Vertebrate cells differentially interpret ciliary and extraciliary cAMP.

Hedgehog pathway components and select G protein-coupled receptors (GPCRs) localize to the primary cilium, an organelle specialized for signal transduction. We investigated whether cells distinguish between ciliary and extraciliary GPCR signaling. To test whether ciliary and extraciliary cyclic AMP (cAMP) convey different information, we engineered optogenetic and chemogenetic tools to control the subcellular site of cAMP generation. Generating equal amounts of ciliary and cytoplasmic cAMP in zebrafish and mammalian cells revealed that ciliary cAMP, but not cytoplasmic cAMP, inhibited Hedgehog signaling. Modeling suggested that the distinct geometries of the cilium and cell body differentially activate local effectors. The search for effectors identified a ciliary pool of protein kinase A (PKA). Blocking the function of ciliary PKA, but not extraciliary PKA, activated Hedgehog signal transduction and reversed the effects of ciliary cAMP. Therefore, cells distinguish ciliary and extraciliary cAMP using functionally and spatially distinct pools of PKA, and different subcellular pools of cAMP convey different information.

An alternative cell cycle coordinates multiciliated cell differentiation.

The canonical mitotic cell cycle coordinates DNA replication, centriole duplication and cytokinesis to generate two cells from one1. Some cells, such as mammalian trophoblast giant cells, use cell cycle variants like the endocycle to bypass mitosis2. Differentiating multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tract, are post-mitotic but generate hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium3,4. Several cell cycle regulators have previously been implicated in specific steps of multiciliated cell differentiation5,6. Here we show that differentiating multiciliated cells integrate cell cycle regulators into a new alternative cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys many canonical cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, cyclin D1, CDK4 and CDK6, which are regulators of mitotic G1-to-S progression, are required to initiate multiciliated cell differentiation. The multiciliation cycle amplifies some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. E2F7, a transcriptional regulator of canonical S-to-G2 progression, is expressed at high levels during the multiciliation cycle. In the multiciliation cycle, E2F7 directly dampens the expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes aberrant acquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, which disrupts centriole maturation and ciliogenesis. We conclude that multiciliated cells use an alternative cell cycle that orchestrates differentiation instead of controlling proliferation.

Flow-Cytometric Analysis and Purification of Airway Epithelial-Cell Subsets.

The human airway epithelium is essential in homeostasis, and epithelial dysfunction contributes to chronic airway disease. Development of flow-cytometric methods to characterize subsets of airway epithelial cells will enable further dissection of airway epithelial biology. Leveraging single-cell RNA-sequencing data in combination with known cell type-specific markers, we developed panels of antibodies to characterize and isolate the major airway epithelial subsets (basal, ciliated, and secretory cells) from human bronchial epithelial-cell cultures. We also identified molecularly distinct subpopulations of secretory cells and demonstrated cell subset-specific expression of low-abundance transcripts and microRNAs that are challenging to analyze with current single-cell RNA-sequencing methods. These new tools will be valuable for analyzing and separating airway epithelial subsets and interrogating airway epithelial biology.

Switching on cilia: transcriptional networks regulating ciliogenesis.

Cilia play many essential roles in fluid transport and cellular locomotion, and as sensory hubs for a variety of signal transduction pathways. Despite having a conserved basic morphology, cilia vary extensively in their shapes and sizes, ultrastructural details, numbers per cell, motility patterns and sensory capabilities. Emerging evidence indicates that this diversity, which is intimately linked to the different functions that cilia perform, is in large part programmed at the transcriptional level. Here, we review our understanding of the transcriptional control of ciliary biogenesis, highlighting the activities of FOXJ1 and the RFX family of transcriptional regulators. In addition, we examine how a number of signaling pathways, and lineage and cell fate determinants can induce and modulate ciliogenic programs to bring about the differentiation of distinct cilia types.

Systematic discovery of novel ciliary genes through functional genomics in the zebrafish.

