Multicilin drives centriole biogenesis via E2f proteins.

Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. Here we show that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, we show that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation.

Rfx2 Stabilizes Foxj1 Binding at Chromatin Loops to Enable Multiciliated Cell Gene Expression.

Cooperative transcription factor binding at cis-regulatory sites in the genome drives robust eukaryotic gene expression, and many such sites must be coordinated to produce coherent transcriptional programs. The transcriptional program leading to motile cilia formation requires members of the DNA-binding forkhead (Fox) and Rfx transcription factor families and these factors co-localize to cilia gene promoters, but it is not clear how many cilia genes are regulated by these two factors, whether these factors act directly or indirectly, or how these factors act with specificity in the context of a 3-dimensional genome. Here, we use genome-wide approaches to show that cilia genes reside at the boundaries of topological domains and that these areas have low enhancer density. We show that the transcription factors Foxj1 and Rfx2 binding occurs in the promoters of more cilia genes than other known cilia transcription factors and that while Rfx2 binds directly to promoters and enhancers equally, Foxj1 prefers direct binding to enhancers and is stabilized at promoters by Rfx2. Finally, we show that Rfx2 and Foxj1 lie at the anchor endpoints of chromatin loops, suggesting that target genes are activated when Foxj1 bound at distal sites is recruited via a loop created by Rfx2 binding at both sites. We speculate that the primary function of Rfx2 is to stabilize distal enhancers with proximal promoters by operating as a scaffolding factor, bringing key regulatory domains bound by Foxj1 into close physical proximity and enabling coordinated cilia gene expression.

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

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.

The hydrolethalus syndrome protein HYLS-1 links core centriole structure to cilia formation.

Centrioles are subcellular organelles composed of a ninefold symmetric microtubule array that perform two important functions: (1) They build centrosomes that organize the microtubule cytoskeleton, and (2) they template cilia, microtubule-based projections with sensory and motile functions. We identified HYLS-1, a widely conserved protein, based on its direct interaction with the core centriolar protein SAS-4. HYLS-1 localization to centrioles requires SAS-4 and, like SAS-4, HYLS-1 is stably incorporated into the outer centriole wall. Unlike SAS-4, HYLS-1 is dispensable for centriole assembly and centrosome function in cell division. Instead, HYLS-1 plays an essential role in cilia formation that is conserved between Caenorhabditis elegans and vertebrates. A single amino acid change in human HYLS1 leads to a perinatal lethal disorder termed hydrolethalus syndrome, and we show that this mutation impairs HYLS-1 function in ciliogenesis. HYLS-1 is required for the apical targeting/anchoring of centrioles at the plasma membrane but not for the intraflagellar transport-dependent extension of the ciliary axoneme. These findings classify hydrolethalus syndrome as a severe human ciliopathy and shed light on the dual functionality of centrioles, defining the first stably incorporated centriolar protein that is not required for centriole assembly but instead confers on centrioles the capacity to initiate ciliogenesis.

Systematic Analysis of CCNO Variants in a Defined Population: Implications for Clinical Phenotype and Differential Diagnosis.

Reduced generation of multiple motile cilia (RGMC) is a novel chronic destructive airway disease within the group of mucociliary clearance disorders with only few cases reported. Mutations in two genes, CCNO and MCIDAS, have been identified as a cause of this disease, both leading to a greatly reduced number of cilia and causing impaired mucociliary clearance. This study was designed to identify the prevalence of CCNO mutations in Israel and further delineate the clinical characteristics of RGMC. We analyzed 170 families with mucociliary clearance disorders originating from Israel for mutations in CCNO and identified two novel mutations (c.165delC, p.Gly56Alafs*38; c.638T>C, p.Leu213Pro) and two known mutations in 15 individuals from 10 families (6% prevalence). Pathogenicity of the missense mutation (c.638T>C, p.Leu213Pro) was demonstrated by functional analyses in Xenopus. Combining these 15 patients with the previously reported CCNO case reports revealed rapid deterioration in lung function, an increased prevalence of hydrocephalus (10%) as well as increased female infertility (22%). Consistent with these findings, we demonstrate that CCNO expression is present in murine ependyma and fallopian tubes. CCNO is mutated more frequently than expected from the rare previous clinical case reports, leads to severe clinical manifestations, and should therefore be considered an important differential diagnosis of mucociliary clearance disorders.

Bbof1 is required to maintain cilia orientation.

