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1.  Coordinated genomic control of ciliogenesis and cell movement by RFX2 
eLife  2014;3:e01439.
The mechanisms linking systems-level programs of gene expression to discrete cell biological processes in vivo remain poorly understood. In this study, we have defined such a program for multi-ciliated epithelial cells (MCCs), a cell type critical for proper development and homeostasis of the airway, brain and reproductive tracts. Starting from genomic analysis of the cilia-associated transcription factor Rfx2, we used bioinformatics and in vivo cell biological approaches to gain insights into the molecular basis of cilia assembly and function. Moreover, we discovered a previously un-recognized role for an Rfx factor in cell movement, finding that Rfx2 cell-autonomously controls apical surface expansion in nascent MCCs. Thus, Rfx2 coordinates multiple, distinct gene expression programs in MCCs, regulating genes that control cell movement, ciliogenesis, and cilia function. As such, the work serves as a paradigm for understanding genomic control of cell biological processes that span from early cell morphogenetic events to terminally differentiated cellular functions.
eLife digest
Cells that have hundreds of tiny hair-like structures called cilia on their surface have important roles in our airways and also in the brain and reproductive system. By beating in a coordinated manner, the cilia cause fluid to flow in a particular direction. The development of these multiciliated cells is a complex process in which genes are expressed as proteins, with this gene expression being regulated by other proteins called transcription factors.
In invertebrates the development of the cilia is controlled by transcription factors from the RFX family, which also appear to be important for development of cilia in vertebrates. However, the details of this process—in particular, the identities of the genes that are involved and how their functions are related—are not well understood in vertebrates.
Chung et al. have sought to remedy this by analyzing the network of genes whose expression is controlled by the transcription factor Rfx2 in vertebrates. The results showed that the genes controlled by Rfx2 were involved in all aspects of cilia, including several genes that are known to be mutated in diseases caused by abnormal cilia. Chung et al. also identified genes that were not previously thought to be relevant to cilia.
As multiciliated cells are developing, but before they can generate cilia, they must first migrate from the bottom of the epithelium, the layer of tissue in which they function, to the top of this layer. Chung et al. found that Rfx2 was also involved in this process.
The approach taken by Chung et al.—which involved a combination of RNA sequence analysis, examination of Rfx2 binding sites on chromosomes, computational predictions of protein interactions and in vivo cellular imaging—could be used to perform similar systems-level analyses of other developmental and biological processes.
PMCID: PMC3889689  PMID: 24424412
cilia; multiciliated cells; mucociliary epithelium; cilia beating; Rfx2; genomics; ttc29; ribc2; nme5; protofilament ribbon; Xenopus
2.  The Small GTPase Rsg1 is important for the cytoplasmic localization and axonemal dynamics of intraflagellar transport proteins 
Cilia  2013;2:13.
Cilia are small, microtubule-based protrusions important for development and homeostasis. We recently demonstrated that the planar cell polarity effector protein Fuz is a critical regulator of axonemal intraflagellar transport dynamics and localization. Here, we report our findings on the role of the small GTPase Rsg1, a known binding partner of Fuz, and its role in the dynamics and cytoplasmic localization of intraflagellar transport proteins.
We find that Rsg1 loss of function leads to impaired axonemal IFT dynamics in multiciliated cells. We further show that Rsg1 is required for appropriate cytoplasmic localization of the retrograde IFT-A protein IFT43. Finally, we show that Rsg1 governs the apical localization of basal bodies, the anchoring structures of cilia.
Our data suggest that Rsg1 is a regulator of multiple aspects of ciliogenesis, including apical trafficking of basal bodies and the localization and dynamics intraflagellar transport proteins.
PMCID: PMC3850895  PMID: 24192041
Cilia; Fuz; IFT; PCP; Rsg1
3.  Control of vertebrate intraflagellar transport by the planar cell polarity effector Fuz 
The Journal of Cell Biology  2012;198(1):37-45.