Cilia are microtubule-based hair-like organelles that play many important roles in development and physiology, and are implicated in a rapidly expanding spectrum of human diseases, collectively termed ciliopathies. Primary ciliary dyskinesia (PCD), one of the most prevalent of ciliopathies, arises from abnormalities in the differentiation or motility of the motile cilia. Despite their biomedical importance, a methodical functional screen for ciliary genes has not been carried out in any vertebrate at the organismal level. We sought to systematically discover novel motile cilia genes by identifying the genes induced by Foxj1, a winged-helix transcription factor that has an evolutionarily conserved role as the master regulator of motile cilia biogenesis. Unexpectedly, we find that the majority of the Foxj1-induced genes have not been associated with cilia before. To characterize these novel putative ciliary genes, we subjected 50 randomly selected candidates to a systematic functional phenotypic screen in zebrafish embryos. Remarkably, we find that over 60% are required for ciliary differentiation or function, whereas 30% of the proteins encoded by these genes localize to motile cilia. We also show that these genes regulate the proper differentiation and beating of motile cilia. This collection of Foxj1-induced genes will be invaluable for furthering our understanding of ciliary biology, and in the identification of new mutations underlying ciliary disorders in humans.

A function for the Joubert syndrome protein Arl13b in ciliary membrane extension and ciliary length regulation.

Cilia perform a variety of functions in a number of developmental and physiological contexts, and are implicated in the pathogenesis of a wide spectrum of human disorders. While the ciliary axoneme is assembled by intraflagellar transport, how ciliary membrane length is regulated is not completely understood. Here, we show that zebrafish embryos as well as mammalian cells overexpressing the ciliary membrane protein Arl13b, an ARF family small GTPase that is essential for ciliary differentiation, showed pronounced increase in ciliary length. Intriguingly, this increase in cilia length occurred as a function of the amounts of overexpressed Arl13b. While the motility of Arl13b overexpressing excessively long motile cilia was obviously disrupted, surprisingly, the abnormally long immotile primary cilia seemed to retain their signaling capacity. arl13b is induced by FoxJ1 and Rfx, and these ciliogenic transcription factors are unable to promote ciliary length increase when Arl13b activity is inhibited. Conversely, overexpression of Arl13b was sufficient to restore ciliary length in zebrafish embryos deficient in FoxJ1 function. We show that Arl13b increases cilia length by inducing protrusion of the ciliary membrane, which is then followed by the extension of the axonemal microtubules. Using mutant versions of Arl13b, one of which has been shown to be causative of the ciliopathy Joubert syndrome, we establish that the GTPase activity of the protein is essential for ciliary membrane extension. Taken together, our findings identify Arl13b as an important effector of ciliary membrane biogenesis and ciliary length regulation, and provide insights into possible mechanisms of dysfunction of the protein in Joubert syndrome.

Mutations in CCDC11, which encodes a coiled-coil containing ciliary protein, causes situs inversus due to dysmotility of monocilia in the left-right organizer.

In vertebrates, establishment of left-right (LR) asymmetry is dependent on cilia-driven fluid flow within the LR organizer. Mutations in CCDC11 disrupt LR asymmetry in humans, but how the gene functions in LR patterning is presently unknown. We describe a patient with situs inversus totalis carrying homozygous loss-of-function mutations in CCDC11. We show that CCDC11 is an axonemal protein in respiratory cilia, but is largely dispensable for their structure and motility. To investigate the role of CCDC11 in LR development, we studied the zebrafish homolog of the gene. Like in human respiratory cilia, loss of Ccdc11 causes minor defects in the motility of zebrafish kidney cilia, although the protein localizes to their axonemes and base. By contrast, Ccdc11 localizes exclusively to the basal bodies of cilia within Kupffer's vesicle, the organ of laterality of teleost fishes, and within the spinal canal. Moreover, the rotational motion of the cilia in these tissues of ccdc11-deficient embryos was strongly impaired. Our findings demonstrate that CCDC11 has a conserved essential function in cilia of the vertebrate LR organizer. To the best of our knowledge, this is the first ciliary component, which has a differential localization and function in different kinds of motile cilia.