Multiciliate cells (MCCs) are highly specialized epithelial cells that employ hundreds of motile cilia to produce a vigorous directed flow in a variety of organ systems. The production of this flow requires the establishment of planar cell polarity (PCP) whereby MCCs align hundreds of beating cilia along a common planar axis. The planar axis of cilia in MCCs is known to be established via the PCP pathway and hydrodynamic cues, but the downstream steps required for cilia orientation remain poorly defined. Here, we describe a new component of cilia orientation, based on the phenotypic analysis of an uncharacterized coiled-coil protein, called bbof1. We show that the expression of bbof1 is induced during the early phases of MCC differentiation by the master regulator foxj1. MCC differentiation and ciliogenesis occurs normally in embryos where bbof1 activity is reduced, but cilia orientation is severely disrupted. We show that cilia in bbof1 mutants can still respond to patterning and hydrodynamic cues, but lack the ability to maintain their precise orientation. Misexpression of bbof1 promotes cilia alignment, even in the absence of flow or in embryos where microtubules and actin filaments are disrupted. Bbof1 appears to mediate cilia alignment by localizing to a polar structure adjacent to the basal body. Together, these results suggest that bbof1 is a basal body component required in MCCs to align and maintain cilia orientation in response to flow.

Myb promotes centriole amplification and later steps of the multiciliogenesis program.

The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.

Specification of ion transport cells in the Xenopus larval skin.

Specialized epithelial cells in the amphibian skin play important roles in ion transport, but how they arise developmentally is largely unknown. Here we show that proton-secreting cells (PSCs) differentiate in the X. laevis larval skin soon after gastrulation, based on the expression of a `kidney-specific' form of the H(+)v-ATPase that localizes to the plasma membrane, orthologs of the Cl(-)/HCO(-)(3) antiporters ae1 and pendrin, and two isoforms of carbonic anhydrase. Like PSCs in other species, we show that the expression of these genes is likely to be driven by an ortholog of foxi1, which is also sufficient to promote the formation of PSC precursors. Strikingly, the PSCs form in the skin as two distinct subtypes that resemble the alpha- and beta-intercalated cells of the kidney. The alpha-subtype expresses ae1 and localizes H(+)v-ATPases to the apical plasma membrane, whereas the beta-subtype expresses pendrin and localizes the H(+)v-ATPase cytosolically or basolaterally. These two subtypes are specified during early PSC differentiation by a binary switch that can be regulated by Notch signaling and by the expression of ubp1, a transcription factor of the grainyhead family. These results have implications for how PSCs are specified in vertebrates and become functionally heterogeneous.

A positive feedback mechanism governs the polarity and motion of motile cilia.

Ciliated epithelia produce fluid flow in many organ systems, ranging from the respiratory tract where it clears mucus to the ventricles of the brain where it transports cerebrospinal fluid. Human diseases that disable ciliary flow, such as primary ciliary dyskinesia, can compromise organ function or the ability to resist pathogens, resulting in recurring respiratory infections, otitis, hydrocephaly and infertility. To create a ciliary flow, the cilia within each cell need to be polarized coordinately along the planar axis of the epithelium, but how polarity is established in any ciliated epithelia is not known. Here we analyse the developmental mechanisms that polarize cilia, using the ciliated cells in the developing Xenopus larval skin as a model system. We show that cilia acquire polarity through a sequence of events, beginning with a polar bias set by tissue patterning, followed by a refinement phase. Our results indicate that during refinement, fluid flow is both necessary and sufficient in determining cilia polarity. These findings reveal a novel mechanism in which tissue patterning coupled with fluid flow act in a positive feedback loop to direct the planar polarity of cilia.

Mechanical strain breaks planar symmetry in embryonic epithelia via polarized microtubules.

Mechanical strain can act as a global cue to orient the core planar cell polarity pathway (Fz-PCP) in developing epithelia, but how strain directs a Fz-PCP vector is not known. Here we use live cell imaging of apical microtubules (MTs) and components of the Fz-PCP pathway to analyze epithelial cells in Xenopus embryos as they respond to anisotropic mechanical strain and form a Fz-PCP axis. We find that a Fz-PCP axis can be detected approximately 40 min after the application of strain. By contrast, the density and length of apical MTs increases rapidly (5-10 min) in response to strain, independently of Fz-PCP. These early-forming apical MTs are planar polarized: they align to the strain axis and display a marked bias in plus-end orientation that invariably points towards the cell edge opposite the direction of strain application. We show that these MTs can promote the vectorial transport of Dvl3-GFP containing vesicles along the apical surface in a directed manner, perhaps explaining why PCP signaling fails when MTs are disrupted. Finally, we provide evidence that the Fz-PCP axis feeds back after an hour to stabilize oriented apical MTs. These results provide insights into how mechanical strain acts as a developmental cue within the appropriate time frame and with the appropriate vector to promote planar axis formation.

Emi2 enables centriole amplification during multiciliated cell differentiation.