The planar cell polarity effector Fuz is required for normal particle dynamics of the intraflagellar transport system, specifically in the retrograde transport of proteins.
Cilia play key roles in development and homeostasis, and defects in cilia structure or function lead to an array of human diseases. Ciliogenesis is accomplished by the intraflagellar transport (IFT) system, a set of proteins governing bidirectional transport of cargoes within ciliary axonemes. In this paper, we present a novel platform for in vivo analysis of vertebrate IFT dynamics. Using this platform, we show that the planar cell polarity (PCP) effector Fuz was required for normal IFT dynamics in vertebrate cilia, the first evidence directly linking PCP to the core machinery of ciliogenesis. Further, we show that Fuz played a specific role in trafficking of retrograde, but not anterograde, IFT proteins. These data place Fuz in the small group of known IFT effectors outside the core machinery and, additionally, identify Fuz as a novel cytoplasmic effector that differentiates between the retrograde and anterograde IFT complexes.
PMCID: PMC3392940  PMID: 22778277
4.  Fuz Mutant Mice Reveal Shared Mechanisms between Ciliopathies and FGF-Related Syndromes 
Developmental Cell  2013;25(6):623-635.
Ciliopathies are a broad class of human disorders with craniofacial dysmorphology as a common feature. Among these is high arched palate, a condition that affects speech and quality of life. Using the ciliopathic Fuz mutant mouse, we find that high arched palate does not, as commonly suggested, arise from midface hypoplasia. Rather, increased neural crest expands the maxillary primordia. In Fuz mutants, this phenotype stems from dysregulated Gli processing, which in turn results in excessive craniofacial Fgf8 gene expression. Accordingly, genetic reduction of Fgf8 ameliorates the maxillary phenotypes. Similar phenotypes result from mutation of oral-facial-digital syndrome 1 (Ofd1), suggesting that aberrant transcription of Fgf8 is a common feature of ciliopathies. High arched palate is also a prevalent feature of fibroblast growth factor (FGF) hyperactivation syndromes. Thus, our findings elucidate the etiology for a common craniofacial anomaly and identify links between two classes of human disease: FGF-hyperactivation syndromes and ciliopathies.
•A genetic model for high arched palate, commonly seen in human craniofacial syndromes•In ciliopathic mice, Fgf8 overexpression leads to cranial neural crest hyperplasia•Enlargement of the maxillary primordia underlies high arched palate in Fuz mutants•An etiological link between ciliopathies and FGF-hyperactivation syndromes
High arched palate is common to many human disorders, including ciliopathies and craniosynostosis syndromes. Tabler et al. develop and analyze a genetic model of high arched palate; they conclude that embryonic changes in neural crest and fibroblast growth factor signaling underlie this unusual phenotype.
PMCID: PMC3697100  PMID: 23806618
6.  Planar Cell Polarity Acts Through Septins to Control Collective Cell Movement and Ciliogenesis 
Science (New York, N.Y.)  2010;329(5997):1337-1340.
The planar cell polarity (PCP) signaling pathway governs collective cell movements duringvertebrate embryogenesis, and certain PCP proteins are also implicated in the assembly ofcilia. The septins are cytoskeletal proteins controlling behaviors such as cell division and migration. Here, we identified control of septin localization by the PCP protein Fritz as a crucial control point for both collective cell movement and ciliogenesis in Xenopus embryos. We also linked mutations in human Fritz to Bardet-Biedl and Meckel-Gruber syndromes, a notable link given that other genes mutated in these syndromes also influence collective cell movement and ciliogenesis. These findings shed light on the mechanisms by which fundamental cellular machinery, such as the cytoskeleton, is regulated during embryonic development and human disease.
PMCID: PMC3509789  PMID: 20671153
7.  Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting Agent 
PLoS Biology  2012;10(8):e1001379.
Analysis of a genetic module repurposed between yeast and vertebrates reveals that a common antifungal medication is also a potent vascular disrupting agent.
Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans.