An RFX transcription factor regulates ciliogenesis in the closest living relatives of animals.

Cilia allowed our protistan ancestors to sense and explore their environment, avoid predation, and capture bacterial prey.1,2,3 Regulated ciliogenesis was likely critical for early animal evolution,2,4,5,6 and in modern animals, deploying cilia in the right cells at the right time is crucial for development and physiology. Two transcription factors, RFX and FoxJ1, coordinate ciliogenesis in animals7,8,9 but are absent from the genomes of many other ciliated eukaryotes, raising the question of how the regulation of ciliogenesis in animals evolved.10,11 By comparing the genomes of animals with those of their closest living relatives, the choanoflagellates, we found that the genome of their last common ancestor encoded at least three RFX paralogs and a FoxJ1 homolog. Disruption of the RFX homolog cRFXa in the model choanoflagellate Salpingoeca rosetta resulted in delayed cell proliferation and aberrant ciliogenesis, marked by the collapse and resorption of nascent cilia. In cRFXa mutants, ciliogenesis genes and foxJ1 were significantly downregulated. Moreover, the promoters of S. rosetta ciliary genes are enriched for DNA motifs matching those bound by the cRFXa protein in vitro. These findings suggest that an ancestral cRFXa homolog coordinated ciliogenesis in the progenitors of animals and choanoflagellates and that the selective deployment of the RFX regulatory module may have been necessary to differentiate ciliated from non-ciliated cell types during early animal evolution.

Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells.

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. Here, we show that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. We have identified the in vivo targets of Prospero throughout the entire genome. We show that Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. We further show that in the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation.

Specification of vertebrate slow-twitch muscle fiber fate by the transcriptional regulator Blimp1.

Skeletal muscles of vertebrates are typically composed of slow- and fast-twitch fibers that differ in their morphology, gene expression profiles, contraction speeds, metabolic properties and patterns of innervation. During myogenesis, how muscle precursors are induced to mature into distinct slow- or fast-twitch fiber-types is inadequately understood. We have previously shown that within the somites of the zebrafish embryo, the activity of the zinc finger and SET domain-containing transcriptional regulator Blimp1 is essential for the specification of slow muscle fibers. Here, we have investigated the mechanism by which Blimp1 programs myoblasts to adopt the slow-twitch fiber fate. In slow myoblasts, expression of the Blimp1 protein is transient, and precedes the expression of slow muscle-specific differentiation genes. We demonstrate that the competence of somitic myoblasts to commit to the slow lineage in response to Blimp1 changes as a function of developmental time. Furthermore, we provide evidence that mammalian Blimp1 can recapitulate the slow myogenic program in zebrafish, suggesting that zebrafish Blimp1 can recognize the same consensus DNA sequence that is bound by the mammalian protein. Finally, we show that zebrafish Blimp1 can repress the expression of fast muscle-specific myosin light chain, mylz2, through direct binding near the promoter of this gene, indicating that an important function of the transcriptional activity of Blimp1 in slow muscle development is the suppression of fast muscle-specific gene expression. Taken together, these findings provide new insights into the molecular basis of vertebrate muscle fiber-type specification, and underscore Blimp1 as the central determinant of this process.

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

amontillado, the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development.

Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.

Turning back the clock on neural progenitors.

Drosophila neural progenitor cells, or neuroblasts, alter their transcriptional profile over time to produce different neural cell types. A recent paper by Pearson and Doe shows that older neuroblasts can be reprogrammed to behave like young neuroblasts, and to produce early neural cell types, simply by expressing the transcription factor, Hunchback. The authors show that competence to respond to Hunchback diminishes over time. Manipulating neural progenitors in this way may have important implications for therapeutic uses of neural stem cells.