THE CILIATED CELL TRANSCRIPTOME
A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
Ramona Hoh March 2010
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© 2010 by Ramona Amy Hoh. All Rights Reserved. Re-distributed by Stanford University under license with the author.
This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/
This dissertation is online at: http://purl.stanford.edu/sk794dv5857
ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Timothy Stearns, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Mark Krasnow
I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Maxence Nachury
I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.
William Nelson
Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education
This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives.
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Abstract Multiciliated cells of the respiratory epithelium are unique in that they generate hundreds of modified centrioles called basal bodies per cell. Each basal body anchors a motile cilium at the cell apical surface, and coordinated beating of motile cilia is vital for protecting from airway infection and for respiratory function. We used mice expressing GFP from the promoter of a ciliated cell-specific gene, FOXJ1, to obtain sorted populations of ciliating cells for transcriptional analysis. In addition to successfully identifying candidates found in other proteomics and genomics studies of motile and nonmotile cilia, approximately half of the significantly upregulated genes identified here have not yet been linked to cilia, and of those a third of are currently uncharacterized. We identified several genes associated with human diseases. These include FTO, which has been linked to human obesity, and DYX1C1, which is a candidate gene for developmental dyslexia. Interestingly, FTO localizes to cilia and Dyx1c1-GFP localizes to cilia and centrosomes, establishing novel links between cilia and two genetic diseases with poorly understood cellular and molecular etiology. Finally, we identified a number of transcription factors that are differentially expressed in ciliating mouse tracheal epithelial cells, including the proto-oncogene c- myb. We show that C-myb is expressed specifically in ciliating cells, and that this expression is temporally restricted to early in the differentiation process. These results suggest a role for the leukemogenic transcription factor C-myb in ciliated cell differentiation.
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Acknowledgments Thanks to Tim Stearns for his mentorship and support, and for giving me the freedom to try some pretty ridiculous things. Maybe some day we will find RNA at the centrosome. The Stearns lab was a fantastic group of people to learn science from. Special thanks to Anna Ballew for being a wonderful baymate and for all our insightful discussions about science, to Tim Stowe for working with me to publish the microarray data, and to Eszter Vladar, for MTEC protocols and advice. Thanks to Dan Van de Mark for his work on the promoter project during his rotation. Thanks to my committee members for all of their helpful advice, and to Fraser Tan, Adam Adler and Matt Saunders, my collaborators at Stanford and elsewhere. Special thanks to Bernie Daigle for his expertise on the design and analysis of microarray experiments, and to Allyson O’Donnell and Clara Bermejo, two ass-kicking postdocs who are also the most wonderful of friends. Thanks to Bryan Guillemette, for help with dataset annotation and for being so very dear. Finally, thanks to my parents, who always encouraged me to think for myself and make my own decisions, and who supported me lovingly in all of them.
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Table of Contents Chapter 1: Introduction 1 References 10
Chapter 2: Transcriptional profiling of ciliogenesis 14 Abstract 15 Introduction 15 Results 17 Discussion 22 Figures 24 Methods and Materials 37 References 40
Chapter 3: Novel localization of candidate genes 42 Abstract 43 Results 43 Discussion 56 Figures 64 Methods and Materials 81 References 83
Chapter 4: Transcriptional Regulation of Ciliogenesis 92 Abstract 93 Introduction 93 Results 95 Discussion 98 Figures 100 Methods and Materials 105 References 106
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List of Illustrations
Chapter 1: Introduction Chapter 2: Transcriptional profiling of ciliogenesis Figure 1: Microarray analysis of ciliating mouse tracheal epithelial cells 24 Figure 2: Microarray hybridizations and design matrices for differential expression analysis 25 Figure 3: Differentially expressed genes in MTECs, and the effect of subtractive analysis 26 Figure 4: Identification of known and putative ciliary and basal body genes in the ALI+4 transcriptome 27 Figure 5: Identification of known and putative ciliary and basal body genes in the ALI+12 transcriptome 32 Figure 6: Identification of putative and validated FoxJ1 target genes in the MTEC transcriptome 35 Figure 7: GO Term analysis of genes upregulated during ciliogenesis 36
Chapter 3: Novel localization of candidate genes Figure 1: Expression of centrosome and centriole related genes in ciliated MTECs 64 Figure 2: Expression of genes encoding candidate proteins in ciliated MTECs 65 Figure 3: Mdm1-GFP localizes to the centrosome and the primary cilium 66 Figure 4: Dyx1c1-GFP localizes to the centrosome in NIH-3T3 cells 67 Figure 5: KIAA0319-GFP localizes to the centrosome in NIH-3T3 cells 68 Figure 6: Genes associated with neuronal migration and developmental dyslexia are upregulated in ciliated MTECs 69 Figure 7: Expression of genes encoding heterotrimeric G-proteins and associated molecules in MTECs 70 Figure 8: Gnb4 is a cilium associated protein 71 Figure 9: Mlf1 localizes to motile cilia 72
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Figure 10: Fatso is a cilium associated protein 73 Figure 11: Fatso is a microtubule associated protein 74 Figure 12: Whsc1 expression in ciliated cells 75 Figure 13: Expression of human disease gene homologs during ciliogenesis in MTECs 76 Figure 14: Expression of small GTPases and modifying proteins during ciliogenesis 77 Figure 15: Upregulated taste receptor genes in ciliated MTECS 78 Figure 16: Expression of olfactory receptor genes in ciliated MTECs 79 Figure 17: Expression of vomeronasal receptors in ciliated MTECs 80
Chapter 4: Transcriptional Regulation of Ciliogenesis Figure 1:Temporal expression profiles of genes encoding regulators of DNA dependent transcription during ciliogenesis 100 Figure 2: C-myb expression before and during ciliogenesis 101 Figure 3: Restriction of c-myb expression during ciliogenesis 102 Figure 4: Promoter analysis of differentially expressed genes during early ciliogenesis 103 Figure 5: Experimental flowchart for characterizing c-myb knockdown phenotype 104
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Chapter 1: Introduction
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Introduction Cilia structure and function Cilia and flagella are microtubule-based structures that project outward from the cell surface and have important sensory and motile functions. They are composed of a microtubule-based axoneme that projects from a modified centriole called a basal body. The axoneme is surrounded by a specialized plasma membrane that is continuous with the plasma membrane of the cell body. At the distal end of the basal body is the transition zone, where the triplet microtubule basal body extends into the doublet microtubule axoneme. Transition fibers link the transition zone of the centriole to a specialized region of the plasma membrane called the “ciliary necklace” (Gilula and Satir, 1972; Sorokin, 1962). It has been proposed that this region acts akin to the nuclear pore complexes of the nuclear envelope to selectively allow transport in and out of the ciliary compartment (Deane et al., 2001; Rosenbaum and Witman, 2002). Cilia are commonly grouped into two broad classifications. Motile cilia generally have a “9+2” structure, which refers to the nine doublet microtubules and a central pair that make the axoneme. The nine outer doublets are composed of A and B subfibers that are connected by nexin linkages. Motility is conferred by the sliding of doublet microtubules relative to each other and this requires the central pair microtubules, outer and inner dynein arms that project from the A tubule and reversibly bind the neighboring B-tubule, and by radial spokes that connect the A tubule of the outer doublet to the central pair. In mammals, motile cilia are found on cells of the airway epithelium, brain ventricles, oviduct, sperm and embryonic node. Each of these, with the exception of sperm and the ciliated cells of the embryonic node, possesses hundreds of motile cilia, and their coordinated beating is vital for movement of fluid and particles over the surface of the epithelium. In the node, motile cilia are unique in that they lack a central pair but are able to rotate due to the activity of ciliary dynein, and this movement generates nodal flow that it critical for the establishment of left-right asymmetry (Hirokawa et al., 2006). The archetypal “9+0” primary cilium is distinct from the motile cilium in that it lacks a central pair and the
2 dynein arms required for ciliary motility, and only one is found per cell. Primary cilia have been found on almost every cell examined in the human body with the exception of the myeloid and lymphoid lineages. Once thought to be vestigial, primary cilia are now known to be important for detecting chemical and mechanical stimuli. Examples of specialized types of cilia are the olfactory receptor cilium, mechanosensing cilia in the kidney tubule epithelium and in bone, and the connecting cilium of photoreceptor sensory neurons. In addition, important developmental signaling pathways such as Sonic hedgehog, platelet-derived growth factor receptor (PDGFR) alpha, polycystin and Wnt are transduced through primary cilia (Berbari et al., 2009). Collectively, diseases or developmental defects caused by an underlying defect in the assembly or function of 9+2 or 9+0 cilia, or both, are termed ciliopathies. Major phenotypes associated with defects in nonmotile cilia include retinal degeneration, obesity, polydactyly, polycystic kidney disease (PKD) and mental retardation (Badano et al., 2006). Immobility of 9+2 cilia has been linked causally to the failure of embryonic turning resulting in embryonic lethality, respiratory failure, sterility, hydrocephalus and randomized left-right asymmetry. The pathologies caused by cilia defects are clearly diverse and reflect the important roles that cilia play in multiple tissues in the body.
Cilia formation and basal body generation Vertebrate ciliogenesis was first described by electron microscopy (EM) examination of ciliated tissues such as the multiciliated epithelia of rat airway epithelium (Sorokin, 1968) and Xenopus laevis skin and trachea (Steinman, 1968). Such early studies described ciliogenesis as proceeding in a series of steps: 1) cytoplasmic generation of basal bodies, where they acquire appendages required for basal body function, and the association of basal bodies with vesicles 2) basal body migration to the surface of the cell, where vesicles fuse with the plasma membrane, allowing the basal body to dock 3) and ciliary membrane growth and axoneme extension from the basal body. How ciliary length is then maintained is not well characterized in vertebrates, but in Chlamydomonas, this is achieved by balanced rates of assembly and disassembly (Marshall et al., 2005).
3 Intraflagellar Transport Protein synthesis does not occur inside the cilium, so components for cilia growth and function must be trafficked there. This is accomplished through intraflagellar transport (IFT), which was first identified in the unicellular flagellated algae Chlamydomonas reinhaardtii (Kozminski et al., 1993). IFT particles consist of “core” structural protein complexes, molecular motors and adaptor proteins, and there are two types of IFT: anterograde and retrograde. In vertebrates, the anterograde IFT complex consists of IFT particles B and the microtubule plus-end directed heterotrimeric kinesin-II complex consisting of KIF3A, KIF3B and KAP. Retrograde IFT complexes consist of IFT particles A and cytoplasmic dynein 2. IFT complex B and A are composed of at least 10 and 6 proteins, respectively, and are thought to facilitate cargo transport through interaction with protein binding domains (Scholey, 2008). Accumulations of IFT particles have been detected at transition fibers, leading to the hypothesis that docking at the transition zone might also be an important regulatory step for transport in and out of the cilium (Deane et al., 2001).
Basal body generation in cycling cells and multiciliated epithelia There are two unifying features common to all cilia and flagella: their axonemes are microtubule based, and they are anchored at their proximal ends by basal bodies. Different pathways exist for the generation of basal bodies. In cells that make a single cilium, the basal body derives from the mother centriole, which is duplicated according to the canonical centrosome cycle of dividing cells. In cells that make multiple cilia, basal bodies are generated according to two mechanisms: the canonical templated pathway and the acentrosomal non-templated pathway. In the latter, basal bodies arise de novo, and rather than a single centriole growing off the side of a pre-existing mother centriole as in the templated pathway, multiple basal bodies grow from an electron-dense mass termed the deuterosome, which are present in multiples in the cytoplasm (Dawe et al., 2007). Another difference is that centriole duplication and maturation occurs during G0 in multiciliated cells, which are terminally differentiated, whereas in most cells in the body centrioles are duplicated once per cell cycle and only in S-phase, and mature later in the cell cycle.
4 A core set of centriolegenesis proteins active in cycling cells has been defined. Polo-like kinase 4 (Plk4) is the most important regulator of centriolegenesis in that it is essential for centriolegenesis in human and flies and its expression is sufficient to induce the de novo formation of centrioles in Drosophila and the amplification of centrioles in human tissue culture cells (for review, see (Bettencourt-Dias and Glover, 2009). Overexpression of Sas-6 has also been demonstrated to produce extra centrioles, and both Sas-6 and Sas-4 are required for basal body duplication in human, fly and worm. It is not known how de novo centriolegenesis is regulated, or whether the molecules required for ciliogenesis in normal cells are also required for acentriolar formation of basal bodies. Depletion of Sas-6 prevents basal body duplication (Vladar and Stearns, 2007), but the role of other key centriolegenesis regulators Plk4 and Sas-4 are unknown. Recently, overexpression of Sas-6, Sas4 and Plk4 in CHO hamster cells was reported to induce the formation of dense aggregates that superficially resembled deuterosomes in that they were amorphous and were found surrounded by centrioles in the “centriolar flower” arrangement (Kuriyama, 2009). No components of deuterosomes have been identified however, and without markers it is difficult to know whether the “fibrillar aggregates” are bona fide deuterosomes. It has also been reported that depletion of the centriolar satellite protein PCM1 greatly reduces ciliogenesis in the hTert-RPE1cell line (Nachury et al., 2007), an immortalized cell line derived from retinal pigment epithelium cells, but the same effect was not observed in multiciliated cells despite near complete knockdown (Vladar and Stearns, 2007). This may be one example of differences in ciliogenesis in primary ciliated cells and multiciliated cells. The coiled coil protein CP110 has also been identified as being required for centriole biogenesis. CP110 localizes to the distal end of daughter centrioles in cycling cells, and overexpression of this protein was able to induce centriole formation in human cells (Kleylein-Sohn et al, 2007). CP110 and its interacting partner Cep97 have now been implicated in preventing untimely cilia formation in cycling cells. The finding that CP110/Cep97 overexpression could block ciliogenesis in serum-starved NIH 3T3 cells, and that their depletion resulted in the formation of cilia-like bodies in
5 cells outside of G0, was the first indication that the switch between centriole and basal body function is controlled by a negative regulatory mechanism (Spektor et al., 2007; Tsang et al., 2008). It is unknown whether a similar mechanism exists for controlling basal body function in multiciliated cells. Save for the brief period early in ciliogenesis when centrioles are migrating through the cytoplasm towards the apical cell surface, there is presumably no need in this cell type to block basal body function.
Cell cycle regulation of basal body generation in multiciliated epithelia In most cells, the centrosome and chromosome cycles are inextricably linked by a host of checkpoints and regulatory molecules. Multiciliated cells are unusual in that although the cells are terminally differentiated and new DNA is not synthesized, centrioles are able to replicate. How this unique cell cycle state is achieved is not fully understood. Just as centrosome duplication is prevented in normal cycling cells {Matsumoto, 1999 #277}, treatment of mouse tracheal epithelial cells (MTECs) grown in primary culture with CDK inhibitors just after the induction of differentiation prevents basal body duplication (Vladar, 2007). CDK2 activators cyclin E and cyclin A were shown to be upregulated at the mRNA and protein levels and localized to the nucleus in ciliating cells, suggesting that active Cdk2 is present during ciliogenesis and that it is activated during ciliogenesis by either cyclin A or cyclin E, or both. However, it has not been confirmed that CDK2, cyclin A or cyclin E are required for basal body duplication.
Centrosomes, cilia and human disease Misregulation of centrosomal genes have been associated with multiple human diseases, such as primary autosomal recessive microcephaly (Kaindl et al., 2009), lissencephaly (Tsai et al., 2007), primordial dwarfism (Rauch et al., 2008) and Seckel syndrome (Griffith et al., 2008), and most notably, a wide range of cancers. The idea that aberrant numbers of centrosomes can cause cancer was proposed long ago by Theodore Boveri, who postulated that cell division with monopolar or multipolar spindles and the resulting chromosome mis-segregation could lead to the loss or gain
6 of genes and tumorigenicity (Boveri, 2008). Although many cancers are associated with high rates of extra centrosomes (Nigg, 2006), and centrosome amplification is associated with genetic instability (Brinkley, 2001), Boveri’s hypothesis is complicated by the fact that at least some cell lines are able to cluster their centrosomes to ensure bipolar mitosis (Quintyne et al., 2005; Ring et al., 1982). More recently, experiments in flies engineered to overduplicate their centrioles by the overexpression of SAK, the functional ortholog of Plk4, suggest that centrosomes might lead to cancer through another mechanism. Although on average mitosis was delayed, the majority of the time cell division proceeded normally in somatic tissues of these flies. However, the asymmetric divisions of neural stem cells were perturbed, resulting in an expansion of the progenitor pool, and transplantation of tissue from SAK-overexpressing flies was sufficient to initiate tumorigenesis in wild type hosts, suggesting that the mechanism for centrosome-induced tumorigenicity might be disrupted asymmetric stem cell divisions (Basto et al., 2008). These experiments were a direct demonstration that, in flies, having too many centrosomes could lead to cancer. Conversely, flies lacking centrioles developed into adults with near-normal morphology. The most striking phenotype in these mutant flies was the complete inability to make cilia, which resulted in defective sensory neurons, a severe lack of coordination and early death (Basto et al., 2006). These results directly supported the viewpoint that ancestral purpose of centrioles is to make cilia, a hypothesis that stems from the fact that centrioles are conserved in all higher eukaryotes excluding nonciliated organisms such as plants and most yeasts (Marshall, 2009). Accordingly, diseases that are attributed to centriole defects or altered centriole number might actually be diseases of cilia formation or function (Nigg and Raff, 2009). As discussed, diseases of defective cilia assembly or function are termed ciliopathies. Phenotypically, as well as genetically, the ciliopathies overlap; defects in a single cilia gene can cause multiple symptoms, and a single disease might be caused by mutations in any number of cilia genes (for review, see (Cardenas-Rodriguez and Badano, 2009)). For instance, Bardet-Biedl Syndrome is a pleiotropic disease with clinical phenotypes that include obesity, retinal degeneration, mental
7 retardation/developmental delay, kidney dysfunction, hypogonadism and polydactyly, and less frequently neurocognitive impairment, hearing loss, craniofacial abnormalities, diabetes mellitus, and defects in cardiovascular and hepatic function. Mutations in 14 genes have been linked genetically to Bardet-Biedl Syndrome (BBS) and the proteins encoded by these genes localize predominantly to centrosomes, basal bodies or cilia. The BBSome, a stoichiometric complex of BBS1/2/4/5/7/8/9, promotes the trafficking of vesicles to the cilium for ciliary membrane extension (Nachury et al., 2007), thus linking the BBS disease phenotypes to cilia defects. Some BBS genes are mutated in multiple ciliopathies; BBS2 and BBS4 for example are mutated in Bardet-Biedl syndrome and Meckel-Gruber syndrome, a disease characterized by polydactyly, kidney and liver dysfunction and central nervous system malformations, and BBS6 is mutated in Bardet-Biedl syndrome, Meckel-Gruber syndrome, and McKusick-Kaufman syndrome, as disease characterized by heart malformations and polydactyly (Zaghloul and Katsanis, 2009). The ciliopathic phenotypes are understood to varying degrees at a mechanistic level. One of the least well understood phenotypes in Bardet-Biedl syndrome and other ciliopathies is obesity. BBS proteins have been implicated in regulating energy homeostasis because of the prevalence of obesity in Bardet-Biedl syndrome patients. Bbs2 (-/-), Bbs4(-/-), and Bbs6(-/-) mice are unable to respond to leptin by reducing food intake (Seo et al., 2009), and Bbs4(-/-) and Bbs6(-/-) mice also have increased leptin levels in the blood (Eichers et al., 2006; Fath et al., 2005). This may be related to IFT, since Kif3a and Ift88 mutants also exhibited obesity and increased blood leptin and insulin (Davenport et al., 2007). There is also evidence of defects in neurons involved in control of feeding. In Bbs4(-/-) and Bbs2(-/-) knockout mice, the G- protein coupled receptor melanin-concentrating hormone receptor 1 (MCHR1), which regulates feeding behavior, is mislocalized (Berbari et al., 2008). Furthermore, deletion of Kif3a of Ift88 specifically in pro-opiomelanocortin (POMC)-expressing cells in the hypothalamus largely prevented cilia formation and resulted in increased hyperphagia and weight gain, albeit not to the extent seen in whole animal knockouts (Davenport et al., 2007). Taken together, the results suggest that POMC neurons lacking cilia are unable to respond to leptin signaling, resulting in a defect in eating
8 control as well as constitutively high levels of circulating leptin. Additionally, a second mechanism for cilia-mediated energy control may be regulation of fat cell differentiation. A recent report showed the presence of transient primary cilia early during adipogenesis and demonstrated that BBS knockdown prevented cilia formation and adipocyte differentiation {Marion, 2009 #488}. The diseases associated with defects in motile cilia tend to be more straightforward due to the fact that motile cilia are primary known for their role in moving fluids. Ciliary beat frequency (CBF) is probably the best-studied aspect of motile cilia biology, as defects in beating have numerous effects on human health, as discussed. It has been known for some time that CBF can change in response to exogenous signals. The many biological and chemical compounds that can affect CBF include prostaglandin (Verdugo, 1980), extracellular ATP (Okada et al., 2006), cytokines (Jain et al., 1995), cigarette smoke (Stanley et al., 1986), ethanol (Sisson, 1995) and Vicks VapoRub (Abanses et al., 2009). Many neurotransmitters have been reported to modulate CBF, although the pathways are complex since effects seem to differ between species. CBF-altering neurotransmitters include acetylcholine (Tamaoki et al., 1995; Zagoory et al., 2001), noradrenalin (Maruyama, 1984), substance P (Schuil et al., 1995) and serotonin (Konig et al., 2009; Nguyen et al., 2001). Recently it was found that motile cilia on mammalian airway epithelial cells are also sites for G-protein coupled receptor proteins, and that motile cilia alter their beating in response to exposure to bitter taste compounds (Shah et al., 2009). Although the functions ascribed to motile cilia and primary cilia have historically been separate, this finding suggests that there may be more overlap than previously thought.
9 References
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12 .$=&-#9=.<$(X.=B(+`M576'(#(;-<=&.$([email protected].&$=(.$(B4/#$(9.>.#-?(0.%&#%&*(H#+) !#22*(CEFCIQH7Q*( 8&-04P<'(M*(C7I6*(+#5hH0&;&$0&$=(B<-/<$#>(%=./4>#=.<$(<@(9.>.#-?(#9=.R.=?*( 56%(4#*(5IDFQNGHE*( 8>#0#-'(`*2*(566Q*(+&$=-.<>&(#%%&/">?(.$(9.>.#=&0(&;.=B&>.#>(9&>>%*(F'(^&;#-=/&$=(<@( W&$&=.9%*(8<>*(MB*^(^.%%&-=#=.<$*(,=#$@<-0(e$.R&-%.=?*(CGC*( 8>#0#-'(`*2*'(#$0(O*(,=&#-$%*(566Q*(L<>&94>#-(9B#-#9=&-.T#=.<$(<@(9&$=-.<>&( #%%&/">?(.$(9.>.#=&0(&;.=B&>.#>(9&>>%*(;)!#22):/-2*(CQIFDCHG5*( c#PB><4>'(K*!*'(#$0(K*(2#=%#$.%*(5667*(L&9B#$.%=.9(.$%.PB=%(.$=<(1#-0&=H1.&0>( %?$0-(9.>.<;#=B?*(;)!2/')F'+#$%*(CC7FG5IHDQ*( c#P<<-?'(Y*'(!*(1-#./#$'(J*(WB&"&-'(#$0(c*(M-.&>*(566C*(3<>&(<@(9#>9.4/(#$0( 9#>/<04>.$(.$(9.>.#-?(%=./4>#=.<$(.$049&0("?(#9&=?>9B<>.$&*(&.);)3">$/-2) !#22)3">$/-2*(5I6F+C66H7*(
13
Chapter 2: Transcriptional profiling of ciliogenesis
! 14 Abstract Motile cilia are crucial for human health, and recent evidence in mammals suggests that they possess sensory functions, like primary cilia. Efforts are underway to catalog the structural and functional components of these important organelles. We perform transcriptional assays on in vitro differentiated cultures of mouse tracheal epithelial cells (MTECs), a complex mixture of cells containing multiciliated cells as well as goblet cells and basal cells. We use as our source of cells a mouse that expresses GFP from the FOXJ1 ciliated cell-specific promoter and we show that this allows us to 1) obtain a pure population of ciliated cells for our analysis, and 2) analytically subtract out genes unrelated to ciliogenesis by comparing the ciliated cell transcriptome to that of other epithelial cells in MTEC cultures. Assaying two timepoints during early and late ciliogenesis identifies 873 genes that are differentially expressed in ciliated cells. This dataset will inform future efforts to understand the differentiation process of multiciliated cells, and how motile cilia are made and function.
Introduction There have been many large-scale studies performed in the last 10 years to identify structural components of centrosomes and cilia and the regulatory molecules that control their formation. The cilia proteome is an online, searchable compendium of 11 such studies: 2 comparative genomics studies, 2 promoter analyses, 1 transcriptional analysis and 6 protein analyses (Gherman et al., 2006). The comparative genomics studies defined a cilia and basal body proteome by identifying genes that are common to ciliated organisms (H.sapiens, C.elegans, D.melanogaster, C.reinhardtii, T.brucei, P.falciparum) but missing from non-ciliated organisms (A.thaliana, D.discoideum, S.cerevisiae) (Avidor-Reiss et al., 2004; Li et al., 2004). The X-box motif is the recognition site for ciliogenic Rfx transcription factors (see Chapter 4), and two studies have identified C.elegans genes that contain this motif in their promoter regions (Blacque et al., 2005; Efimenko et al., 2005). The transcriptional readout during flagellar regrowth in C.reinhardtii was analyzed (Stolc et al., 2005), and the remaining 6 studies were mass spectrometry analyses of purified
! 15 ciliary axonemes from human bronchial epithelium (Ostrowski et al., 2002), interphase centrosomes from a transformed human cell line (Andersen et al., 2003), mouse photoreceptor sensory cilia (Liu et al., 2007), Chlamydomonas centrioles (Keller et al., 2005) and flagella with associated basal bodies (Pazour et al., 2005), and Trypanosome flagella (Broadhead et al., 2006). Integration of these studies in the ciliary proteome has created a list of more than 1200 unique human orthologs (Gherman et al., 2006), but we believe that the list is likely incomplete for many reasons. The comparative genomics studies eliminate genes that are involved in ciliogenesis but are involved in other cellular functions possessed by non-ciliated organisms (for instance, actin cytoskeleton remodeling). The human centrosome proteome is an excellent resource for the study of novel centrosome components, but it may not include many proteins that have basal body functions because the cell types used for purification does not usually make cilia. The mouse photoreceptor sensory neuron proteome does not include proteins required for ciliary motility or basal body duplication, and the preparation of motile cilia from human respiratory epithelium for the proteomics analysis removed membranes and membrane components. Finally, there are pathways for signaling and human diseases that cannot be recapitulated in non-mammalian systems. To identify additional genes associated with mammalian motile cilia formation and basal body duplication, we performed transcriptional analyses on ciliated mouse tracheal epithelial cells (MTECs). We were particularly interested in capturing cells at the time when they were actively duplicating centrioles, so we utilized an established method for the in vitro differentiation of MTECs that recapitulates wounding of the airway epithelium (You et al., 2002). Previous characterization of ciliogenesis in these cultures demonstrated that the establishment of ALI could be used to confer a measure of synchronization on the ciliating cells, however crude, and that ciliogenesis in these cultures occurred in discrete temporal steps measureable by immunofluorescence light microscopy (Vladar and Stearns, 2007). Differentiation progresses as follows: MTECs dissociated from the trachea grow and divide to repopulate the filter; once the culture is confluent the cells become contact inhibited, at which point they possess a long primary cilium; basal bodies are generated and
! 16 migrate to the apical cytoplasm; and finally, ciliary axonemes extend from apically docked basal bodies. A study using a similar ALI culture system for differentiation of human airway epithelial cells demonstrated that measurable gene expression changes occur over the timecourse of ciliogenesis, and indeed, this study identified groups of functionally similar genes with distinct expression patterns (Ross et al., 2007), indicating that our approach was a workable one. However, our ability to sort MTEC cultures into ciliated and non-ciliated populations allowed us to focus specifically on ciliated cells and ciliogenesis, whereas the prior study was a more general survey of the mixed population of cells in the mucociliary epithelium.
Results Identification of the ciliated cell transcriptome To identify genes that were differentially expressed in ciliated cells, we performed microarrays on FACS sorted samples from MTEC cultures derived from mice expressing GFP from a FoxJ1 ciliated-cell specific promoter (Fig.1a). MTECs were harvested at 12 days after establishment of the air-liquid interface (ALI), a treatment that induces differentiation of ciliated cells in the columnar epithelium (You et al). At this time point, the majority of cells expressing GFP are fully ciliated with the occasional cell at an earlier stage of ciliogenesis (Vladar et al 2007). Although between 20-60% of the surface cells were ciliated, the total percentage of ciliated cells in the FACS-sorted material was generally <5% (data not shown). To remove non- ciliated cells from our analysis, we FACS sorted single-cell suspensions of MTEC cultures into GFP+ and GFP- populations (Fig.1b). After sorting, 90-95% of cells in the GFP+ population had observable cilia and/or duplicated basal bodies by acetylated alpha tubulin staining (Fig.1c). Fewer than 1% of cells recovered in the GFP- population stained positive for these markers. We reasoned that since ciliogenesis proceeds through discrete stages such as basal body duplication, docking, and axoneme formation (Vladar and Stearns, 2007) there would be accompanying temporal changes in the transcriptional profiles of cells at different stages of ciliogenesis. This is supported by the observation that gene
! 17 expression profiles of mucociliary differentiation in human bronchial epithelial cell cultures, which contain basal, secretory and ciliated cells, change significantly over the course of the 4-week differentiation period (Ross, Dailey et al. 2007). To enrich for genes encoding structural and regulatory proteins involved in basal body duplication, we harvested GFP-expressing cells at a second time point four days after ALI (ALI+4). RNA was extracted from the sorted cell populations. Sorted populations were high purity but low yield so RNA was amplified prior to labeling and hybridization. Experimental samples were hybridized against a commercially available reference pool of mouse RNAs that was also amplified to control for potential biases in this step. Hybridizations consisted of either a GFP+ or GFP- sample against a universal reference (Fig. 2a). The differential expression of individual genes was calculated in comparison to the universal RNA reference.
After applying a fold-change cutoff of M>1.0 or M<-1.0 (where M = log2 ratio experimental/reference), and adjusted p-value filters p<0.05, p<0.01 or p<0.001, we obtained the number of significantly differentially expressed genes. In general, there were far more differentially expressed genes at ALI+4 than at ALI+12. 4255 genes were upregulated at ALI+4, and 959 at ALI+12 (adjusted p-value <0.05). 3748 genes were downregulated at ALI+4, and 330 at ALI+12 (adjusted p-value < 0.05). The results are summarized in Fig. 3a.
Filtering gene lists by subtraction Datasets from previous microarray studies performed on ciliated cell cultures were enriched in genes involved in epithelial processes such as cell adhesion, apical- basal polarity, and components of tight junction proteins (Chhin et al., 2008; Ross et al., 2007). Ciliogenesis undoubtedly requires the activity of some of these proteins, but we were interested in identifying molecules that were required specifically for ciliogenesis. We reasoned that by subtracting the transcriptional profiles of FoxJ1- GFP positive cells against GFP- cells from the same cultures we might remove genes that are generally expressed in tracheal epithelia. We also sought to remove the handling effects from culture and harvesting protocols.
