To investigate how intraflagellar transport protein 122 (IFT122) regulates murine embryonic palate mesenchymal cell (mEPMC) proliferation via the Sonic Hedgehog (Shh) pathway and its dependency on primary cilia integrity, elucidating mechanisms underlying cleft palate pathogenesis.
Design
Intraflagellar transport protein 122 was silenced in E14.5-derived mEPMCs using lentiviral shRNA. Primary cilia morphology (immunofluorescence), Shh signaling components (Smo, Gli3 via qPCR/Western blot), and proliferation markers (CCK-8 assay, Proliferating Cell Nuclear Antigen [PCNA], Cyclin D1) were analyzed. Rescue experiments employed the Smoothened agonist (SAG) to activate Shh signaling.
Main Outcome Measure
Cilia incidence and length; Smo/Gli3 mRNA and protein expression; Gli3A/Gli3R ratio; cell proliferation rates (OD values, PCNA, Cyclin D1); SAG-mediated rescue effects.
Results
Intraflagellar transport protein 122 knockdown reduced cilia incidence (10.2% vs 2.4%, P < .01) and length (7.8 μm vs 3.4 μm, P < .01), impaired Smo trafficking (mRNA↓42%, protein↓50%), suppressed Gli3A/Gli3R ratio (↓62%), and inhibited proliferation (PCNA/Cyclin D1↓40%-60%, P < .01). Smoothened agonist partially restored Smo expression (↑1.8×), Gli3 activation, and proliferation (P < .05), confirming cilia-dependent Shh regulation.
Conclusion
Intraflagellar transport protein 122 maintains primary cilia structure to enable Shh-driven mEPMC proliferation. Its deficiency disrupts cilia integrity and Shh signaling, linking ciliary dysfunction to cleft palate. Partial rescue by SAG validates the mechanism, highlighting therapeutic potential for targeting cilia-Shh axis defects.
EversonJLFinkDMChungHMSunMR LipinskiRJ.Identification of sonic hedgehog-regulated genes and biological processes in the cranial neural crest mesenchyme by comparative transcriptomics. BMC Genomics. 2018. doi:https://doi.org/10.1186/s12864-018-4885-5
AnvarianZMykytynKMukhopadhyaySPedersenLBChristensenST.Cellular signalling by primary cilia in development, organ function and disease. Nat Rev Nephrol. 2019;15(4):199-219. doi:https://doi.org/10.1038/s41581-019-0116-9
9.
SchouKBPedersenLBChristensenST. Ins and outs of GPCR signaling in primary cilia. EMBO Rep. 2015;16(9):1099-1113. doi:https://doi.org/10.15252/embr.201540530
10.
AlsolamiMKuhnsSAlsulamiMBlacqueOE.ERICH3 in primary cilia regulates cilium formation and the localisations of ciliary transport and sonic hedgehog signaling proteins. Sci Rep. 2019;9(1):16519. doi:https://doi.org/10.1038/s41598-019-52830-1
NachuryMVLoktevAVZhangQWestlakeCJPeränenJMerdesASlusarskiDCSchellerRHBazanZFSheffieldVC, et al.A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 2007;129(6):1201-1213. doi:https://doi.org/10.1016/j.cell.2007.03.053
13.
PedersenLBRosenbaumJL. Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling. Curr Top Dev Biol. 2008;85:23-61. doi:https://doi.org/10.1016/S0070-2153(08)00802-8
MeleppattuSZhouHDaiJGuiMBrownA.Mechanism of IFT-A polymerization into trains for ciliary transport. Cell. 2022;185(26):4986-4998.e12. doi:https://doi.org/10.1016/j.cell.2022.11.033
16.
NakayamaKKatohY. Ciliary protein trafficking mediated by IFT and BBSome complexes with the aid of kinesin-2 and dynein-2 motors. J Biochem. 2018;163(3):155-164. doi:https://doi.org/10.1093/jb/mvx087
17.
MillerKAAh-CannCJWelfareMFTanTYPopeKCaruanaGFreckmannMLSavarirayanRBertramJFDobbieM, et al.Cauli: a mouse strain with an Ift140 mutation that results in a skeletal ciliopathy modelling Jeune syndrome. PLoS Genet. 2013;9(8):e1003746. doi:https://doi.org/10.1371/journal.pgen.1003746
18.
LiemKFJrAsheAHeMSatirPMoranJBeierDWickingCAndersonKV.The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking. J Cell Biol. 2012;197(6):789-800. doi:https://doi.org/10.1083/jcb.201110049
19.
TakaharaMKatohYNakamuraKHiranoTSugawaMTsurumiYNakayamaK.Ciliopathy-associated mutations of IFT122 impair ciliary protein trafficking but not ciliogenesis. Hum Mol Genet. 2018;27(3):516-528. doi:https://doi.org/10.1093/hmg/ddx421
20.
AlazamiAMSeidahmedMZAlzahraniF, Mohammed AO, Alkuraya FS.Novel IFT122 mutation associated with impaired ciliogenesis and cranioectodermal dysplasia. Mol Genet Genomic Med. 2014;2(2):103-106. doi:https://doi.org/10.1002/mgg3.44
21.
QinJLinYNormanRXKoHWEggenschwilerJT.Intraflagellar transport protein 122 antagonizes sonic hedgehog signaling and controls ciliary localization of pathway components. Proc Natl Acad Sci U S A. 2011;108(4):1456-1461. doi:https://doi.org/10.1073/pnas.1011410108
22.
HiranoTKatohYNakayamaK. Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein-coupled receptors. Mol Biol Cell. 2017;28(3):429-439. doi:https://doi.org/10.1091/mbc.E16-11-0813
23.
BriscoeJThérondPP. The mechanisms of hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol. 2013;14(7):416-429. doi:https://doi.org/10.1038/nrm3598
24.
Walczak-SztulpaJEggenschwilerJOsbornDBrownDAEmmaFKlingenbergCHennekamRCTorreGGarshasbiMTzschachA, et al.Cranioectodermal dysplasia, sensenbrenner syndrome, is a ciliopathy caused by mutations in the IFT122 gene. Am J Hum Genet. 2010;86(6):949-956. doi:https://doi.org/10.1016/j.ajhg.2010.04.012
WangQKurosakaHKikuchiMNakayaATrainorPAYamashiroT.Perturbed development of cranial neural crest cells in association with reduced sonic hedgehog signaling underlies the pathogenesis of retinoic-acid-induced cleft palate. Dis Model Mech. 2019;12(10):dmm040279. doi:https://doi.org/10.1242/dmm.040279
28.
ShinJOSongJChoiHSLeeJLeeKKoHWBokJ.Activation of sonic hedgehog signaling by a smoothened agonist restores congenital defects in mouse models of endocrine-cerebro-osteodysplasia syndrome. EBioMedicine. 2019;49:305-317. doi:https://doi.org/10.1016/j.ebiom.2019.10.016
29.
MukhopadhyaySWenXChihBNelsonCDLaneWSScalesSJJacksonPK.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):2180-2193. doi:https://doi.org/10.1101/gad.1966210
30.
BaeYKKimGHKwonJHKimMChoiSJOhWUmSJinHJ.Primary cilia mediate Wnt5a/β-catenin signaling to regulate adipogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells following calcium induction. Tissue Eng Regen Med. 2020;17(2):193-202. doi:https://doi.org/10.1007/s13770-019-00237-4
