Foxn4 promotes gene expression required for the formation of multiple motile cilia

Abstract

Multiciliated cell (MCC) differentiation involves extensive organelle biogenesis required to extend hundreds of motile cilia. Key transcriptional regulators known to drive the gene expression required for this organelle biogenesis are activated by the related coiled-coil proteins Multicilin and Gemc1. Here we identify foxn4 as a new downstream target of Multicilin required for MCC differentiation in Xenopus skin. When Foxn4 activity is inhibited in Xenopus embryos, MCCs show transient ciliogenesis defects similar to those seen in mutants of Foxj1, a known key regulator of genes required for motile ciliation. RNAseq analysis indicates that Foxn4 co-activates some Foxj1 target genes strongly and many Foxj1 targets weakly. ChIPseq suggests that whereas 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 than in single mutants. In contrast to Foxj1, Foxn4 is not required to extend a single motile cilium by cells involved in left-right patterning. 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 multiple motile cilia.

What we can learn from a tadpole about ciliopathies and airway diseases: Using systems biology in Xenopus to study cilia and mucociliary epithelia

“…Importantly, MO‐mediated knockdown has proven to work specifically and efficiently in Xenopus , and defects due to upregulation of p53 expression in response to MO injection, as reported for zebrafish, was not observed in the frog (Blum, De Robertis, Wallingford, & Niehrs, ). Recent direct comparisons between MO and CRISPR/Cas9 knockout of transcription factors in Xenopus by RNA‐sequencing (RNA‐seq) further support the specificity of both the transcriptional changes and downstream morphant phenotypes in frog embryos (Campell, Quigley, & Kintner, ). The availability of high‐quality genome sequences in both X. laevis (Session et al, ) and X. tropicalis (Hellsten et al, ) also makes it easy to design specific guide RNAs (gRNAs) for genome editing, and analysis of potential off‐target effects by RNA‐seq in CRISPR/Cas9‐manipulated Xenopus embryos revealed extremely efficient effects on the targeted genes (>90% of transcripts expressed from the targeted genes were mutated) and a lack of off‐target effects in transcripts harboring potential off‐target sequences with as few as 1 to 5 mismatches (Campell et al, ).…”

“…Recent direct comparisons between MO and CRISPR/Cas9 knockout of transcription factors in Xenopus by RNA‐sequencing (RNA‐seq) further support the specificity of both the transcriptional changes and downstream morphant phenotypes in frog embryos (Campell, Quigley, & Kintner, ). The availability of high‐quality genome sequences in both X. laevis (Session et al, ) and X. tropicalis (Hellsten et al, ) also makes it easy to design specific guide RNAs (gRNAs) for genome editing, and analysis of potential off‐target effects by RNA‐seq in CRISPR/Cas9‐manipulated Xenopus embryos revealed extremely efficient effects on the targeted genes (>90% of transcripts expressed from the targeted genes were mutated) and a lack of off‐target effects in transcripts harboring potential off‐target sequences with as few as 1 to 5 mismatches (Campell et al, ). While efficient, targeted indels generated by this manipulation were highly variable, suggesting that Cas9 lesions take place not in single‐cell embryos but after a number of cell divisions, as suggested by others working in zebrafish (Campell et al, , Hruscha, Krawitz, Rechenberg, Heinrich, & Hecht, ).…”

Single Cell RNAseq Reveals A Critical Role of Chloride Channels in Airway Development

“…For example, a conserved precursor state for ciliated cells is marked by Foxn4 and the novel marker Shisa8 . Foxn4 is required to promote motile cilia formation in Xenopus epidermis (Campbell et al, 2016), and its expression is very transient during frog embryogenesis (Briggs et al, 2018). Our data indicate that Foxn4 expression peaks transiently at E16 in the mouse trachea as ciliated cells begin to emerge, supporting a central role of Foxn4 in promoting motile ciliogenesis in mammals.…”

“…At E16, ciliated cells consist of three distinct states that are all marked by Foxj1 , including one (state I, Figure 5A) with high Foxn4 expression, a transcriptional factor required for motile cilia formation in Xenopus epidermis and in cultured human bronchial epithelial cells (Campbell et al, 2016;Plasschaert et al, 2018). In addition to Foxn4 , cells from state I also express Ccno ,…”

The transcriptional signature associated with human motile cilia

“…Moreover, in conjunction with other transcriptional regulators, such as HNF1B and SOX5, further ciliary diversity is introduced for mechanosensory renal cilia and bronchiolar cilia, respectively (Gresh et al, 2004, Kiselak et al, 2010). Proteomic profiling studies have sought to define the components of motile cilia by dysregulating such transcriptional regulators and analysing the proteome of isolated cilia preparations using mass spectrometry (Choksi et al, 2014a, Blackburn et al, 2017, Ostrowski et al, 2002, El Zein et al, 2009, Campbell et al, 2016). Each of these studies has produced a list of cilia-associated proteins and accordingly a number of databases have been established.…”