! 18 Subtractive analysis was performed on the ALI+4 and ALI+12 arrays (Fig. 2b). As expected, far fewer differentially expressed genes were identified in ciliated cells when compared to GFP-cells than when compared to the mouse universal reference (Fig. 3a). 791 genes were significantly upregulated at ALI+4 with respect to GFP- cells, and 105 genes at ALI+12 (M>1.0, adjusted p-value p<0.05). 182 genes were downregulated at ALI+4, and only 5 genes were downregulated at ALI+12 (M < -1, adjusted p-value p<0.05). We compared the overlap of genes in ALI4, ALI12 and GFP- that were differentially expressed to ensure that genes common to GFP+ and GFP- cells were being removed (Fig. 3b). Although there was a large overlap between upregulated genes at ALI+4 and ALI+12 more than half of all significantly upregulated genes at ALI+4 are not upregulated at ALI+12. Similarly, the majority of genes downregulated at ALI+4 are not downregulated at ALI+12 or in the GFP- cells. This confirms that ciliating cells at 4 and 12 days post-ALI have unique transcriptional profiles that are different from non-ciliating cells.
Internal validation of microarray data To assess the quality of our microarray data we selected genes that were upregulated 10-fold or more and searched for them against a ciliary proteome database- a collection of genes identified as ‘ciliary’ through proteomic, genomic and comparative genomic studies (Gherman et al, 2006). In the early dataset 314 genes were identified that met this criteria, 44% of which were found in one or more studies in the ciliary proteome (Fig. 4). 125 genes were 10-fold or more upregulated in the late dataset, and 54% of these genes were found in the proteome (Fig. 5). Secondly, we compared the results of our analysis more closely to the two most similar large-scale studies available for comparison: the first, a proteomic analysis of purified ciliary axonemes {Ostrowski, 2002 #213} that is included in the Cilia Proteome, and the second, a transcriptional analysis of ciliogenesis (Ross et al., 2007). Both used as their source material primary cultures of human bronchial epithelial cells (HBECs) differentiated under ALI conditions. The second study differs from ours in that they did not have the ability to sort HBECs into ciliated and non-ciliated populations, so samples from whole cultures were used in the
! 19 hybridizations. The mouse orthologs of proteins from the axonemal purification were identified by non-reciprocal BLAST searching using the published accession numbers for unique genes. 206 accession numbers were entered into the NCBI BLAST database. 5 protein accession numbers were not recovered. The cognate genes for 201 mouse proteins were obtained from NCBI. Of these, 36 were not found on the MEEBO arrays. 23 duplicate genes were found and removed. Of the remaining 141 genes encoding mouse orthologs of HBEC axonemal proteins, 93 (66%) were upregulated more than 2-fold in ciliated MTECs with respect to nonciliated cells. Only 49/301 (16%) genes were upregulated more than 2-fold in the HBEC transcriptome, which included in the analysis entire gene families (ie., alpha and beta tubulin, 14-3-3) when individual members were not identified in the proteomic data (Ross et al., 2007). These genes were not included in our analysis, accounting for the difference in total genes assayed. This confirms that our transcriptional profiling of ciliogenesis improves upon a similar, pre-existing dataset that was created without the use of cell sorting. Our isolation of ciliated cells from the non-ciliated cells in MTEC cultures relied upon the use of EGFP expressed from the FoxJ1 promoter. Our microarray analysis successfully identified FoxJ1 as a significantly upregulated gene at both time points. Putative FoxJ1 targets have been identified by ChIP analysis and microarray study of Xenopus embryos (Stubbs et al., 2008; Yu et al., 2008). As a third means of validating our dataset we searched for upregulation of putative FoxJ1 targets. The mouse orthologs of 43 of 86 putative FoxJ1 targets were upregulated (M>1.0) in MTECs, including Dnahc9, Wdr78, Efhc1, Cetn2 and Tekt1 (Fig. 6). Only 8 target genes had been previously identified in the 11 studies comprising the Cilia Proteome. This suggests that many of the genes regulated by FoxJ1 may encode proteins that are not localized to basal bodies or cilia and likely have roles in cellular pathways outside of ciliogenesis.
GO Term analysis Finally, we performed GO term analysis on the subtracted transcriptomes from ALI+4 and ALI+12 (Fig. 7). Genes that were upregulated 5-fold or greater in ciliated
! 20 cells with respect to non-ciliated cells were submitted to GoTermFinder (go.princeton.edu/cgi-bin/GoTermFinder) to identify terms that were enriched in ciliated cell transcriptomes relative to the genome. 730 and 514 genes met the criteria for the ALI4 time point and the ALI 12 time point, respectively. ALI+4 and ALI+12 were, by GO Term analysis, largely similar. Many of the overrepresented (p<0.05) GO Terms detected in both ALI+4 and ALI+12 transcriptomes were obviously associated with cilia: microtubule-based process, microtubule cytoskeleton and cilium cellular compartments, and motor activity molecular function. These terms were not similarly enriched amongst genes upregulated 5-fold or more in the non-ciliated cell (GFP-) transcriptome. Biological process terms that were enriched in the non-ciliated cells (GFP-) included tissue, epithelium and tube development and oxidation- reduction biological processes, and cell-cell junctions, apical junction complex, extracellular matrix and basement membrane cellular component terms. That these epithelial and tissue terms did not appear in the ALI+4 and ALI+12 datasets validates our subtractive transcriptomic analysis of ciliated cells. Interestingly, there were notable differences between ALI+4 and ALI+12 as well. The spermatogenesis biological process was highly overrepresented, consistent with other genomic and proteomic studies of cilia (Chhin et al., 2008; McClintock et al., 2008; Sammeta et al., 2007; Stolc et al., 2005), but at ALI+4 and not ALI+12. Similarly, ALI+4 was enriched in the centrosome cellular compartment term and the fat cell differentiation biological process, but these were not enriched in ALI+12. The upregulated genes from the latter category included most of the BBS genes, consistent with the role of the BBSome in vesicular trafficking to the basal bodies and cilium (Blacque and Leroux, 2006; Leroux, 2007; Nachury et al., 2007). Surprisingly, cilium assembly and left/right asymmetry GO terms were enriched at ALI+4 also, but not ALI+12. This suggests that at ALI+ 4 cells have upregulated mRNAs for duplication of basal bodies but also for ciliary components in anticipation of ciliary formation. This effect was also observed during flagellar regrowth in C.reinhardtii, in which genes upregulated early after de-flagellation were involved in ciliary regrowth, while genes upregulated later were involved in cilia maintenance (Stolc et al., 2005). Interestingly,
! 21 ALI+12 but not ALI+4 was enriched for growth factor binding and cyclin-dependent protein kinase activity molecular function terms, as well as anterior/posterior pattern formation, and brain development. We speculate that the ALI+4 transcriptome is enriched in genes involved in making basal bodies and cilia, and that ALI+12 is enriched in genes for functions that have been linked to mature cilia, such as signaling.
Discussion We have characterized the transcriptional profile of mouse ciliated tracheal epithelial cells using a system for in vitro culture. We utilized a transgenic mouse line expressing GFP from the FoxJ1 ciliated cell specific promoter to obtain highly pure populations of cells at two time points after the induction of ciliogenesis by ALI establishment. Two datasets have been generated that describe the multiciliated cell transcriptome during early ciliogenesis (ALI+4) when basal bodies are being duplicated, and during late ciliogenesis (ALI+12) when cilia are being maintained. The transcriptomes can be viewed in comparison with non-ciliated cells in the MTEC cultures or a reference pool of mouse RNAs. The former can be used to examine genes that are important for multiciliated cell differentiation, but may also be important in the non-ciliated cells of airway epithelium. The latter can be used to isolate genes that are solely associated with motile cilia. Refining the list in this way successfully removed epithelial genes form consideration. The ciliated cell transcriptome was validated in four ways: 1) by analysis of genes that were upregulated 10-fold or more, using the cilia proteome as a source of ciliary components; 2) by comparison to proteomic and transcriptomic analyses of motile cilia axonemes and mucociliary differentiation, respectively, from human airway epithelia; 3) by comparison to a list of putative and known FoxJ1 target genes from Xenopus, and 4) by GO term analysis. Applying an arbitrary significance cutoff of 2-fold differential expression generated a list of about 800 “candidate” centrosomal and ciliary genes of which nearly half of the most upregulated genes have been linked to cilia/basal bodies by at least one other study in the Ciliary Proteome. The following chapters will describe
! 22 the characterization of a selection of genes that are upregulated in the ciliated MTEC transcriptome.
! 23 a b FoxJ1/EGFP mice FoxJ1/EGFP- FoxJ1/EGFP+ 1000 800 remove tracheas 600 400 200
MTECs grown at ALI Forward Scatter 0 GFP- GFP+ 1 1 10 10 100 100
(air liquid interface) 1000 1000 10000 10000 FACs sort FITC
non- c ciliating ciliating cells cells microarrays GFP- GFP+ vs vs
reference reference ciliated cell non-ciliated cell transcriptome transcriptome
subtracted ciliated cell transcriptome
Figure 1. Microarray analysis of ciliating mouse tracheal epithelial cells. (a) Experimental flowchart detailing microarray analysis. (b) FACS analysis of cells dissociated from a wild type mouse trachea (left panel) and a FoxJ1/EGFP mouse trachea. The red and blue rectangles are representative gates used to sort GFP+ and GFP- populations, respectively. (c) Sorted cell populations were stained with DAPI (blue) and acetylated alpha tubulin/gamma tubulin antibody (green) to detect cilia and basal bodies. After sorting, 90-95% of cells in the GFP+ population harvested at ALI+12 had observable cilia and or duplicated basal bodies by acetylated alpha tubulin staining. In contrast, fewer than 1% of cells recovered in the GFP- population stained positive for these markers, although some had a single primary cilium (arrowhead).
24 a loop design common reference design
ciliating cell ciliating cell ciliated cell non-ciliated cell
ciliated cell non-ciliated cell reference reference reference can easily extend experiment • minimal # of arrays needed • • direct comparison • less RNA needed b Late Timepoint Design Matrix for Late Timepoint Cy3 Cy5 Baseline Ciliated Array 1 Ref GFP+ (ALI+12) 1 1 Array 2 GFP+ (ALI+12 Ref -1 -1 Array 3 GFP+ (ALI+12) Ref -1 -1 Array 9 Ref GFP- 1 0 Array 10 Ref GFP- 1 0 Array 11 Ref GFP- 1 0 Early Timepoint Design Matrix for Early Timepoint Cy3 Cy5 Baseline Ciliated Array 4 Ref GFP+ (ALI+4) 1 1 Array 5 Ref GFP+ (ALI+4) 1 1 Array 6 GFP+ (ALI+4), dyeswap Ref -1 -1 Array 7 GFP+ (ALI+4), dyeswap Ref -1 -1 Array 8 GFP+ (ALI+4) Ref -1 -1 Array 9 Ref GFP- 1 0 Array 10 Ref GFP- 1 0 Array 11 Ref GFP- 1 0 Figure 2. Microarray hybridizations and design matrices for differential expression analysis. a) Schematic outlining the hybidizations for two microarray experimental designs. The common reference design was chosen. b) Experimental samples and hybridizations are shown on the left half of the chart. The right side of he chart shows the matrices that were applied in LIMMA during analysis. The matrices were designed for grouping differentially expressed genes into “baseline” and “ciliated” effects. A score of 1 indicates that differentially regulated genes from the given experiment participate in the indicated effect. The - indicates a dyeswap experiment. The 0 notation indicates that genes that are differentially regulated do not participate in the condition. Therefore, according to the late timepoint matrix, the baseline group will consist of genes that are differentially regulated in GFP+ arrays and GFP- arrays, and the ciliated group will only contain the genes that are differentially expressed.
25 a
Non-Subtracted Subtracted
genes genes
b Non-Subtracted Subtracted
ALI+4 ALI+12 ALI+4 ALI+12 GFP-
GFP- GFP-
Figure 3. Differentially expressed genes in MTECs, and the effect of subtractive analysis. a) The number of differentially expressed genes before and after subtraction. Genes in the upregulated categories were filtered for M>1. Genes in the downregulated categories were filered for M<-1. Subtraction against non-ciliated cells (GFP-) decreases the number of genes that pass the M and p-value filters for differential expression in ALI 4 and ALI 12. b) Overlap of upregulated genes before and after subtraction. Genes with M>1.0 and p<0.05 were selected from ALI 4, ALI 12 and GFP- vs reference arrays. The left panel shows the overlap of genes that are upregulated in ciliated cells at ALI 4 and ALI 12 with genes that are upregulated in non-ciliated cells (GFP-). The right panel confirms that the overlap is reduced when the ALI 4 and ALI 12 tran- scripional data are subtracting against the non-ciliated cell data. The GFP- group in both panels represents upregulated genes in non-ciliated cells relative to the reference RNA.
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Lrdd NM_022654 3.69 12.93 1.96E-05 6.09E-03
Kif9 NM_010628 4.53 23.05 1.06E-05 4.97E-03 Ift74 NM_026319 3.39 10.45 2.31E-04 1.86E-02 1
Myl4 NM_010858 4.90 29.89 2.62E-05 6.54E-03
Bbs5 NM_028284 4.14 17.59 9.72E-06 4.97E-03 1 1
Pim2 NM_138606 4.01 16.16 6.80E-06 4.25E-03
Plk4 NM_011495 5.15 35.45 1.72E-05 5.70E-03
Ift81 NM_009879 3.66 12.61 2.12E-05 6.17E-03 1 1 1
Cenpj XM_127861 3.68 12.86 3.89E-05 7.44E-03
Mns1 NM_008613 7.28 155.61 8.23E-07 2.26E-03 1
Rage NM_011973 3.60 12.17 1.13E-04 1.23E-02
Ttc26 NM_153600 3.55 11.73 1.82E-05 5.82E-03 1 1 1 1
Kif3a NM_008443 3.34 10.15 3.30E-05 6.86E-03 1 Ube2t NM_026024 3.34 10.09 6.36E-04 3.44E-02
Stk33 XM_358897 3.56 11.79 8.24E-05 1.07E-02
Cdkl1 NM_183294 3.92 15.13 1.73E-05 5.70E-03
Cdkl2 NM_016912 4.09 17.07 1.03E-04 1.18E-02
Efhc1 XM_129694 4.54 23.29 2.45E-04 1.92E-02 1
Lrrc18 NM_026253 4.27 19.25 2.72E-05 6.56E-03
Lrrc34 XM_487721 6.57 94.96 1.37E-05 5.62E-03
Ribc1 NM_025660 5.39 41.79 3.89E-06 3.14E-03 1
Lrrc23 NM_013588 3.56 11.77 4.47E-03 1.04E-01 1
Lrrc50 NM_026648 4.11 17.25 9.38E-04 4.25E-02 1
Fank1 NM_025850 4.36 20.49 5.63E-07 2.26E-03
Nphp4 NM_153424 3.53 11.53 3.79E-04 2.50E-02 1 1
Dnali1 NM_175223 4.38 20.79 2.22E-05 6.25E-03 1
Lrrc48 NM_029044 4.66 25.24 1.56E-03 5.66E-02 1
Spag6 NM_015773 5.26 38.30 5.29E-07 2.26E-03 1
Wdr63 NM_172864 4.33 20.11 6.49E-05 9.69E-03 1
Mast3 NM_199308 4.00 16.05 3.09E-05 6.62E-03
Wdr78 NM_146254 5.24 37.80 8.57E-05 1.08E-02 1
Wdr38 XM_484992 3.66 12.60 3.07E-05 6.62E-03
Ccdc96 NM_025725 4.22 18.64 1.83E-05 5.82E-03 1
Wdr16 NM_027963 3.73 13.26 2.14E-03 6.74E-02 1 1 1
Wdr69 NM_027725 4.23 18.75 1.63E-05 5.70E-03
Txndc6 XM_135038 3.35 10.19 1.53E-02 2.08E-01 1
Ccdc39 NM_026222 6.15 71.07 2.85E-06 2.49E-03
Dnahc9 XM_110968 4.55 23.51 3.03E-05 6.62E-03 1
Dnahc8 NM_013811 4.71 26.20 9.78E-06 4.97E-03 1
Ccdc41 NM_029852 4.53 23.06 2.50E-05 6.37E-03
Ccdc18 XM_144475 5.12 34.85 1.41E-05 5.68E-03
Tuba1a NM_011653 4.12 17.34 1.11E-03 4.65E-02
Cep135 NM_199032 3.73 13.28 2.75E-04 2.08E-02 1 1
Gene.Symbol Accession MChange Fold P.Value adj.P.Val Li Efimenko Stolc OstrowskiPazour Avidor-ReissKeller Andersen Blacque Broadhead Liuhits #
Gene.Name
tubulin, alpha 1A alpha tubulin,
renal tumor antigen tumor renal
centromere protein J protein centromere
WD repeat domain 63 domain repeat WD
WD repeat domain 78 domain repeat WD
WD repeat domain 38 domain repeat WD
WD repeat domain 16 domain repeat WD
WD repeat domain 69 domain repeat WD
proviral integration site 2 site integration proviral
kinesin family member 9 member family kinesin
centrosomal protein 135 protein centrosomal
myosin, light polypeptide 4 polypeptide light myosin,
kinesin family member 3A member family kinesin
serine/threonine kinase 33 kinase serine/threonine
sperm associated antigen 6 antigen associated sperm
polo-like kinase 4 (Drosophila) 4 kinase polo-like
coiled-coil domain containing 96 containing domain coiled-coil
leucine rich repeat containing 18 containing repeat rich leucine
leucine rich repeat containing 23 containing repeat rich leucine
leucine rich repeat containing 50 containing repeat rich leucine
thioredoxin domain containing 6 containing domain thioredoxin
leucine rich repeat containing 48 containing repeat rich leucine
coiled-coil domain containing 39 containing domain coiled-coil
coiled-coil domain containing 41 containing domain coiled-coil
Leucine rich repeat containing 34 containing repeat rich Leucine
dynein, axonemal, heavy chain 9 chain heavy axonemal, dynein,
dynein, axonemal, heavy chain 8 chain heavy axonemal, dynein,
coiled-coil domain containing 18 18 containing domain coiled-coil
Bardet-Biedl syndrome 5 (human) 5 syndrome Bardet-Biedl
RIB43A domain with coiled-coils 1 coiled-coils with domain RIB43A
tetratricopeptide repeat domain 26 domain repeat tetratricopeptide
leucine-rich and death domain containing domain death and leucine-rich
EF-hand domain (C-terminal) containing 1 containing (C-terminal) domain EF-hand
meiosis-specific nuclear structural protein 1 protein structural nuclear meiosis-specific
ubiquitin-conjugating enzyme E2T (putative) E2T enzyme ubiquitin-conjugating
nephronophthisis 4 (juvenile) homolog (human) homolog (juvenile) 4 nephronophthisis
fibronectin type 3 and ankyrin repeat domains 1 domains repeat ankyrin and 3 type fibronectin
Microtubule associated serine/threonine kinase 3 kinase serine/threonine associated Microtubule
dynein, axonemal, light intermediate polypeptide 1 polypeptide intermediate light axonemal, dynein,
intraflagellar transport 74 homolog (Chlamydomonas) homolog 74 transport intraflagellar
cyclin-dependent kinase-like 1 (CDC2-related kinase) (CDC2-related 1 kinase-like cyclin-dependent
cyclin-dependent kinase-like 2 (CDC2-related kinase) (CDC2-related 2 kinase-like cyclin-dependent
TNF receptor-associated factor 3 interacting protein 1 protein interacting 3 factor receptor-associated TNF Traf3ip1 XM_129927 4.39 20.96 1.24E-06 2.26E-03 1 1 1
intraflagellar transport 81 homolog (Chlamydomonas) homolog 81 transport intraflagellar
serine/threonine kinase 36 (fused homolog, Drosophila) homolog, (fused 36 kinase serine/threonine Stk36 NM_175031 4.80 27.89 2.89E-06 2.49E-03
tubulin polymerization-promoting protein family member 3 member family protein polymerization-promoting tubulin Tppp3 NM_026481 4.25 19.06 1.43E-03 5.42E-02 1 1
NIMA (never in mitosis gene a)-related expressed kinase 5 kinase expressed a)-related gene mitosis in (never NIMA Nek5 NM_177898 3.33 10.07 3.82E-03 9.62E-02
NIMA (never in mitosis gene a)-related expressed kinase 11 kinase expressed a)-related gene mitosis in (never NIMA Nek11 NM_172461 3.85 14.38 2.83E-06 2.49E-03 dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3 kinase regulated tyrosine-(Y)-phosphorylation dual-specificity Dyrk3 NM_145508 4.35 20.34 9.60E-06 4.97E-03
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Kit NM_021099 3.40 10.59 1.65E-04 1.53E-02
Fhl1 NM_010211 5.03 32.75 1.57E-04 1.50E-02
Mlf1 NM_010801 5.40 42.37 3.78E-05 7.33E-03
Agr3 NM_207531 6.56 94.58 8.70E-07 2.26E-03
Fgl1 NM_145594 3.87 14.65 3.65E-04 2.46E-02
Ttc25 XM_126529 4.88 29.40 1.07E-05 4.97E-03
Rtdr1 XM_354544 4.89 29.75 2.21E-06 2.49E-03
Ttc18 XM_127606 5.19 36.41 3.39E-05 6.96E-03
Foxj1 NM_008240 3.47 11.09 6.06E-04 3.34E-02
Bbs9 NM_181316 4.43 21.57 1.20E-05 5.37E-03
Lrrc6 NM_019457 5.99 63.36 5.31E-06 3.74E-03 1
Kcnrg NM_206974 4.36 20.52 2.07E-04 1.74E-02
Bbs7 NM_027810 3.59 12.02 1.73E-05 5.70E-03 1 1
Hydin NM_172916 4.30 19.69 6.04E-06 4.04E-03 1
Klhdc9 XM_287054 4.90 29.81 8.91E-07 2.26E-03 1
Cep76 XM_129027 3.61 12.25 5.37E-05 8.88E-03 1
Mdm1 NM_148922 6.40 84.25 1.75E-06 2.47E-03
Armc3 XM_130012 4.16 17.85 2.31E-04 1.86E-02
Sass6 XM_131155 3.46 10.97 8.63E-05 1.09E-02 1
Ddah1 NM_026993 4.01 16.08 1.98E-04 1.69E-02 1
Cep97 NM_028815 4.02 16.18 3.67E-05 7.24E-03
Ypel1 NM_023249 3.62 12.28 4.10E-05 7.60E-03 1
Morn1 XM_131829 3.70 12.97 2.92E-05 6.58E-03
Cep78 NM_198019 4.15 17.76 6.66E-05 9.72E-03
Anubl1 XM_132758 3.52 11.46 2.99E-05 6.62E-03
Ruvbl1 NM_019685 3.58 11.92 1.32E-04 1.35E-02
Spag1 NM_012031 3.53 11.54 4.38E-05 7.86E-03
Ropn1l NM_145852 5.90 59.63 8.20E-06 4.61E-03
Efcab1 NM_025769 3.93 15.25 3.39E-06 2.80E-03
Cdh26 NM_198656 3.52 11.47 3.14E-05 6.65E-03
Mdh1b NM_029696 5.67 51.01 1.00E-05 4.97E-03
Osbpl6 NM_145525 4.51 22.86 6.67E-06 4.24E-03
Tsga14 NM_031998 3.63 12.37 1.12E-04 1.23E-02
Dusp18 NM_173745 3.59 12.04 3.09E-05 6.62E-03 1
Ccdc11 XM_128864 4.33 20.10 1.64E-05 5.70E-03
Cep152 XM_130551 3.85 14.42 7.54E-05 1.03E-02
Ccdc65 NM_153518 3.57 11.90 8.42E-05 1.08E-02 1
Ankrd42 NM_028665 3.55 11.74 2.86E-04 2.14E-02
Tmem67 NM_177861 4.41 21.32 1.33E-06 2.26E-03
Ccdc113 NM_172914 4.53 23.16 4.76E-04 2.91E-02 1
Ccdc114 XM_133435 3.62 12.33 3.02E-05 6.62E-03 1
Gene.Symbol Accession MChange Fold P.Value adj.P.Val Li Efimenko Stolc OstrowskiPazour Avidor-ReissKeller Andersen Blacque Broadhead Liuhits #
4933434I06Rik XM_283704 4.82 28.32 5.94E-04 3.29E-02 1
4932425I24Rik XM_489540 6.73 106.00 2.02E-05 6.10E-03 1
D630013G24Rik XM_357326 4.74 26.68 1.72E-07 2.23E-03
1700008D07Rik XM_485969 4.03 16.31 5.63E-04 3.19E-02 1
1700026D08Rik NM_029335 4.34 20.28 5.67E-05 8.99E-03
Gene.Name
kit oncogene kit
ropporin 1-like ropporin
forkhead box J1 box forkhead
cadherin-like 26 cadherin-like
RuvB-like protein 1 protein RuvB-like
centrosomal protein 76 protein centrosomal
centrosomal protein 97 protein centrosomal
centrosomal protein 78 protein centrosomal
testis specific gene A14 gene specific testis
fibrinogen-like protein 1 protein fibrinogen-like
hydrocephalus inducing hydrocephalus
centrosomal protein 152 protein centrosomal
kelch domain containing 9 containing domain kelch
myeloid leukemia factor 1 factor leukemia myeloid
ankyrin repeat domain 42 domain repeat ankyrin
yippee-like 1 (Drosophila) 1 yippee-like
transmembrane protein 67 protein transmembrane
MORN repeat containing 1 containing repeat MORN
sperm associated antigen 1 antigen associated sperm
potassium channel regulator channel potassium
armadillo repeat containing 3 containing repeat armadillo
four and a half LIM domains 1 domains LIM half a and four
oxysterol binding protein-like 6 protein-like binding oxysterol
dual specificity phosphatase 18 phosphatase specificity dual
RIKEN cDNA 4933434I06 gene 4933434I06 cDNA RIKEN
RIKEN cDNA 4932425I24 gene 4932425I24 cDNA RIKEN
coiled-coil domain containing 11 containing domain coiled-coil
coiled-coil domain containing 65 containing domain coiled-coil
RIKEN cDNA 1700008D07 gene 1700008D07 cDNA RIKEN
RIKEN cDNA 1700026D08 gene 1700026D08 cDNA RIKEN
RIKEN cDNA D630013G24 gene gene D630013G24 cDNA RIKEN
tetratricopeptide repeat domain 25 domain repeat tetratricopeptide
tetratricopeptide repeat domain 18 domain repeat tetratricopeptide
coiled-coil domain containing 113 containing domain coiled-coil
coiled-coil domain containing 114 containing domain coiled-coil
Bardet-Biedl syndrome 9 (human) 9 syndrome Bardet-Biedl
EF hand calcium binding domain 1 domain binding calcium hand EF
Bardet-Biedl syndrome 7 (human) 7 syndrome Bardet-Biedl
rhabdoid tumor deletion region gene 1 gene region deletion tumor rhabdoid
leucine rich repeat containing 6 (testis) 6 containing repeat rich leucine
spindle assembly 6 homolog (C. elegans) (C. homolog 6 assembly spindle
malate dehydrogenase 1B, NAD (soluble) NAD 1B, dehydrogenase malate
dimethylarginine dimethylaminohydrolase 1 dimethylaminohydrolase dimethylarginine
transformed mouse 3T3 cell double minute 1 minute double cell 3T3 mouse transformed
Anterior gradient homolog 3 (Xenopus laevis) (Xenopus 3 homolog gradient Anterior