Massive centriole amplification during multiciliated cell (MCC) differentiation is a notable example of organelle biogenesis. This process is thought to be enabled by a derived cell cycle state, but the key cell cycle components required for centriole amplification in MCC progenitors remain poorly defined. Here, we show that emi2 (fbxo43) expression is up-regulated and acts in MCC progenitors after cell cycle exit to transiently inhibit anaphase-promoting complex/cyclosome (APC/C)cdh1 activity. We find that this inhibition is required for the phosphorylation and activation of a key cell cycle kinase, plk1, which acts, in turn, to promote different steps required for centriole amplification and basal body formation, including centriole disengagement, apical migration, and maturation into basal bodies. This emi2-APC/C-plk1 axis is also required to down-regulate gene expression essential for centriole amplification after differentiation is complete. These results identify an emi2-APC/C-plk1 axis that promotes and then terminates centriole assembly and basal body formation during MCC differentiation.

The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands.

Mutations in the DSL (Delta, Serrate, Lag2) Notch (N) ligand Delta-like (Dll) 3 cause skeletal abnormalities in spondylocostal dysostosis, which is consistent with a critical role for N signaling during somitogenesis. Understanding how Dll3 functions is complicated by reports that DSL ligands both activate and inhibit N signaling. In contrast to other DSL ligands, we show that Dll3 does not activate N signaling in multiple assays. Consistent with these findings, Dll3 does not bind to cells expressing any of the four N receptors, and N1 does not bind Dll3-expressing cells. However, in a cell-autonomous manner, Dll3 suppressed N signaling, as was found for other DSL ligands. Therefore, Dll3 functions not as an activator as previously reported but rather as a dedicated inhibitor of N signaling. As an N antagonist, Dll3 promoted Xenopus laevis neurogenesis and inhibited glial differentiation of mouse neural progenitors. Finally, together with the modulator lunatic fringe, Dll3 altered N signaling levels that were induced by other DSL ligands.

Retinoic acid regulation of the Mesp-Ripply feedback loop during vertebrate segmental patterning.

The Mesp bHLH genes play a conserved role during segmental patterning of the mesoderm in the vertebrate embryo by specifying segmental boundaries and anteroposterior (A-P) segmental polarity. Here we use a xenotransgenic approach to compare the transcriptional enhancers that drive expression of the Mesp genes within segments of the presomitic mesoderm (PSM) of different vertebrate species. We find that the genomic sequences upstream of the mespb gene in the pufferfish Takifugu rubripes (Tr-mespb) are able to drive segmental expression in transgenic Xenopus embryos while those from the Xenopus laevis mespb (Xl-mespb) gene drive segmental expression in transgenic zebrafish. In both cases, the anterior segmental boundary of transgene expression closely matches the expression of the endogenous Mesp genes, indicating that many inputs into segmental gene expression are highly conserved. By contrast, we find that direct retinoic acid (RA) regulation of endogenous Mesp gene expression is variable among vertebrate species. Both Tr-mespb and Xl-mespb are directly upregulated by RA, through a complex, distal element. By contrast, RA represses the zebrafish Mesp genes. We show that this repression is mediated, in part, by RA-mediated activation of the Ripply genes, which together with Mesp genes form an RA-responsive negative feedback loop. These observations suggest that variations in a direct response to RA input may allow for changes in A-P patterning of the segments in different vertebrate species.

Regulation of segmental patterning by retinoic acid signaling during Xenopus somitogenesis.

Somites, the segmented building blocks of the vertebrate embryo, arise one by one in a patterning process that passes wavelike along the anteroposterior axis of the presomitic mesoderm (PSM). We have studied this process in Xenopus embryos by analyzing the expression of the bHLH gene, Thylacine1, which is turned on in the PSM as cells mature and segment, in a pattern that marks both segment boundaries and polarity. Here, we show that this segmental gene expression involves a PSM enhancer that is regulated by retinoic acid (RA) signaling at two levels. RA activates Thylacine1 expression in rostral PSM directly. RA also activates Thylacine1 expression in the caudal PSM indirectly by inducing the expression of MKP3, an inhibitor of the FGF signaling pathway. RA signaling is therefore a major contributor to segmental patterning by promoting anterior segmental polarity and by interacting with the FGF signaling pathway to position segmental boundaries.

Mechanical Strain Determines Cilia Length, Motility, and Planar Position in the Left-Right Organizer.

The Xenopus left-right organizer (LRO) breaks symmetry along the left-right axis of the early embryo by producing and sensing directed ciliary flow as a patterning cue. To carry out this process, the LRO contains different ciliated cell types that vary in cilia length, whether they are motile or sensory, and how they position their cilia along the anterior-posterior (A-P) planar axis. Here, we show that these different cilia features are specified in the prospective LRO during gastrulation, based on anisotropic mechanical strain that is oriented along the A-P axis, and graded in levels along the medial-lateral axis. Strain instructs ciliated cell differentiation by acting on a mesodermal prepattern present at blastula stages, involving foxj1. We propose that differential strain is a graded, developmental cue, linking the establishment of an A-P planar axis to cilia length, motility, and planar location during formation of the Xenopus LRO.

