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• W1994857963 abstract "Recent findings indicate that mammalian Sonic hedgehog (Shh) signal transduction occurs within primary cilia, although the cell biological mechanisms underlying both Shh signaling and ciliogenesis have not been fully elucidated. We show that an uncharacterized TBC domain-containing protein, Broad-minded (Bromi), is required for high-level Shh responses in the mouse neural tube. We find that Bromi controls ciliary morphology and proper Gli2 localization within the cilium. By use of a zebrafish model, we further show that Bromi is required for proper association between the ciliary membrane and axoneme. Bromi physically interacts with cell cycle-related kinase (CCRK), whose Chlamydomonas homolog regulates flagellar length. Biochemical and genetic interaction data indicate that Bromi promotes CCRK stability and function. We propose that Bromi and CCRK control the structure of the primary cilium by coordinating assembly of the axoneme and ciliary membrane, allowing Gli proteins to be properly activated in response to Shh signaling. Recent findings indicate that mammalian Sonic hedgehog (Shh) signal transduction occurs within primary cilia, although the cell biological mechanisms underlying both Shh signaling and ciliogenesis have not been fully elucidated. We show that an uncharacterized TBC domain-containing protein, Broad-minded (Bromi), is required for high-level Shh responses in the mouse neural tube. We find that Bromi controls ciliary morphology and proper Gli2 localization within the cilium. By use of a zebrafish model, we further show that Bromi is required for proper association between the ciliary membrane and axoneme. Bromi physically interacts with cell cycle-related kinase (CCRK), whose Chlamydomonas homolog regulates flagellar length. Biochemical and genetic interaction data indicate that Bromi promotes CCRK stability and function. We propose that Bromi and CCRK control the structure of the primary cilium by coordinating assembly of the axoneme and ciliary membrane, allowing Gli proteins to be properly activated in response to Shh signaling. Potent Hedgehog signaling in mice requires Broad-minded (Bromi) function Bromi promotes coordinated assembly of the ciliary membrane and axoneme Bromi forms a complex with cell cycle-related kinase and promotes its stability Cell cycle-related kinase and Bromi similarly regulate ciliary assembly Signaling by the Hedgehog (Hh) family of secreted ligands is used in vertebrate embryonic development to control cellular identity, proliferation, differentiation, and survival (Ingham, 2008Ingham P.W. Hedgehog signalling.Curr. Biol. 2008; 18: R238-R241Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, Varjosalo and Taipale, 2008Varjosalo M. Taipale J. Hedgehog: functions and mechanisms.Genes Dev. 2008; 22: 2454-2472Crossref PubMed Scopus (872) Google Scholar). Sonic hedgehog (Shh) acts as a morphogen in specifying cell fates within the developing neural tube; graded concentrations of the ligand direct cells to adopt distinct identities along the dorsal-ventral axis (Dessaud et al., 2008Dessaud E. McMahon A.P. Briscoe J. Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network.Development. 2008; 135: 2489-2503Crossref PubMed Scopus (486) Google Scholar, Ericson et al., 1997Ericson J. Rashbass P. Schedl A. Brenner-Morton S. Kawakami A. van Heyningen V. Jessell T.M. Briscoe J. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling.Cell. 1997; 90: 169-180Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar). How the quantitative information, represented by Shh ligand concentration, is accurately transduced by the signaling pathway to control gene expression patterns is an area of active research. The molecular mechanism of Hh signaling transduction is understood best in Drosophila. In fruit flies, Hh ligands bind to their cell surface receptor, Patched. This binding prevents Patched from inhibiting a second transmembrane protein, Smoothened, allowing the Hh pathway to be activated (Jia and Jiang, 2006Jia J. Jiang J. Decoding the Hedgehog signal in animal development.Cell. Mol. Life Sci. 2006; 63: 1249-1265Crossref PubMed Scopus (81) Google Scholar). Smoothened controls the activity of the downstream transcription factor Cubitus interruptus (Ci) through a cytoplasmic complex containing the atypical kinesin Costal 2 (Cos2), Fused (Fu) kinase, and Ci (Lum et al., 2003Lum L. Zhang C. Oh S. Mann R.K. von Kessler D.P. Taipale J. Weis-Garcia F. Gong R. Wang B. Beachy P.A. Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2.Mol. Cell. 2003; 12: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, Ruel et al., 2003Ruel L. Rodriguez R. Gallet A. Lavenant-Staccini L. Therond P.P. Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog.Nat. Cell Biol. 2003; 5: 907-913Crossref PubMed Scopus (158) Google Scholar). In addition, Suppressor of fused (Su(fu)) regulates the nuclear localization of Ci, although Su(fu) in Drosophila is dispensable for Hh signaling on its own (Methot and Basler, 2000Methot N. Basler K. Suppressor of fused opposes hedgehog signal transduction by impeding nuclear accumulation of the activator form of Cubitus interruptus.Development. 2000; 127: 4001-4010PubMed Google Scholar). Ci acts both as a transcriptional activator and repressor of Hh target genes, depending on its proteolysis, which is regulated by the pathway (Methot and Basler, 1999Methot N. Basler K. Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus.Cell. 1999; 96: 819-831Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). The molecular basis of Hh signaling in mammals is similar to that in Drosophila, although there are important differences. In mice, Hh ligands signal via Patched and Smoothened homologs (Ptch1 and Smo) to control three Ci homologs, Gli1, Gli2, and Gli3. However, the functional significance of Fu and Su(fu) homologs in Hh signaling appears to have changed during evolution (Svard et al., 2006Svard J. Heby-Henricson K. Persson-Lek M. Rozell B. Lauth M. Bergstrom A. Ericson J. Toftgard R. Teglund S. Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway.Dev. Cell. 2006; 10: 187-197Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, Varjosalo et al., 2006Varjosalo M. Li S.P. Taipale J. Divergence of hedgehog signal transduction mechanism between Drosophila and mammals.Dev. Cell. 2006; 10: 177-186Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, Wilson et al., 2009Wilson C.W. Nguyen C.T. Chen M.H. Yang J.H. Gacayan R. Huang J. Chen J.N. Chuang P.T. Fused has evolved divergent roles in vertebrate Hedgehog signalling and motile ciliogenesis.Nature. 2009; 459: 98-102Crossref PubMed Scopus (97) Google Scholar) and, although Cos2 homologs regulate Hh signaling in mammals and Drosophila, their biochemical functions may differ (Endoh-Yamagami et al., 2009Endoh-Yamagami S. Evangelista M. Wilson D. Wen X. Theunissen J.W. Phamluong K. Davis M. Scales S.J. Solloway M.J. de Sauvage F.J. Peterson A.S. The mammalian Cos2 homolog Kif7 plays an essential role in modulating Hh signal transduction during development.Curr. Biol. 2009; 19: 1320-1326Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, Liem et al., 2009Liem Jr., K.F. He M. Ocbina P.J. Anderson K.V. Mouse Kif7/Costal2 is a cilia-associated protein that regulates Sonic hedgehog signaling.Proc. Natl. Acad. Sci. USA. 2009; 106: 13377-13382Crossref PubMed Scopus (190) Google Scholar). The role of intraflagellar transport (IFT) and primary cilia in mammalian Hh signaling represents another key difference (Eggenschwiler and Anderson, 2007Eggenschwiler J.T. Anderson K.V. Cilia and developmental signaling.Annu. Rev. Cell Dev. Biol. 2007; 23: 345-373Crossref PubMed Scopus (396) Google Scholar). During mouse development, many cell types generate primary cilia, including those responding to Hh signals. Mutations disrupting different aspects of ciliogenesis have distinct effects on the Hh pathway, interfering with steps downstream of Shh and Ptch1 (Caspary et al., 2007Caspary T. Larkins C.E. Anderson K.V. The graded response to Sonic Hedgehog depends on cilia architecture.Dev. Cell. 2007; 12: 767-778Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, Huangfu and Anderson, 2005Huangfu D. Anderson K.V. Cilia and Hedgehog responsiveness in the mouse.Proc. Natl. Acad. Sci. USA. 2005; 102: 11325-11330Crossref PubMed Scopus (606) Google Scholar, Huangfu et al., 2003Huangfu D. Liu A. Rakeman A.S. Murcia N.S. Niswander L. Anderson K.V. Hedgehog signalling in the mouse requires intraflagellar transport proteins.Nature. 