AN1, ubiquitin-like, homolog (Xenopus laevis) (Xenopus homolog ubiquitin-like, AN1,
phosphatidylinositol-4-phosphate 5-kinase, type 1 beta 1 type 5-kinase, phosphatidylinositol-4-phosphate Pip5k1b NM_008846 3.88 14.71 1.63E-05 5.70E-03
intraflagellar transport 122 homolog (Chlamydomonas) homolog 122 transport intraflagellar Ift122 NM_031177 3.44 10.86 6.16E-05 9.52E-03 1
calcium channel, voltage-dependent, T type, alpha 1H subunit 1H alpha type, T voltage-dependent, channel, calcium Cacna1h NM_021415 4.81 27.97 4.37E-06 3.23E-03 1 echinoderm microtubule associated protein like 1 transcript variant 1 variant transcript 1 like protein associated microtubule echinoderm Eml1 XM_127139 4.82 28.31 8.82E-05 1.10E-02 1
28
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
1
1
1
1
5.20 36.76 1.38E-05 5.62E-03
Atr XM_147046 3.45 10.89 1.02E-04 1.18E-02
Ckb NM_021273 5.14 35.22 1.06E-06 2.26E-03
Ddo NM_027442 5.86 58.05 7.63E-08 2.23E-03
Rshl3 XM_137041 5.10 34.28 3.56E-05 7.11E-03
Ttc16 NM_177384 5.30 39.43 2.49E-06 2.49E-03
Strbp NM_176932 3.43 10.79 8.42E-05 1.08E-02
Stox1 XM_125685 5.85 57.50 4.76E-07 2.26E-03
Dna2 NM_177372 3.79 13.87 1.87E-04 1.64E-02
Tctn3 NM_026260 3.65 12.58 1.70E-05 5.70E-03
Dydc2 XM_127662 5.40 42.35 6.19E-07 2.26E-03 Efhd1 NM_028889 3.50 11.29 9.43E-04 4.26E-02
Ccna1 NM_007628 5.21 36.89 6.49E-06 4.24E-03
Lrrc67 NM_145692 6.02 64.76 1.37E-07 2.23E-03
Wdr65 NM_026789 5.66 50.73 1.18E-06 2.26E-03
Itga10 XM_112192 3.49 11.23 2.81E-05 6.56E-03
Acox2 NM_053115 3.72 13.18 7.43E-04 3.72E-02
Calml4 NM_138304 5.41 42.52 2.05E-05 6.10E-03
Pih1d2 NM_028300 5.52 46.01 1.01E-06 2.26E-03
Tagap NM_145968 3.80 13.91 3.78E-05 7.33E-03
Spag5 NM_017407 3.69 12.88 1.09E-04 1.22E-02
Lrrc36 XM_194421 4.12 17.42 2.84E-06 2.49E-03
Spa17 NM_011449 4.05 16.52 1.55E-05 5.70E-03
Dixdc1 NM_178118 3.64 12.47 7.09E-04 3.63E-02
Cadm1 NM_018770 3.49 11.22 1.64E-04 1.53E-02
Sorbs1 NM_009166 3.65 12.54 2.74E-05 6.56E-03
Neurl1a NM_021360 5.75 53.65 2.09E-06 2.49E-03
Dnahc2 NM_177617 6.36 82.36 1.11E-06 2.26E-03
Sccpdh NM_178653 3.50 11.28 1.29E-04 1.33E-02
Chchd6 NM_025351 3.34 10.15 1.16E-04 1.24E-02
Ccdc15 XM_146705 3.43 10.80 3.73E-05 7.30E-03
Tspan2 NM_027533 3.83 14.20 2.52E-06 2.49E-03
Arhgdig NM_008113 3.90 14.94 7.81E-05 1.05E-02
Ccdc89 XM_133591 4.32 19.98 1.06E-05 4.97E-03
Ubxn11 NM_026257 4.57 23.69 1.13E-05 5.16E-03 1
Aldh3b1 NM_026316 4.45 21.80 8.12E-05 1.07E-02
Dennd5b NM_177192 3.61 12.18 3.24E-05 6.81E-03
Hist2h2aa1 NM_013549 3.72 13.22 1.08E-04 1.22E-02
Gene.Symbol Accession MChange Fold P.Value adj.P.Val Li Efimenko Stolc OstrowskiPazour Avidor-ReissKeller Andersen Blacque Broadhead Liuhits #
1700001L19Rik XM_127414 5.05 33.20 1.82E-05 5.82E-03
1700026L06Rik XM_130129 5.25 38.05 5.10E-05 8.59E-03
1700013F07Rik XM_131080 6.06 66.83 5.88E-06 4.04E-03
4930504H06Rik XM_126856 5.89 59.27 3.18E-06 2.68E-03
4833427G06Rik NM_177702 5.61 48.72 2.63E-05 6.54E-03 1700028P14Rik NM_026188 5.93 61.13 1.34E-06 2.26E-03
A330021E22Rik NM_172447 5.87 58.42 5.20E-06 3.74E-03
cyclin A1 cyclin
Gene.Name
tetraspanin 2 tetraspanin
storkhead box 1 box storkhead
calmodulin-like 4 calmodulin-like
integrin, alpha 10 10 alpha integrin,
D-aspartate oxidase D-aspartate
creatine kinase, brain kinase, creatine
WD repeat domain 65 domain repeat WD
radial spokehead-like 3 spokehead-like radial
UBX domain protein 11 protein domain UBX
histone cluster 2, H2aa1 2, cluster histone
cell adhesion molecule 1 molecule adhesion cell
DIX domain containing 1 containing domain DIX
tectonic family member 3 member family tectonic
PIH1 domain containing 2 containing domain PIH1
DPY30 domain containing 2 containing domain DPY30
sperm associated antigen 5 antigen associated sperm
EF hand domain containing 1 containing domain hand EF
sperm autoantigenic protein 17 protein autoantigenic sperm
RIKEN cDNA 1700001L19 gene 1700001L19 cDNA RIKEN
RIKEN cDNA 1700026L06 gene 1700026L06 cDNA RIKEN
coiled-coil domain containing 15 containing domain coiled-coil
RIKEN cDNA 1700013F07 gene 1700013F07 cDNA RIKEN
RIKEN cDNA 1700028P14 gene 1700028P14 cDNA RIKEN RIKEN cDNA 4833427G06 gene 4833427G06 cDNA RIKEN
leucine rich repeat containing 67 containing repeat rich leucine
RIKEN cDNA A330021E22 gene A330021E22 cDNA RIKEN
RIKEN cDNA 4930504H06 gene 4930504H06 cDNA RIKEN
coiled-coil domain containing 89 containing domain coiled-coil
dynein, axonemal, heavy chain 2 chain heavy axonemal, dynein,
leucine rich repeat containing 36 containing repeat rich leucine
tetratricopeptide repeat domain 16 domain repeat tetratricopeptide
neuralized homolog 1A (Drosophila) 1A homolog neuralized
DENN/MADD domain containing 5B containing domain DENN/MADD
sorbin and SH3 domain containing 1 containing domain SH3 and sorbin
ataxia telangiectasia and rad3 related rad3 and telangiectasia ataxia
saccharopine dehydrogenase (putative) dehydrogenase saccharopine
spermatid perinuclear RNA binding protein binding RNA perinuclear spermatid
DNA replication helicase 2 homolog (yeast) homolog 2 helicase replication DNA
acyl-Coenzyme A oxidase 2, branched chain branched 2, oxidase A acyl-Coenzyme
Rho GDP dissociation inhibitor (GDI) gamma (GDI) inhibitor dissociation GDP Rho
aldehyde dehydrogenase 3 family, member B1 member family, 3 dehydrogenase aldehyde
T-cell activation Rho GTPase-activating protein GTPase-activating Rho activation T-cell
coiled-coil-helix-coiled-coil-helix domain containing 6 containing domain coiled-coil-helix-coiled-coil-helix
dyslexia susceptibility 1 candidate 1 homolog (human) homolog 1 candidate 1 susceptibility dyslexia Dyx1c1 NM_026314 4.08 16.93 1.46E-05 5.70E-03 1
translin-associated factor X (Tsnax) interacting protein 1 protein interacting (Tsnax) X factor translin-associated Tsnaxip1 NM_024445
cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial 2, kinase (UMP-CMP) monophosphate cytidine Cmpk2 NM_020557 5.83 56.92 2.26E-06 2.49E-03 DNA segment, Chr 15, Wayne State University 169, expressed 169, University State Wayne 15, Chr segment, DNA D15Wsu169e NM_198420 4.01 16.13 2.82E-05 6.56E-03
29 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0
7.46E-05 1.03E-02
Nin NM_008697 3.65 12.57 8.58E-05 1.08E-02
Cklf NM_029295 3.71 13.11 5.56E-04 3.18E-02
Tekt2 NM_011902 3.81 13.98 1.05E-03 4.52E-02 Plch1 NM_183191 3.93 15.22 1.10E-04 1.22E-02
Zfp98 NM_016793 3.87 14.61 1.19E-05 5.35E-03 Cxcr4 NM_009911 3.69 12.89 5.80E-04 3.24E-02
Styxl1 NM_029659 3.77 13.63 6.86E-05 9.93E-03 cspp1 XM_129366 3.75 13.44 2.26E-05 6.27E-03
Morc1 NM_010816 3.82 14.16 3.09E-04 2.24E-02 Morn5 XM_130188 3.68 12.81 7.72E-04 3.79E-02 Tcp11 NM_013687 3.91 15.07 6.57E-04 3.51E-02
Wfdc3 XM_355365 3.73 13.28 9.61E-05 1.14E-02
Dnaic1 NM_175138 3.66 12.66 7.31E-05 1.02E-02 Zfp423 NM_033327 3.69 12.86 3.09E-05 6.62E-03
Ckap2l NM_181589 3.72 13.18 1.11E-04 1.23E-02
Fbxo16 NM_015795 4.02 16.26 2.74E-06 2.49E-03
Hspb11 XM_204090 3.86 14.47 3.34E-05 6.89E-03
Cep120 NM_178686 3.72 13.18 5.47E-05 8.88E-03
Spata17 NM_028848 3.79 13.85 4.31E-06 3.23E-03 Dnajb13 NM_153527 4.02 16.25 3.21E-04 2.29E-02
Rundc3b NM_198620 3.77 13.63 3.78E-04 2.50E-02
Fam161b NM_172581 3.81 14.01 2.74E-04 2.07E-02 Fam167a NM_177628 3.92 15.18 1.35E-05 5.62E-03 Fam154b NM_177894 3.99 15.89 1.29E-04 1.32E-02
Arhgap11a NM_181416 3.95 15.47 3.91E-04 2.56E-02
Gene.Symbol Accession M Fold Change P.Value adj.P.Val Li Efimenko Stolc OstrowskiPazour Avidor-ReissKeller Andersen Blacque Broadhead Liu # hits
1700010I14Rik NM_025851 3.83 14.22 1.28E-05 5.62E-03
1700086L19Rik XM_358420 3.74 13.39 1.30E-05 5.62E-03
4932411E22Rik NM_172534 3.871700088E04Rik NM_138581 14.60 3.84 1.26E-042010300C02Rik 1.31E-02 XM_129808 14.33 3.81 1.67E-04 1.53E-02 14.05 4.91E-04 2.98E-02 1700001C02Rik NM_029285 3.81 13.98 1.88E-04 1.64E-02 1700054A03Rik XM_148917 3.80 13.894930452B06Rik NM_028934 3.78 1.01E-05 4.97E-03 13.75 1.51E-05 5.70E-03 1110034A24Rik NM_027269 3.98 15.75 7.14E-06 4.35E-03 6430537H07Rik NM_178689 3.78 13.73 2.87E-05 6.56E-03 6820408C15Rik NM_177656 3.776330503K22Rik NM_182995 13.65 3.76 4.24E-04 2.69E-02 13.54 1.76E-04 1.60E-02 9530077C05Rik NM_026739 3.75 13.41 5.47E-05 8.88E-03
C530043A13Rik NM_176897 3.67 12.68 2.26E-05 6.27E-03 1700024G13Rik XM_147820 3.88 14.69A430083B19Rik NM_177624 3.84 1.02E-05 4.97E-03 14.35 8.54E-05 1.08E-02 1200014M14Rik NM_026173 3.98 15.82 6.96E-04 3.60E-02 5133400G04Rik NM_027733 3.99 15.92 2.22E-05 6.25E-03
tektin 2
Gene.Name
microrchidia 1
F-box protein 16
t-complex protein 11
zinc finger protein 98
chemokine-like factor
zinc finger protein 423
phospholipase C, eta 1
centrosomal protein 120
MORN repeat containing 5
RUN domain containing 3B
RIKEN cDNA 1700010I14 gene RIKEN cDNA spermatogenesis associated 17
RIKEN cDNA 1700086L19 gene RIKEN cDNA
RIKEN cDNA 4932411E22 gene 4932411E22 RIKEN cDNA 1700088E04 gene RIKEN cDNA RIKEN cDNA 4930452B06 gene RIKEN cDNA RIKEN cDNA 1110034A24 gene 1110034A24 RIKEN cDNA 1700054A03 gene RIKEN cDNA RIKEN cDNA 6330503K22 gene RIKEN cDNA
RIKEN cDNA 2010300C02 gene RIKEN cDNA RIKEN cDNA 1700024G13 gene RIKEN cDNA A430083B19 gene RIKEN cDNA 1700001C02 gene RIKEN cDNA RIKEN cDNA 6430537H07 gene RIKEN cDNA RIKEN cDNA 5133400G04 gene RIKEN cDNA 6820408C15 gene RIKEN cDNA RIKEN cDNA 9530077C05 gene RIKEN cDNA
RIKEN cDNA 1200014M14 gene RIKEN cDNA
RIKEN cDNA C530043A13 gene RIKEN cDNA
WAP four-disulfide core domain 3 WAP
Rho GTPase activating protein 11A
chemokine (C-X-C motif) receptor 4
cytoskeleton associated protein 2-like
dynein, axonemal, intermediate chain 1
serine/threonine/tyrosine interacting-like 1
family with sequence similarity 161, member B family with sequence similarity 167, member A family with sequence similarity 167, member family with sequence similarity 154, member B
DnaJ (Hsp40) related, subfamily B, member 13
heat shock protein family B (small), member 11
centrosome and spindle pole associated protein 1
inturned planar homolog cell polarity (Drosophila) effector Intu NM_175515 3.68 12.78 2.11E-05 6.17E-03
spermatogenesis associated glutamate (E)-rich protein 4d Speer4d NM_025759 3.79 13.86 7.45E-06 4.38E-03 spermatogenesis associated glutamate (E)-rich protein 4e Speer4e XM_486542 3.71 13.12 2.46E-05 6.36E-03
dysbindin (dystrobrevin binding protein 1) domain containing 1 Dbndd1 NM_028146 3.65 12.57
proteasome (prosome, macropain) 26S subunit, ATPase 3, interacting proteinPsmc3ipATPase proteasome (prosome, NM_008949 macropain) 26S subunit, 3.78 13.75 1.61E-04 1.51E-02
oasu ag odcac acu-ciae hne,sbaiyM eamme KnbN_2213.71potassium large conductance calcium-activated channel, subfamily M, beta member 2Kcnmb2NM_028231 13.07 6.51E-04 3.49E-02
30
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Irf8 NM_008320 3.56 11.78 5.45E-05 8.88E-03
Cby1 NM_028634 3.43 10.80 2.28E-04 1.85E-02
Ttll6 NM_172799 3.61 12.20 6.94E-05 9.97E-03
Polg2 NM_015810 3.32 10.01 3.90E-05 7.44E-03
Nags NM_178053 3.54 11.66 1.76E-04 1.60E-02 Cytip NM_139200 3.57 11.85 1.81E-03 6.09E-02
Usp11 NM_145628 3.35 10.18 3.43E-05 6.97E-03
Acot1 NM_012006 3.59 12.03 3.96E-05 7.49E-03
Rsad2 NM_021384 3.46 10.99 6.61E-04 3.51E-02
Dzip1l NM_028258 3.52 11.44 2.85E-05 6.56E-03
Igsf11 NM_170599 3.58 11.95 1.36E-04 1.39E-02
Spink2 NM_183284 3.45 10.91 3.57E-05 7.11E-03
Enpp4 NM_199016 3.55 11.71 1.65E-04 1.53E-02
Rbm20 XM_140742 3.40 10.55 8.43E-04 4.00E-02
Grb14 NM_016719 3.60 12.09 2.89E-05 6.56E-03
Spire1 NM_176832 3.59 12.07 1.09E-03 4.61E-02
Rragd NM_027491 3.63 12.40 9.52E-06 4.97E-03
Acyp1 NM_025421 3.63 12.40 1.61E-05 5.70E-03 Fbxo36 NM_025386 3.44 10.83 1.03E-03 4.47E-02
Tcea3 NM_011542 3.64 12.43 2.32E-05 6.29E-03
Fam81a XM_135000 3.32 10.01 1.23E-04 1.29E-02
Pnma1 XM_127024 3.58 11.95 9.23E-05 1.12E-02
Ccdc14 NM_172824 3.63 12.36 2.17E-04 1.80E-02
Mycbpap NM_170671 3.48 11.19 9.96E-05 1.17E-02
Slc27a2 NM_011978 3.65 12.54 4.05E-05 7.55E-03
Fam118a NM_133750 3.61 12.23 7.88E-05 1.05E-02
D0H4S114 NM_053078 3.62 12.26 3.27E-04 2.32E-02
Ncrna00166 NM_025506 3.45 10.89 4.01E-03 9.87E-02
Gene.Symbol Accession MChange Fold P.Value adj.P.Val Li Efimenko Stolc Ostrowski Pazour Avidor-Reiss Keller Andersen Blacque Broadhead Liuhits #
1700023L04Rik XM_485737 3.34 10.10 1.54E-04 1.49E-02
2610028H24Rik XM_483921 3.39 10.48 1.11E-03 4.66E-02
1110049B09Rik XM_126902 3.39 10.50 2.62E-04 2.01E-02
1700025K23Rik NM_183254 3.39 10.51 6.36E-04 3.44E-02
6030470M02Rik NM_177119 3.42 10.71 1.35E-05 5.62E-03
E030019B06Rik XM_133957 3.43 10.81 1.81E-04 1.62E-02
1700003E16Rik XM_132615 3.46 10.97 1.95E-03 6.35E-02
1700040L02Rik NM_028491 3.57 11.89 9.36E-05 1.13E-02
1500015O10Rik NM_024283 3.47 11.11 2.45E-04 1.92E-02
3110040M04Rik XM_484925 3.55 11.72 3.48E-04 2.41E-02
4930579J09Rik NM_133689 3.64 12.50 1.28E-03 5.08E-02
0610010F05Rik NM_027860 3.62 12.27 9.07E-05 1.12E-02
B230373P09Rik NM_177336 3.59 12.01 4.49E-04 2.79E-02
Gene.Name
F-box protein 36 protein F-box
acyl-CoA thioesterase 1 thioesterase acyl-CoA
MYCBP associated protein associated MYCBP
RIKEN cDNA 0610010F05 cDNA RIKEN
N-acetylglutamate synthase N-acetylglutamate
Ras-related GTP binding D binding GTP Ras-related non-protein coding RNA 166 RNA coding non-protein
RNA binding motif protein 20 protein motif binding RNA
paraneoplastic antigen MA1 antigen paraneoplastic
interferon regulatory factor 8 factor regulatory interferon
DAZ interacting protein 1-like protein interacting DAZ
ubiquitin specific peptidase 11 peptidase specific ubiquitin
spire homolog 1 (Drosophila) 1 homolog spire
chibby homolog 1 (Drosophila) 1 homolog chibby
cytohesin 1 interacting protein interacting 1 cytohesin
RIKEN cDNA 1700023L04 gene 1700023L04 cDNA RIKEN
DNA segment, human D4S114 human segment, DNA
RIKEN cDNA 1110049B09 gene 1110049B09 cDNA RIKEN
RIKEN cDNA 1700025K23 gene 1700025K23 cDNA RIKEN
RIKEN cDNA E030019B06 gene E030019B06 cDNA RIKEN
RIKEN cDNA 1700003E16 gene 1700003E16 cDNA RIKEN
RIKEN cDNA 6030470M02 gene 6030470M02 cDNA RIKEN
RIKEN cDNA 1700040L02 gene 1700040L02 cDNA RIKEN
RIKEN cDNA 2610028H24 gene gene 2610028H24 cDNA RIKEN
RIKEN cDNA 1500015O10 gene 1500015O10 cDNA RIKEN
coiled-coil domain containing 14 containing domain coiled-coil
RIKEN cDNA 3110040M04 gene 3110040M04 cDNA RIKEN
RIKEN cDNA B230373P09 gene B230373P09 cDNA RIKEN
RIKEN cDNA 4930579J09 gene 4930579J09 cDNA RIKEN
serine peptidase inhibitor, Kazal type 2 type Kazal inhibitor, peptidase serine
growth factor receptor bound protein 14 protein bound receptor factor growth
transcription elongation factor A (SII), 3 (SII), A factor elongation transcription
immunoglobulin superfamily, member 11 member superfamily, immunoglobulin
family with sequence similarity 81, member A member 81, similarity sequence with family
tubulin tyrosine ligase-like family, member 6 member family, ligase-like tyrosine tubulin
family with sequence similarity 118, member A member 118, similarity sequence with family
acylphosphatase 1, erythrocyte (common) type (common) erythrocyte 1, acylphosphatase
radical S-adenosyl methionine domain containing 2 containing domain methionine S-adenosyl radical
ectonucleotide pyrophosphatase/phosphodiesterase 4 pyrophosphatase/phosphodiesterase ectonucleotide
polymerase (DNA directed), gamma 2, accessory subunit accessory 2, gamma directed), (DNA polymerase
solute carrier family 27 (fatty acid transporter), member 2 member transporter), acid (fatty 27 family carrier solute protein tyrosine phosphatase-like (proline instead of catalytic arginine), member a member arginine), catalytic of instead (proline phosphatase-like tyrosine protein Ptpla NM_013935 3.47 11.07 1.55E-04 1.49E-02 Figure 4. Identification of known and putative ciliary and basal body genes in the ALI+4 transcriptome. 314 genes were upregulated 10-fold or greater at Figure 4. Identification of known and putative ciliary basal body genes in the indicate the presence or to ‘Liu’ The columns ‘Li’ ALI+4 with reference to non-ciliated MTECs. M values are equal the fold change in log (base 2). The number of studies that have identi- indicates present. ‘1’ datasets comprising the cilia proteome (www.ciliaproteome.org). absence of genes in the 11 ALI+12 have been identified in at least one other study the cilia fied a gene is tallied in the column ‘# hits’. 138 of 314 upregulated genes (44%) at proteome.
31
3
3
3
3
3
4 4
5
5
5
6
6
7
7
8
12 12
12
12
12
12
12
12
12
12
13
13
13
13
15
15
15
15
16 17
17
17
18
1 1 3
1
1
1 1 4
1
1 1 6
1
1 1 6
1
1
1 1 7
1
1
1
1
1 1 1 5
1
1
1
1
1
1 1 1 1 7
1
1 1 1 1 8
1 1 1 1 8
1 1 1 1 9
1
1
1
1
1
1 1 1
1 1 1
1
1
1
1
1
1
1
1
1
1
1
1
1
11
1
11
1
11
1
1
1 1 1 1 1
1 1 1 1 1
1 1 1 1 1 1 1 9
11
1
1 1 1
11
1 1 1
1 1 1 1 1 1 1 1
1
1
1
1
1
1 1 1
1
1 1 1
1 1 1
1 1 1
1 1 1 1
1 1 1
1 1 1 1
1
1 1 1
1 1 1 1 1
1 1 1 1 1
1 1 1 1
1
1
1
11
11
1
1
11
1
1
Mlf1 NM_010801 4.75 26.82 1.30E-06 2.45E-03
Fgl2 NM_008013 3.34 10.12 6.58E-04 1.24E-01
Plk4 NM_011495 4.44 21.65 6.05E-06 7.34E-03 1 1
Ift81 NM_009879 3.57 11.86 2.74E-04 7.34E-02 1 1 1
Myl4 NM_010858 3.91 15.05 6.62E-05 3.17E-02
Eml1 XM_127139 3.73 13.23 1.45E-04 5.01E-02 1
Lrrc6 NM_019457 4.18 18.07 2.05E-05 1.48E-02 1
Bbs5 NM_028284 3.38 10.38 5.74E-04 1.16E-01 1 1
Ptprc NM_011210 3.55 11.72 2.93E-04 7.45E-02
Kif27 NM_175214 4.29 19.53 1.22E-05 1.08E-02
Tekt1 NM_011569 3.35 10.16 6.44E-04 1.22E-01 1
Tekt4 XM_128462 4.54 23.21 3.71E-06 5.53E-03 1
Mns1 NM_008613 7.41 170.03 4.12E-14 1.60E-09 1
Cdkl2 NM_016912 3.37 10.35 5.84E-04 1.17E-01
Dyrk3 NM_145508 3.57 11.84 2.76E-04 7.34E-02
Prkcb NM_008855 3.62 12.28 2.24E-04 6.74E-02
Efhc1 XM_129694 4.04 16.46 3.77E-05 2.12E-02 1
Ribc1 NM_025660 4.23 18.70 1.64E-05 1.30E-02 1
Mdm1 NM_148922 6.17 72.19 3.04E-10 3.93E-06
Morn3 XM_132350 3.46 11.03 4.13E-04 9.41E-02
Mast3 NM_199308 3.61 12.21 2.32E-04 6.87E-02
Lrrc34 XM_487721 4.42 21.39 6.57E-06 7.72E-03
Lrrc50 NM_026648 3.62 12.31 2.21E-04 6.69E-02 1
Lrrc23 NM_013588 4.12 17.41 2.62E-05 1.69E-02 1
Tppp3 NM_026481 4.17 17.99 2.12E-05 1.49E-02 1 1
Dnali1 NM_175223 4.19 18.28 1.90E-05 1.42E-02 1
Lrrc48 NM_029044 4.46 22.06 5.30E-06 6.86E-03 1
Cep78 NM_198019 3.36 10.24 6.20E-04 1.21E-01
Ddah1 NM_026993 3.74 13.33 1.39E-04 4.97E-02 1
Spag6 NM_015773 3.71 13.05 1.57E-04 5.35E-02 1
Cdh11 NM_009866 7.21 148.36 1.89E-13 3.66E-09
Wdr78 NM_146254 4.74 26.75 1.33E-06 2.45E-03 1
Mdh1b NM_029696 4.16 17.84 2.24E-05 1.52E-02
Wdr63 NM_172864 3.36 10.29 6.04E-04 1.20E-01 1
Ruvbl1 NM_019685 3.79 13.87 1.09E-04 4.42E-02
Osbpl6 NM_145525 3.33 10.09 6.71E-04 1.24E-01
Ccdc65 NM_153518 3.44 10.87 4.47E-04 9.91E-02 1
Ccdc39 NM_026222 4.90 29.92 5.72E-07 1.48E-03
Ccdc18 XM_144475 4.00 15.99 4.52E-05 2.44E-02
Ccdc41 NM_029852 4.61 24.34 2.64E-06 4.37E-03
Pip5k1b NM_008846 3.55 11.71 2.94E-04 7.45E-02
Cep135 NM_199032 3.57 11.85 2.75E-04 7.34E-02 1 1
Ccdc113 NM_172914 3.55 11.75 2.88E-04 7.45E-02 1
Ccdc114 XM_133435 3.36 10.28 6.08E-04 1.20E-01 1
Cacna1h NM_021415 4.19 18.30 1.89E-05 1.42E-02 1
Gene.Symbol Accession MChange Fold P.Value adj.P.Val Li Efimenko Stolc Ostrowski Pazour Avidor-Reiss Keller Andersen Blacque Broadhead Liuhits #
4933434I06Rik XM_283704 4.20 18.41 1.82E-05 1.41E-02 1
4932425I24Rik XM_489540 5.84 57.41 2.53E-09 2.45E-05 1
1700008D07Rik XM_485969 3.69 12.88 1.70E-04 5.62E-02 1
1700026D08Rik NM_029335 4.67 25.38 1.95E-06 3.43E-03
tektin 1 tektin
tektin 4 tektin
cadherin 11 cadherin
Gene.Name
RuvB-like protein 1 protein RuvB-like
WD repeat domain 78 domain repeat WD
WD repeat domain 63 domain repeat WD
protein kinase C, beta C, kinase protein
centrosomal protein 78 protein centrosomal
fibrinogen-like protein 2 protein fibrinogen-like
centrosomal protein 135 protein centrosomal
myeloid leukemia factor 1 factor leukemia myeloid
kinesin family member 27 member family kinesin
myosin, light polypeptide 4 polypeptide light myosin,
MORN repeat containing 3 containing repeat MORN
sperm associated antigen 6 antigen associated sperm
oxysterol binding protein-like 6 protein-like binding oxysterol
polo-like kinase 4 (Drosophila) 4 kinase polo-like
RIKEN cDNA 4933434I06 gene 4933434I06 cDNA RIKEN
RIKEN cDNA 4932425I24 gene 4932425I24 cDNA RIKEN
coiled-coil domain containing 65 containing domain coiled-coil
coiled-coil domain containing 39 containing domain coiled-coil
coiled-coil domain containing 18 containing domain coiled-coil
coiled-coil domain containing 41 containing domain coiled-coil
leucine rich repeat containing 50 containing repeat rich leucine
leucine rich repeat containing 23 containing repeat rich leucine
leucine rich repeat containing 48 containing repeat rich leucine
RIKEN cDNA 1700026D08 gene 1700026D08 cDNA RIKEN
RIKEN cDNA 1700008D07 gene gene 1700008D07 cDNA RIKEN
Leucine rich repeat containing 34 containing repeat rich Leucine
coiled-coil domain containing 113 containing domain coiled-coil
coiled-coil domain containing 114 containing domain coiled-coil
Bardet-Biedl syndrome 5 (human) 5 syndrome Bardet-Biedl
RIB43A domain with coiled-coils 1 coiled-coils with domain RIB43A
leucine rich repeat containing 6 (testis) 6 containing repeat rich leucine
malate dehydrogenase 1B, NAD (soluble) NAD 1B, dehydrogenase malate
EF-hand domain (C-terminal) containing 1 containing (C-terminal) domain EF-hand
dimethylarginine dimethylaminohydrolase 1 dimethylaminohydrolase dimethylarginine
meiosis-specific nuclear structural protein 1 protein structural nuclear meiosis-specific
transformed mouse 3T3 cell double minute 1 minute double cell 3T3 mouse transformed
protein tyrosine phosphatase, receptor type, C type, receptor phosphatase, tyrosine protein
Microtubule associated serine/threonine kinase 3 kinase serine/threonine associated Microtubule
dynein, axonemal, light intermediate polypeptide 1 polypeptide intermediate light axonemal, dynein,
cyclin-dependent kinase-like 2 (CDC2-related kinase) (CDC2-related 2 kinase-like cyclin-dependent
intraflagellar transport 81 homolog (Chlamydomonas) homolog 81 transport intraflagellar
phosphatidylinositol-4-phosphate 5-kinase, type 1 beta 1 type 5-kinase, phosphatidylinositol-4-phosphate
tubulin polymerization-promoting protein family member 3 member family protein polymerization-promoting tubulin
calcium channel, voltage-dependent, T type, alpha 1H subunit 1H alpha type, T voltage-dependent, channel, calcium
dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 3 kinase regulated tyrosine-(Y)-phosphorylation dual-specificity echinoderm microtubule associated protein like 1, transcript variant 1 variant transcript 1, like protein associated microtubule echinoderm
32
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0
0
0
0 0
0
0 0
0
0
0 0
0
0
0
1
1
1
1
1
1
1
1
11
11
11
11
11
11
11
11
11
11
1
1
1
1
1
1
1
Ckb NM_021273 4.99 31.80 3.57E-07 1.15E-03
C1s NM_144938 3.51 11.42 3.40E-04 8.39E-02
Myb NM_010848 4.33 20.05 1.02E-05 9.46E-03
Tln1 NM_011602 3.77 13.67 1.19E-04 4.61E-02
Plek NM_019549 5.17 36.04 1.33E-07 5.73E-04
Fhl1 NM_010211 5.00 32.06 3.36E-07 1.15E-03
Agr3 NM_207531 5.71 52.33 5.77E-09 3.73E-05
Cytip NM_139200 3.83 14.18 9.53E-05 4.06E-02
Rbks NM_153196 3.87 14.59 8.02E-05 3.70E-02
Pdxk XM_488563 3.60 12.09 2.45E-04 6.99E-02
Ccno XM_127523 4.78 27.44 1.10E-06 2.36E-03
Cd36 NM_007643 4.15 17.79 2.28E-05 1.52E-02
Cd53 NM_007651 3.34 10.11 6.65E-04 1.24E-01
Ttc18 XM_127606 3.55 11.75 2.89E-04 7.45E-02
Rtdr1 XM_354544 3.99 15.84 4.80E-05 2.52E-02
Ttc25 XM_126529 4.06 16.69 3.45E-05 2.04E-02
Lhfpl2 NM_172589 4.05 16.59 3.58E-05 2.04E-02
Dydc2 XM_127662 4.16 17.86 2.22E-05 1.52E-02
Glcci1 NM_178072 4.37 20.73 8.18E-06 8.62E-03
Lrrc67 NM_145692 4.93 30.50 4.93E-07 1.37E-03
Lrrc36 XM_194421 3.36 10.26 6.12E-04 1.20E-01
Morn5 BC036965 3.83 14.19 9.50E-05 4.06E-02
Rsph1 NM_025290 5.60 48.61 1.10E-08 6.08E-05
Wdr65 NM_026789 3.79 13.82 1.11E-04 4.44E-02
Calml4 NM_138304 5.24 37.73 9.20E-08 4.46E-04
Cmpk2 NM_020557 4.10 17.13 2.91E-05 1.80E-02
Vpreb3 NM_009514 4.48 22.34 4.86E-06 6.60E-03
Ropn1l NM_145852 4.24 18.88 1.54E-05 1.24E-02
Tm4sf1 NM_008536 4.75 26.91 1.27E-06 2.45E-03
Sccpdh NM_178653 3.47 11.07 4.04E-04 9.38E-02
Mtap1b NM_008634 3.50 11.32 3.56E-04 8.57E-02
Dnahc2 NM_177617 3.78 13.72 1.17E-04 4.57E-02
Arhgdig NM_008113 3.85 14.43 8.59E-05 3.87E-02
Ubxn11 NM_026257 4.37 20.74 8.13E-06 8.62E-03 1
Neurl1a NM_021360 4.81 28.13 9.10E-07 2.08E-03
Aldh3b1 NM_026316 4.27 19.27 1.34E-05 1.16E-02
Tsnaxip1 NM_024445 4.96 31.21 4.13E-07 1.23E-03
Gramd1b NM_172768 3.57 11.88 2.71E-04 7.34E-02
Gene.Symbol Accession MChange Fold P.Value adj.P.Val Li Efimenko Stolc Ostrowski Pazour Avidor-Reiss Keller Andersen Blacque Broadhead Liuhits #
3100002J23Rik XM_484029 4.10 17.11 2.93E-05 1.80E-02
