AbstractPrimary cilia are sensory organelles that are essential for eukaryotic development and health. These antenna-like structures are synthesized by intraflagellar transport protein complexes, IFT-B and IFT-A, which mediate bi-directional protein trafficking along the ciliary axoneme. Here using mouse embryonic fibroblasts (MEF), we investigate the ciliary roles of two mammalian orthologues ofChlamydomonasIFT-A gene,IFT139, namelyThm1(also known asTtc21b) andThm2 (Ttc21a). Thm1loss causes perinatal lethality, andThm2loss allows survival into adulthood. At E14.5, the number ofThm1;Thm2double mutant embryos is lower than that for a Mendelian ratio, indicating deletion ofThm1andThm2causes mid-gestational lethality. We examined the ciliary phenotypes of mutant MEF.Thm1-mutant MEF show decreased cilia assembly, shortened primary cilia, a retrograde IFT defect for IFT and BBS proteins, and reduced ciliary entry of membrane-associated proteins.Thm1-mutant cilia also show a retrograde transport defect for the Hedgehog transducer, Smoothened, and an impaired response to Smoothened agonist, SAG.Thm2-null MEF show normal ciliary dynamics and Hedgehog signaling, but additional loss of aThm1allele impairs response to SAG. Further,Thm1;Thm2double mutant MEF show enhanced cilia disassembly, and relative toThm1-null MEF, increased impairment of IFT81 retrograde transport and of INPP5E ciliary import. Thus,Thm1andThm2have unique and redundant roles in MEF.Thm1regulates cilia assembly, and together withThm2, cilia disassembly. Moreover,Thm1alone and together withThm2, regulates ciliary protein trafficking, Hedgehog signaling, and embryogenesis. These findings shed light on mechanisms underlyingThm1-,Thm2- or IFT-A-mediated ciliopathies.