2003; 426: 83-87Crossref PubMed Scopus (975) Google Scholar, Tran et al., 2008Tran P.V. Haycraft C.J. Besschetnova T.Y. Turbe-Doan A. Stottmann R.W. Herron B.J. Chesebro A.L. Qiu H. Scherz P.J. Shah J.V. et al.THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia.Nat. Genet. 2008; 40: 403-410Crossref PubMed Scopus (237) Google Scholar). Complementing the genetic data are cell biological studies showing that several key components of the mammalian Hh pathway, Ptch1, Smo, Gli2, Gli3, and Su(fu), localize to primary cilia (Corbit et al., 2005Corbit K.C. Aanstad P. Singla V. Norman A.R. Stainier D.Y. Reiter J.F. Vertebrate Smoothened functions at the primary cilium.Nature. 2005; 437: 1018-1021Crossref PubMed Scopus (1004) Google Scholar, Haycraft et al., 2005Haycraft C.J. Banizs B. Aydin-Son Y. Zhang Q. Michaud E.J. Yoder B.K. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.PLoS Genet. 2005; 1: e53Crossref PubMed Scopus (629) Google Scholar, Rohatgi et al., 2007Rohatgi R. Milenkovic L. Scott M.P. Patched1 regulates hedgehog signaling at the primary cilium.Science. 2007; 317: 372-376Crossref PubMed Scopus (947) Google Scholar). Despite these findings, how the regulatory cascade proceeds within a ciliary context remains obscure. As with Hh signaling, our understanding of ciliogenesis has seen significant advances, although many questions remain. The assembly of these structures is clinically important, as they are involved in a group of human disorders, the so-called “ciliopathies,” such as polycystic kidney disease and Bardet-Biedl syndrome (BBS; Quinlan et al., 2008Quinlan R.J. Tobin J.L. Beales P.L. Modeling ciliopathies: primary cilia in development and disease.Curr. Top. Dev. Biol. 2008; 84: 249-310Crossref PubMed Scopus (101) Google Scholar). Much of what we understand about ciliogenesis comes from research on the green algae Chlamydomonas reinhardtii. During interphase, Chlamydomonas cells generate a pair of motile flagella by strategies very similar to those acting in metazoan ciliogenesis. Studies in Chlamydomonas have highlighted the importance of IFT, which allows bidirectional microtubule-based movement of cargo to and from the tips of growing cilia and flagella (Pedersen and Rosenbaum, 2008Pedersen L.B. Rosenbaum J.L. Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling.Curr. Top. Dev. Biol. 2008; 85: 23-61Crossref PubMed Scopus (373) Google Scholar). While the composition and movement of IFT protein complexes and the structure of axonemes have been studied in detail, other aspects of ciliogenesis are less clear. One example is how the lengths of cilia and flagella are controlled. The effects on flagellar length and growth rate after amputation of a single flagellum have led investigators to propose that Chlamydomonas actively monitor and regulate flagellar length (Rosenbaum et al., 1969Rosenbaum J.L. Moulder J.E. Ringo D.L. Flagellar elongation and shortening in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis and assembly of flagellar proteins.J. Cell Biol. 1969; 41: 600-619Crossref PubMed Scopus (347) Google Scholar). More recently, genetic and RNAi studies have identified several key regulators of the processes (Wilson et al., 2008Wilson N.F. Iyer J.K. Buchheim J.A. Meek W. Regulation of flagellar length in Chlamydomonas.Semin. Cell Dev. Biol. 2008; 19: 494-501Crossref PubMed Scopus (38) Google Scholar). Among these are three proteins, one kinase related to cyclin-dependent kinases (CDKs), long flagella 2 protein (LF2p), and two proteins of unknown function, LF1p and LF3p, which comprise a cytoplasmic length regulatory complex (LRC; Barsel et al., 1988Barsel S.E. Wexler D.E. Lefebvre P.A. Genetic analysis of long-flagella mutants of Chlamydomonas reinhardtii.Genetics. 