1700026L06Rik XM_130129 5.02 32.54 2.99E-07 1.15E-03
1190002A17Rik XM_130050 3.79 13.86 1.09E-04 4.42E-02
4930504H06Rik XM_126856 3.92 15.14 6.37E-05 3.09E-02
4930451C15Rik XM_148428 3.93 15.26 6.06E-05 2.98E-02
1700028P14Rik NM_026188 4.38 20.83 7.89E-06 8.62E-03
2010300C02Rik XM_129808 4.48 22.29 4.93E-06 6.60E-03
E030011K20Rik XM_149293 3.79 13.80 1.12E-04 4.44E-02
4833427G06Rik NM_177702 4.33 20.11 1.00E-05 9.46E-03
A330021E22Rik NM_172447 5.81 55.92 3.20E-09 2.48E-05
D630013G24Rik XM_357326 4.11 17.30 2.73E-05 1.74E-02
talin 1 talin
cyclin O cyclin
pleckstrin
ribokinase
Gene.Name
CD36 antigen CD36
CD53 antigen CD53
ropporin 1-like ropporin
calmodulin-like 4 calmodulin-like
creatine kinase, brain kinase, creatine
WD repeat domain 65 domain repeat WD
UBX domain protein 11 protein domain UBX
pre-B lymphocyte gene 3 gene lymphocyte pre-B
myeloblastosis oncogene myeloblastosis
MORN repeat containing 5 containing repeat MORN
DPY30 domain containing 2 containing domain DPY30
GRAM domain containing 1B containing domain GRAM
four and a half LIM domains 1 domains LIM half a and four
cytohesin 1 interacting protein interacting 1 cytohesin
RIKEN cDNA 3100002J23 gene 3100002J23 cDNA RIKEN
RIKEN cDNA 1700026L06 gene 1700026L06 cDNA RIKEN
RIKEN cDNA 1190002A17 gene 1190002A17 cDNA RIKEN
RIKEN cDNA 1700028P14 gene 1700028P14 cDNA RIKEN
leucine rich repeat containing 67 containing repeat rich leucine
leucine rich repeat containing 36 containing repeat rich leucine
RIKEN cDNA E030011K20 gene E030011K20 cDNA RIKEN
RIKEN cDNA 4930504H06 gene 4930504H06 cDNA RIKEN
RIKEN cDNA 4930451C15 gene 4930451C15 cDNA RIKEN
RIKEN cDNA 4833427G06 gene 4833427G06 cDNA RIKEN
RIKEN cDNA 2010300C02 gene 2010300C02 cDNA RIKEN
RIKEN cDNA A330021E22 gene A330021E22 cDNA RIKEN
dynein, axonemal, heavy chain 2 chain heavy axonemal, dynein,
RIKEN cDNA D630013G24 gene gene D630013G24 cDNA RIKEN
microtubule-associated protein 1B protein microtubule-associated
tetratricopeptide repeat domain 18 domain repeat tetratricopeptide
tetratricopeptide repeat domain 25 domain repeat tetratricopeptide
Glucocorticoid induced transcript 1 transcript induced Glucocorticoid
lipoma HMGIC fusion partner-like 2 partner-like fusion HMGIC lipoma
neuralized homolog 1A (Drosophila) 1A homolog neuralized
rhabdoid tumor deletion region gene 1 gene region deletion tumor rhabdoid
saccharopine dehydrogenase (putative) dehydrogenase saccharopine
transmembrane 4 superfamily member 1 member superfamily 4 transmembrane
Pyridoxal (pyridoxine, vitamin B6) kinase B6) vitamin (pyridoxine, Pyridoxal
complement component 1, s subcomponent s 1, component complement
Rho GDP dissociation inhibitor (GDI) gamma (GDI) inhibitor dissociation GDP Rho
Anterior gradient homolog 3 (Xenopus laevis) (Xenopus 3 homolog gradient Anterior
aldehyde dehydrogenase 3 family, member B1 member family, 3 dehydrogenase aldehyde
radial spoke head 1 homolog (Chlamydomonas) homolog 1 head spoke radial
translin-associated factor X (Tsnax) interacting protein 1 protein interacting (Tsnax) X factor translin-associated cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial 2, kinase (UMP-CMP) monophosphate cytidine
33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
XM_129176 3.73 13.31 1.40E-04 4.97E-02 XM_133920 3.73 13.29 1.41E-04 4.97E-02 NM_178671 3.68 12.86 1.71E-04 5.62E-02 NM_027442 3.66 12.61 1.92E-04 6.11E-02
NM_011725 3.60 12.16 2.37E-04 6.91E-02 XM_355478 3.55 11.69 2.97E-04 7.45E-02 NM_025961 3.47 11.08 4.02E-04 9.38E-02 NM_029815 3.46 11.03 4.12E-04 9.41E-02 NM_009259 3.45 10.92 4.35E-04 9.80E-02
XM_133659 3.41 10.66 4.96E-04 1.08E-01 NM_009185 3.41 10.61 5.09E-04 1.08E-01
NM_199016 3.40 10.58 5.19E-04 1.08E-01 NM_007901 3.39 10.48 5.46E-04 1.12E-01 XM_125685 3.38 10.43 5.62E-04 1.15E-01 XM_137041 3.36 10.28 6.07E-04 1.20E-01 NM_013655 3.34 10.11 6.62E-04 1.24E-01
NM_013687 3.33 10.03 6.92E-04 1.25E-01 NM_007860 3.32 10.02 6.96E-04 1.25E-01 NM_175386 3.32 10.02 6.97E-04 1.25E-01
Xlr
Stil
Spn Ddo
Lhfp
Dio1
Cbe1
Gatm
Stox1 Rshl3
Kndc1
S1pr1 Bcas1
Tcp11
Enpp4 Mpeg1
Cxcl12
Ubxn10
Ccdc81
Tctex1d2 NM_025329 3.34 10.10 6.68E-04 1.24E-01
Gene.Symbol Accession M Fold Change P.Value adj.P.Val Li Efimenko Stolc Ostrowski Pazour Avidor-Reiss Keller Andersen Blacque Broadhead Liu # hits
D15Wsu169e NM_198420 3.65 12.57 1.95E-04 6.16E-02
1700013F07Rik XM_131080 3.65 12.52 2.00E-04 6.27E-02 1700019L03Rik XM_484987 3.41 10.60 5.12E-04 1.08E-01 6820408C15Rik NM_177656 3.74 13.36 1.37E-04 4.97E-02
4933404M02Rik NM_025744 3.44 10.88 4.44E-04 9.90E-02
sialophorin
Gene.Name
storkhead box 1
D-aspartate oxidase
t-complex protein 11
UBX domain protein 10
radial spokehead-like 3
Scl/Tal1 interrupting locus Scl/Tal1
Tctex1 domain containing 2 Tctex1
lipoma HMGIC fusion partner
Ciliated bronchial epithelium 1
macrophage expressed gene 1
coiled-coil domain containing 81 RIKEN cDNA 1700013F07 RIKENgene cDNA RIKEN cDNA 1700019L03 RIKENgene cDNA
RIKEN cDNA 6820408C15RIKEN gene cDNA
deiodinase, iodothyronine, type I
RIKEN cDNA 4933404M02RIKEN gene cDNA
chemokine (C-X-C motif) ligand 12
sphingosine-1-phosphate receptor 1
breast carcinoma amplified sequence 1
X-linked lymphocyte-regulated complex
ectonucleotide pyrophosphatase/phosphodiesterase 4
kinase non-catalytic C-lobe domain (KIND) containing 1
DNA segment, State Chr University15, Wayne DNA 169, expressed
glycine amidinotransferase (L-arginine:glycine amidinotransferase) Figure 5. Identification of known and putative ciliary and basal body genes in the ALI+12 transcriptome. 135 genes were Figure 5. Identification of known and putative ciliary basal body genes in the ALI+12 with reference to non-ciliated MTECs. M values are equal the fold change in log upregulated 10-fold or greater at datasets comprising the cilia proteome indicate the presence or absence of genes in 11 to ‘Liu’ The columns ‘Li’ (base 2). The number of studies that have identified a gene is tallied in the column ‘# indicates present. ‘1’ (www.ciliaproteome.org). ALI+12 have been identified in at least one other study the cilia proteome. hits’. 75 of the 135 upregulated genes (56%) at
34 Gene Name RefSeq mRNA Gene Symbol Mouse Ortholog ALI+4 ALI+12 Ciliary Proteome MDC2 NM_004194 ADAM22 ADENYLATE KINASE 5 NM_012093 AK5 ADENYLATE KINASE 7 NM_152327 AK7 1 ANKYRIN REPEAT DOMAIN 45 NM_198493 ANKRD45 1 ARMADILLO REPEAT CONTAINING 4 NM_018076 ARMC4 ARYL-HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR 2 NM_014862 ARNT2 HYPOTHETICAL PROTEIN MGC40178 NM_152325 C13ORF26 4930588N13Rik 1 CHROMOSOME 14 OPEN READING FRAME 45 NM_025057 C14ORF45 2900006K08Rik CHROMOSOME 14 OPEN READING FRAME 50 NM_172365 C14ORF50 Gm70 CHROMOSOME 15 OPEN READING FRAME 26 NM_173528 C15ORF26 1700026D08Rik 11 GENE TRAP LOCUS 3 (MOUSE) NM_013242 C16ORF80 Gtl3 XM_357260 C20orf28 Spef1 1 CHROMOSOME 6 OPEN READING FRAME 206 NM_152732 C6ORF206 1700027N10Rik CHROMOSOME 6 OPEN READING FRAME 97 NM_025059 C6ORF97 Gm221 CHROMOSOME 8 OPEN READING FRAME 70 NM_016010 C8ORF70 Fam164A 11 CHROMOSOME 9 OPEN READING FRAME 116 NM_144654 C9ORF116 1700007K13Rik CHROMOSOME 9 OPEN READING FRAME 68 NM_001039395 C9ORF68 4430402I18Rik 1 CHROMOSOME 9 OPEN READING FRAME 98 NM_152572 C9ORF98 1190002A17Rik 11 HYPOTHETICAL PROTEIN FLJ40365 NM_173482 CCDC105 COILED-COIL DOMAIN CONTAINING 19 NM_012337 CCDC19 11 COILED-COIL DOMAIN CONTAINING 27 NM_152492 CCDC27 COILED-COIL DOMAIN CONTAINING 63 NM_152591 CCDC63 COILED-COIL DOMAIN CONTAINING 78 NM_173476 CCDC78 HYPOTHETICAL PROTEIN FLJ10786 NM_018219 CCDC87 1 HYPOTHETICAL PROTEIN FLJ90575 NM_153376 CCDC96 1 CENTRIN 2 NM_019405 CETN2 11 1 CYTOCHROME P450, FAMILY 1, SUBFAMILY A, POLYPEPTIDE 1 NM_000499 CYP1A1 DOUBLECORTIN DOMAIN CONTAINING 2 NM_016356 DCDC2 SIMILAR TO DOUBLECORTIN DOMAIN-CONTAINING PROTEIN 2 (RU2S PROTEIN) XM_940631 DCDC2B DYNEIN, AXONEMAL, HEAVY POLYPEPTIDE 8 NM_001371 DNAH8 DYNEIN, AXONEMAL, HEAVY POLYPEPTIDE 9 NM_004662 DNAH9 Dnahc9 11 1 DYNEIN, AXONEMAL, INTERMEDIATE POLYPEPTIDE 1 NM_012144 DNAI1 Dnaic1 11 DYNEIN, AXONEMAL, LIGHT INTERMEDIATE POLYPEPTIDE 1 NM_003462 DNALI1 11 DPY30 DOMAIN CONTAINING 1 NM_138812 DYDC1 11 DYNEIN, LIGHT CHAIN, ROADBLOCK-TYPE 2 NM_130897 DYNLRB2 11 HYPOTHETICAL PROTEIN FLJ10466 NM_018100 EFHC1 11 FAMILY WITH SEQUENCE SIMILARITY 92, MEMBER B NM_198491 FAM92B F-BOX PROTEIN 31 NM_024735 FBXO31 FILAMIN C, GAMMA (ACTIN BINDING PROTEIN 280) NM_001458 FLNC GUANOSINE MONOPHOSPHATE REDUCTASE 2 NM_001002002 GMPR2 G PROTEIN-COUPLED RECEPTOR 156 NM_153002 GPR156 HOMEOBOX A1 NM_153620 HOXA1 HOMEOBOX A3 NM_153632 HOXA3 IQ MOTIF CONTAINING WITH AAA DOMAIN NM_024726 IQCA 1 IQ MOTIF CONTAINING H NM_001031715 IQCH 1 JANUS KINASE 2 (A PROTEIN TYROSINE KINASE) NM_004972 JAK2 1 KIAA1370 NM_019600 KIAA1370 BC031353 11 KINESIN FAMILY MEMBER 9 NM_182903 KIF9 11 LUTEINIZING HORMONE BETA POLYPEPTIDE NM_000894 LHB 1 HYPOTHETICAL LOC342346 XM_296817 LOC342346 4930562C15Rik XM_203398 LOC463933 4930415F15Rik LEUCINE RICH REPEAT CONTAINING 27 NM_030626 LRRC27 LEUCINE RICH REPEAT CONTAINING 34 NM_153353 LRRC34 NM_026648 LRRC50 11 LEUCINE RICH REPEAT CONTAINING 51 NM_145309 LRRC51 NIMA (NEVER IN MITOSIS GENE A)-RELATED KINASE 5 NM_199289 NEK5 11 NON-METASTATIC CELLS 5, PROTEIN EXPRESSED IN (NUCLEOSIDE-DIPHOSPHATE KINASE) NM_003551 NME5 11 OUTER DENSE FIBER OF SPERM TAILS 3 NM_053280 ODF3 11 PHOSPHODIESTERASE 4D, CAMP-SPECIFIC (PHOSPHODIESTERASE E3 DUNCE HOMOLOG, DROSOPHILA) NM_006203 PDE4D RENAL TUMOR ANTIGEN NM_014226 RAGE 11 ROPPORIN 1-LIKE NM_031916 ROPN1L 11 RADIAL SPOKEHEAD-LIKE 1 NM_031255 RSHL1 RADIAL SPOKEHEAD-LIKE 2A NM_025789 RSHL2A 11 RADIAL SPOKEHEAD-LIKE 3 NM_001010892 RSHL3 11 RHABDOID TUMOR DELETION REGION GENE 1 NM_014433 RTDR1 RETICULON 1 NM_021136 RTN1 SPERM AUTOANTIGENIC PROTEIN 17 NM_017425 SPA17 11 SPERM ASSOCIATED ANTIGEN 6 NM_172242 SPAG6 11 SPERM ASSOCIATED ANTIGEN 8 NM_172312 SPAG8 SPECTRIN REPEAT CONTAINING, NUCLEAR ENVELOPE 1 NM_182961 SYNE1 1 TCTEX1 DOMAIN CONTAINING 1 NM_152665 TCTEX1D1 TEKTIN 1 NM_053285 TEKT1 11 TEKTIN 2 (TESTICULAR) NM_014466 TEKT2 11 1 TEKTIN 3 NM_031898 TEKT3 1 1 TEKTIN 4 NM_144705 TEKT4 11 TESTIS/PROSTATE/PLACENTA-EXPRESSED PROTEIN, ISOFORM 2 NM_199456 TEPP 1700055M20Rik TRANSMEMBRANE 4 L SIX FAMILY MEMBER 1 NM_014220 TM4SF1 11 TRANSMEMBRANE 4 L SIX FAMILY MEMBER 18 NM_138786 TM4SF18 TESTIS SPECIFIC GENE A2 NM_025290 TSGA2 TETRATRICOPEPTIDE REPEAT DOMAIN 12 NM_017868 TTC12 1 WD REPEAT DOMAIN 16 NM_001037306 WDR16 11 NA NA WDR38 WD REPEAT DOMAIN 49 NM_178824 WDR49 WD REPEAT DOMAIN 63 NM_145172 WDR63 11 WD REPEAT DOMAIN 66 NM_144668 WDR66 1 WD REPEAT DOMAIN 69 NM_178821 WDR69 1 WD REPEAT DOMAIN 78 NM_146254 WDR78 11 1 Total 42 31 8 Figure 6. Identification of putative and validated FoxJ1 target genes in MTEC transcriptome. The human orthologs of FoxJ1 target genes identifiied in Stubbs et al, 2008, and Yu et al, 2008, are given in the leftmost column. The mouse ortholog, if different from the human ortholog, is given in column titled ‘mouse ortholog’. A ‘1’ in the ALI+4, ALI+12 or Cilia Proteome column indicates the presence of a gene in that dataset. The MTEC datasets, filtered for upregulated genes (M>1.0), identified 43 of 86 candidate FoxJ1 target genes identified in other studies. In comparison, the combined datasets in the ciliary proteome identified 8.
35 Biological Process ALI4 (p-value) ALI12 (p-value) GFP- (p-value) spermatogenesis 1.86E-09 >0.05 >0.05 microtubule-based process 2.31E-08 7.03E-05 >0.05 determination of left/right symmetry 1.66E-05 >0.05 >0.05 M phase 5.70E-05 >0.05 >0.05 cell cycle 9.70E-05 6.67E-03 >0.05 cilium assembly 1.52E-04 >0.05 >0.05 protein-DNA complex assembly 4.97E-04 >0.05 >0.05 chromatin assembly 2.15E-03 1.13E-04 4.52E-16 cell differentiation 5.85E-03 >0.05 >0.05 protein modification process 4.93E-02 3.02E-02 >0.05 fat cell differentiation 4.94E-02 >0.05 >0.05 immune system process >0.05 1.20E-07 >0.05 cell adhesion >0.05 2.60E-05 3.36E-06 anterior/posterior pattern formation >0.05 4.15E-04 >0.05 phosphorus metabolic process >0.05 1.12E-03 >0.05 embryonic development >0.05 6.43E-03 2.77E-05 brain development >0.05 3.78E-02 >0.05 tissue development >0.05 >0.05 5.55E-14 epithelium development >0.05 >0.05 4.15E-12 neuron differentiation >0.05 >0.05 2.66E-07 oxidation reduction >0.05 >0.05 1.22E-06 tube development >0.05 >0.05 2.34E-05 respiratory system development >0.05 >0.05 8.11E-05 positive regulation of transcription, DNA-dependent >0.05 >0.05 2.17E-02
Component ALI4 (p-value) ALI12 (p-value) GFP- (p-value) microtubule cytoskeleton 1.53E-22 1.53E-22 >0.05 cilium 2.05E-19 1.44E-06 >0.05 centrosome 1.42E-03 >0.05 >0.05 cell-cell junction >0.05 >0.05 3.51E-07 extracellular matrix >0.05 >0.05 1.91E-06 apical junction complex >0.05 >0.05 2.70E-07 intermediate filament >0.05 >0.05 5.63E-03 basement membrane >0.05 >0.05 3.89E-02
Molecular Function ALI4 (p-value) ALI12 (p-value) GFP- (p-value) ATP binding 1.77E-10 4.53E-04 >0.05 kinase activity 1.17E-08 1.45E-05 >0.05 motor activity 9.99E-04 3.36E-02 >0.05 tubulin-tyrosine ligase activity 2.71E-03 >0.05 >0.05 DNA binding 3.16E-02 >0.05 >0.05 growth factor binding >0.05 6.54E-04 >0.05 cyclin-dependent protein kinase activity >0.05 1.80E-02 >0.05 calcium ion binding >0.05 1.95E-02 1.08E-02
Figure 7. GO term analysis of genes upregulated during ciliogenesis. Genes upregulated 5-fold or more at ALI 4 and ALI 12 with reference to non-ciliated cells were submitted to GoTermFinder (go.princeton.edu/cgi-bin/GOTermFinder) to identify enriched processes, components and functions. The p-values indicate the significance of the enrichment of GO terms relative to the mouse genome.
36 Methods Animals and Animal Care MTECs were derived from wild-type C3H X C57Bl/6J F1 hybrid or FOXJ1/EGFP transgenic mice (a gift from L. Ostrowski, University of North Carolina at Chapel Hill, Chapel Hill, NC) generated on C3H X C57B1/6J F1 hybrid background (Ostrowski et al., 2003). Heterozygous FoxJ1/EGFP mice were obtained as described (Vladar and Stearns, 20007). PCR genotyping was performed using a forward primer specific to the FoxJ1 promoter region (5’- GCAGGCACCACATACTTATTCGGAGG-3’) and a reverse primer specific to EGFP (5’-CGTCCTTGAAGAAGATGGTGCG-3’). Mice were sacrificed by CO2 anesthesia and tracheas surgically removed. All work was performed in accordance with the Stanford University Administrative Panel for Laboratory Animal Care.
MTEC culture MTECs for microarray experiments were derived from tracheas removed from male and female mice between 6-20 weeks of age from multiple litters. For microarray experiments, MTECs were pooled and cultured on 6-well plates containing Transwell- Clear permeable membrane supports (Corning) for the microarray experiments. Cells dissociated from the tracheas of approximately 12-15 mice were seeded per 6-well plate. Four to six wells of one 6-well plate were pooled at ALI+12 and sorted to obtain RNA for a single biological replicate. Fourteen to sixteen wells at ALI+4 were pooled and sorted for a single biological replicate at ALI+4. Culture and differentiation of MTECs were performed as previously described (Vladar and Stearns, 2007).
FACS To prepare MTECs for FACS, cells were first removed from the filter by incubating at 37°C for 30 minutes in a 1:1 mixture of Cell Dissociation Solution (Sigma-Aldrich) and 0.5% Trypsin/EDTA (Invitrogen). After washing 2X in PBS, cells were resuspended in PBS at 106 cells/mL and passed through a 100 !m nylon
! 37 mesh cell strainer (BD Falcon) to remove clumped cells. GFP+ and GFP- populations were collected in serum. GFP+ cells were sorted based on a 2-fold logarithmic higher FITC intensity over a similar cell population from MTECs derived from wild-type mice FoxJ1-/- MTECs. Sorting was performed courtesy the Stanford Shared FACS Facility on a Vantage Vanford sorter. Data were acquired using CellQuest software.
RNA Extraction Total RNA was extracted using the RNEasy Kit (Qiagen) according to manufacturer’s protocols. Briefly, FACS sorted samples in serum were diluted 5:1 in PBS and centrifuged for 5 minutes at 1200 x g. The pellet was resuspended in 250!L of Buffer RT and spun through a QiaShredder Column (Qiagen) to disrupt cells before proceeding with the rest of the protocol. RNA was eluted in 30!L of RNase free water. Total RNA concentration was measured by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE) and stored at –80°C until amplification.
Probe preparation and hybridization Amplification and coupling of RNA to Cy3/Cy5 was performed with amino allyl MessageAmp II aRNA amplification kit (Ambion) according to the manufacturer’s protocols. Total RNA from MTECs or Universal Mouse Reference RNA (Stratagene) was reverse transcribed using the supplied T7 oligodT primer to produce first-strand cDNA. After second strand synthesis, cDNA was subjected to a single round of in vitro amplification with amino allyl modified UTP. The modified aRNA was purified, dried and coupled to either Cy3 or Cy5 reactive dyes. Dye incorporation and final probe concentration was checked by NanoDrop-1000 and between 2-10 !g labeled RNA was fragmented using RNA Fragmentation Reagent (Ambion) before proceeding to hybridization. Hybridization was done on MEEBO oligonucleotide arrays printed at the Stanford Functional Genomics Facility (www.microarray.org). A list of hybridizations is shown in Fig. 2a. MEEBO slides were post-processed the same day of hybridization. Fluorescently labeled amplified RNA samples from MTECs were hybridized to the array with an amplified reference RNA sample (Stratagene, La Jolla) for 16 hours at 65°C. Arrays were scanned on
! 38 Agilent G2565AA scanner immediately after washing. Features were extracted using GenePix6.0 (Axon Instruments). Detailed microarray protocols are available at http://brownlab.stanford.edu/protocols.html.
Data Analysis Linear Models for MicroArray (LIMMA) LIMMA software(Wettenhall and Smyth, 2004) , an R-based program the employs Empirical Bayes for the analysis of microarray data, was used to identify differentially expressed genes (Smyth, 2004) and for data normalization (Smyth and Speed, 2003). The design matrix used to obtain the subtracted ciliated cell transcriptome is shown in Fig. 2b. LIMMA software is available from the BioConductor website http://www.bioconductor.org/packages/release/bioc/html/limma.html. Genes were clustered by Pearson Correlation using Cluster 3.0 and visualized using TreeView as implemented by SMD (Demeter et al., 2007).
! 39 References
Andersen, J.S., C.J. Wilkinson, T. Mayor, P. Mortensen, E.A. Nigg, and M. Mann. 2003. Proteomic characterization of the human centrosome by protein correlation profiling. Nature. 426:570-4. Avidor-Reiss, T., A.M. Maer, E. Koundakjian, A. Polyanovsky, T. Keil, S. Subramaniam, and C.S. Zuker. 2004. Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell. 117:527-39. Blacque, O.E., and M.R. Leroux. 2006. Bardet-Biedl syndrome: an emerging pathomechanism of intracellular transport. Cell Mol Life Sci. 63:2145-61. Blacque, O.E., E.A. Perens, K.A. Boroevich, P.N. Inglis, C. Li, A. Warner, J. Khattra, R.A. Holt, G. Ou, A.K. Mah, S.J. McKay, P. Huang, P. Swoboda, S.J. Jones, M.A. Marra, D.L. Baillie, D.G. Moerman, S. Shaham, and M.R. Leroux. 2005. Functional genomics of the cilium, a sensory organelle. Curr Biol. 15:935-41. Broadhead, R., H.R. Dawe, H. Farr, S. Griffiths, S.R. Hart, N. Portman, M.K. Shaw, M.L. Ginger, S.J. Gaskell, P.G. McKean, and K. Gull. 2006. Flagellar motility is required for the viability of the bloodstream trypanosome. Nature. 440:224- 7. Chhin, B., J.T. Pham, L. El Zein, K. Kaiser, O. Merrot, and P. Bouvagnet. 2008. Identification of transcripts overexpressed during airway epithelium differentiation. Eur Respir J. 32:121-8. Demeter, J., C. Beauheim, J. Gollub, T. Hernandez-Boussard, H. Jin, D. Maier, J.C. Matese, M. Nitzberg, F. Wymore, Z.K. Zachariah, P.O. Brown, G. Sherlock, and C.A. Ball. 2007. The Stanford Microarray Database: implementation of new analysis tools and open source release of software. Nucleic Acids Res. 35:D766-70. Efimenko, E., K. Bubb, H.Y. Mak, T. Holzman, M.R. Leroux, G. Ruvkun, J.H. Thomas, and P. Swoboda. 2005. Analysis of xbx genes in C. elegans. Development. 132:1923-34. Gherman, A., E.E. Davis, and N. Katsanis. 2006. The ciliary proteome database: an integrated community resource for the genetic and functional dissection of cilia. Nat Genet. 38:961-2. Keller, L.C., E.P. Romijn, I. Zamora, J.R. Yates, 3rd, and W.F. Marshall. 2005. Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes. Curr Biol. 15:1090-8. Leroux, M.R. 2007. Taking vesicular transport to the cilium. Cell. 129:1041-3. Li, J.B., J.M. Gerdes, C.J. Haycraft, Y. Fan, T.M. Teslovich, H. May-Simera, H. Li, O.E. Blacque, L. Li, C.C. Leitch, R.A. Lewis, J.S. Green, P.S. Parfrey, M.R. Leroux, W.S. Davidson, P.L. Beales, L.M. Guay-Woodford, B.K. Yoder, G.D. Stormo, N. Katsanis, and S.K. Dutcher. 2004. Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell. 117:541-52. Liu, Q., G. Tan, N. Levenkova, T. Li, E.N. Pugh, Jr., J.J. Rux, D.W. Speicher, and E.A. Pierce. 2007. The proteome of the mouse photoreceptor sensory cilium complex. Mol Cell Proteomics. 6:1299-317.
! 40 McClintock, T.S., C.E. Glasser, S.C. Bose, and D.A. Bergman. 2008. Tissue expression patterns identify mouse cilia genes. Physiol Genomics. 32:198-206. Nachury, M.V., A.V. Loktev, Q. Zhang, C.J. Westlake, J. Peranen, A. Merdes, D.C. Slusarski, R.H. Scheller, J.F. Bazan, V.C. Sheffield, and P.K. Jackson. 2007. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 129:1201-13. Ostrowski, L.E., K. Blackburn, K.M. Radde, M.B. Moyer, D.M. Schlatzer, A. Moseley, and R.C. Boucher. 2002. A proteomic analysis of human cilia: identification of novel components. Mol Cell Proteomics. 1:451-65. Pazour, G.J., N. Agrin, J. Leszyk, and G.B. Witman. 2005. Proteomic analysis of a eukaryotic cilium. J Cell Biol. 170:103-13. Ross, A.J., L.A. Dailey, L.E. Brighton, and R.B. Devlin. 2007. Transcriptional profiling of mucociliary differentiation in human airway epithelial cells. Am J Respir Cell Mol Biol. 37:169-85. Sammeta, N., T.T. Yu, S.C. Bose, and T.S. McClintock. 2007. Mouse olfactory sensory neurons express 10,000 genes. J Comp Neurol. 502:1138-56. Smyth, G.K. 2004. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 3:Article3. Smyth, G.K., and T. Speed. 2003. Normalization of cDNA microarray data. Methods. 31:265-73. Stolc, V., M.P. Samanta, W. Tongprasit, and W.F. Marshall. 2005. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. Proc Natl Acad Sci U S A. 102:3703-7. Stubbs, J.L., I. Oishi, J.C. Izpisua Belmonte, and C. Kintner. 2008. The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos. Nat Genet. 40:1454-60. Vladar, E.K., and T. Stearns. 2007. Molecular characterization of centriole assembly in ciliated epithelial cells. J Cell Biol. 178:31-42. Wettenhall, J.M., and G.K. Smyth. 2004. limmaGUI: a graphical user interface for linear modeling of microarray data. Bioinformatics. 20:3705-6. You, Y., E.J. Richer, T. Huang, and S.L. Brody. 2002. Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population. Am J Physiol Lung Cell Mol Physiol. 283:L1315-21. Yu, X., C.P. Ng, H. Habacher, and S. Roy. 2008. Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nat Genet. 40:1445-53.
! 41
Chapter 3: Novel localization of candidate genes
42 Abstract Here we describe the localization of candidate ciliary proteins in cycling cells and MTECs using antibody staining and GFP-fusion constructs. Candidate genes were chosen based on a number of criteria: transcriptional upregulation in ciliated MTECs, as well as identification in other ciliary/basal body datasets, physical and genetic interactions with ciliary proteins and/or links to human disease. We describe the novel ciliary or centrosomal localization for 6 candidate proteins, of which 2 have never before been described in a screen for ciliary or centrosomal components. Two genes, MDM1 and FTO are linked to diseases with phenotypes that are part of the clinical spectrum of ciliopathies. Two genes, DYX1C1 and KIAA0319 are associated with dyslexia, which has never been described in patients with cilia defects. The 2 other genes examined, MLF1 and WHSC1, are involved respectively in leukemia and Wolf-Hirschorn syndrome, a multi-organ disease with many of the same features as the ciliopathies.