Author Summary
Yeast cells and vertebrate blood vessels would not seem to have much in common. However, we have discovered that during the course of evolution, a group of proteins whose function in yeast is to maintain cell walls has found an alternative use in vertebrates regulating angiogenesis. This remarkable repurposing of the proteins during evolution led us to hypothesize that, despite the different functions of the proteins in humans compared to yeast, drugs that modulated the yeast pathway might also modulate angiogenesis in humans and in animal models. One compound seemed a particularly promising candidate for this sort of approach: thiabendazole (TBZ), which has been in clinical use as a systemic antifungal and deworming treatment for 40 years. Gratifyingly, our study shows that TBZ is indeed able to act as a vascular disrupting agent and an angiogenesis inhibitor. Notably, TBZ also slowed tumor growth and decreased vascular density in human tumors grafted into mice. TBZ’s historical safety data and low cost make it an outstanding candidate for translation to clinical use as a complement to current anti-angiogenic strategies for the treatment of cancer. Our work demonstrates how model organisms from distant branches of the evolutionary tree can be exploited to arrive at a promising new drug.
PMCID: PMC3423972  PMID: 22927795
8.  A revised model of Xenopus dorsal midline development: Differential and separable requirements for Notch and Shh signaling 
Developmental Biology  2011;352(2):254-266.
The development of the vertebrate dorsal midline (floor plate, notochord, and hypochord) has been an area of classical research and debate. Previous studies in vertebrates have led to contrasting models for the roles of Shh and Notch signaling in specification of the floor plate, by late inductive or early allocation mechanisms, respectively. Here, we show that Notch signaling plays an integral role in cell fate decisions in the dorsal midline of Xenopus laevis, similar to that observed in zebrafish and chick. Notch signaling promotes floor plate and hypochord fates over notochord, but has variable effects on Shh expression in the midline. In contrast to previous reports in frog, we find that Shh signaling is not required for floor plate vs. notochord decisions and plays a minor role in floor plate specification, where it acts in parallel to Notch signaling. As in zebrafish, Shh signaling is required for specification of the lateral floor plate in the frog. We also find that the medial floor plate in Xenopus comprises two distinct populations of cells, each dependent upon different signals for its specification. Using expression analysis of several midline markers, and dissection of functional relationships, we propose a revised allocation mechanism of dorsal midline specification in Xenopus. Our model is distinct from those proposed to date, and may serve as a guide for future studies in frog and other vertebrate organisms.
PMCID: PMC3282588  PMID: 21276789
floor plate; notochord; hypochord; dorsal midline; Notch; Shh
9.  Planar cell polarity signaling, cilia and polarized ciliary beating 
Current opinion in cell biology  2010;22(5):597-604.
Planar cell polarity signaling governs a wide array of polarized cell behaviors in animals. Recent reports now show that PCP signaling is essential for the directional beating of motile cilia. Interestingly, PCP signaling acts in a variety of ciliated cell types that use motile cilia to generate directional fluid flow in very different ways. This review will synthesize these recent papers and place them in context with previous studies of PCP signaling in polarized cellular morphogenesis and collective cell movement.
PMCID: PMC2974441  PMID: 20817501
10.  Embryogenesis and laboratory maintenance of the foam-nesting túngara frogs, genus Engystomops (= Physalaemus) 
The vast majority of embryological research on amphibians focuses on just a single genus of frogs, Xenopus. To attain a more comprehensive understanding of amphibian development, experimentation on non-model frogs will be essential. Here, we report on the early development, rearing, and embryological analysis of túngara frogs (genus Engystomops, also called Physaleamus). The frogs Engystomops pustulosus, Engystomops coloradorum and Engystomops randi construct floating foam-nests with small eggs. We define a table of 23 stages for the developmental period in the foam-nest. Embryos were immunostained against Lim1, neural, and somite-specific proteins and the expression pattern of RetinoBlastoma Binding Protein 6 (RBBP6) was analyzed by in situ hybridization. Due to their brief life-cycle, frogs belonging to the genus Engystomops are attractive for comparative and genetic studies of development.