1988; 118: 637-648Crossref PubMed Google Scholar, Tam et al., 2007Tam L.W. Wilson N.F. Lefebvre P.A. A CDK-related kinase regulates the length and assembly of flagella in Chlamydomonas.J. Cell Biol. 2007; 176: 819-829Crossref PubMed Scopus (71) Google Scholar). In this study, we show that an uncharacterized protein, Broad-minded (Bromi), links activity of the Shh signaling pathway to the coordinated assembly of the ciliary axoneme and membrane. In mouse bromi mutants, the Shh pathway can be properly activated by low and intermediate levels of signals, but high-level responses are blocked. Primary cilia on bromi mutant neural progenitors are abnormally shaped, such that the axonemes appeared curled with Gli2 mislocalizing to their centers. A similar effect on ciliary morphology is seen in zebrafish bromi morphants. Ultrastructural analysis revealed detachment and massive expansion of the ciliary membrane away from the axoneme, indicating that proper assembly and association between the two require Bromi function. We find that Bromi physically interacts with cell cycle-related kinase (CCRK), a mammalian homolog of the LRC component LF2p, and that knockdown of ccrk in zebrafish results in curled cilia like those seen in bromi morphants. We observe a genetic interaction between bromi and ccrk, indicating that the two proteins function in a common mechanism, and our biochemical data suggest that Bromi-CCRK complex formation promotes CCRK stability. We propose that Bromi and CCRK act together to regulate ciliary membrane and axonemal growth, and that the close association between these compartments ensures that Gli proteins can efficiently mediate responses to high levels of Shh signals. A recessive mutation, bromi, was identified in a genetic screen for genes controlling midgestation development in the mouse (Zohn et al., 2005Zohn I.E. Anderson K.V. Niswander L. Using genomewide mutagenesis screens to identify the genes required for neural tube closure in the mouse.Birth Defects Res. A Clin. Mol. Teratol. 2005; 73: 583-590Crossref PubMed Scopus (42) Google Scholar). bromi mutants exhibit exencephaly, poorly developed eyes, and preaxial polydactyly (see Figures S1A–S1E available online). The cephalic ventral midline furrow, which can be seen in exencephalic mouse mutants with normal neural patterning, was absent in bromi mutants, suggesting that ventral neural fates are not properly specified (Figure S1D). These features resembled those of mutants with defects in Hh signaling. To investigate Shh signaling in bromi mutants, we assayed dorsal-ventral neural tube patterning, as this process is tightly controlled by the Shh morphogen (Dessaud et al., 2008Dessaud E. McMahon A.P. Briscoe J. Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network.Development. 2008; 135: 2489-2503Crossref PubMed Scopus (486) Google Scholar). In the Embryonic Day (E) 10.5 bromi mutant neural tube, FoxA2+/Shh+ floor plate cells, specified by high levels of Shh signaling, were absent, Nkx2.2+ V3 interneuron progenitors (p3) were observed in the ventral midline and were variably reduced in number, and HB9+ motor neuron progenitors, specified by intermediate levels of Shh, were expanded ventrally along the midline (Figure 1A). Pax6 expression, which is normally repressed by high-level Shh signaling, expanded ventrally in bromi neural tubes. Expression of Pax7, which is sensitive to the lowest levels of Shh signaling, was unaffected in bromi mutants. We monitored the expression of an independent reporter of Shh signaling activity, Ptch1-LacZ (Goodrich et al., 1997Goodrich L.V. Milenkovic L. Higgins K.M. Scott M.P. Altered neural cell fates and medulloblastoma in mouse patched mutants.Science. 1997; 277: 1109-1113Crossref PubMed Scopus (1366) Google Scholar). In bromi, Ptch1LacZ/+ animals, staining for β-galactosidase activity was diminished in ventral neural progenitors compared with Ptch1LacZ/+ controls, while the dorsal border of the expression domain was not appreciably affected (Figure 1B). These results suggest that bromi neural progenitors respond to low and intermediate levels of Shh signaling, but responses to high levels are blocked. We next asked whether bromi can suppress the phenotype caused by loss of Ptch1. Ptch1 represses Hh target genes in the absence of ligand (Varjosalo and Taipale, 2008Varjosalo M. Taipale J. Hedgehog: functions and mechanisms.Genes Dev. 2008; 22: 2454-2472Crossref PubMed Scopus (872) Google Scholar). Disruption of Ptch1 strongly activates the Hh pathway, resulting in potent ventralization of neural identity (Goodrich et al., 1997Goodrich L.V. Milenkovic L. Higgins K.M. Scott M.P. Altered neural cell fates and medulloblastoma in mouse patched mutants.Science. 