Results Transcriptional regulation of centrosome components during ciliogenesis in ciliated epithelial cells The basal bodies of ciliated epithelial cells are morphologically similar to those in cycling cells, but the extent of the similarity, with respect to composition and mechanism of formation, is unclear. We searched the array dataset for 32 known centrosomal proteins identified in a proteomic purification of human centrosomes (Andersen et al., 2003). Indeed, 25 of 32 were upregulated greater than 2-fold at ALI+4 (Fig. 1a). The seven that were not upregulated were Cep170, Cep192, Cep250/Cnap-1, Cep27, Cep68 and Eb1. Cep-250/Cnap-1 and Cep68 act in centrosome cohesion (Graser et al., 2007; Mayor et al., 2000). Cep27/Haus2 is part of a 8-subunit complex that localizes to the centrosome and regulates mitotic spindle formation (Lawo et al., 2009), and Cep170 also organizes microtubules (Guarguaglini et al., 2005). Cep192 is the mammalian ortholog of C.elegans spd-2, which encodes a centriolar and pericentriolar material (pcm)-localized protein that is important for recruiting microtubule organizers to the
43 pcm (Zhu et al., 2008). Eb1 controls microtubule dynamics (Rogers et al., 2002) (Schroder et al., 2007) and has also been reported to be important for primary cilia formation (Yan et al., 2006). Many of the genes for verified and candidate centrosomal proteins are upregulated in the MTEC dataset, and many of those that were not detected to be significantly upregulated are involved in microtubule organization and dynamics. This might reflect a functional difference between the centriole in cycling cells and basal bodies in ciliated cells. In support by the transcriptional data showing that except for Tubg1 and Gcp3 (encoding gamma tubulin and a gamma tubulin small complex member Gcp3), the other components of the gamma tubulin ring complex do not show significant levels of upregulation relative to nonciliated cells (Fig. 1b). Similarly, the genes for Cep250/Cnap-1 and Cep68 centrosome cohesion proteins were not induced during ciliogenesis.
Centriole duplication genes are upregulated during basal body generation A core set of conserved proteins required for centriolegenesis in cycling cells has been defined, and we asked whether these proteins are upregulated during ciliogenesis. These proteins, Plk4, Sas-6, Sas-4, Cep135, CP110, centrobin, Cep120, epsilon tubulin and delta tubulin, are all associated with centrioles (Bettencourt-Dias and Glover, 2009). All were upregulated more than 2.8-fold at ALI+4 (Fig. 1d), suggesting that centriole formation in cycling cells and basal body duplication in ciliated epithelial cells share a common mechanism. We examined the expression of CP110 and its interacting partner Cep97, which have been implicated in preventing untimely cilia formation in cycling cells {Spektor, 2007 #5;Tsang, 2008 #2}. Both CP110 and Cep97 are cell cycle regulated: they are transcribed in low amounts in quiescent cells and peak in S-G2 (Chen et al., 2002). Interestingly, in multiciliated cells, CP110/RIKEN cDNA 6330503K22 and Cep97/Lrriq2 were both 3.5-fold upregulated at ALI+4 relative to nonciliated cells. CP110 expression dropped slightly at ALI+12, and Cep97 expression increased slightly.
44 Drops in transcription of all core centriolegenesis genes from ALI+4 to ALI+12 were detected, consistent with observations that the induction of expression of other proteins required for basal body duplication is limited to a brief period during ciliated cell differentiation (Ross et al., 2007; Stolc et al., 2005; Vladar and Stearns, 2007). This contrasts with the expression of ciliary components, which generally exhibit a pattern of induction at the beginning of ciliogenesis and then maintenance at a high level of expression (Ross et al., 2007). The CETN4 gene encodes centrin4, which is expressed specifically in ciliated tissues (Gavet et al., 2003). CETN4 was highly upregulated during ALI+4 and then decreased in expression at ALI+12 (Fig. 1e), suggesting that its primary role is to facilitate basal body generation. Centrin-3 localizes to basal bodies in ciliated cells while Centrin-2 appears to localize to basal bodies as well as the proximal axoneme (Laoukili et al., 2000) and this is reflected in the transcriptional data: CETN3 is strongly induced and at the beginning of ciliogenesis and then decreases to the same level as nonciliated cells, while CETN2 induction increases slowly from ALI+4 to ALI+12.
Cell cycle control of basal body duplication One of the unique properties of ciliated epithelial cells is that multiple rounds of centriolegenesis occur in a G0 arrested cytoplasm, unlike in most cells where centrioles are duplicated once and only once in S-phase. To probe the unique cell cycle state of ciliated MTECs, we examined the expression of known cell cycle related regulators of centriolegenesis (Fig. 1c), and compared our results to a previous study also examining the expression of cell cycle regulators in MTECs (Vladar, 2007). This study previously reported that CDK2 showed little difference in expression between ciliated and non-ciliated cells while activators of CDK2 cyclin A and cyclin E were induced at both the transcriptional and translational level. Additionally, qRT-PCR analysis of ciliating MTECs showed that p21 was upregulated about 4-fold at the start of ciliogenesis and this level was maintained through late ciliogenesis. Western blots of p27 showed little difference in protein level between ciliated and nonciliated MTEC populations. All of these expression data were confirmed by our microarray data with the exception of cyclin E1. CCNE1 expression in ciliated cells was about the same as
45 in nonciliated cells, whereas CCNE2 appeared to be slightly downregulated. We also noted that CCNA1 but not CCNA2 is upregulated. Most of the genes that we examined (Fig. 1c) fell below the arbitrary significance level of 2-fold (M > 1 or M<-1) differential expression. Securin (PTTG1) and shugoshin-like 2 (SGOL2) were upregulated in ciliated cells relative to non- ciliated cells. Separase (ESPL1) was not differentially expressed. Shugoshin-like 1 maintains cohesion at sister chromatids (Kitajima et al., 2006) but relatively little is known about shugoshin-2. The SCF/Slimb ubiquitin conjugating complex degrades Plk4 and knockdown of Slimb leads to Plk4 dependent centriole amplification in Drosophila cell lines (Cunha-Ferreira et al., 2009). Both Btrc, the mouse ortholog of Slimb, and Skp2 were >2-fold upregulated in ciliated cells relative to nonciliated cells. Since Skp2/Slmb substrate recognition is tightly controlled by the phosphorylation state of the substrate (Vodermaier, 2004) it is difficult to draw any conclusions about Skp2/Slimb activity from their modest induction.
Novel centrosomal localization of proteins encoded by genes induced during ciliogenesis One goal of this study was to use our microarray data to identify novel centrosome-localized proteins based on their transcriptional induction. An obvious caveat was that, unlike methods that directly purify components from the structure of interest, our microarray array data were certain to identify genes encoding proteins that might be important for ciliogenesis and basal body duplication yet localize elsewhere than the cilium and the basal body. To narrow our search to candidate ciliogenesis genes that would be more likely to be validated by localization, we used a variety of criteria that included their upregulation in MTECs (Fig. 2) as well as their presence in other ciliary datasets without associated validation by localization or function, interactions with proteins known to be involved in ciliogenesis, and mutant phenotypes documented in the literature. To test whether candidate genes were involved in ciliogenesis, we either 1) expressed GFP-tagged fusion proteins in tissue culture cells and observed their localization by indirect immunofluorescence staining
46 or 2) performed antibody staining on MTEC cultures to determine their localization in ciliated epithelial cells.
Mdm1 (mouse double-minute 1) MDM1 was first identified as an amplified gene present on extrachromosomal bodies (double minutes) in NIH 3T3 cells lines (Cahilly-Snyder et al., 1987). Little is known about Mdm1 but it has been reported to localize to the nucleus (Snyder et al., 1988). Recently, a nonsense mutation that results in truncation of the Mdm1 protein was identified in the arrd2 mouse model for age-related retinal degeneration (ARRD) (Chang et al., 2008). ARRD has not been described as a ciliopathy, and the retinal degeneration observed in Mdm1-/- mice differs from that seen in BBS- nulls in its extremely late onset. Nonetheless, Mdm1 was highly upregulated during ciliogenesis, and has been identified in a proteomic purification of human centrosomes (Andersen et al., 2003) and a proteomic analysis of mouse photoreceptor sensory cilia (Liu et al., 2007), so we investigated further. Mdm1 has multiple splice isoforms and two were represented on the MEEBO arrays. Transcript variant 1 was slightly shorter than variant 2 due to a 60 nucleotide deletion at the 3’ end of the transcript. Both were strongly upregulated at ALI+4. Mdm1 possesses a coiled-coil protein interaction domain that is maintained in the truncated protein in arrd2 (Fig. 3a). This domain is found in many proteins that have been associated with ciliogenesis (Pazour et al., 2005). Transfection of NIH-3T3 cells with a plasmid expressing Mdm1-GFP revealed that Mdm1-GFP colocalizes with !- tubulin at the centrosome (Fig. 3b) and is also detected on primary cilia and in the nucleus (Fig. 3c).
Dyx1c1 (Dyslexia Susceptibility 1 Candidate 1) and KIAA0319 Neurocognitive impairments have been observed in individuals with ciliopathies but the mechanistic link between the phenotype and cilia is poorly understood (Zaghloul and Katsanis, 2009). Multiple genetic loci have been linked to susceptibility for one such neurocognitive impairment, dyslexia (Williams and O'Donovan, 2006), and we noted that for three of those regions(Paracchini et al.,
47 2006; Poelmans et al., 2009; Taipale et al., 2003), nine of ten candidate dyslexia genes present were differentially regulated in ciliated cells: DCDC2A, S100B, DYX1C1, KIAA0319, ACOT13, PRMT2, TTRAP, PCNT and ROBO1 (Fig. 6b). Based on this surprising finding, we asked whether the proteins encoded by two, Dyx1c1 and KIAA0319, can localize to cilia. DYX1C1 (Dyslexia Susceptibility Candidate 1), the first reported candidate gene for dyslexia (Taipale et al., 2003), was upregulated more than 16-fold at ALI+4 in MTECs, and was identified in a comparative genomics screen for genes that are found only in organisms that make cilia (Li et al., 2004). In utero RNAi of Dyx1c1 in the lateral ventricles of rat brains causes disruption of neuronal migration, and these mice had corresponding defects in auditory and spatial processing (Threlkeld et al., 2007), which can be an early indicator for dyslexia (Tallal and Benasich, 2002). KIAA0319 was upregulated more than 4-fold at ALI+4, and depletion of KIAA0319 also impairs neuronal migration {Paracchini, 2006 #494}. Interestingly, a number of proteins that are associated with neuronal migration (Fig. 6a) are differentially regulated in ciliated cells. GFP-tagged versions of rat Dyx1c1 (Wang et al., 2006) and mouse KIAA0319 were expressed in NIH3T3 cells to determine their localization. Dyx1c1 contains an N-terminal p23 chaperone domain and three C-terminal TPR domains. Previously it was shown that full-length Dyx1c1-GFP overexpressed in embryonic rat brains localized predominantly to the cytoplasm of neurons, and that a N-terminal construct lacking the TPR repeats was nuclear and cytoplasmic while the C-terminal construct containing the TPR repeats but not the p23 domain was mostly cytoplasmic with a possible concentration at the centrosome (Wang et al., 2006). We tested the same constructs for localization to the centrosome and cilium. Both Dyx1c1-GFP and KIAA0319-GFP colocalized with gamma-tubulin at the centrosome (Fig. 4a, Fig. 5) and furthermore, we detected DYX1C1-GFP in the primary cilium of some cells (Fig. 4b). We found that a 320 amino acid N-terminal fragment localized to the cytoplasm and nucleus (Fig. 4c) and was occasionally observed at the centrosome (Fig. 4d), and a 108 amino acid C-terminal fragment of Dyx1c1 localized to the cytoplasm and the centrosome in NIH 3T3 cells (Fig. 4e). Therefore, consistent with previous reports (Wang et al., 2006), loss of the C-terminal amino acids causes the majority of the
48 truncated protein to localize to the nucleus, whereas full length Dyx1c1 localizes to the centrosome and the primary cilium.
Identification of a ciliary heterotrimeric G protein subunit G-protein coupled signaling is involved in numerous diseases and cellular functions. Since G-protein coupled receptors are localized to motile cilia, we speculated that other components of G-protein coupled signaling pathways might also be present. A number of heterotrimeric G proteins were differentially expressed in MTECs (Fig. 7). One of these, guanine nucleotide binding protein (G protein), beta polypeptide 4 (Gnb4), has been identified in numerous studies in the cilia proteome. Little is known about the specific activity of Gnb4, although it has been associated with various cancers {Riemann, 2007 #337;Riemann, 2009 #338}. MTECs stained with an anti-Gnb4 antibody revealed localization of Gnb4 to motile cilia stained with acetylated alpha tubulin (Fig. 8a). Gnb4 was also detected at the cell junctions of both ciliated and nonciliated cells. A closer look at freshly dissociated cells from mouse trachea showed Gnb4 in the cytoplasm of ciliated and non-ciliated cells (Fig. 8b). Fig. 8a and 8b were performed on MTECs fixed with 4% paraformaldehyde. Methanol fixation removed much of the cytoplasmic and cell junction staining, leaving Gnb4 intact on motile cilia and the primary cilia of neighboring cells (arrows in Fig. 8c). Gnb4 did not stain primary cilia of paraformaldehyde or methanol fixed RPE1 cells (data not shown).
Novel localization of a leukemia disease gene product to cilia Acute myeloid leukemia (AML) is one of four types of leukemia and it is characterized by blood cell deficiency caused by an increase in the number of immature myeloid cells in bone marrow combined with a block in their differentiation. Mutations of nucleophosmin (NPM) are found in 30% of de novo adult acute myeloid leukemia (AML), making them the most common genetic mutation in the disease {Falini, 2007 #360}. NPM is a predominantly nucleolar protein that shuttles between nucleus and cytoplasm. In addition to playing roles in ribosome biogenesis, DNA repair and apoptosis (Falini et al., 2007), nucleophosmin is a Cdk2/cyclinE substrate
49 and proposed regulator of centriole duplication (Okuda et al., 2000). Dissociation of nucleophosmin from the centrosome in G1/S is required for centrosome duplication, and preventing nucleophosmin localization to the centrosome by interference with the Ran-Crm1 pathway results in centrosome amplification (Wang et al., 2005). Therefore, nucleophosmin has both nuclear and cytoplasmic functions. Fusions of NPM to many different genes have been identified in AML. NPM- MLF1 fusions account for less than 1% of AML cases and are caused by a t(3;5)(q25;q35) translocation that is most commonly found in the French-American- British (FAB) classified M6 subtype specifying acute erythroid leukemia, or proliferation of erythroblast precursors. Mlf1 was first identified as Hls7, a gene whose ectopic expression was sufficient to induce spontaneous switching of erythroid cells to cells of the myeloid lineage {Williams, 1999 #439}. Mlf1 is expressed in bone marrow cells but declines rapidly as cells differentiated (Matsumoto et al., 2000). Mlf1 expression is observed in numerous non-hematopoietic tissues however, such as testis, ovary, skeletal muscle, kidney (Yoneda-Kato et al., 1996), as well as the epithelium of the nose and brain ependyma (Hitzler et al., 1999). Mlf1 is a predominantly cytoplasmic protein, but under certain conditions it accumulates in the nucleus (Winteringham et al., 2006; Yoneda-Kato and Kato, 2008). Fusion of NPM and Mlf1 joins the amino-terminal portion of NPM (amino acids 1-175) and all of Mlf1 except for the first 16 amino acids (Yoneda-Kato et al., 1996), and the resulting NPM-Mlf1 protein localizes almost exclusively to the nucleus (Yoneda-Kato et al., 1996). We detected high levels of MLF1 induction in ciliated MTECs. We stained MTECs with a commercial antibody made against Mlf1. Mlf1 was detected on motile cilia of multiciliated cells (Fig. 9a) but not on the primary cilia of neighboring cells (data not shown). Mlf1 labeled the length of the motile cilia in a punctate pattern, and in some cells appeared to be concentrated at the apical tips (Fig. 9b). Mlf1 staining was somewhat variable in ciliating cultures, staining the cytoplasm in some cells and in others appearing weakly punctate or strongly punctate along the entire cilium (data not shown). This difference likely reflects staining of cells at different stages of ciliation.
50
The obesity related gene fto encodes a microtubule and cilium-associated protein Fatso (Fto) was originally identified as one of the six genes present on the 1.6 Mb deletion region of chromosome 8 that were deleted or disrupted in the fused toes (ft) mouse: fto, fts, ftm (fantom, otherwise known as Rpgrip1l), and Irx 3, 5 and 6 (Peters et al., 1999). The fused toes mutation is embryonic lethal between E10 and E14.5 in homozygous animals, and embryos display hallmarks of ciliopathic disease such as syndactyly and polydactyly, neural tube patterning, craniofacial abnormalities and left-right asymmetry defects (Anselme et al., 2007; Gotz et al., 2005; Grotewold and Ruther, 2002; van der Hoeven et al., 1994). From a cilia-centric point of view this was interesting since the phenotypes of the Ft mouse closely resembles those observed in BBS mutants, and it has been shown that the basal body protein RPGRIP1l is responsible for most of these phenotypes, likely due to a deficiency in Shh signaling despite a lack of defects in primary cilium formation (Vierkotten et al., 2007). FTO was originally linked to obesity and obesity-dependent type 2 diabetes by genome-wide association studies of in human patients (Dina et al., 2007; Frayling et al., 2007). Possession of two copies of a common high risk allele predisposes individuals to weighing 3 kg on average more than individuals with both copies of the low risk allele (Frayling et al., 2007). Subsequently, fto knockout mice were described that ate more per unit body mass, exercised less, and were leaner than their wild type counterparts, demonstrating directly that the Fto gene product plays a functional role in controlling energy homeostasis {Frayling, 2007 #453}. Of the six genes present in the Ft deletion (Fig. 10a), Fto, Rpgrip1l and Irx5 were upregulated in ciliated MTECs relative to nonciliated MTECs (Fig.10b). Irx3 was highly upregulated in ciliated MTECs but also in nonciliated cells. Irx6 was downregulated relative to nonciliated cells, and Fts was downregulated in all MTECs. We analyzed the data for genes associated with fat-cell differentiation (GO Term 0045444) by clustering (Fig. 10c). Most of the genes in the upregulated cluster are known cilia/basal body proteins: they included Bardet-Biedl members 2,3,4,6,8 and 9, as well as the gene for chibby, a basal body protein that represses beta-catenin
51 dependent transcription and is required for ciliogenesis in airway epithelium (Voronina et al., 2009). To determine whether fatso also localized to ciliary structures we stained ciliated MTECs with a commercial antibody against fatso. Fatso co-localized to motile cilia marked with the IFT component polaris (Fig. 10d), decorating the length of the cilium (Fig. 10e). In nonciliated cells in MTEC cultures, Fatso was seen at the primary cilium and cytoplasmic microtubules (Fig. 11a) and in the midbodies of rarely observed dividing cells (Fig. 11b). In RPE1 cells, Fatso stained primary cilia and cytoplasmic microtubules (Fig. 11c).
Wolf-Hirschhorn syndrome candidate 1, a histone methyltransferase, is expressed in ciliated cells Whsc1 is one of 5 putative SET2 (Suppressor of variegation [Sur(var)3-9], Enhancer of zeste [E(z)], and Trithorax 2) orthologs in mouse. Whsc1 is also known as Nsd2 (Nuclear receptor-binding SET domain-containing protein) or MM-SET (multiple myeloma-with SET domain protein). Whsc1 is a histone lysine methyltransferase that is thought to repress transcription (Marango et al., 2008) (Nimura et al., 2009). In addition to its SET domain, Whsc1 also possesses a PWWP (proline-tryptophan-tryptophan-proline) domain common in nuclear proteins, a HMG (high mobility group) box, thought to be a DNA binding domain, and PHD (plant homeodomain) zinc fingers (Stec et al., 1998). Deletions in Whsc1 have been found in every known case of Wolf-Hirschhorn Syndrome (WHS), which is characterized by abnormalities in growth and cranio-facial development, learning disability, congenital heart problems and numerous midline defects (Bergemann et al., 2005). Whsc1 is also mutated in t(4;14)(p16;q32) -associated multiple myeloma, which accounts for about 15-20% of all MM cases (Stec et al., 1998). This translocation fuses Whsc1 to the IgH locus, an event observed in all studied case of Wolf-Hirschhorn Syndrome, and the fusion results in overexpression of Whsc1 (Keats et al., 2003). Wolf-Hirschhorn candidate 1 (Whsc1) was identified by global analyses of Xbox containing genes in C.elegans (Blacque et al., 2005) and by comparative genomics of ciliated and nonciliated organisms (Avidor-Reiss et al., 2004), suggesting
52 that it has a conserved role in ciliogenesis. WHSC1 was also upregulated in our MTEC dataset. To confirm our microarray data we stained MTECs using a commercially available rabbit antibody against Whsc1. Whsc1 was detected in nuclei of cells making cilia (Fig. 12b, arrow). Surprisingly, in addition to its nuclear localization, Whsc1 also stained the basal bodies (Fig. 12b, c). We were unable to detect Whsc1 localization to centrioles or cilia in RPE1 tissue culture cells, likely because it is not expressed (data not shown). Three genes, Ezh1, Suc39h1, and Setdb1, which also encode proteins with histone lysine methyltransferase activity, were upregulated (M>1.0) in ciliated cells relative to nonciliated cells (Fig. 12a), but none of the three GO-annotated histone arginine methyltransferases in the mouse genome were (data not shown).
Upregulation of Human Disease Genes in MTECs A number of genes implicated in human disease were upregulated in FoxJ1/EGFP cells during ciliogenesis (Fig.13). Many of these revealed no known association with centrosomes or cilia as determined by Pubmed searches using the gene name/symbol and the words ‘cilia’ or ‘centrosome’. Many of these genes are mutated in various cancers. Some have been linked to defects that are observed in individuals with ciliopathies, such as male infertility (Boll, Morc1), obesity (Hap1), retinal degeneration (RD3, Rp1h). One gene, MMACHC, was upregulated that is linked to kidney disease, but not of the polycystic variety. A number of genes linked to motor neuron disease were identified (Als2cr12, Atcay, Nipa1, Pnkd, Sp7). Other genes linked to types of lateral sclerosis and spastic paralysis seem to localize to the centrosome (Errico et al., 2004; Millecamps et al., 2005; Robay et al., 2006), which may reflect a general role for centrosomes in neuronal development. Similarly, many genes linked to neurological disorders were upregulated. In addition to numerous genes associated with leukemia (Fgfr1op, Mlf1, Myb, Pbx4), a number of the 15 member FANC (Fanconi Anemia) family were upregulated. These genes are thought to mediate replication-coupled DNA repair (Thompson and Hinz, 2009). Interestingly, Grk2 (G-protein coupled receptor kinase 2) was upregulated. Grk2 has been linked to heart failure and positively regulates Smoothened signaling
53 by phosphorylating Smoothened and promoting its internalization (Chen et al., 2004; Meloni et al., 2006). Numerous multi-organ disease genes Hps1, Muted, Porcn, Tulp2, Wfs1, and Sos1 were upregulated. Both Hsp1 and Muted are associated with Hermansky-Pudlak, a disease of hypopigmentation, hemorrhaging, and early death due to lung abnormalities. A second, shorter transcript variant of Muted was not upregulated. Porcn encodes the Porcupine protein, which is mutated in focal dermal hypoplasia. Tulp2 is a member of the Tubby family, and members have been linked to Shh signaling, obesity and cilia (Mak et al., 2006; Norman et al., 2009). Wfs1 is mutated in Wolfram syndrome, which has an interesting combination of phenotypes: Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, and Deafness. Mice lacking Wolframin, the product of Wfs1, also have reduced fertility (Noormets et al., 2009). Sos1, the gene for Son of Sevenless, is mutated in Noonan syndrome, which is characterized by short stature, facial dysmorphism, congenital heart defects, and skeletal anomalies.
Expression of Ras GTPases and effector proteins during ciliogenesis The Rabin8 guanosyl exchange factor (GEF) for Rab8 is associated with the BBsome and is required for BBS4 localization to centriolar satellites and normal cilia formation (Nachury et al., 2007). Rab8GTP, which is found in the cilium and is required for growth of the ciliary membrane, is unable to enter the cilium in cells depleted of Rabin8, suggesting that Rabin8 is required for Rab8GTP to enter the cilium, and that defects in ciliogenesis after Rabin8 depletion are due to a failure of Rab8GTPto enter the cilium {Nachury, 2007 #194}. Probes for Sec2p (Rabin8 GTP exchange factor (GEF)) were not found on the MEEBO arrays. The gene for Rab8 was present but not differentially expressed in ciliated cells relative to nonciliated cells (Fig. 14). This might reflect high levels of Rab8 in nonciliated cells. However, a survey of genes encoding RabGTPases on the MEEBO arrays identified two that are strongly upregulated, Rab28 and Rab36. Four Rab-like proteins, Rabl2a, Rabl3, Rabl4 and Rabl5 were also upregulated. Arl8a, Arl8b and Arl3 from the Arl family of Arf-like GTP-binding proteins were upregulated, as well as Arfgef2, an ADP ribosylation factor guanine nucleotide-exchange factor that has been implicated in vesicle
54 trafficking and neural migration (Sheen et al., 2004). Although we did not detect upregulation of Rho GTPases, Arhgap18, Arhgap24, Arhgap4, Arhgap5 and Arhgdig were. In summary we were unable to detect the transcriptional upregulation of Rab8, the canonical ciliary Rab, but we did detect the upregulation of a number of other small GTPases. The activity of small GTPases is modulated by whether it is bound to GTP (active) or GDP (inactive), and we also detected the upregulation of a subset of Rab effectors.
G-protein coupled receptors are upregulated in ciliated MTECs Searching the set of genes upregulated more than 2-fold in ciliated MTECs, we identified two GPCRs that have neurotransmitters for ligands: opioid receptor, kappa 1 (Oprk1) (M = 1.6 at ALI+12) and dopamine receptor 2 (Drd2) (M = 1.5 at ALI+4). Opiates are known to have a strong repressive effect on breathing though their action on breathing control centers in the brainstem and cortex, and in mechanosensing nerve ends in the alveolar walls and smooth muscle of the trachea and the bronchus (Pattinson, 2008). In the airways, opioid receptors inhibit coughing and bronchoconstriction (Karlsson et al., 1990), as well as mucus secretion by goblet cells (Kuo et al., 1992). Groups have attempted to show that opiates affect CBF using ciliated epithelium obtained from nasal brushes of human patients, but have achieved either negative results (Selwyn et al., 1996) or inconclusive results (Karlsson et al., 1990; Roth et al., 1991; Rutland et al., 1982). On the other hand, dopamine has been shown to decrease CBF in mollusk (Woodward and Willows, 2006) and sea urchin (Wada et al., 1997). Human T2R4, T2R38, T2R43, T2R46 localized to motile cilia in human airway epithelium (Shah et al., 2009). The closest non-reciprocal mouse orthologs to the human proteins encoded by these genes are Tas2r108, which was 1.93-fold upregulated at ALI+12, Tas2r138, which was not on the mouse arrays, and Tas2r120 (best match for both T2R43 and T2R46). Out of the 30 taste receptor genes represented on the arrays, we detected upregulation for 3: Tas2r102, Tas2r107 and Tas2r125 (Fig. 15). The closest non-reciprocal human orthologs of the proteins
55 encoded by these genes are T2R13, T2R10, T2R14. This suggests that the ability of human airway epithelial cells to respond to taste compounds via taste receptors located in the motile cilia of multiciliated cells is preserved in rodents. Olfaction is another form of chemoreception that is thought to be mediated by ciliated olfactory sensory neurons (OSNs) in the main olfactory epithelia (MOE) of the nasal cavities. There are predicted to be over 1000 genes encoding odorant receptors in the mouse genome, compared to less than 400 in the human genome (Niimura and Nei, 2006). The difference seems to arise from a large expansion of genes in the mouse post mouse-human divergence and inactivation of a majority of human odorant receptor genes, which coincided with developments in the human visual apparatus that may have obviated the need for a finely-honed sense of smell (Gilad et al., 2004). Expression of odorant receptors is thought to be limited to the olfactory epithelium (Buck and Axel, 1991). However, out of 920 olfactory receptor genes that are represented on the arrays, 94 were upregulated more than 2-fold (Fig. 16a, b). This might indicate that olfactory receptors are also localized to motile cilia. The vomeronasal organ is an auxiliary odorant sensing organ that is the predominant organ for sensing pheromones. Over 160 genes encoding vomeronasal receptors in the mouse genome have bee identified (Zhang et al., 2004). 134 of these genes are represented on the MEEBO arrays, and 15 were upregulated more than 2- fold. (Fig. 17).
Discussion It is not clear how basal bodies are generated de novo, and this is an important question since the majority of basal bodies in multiciliated epithelial cells are generated in this manner. We have examined the transcriptional profiles of genes encoding proteins that play conserved roles in centriolegenesis in normal cells, and have found that all of these genes are upregulated, suggesting that they are required for de novo centriolegenesis as well. We confirmed previous findings that cyclin A is upregulated during ciliogenesis (Vladar, E.K., unpublished data.). Interestingly, the mouse ortholog of a human cyclin A binding protein, Klhdc9, was highly upregulated
56 in our dataset (M = 4.9 at ALI+4, M = 2.2 at ALI+12), and like Ccna1, its expression decreased greatly at ALI12 after most of basal body duplication has occurred. Recently, CP110, which is required for centriolegenesis, has also been implicated in negatively regulating primary cilium formation through its association with Cep97, constituting a regulatory “switch” between centriole and basal body functions of centrioles. We found that both CP110 and Cep97 were highly upregulated in ciliating cells. Why Cep97, a protein whose only known function is to negatively regulate cilia formation, is upregulated in ciliating cells is unclear. It would be interesting to determine whether the temporal separation of centrosome and basal body functions is replaced by a spatial separation in multiciliated cells, ie., whether CP110/Cep97 can be detected at nascent centrioles in the cytoplasm and/or basal bodies docked at the apical surface. We identified novel localization to the centrosome in tissue culture cells for 3 products of candidate genes upregulated in the MTEC ciliated cell transcriptome. We also established novel localization for 4 candidate genes to cilia and basal bodies of multiciliated epithelial cells. Out of 7 proteins, 2 had not previously been found in a ciliary database. 2 highly upregulated ciliogenesis candidates that were tested did not localize as GFP-fusions to centrosomes or cilia in tissue culture cells: Mns1 and Lrguk.
Cilia and human disease In our study we have identified genes upregulated during ciliogenesis representing phenotypically diverse diseases, many of which have never been linked to defects in motile or primary cilia. Future work will determine whether these disease proteins are involved in ciliogenesis and whether the pathologies caused by having defective copies of these genes are linked to proper cilia formation. Retinopathy is a symptom that is observed in many ciliopathic diseases, and mutations in genes encoding ciliary and centrosomal proteins have been linked to retinopathy. We have shown that Mdm1, mutated in the arrd2 mouse model for age-related retinal degeneration, is highly expressed in multiciliated cells and can localize to the primary cilium. Interestingly, Mdm1 was also detected in the nucleus, suggesting a potential
57 function in signaling. The truncation that occurs in the arrd2 mutant mouse has been defined and it will be important to determine whether this prevents Mdm1 localization to the cilium and what effect if any it has on maintaining a cilium. It has been suggested that the source of ciliopathic retinal degeneration is a defect of the connecting cilium that links the outer segment to the inner segment, and that this defect might be caused by 1) defective vesicular transport, in the case of BBS mutation, or 2) defective transport through the connecting cilium, in the case of mutations of IFT (Zaghloul and Katsanis, 2009). It will be interesting to determine whether Mdm1 interacts with any of these transport pathways.