PMCID: PMC2934778  PMID: 19384855
Gastrulation modes; somitogenesis; neural development; Colostethus machalilla; Engystomops coloradorum; Engystomops randi; Engystomops pustulosus; Gastrotheca riobambae
11.  Diversification of the expression patterns and developmental functions of the Dishevelled gene family during chordate evolution 
Dishevelled (Dvl) proteins are key transducers of Wnt signaling encoded by members of a multi-gene family in vertebrates. We report here the divergent, tissue-specific expression patterns for all three Dvl genes in Xenopus embryos, which contrast dramatically with their expression patterns in mice. Moreover, we find that the expression patterns of Dvl genes in the chick diverge significantly from those of Xenopus. In addition, in hemichordates, an outgroup to chordates, we find that the one Dvl gene is dynamically expressed in a tissue-specific manner. Using knockdowns, we find that Dvl1 and Dvl2 are required for early neural crest specification and for somite segmentation in Xenopus. Most strikingly, we report a novel role for Dvl3 in the maintenance of gene expression in muscle and in the development of the Xenopus sclerotome. These data demonstrate that the expression patterns and developmental functions of specific Dvl genes have diverged significantly during chordate evolution.
PMCID: PMC2782374  PMID: 19618470
12.  Changes in localization and expression levels of Shroom2 and spectrin contribute to variation in amphibian egg pigmentation patterns 
Development genes and evolution  2009;219(6):319-330.
One contributing factor in the worldwide decline in amphibian populations is thought to be exposure of eggs to UV light. Enrichment of pigment in the animal hemisphere of eggs laid in the sunlight defends against UV damage, but little is known about the cell biological mechanisms controlling such polarized pigment patterns. Even less is known about how such mechanisms were modified during evolution to achieve the array of amphibian egg pigment patterns. Here, we show that ectopic expression of the γ-tubulin regulator, Shroom2, is sufficient to induce co-accumulation of pigment granules, spectrin, and dynactin in Xenopus blastomeres. Shroom2 and spectrin are enriched and co-localize specifically in the pigmented animal hemisphere of Xenopus eggs and blastulae. Moreover, Shroom2 mRNA is expressed maternally at high levels in Xenopus. By contrast to Xenopus, eggs and blastulae of Physalaemus pustulosus have very little surface pigmentation. Rather, we find that pigment is enriched in the perinuclear region of these embryos, where it co-localizes with spectrin. Moreover, maternal Shroom2 mRNA was barely detectable in Physaleamus, though zygotic levels were comparable to Xenopus. We therefore suggest that a Shroom2/spectrin/dynactin-based mechanism controls pigment localization in amphibian eggs, and that variation in maternal Shroom2 mRNA levels accounts in part for variation in amphibian egg pigment patterns during evolution.
PMCID: PMC2902998  PMID: 19554350
Shroom2; Spectrin; pigmentation; melanosome; Physalaemus
13.  The planar cell polarity effector Fuz is essential for targeted membrane trafficking, ciliogenesis, and mouse embryonic development 
Nature cell biology  2009;11(10):1225-1232.
The planar cell polarity (PCP) signaling pathway is essential for embryonic development because it governs diverse cellular behaviors, and the “core PCP” proteins, such as Dishevelled and Frizzled, have been extensively characterized1–4. By contrast, the “PCP effector” proteins, such as Intu and Fuz, remain largely unstudied5, 6. These proteins are essential for PCP signaling, but they have never been investigated in a mammal and their cell biological activities remain entirely unknown. We report here that Fuz mutant mice display neural tube defects, skeletal dysmorphologies, and Hedgehog signaling defects stemming from disrupted ciliogenesis. Using bioinformatics and imaging of an in vivo mucociliary epithelium, we establish a central role for Fuz in membrane trafficking, showing that Fuz is essential for trafficking of cargo to basal bodies and to the apical tips of cilia. Fuz is also essential for exocytosis in secretory cells. Finally, we identify a novel, Rab-related small GTPase as a Fuz interaction partner that is also essential for ciliogenesis and secretion. These results are significant because they provide novel insights into the mechanisms by which developmental regulatory systems like PCP signaling interface with fundamental cellular systems such as the vesicle trafficking machinery.