1997; 277: 1109-1113Crossref PubMed Scopus (1366) Google Scholar). In contrast to Ptch1 mutants, patterning in bromi,Ptch1 double-mutant neural tubes was dorsalized as in bromi single mutants. The double-mutant neural tubes failed to express Shh and FoxA2, and they maintained ventral Pax6 expression (Figure 2). Consistent with our hypothesis that the bromi mutation blocks responses to high, but not low, levels of Shh signaling, neural progenitors in the double mutants ectopically expressed Nkx6.1, a marker sensitive to both high and low levels of Shh signaling (Figure S2A). Moreover, bromi,Ptch1 double mutants exhibited ectopic Ptch1-LacZ activity throughout most of the embryo, but it was significantly weaker than that observed in Ptch1 single mutants (Figure S2B). We next investigated the relationship between bromi and Rab23. In mice, disruption of Rab23 causes activation of the Shh pathway and cell-autonomous ventralization of neural identity (Eggenschwiler et al., 2001Eggenschwiler J.T. Espinoza E. Anderson K.V. Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway.Nature. 2001; 412: 194-198Crossref PubMed Scopus (302) Google Scholar). This effect occurs independently of Shh and Smo, but it is efficiently suppressed in Rab23,Gli2 double mutants, indicating that Gli2 activity is unrestrained in Rab23 mutants (Eggenschwiler et al., 2006Eggenschwiler J.T. Bulgakov O.V. Qin J. Li T. Anderson K.V. Mouse Rab23 regulates hedgehog signaling from smoothened to Gli proteins.Dev. Biol. 2006; 290: 1-12Crossref PubMed Scopus (110) Google Scholar). In contrast to Rab23opb2 mutants, bromi,Rab23opb2 double mutants lacked a Shh+/FoxA2+ floor plate, and their neural patterning was identical to that of bromi single mutants (Figure S2C). These data suggest that bromi acts at a step downstream of Shh, Ptch1, Smo, and Rab23, and is required for Gli2 to potently activate Shh targets. If the effects of bromi on cell identity are through Shh signaling, increasing pathway activity at a step further downstream should rescue the bromi patterning phenotype. Gli3 plays a dual role as an activator and repressor of Shh target genes. In the presence or absence of Shh signals, Gli3 is converted into a transcriptional activator, Gli3(Act), or it is processed into a repressor form, Gli3(Rep), respectively (Wang et al., 2000Wang B. Fallon J.F. Beachy P.A. Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb.Cell. 2000; 100: 423-434Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar). A Gli3 null mutation (Gli3xt) causes modestly elevated pathway activity in the neural tube due to the loss of Gli3(Rep) (Persson et al., 2002Persson M. Stamataki D. te Welscher P. Andersson E. Bose J. Ruther U. Ericson J. Briscoe J. Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity.Genes Dev. 2002; 16: 2865-2878Crossref PubMed Scopus (244) Google Scholar). We found that increasing Shh pathway activity in bromi mutants by disrupting Gli3 (Figure 3A) was sufficient to restore ventral neural patterning, including specification of the floor plate, in bromi,Gli3xt double mutants. Thus, bromi neural progenitors are capable of adopting the most ventral of neural cell identities, provided that the Shh pathway is sufficiently activated. The bromi neural patterning phenotype was similar to that of Gli2 null mutants (Matise et al., 1998Matise M.P. Epstein D.J. Park H.L. Platt K.A. Joyner A.L. Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system.Development. 