers S. Gli proteins in development and disease. Annu Rev Cell Dev Biol. 2011;27:513–37. Epub 2011/08/02. doi: 10.1146/annurev-cellbio-092910-154048 21801010.

19. Stottmann RW, Tran PV, Turbe-Doan A, Beier DR. Ttc21b is required to restrict sonic hedgehog activity in the developing mouse forebrain. Dev Biol. 2009;335(1):166–78. Epub 2009/09/08. doi: 10.1016/j.ydbio.2009.08.023 19732765.

20. Doetschman T. Influence of genetic background on genetically engineered mouse phenotypes. Methods Mol Biol. 2009;530:423–33. Epub 2009/03/07. doi: 10.1007/978-1-59745-471-1_23 19266333.

21. Mackay TF. Epistasis and quantitative traits: using model organisms to study gene-gene interactions. Nat Rev Genet. 2014;15(1):22–33. Epub 2013/12/04. doi: 10.1038/nrg3627 24296533.

22. Herron BJ, Lu W, Rao C, Liu S, Peters H, Bronson RT, et al. Efficient generation and mapping of recessive developmental mutations using ENU mutagenesis. Nat Genet. 2002;30(2):185–9. Epub 2002/01/31. doi: 10.1038/ng812 11818962.

23. Dixon J, Dixon MJ. Genetic background has a major effect on the penetrance and severity of craniofacial defects in mice heterozygous for the gene encoding the nucleolar protein Treacle. Dev Dyn. 2004;229(4):907–14. Epub 2004/03/26. doi: 10.1002/dvdy.20004 15042714.

24. Hide T, Hatakeyama J, Kimura-Yoshida C, Tian E, Takeda N, Ushio Y, et al. Genetic modifiers of otocephalic phenotypes in Otx2 heterozygous mutant mice. Development. 2002;129(18):4347–57. Epub 2002/08/17. 12183386.

25. Mukhopadhyay P, Brock G, Webb C, Pisano MM, Greene RM. Strain-specific modifier genes governing craniofacial phenotypes. Birth Defects Res A Clin Mol Teratol. 2012;94(3):162–75. Epub 2012/03/01. doi: 10.1002/bdra.22890 22371338.

26. Percival CJ, Marangoni P, Tapaltsyan V, Klein O, Hallgrimsson B. The Interaction of Genetic Background and Mutational Effects in Regulation of Mouse Craniofacial Shape. G3 (Bethesda). 2017;7(5):1439–50. Epub 2017/03/11. doi: 10.1534/g3.117.040659 28280213.

27. Morgan AP, Fu CP, Kao CY, Welsh CE, Didion JP, Yadgary L, et al. The Mouse Universal Genotyping Array: From Substrains to Subspecies. G3 (Bethesda). 2015;6(2):263–79. Epub 2015/12/20. doi: 10.1534/g3.115.022087 26684931.

28. Broman KW, Sen S. A guide to QTL mapping with R/qtl. Dordrecht: Springer; 2009. xv, 396 p. p.

29. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4(7):1073–81. Epub 2009/06/30. doi: 10.1038/nprot.2009.86 19561590.

30. Greene ND, Stanier P, Copp AJ. Genetics of human neural tube defects. Hum Mol Genet. 2009;18(R2):R113–29. Epub 2009/10/08. doi: 10.1093/hmg/ddp347 19808787.

31. Bell SP. The origin recognition complex: from simple origins to complex functions. Genes Dev. 2002;16(6):659–72. Epub 2002/03/27. doi: 10.1101/gad.969602 11914271.

32. Schneider E, Ryan TJ. Gamma-glutamyl hydrolase and drug resistance. Clin Chim Acta. 2006;374(1–2):25–32. Epub 2006/07/25. doi: 10.1016/j.cca.2006.05.044 16859665.

33. Ma S, Kwon HJ, Johng H, Zang K, Huang Z. Radial glial neural progenitors regulate nascent brain vascular network stabilization via inhibition of Wnt signaling. PLoS Biol. 2013;11(1):e1001469. Epub 2013/01/26. doi: 10.1371/journal.pbio.1001469 23349620.

34. Wheway G, Schmidts M, Mans DA, Szymanska K, Nguyen TT, Racher H, et al. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nat Cell Biol. 2015;17(8):1074–87. Epub 2015/07/15. doi: 10.1038/ncb3201 26167768.

35. Kawasawa Y, Kume K, Nakade S, Haga H, Izumi T, Shimizu T. Brain-specific expression of novel G-protein-coupled receptors, with homologies to Xenopus PSP24 and human GPR45. Biochem Biophys Res Commun. 2000;276(3):952–6. Epub 2000/10/12. doi: 10.1006/bbrc.2000.3569 11027574.