Multicilin and activated E2f4 induce multiciliated cell differentiation in primary fibroblasts.

Multiciliated cells (MCCs) are specialized epithelial cells that project hundreds of motile cilia. To form these cilia, MCCs differentiate by dramatically expanding centriole number, using assembly factors required for centriole duplication during the cell cycle and multiple, novel assembly sites, called the deuterosome. The small coiled-coil protein, Multicilin, acting in a complex with the E2F proteins can initiate multiciliated cell differentiation, but reportedly only in a limited range of epithelial progenitors. To examine the nature of this restricted activity, we analyzed Multicilin activity in primary mouse embryonic fibroblasts (MEFs), a cell type distant from the epithelial lineages where MCCs normally arise. We show that Multicilin transcriptional activity is markedly attenuated in MEFs, where it induces only limited centriole expansion in a small fraction of cells. We further show that this transcriptional block is largely bypassed by expressing Multicilin along with a form of E2f4 where a generic activation domain from HSV1 VP16 (E2f4VP16) is fused to the carboxy terminus. MEFs respond to Multicilin and E2f4VP16 by undergoing massive centriole expansion via the deuterosome pathway, recapitulating a temporal sequence of organelle biogenesis that occurs in epithelial progenitors during MCC differentiation. These results suggest that the pattern of organelle biogenesis occurring in differentiating MCCs is largely determined by the transcriptional changes induced by Multicilin.

Mechanical strain determines the axis of planar polarity in ciliated epithelia.

Epithelia containing multiciliated cells align beating cilia along a common planar axis specified by the conserved planar cell polarity (PCP) pathway. Specification of the planar axis is also thought to require a long-range cue to align the axis globally, but the nature of this cue in ciliated and other epithelia remains poorly understood. We examined this issue using the Xenopus larval skin, where ciliary flow aligns to the anterior-posterior (A-P) axis. We first show that a planar axis initially arises in the developing skin during gastrulation, based on the appearance of polarized apical microtubules and cell junctions with increased levels of stable PCP components. This axis also arises in severely ventralized embryos, despite their deficient embryonic patterning. Because ventralized embryos still gastrulate, producing a mechanical force that strains the developing skin along the A-P axis, we asked whether this strain alone drives global planar patterning. Isolated skin explanted before gastrulation lacks strain and fails to acquire a global planar axis but responds to exogenous strain by undergoing cell elongation, forming polarized apical microtubules, and aligning stable components of the PCP pathway orthogonal to the axis of strain. The planar axis in embryos can be redirected by applying exogenous strain during a critical period around gastrulation. Finally, we provide evidence that apical microtubules and the PCP pathway interact to align the planar axis. These results indicate that oriented tissue strain generated by the gastrulating mesoderm plays a major role in determining the global axis of planar polarity of the developing skin.

MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia.

Reduced generation of multiple motile cilia (RGMC) is a rare mucociliary clearance disorder. Affected persons suffer from recurrent infections of upper and lower airways because of highly reduced numbers of multiple motile respiratory cilia. Here we report recessive loss-of-function and missense mutations in MCIDAS-encoding Multicilin, which was shown to promote the early steps of multiciliated cell differentiation in Xenopus. MCIDAS mutant respiratory epithelial cells carry only one or two cilia per cell, which lack ciliary motility-related proteins (DNAH5; CCDC39) as seen in primary ciliary dyskinesia. Consistent with this finding, FOXJ1-regulating axonemal motor protein expression is absent in respiratory cells of MCIDAS mutant individuals. CCNO, when mutated known to cause RGMC, is also absent in MCIDAS mutant respiratory cells, consistent with its downstream activity. Thus, our findings identify Multicilin as a key regulator of CCNO/FOXJ1 for human multiciliated cell differentiation, and highlight the 5q11 region containing CCNO and MCIDAS as a locus underlying RGMC.

Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia.

Using a whole-exome sequencing strategy, we identified recessive CCNO (encoding cyclin O) mutations in 16 individuals suffering from chronic destructive lung disease due to insufficient airway clearance. Respiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects. Careful subcellular analyses as well as in vitro ciliogenesis experiments in CCNO-mutant cells showed defective mother centriole generation and placement. Morpholino-based knockdown of the Xenopus ortholog of CCNO also resulted in reduced MMC and centriole numbers in embryonic epidermal cells. CCNO is expressed in the apical cytoplasm of multiciliated cells and acts downstream of multicilin, which governs the generation of multiciliated cells. To our knowledge, CCNO is the first reported gene linking an inherited human disease to reduced MMC generation due to a defect in centriole amplification and migration.