1998; 125: 2759-2770Crossref PubMed Google Scholar). However, whereas floor plate and V3 progenitors are lost entirely in Gli2,Gli3xt double mutants (Bai et al., 2004Bai C.B. Stephen D. Joyner A.L. All mouse ventral spinal cord patterning by hedgehog is Gli dependent and involves an activator function of Gli3.Dev. Cell. 2004; 6: 103-115Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar), these cell types were rescued in bromi,Gli3xt mutants, indicating that Gli2 retains some function in bromi mutants (Figure 3A). Consistent with this hypothesis, disruption of Gli2 in bromi mutants weakly exacerbated the bromi phenotype; Nkx2.2+ p3 progenitors were not observed in any of three bromi,Gli2 double mutants, whereas Nkx2.2+ cells were observed in all six Gli2 and bromi single mutants analyzed (21 ± 8 and 16 ± 5 cells/section, respectively; Figure 3B). Western blotting of bromi and wild-type extracts showed that the amount of processed Gli3 (Gli3(Rep)) was comparable between genotypes, although a greater amount of unprocessed full-length Gli3 was observed in the mutant samples (Figure S3A). A similar phenomenon has been observed in the ciliogenesis mutant ftm (Vierkotten et al., 2007Vierkotten J. Dildrop R. Peters T. Wang B. Ruther U. Ftm is a novel basal body protein of cilia involved in Shh signalling.Development. 2007; 134: 2569-2577Crossref PubMed Scopus (148) Google Scholar). Disruption of Gli3(Rep) activity may explain the polydactylous phenotype of bromi mutants. However, because the expression patterns of Shh- and Gli3-dependent genes in the bromi mutant limbs were not dramatically affected (Figure S1F), such an effect on Gli3(Rep) function would be subtle. As steady-state Gli2 levels appeared normal in bromi mutants (Figure S3B), Bromi more likely regulates Gli2 activity rather than its synthesis or stability. The data suggest that the activator functions of Gli2 and Gli3 are less potent in the bromi mutant neural tube, and that disruption of Gli3(Rep) can compensate for this defect. We identified the bromi gene by meiotic recombinant mapping (Figure 4A). Sequencing of genes in the interval revealed an A-to-G transition in the splice acceptor site of exon 11 in the uncharacterized gene C6orf170 (D630037F22Rik). Mutant transcripts make use of a cryptic splice acceptor in exon 11 that shifts the reading frame and results in a nonsense codon immediately downstream (Figures 4B and 4C). We raised polyclonal antisera against Bromi and performed western blotting. A species of the predicted size (∼140 kDa) was observed in wild-type, but not bromi mutant, embryonic extracts (Figure 4D). A gene-trap insertion in C6orf170, RRF165 failed to complement bromi, showing the bromi phenotype in bromi/RRF165 transheterozygotes (Figure S4A), and RRF165 homozygotes showed a patterning phenotype identical to that of bromi mutants (Figure S4B). Thus, bromi disrupts C6orf170 and appears to be a null allele. Bromi homologs are restricted to chordates, and are predicted to contain a Tre-2, Bub2, and Cdc16 (TBC) domain near their C termini. Proteins containing TBC domains typically function as GTPase-activating proteins (GAPs) for Rab GTPases (Rab-GAPs), functioning in membrane trafficking (Grosshans et al., 2006Grosshans B.L. Ortiz D. Novick P. Rabs and their effectors: achieving specificity in membrane traffic.Proc. Natl. Acad. Sci. USA. 2006; 103: 11821-11827Crossref PubMed Scopus (772) Google Scholar). Sequence comparison of the Bromi TBC domain with those of known Rab-GAPs (data not shown) revealed that Bromi lacks two key residues, an arginine and a glutamine in the catalytic finger motifs, essential for Rab-GAP activity (Pan et al., 2006Pan X. Eathiraj S. Munson M. Lambright D.G. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism.Nature. 2006; 442: 303-306Crossref PubMed Scopus (232) Google Scholar). Neither Bromi nor its TBC domain showed an interaction with any among a panel of 60 GTP-locked Rab proteins (Figure S4D) in a yeast two-hybrid assay previously used to identify Rab-GAPs (Itoh et al., 2006Itoh T. Satoh M. Kanno E. Fukuda M. Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activity.Genes Cells. 