Cilia and cancer A causative link between altered centrosome number and cancer has long been proposed, and recent studies in mutant flies with altered centrosome number directly support this (Basto et al., 2008; Castellanos et al., 2008). Cilia may also be causally linked to cancer; polycystic kidneys, for example, are a hallmark of ciliopathy. Indeed, work with mouse models has shown that cilia defects can alternatively enhance or block tumorigenic effects from the hedgehog signaling pathway (Han et al., 2009). Our results show that Mlf1, a protein associated with myeloproliferative disorder when misexpressed or when fused to nuclephosmin by genetic translocation, is upregulated in multiciliated cells. Surprisingly, an antibody directed against Mlf1 stained motile and primary cilia in MTECs. How the ciliary localization of Mlf1 might relate to leukemia is unclear since lymphoid and myeloid cells do not make primary cilia, but it is not inconceivable that Mlf1 might play roles outside of cilia, as has been demonstrated for IFT20 (Finetti et al., 2009). Physical interactions between Mlf1 and MRJ, a chaperone protein from the DnaJ/Hsp40 family (Li et al., 2008), dynein light chain 8 and 14-3-3zeta (Lim et al., 2002) have been detected, but little is known about the cellular function of Mlf1 except that Mlf1 has been linked to cell cycle arrest and differentiation. In cycling cells it has been reported that altering Mlf1 levels can interfere with the cell cycle and differentiation and that this can occur in two ways: by preventing cells from exiting the cell cycle in a manner linked to accumulation of p27KIP1 (Winteringham et al., 2004) and by inducing p53-dependent G1/G0 arrest
58 (Yoneda-Kato et al., 2005). In addition to mediating hedgehog signaling, other evidence suggests that cilia are required for cell cycle progression (for review, see (Pan and Snell, 2007; Snell and Golemis, 2007)). Multiciliated cells are unique in that the DNA and centriole duplication cycles are uncoupled. It will be interesting to determine whether depletion of Mlf1 in these cells will relieve the block to DNA replication, as suggested by a positive regulatory effect on cell cycle arrest, or prevent centriole duplication. G-protein signaling has been linked to cell cycle regulation and cancer. We observed Gnb4 at both motile and primary cilia, suggesting that it may have a role in cilium formation or some signaling pathway that is common to both motile and primary cilia. A number of other heterotrimeric G protein subunits were also upregulated, so it seems unlikely that Gnb4 is the only family member present in cilia. This does however underscore the recent finding that motile cilia are chemosensory, and it opens a path for discovery of novel signaling pathways that operate through motile and non-motile cilia.
Cilia and obesity Obesity is one of the most common and yet least well understood clinical phenotypes of the ciliopathies. In this study, we identify a new cilium and microtubule associated protein that has been linked significantly to obesity: Fatso. The FTO gene is historically important because the first SNP variants linked to human obesity were identified in FTO (Speliotes, 2009). Whether a defect in the protein Fatso is directly involved in causing obesity is complicated by the fact that human SNPs linked to obesity are intronic, and that FTO may be co-transcriptionally regulated along with RPGRIP1l, which lies directly upstream of it. However, mouse models in which the second and third exons were replaced with a neomycin cassette or containing a point mutation in a conserved C-terminal region both exhibited alterations in body mass when controlled for food intake and activity, supporting a role for Fatso in energy regulation. Mechanistically, it is not known how Fatso might affect body mass, but Fatso possesses nucleic acid demethylase activity and some members of the same family are
59 known to mediate DNA damage repair. Fatso expressed exogenously in Cos7 cells localizes to the nucleus (Gerken et al., 2007). Taken together, this suggests that it might be important for maintaining genome integrity, but Fatso substrates have not yet been identified. In this study we have shown that Fatso is expressed in multiciliated cells of the tracheal epithelium and in fact localizes to motile and primary cilia. This raises a new possibility that Fatso may be important for cilia function, and that its effect on energy regulation might function through the cilium. Recently, a consanguineous multiplex family was identified whose affected individuals carry a point mutation in a conserved C-terminal catalytic region of FTO. Individuals homozygous for this mutation had multiple neurological defects, including hydrocephaly, microcephaly and lissencephaly, as well as neurosensory deafness and increased early mortality from infection or unidentified causes (Boissel et al., 2009). Hydrocephaly and deafness are associated with defects in ciliary motility and sensory cilia, respectively, and are consistent with a role for Fatso in cilia function. We also note that Fatso, in addition to localizing to cilia, is found on other microtubule based structures, suggesting that it may generally modify microtubules and thus impact multiple processes that involve microtubules, which could explain other phenotypes observed in human patients.
Potential identification of a novel ciliopathy The histone modifying protein Whsc1 was detected in nuclei of a subset of ciliating cells. This is consistent with its demonstrated role in DNA binding and histone modification. That it was detected specifically in ciliating cells suggests that Whsc1 regulates genes required for ciliated cell differentiation. Wolf-Hirschhorn syndrome is characterized by a number of defects that together do not strongly imply ciliary dysfunction, but the major causes of mortality, being congenital defects, anoxia at birth and respiratory infection {Shannon, 2001 #489}, do. Therefore, we speculate that Whsc1 is important for the generation of motile cilia in the airway epithelium, which is supported by the detection of Whsc1 in ciliated MTECs but not nonciliated MTECs or RPE1 cells. Unexpectedly, we also found Whsc1 at basal bodies. This suggests that Whsc1 may have roles in ciliogenesis other than transcriptional control.
60 Protein methylation is an important post-translational modification that regulates a number of cellular processes, and there is a precedent for the localization of histone methylases to motile cilia (Schneider et al., 2008). Whsc1 may represent a new member of a class of proteins involved in post-translational regulation of cilia and basal body components.
Cilia and dyslexia Our data provide the first evidence linking a dyslexia candidate gene to cilia. Neuronal disorders observed in patients with ciliopathies range from central nervous system defects to neurocognitive impairments and sensorineural disorders, with mental retardation as the most common feature (Cardenas-Rodriguez and Badano, 2009), but to the best of our knowledge, incidence of dyslexia has not been documented. Currently the molecular basis for dyslexia is unknown, but four candidates genes identified by genetic linkage, DYX1C1, KIAA0139 and DCDC2 (reviewed in (Gabel et al., 2009)), have been shown to be required for neuronal migration in the cortex, providing one possible mechanism for development of the disease. Here we have shown that that GFP-tagged Dyx1c1 and KIAA0319 are capable of localizing to the centrosome and the cilium, which suggests that these proteins may play ciliary roles. The protein structure of Dyx1c1 offers few clues to what roles it may be playing. Previously it was shown that the C-terminal TPR containing region of Dyx1c1 was necessary and sufficient to for rescuing the neuronal migration phenotype observed in in utero RNAi-treated rat brains (Wang et al., 2006). Our findings confirm that Dyx1c1 is a centrosomal protein and that loss of the 108 C-terminal amino acids bearing TPR repeats allowed the overexpressed protein to enter the nucleus, but did not prevent association with the centrosome. This suggests that centrosomal localization of Dyx1c1-GFP alone is not sufficient to complement the loss of full-length Dyx1c1, and that the TPR protein binding motifs in the C-terminus are also required, perhaps to recruit additional factors to the centrosome. Centrosome positioning and movement is thought to be vital for the interkinetic nuclear movements during neuronal migration, and complexes of proteins
61 that are required for neuronal migration, including Lis1, dynein and Par6alpha, localize to the centrosome (Higginbotham and Gleeson, 2007). It has been suggested that Dyx1c1 might interact with these complexes at the centrosome to ensuring proper neuronal migration and that this could be the mechanism by which Dyx1c1 is linked to dyslexia, but it has not been demonstrated that Dyx1c1 interacts with any of these proteins endogenously (Wang et al., 2006). Alternatively, the localization of Dyx1c1- GFP to the centrosome and cilium suggest that it might interact with proteins that are important for cilia. We observed Dyx1c1-GFP on the cilia of some, but not all transfected NIH 3T3 cells, so it seems unlikely that it would be required for cilium formation, but its ability to localize to the cilium suggests that it might be important for cilia function. KIAA0319 possesses multiple copies of the PKD domain that is found in the extracellular portion of polycystin (Hughes et al., 1995), a membrane protein that interacts with polycystin-2 in the primary cilium of kidney epithelial cells to mediate fluid sensing. Although we did not detect ciliary localization in NIH 3T3 cells overexpressing KIAA0319-GFP, the promoter of KIAA0319 contains a binding site for the RFX family of transcription factors, which includes the ciliogenesis regulators Rfx2 and Rfx3. Different splice isoforms of KIAA0319 have been found to localize to the plasma membrane or be secreted (Velayos-Baeza, 2008 #655}, and KIAA0319 appears to be subject to clathrin-mediated endocytosis (Levecque et al., 2009). It is tempting to speculate that KIAA0319 might participate in cilium-mediated signaling, and that blocks in this function might contribute to dyslexia. Interestingly, the KIAA0319 homolog KIAA0319l, which was not significantly upregulated in our MTEC dataset, is present in a separate dyslexia disease locus (Couto et al., 2008). Future work will determine whether KIAA0319l is also a component of centrosomes or cilia. In addition to Dyx1c1 and KIAA0319, 9 other genes found on dyslexia susceptibility loci were upregulated in MTECs. One of these, DCDC2a, is related through its microtubule-binding doublecortin (DCX) domain to the retinitis pigmentosa protein RP1, a component of the photoreceptor connecting cilium (Liu et al., 2004), suggesting that DCDC2a may also be a ciliary protein. The PCM protein
62 pericentrin, which has been linked to human dwarfism and microcephaly (Rauch et al., 2008), was upregulated. S100beta is a member of the S100 family of 2 EF-hand calcium-binding motifs containing proteins and has been reported to colocalize with centrosomes and microtubules in the brain (Sorci et al., 1998). Them2 and Ttrap2 are encoded by genes in the same dyslexia linkage locus as KIAA0319 and Dcdc2a (Londin et al., 2003). Thioesterase superfamily member 2 (Them2) is thought to catalyze the hydrolysis of medium-to-long-chain-acyl-CoA thioesters (Cao et al., 2009; Wei et al., 2009) and has been reported to localize to microtubules in tissue culture cells (Cheng et al., 2006). Traf and TNF receptor associated protein (Ttrap) is a member of a divalent cation-dependent phosphodiesterases, and knockdown of Ttrap in zebrafish results in randomized left-right axis determination {Esguerra, 2007 #299}. Genetic evidence link DYX1C1, KIAA0319, and DCDC2a most strongly to dyslexia, but it is possible that multiple genes from the associated regions are involved. That 7 out of the 10 dyslexia candidate genes from 3 genetic loci are upregulated during ciliogenesis and either localize to the centrosome (pericentrin, Dyx1c1, KIAA0319, S100b), have a putative interaction with microtubules (Dcdc2a, Them2) or when mutated display phenotypes associated with defective cilia (Ttrap), strongly suggests that cilia dysfunction may underlie dyslexia. How cilia might be linked mechanistically to dyslexia is unclear. There is currently no established role for cilia in neuronal migration. Three separate studies showed that 1) neuronal migration in zebrafish is dependent upon functional PCP (Jessen et al., 2002) (Carreira-Barbosa et al., 2003), 2) some PCP effectors are required for mammalian ciliogenesis (Park et al., 2008; Zeng et al.), and 3) BBS genes interact genetically with the PCP gene Vangl (Ross et al., 2005); taken together, these studies link cilia to neuronal migration through the planar cell polarity pathway. It has also been demonstrated that depletion of polaris/IFT88 impairs neuronal migration from the subventricular zone to the olfactory bulb, but it is not clear whether this is due to the movement of cerebrospinal fluid by motile ependymal cilia or (Sawamoto et al., 2006) or to an effect on primary cilia in the subventricular zone.
63 a ALI+4 ALI+12 GFP- b ALI+4 ALI+12 GFP-
Eb1 Tubgcp2 Cep170 Tubg1 Cep170 Tubgcp3 Cep1 Tubgcp4 Cep27 Tubgcp5 Cep2 Tubg2
Cep192 ring complex Tubgcp6 Cep1 gamma tubulin Cep68 Cep250 ALI+4 ALI+12 GFP- Cep1 c Cdk5rap2 Skp1a Cep70 Plk2 Ofd1 Ccne2 Cep57 Ccna2 Cep63 Trp53 Cep72 Cdk2 Cntrob Chek1 Cep1 Npm1 Odf2 Cdc2a Cep120 Sgol1 Cep97 Ccne1 Cep76 Brca1 Cep135 Sgol2 centrosome Pcm1 Ccna1 cell cycle related Sfi1 Bttrc Nin Pttg1 Cep152 Cul1 Cep78 Espl1 Hyls1 Plk1 Poc5 Skp2 Cep55 Cdc14b Cep164 Cep350 Crocc Pcnt <-5.65 1:1 <5.65 Cep290 d e
Cep120 Centrobin Cetn4 Tubd1
Tube1 Cetn3 GFP- Cep135 ALI12 ALI12 CP110 Cetn2 ALI4 ALI4 Cenpj Cetn1 Sass6
Plk4 -2 0 2 4 6
0 1 2 3 4 log 2 log 2 Figure 1. Expression of centrosome and centriole related genes in ciliated MTECs. Heat maps depict the expression of genes encoding (a) known and putative centrosomal proteins, (b) the gamma tubulin ring complex, and (c) cell cycle regulators important for centriole formation during ciliogenesis. Genes are shown in rows and each column represents one single array. Data are zero transformed against non-ciliated (GFP-) cells and clustered. The scale indicates fold upregulation. Grey boxes represent gene spots that failed to pass quality control filters for the indicated array. The bar graphs show the expression of genes encoding key regulators of centriole duplication (d) and the mouse centrin family (e) at ALI+4 and ALI+12, relative to non-ciliated (GFP-) cells. x-values are shown in log (base 2).
64 Gene Name Symbol RefSeq ALI+4 ALI+12 transformed mouse 3T3 cell Mdm1 NM_010785 6.1 4.5 double minute 1, variant 1 transformed mouse 3T3 cell Mdm1 NM_148922 2.1 1.8 double minute 1, variant 2 dyslexia susceptibility 1 candidate 1 Dyx1c1 NM_026314 4.1 2.7 homolog KIAA0319 KIAA0319 XM_111397 2.1 0.9
Figure 2. Expression of genes encoding candidate proteins in ciliated MTECs. M values are given in log (base 2) relative to nonciliated cells.
65 a
322 355 708 Mdm1 coiled coil
322 355 398 Mdm1 coiled coil (arrd2)
b Mdm1-GFP !-tubulin Mdm1-GFP !-tubulin DNA
c Mdm1-GFP tubulinac Mdm1-GFP tubulin ac DNA
Figure 3. Schematic of Mdm1 full-length and arrd2 protein (a). Mdm1- GFP localized to the centrosome (b) as well as the cilium and nucleus (c) in NIH-3T3 cells. Insets show a magnified image of centrosomes and the primary cilium. Images were offest as indicate by the circles in the insets.
66 a Dyx1c1-GFP !-tubulin Dyx1c1-GFP !-tubulin DNA
b ac Dyx1c1-GFP tubulin Dyx1c1-GFP tubulin ac DNA
c N-Dyx1c1-GFP !-tubulin N-Dyx1c1-GFP !-tubulin DNA
d ac N-Dyx1c1-GFP tubulin N-Dyx1c1-GFP tubulin ac DNA
e C-Dyx1c1-GFP tubulinac C-Dyx1c1-GFP tubulin ac DNA
Figure 4. Dyx1c1-GFP localizes to the centrosome in NIH 3T3 cells. NIH 3T3 cells were transfected with full length, N-terminal and C-terminal deletion constructs of Dyx1c1 fused to GFP that were obtained from J.J.Loturco. Full length Dyx1c1-GFP localizes to the centrosome (a) and in some cells the primary cilium (b). An N-terminal construct fused to GFP (N-Dyx1c1-GFP) localized to the nucleus and cytoplasm (c) and was occasionally observed at the centrosome (d). A C-terminal construct fused to GFP (C-Dyx1c1-GFP) localized to the cytoplasm and centrosome (e). Insets show positively staining regions at higher magnification. Green channel, GFP (a-e). Red channel, !-tubulin (a,c); acetylated tubulin (b,d,e).
67 KIAA0319-GFP !-tubulin KIAA0319-GFP !-tubulin DNA
Figure 5. KIAA0319-GFP localizes to the centrosome in NIH-3T3 cells. The inset shows a magnified image of centrosomes.
68 a b ALI+4 ALI+12 GFP-
Cck S100b Nr2f1 Cxcl12 Prmt2 Pax6 Pcnt
Myh10 Dip2a Pex5 Lmx1b Nrsn1 ALI+12 Barhl1 Ttrap
Nr2f2 Them2 ALI+4 Neurog2 Top2b KIAA0319 Reln Dcdc2a Ptk2 Robo1 Top2b Pafah1b1 Dyx1c1
Dcdc2a -4 -2 0 2 4 6 Cxcr4 Phox2b log 2 Wnt2 Cdk5 Esr2 Dyx1c1 KIAA0319 Pex7 Robo3 Cckar Apbb1 Arx Figure 6 . Genes associated wih neuronal Titf1 migration and developmental dyslexia are Pxmp3 Ywhae upregulated in ciliated MTECs. Genes anno- Nr4a2 tated with the neuronal migration GO term (GO: Prkg1 Fezf1 0001764) plus Dyx1c1, KIAA0319 and Dcdc2a Sox1 were retrieved and clustered (a). Data were Ndel1 zero-transformed against nonciliated (GFP-) Ndn Tlx3 cells. The expression of genes associated with Dab1 dyslexia are shown in (b). M values are shown Mdga1 in log (base 2) for ciliated cells at ALI+4 and Gfra3 Ascl1 ALI+12 relative to nonciliated cells. Vax1 Twist1 Fyn Dckl1 Neurod4 Asz1 Psen1 Met Ccr4 Nav1 Atoh1 Gja1 Tes Chl1 Itga3 Satb2 Dcx Ntn1 Apbb2 Katna1 Bax Capza2 Prrxl1 Cttnbp2 Nde1 Dcc <-5.65 1:1 <5.65
69 ALI+4 ALI+12 GFP- Gnas Gng4 Gng12 Gnb1 Gng3 Rsg7 Gna15 Rgs6 Gng11 Gnao1 Gng7 Gngt2 Gng8 Gng5 Gnb5 Gnaq Gnb4 Gnai3 Gna13 Gnb3 Gnat2 Gng13 Gna11 Gnat1 Rgs9 Rgs11 Gna12 Gnaz Gng10
<-5.65 1:1 <5.65
Figure 7. Expression of genes encoding heterotrimeric G-proteins and associated molecules in MTECs. Data were zero transformed against nonciliated (GFP-) cells.
70 a tubulin Gnb4 tubulinmerge Gnb4 DAPI
b tubulin Gnb4 tubulinmerge Gnb4 DAPI
c Gnb4 Gnb4
DAPI MtOH
Figure 8. Gnb4 is a cilium associated protein. (a,b) MTECs were fixed with 4% PFA and stained with antibodies to Gnb4 (green), acetylated alpha tubulin (red) and DAPI (blue). Gnb4 localized to motile cilia labelled with acetylated alpha tubulin as well as cell junctions of ciliated and nonciliated MTECs (a). Gnb4 was detected in the cyto- plasm as well as motile cilia of freshly dissociated ciliated cells from mouse trachea (b). Methanol fixation removed much of the cytoplasmic and cell junction staining (c, compare with b). Gnb4 was detected on primary cilia of neighboring cells that were not multiciliated (arrows).
71 a polaris Mlf1 Mlf1 polaris DNA
b Gnb4 Mlf1 Mlf1 Gnb4 DNA
Figure 9. Mlf1 localizes to motile cilia. Mlf1 stained the motile cilia of ciliated MTECs as detected by polaris (a). Concentrations of Mlf1 staining at the apical tips of motile cilia (b) were occaionally detected.
72 ALI+4 ALI+12 GFP- a c Cepbp Rpgripl1 Mkks Socs7 Ttc8 Bbs9 b Bbs4 Irx6 Med1 Irx5 Bbs2 GFP- Irx3 Bbs7 ALI 12 Pgea1 Rpgrip1l ALI 4 Arl6 Fts Tbl1x
Fto Steap4 Socs1 -2 0 2 4 6 Mettl8 log 2 Sdf4 Gpx1 Gsk3b fatso Nocl3 fatso Trim32 Retn Runx1t1
<-5.65 1:1 <5.65
d polaris fatso polaris fatso DAPI
e Abcam fatso Abcam fatso DAPI
polaris fatso merge
Figure 10. Fatso is a cilium associated protein. a) The ft delete region. Adapted from Peters et al., 2002. b) Four genes present in the ft delete region were upregulated in ciliated MTECs. Values shown are relative to a pool of mouse reference RNAs . c) Expression of genes involved in fat cell differentia- tion. Data are zero transformed against GFP- and clustered. d) Fatso localizes to cilia, as detected by polaris staining, in MTEC cultures. e) Staining of dissocaiated MTECs revealed that Fatso localizes along the length of motile cilia.
73 a polaris fatso polaris fatso DAPI
Abcam fatso b polaris fatso polaris fatso DAPI
c polaris fatso polaris fatso DAPI
Figure 11. Fatso is a microtubule associated protein. Fatso stained primary cilia and cytoplasmic microtubles of MTECs (a), as well as a rare midbody (b). Fatso also stained primary cilia and cytoplasmic microtubules in RPE1 cells (c).
74 ALI+4 ALI+12 GFP- a Suv39h2 Ezh2 Setd7 Ehmt1 Ash1l Ehmt2 Smyd3 Suv39h1 DAPI Dot1l Ezh1 Setdb1 Whsc1 Setd8 <-5.65 1:1 <5.65 b tubulin Whsc1 tubulin Whsc1 DAPI
c tubulin Whsc1 tubulin Whsc1 DAPI
Figure 12. Whsc1 expression in ciliated cells. a) Expression of genes encoding histone methylases during ciliogenesis. Data are zero transformed against noncili- ated cells (GFP-). Genes are clustered according to a Pearson non-centered distance metric. Some genes appear more than once because they are represented by more than one probe on the arrays. Whsc1 localized to basal bodies at the cell surface and the nucleus (arrow) in mutliciliated cells (b). A side view of a single ciliated cell, showed Whsc1 labeling at basal bodies and in the cytoplasm (c).
75 ALI+4 ALI+12 GFP- ALI+4 ALI+12 GFP-
Fancb Morc1
Fanci Boll male male
Fancd2 infertility
amemia Fancl
Spg7 Nipa1 Mtss1 Als2 Prcc Spast Nov Als2cr12 Brms1 Spg20 Blcap
motor neuron motor Atcay Tsg101 Pnkd Ss18 Nras Yes1 Ict1 Dmpk Pttg1 Sgcb
Frat2 Dmwd muscular St7 dystrophy Vbp1 Wfs1 Pim2 Wbscr16 Steap1 Homer1
cancer Sdccag8 Dyx1c1 Fhit Fxr1 Bcas1 Fxr2 Stk30 Cdr2 Casc1 Tulp2 Kit Wbscr22 Casc4 Fmr1 Maged2 neurological disorder Tsc2 Tusc1 Etaa1 Akt3 Ect2 Agc1 Mycl1 Sos1
Hic2 skeletal
Sdccag3 abnormalities Mtus1 Fosb Hps1 Mia3 Muted Casc5 Porcn Tulp2 Whsc1l1 Arvcf Whsc1 Clptm1 disease
palate Whsc1 multi-organ multi-organ
Wfs1 cleft lip and and lip cleft Sos1
Gprk2l Mmachc <-5.65 1:1 <5.65
Mmachc
kidney kidney dysfunction
Figure 13. Expression of human disease gene homologs during ciliogenesis in MTECs. Only genes that encode proteins that have not been reported to be impor- tant for cilium formation or to localize to the centrosome or cilium are shown. Data were zero transformed against GFP- (nonciliated) cells and clustered. Some genes are appear more than once because they are represented multiple times on the arrays.
76 ALI+4 ALI+12 GFP- ALI+4 ALI +12 GFP- ALI+4 ALI+12 GFP-
Rab11b Rhob Arhgap15 Rab11a Rhoq Racgap1 Rab24 Rhof Racgap1 Rab4b Rho Arhgap6 Rab3c Rhobtb3 Arhgef6 Rabl2a Rhoa Prr5 Rab25 Rhod Arhgap9 Rab15 Rhoj Arhgap6 Rab11fip1 Rnd3 Arhgap25 Rab9b Rhoc Cdc42ep2 Rabl3 Rhog Arhgdib Rab2b Rhov Arhgef17 Rab3a Rac3 Arhgap10 Rab3ip Cdc42 Cdc42ep5 Rab11fip4 Rac1 Arhgef18 Rab36 Rhobtb2 Arhgef11
Rabl5 gtpases rho Rac2 Arhgef16 Rabl4 Rhot1 Arhgef12 Rab40c Rhot2 Arhgef5 Rab28 Rhobtb2 Arhgap5 Rab11fip5 Rnd1 Arhgef2 Rab5c Rhou Arhgef1 Rab35 Rhobtb1 Arhgef7 Rab26 Rnd1 Arhgef19 Rab71l Rhoh Arhgef3 Rab39b Arhgap19 Rab33b Arf6 Arhgap4 Rab17 Arfrp1 Arhgap20 Rabac1 Arl4c rho gtpase gap, gef Arhgap18 Rab1b Arf5 Arhgdig Rab4a Arl2 Cdc42ep4 Rab37 Arl5a Arhgap24 Rab5b Arl15 Cdc42ep3 Rab7 Arf3 Arhgap21 Rab22a Arl9 Arhgap12 Rab8a Arl11 Arhgdia Rab38 Exosc8 Arhgap1 rab gtpases rab Rab13 Arl5c Arhgef10 Rab11fip2 Arf4 Arhgap17 Arfl4 Rab40b gpases arf Rab19 Arl4a Arl10 Arl6ip5 Rab27b Atl2 Rab6ip2 Arl3 Arf2 Arfgef1 Rab5a Arl6ip3 Rab10 Arl5b Arl8b Arl6ip6 Rab21 Arl6ip4 Rab8b Arl6 Arl8a Arl2bp Rab9 Arfgap3 Rab20 Arl1 Arf1 Arfgef2
Rab6b arl/arf gap, gef Arl13a Arfgef1 Dendd5a Arl6ip1 Rab6ip2 Arfgap1 Rab39 Rabgef1 Erc1 Rabgap1 Rab3b Rabggtb Rab32 <-5.65 1:1 <5.65
gap, gef Rabggta Rab3d rab gtpasae Rabgap1l Rab11fip3 Rab27a Rab2a Rab14 Figure 14. Expression of small GTPases and modifying Rab6ip2 proteins during ciliogenesis. Data were zero trans- Rab28 Rab34 formed against nonciliated (GFP-) cells and clustered. Rab3il1 Rab12 Some genes appear multiple times because they are Rab23 Rab33a represented by multiple probes on the arrays.
77 Name Symbol Refseq M (ALI+4) M (ALI+12) taste receptor, type 2, member 102 Tas2r102 NM_199153 1.65 1.43 taste receptor, type 2, member 107 Tas2r107 NM_199154 1.47 1.81 taste receptor, type 2, member 125 Tas2r125 NM_207027 1.57 1.64
Figure 15. Upregulated taste receptor genes in ciliated MTECs. 3 genes encoding taste receptors were upregulated (M>1.0) in ciliated cells at either ALI+4 or ALI+12, or at both. M values shown are with respect to a pool of mouse reference RNAs, and are given in log (base 2).
78 a Symbol Refseq M (ALI+4) M (ALI+12) Symbol Refseq M (ALI+4) M (ALI+12) Olfr1337 NM_146309 1.92 1.30 Olfr117 NM_207155 0.91 1.43 Olfr20 NM_146923 1.88 1.42 Olfr1324 NM_146292 0.90 1.01 Olfr878 NM_146798 1.74 1.48 Olfr806 NM_146553 0.90 1.85 Olfr849 NM_146527 1.63 1.27 Olfr473 NM_146775 0.90 1.01 Olfr320 NM_207230 1.63 1.83 Olfr683 NM_147045 0.89 1.48 Olfr101 NM_146834 1.52 1.73 Olfr1022 NM_146589 0.87 1.16 Olfr1507 NM_020512 1.51 1.29 Olfr196 NM_146779 0.86 1.30 Olfr690 NM_020290 1.49 1.45 Olfr909 NM_146873 0.85 1.50 Olfr1133 NM_146351 1.48 1.47 Olfr482 NM_146733 0.84 1.04 Olfr986 NM_146615 1.47 1.43 Olfr1167 NM_146294 0.83 1.11 Olfr1272 NM_146980 1.46 1.48 Olfr1126 NM_146837 0.83 1.29 Olfr644 NM_147121 1.45 1.21 Olfr1280 NM_146908 0.82 1.45 Olfr1218 NM_146818 1.41 1.54 Olfr741 NM_207133 0.81 1.45 Olfr466 NM_146819 1.37 1.70 Olfr1508 NM_020513 0.80 1.25 Olfr338 NM_146947 1.36 1.45 Olfr459 NM_146576 0.78 1.14 Olfr1475 NM_146301 1.29 1.79 Olfr1420 NM_146410 0.77 1.02 Olfr90 NM_146477 1.28 1.37 Olfr1445 NM_146699 0.77 1.06 Olfr552 NM_147102 1.27 0.70 Olfr694 NM_146452 0.74 1.15 Olfr1277 NM_146396 1.26 1.42 Olfr27 NM_146829 0.72 1.17 Olfr983 NM_146827 1.25 1.78 Olfr876 NM_146883 0.69 1.21 Olfr1084 NM_207135 1.24 1.37 Olfr1412 NM_146277 0.69 1.27 Olfr855 NM_146524 1.23 0.75 Olfr645 NM_207144 0.68 1.39 Olfr58 NM_011001 1.21 1.69 Olfr1391 NM_146468 0.68 1.27 Olfr275 NM_146858 1.20 1.46 Olfr881 NM_146418 0.66 1.15 Olfr262 NM_146688 1.20 -0.03 Olfr1433 NM_146685 0.65 1.12 Olfr448 NM_146273 1.18 0.95 Olfr677 NM_146358 0.64 1.23 Olfr464 NM_146412 1.18 1.35 Olfr558 NM_147093 0.63 1.05 Olfr1459 NM_146689 1.17 1.41 Olfr232 NM_146686 0.63 1.30 Olfr402 NM_146708 1.17 1.29 Olfr777 NM_146544 0.63 1.17 Olfr518 NM_146306 1.16 1.40 Olfr1269 NM_146342 0.62 1.04 Olfr478 NM_146734 1.13 1.67 Olfr557 NM_146361 0.58 1.00 Olfr1510 NM_146431 1.11 0.73 Olfr51 NM_146909 0.55 1.10 Olfr1442 NM_146697 1.11 1.21 Olfr981 NM_146286 0.52 1.06 Olfr1180 NM_146918 1.10 1.35 Olfr507 NM_146743 0.50 1.24 Olfr1283 NM_207236 1.09 1.09 Olfr100 NM_207673 0.44 1.61 Olfr444 NM_146656 1.08 1.16 Olfr32 NM_010980 0.42 1.34 Olfr1132 NM_146836 1.06 1.33 Olfr920 NM_146787 0.40 1.14 Olfr154 NM_013728 1.06 1.52 Olfr618 NM_147047 0.38 1.18 Olfr969 NM_146826 1.06 1.40 Olfr710 NM_146601 0.36 1.21 Olfr875 NM_146749 1.03 1.01 Olfr899 NM_146479 0.24 1.05 Olfr924 NM_207560 1.03 1.10 Olfr666 NM_147096 0.24 1.01 Olfr403 NM_207622 0.99 1.07 Olfr733 NM_146663 0.23 1.11 Olfr592 NM_207556 0.97 1.18 Olfr971 NM_146614 0.22 1.46 Olfr806 NM_146553 0.96 1.39 Olfr604 NM_147070 0.09 1.10 Olfr508 NM_146773 0.96 1.66 Olfr747 NM_207156 0.05 1.33 Olfr394 NM_147007 0.94 1.12 Olfr767 NM_146318 -0.04 1.44 Olfr820 NM_146675 0.94 1.19 Olfr78 NM_130866 -0.57 1.34 Olfr780 NM_146284 0.92 1.07
b 2.00 Olfr806 Figure 16. Expression of olfactory receptor Olfr1475 Olfr320 Olfr 101 genes in ciliated MTECs. a) 94 genes Olfr20 1.50 encoding olfactory receptors were upregu-
1.00 lated (M>1.0) in ciliated cells at either ALI+4 gene or ALI+12, or at both. M values shown are 0.50 M at ALI+12 with respect to a pool of mouse reference
0.00 RNAs, and are given in log (base 2). b) -1.00 -0.50 0.00 0.50 1.00 1.50 2.00 2.50 Scatter plot highlighting some of the most -0.50 M at ALI+4 upregulated genes.
