Foxn4 promotes gene expression required for multiple motile cilia formation

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. D ev el o pm en t • A dv an ce a rt ic le Introduction The multiciliated cell (MCC) is a specialized epithelial cell type that employs hundreds of motile cilia in order to produce robust fluid flow along lumenal surfaces (Brooks and Wallingford, 2014). To undergo multiple motile ciliation, MCC differentiation requires large changes in gene expression that enables large numbers of complex organelles to form (Quigley and Kintner, submitted). Analysis of this gene expression can provide insights into the macromolecular complexes required for cilia assembly and function as well as how motile cilia are impaired in human ciliopathies such as primary ciliary dyskinesia (PCD) (Choksi et al., 2014). Two related coiled-coil proteins, Multicilin and Gemc1, are both necessary and sufficient to initiate MCC differentiation, acting as co-regulators in a transcriptional complex with a subset of the E2F proteins (Arbi et al., 2016; Kyrousi et al., 2015; Ma et al., 2014; Stubbs et al., 2012; Terre et al., 2016; Zhou et al., 2015). The complex between Multicilin, E2f4 and DP1 (EDM) in particular appears to directly activate gene expression required for centriolar biogenesis (Ma et al., 2014), thus driving a novel form of organelle assembly that enables MCCs to form the hundreds of basal bodies required for multiple ciliation. Multicilin and Gemc1 also initiate gene expression required for motile cilium formation during MCC differentiation, but this occurs indirectly via their ability to activate the expression of Foxj1, a key regulator of genes required for motile axoneme extension in combination with the RFX factors (Choksi et al., 2014). Foxj1 is also required to induce a single motile cilium to form on cells located within a structure called the left-right organizer (LRO) in the early embryo (Brody et al., 2000; Stubbs et al., 2008). Thus, one transcriptional program (predominantly dependent on Foxj1) is required for motile cilium biogenesis regardless of whether this program operates in cells with one or many cilia while a second transcriptional program (predominantly dependent on Gemc1/Multicilin) is required specifically in MCCs for centriole biogenesis to increase cilia number. D ev el o pm en t • A dv an ce a rt ic le Current models do not fully explain how a MCC is capable of forming hundreds of motile cilia, not just the one cilium that Foxj1 induces at the LRO. Biogenesis of cilia can be sensitive to the levels of transcriptional activity driving cilia gene expression. For example, the length of cilia ectopically induced by Foxj1 is dependent on the levels of Foxj1 ectopically expressed (Stubbs et al., 2008). In addition, especially broad transcriptional activity may be required to drive the relatively large number of genes upregulated during MCC differentiation. For example, around 800 genes are markedly upregulated in differentiating MCCs whereas around 150 genes are upregulated in skin ionocytes, based on extensive RNAseq analysis of Xenopus skin progenitors (Quigley and Kintner, submitted). Thus gene expression required for multiple cilia formation may require high levels of Foxj1 and the RFX factor expression, uniquely driven to these levels in MCCs by Multicilin/Gemc1, acting directly or perhaps indirectly via Tp73 (Nemajerova et al., 2016). Alternatively, Multicilin/Gemc1 could facilitate Foxj1’s action in MCCs by activating additional regulators. One precedent for the latter possibility is that Multicilin activates the expression of Myb, which is clearly required for MCC differentiation in the lung (Tan et al., 2013). However, Myb targets and mechanism of action during MCC differentiation remain unclear, especially in light of the finding that it is also required for the differentiation of other lung cell types (Pan et al., 2014). Thus, additional regulators downstream of Multicilin/Gemc1 may be required to shape gene expression during MCC differentiation, to produce the relatively broad, high levels of gene expression required for multiple motile cilia to form. Here we identify and characterize foxn4, which encodes another forkhead transcription factor, as a gene strongly upregulated during MCC differentiation in the Xenopus larval skin. Although Foxn4 expression occurs in MCC progenitors in the mouse lung, the analysis of conditional Foxn4 mutants failed to detect a gross MCC phenotype (Hoh et al., 2012 ; Li and Xiang, 2011; Treutlein et al., 2014). We nonetheless examined Foxn4 function further in Xenopus skin MCCs, using both morpholino knockdown and D ev el o pm en t • A dv an ce a rt ic le Cas9/CRISPR mutagenesis. Both approaches produce the same phenotype where MCCs initiate centriole biogenesis but then show defects in basal body docking and cilia extension, similar to those observed in Foxj1 mutants. This cilia extension phenotype, however, mostly, but not completely, recovers over time, resulting in a marked subset of MCCs that are not fully differentiated. The RNAseq analysis of mutant phenotypes and the ChiPseq analysis of Foxn4 binding suggest that Foxn4 is present at MCC gene promoters where it regulates a subset of Foxj1 transcriptional targets strongly and many Foxj1 regulated genes weakly. Consistent with this co-regulation, disrupting both Foxn4 and Foxj1 with Cas9/CRISPR leads to an even more extreme phenotype than that observed when each is targeted alone. Finally, we show that Foxn4 is apparently not required for motile cilia formation in the Xenopus LRO where Foxj1 plays a critical role. Together, these findings indicate that Foxn4 functions to ensure robust gene expression during MCC differentiation, thus allowing large numbers of specialized organelles to be assembled in a timely fashion. D ev el o pm en t • A dv an ce a rt ic le