Cilia are specialized organelles that extend from plasma membrane, functioning as antennas for signal transduction and are involved in embryonic morphogenesis. Dysfunction of cilia lead to many developmental defects, including neural tube defects (NTDs). HeterodimerWDR60-WDR34(WD repeat domain 60 and 34) are intermediate chains of motor protein dynein-2, which play important roles in ciliary retrograde transport. It has been reported that disruption ofWdr34in mouse model results in NTDs and defects of Sonic Hedgehog (SHH) signaling. However, noWdr60deficiency mouse model has been reported yet. In this study, piggyBac (PB) transposon is used to interfereWdr60andWdr34expression respectively to establishWdr60PB/PBandWdr34PB/PBmouse models. We found that the expression ofWdr60orWdr34is significantly decreased in the homozygote mice.Wdr60homozygote mice die around E13.5 to E14.5, whileWdr34homozygote mice die around E10.5 to E11.5.WDR60is highly expressed in the head region at E10.5 andWdr60PB/PBembryos have head malformation. RNAseq and qRT-PCR experiments revealed that Sonic Hedgehog signaling is also downregulated inWdr60PB/PBhead tissue, demonstrating that WDR60 is also required for promoting SHH signaling. Further experiments on mouse embryos also revealed that the expression levels of planar cell polarity (PCP) components such as CELSR1 and downstream signal molecule c-Jun were downregulated inWDR34homozygotes compared to wildtype littermates. Coincidently, we observed much higher ratio of open cranial and caudal neural tube inWdr34PB/PBmice. CO-IP experiment showed thatWDR60andWDR34both interact with IFT88, but onlyWDR34interacts with IFT140. Taken together,WDR60andWDR34play overlapped and distinct functions in modulating neural tube development.