PMCID: PMC2755648  PMID: 19767740
14.  Wnt9b signaling regulates planar cell polarity and kidney tubule morphogenesis 
Nature genetics  2009;41(7):793-799.
Although many vertebrate organs, such as kidneys, lungs and liver, are composed of epithelial tubules, little is known of the mechanisms that establish the length or diameter of these tubules. In the kidney, defects in the establishment and/or maintenance of tubule diameter are associated with one of the most common inherited human disorders, polycystic kidney disease. Here, we show that attenuation of Wnt9b signaling during kidney morphogenesis affects the planar cell polarity of the epithelium and leads to tubules with significantly increased diameter. Although previous studies showed that polarized cell divisions maintain the diameter of postnatal kidney tubules, we find cell divisions are randomly oriented during embryonic development. Our data suggest that diameter is established during early morphogenetic stages by convergent extension processes and maintained by polarized cell divisions. Wnt9b, signaling through the non-canonical Rho/Jnk branch of the Wnt pathway, is necessary for both of these processes.
PMCID: PMC2761080  PMID: 19543268
15.  Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells 
Nature genetics  2008;40(7):871-879.
The planar cell polarity (PCP) signaling system governs many aspects of polarized cell behavior. Here, we use an in vivo model of vertebrate mucociliary epithelial development to show that Dishevelled (Dvl) is essential for the apical positioning of basal bodies. We find that Dvl and Inturned mediate the activation of the Rho GTPase specifically at basal bodies, and that these three proteins together mediate the docking of basal bodies to the apical plasma membrane. Moreover, we find that the docking involves a Dvl-dependent association of basal bodies with membrane-bound vesicles and with the vesicle-trafficking protein, Sec8. Once docked, Dvl and Rho are once again required for the planar polarization of basal bodies that underlies directional beating of cilia. These results demonstrate novel functions for PCP signaling components and suggest that a common signaling appratus governs both apical docking and planar polarization of basal bodies.
PMCID: PMC2771675  PMID: 18552847
16.  The Shroom family proteins play broad roles in the morphogenesis of thickened epithelial sheets 
Thickened epithelial sheets are found in most organ systems, but the mechanisms governing their morphogenesis remain poorly defined. We show here that Shroom family proteins are broadly involved in generating thickened epithelial sheets. Through in situ hybridization, we report temporal and spatial expression patterns of the four Shroom family members during early Xenopus development from oocytes to tadpole stages. Shroom1 and 2 mRNAs are maternally expressed, while Shroom3 and Shroom4 are zygotic transcripts. During later development, all four Shroom family proteins are broadly expressed in developing epithelial organs, and the epithelial cells that express Shrooms are elongated. Moreover, we show that ectopic expression of Shroom2, like Shroom3, is able to increase cell height and that loss of Shroom2 function results in a failure of cell elongation in the neural epithelium. These data suggest that Shroom family proteins play an important role in the morphogenesis of several different embryonic epithelial tissues.
PMCID: PMC2699254  PMID: 19384856
17.  Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development 
Developmental biology  2007;312(1):115-130.
Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. \Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.
PMCID: PMC2225594  PMID: 17961536
18.  A new standard nomenclature for proteins related to Apx and Shroom 
BMC Cell Biology  2006;7:18.
Shroom is a recently-described regulator of cell shape changes in the developing nervous system. This protein is a member of a small family of related proteins that are defined by sequence similarity and in most cases by some link to the actin cytoskeleton. At present these proteins are named Shroom, APX, APXL, and KIAA1202. In light of the growing interest in this family of proteins, we propose here a new standard nomenclature.
PMCID: PMC1481537  PMID: 16615870

Results 1-18 (18)