79 Name Symbol RefSeq M (ALI+4) M (ALI+I12) vomeronasal 1 receptor, A5 V1ra5 NM_053220 1.25 1.18 vomeronasal 1 receptor, B7 V1rb7 NM_053228 0.97 1.53 vomeronasal 1 receptor, C17 V1rc17 NM_134172 1.03 1.49 vomeronasal 1 receptor, C21 V1rc21 NM_134176 -0.07 1.26 vomeronasal 1 receptor, C8 V1rc8 NM_053238 1.15 1.40 vomeronasal 1 receptor, D14 V1rd14 NM_030736 0.42 1.22 vomeronasal 1 receptor, D19 V1rd19 NM_207619 0.57 1.17 vomeronasal 1 receptor, D4 V1rd4 NM_030739 0.99 1.75 vomeronasal 1 receptor, D4 V1rd4 NM_030739 1.10 1.22 vomeronasal 1 receptor, D6 V1rd6 NM_030738 0.79 1.27 vomeronasal 1 receptor, E12 V1re12 NM_134231 1.14 1.48 vomeronasal 1 receptor, E4 V1re4 NM_134193 0.82 1.15 vomeronasal 1 receptor, F4 V1rf4 NM_134201 1.36 1.74 vomeronasal 1 receptor, F5 V1rf5 NM_134202 1.45 1.65 vomeronasal 1 receptor, H11 V1rh11 NM_134236 1.29 2.05 vomeronasal 1 receptor, I10 V1ri10 NM_134245 1.56 1.96
Figure 17. Expression of vomeronasal receptors in ciliated MTECs. 15 genes encoding vomeronasal receptors were upregulated (M>1.0) in ciliated cells at either ALI+4 or ALI+12, or at both. M values shown are with respect to a pool of mouse reference RNAs, and are given in log (base 2). V1rd4 was repre- sented by two probes on the MEEBO arrays. Both probes are shown.
80 Materials and Methods Clustering Gene clustering analysis was performed with Cluster and TreeView programs as implemented by the Stanford Microarray Database website. A Pearson non- centered distance algorithm was used for gene clustering. Values for each timepoint were zero transformed against the mean of the values for the three arrays from nonciliated (FoxJ1/EGFP-) cells.
Plasmids The full-length and N- and C terminal rat Dyx1c1-GFP fusions in the pCAGGS plasmid were the gift of Dr. J.J.LoTurco. Mouse KIAA0319 was PCR amplified from a plasmid bearing the cDNA (Accession BC115940). Mdm1 was PCR amplified from a cDNA library generated from MTEC cultures harvested at ALI+10. The cDNA library was generated using Superscript II RT (Invitrogen) according to the manufacturer’s protocols. PCR products were cloned in frame to the lentiviral transfer vector pRRL.sin-18.PPT.PGK.IRES.GFP.pre {Follenzi, 2000 #544} using either the AgeI site or the AgeI site in combination with BamHI. All PCR generated clones were sequenced to ensure in frame fusion to GFP.
Cell Culture and Transfection NIH 3T3 and RPE1 cells were cultured in DMEM with 10% FBS (Invitrogen). For transfection, cells were plated onto poly-L-lysine coated coverslips at 80% confluency the night before transfection. The day of transfection, cells were rinsed 2X with PBS and incubated with plasmid plus Lipofectamine 2000 (Invitrogen) for 6 hours before removing and adding fresh media. Cells were imaged 24-48 hours later.
Indirect Immunofluorescence Unless otherwise stated, NIH 3T3 cells and MTECs were prepared for indirect IF by washing twice with PBS and fixing in 4% PFA at room temperature for 10 minutes. For some antigens, cells were fixed in methanol at -20°C for 10 minutes. After fixation, cells were rinsed with PBS and blocked in PBS-BT (PBS + 3% BSA +
81 0.1% Triton X-100) for 30 minutes at room temperature. MTEC filters were cut at this point from the solid supports for staining. MTECs were incubated with primary antibody for 1 hour at room temperature, while tissue culture cells were incubated for 30 minutes at room temperature. Incubations with secondary antibodies were for 30 minutes at room temperature for all cells. To visualize DNA, cells were incubated with 4',6-diamidino-2-phenylindole (DAPI, Molecular Probes) for 5 minutes at room temperature using a 1 !g/mL dilution for NIH 3T3 cells and a 10 !g/mL dilution for MTECs. In between incubations, cells were washed 3x with PBS-BT for 5 minutes each. The primary antibodies used were mouse anti-acetylated !-tubulin (1:2000, Sigma), mouse anti-"-tubulin (1:500, Sigma), mouse anti-Mlf1 (1:400, Abnova), rabbit anti-Whsc1 (1:400, Sigma), rabbit anti-Gnb4 (1:400, Santa Cruz Biotechnology), mouse anti-FTO (1:400, Abcam) and rabbit anti-polaris (1:500, gift from Bradley Yoder). The secondary antibodies used were Alexa 488 and 594-conjugated goat anti- mouse and goat anti-rabbit (1:200, Invitrogen). All antibodies were diluted in PBS- BT. Cells were then observed under a Zeiss Axiovert 200M microscope (Zeiss, Thornwood, NJ) using a 100 x oil-immersion objective.
82 References Andersen, J.S., C.J. Wilkinson, T. Mayor, P. Mortensen, E.A. Nigg, and M. Mann. 2003. Proteomic characterization of the human centrosome by protein correlation profiling. Nature. 426:570-4. Anselme, I., C. Laclef, M. Lanaud, U. Ruther, and S. Schneider-Maunoury. 2007. Defects in brain patterning and head morphogenesis in the mouse mutant Fused toes. Dev Biol. 304:208-20. Avidor-Reiss, T., A.M. Maer, E. Koundakjian, A. Polyanovsky, T. Keil, S. Subramaniam, and C.S. Zuker. 2004. Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell. 117:527-39. Basto, R., K. Brunk, T. Vinadogrova, N. Peel, A. Franz, A. Khodjakov, and J.W. Raff. 2008. Centrosome amplification can initiate tumorigenesis in flies. Cell. 133:1032-42. Bergemann, A.D., F. Cole, and K. Hirschhorn. 2005. The etiology of Wolf-Hirschhorn syndrome. Trends Genet. 21:188-95. Bettencourt-Dias, M., and D.M. Glover. 2009. SnapShot: centriole biogenesis. Cell. 136:188-188 e1. Blacque, O.E., E.A. Perens, K.A. Boroevich, P.N. Inglis, C. Li, A. Warner, J. Khattra, R.A. Holt, G. Ou, A.K. Mah, S.J. McKay, P. Huang, P. Swoboda, S.J. Jones, M.A. Marra, D.L. Baillie, D.G. Moerman, S. Shaham, and M.R. Leroux. 2005. Functional genomics of the cilium, a sensory organelle. Curr Biol. 15:935-41. Boissel, S., O. Reish, K. Proulx, H. Kawagoe-Takaki, B. Sedgwick, G.S. Yeo, D. Meyre, C. Golzio, F. Molinari, N. Kadhom, H.C. Etchevers, V. Saudek, I.S. Farooqi, P. Froguel, T. Lindahl, S. O'Rahilly, A. Munnich, and L. Colleaux. 2009. Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations. Am J Hum Genet. 85:106-11. Buck, L., and R. Axel. 1991. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell. 65:175-87. Cahilly-Snyder, L., T. Yang-Feng, U. Francke, and D.L. George. 1987. Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line. Somat Cell Mol Genet. 13:235-44. Cao, J., H. Xu, H. Zhao, W. Gong, and D. Dunaway-Mariano. 2009. The mechanisms of human hotdog-fold thioesterase 2 (hTHEM2) substrate recognition and catalysis illuminated by a structure and function based analysis. Biochemistry. 48:1293-304. Cardenas-Rodriguez, M., and J.L. Badano. 2009. Ciliary biology: understanding the cellular and genetic basis of human ciliopathies. Am J Med Genet C Semin Med Genet. 151C:263-80. Carreira-Barbosa, F., M.L. Concha, M. Takeuchi, N. Ueno, S.W. Wilson, and M. Tada. 2003. Prickle 1 regulates cell movements during gastrulation and neuronal migration in zebrafish. Development. 130:4037-46.
83 Castellanos, E., P. Dominguez, and C. Gonzalez. 2008. Centrosome dysfunction in Drosophila neural stem cells causes tumors that are not due to genome instability. Curr Biol. 18:1209-14. Chang, B., M.N. Mandal, V.R. Chavali, N.L. Hawes, N.W. Khan, R.E. Hurd, R.S. Smith, M.L. Davisson, L. Kopplin, B.E. Klein, R. Klein, S.K. Iyengar, J.R. Heckenlively, and R. Ayyagari. 2008. Age-related retinal degeneration (arrd2) in a novel mouse model due to a nonsense mutation in the Mdm1 gene. Hum Mol Genet. 17:3929-41. Chen, W., X.R. Ren, C.D. Nelson, L.S. Barak, J.K. Chen, P.A. Beachy, F. de Sauvage, and R.J. Lefkowitz. 2004. Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2. Science. 306:2257-60. Chen, Z., V.B. Indjeian, M. McManus, L. Wang, and B.D. Dynlacht. 2002. CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells. Dev Cell. 3:339-50. Cheng, Z., S. Bao, X. Shan, H. Xu, and W. Gong. 2006. Human thioesterase superfamily member 2 (hTHEM2) is co-localized with beta-tubulin onto the microtubule. Biochem Biophys Res Commun. 350:850-3. Couto, J.M., L. Gomez, K. Wigg, T. Cate-Carter, J. Archibald, B. Anderson, R. Tannock, E.N. Kerr, M.W. Lovett, T. Humphries, and C.L. Barr. 2008. The KIAA0319-like (KIAA0319L) gene on chromosome 1p34 as a candidate for reading disabilities. J Neurogenet. 22:295-313. Cunha-Ferreira, I., A. Rodrigues-Martins, I. Bento, M. Riparbelli, W. Zhang, E. Laue, G. Callaini, D.M. Glover, and M. Bettencourt-Dias. 2009. The SCF/Slimb ubiquitin ligase limits centrosome amplification through degradation of SAK/PLK4. Curr Biol. 19:43-9. Dina, C., D. Meyre, S. Gallina, E. Durand, A. Korner, P. Jacobson, L.M. Carlsson, W. Kiess, V. Vatin, C. Lecoeur, J. Delplanque, E. Vaillant, F. Pattou, J. Ruiz, J. Weill, C. Levy-Marchal, F. Horber, N. Potoczna, S. Hercberg, C. Le Stunff, P. Bougneres, P. Kovacs, M. Marre, B. Balkau, S. Cauchi, J.C. Chevre, and P. Froguel. 2007. Variation in FTO contributes to childhood obesity and severe adult obesity. Nat Genet. 39:724-6. Errico, A., P. Claudiani, M. D'Addio, and E.I. Rugarli. 2004. Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon. Hum Mol Genet. 13:2121-32. Falini, B., I. Nicoletti, N. Bolli, M.P. Martelli, A. Liso, P. Gorello, F. Mandelli, C. Mecucci, and M.F. Martelli. 2007. Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias. Haematologica. 92:519-32. Finetti, F., S.R. Paccani, M.G. Riparbelli, E. Giacomello, G. Perinetti, G.J. Pazour, J.L. Rosenbaum, and C.T. Baldari. 2009. Intraflagellar transport is required for polarized recycling of the TCR/CD3 complex to the immune synapse. Nat Cell Biol. 11:1332-9. Frayling, T.M., N.J. Timpson, M.N. Weedon, E. Zeggini, R.M. Freathy, C.M. Lindgren, J.R. Perry, K.S. Elliott, H. Lango, N.W. Rayner, B. Shields, L.W. Harries, J.C. Barrett, S. Ellard, C.J. Groves, B. Knight, A.M. Patch, A.R. Ness, S. Ebrahim, D.A. Lawlor, S.M. Ring, Y. Ben-Shlomo, M.R. Jarvelin, U. Sovio,
84 A.J. Bennett, D. Melzer, L. Ferrucci, R.J. Loos, I. Barroso, N.J. Wareham, F. Karpe, K.R. Owen, L.R. Cardon, M. Walker, G.A. Hitman, C.N. Palmer, A.S. Doney, A.D. Morris, G.D. Smith, A.T. Hattersley, and M.I. McCarthy. 2007. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science. 316:889-94. Gabel, L.A., C.J. Gibson, J.R. Gruen, and J.J. Loturco. 2009. Progress towards a cellular neurobiology of reading disability. Neurobiol Dis. Gavet, O., C. Alvarez, P. Gaspar, and M. Bornens. 2003. Centrin4p, a novel mammalian centrin specifically expressed in ciliated cells. Mol Biol Cell. 14:1818-34. Gerken, T., C.A. Girard, Y.C. Tung, C.J. Webby, V. Saudek, K.S. Hewitson, G.S. Yeo, M.A. McDonough, S. Cunliffe, L.A. McNeill, J. Galvanovskis, P. Rorsman, P. Robins, X. Prieur, A.P. Coll, M. Ma, Z. Jovanovic, I.S. Farooqi, B. Sedgwick, I. Barroso, T. Lindahl, C.P. Ponting, F.M. Ashcroft, S. O'Rahilly, and C.J. Schofield. 2007. The obesity-associated FTO gene encodes a 2- oxoglutarate-dependent nucleic acid demethylase. Science. 318:1469-72. Gilad, Y., M. Przeworski, and D. Lancet. 2004. Loss of olfactory receptor genes coincides with the acquisition of full trichromatic vision in primates. PLoS Biol. 2:E5. Gotz, K., J. Briscoe, and U. Ruther. 2005. Homozygous Ft embryos are affected in floor plate maintenance and ventral neural tube patterning. Dev Dyn. 233:623- 30. Graser, S., Y.D. Stierhof, and E.A. Nigg. 2007. Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion. J Cell Sci. 120:4321-31. Grotewold, L., and U. Ruther. 2002. The Fused toes (Ft) mouse mutation causes anteroposterior and dorsoventral polydactyly. Dev Biol. 251:129-41. Guarguaglini, G., P.I. Duncan, Y.D. Stierhof, T. Holmstrom, S. Duensing, and E.A. Nigg. 2005. The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles. Mol Biol Cell. 16:1095-107. Han, Y.G., H.J. Kim, A.A. Dlugosz, D.W. Ellison, R.J. Gilbertson, and A. Alvarez- Buylla. 2009. Dual and opposing roles of primary cilia in medulloblastoma development. Nat Med. 15:1062-5. Higginbotham, H.R., and J.G. Gleeson. 2007. The centrosome in neuronal development. Trends Neurosci. 30:276-83. Hitzler, J.K., D.P. Witte, N.A. Jenkins, N.G. Copeland, D.J. Gilbert, C.W. Naeve, A.T. Look, and S.W. Morris. 1999. cDNA cloning, expression pattern, and chromosomal localization of Mlf1, murine homologue of a gene involved in myelodysplasia and acute myeloid leukemia. Am J Pathol. 155:53-9. Hughes, J., C.J. Ward, B. Peral, R. Aspinwall, K. Clark, J.L. San Millan, V. Gamble, and P.C. Harris. 1995. The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains. Nat Genet. 10:151-60. Jessen, J.R., J. Topczewski, S. Bingham, D.S. Sepich, F. Marlow, A. Chandrasekhar, and L. Solnica-Krezel. 2002. Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements. Nat Cell Biol. 4:610-5.
85 Karlsson, J.A., A.S. Lanner, and C.G. Persson. 1990. Airway opioid receptors mediate inhibition of cough and reflex bronchoconstriction in guinea pigs. J Pharmacol Exp Ther. 252:863-8. Keats, J.J., T. Reiman, C.A. Maxwell, B.J. Taylor, L.M. Larratt, M.J. Mant, A.R. Belch, and L.M. Pilarski. 2003. In multiple myeloma, t(4;14)(p16;q32) is an adverse prognostic factor irrespective of FGFR3 expression. Blood. 101:1520- 9. Kitajima, T.S., T. Sakuno, K. Ishiguro, S. Iemura, T. Natsume, S.A. Kawashima, and Y. Watanabe. 2006. Shugoshin collaborates with protein phosphatase 2A to protect cohesin. Nature. 441:46-52. Kuo, H.P., J.A. Rohde, P.J. Barnes, and D.F. Rogers. 1992. Differential inhibitory effects of opioids on cigarette smoke, capsaicin and electrically-induced goblet cell secretion in guinea-pig trachea. Br J Pharmacol. 105:361-6. Laoukili, J., E. Perret, S. Middendorp, O. Houcine, C. Guennou, F. Marano, M. Bornens, and F. Tournier. 2000. Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro. J Cell Sci. 113 ( Pt 8):1355-64. Lawo, S., M. Bashkurov, M. Mullin, M.G. Ferreria, R. Kittler, B. Habermann, A. Tagliaferro, I. Poser, J.R. Hutchins, B. Hegemann, D. Pinchev, F. Buchholz, J.M. Peters, A.A. Hyman, A.C. Gingras, and L. Pelletier. 2009. HAUS, the 8- subunit human Augmin complex, regulates centrosome and spindle integrity. Curr Biol. 19:816-26. Levecque, C., A. Velayos-Baeza, Z.G. Holloway, and A.P. Monaco. 2009. The dyslexia-associated protein KIAA0319 interacts with adaptor protein 2 and follows the classical clathrin-mediated endocytosis pathway. Am J Physiol Cell Physiol. 297:C160-8. Li, J.B., J.M. Gerdes, C.J. Haycraft, Y. Fan, T.M. Teslovich, H. May-Simera, H. Li, O.E. Blacque, L. Li, C.C. Leitch, R.A. Lewis, J.S. Green, P.S. Parfrey, M.R. Leroux, W.S. Davidson, P.L. Beales, L.M. Guay-Woodford, B.K. Yoder, G.D. Stormo, N. Katsanis, and S.K. Dutcher. 2004. Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell. 117:541-52. Li, Z.F., X. Wu, Y. Jiang, J. Liu, C. Wu, M. Inagaki, I. Izawa, A.P. Mizisin, E. Engvall, and G.D. Shelton. 2008. Non-pathogenic protein aggregates in skeletal muscle in MLF1 transgenic mice. J Neurol Sci. 264:77-86. Lim, R., L.N. Winteringham, J.H. Williams, R.K. McCulloch, E. Ingley, J.Y. Tiao, J.P. Lalonde, S. Tsai, P.A. Tilbrook, Y. Sun, X. Wu, S.W. Morris, and S.P. Klinken. 2002. MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1. J Biol Chem. 277:40997-1008. Liu, Q., G. Tan, N. Levenkova, T. Li, E.N. Pugh, Jr., J.J. Rux, D.W. Speicher, and E.A. Pierce. 2007. The proteome of the mouse photoreceptor sensory cilium complex. Mol Cell Proteomics. 6:1299-317. Liu, Q., J. Zuo, and E.A. Pierce. 2004. The retinitis pigmentosa 1 protein is a photoreceptor microtubule-associated protein. J Neurosci. 24:6427-36.
86 Londin, E.R., H. Meng, and J.R. Gruen. 2003. A transcription map of the 6p22.3 reading disability locus identifying candidate genes. BMC Genomics. 4:25. Mak, H.Y., L.S. Nelson, M. Basson, C.D. Johnson, and G. Ruvkun. 2006. Polygenic control of Caenorhabditis elegans fat storage. Nat Genet. 38:363-8. Marango, J., M. Shimoyama, H. Nishio, J.A. Meyer, D.J. Min, A. Sirulnik, Y. Martinez-Martinez, M. Chesi, P.L. Bergsagel, M.M. Zhou, S. Waxman, B.A. Leibovitch, M.J. Walsh, and J.D. Licht. 2008. The MMSET protein is a histone methyltransferase with characteristics of a transcriptional corepressor. Blood. 111:3145-54. Matsumoto, N., N. Yoneda-Kato, T. Iguchi, Y. Kishimoto, T. Kyo, H. Sawada, E. Tatsumi, and S. Fukuhara. 2000. Elevated MLF1 expression correlates with malignant progression from myelodysplastic syndrome. Leukemia. 14:1757- 65. Mayor, T., Y.D. Stierhof, K. Tanaka, A.M. Fry, and E.A. Nigg. 2000. The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion. J Cell Biol. 151:837-46. Meloni, A.R., G.B. Fralish, P. Kelly, A. Salahpour, J.K. Chen, R.J. Wechsler-Reya, R.J. Lefkowitz, and M.G. Caron. 2006. Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2. Mol Cell Biol. 26:7550-60. Millecamps, S., B.J. Gentil, F. Gros-Louis, G. Rouleau, and J.P. Julien. 2005. Alsin is partially associated with centrosome in human cells. Biochim Biophys Acta. 1745:84-100. Nachury, M.V., A.V. Loktev, Q. Zhang, C.J. Westlake, J. Peranen, A. Merdes, D.C. Slusarski, R.H. Scheller, J.F. Bazan, V.C. Sheffield, and P.K. Jackson. 2007. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 129:1201-13. Niimura, Y., and M. Nei. 2006. Evolutionary dynamics of olfactory and other chemosensory receptor genes in vertebrates. J Hum Genet. 51:505-17. Nimura, K., K. Ura, H. Shiratori, M. Ikawa, M. Okabe, R.J. Schwartz, and Y. Kaneda. 2009. A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to Wolf- Hirschhorn syndrome. Nature. 460:287-91. Noormets, K., S. Koks, A. Kavak, A. Arend, M. Aunapuu, A. Keldrimaa, E. Vasar, and V. Tillmann. 2009. Male mice with deleted Wolframin (Wfs1) gene have reduced fertility. Reprod Biol Endocrinol. 7:82. Norman, R.X., H.W. Ko, V. Huang, C.M. Eun, L.L. Abler, Z. Zhang, X. Sun, and J.T. Eggenschwiler. 2009. Tubby-like protein 3 (TULP3) regulates patterning in the mouse embryo through inhibition of Hedgehog signaling. Hum Mol Genet. 18:1740-54. Okuda, M., H.F. Horn, P. Tarapore, Y. Tokuyama, A.G. Smulian, P.K. Chan, E.S. Knudsen, I.A. Hofmann, J.D. Snyder, K.E. Bove, and K. Fukasawa. 2000. Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication. Cell. 103:127-40. Pan, J., and W. Snell. 2007. The primary cilium: keeper of the key to cell division. Cell. 129:1255-7. Paracchini, S., A. Thomas, S. Castro, C. Lai, M. Paramasivam, Y. Wang, B.J. Keating, J.M. Taylor, D.F. Hacking, T. Scerri, C. Francks, A.J. Richardson, R. Wade-
87 Martins, J.F. Stein, J.C. Knight, A.J. Copp, J. Loturco, and A.P. Monaco. 2006. The chromosome 6p22 haplotype associated with dyslexia reduces the expression of KIAA0319, a novel gene involved in neuronal migration. Hum Mol Genet. 15:1659-66. Park, T.J., B.J. Mitchell, P.B. Abitua, C. Kintner, and J.B. Wallingford. 2008. Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells. Nat Genet. 40:871-9. Pattinson, K.T. 2008. Opioids and the control of respiration. Br J Anaesth. 100:747- 58. Pazour, G.J., N. Agrin, J. Leszyk, and G.B. Witman. 2005. Proteomic analysis of a eukaryotic cilium. J Cell Biol. 170:103-13. Peters, T., K. Ausmeier, and U. Ruther. 1999. Cloning of Fatso (Fto), a novel gene deleted by the Fused toes (Ft) mouse mutation. Mamm Genome. 10:983-6. Poelmans, G., J.J. Engelen, J. Van Lent-Albrechts, H.J. Smeets, E. Schoenmakers, B. Franke, J.K. Buitelaar, M. Wuisman-Frerker, W. Erens, J. Steyaert, and C. Schrander-Stumpel. 2009. Identification of novel dyslexia candidate genes through the analysis of a chromosomal deletion. Am J Med Genet B Neuropsychiatr Genet. 150B:140-7. Rauch, A., C.T. Thiel, D. Schindler, U. Wick, Y.J. Crow, A.B. Ekici, A.J. van Essen, T.O. Goecke, L. Al-Gazali, K.H. Chrzanowska, C. Zweier, H.G. Brunner, K. Becker, C.J. Curry, B. Dallapiccola, K. Devriendt, A. Dorfler, E. Kinning, A. Megarbane, P. Meinecke, R.K. Semple, S. Spranger, A. Toutain, R.C. Trembath, E. Voss, L. Wilson, R. Hennekam, F. de Zegher, H.G. Dorr, and A. Reis. 2008. Mutations in the pericentrin (PCNT) gene cause primordial dwarfism. Science. 319:816-9. Robay, D., H. Patel, M.A. Simpson, N.A. Brown, and A.H. Crosby. 2006. Endogenous spartin, mutated in hereditary spastic paraplegia, has a complex subcellular localization suggesting diverse roles in neurons. Exp Cell Res. 312:2764-77. Rogers, S.L., G.C. Rogers, D.J. Sharp, and R.D. Vale. 2002. Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J Cell Biol. 158:873-84. Ross, A.J., L.A. Dailey, L.E. Brighton, and R.B. Devlin. 2007. Transcriptional profiling of mucociliary differentiation in human airway epithelial cells. Am J Respir Cell Mol Biol. 37:169-85. Ross, A.J., H. May-Simera, E.R. Eichers, M. Kai, J. Hill, D.J. Jagger, C.C. Leitch, J.P. Chapple, P.M. Munro, S. Fisher, P.L. Tan, H.M. Phillips, M.R. Leroux, D.J. Henderson, J.N. Murdoch, A.J. Copp, M.M. Eliot, J.R. Lupski, D.T. Kemp, H. Dollfus, M. Tada, N. Katsanis, A. Forge, and P.L. Beales. 2005. Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates. Nat Genet. 37:1135-40. Roth, Y., E.F. Aharonson, H. Teichtahl, G.L. Baum, Z. Priel, and M. Modan. 1991. Human in vitro nasal and tracheal ciliary beat frequencies: comparison of sampling sites, combined effect of medication, and demographic relationships. Ann Otol Rhinol Laryngol. 100:378-84.
88 Rutland, J., W. Griffin, and P.J. Cole. 1982. An in vitro model for studying the effects of pharmacological agents on human ciliary beat frequency: effects of lignocaine. Br J Clin Pharmacol. 13:679-83. Sawamoto, K., H. Wichterle, O. Gonzalez-Perez, J.A. Cholfin, M. Yamada, N. Spassky, N.S. Murcia, J.M. Garcia-Verdugo, O. Marin, J.L. Rubenstein, M. Tessier-Lavigne, H. Okano, and A. Alvarez-Buylla. 2006. New neurons follow the flow of cerebrospinal fluid in the adult brain. Science. 311:629-32. Schneider, M.J., M. Ulland, and R.D. Sloboda. 2008. A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption. Mol Biol Cell. 19:4319-27. Schroder, J.M., L. Schneider, S.T. Christensen, and L.B. Pedersen. 2007. EB1 is required for primary cilia assembly in fibroblasts. Curr Biol. 17:1134-9. Selwyn, D.A., J.H. Raphael, D.G. Lambert, and J.A. Langton. 1996. Effects of morphine on human nasal cilia beat frequency in vitro. Br J Anaesth. 76:274-7. Shah, A.S., Y. Ben-Shahar, T.O. Moninger, J.N. Kline, and M.J. Welsh. 2009. Motile Cilia of Human Airway Epithelia Are Chemosensory. Science. Sheen, V.L., V.S. Ganesh, M. Topcu, G. Sebire, A. Bodell, R.S. Hill, P.E. Grant, Y.Y. Shugart, J. Imitola, S.J. Khoury, R. Guerrini, and C.A. Walsh. 2004. Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex. Nat Genet. 36:69-76. Snell, W.J., and E.A. Golemis. 2007. A ciliary timer for S-phase entry. Nat Rev Mol Cell Biol. Snyder, L.C., S.P. Trusko, N. Freeman, J.R. Eshleman, S.S. Fakharzadeh, and D.L. George. 1988. A gene amplified in a transformed mouse cell line undergoes complex transcriptional processing and encodes a nuclear protein. J Biol Chem. 263:17150-8. Sorci, G., A.L. Agneletti, R. Bianchi, and R. Donato. 1998. Association of S100B with intermediate filaments and microtubules in glial cells. Biochim Biophys Acta. 1448:277-89. Speliotes, E.K. 2009. Genetics of common obesity and nonalcoholic fatty liver disease. Gastroenterology. 136:1492-5. Stec, I., T.J. Wright, G.J. van Ommen, P.A. de Boer, A. van Haeringen, A.F. Moorman, M.R. Altherr, and J.T. den Dunnen. 1998. WHSC1, a 90 kb SET domain-containing gene, expressed in early development and homologous to a Drosophila dysmorphy gene maps in the Wolf-Hirschhorn syndrome critical region and is fused to IgH in t(4;14) multiple myeloma. Hum Mol Genet. 7:1071-82. Stolc, V., M.P. Samanta, W. Tongprasit, and W.F. Marshall. 2005. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. Proc Natl Acad Sci U S A. 102:3703-7. Taipale, M., N. Kaminen, J. Nopola-Hemmi, T. Haltia, B. Myllyluoma, H. Lyytinen, K. Muller, M. Kaaranen, P.J. Lindsberg, K. Hannula-Jouppi, and J. Kere. 2003. A candidate gene for developmental dyslexia encodes a nuclear tetratricopeptide repeat domain protein dynamically regulated in brain. Proc Natl Acad Sci U S A. 100:11553-8.