36. Lee DK, George SR, Cheng R, Nguyen T, Liu Y, Brown M, et al. Identification of four novel human G protein-coupled receptors expressed in the brain. Brain Res Mol Brain Res. 2001;86(1–2):13–22. Epub 2001/02/13. doi: 10.1016/s0169-328x(00)00242-4 11165367.

37. Michelsen K, Yuan H, Schwappach B. Hide and run. Arginine-based endoplasmic-reticulum-sorting motifs in the assembly of heteromultimeric membrane proteins. EMBO Rep. 2005;6(8):717–22. Epub 2005/08/03. doi: 10.1038/sj.embor.7400480 16065065.

38. Bockaert J, Marin P, Dumuis A, Fagni L. The ‘magic tail’ of G protein-coupled receptors: an anchorage for functional protein networks. FEBS Lett. 2003;546(1):65–72. Epub 2003/06/28. doi: 10.1016/s0014-5793(03)00453-8 12829238.

39. Duden R. ER-to-Golgi transport: COP I and COP II function (Review). Mol Membr Biol. 2003;20(3):197–207. Epub 2003/08/02. doi: 10.1080/0968768031000122548 12893528.

40. McMahon HT, Mills IG. COP and clathrin-coated vesicle budding: different pathways, common approaches. Curr Opin Cell Biol. 2004;16(4):379–91. Epub 2004/07/21. doi: 10.1016/ 15261670.

41. Yuan H, Michelsen K, Schwappach B. 14-3-3 dimers probe the assembly status of multimeric membrane proteins. Curr Biol. 2003;13(8):638–46. Epub 2003/04/18. doi: 10.1016/s0960-9822(03)00208-2 12699619.

42. Zerangue N, Malan MJ, Fried SR, Dazin PF, Jan YN, Jan LY, et al. Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells. Proc Natl Acad Sci U S A. 2001;98(5):2431–6. Epub 2001/02/28. doi: 10.1073/pnas.051630198 11226256.

43. Kundu K, Backofen R. Cluster based prediction of PDZ-peptide interactions. BMC Genomics. 2014;15 Suppl 1:S5. Epub 2014/02/26. doi: 10.1186/1471-2164-15-S1-S5 24564547.

44. Kundu K, Costa F, Backofen R. A graph kernel approach for alignment-free domain-peptide interaction prediction with an application to human SH3 domains. Bioinformatics. 2013;29(13):i335–43. Epub 2013/07/03. doi: 10.1093/bioinformatics/btt220 23813002.

45. Kundu K, Mann M, Costa F, Backofen R. MoDPepInt: an interactive web server for prediction of modular domain-peptide interactions. Bioinformatics. 2014;30(18):2668–9. Epub 2014/05/30. doi: 10.1093/bioinformatics/btu350 24872426.

46. Saksela K, Permi P. SH3 domain ligand binding: What’s the consensus and where’s the specificity? FEBS Lett. 2012;586(17):2609–14. Epub 2012/06/20. doi: 10.1016/j.febslet.2012.04.042 22710157.

47. Klink BU, Zent E, Juneja P, Kuhlee A, Raunser S, Wittinghofer A. A recombinant BBSome core complex and how it interacts with ciliary cargo. Elife. 2017;6. Epub 2017/11/24. doi: 10.7554/eLife.27434 29168691.

48. Kroeze WK, Sassano MF, Huang XP, Lansu K, McCorvy JD, Giguere PM, et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat Struct Mol Biol. 2015;22(5):362–9. Epub 2015/04/22. doi: 10.1038/nsmb.3014 25895059.

49. Drinkwater NR, Gould MN. The long path from QTL to gene. PLoS Genet. 2012;8(9):e1002975. Epub 2012/10/11. doi: 10.1371/journal.pgen.1002975 23049490.

50. Flint J, Valdar W, Shifman S, Mott R. Strategies for mapping and cloning quantitative trait genes in rodents. Nat Rev Genet. 2005;6(4):271–86. Epub 2005/04/02. doi: 10.1038/nrg1576 15803197.

51. Morgan AP. argyle: An R Package for Analysis of Illumina Genotyping Arrays. G3 (Bethesda). 2015;6(2):281–6. Epub 2015/12/20. doi: 10.1534/g3.115.023739 26684930.

Genetika Reprodukční medicína

Článek vyšel v časopise

PLOS Genetics

2019 Číslo 11

Nejčtenější v tomto čísle
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se