Foxn4 promotes gene expression required for multiple motile cilia formation

Abstract

Multiciliated cell (MCC) differentiation involves extensive organelle biogenesis required to extend hundreds of motile cilia. Key transcriptional regulators have been identified that drive gene expression required for organelle biogenesis during MCC differentiation, which are activated by, and act downstream, of two related, small coiled-coil proteins, Multicilin and Gemc1. Here we identify foxn4 as a new downstream target of Multicilin required for MCC differentiation in the Xenopus skin. When Foxn4 activity is inhibited in Xenopus embryos, MCCs show transient ciliogenesis defects similar to those observed in mutants in Foxj1, a known key regulator of genes required for motile ciliation. RNAseq analysis indicates that Foxn4 is required to co-activate some Foxj1 target genes strongly and many Foxj1 targets weakly. ChIPseq suggests that while Foxn4 and Foxj1 frequently bind to different targets at distal enhancers, they largely bind together at MCC gene promoters. Consistent with this co-regulation, cilia extension by MCCs is more severely compromised in foxn4 and foxj1 double mutants compared to single mutants. Finally, in contrast to Foxj1, Foxn4 is not required to extend a single motile cilium by cells involved in left-right patterning. Together these results indicate that Foxn4 complements Foxj1 transcriptionally during MCC differentiation, thereby shaping the levels of gene expression required for the timely and complete biogenesis of cilia in large numbers.