Regulatory factor X (RFX)-family proteins are transcription factors involved in ciliogenesis in worms, flies and mice. In mice, it has been shown that RFX3 regulates the growth of primary cilia. However, further investigation of the biological role of RFX3 has been a challenge because Rfx3–/– mouse embryos die at birth, and ciliogenesis is completed only postnatally. Bénédicte Durand and colleagues () now provide new evidence that RFX3 also has a crucial role in the biogenesis of motile cilia. Here, they describe a novel primary-cell culture system that allows multiciliated ependymal cells to be differentiated from E18.5 neural stem cells in vitro. Using this system, they show that Rfx3–/– cultures exhibit a marked decrease in the number of multiciliated cells compared with wild-type cultures, and that cilia in Rfx3–/– cultures are significantly shorter. Furthermore, ciliary beating efficiency is defective in Rfx3–/– cultures, indicating that RFX3 is required for both ciliary biogenesis and motility. The authors then use PCR and ChIP experiments to show that RFX3 is required for the optimal expression of genes involved in ciliary assembly and motility. As Rfx3–/– cultures also show a modest decrease in the expression of FOXJ1, another transcription factor thought to be central for ciliogenesis, the authors propose that RFX3 and FOXJ1 might work together to regulate the expression of other ciliary genes.
