Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.

Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.

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33Motile cilia serve vital functions in development, homeostasis and regeneration. We recently 34 demonstrated that TAp73 is an essential transcriptional regulator of respiratory multiciliogenesis. 35Here, we show that TAp73 is expressed in multiciliated cells (MCCs) of diverse tissues. Analysis 36 of TAp73 mutant animals revealed that TAp73 regulates Foxj1, Rfx2, Rfx3, axonemal dyneins 37 Dnali1 and Dnai1, plays a pivotal role in the generation of MCCs in male and female reproductive 38 ducts, and contributes to fertility. However, the function of MCCs in the brain appears to be 39 preserved despite the loss of TAp73, and robust activity of cilia-related networks is maintained in 40 the absence of TAp73. Notably, TAp73 loss leads to distinct changes in ciliogenic microRNAs: 41 miR34bc expression is reduced, whereas the miR449 cluster is induced in diverse multiciliated 42 epithelia. Among different MCCs, choroid plexus (CP) epithelial cells in the brain display prominent 43 miR449 expression, whereas brain ventricles exhibit significant increase in miR449 levels along 44 with an increase in the activity of ciliogenic E2F4/MCIDAS circuit in TAp73 mutant animals. 45Conversely, E2F4 induces robust transcriptional response from miR449 genomic regions. To 46 address whether increased miR449 levels in the brain maintain the multiciliogenesis program in 47 the absence of TAp73, we deleted both TAp73 and miR449 in mice. Although loss of miR449 48 alone led to a mild ciliary defect in the CP, more pronounced ciliary defects and hydrocephalus 49 were observed in the brain lacking both TAp73 and miR449. In contrast, miR449 loss in other 50MCCs failed to enhance ciliary defects associated with TAp73 loss. Together, our study shows 51 that, in addition to the airways, TAp73 is essential for generation of MCCs in male and female 52 reproductive ducts, whereas miR449 and TAp73 complement each other to support 53 multiciliogenesis and CP development in the brain. 54

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