89 Tallal, P., and A.A. Benasich. 2002. Developmental language learning impairments. Dev Psychopathol. 14:559-79. Thompson, L.H., and J.M. Hinz. 2009. Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: Mechanistic insights. Mutat Res. 668:54-72. Threlkeld, S.W., M.M. McClure, J. Bai, Y. Wang, J.J. LoTurco, G.D. Rosen, and R.H. Fitch. 2007. Developmental disruptions and behavioral impairments in rats following in utero RNAi of Dyx1c1. Brain Res Bull. 71:508-14. van der Hoeven, F., T. Schimmang, A. Volkmann, M.G. Mattei, B. Kyewski, and U. Ruther. 1994. Programmed cell death is affected in the novel mouse mutant Fused toes (Ft). Development. 120:2601-7. Vierkotten, J., R. Dildrop, T. Peters, B. Wang, and U. Ruther. 2007. Ftm is a novel basal body protein of cilia involved in Shh signalling. Development. 134:2569- 77. Vladar, E.K. 2007. Centriole assembly in ciliated epithelial cells. In Department of Genetics. Vol. Ph.D Dissertation. Stanford University. 141. Vladar, E.K., and T. Stearns. 2007. Molecular characterization of centriole assembly in ciliated epithelial cells. J Cell Biol. 178:31-42. Vodermaier, H.C. 2004. APC/C and SCF: controlling each other and the cell cycle. Curr Biol. 14:R787-96. Voronina, V.A., K. Takemaru, P. Treuting, D. Love, B.R. Grubb, A.M. Hajjar, A. Adams, F.Q. Li, and R.T. Moon. 2009. Inactivation of Chibby affects function of motile airway cilia. J Cell Biol. 185:225-33. Wada, Y., Y. Mogami, and S. Baba. 1997. Modification of ciliary beating in sea urchin larvae induced by neurotransmitters: beat-plane rotation and control of frequency fluctuation. J Exp Biol. 200:9-18. Wang, W., A. Budhu, M. Forgues, and X.W. Wang. 2005. Temporal and spatial control of nucleophosmin by the Ran-Crm1 complex in centrosome duplication. Nat Cell Biol. 7:823-30. Wang, Y., M. Paramasivam, A. Thomas, J. Bai, N. Kaminen-Ahola, J. Kere, J. Voskuil, G.D. Rosen, A.M. Galaburda, and J.J. Loturco. 2006. DYX1C1 functions in neuronal migration in developing neocortex. Neuroscience. 143:515-22. Wei, J., H.W. Kang, and D.E. Cohen. 2009. Thioesterase superfamily member 2 (Them2)/acyl-CoA thioesterase 13 (Acot13): a homotetrameric hotdog fold thioesterase with selectivity for long-chain fatty acyl-CoAs. Biochem J. 421:311-22. Williams, J., and M.C. O'Donovan. 2006. The genetics of developmental dyslexia. Eur J Hum Genet. 14:681-9. Winteringham, L.N., R. Endersby, S. Kobelke, R.K. McCulloch, J.H. Williams, J. Stillitano, S.M. Cornwall, E. Ingley, and S.P. Klinken. 2006. Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule. J Biol Chem. 281:38791-800. Winteringham, L.N., S. Kobelke, J.H. Williams, E. Ingley, and S.P. Klinken. 2004. Myeloid Leukemia Factor 1 inhibits erythropoietin-induced differentiation, cell cycle exit and p27Kip1 accumulation. Oncogene. 23:5105-9.
90 Woodward, O.M., and A.O. Willows. 2006. Dopamine modulation of Ca(2+) dependent Cl(-) current regulates ciliary beat frequency controlling locomotion in Tritonia diomedea. J Exp Biol. 209:2749-64. Yan, X., R. Habedanck, and E.A. Nigg. 2006. A complex of two centrosomal proteins, CAP350 and FOP, cooperates with EB1 in microtubule anchoring. Mol Biol Cell. 17:634-44. Yoneda-Kato, N., and J.Y. Kato. 2008. Shuttling imbalance of MLF1 results in p53 instability and increases susceptibility to oncogenic transformation. Mol Cell Biol. 28:422-34. Yoneda-Kato, N., A.T. Look, M.N. Kirstein, M.B. Valentine, S.C. Raimondi, K.J. Cohen, A.J. Carroll, and S.W. Morris. 1996. The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1. Oncogene. 12:265-75. Yoneda-Kato, N., K. Tomoda, M. Umehara, Y. Arata, and J.Y. Kato. 2005. Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3. EMBO J. 24:1739-49. Zaghloul, N.A., and N. Katsanis. 2009. Mechanistic insights into Bardet-Biedl syndrome, a model ciliopathy. J Clin Invest. 119:428-37. Zeng, H., A.N. Hoover, and A. Liu. PCP effector gene Inturned is an important regulator of cilia formation and embryonic development in mammals. Dev Biol. Zhang, X., I. Rodriguez, P. Mombaerts, and S. Firestein. 2004. Odorant and vomeronasal receptor genes in two mouse genome assemblies. Genomics. 83:802-11. Zhu, F., S. Lawo, A. Bird, D. Pinchev, A. Ralph, C. Richter, T. Muller-Reichert, R. Kittler, A.A. Hyman, and L. Pelletier. 2008. The mammalian SPD-2 ortholog Cep192 regulates centrosome biogenesis. Curr Biol. 18:136-41.
91
Chapter 4: Transcriptional regulation of ciliogenesis
92 Abstract Cell differentiation is often accompanied and effected by changes in transcriptional programs. Given the dramatic transformation the cells undergo during ciliogenesis, we predicted that many transcription factors and regulators of transcription would be differentially expressed. Here we describe the initial validation and characterization of a candidate transcription factor from the MTEC dataset, as well as the results of a promoter assay to identify novel motifs that are enriched in genes differentially regulated during ciliogenesis. Understanding how multiciliated cell differentiation is regulated might also further our understanding of how primary cilia are made, as well as being medically useful for pulmonary and developmental research.
Introduction FoxJ1 is a transcription factor that is expressed primarily in cells that make motile cilia, such as the ciliated cells of the respiratory epithelium, oviduct, brain ependyma, choroid plexus, testis and embryonic node (Hackett, Brody et al. 1995; Lim, Zhou et al. 1997; Blatt, Yan et al. 1999; Brody, Yan et al. 2000). FoxJ1 was dubbed a “master regulator“ of ciliogenesis following reports that in Xenopus embryos and zebrafish, overexpression of FoxJ1 was sufficient to induce the formation of ectopic motile cilia in cells that normally make only a single nonmotile cilium. (Stubbs, Oishi et al. 2008; Yu, Ng et al. 2008). These studies identified putative FoxJ1 targets, many of which encode proteins involved in ciliary motility, such as motor proteins, central apparatus proteins, and components of radial spokes, as well as general axonemal components. FoxJ1 is also required for motile ciliogenesis, as mice lacking FoxJ1 have situs inversus due to a failure to make nodal cilia, and hydrocephaly (Chen, Knowles et al. 1998), and MTECs derived from FoxJ1 null mice do not produce cilia. In these FoxJ1-/- cells, the block to ciliogenesis occurs at the docking step; duplicated basal bodies migrate apically but fail to dock at the ciliary membrane due to a defect in apical actin organization {Huang, 2003 #505}. This effect on apical actin was subsequently linked to RhoA activity, which can be activated by FoxJ1 overexpression (Pan, You et al. 2007). The fact that basal bodies
93 overduplicate indicates that even in the absence of FoxJ1, cells are able to commit to the ciliated cell differentiation pathway. Therefore, although FoxJ1 may be a master regulator of components that confer ciliary motility, there must be other factors controlling the commitment step. Members of the Rfx (regulatory factor binding to the X box)-type transcription factors have also been implicated in control of ciliogenesis. There are now seven described mammalian members, Rfx1-7, whereas one has been identified in yeast and C.elegans, two in Drosophila (Aftab et al, 2008; Emery et al, 1996). Of the mammalian Rfx family members, Rfx3 is the most well established regulator of ciliogenesis. Deletion of Rfx3 in mouse prevents formation of 9+2 nodal cilia (Bonnafe et al, 2004), as well as the multicilia of the brain ependyma and choroid plexus (Baas et al, 2006), resulting in left-right asymmetry defects and hydrocephalus. Rfx3 has also been shown to have a role in primary cilia formation in pancreatic cell lineages (Ait-Lounis et al, 2007). Recently, genes required for ciliary motility have been identified as Rfx3 targets by assaying transcriptional changes in Rfx3-/- derived primary cultures of ependymal epithelium (El Zein, Ait-Lounis et al. 2009). In some cases Rfx3 was shown to bind these promoter sequences, indicating direct regulation by Rfx3. Interestingly, the promoter of FoxJ1 was one of those that was bound, indicating that Rfx3 may lie upstream of FoxJ1. However, FoxJ1 is likely regulated by other factors, since cells in Rfx3/- derived MTEC cultures are still observed that make 9+2 cilia, albeit in reduced numbers, that beat. In zebrafish, injection of anti-Rfx2 morpholinos results in an absence of motile cilia in the pronephric duct (unpublished data, Liu et al, 2007), but it is not known whether the same requirement for Rfx2 exists in mammalian multicilia formation. In mammals, Rfx2 is highly expressed in the testis, and Rfx2 binds and is able to regulate in vitro the promoter of at least one gene involved in spermatogenesis (VanWert, Wolfe et al. 2008). Rfx4 is also highly expressed in testis (Morotomi-Yano et al, 2002) as well as brain, and mutation of Rfx4 has been linked to improper formation of dorsal midline brain structures (Blackshear et al, 2003). Rfx5 regulates major histocompatability class II gene expression. Human Rfx6 and Rfx7 were recently identified by phylogenetic analysis, and although nothing is known yet about their
94 function Rfx6 expression appears to be limited to pancreatic islets, suggesting a tissue specific role for this gene (Aftab et al, 2008). Many more distal regulators of ciliogenesis have been identified. Blockage of Fgfr signaling in zebrafish and Xenopus leads to downregulation of FoxJ1 and Rfx2, a decrease of cilia length and resulting left-right asymmetry defects (Neugebauer et al, 2009). Disruption of the mouse homeobox gene Noto also causes left-right asymmetry defects, and mutant embryos have decreased expression of FoxJ1 and Rfx3 (Beckers, Alten et al. 2007). It is not known if the Fgfr1 transcriptional activator or Noto directly bind FoxJ1 and Rfx2 promoter regions, or whether transcriptional regulation of these genes is effected far downstream.
Results Identification of transcription factors upregulated during ciliogenesis We examined the expression of key regulators of ciliogenesis in our MTEC dataset (Fig. 1b). FoxJ1 was strongly upregulated at ALI+4 and ALI+12, consistent with its requirement for motile cilia formation and maintenance, and in agreement with previous results (Vladar and Stearns, 2007). Neither FoxJ1 homologs FoxJ2 or FoxJ3 were significantly induced. Rfx3 was upregulated during early ciliogenesis, and expression dropped slightly at the later timepoint, consistent with findings from other works. Rfx2 was also induced, and more strongly induced than Rfx3, suggesting that it too plays an important role in the differentiation of ciliated MTECs. Surprisingly, Rfx5, which has no prior link to ciliogenesis, was also was significantly upregulated (M = 1.5 at ALI+4) in ciliated cells. None of the other members of the family were upregulated significantly in FoxJ1/EGFP positive cells, although Rfx6 and Rfx7 were only recently identified and are not found on the arrays. To identify transcription factors that may be involved in ciliogenesis, we analyzed the expression pattern of genes with the DNA dependent transcription activity GO term annotation (GO term GO:0006351) by clustering (Fig. 1a). Seven genes clustered closely with FoxJ1: general transcription factor 2b (Gtf2b), amyloid beta (A4) precursor protein-binding family B member 1 (Apbb1/Fe65), zinc finger protein 191 (Zfp191), intraflagellar transport homolog 57 (Ift57/Hippi), cellular
95 myeloblastoma (c-myb), enhancer of zeste homolog 1 (Ezh1) and interleukin receptor- associated kinase (IRAK) -1 binding protein 1 (Irak1bp1/SIMPL), and Rfx2. Ift57, also known as Huntington interacting protein-1 protein interactor (hippi), was initially known for its role in regulating apoptosis in response to neuronal stress caused by polyglutamine expansion of the protein huntingtin (htt) in Huntington Disease (Gervais et al, 2002). It was later shown that IFT57/hippi interacted with components of the intraflagellar complex transport machinery (Baker et al, 2003), and that the knockout mice were defective in making nodal cilia (Houde et al, 206). The D. melanogaster ortholog of IFT57 is regulated by Rfx genes (Laurencon et al, 2007). Ezh1 is a polycomb group chromatin inhibitor that seems to be involved in preventing embryonic stem cell differentiation (Ho and Crabtree, 2008). Apbb1/Fe65 is a transcriptional activator that binds amyloid beta-protein precursor (APP), a protein important in the pathogenesis of Alzheimer disease (McLoughlin and Miller at al, 2008). Irak1bp1 (interleukin receptor-associated kinase (IRAK)-1 binding protein 1) has been implicated in immune response regulation through the Toll-like receptor signaling pathway (Ho and Crabtree, 2008). Zfp191 helps repress differentiation in neural stem cells (Khalfallah et al., 2009). Gt2fb (general transcription factor II B) is a subunit of the RNA polymerase II pre-initiation complex. Finally, the c-myb proto-oncogene encodes a transcription factor that is required for development of the hematopoietic cell lineage, colonic crypts and subventricular zones of the brain (Ramsay and Gonda, 2008). In these tissues, c-myb is high in progenitor cells and low in differentiated cells, suggesting that c-myb in physiological quantities acts to maintain the progenitor population. In the blood, c- myb is required for differentiation of cells in both the lymphoid and erythroid lineages (Mucenski et al., 1991). In the ependyma, deletion of Myb resulted in hydrocephalus, and ependymal cells had fewer and shorter cilia (Malaterre et al., 2008). There are three Myb family members in mouse: Mybl1 (A-myb), Mybl2 (B- myb), and Myb (C-myb), c-myb was the only member upregulated in ciliated cells (Fig. 1b). The following work was done in conjunction with Fraser Tan and the lab of Mark Krasnow. To test whether c-myb is expressed in mouse tracheal epithelial cells, MTECS were stained with an antibody against c-myb. Cultures were stained before
96 and after establishment of ALI. At ALI-7, two days after plating primary tracheal cells onto filters, all cells exhibited low levels of nuclear c-myb expression (Fig. 2a). FoxJ1 was not detected at this timepoint, save in a few isolated ciliated cells that sat down with the cultures. At ALI+0, the day of ALI establishment, nuclear c-myb expression was gone (Fig. 2a). Interestingly, nuclear FoxJ1 was detected in a small number of cells, indicating that, as in embryos, MTECS are able to initiate the ciliogenesis program even without the creation of an air liquid interface. By ALI+4, nuclear c-myb was observed in a subset of MTECs, all of which costained with FoxJ1 (Fig. 2c). This contrasted with the expression of an antibody that detects all three members of the mouse myb family members (pan-myb). This antibody detected nuclear signal in all cells, both FoxJ1 positive and FoxJ1 negative (Fig. 2d). We performed a timecourse, counting c-myb positive and FoxJ1 positive cells at ALI+0, 2, 3, 4, 5 and 6. At all timepoints, all c-myb positive cells were also FoxJ1 positive (data not shown). The reverse did not hold true, suggesting that FoxJ1 is induced in ciliated cells before c-myb. We performed a triple stain of MTECs with an antibody against acetylated alpha tubulin to stain cilia, and antibodies against c-myb and FoxJ1. The goal was to place c-myb expression relative to ciliogenesis, so we assayed MTECs at ALI+8. Interestingly, we found that although surrounding FoxJ1 positive/c-myb negative cells were ciliated, cilia were never found on cells expressing c-myb (Fig. 3a). Even in very mature cultures that were assayed after 19 days at ALI, c-myb expressing cells that also were ciliated were never found (Fig. 3b).
Identification of novel promoter motifs in genes upregulated during ciliogenesis In collaboration with Adam Adler, formerly of the Howard Chang lab, we performed a promoter analysis on genes upregulated or downregulated during early ciliogenesis. We chose to use the ALI+4 timepoint for the analysis because this was when the most transcriptional changes seemed to be occurring (see Chapter 2) and because we were most interesting in defining early events in ciliogenesis, such as basal body duplication. Genes that were more than 2-fold up or down regulated with a p value <0.05 were selected for the analysis. Upregulated and downregulated genes
97 from the MTEC data were compared to two other datasets: the cilia proteome database (Gherman et al., 2006) and a transcriptional analysis of flagellar regrowth in Chlamydomonas (Stolc et al., 2005). The non-reciprocal best human orthologs of mouse and Chlamydomonas genes from the MTEC dataset and Stolc dataset were used, respectively. In total, 788 orthologs from the cilia proteome, 83 from the Chlamydomonas set, 606 from the upregulated genes from MTECs and 328 from the downregulated genes from MTECs were compared. Fig. 4a shows the enrichment of promoter motifs in the various datasets. The level of enrichment of the motif is represented by the depth of color in the intersecting square. Deeper color indicates greater similarity. Three motifs were identified that were enriched in both MTEC datasets and the cilia proteome dataset. The most highly enriched of these were the RFX family X-box motif, validating our analysis, and CYTGCAAY, which does not have a known cognate transcription factor currently. Four other motifs were identified that were not enriched significantly amongst the cilia proteome genes but were enriched in genes upregulated in MTECs. Of these, one is a binding site for Mif1 (Mus musculus inhibitor of four), thought to be a Kruppel related transcription factor (Nikulina et al., 2006). Mif1 was not present on the MEEBO datasets. One enriched motif was identified for the downregulated genes, Nrf1. Nrf1 is involved in the transcriptional activation of the antioxidant response {Ohtsuji, 2008 #542}. Most motifs were not enriched in the Stolc set, save for the ETF/HFH3 binding site. This was unique in its enrichment in the Stolc set. Fig. 4b lists genes with motifs for Rfx, Mif1 and Etf/Hfh3.
Discussion In collaboration with Fraser Tan and the Mark Krasnow lab, we have determined that c-myb, but not a-myb and b-myb, are expressed specifically in cells committed to differentiation as indicated by FoxJ1 expression. After several days in culture but before establishment of the air liquid interface FoxJ1 can be seen in nuclei but not c-myb, suggesting that during ciliogenesis FoxJ1 is induced before c-myb. Another interpretation is that c-myb, which has a very short half-life (Ramsay and Gonda, 2008), has already performed its role and has been degraded by the time most
98 cells begin to express FoxJ1. Indeed, 2 days after cultures before contact inhibition is established, c-myb was weakly expressed in cell nuclei, suggesting that either the ”transcribe and run” model is correct, or that c-myb may play duel roles at different timepoints in the MTEC cultures: the first, during the proliferative stage, and the second, during differentiation. Depletion or interference with c-myb by expression of shRNAs against c-myb in MTECs could be performed to address this issue. There are ideally two possible outcomes to such an experiment: either 1) c-myb knockdown prevents FoxJ1 expression, in which case FoxJ1 is downstream of c-myb, or 2) c-myb knockdown does not prevent FoxJ1 expression, in which case FoxJ1 would either be upstream of c-myb or be regulated along a different genetic pathway. It is interesting that, post-contact inhibition, we only observe c-myb in cells that are FoxJ1 positive but lack cilia. This suggests that c-myb is temporally restricted to a brief period around centriolegenesis. It will be important to determine whether c- myb is expressed during basal body duplication, and whether downregulation of c- myb is required for cilia formation. A flowchart detailing possible future experiments for the characterization of c-myb with respect to centriolegenesis and ciliogenesis is shown in Fig.5.
99
a ALI+4 ALI+12 GFP- Gtf2b FoxJ1 Apbb1 Zfp191 Ift57 Rfx2 Myb Ezh1 Irak1bp1 <-5.65 1:1 <5.65
b Rfx6 Foxj3 Rfx5
GFP- Rfx4 GFP- Foxj2 ALI12 ALI12 Rfx3 ALI4 ALI4 Rfx2 Foxj1 Rfx1
-1 0 1 2 3 4 5 -2 -1 0 1 2 3 4
Mybl2
GFP-
Mybl1 ALI12
ALI4
Myb
-2 -1 0 1 2 3
Figure 1. Temporal expression profiles of genes encoding regulators of DNA dependent transcription during ciliogenesis. a) Data were clustered using a Pearson non-centered distance metric and zero transformed against non-ciliated (GFP-) cells. Grey indicates missing data for that timepoint. Genes that clustered tightly with FoxJ1 include Rfx2, Ift57, and Myb. b) Gene expression patterns for FoxJ, Rfx and Myb family members. Tran- script levels are shown relative to a universal mouse reference RNA.
100 a FoxJ1 c-myb DAPI FoxJ1 c-myb
DAPI ALI -7 ALI b FoxJ1 c-myb DAPI FoxJ1 c-myb
DAPI ALI+ 0 ALI+ c FoxJ1 c-myb DAPI FoxJ1 c-myb
DAPI ALI +4 ALI d FoxJ1 pan-myb DAPI FoxJ1 pan-myb
DAPI ALI +4 ALI
Figure 2. C-myb expression before and during ciliogenesis. MTECs cultured in vitro were fixed with 4% PFA and stained for c-myb before establishment of ALI (a), 4 days (b) or 12 days (c) post-ALI. C-myb is expressed in a subset of cells that are all FoxJ1 positive. In contrast, an antibody detecting a-myb, b-myb and c-myb (”pan-myb”) recognizes all cells in MTEC cultures (d).
101 a FoxJ1 c-myb tubulin ac tubulin ac c-myb
FoxJ1 ALI +8 ALI b FoxJ1 c-myb tubulin ac tubulin ac c-myb
FoxJ1 FoxJ1 ALI +19 ALI
Figure 3. Restriction of c-myb expression during ciliogenesis. MTEC cultures were fixed at a) 8 days or b) 19 days post-ALI establishment were stained with antibodies to FoxJ1 (red), c-myb (green) and acetylated alpha tubulin (blue) to mark cilia. c-myb expression was not observed in cells that had motile cilia.
102
a
Stolc Transcriptome Stolc
Cilia Proteome Cilia
ALI+4 Downregulated ALI+4 ALI+4 Upregulated ALI+4 CYTAGCAAY (RFX1) GTTRYCATRR RYTGCNNRGN (MIF1) GTTNYYNNGTTNA GTTGNYNNRGNAAC RYTGCNWTGGNR RCGCANGCGY (NRF1) (ETF/HFH3)
b RFX1 (p = 9.5E-10) MIF1 (p = 5.2E-7) ETF/HFH3 (p = 1.1E-4) LLID SYMBOL LLID SYMBOL LLID SYMBOL 7802 DNALI1 27148 STK36 8613 PPAP2B 22924 MAPRE3 9778 KIAA0232 95 ACY1 55471 PRO1853 55296 TBC1D19 604 BCL6 112942 CCDC104 23552 CCRK 55214 LEPREL1 84058 WDR54 79896 THNSL1 2150 F2RL1 4867 NPHP1 143241 DYDC1 9314 KLF4 64147 KIF9 26123 C10orf61 143686 SESN3 133015 C4orf28 81490 PTDSS2 1112 CHES1 6691 SPINK2 160777 CCDC60 64919 BCL11B 114327 EFHC1 123016 TTC8/BBS8 51226 COPZ2 84498 FAM120B 5891 RAGE 1265 CNN2 84310 MGC11257 57707 KIAA1609 2769 GNA15 2645 GCK 2622 GAS8 23474 ETHE1 5577 PRKAR2B 83538 TTC25 7515 XRCC1 79645 EFCAB1 312 ANXA13 23639 LRRC6 Figure 4. Promoter analysis of differentially 114987 WDR31 79829 NAT11 expressed genes during early ciliogenesis. a) 60494 CCDC81 Promoter motifs enriched (p<0.05) in ciliary datasets. 196403 DTX3 51199 NIN The darkness of the color indicates the amount of 161835 FSIP1 54839 LRRC49 similarity. Known transcription factors are shown in 84942 WDR73 parentheses. b) Differentially expressed genes 54768 HYDIN 9851 KIAA0753 containing RFX1, Mif1 or ETF/HFH3 motifs in their 8787 RGS9 promoters. The locuslink ID (LLID) is given next to 80776 MGC4093 129138 ANKRD54 the gene symbol. Data are courtesy Adam Adler.
103 Primary MTEC culture with c-myb shRNA knockdown
makes cilia? Yes No
Are cilia motile? Is FoxJ1 expressed? Yes No Yes No
Is cilia beat Are structures required for Are basal C-myb is required for frequency motility present? ie., bodies present? FoxJ1 expression (CBF) normal? dynein arms, radial spokes, central pair. Yes No No Yes No Are they apically localized? Yes No C-myb is Intracellular calcium required for changes upon Properly basal body exposure to CBF- oriented? duplication. altering stimuli? (basal feet) No
Is the apical actin cytoskeleton intact? No
Are proteins required for affected structures expressed (protein/mRNA)? Yes
C-myb is required for expression of Gene A, Gene B, etc.
Figure 5. Experimental flowchart for characterizing c-myb knockdown phenotype.
104
Materials and Methods Tissue culture and Immunofluorescence Cell culture, transfection and indirect immunofluorescence protocols were described in Chapter 3. The mouse monoclonal FoxJ1 antibody was a gift from Dr. Steve Brody. The c-19 c-myb antibody (sc-517) was a gift from Dr. Mark Krasnow. The pan-myb antibody (Bgl-Nsi) detects murine A-myb, B-myb and C-myb and was the gift of Dr. Joe Lipsick.
105 References
El Zein, L., A. Ait-Lounis, L. Morle, J. Thomas, B. Chhin, N. Spassky, W. Reith, and B. Durand. 2009. RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies. J Cell Sci. Gherman, A., E.E. Davis, and N. Katsanis. 2006. The ciliary proteome database: an integrated community resource for the genetic and functional dissection of cilia. Nat Genet. 38:961-2. Ho, L., and G.R. Crabtree. 2008. An EZ mark to miss. Cell Stem Cell. 3:577-8. Khalfallah, O., P. Ravassard, C.S. Lagache, C. Fligny, A. Serre, E. Bayard, N. Faucon-Biguet, J. Mallet, R. Meloni, and J. Nardelli. 2009. Zinc finger protein 191 (ZNF191/Zfp191) is necessary to maintain neural cells as cycling progenitors. Stem Cells. 27:1643-53. Malaterre, J., T. Mantamadiotis, S. Dworkin, S. Lightowler, Q. Yang, M.I. Ransome, A.M. Turnley, N.R. Nichols, N.R. Emambokus, J. Frampton, and R.G. Ramsay. 2008. c-Myb is required for neural progenitor cell proliferation and maintenance of the neural stem cell niche in adult brain. Stem Cells. 26:173-81. Mucenski, M.L., K. McLain, A.B. Kier, S.H. Swerdlow, C.M. Schreiner, T.A. Miller, D.W. Pietryga, W.J. Scott, Jr., and S.S. Potter. 1991. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell. 65:677-89. Nikulina, K., M. Bodeker, J. Warren, P. Matthews, and T.P. Margolis. 2006. A novel Kruppel related factor consisting of only a KRAB domain is expressed in the murine trigeminal ganglion. Biochem Biophys Res Commun. 348:839-49. Ramsay, R.G., and T.J. Gonda. 2008. MYB function in normal and cancer cells. Nat Rev Cancer. 8:523-34. Stolc, V., M.P. Samanta, W. Tongprasit, and W.F. Marshall. 2005. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. Proc Natl Acad Sci U S A. 102:3703-7.
106 Stubbs, J.L., I. Oishi, J.C. Izpisua Belmonte, and C. Kintner. 2008. The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos. Nat Genet. 40:1454-60. VanWert, J.M., S.A. Wolfe, and S.R. Grimes. 2008. Binding of RFX2 and NF-Y to the testis-specific histone H1t promoter may be required for transcriptional activation in primary spermatocytes. J Cell Biochem. 104:1087-101. Yu, X., C.P. Ng, H. Habacher, and S. Roy. 2008. Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nat Genet. 40:1445-53.
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Chapter 5: Future Directions
108 Future Experiments We have generated a ciliated transcriptome through expression profiling of in vitro differentiated primary mouse tracheal epithelial cultures. By sorting cells into FoxJ1/EGFP+ and FoxJ1/EGFP- populations and then subtracting their transcriptomes, we have sought to identify genes that involve in ciliogenesis while excluding background epithelial, tissue and culture related genes. By some metrics, our dataset has outperformed the collective datasets in the ciliary proteome at identifying genes associated with ciliogenesis. This ciliated cell transcriptome will hopefully be useful for those wishing to study primary and motile ciliogenesis, signaling, differentiation and human disease. The experiment was designed using indirect hybridization of sample vs. reference, instead of a direct hybridization of sample vs. sample, so that arrays performed with different tissues or conditions could be compared to the MTEC data without requiring more arrays with MTEC samples. In the future, more arrays could be performed on FoxJ1/EGFP expressing cells from oviduct or brain and then compared to the MTEC transcriptome to further refine the list of genes common to all multiciliated cells. This could also be used to identify genes that may be important for the tissue-specific physiological roles that motile cilia may play, as exemplified by the localization of receptors for bitter taste sensing in motile cilia of the trachea, which are important for airway function and host airway defense. We have established novel localization of a number of known and putative human disease genes to the centrosome. These were all selected based on their upregulation during ciliogenesis in tracheal epithelial cells and had never been localized to the centrosome before. This novel localization is a further validation of our microarray data. It will be interesting to determine how these novel centrosomal proteins are involved in ciliogenesis in multiciliated cells, whether it be in centriole duplication, transport, cilium formation, cell cycle control or signaling. We have also established novel localization of a heterotrimeric G protein subunit and fatso, a protein strongly linked to human obesity, to both primary and motile cilia. In the case of Gnb4, it is the first heterotrimeric G protein subunit localized to motile cilia and will likely not be the last given the upregulation of
109 numerous other subunits during ciliogenesis. Fatso is significantly associated with human obesity, and the knockout mouse has defects in energy homeostasis that lead to abnormal leanness. How fatso is regulating energy balance is almost completely unknown, and the finding that it localizes to cilia and the microtubule cytoskeleton suggests that it may be acting through cilia. Similarly, the mechanism underlying cilia-related obesity is unknown. Fatso may be a crucial link between the two. Two proteins, Mlf1 and Whsc1, were localized only to motile cilia and nuclei/basal bodies or respectively, in multiciliated cells. There is no known link between AML and cilia. It will be important to determine how Mlf1 contributes to ciliogenesis, and whether Mlf1, with its reported function in cell cycle control, is important for basal body duplication in multiciliated cells or for blocking DNA replication. Whsc1 is a candidate gene present in almost all known cases of Wolf- Hirschhorn syndrome, whose sufferers often die of respiratory infection and anoxia. These phenotypes suggest motile cilia dysfunction, which would be consistent with Whsc1 expression in multiciliated airway epithelial cells. There are a number of human disease genes that are upregulated during ciliogenesis that have not been validated. Some, like Wolfram syndrome, exhibit some obvious characteristics of ciliopathies. It will be interesting to determine whether their causative genes are important for making functional cilia, or whether cilia are defective in organisms with the disease. There are still many questions about what makes cells decide to become multiciliated. In collaboration with Fraser Tan and the lab of Mark Krasnow we have identified a novel role for the c-myb transcription factor in the differentiation of airway epithelial cells. C-myb exhibits a strikingly restricted expression pattern that implies tight regulation. It will be important to determine through functional assays how c-myb is involved in ciliogenesis, and whether this role is preserved in multiciliated cells on other tissues in the body. Finally, analysis by Adam Adler formerly of the Howard Chang lab has identified promoter motifs enriched in genes differentially expressed during ciliogenesis. For some of these their cognate transcription factors are known and have never been described before with relation to cilia, and it will be important to validate
110 these findings. Others are not yet associated with transcription factors, and it could be that these motifs represent novel transcription factors that are also required for ciliogenesis. Identification of a set of genes that are sufficient to induce basal body overduplication and motile cilia formation in undifferentiated airway epithelial cells would be a great advancement in our understanding of the development of the airway epithelium, and it would have enormous therapeutic potential.
111