2006; 11: 1023-1037Crossref PubMed Scopus (112) Google Scholar). As Bromi functions in ciliogenesis (see below), we tested whether it coimmunoprecipitates with FLAG-tagged Rabs 8a, 17, and 23, which have been implicated in the control of ciliogenesis (Yoshimura et al., 2007Yoshimura S. Egerer J. Fuchs E. Haas A.K. Barr F.A. Functional dissection of Rab GTPases involved in primary cilium formation.J. Cell Biol. 2007; 178: 363-369Crossref PubMed Scopus (268) Google Scholar), but found no evidence for such interactions (Figure S4E). Collectively, these data suggest that Bromi does not function as a classical Rab-GAP. Because primary cilia are thought to play a key role in mouse Hh signal transduction, we investigated ciliogenesis in bromi mutants by scanning electron microscopy (SEM) and confocal microscopy. Cilia of the wild-type neuroepithelium were short and straight, projecting apically into the neural tube lumen (Figure 5A). In contrast, bromi neuroepithelial cilia, which were generated at the same frequency as wild-type (Figure 5B), appeared bulbous or spherical (Figure 5A). Staining with Arl13b, IFT88, and acetylated α-tubulin antibodies revealed structures resembling open rings when viewed from the apical surface (Figures 5C and 5D). We suggest that these ring-shaped structures represent curled axonemes enveloped by dilated ciliary membranes, although it is also possible that Arl13b, IFT88, and acetylated α-tubulin localize away from axonemes, adjacent to the dilated ciliary membranes. As the genetic data suggested that the function of the Gli activators is impaired in bromi mutants, we examined the localization of Gli2 within cilia (Figure 5D). In wild-type samples, Gli2 staining was concentrated at cilia tips and colocalized with Arl13b and IFT88. In contrast, Gli2 staining was largely concentrated in the centers of the bromi mutant cilia, encircled by rings of Arl13b and IFT88 staining. Colabeling with gamma tubulin antibodies indicated that Gli2 localized within the mutant cilia rather than to the basal bodies. Cilia from neural progenitors are short and therefore difficult to study with high spatial resolution. For this reason, we further investigated Bromi's role in ciliogenesis by using another system, zebrafish kidney tubules, where longer cilia could be better visualized. We investigated the function of the zebrafish bromi homolog (si:dkey-233p4.1) with morpholinos (MOs) directed against the start site (bromi-AUG) and a splice site (bromi-e8i8) of the gene. Injection of either MO resulted in morphants that were viable during embryonic and early larval stages, but exhibited curvature of the body axis and hydrocephalus (Figure 6A). RT-PCR confirmed that splicing of zebrafish bromi was disrupted by the bromi-e8i8 MO (Figure S5A). We analyzed the cilia from distal kidney tubules, which are long and easily imaged by confocal microscopy with antibodies against acetylated α-tubulin (Figure 6B). Interestingly, the cilia seen in the majority of bromi-AUG and bromi-e8i8 morphants (20/31 and 8/11, respectively) exhibited pronounced curling, often forming rings. This ciliary phenotype resembled that seen in the bromi mouse mutant neuroepithelium, suggesting that Bromi plays a qualitatively similar function in controlling axonemal shape in both systems. To gain a better understanding of the ultrastructure of these cilia, we performed transmission electron microscopy (TEM). The cilia of bromi morphants showed a striking defect: their ciliary membranes were detached from axonemes along one side and dramatically expanded within the kidney tubule lumens (Figures 6D and 6E). The large space between the expanded membranes and axonemes appeared empty. In contrast, ciliary membranes were tightly associated along the periphery of the ciliary axonemes in control fish (Figure 6C). The axonemes of the morphant cilia showed the characteristic structure of nine doublet microtubules surrounding a pair of central microtubules, indicating that morphant axonemes were largely intact. These defects indicate that coordinated assembly of the ciliary membrane and the axoneme is lost in the bromi morphants. To gain insight into the molecular mechanism by w" @default.
• W1994857963 created "2016-06-24" @default.
• W1994857963 creator A5003179214 @default.
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• W1994857963 date "2010-02-01" @default.
• W1994857963 modified "2023-10-18" @default.
• W1994857963 title "Broad-Minded Links Cell Cycle-Related Kinase to Cilia Assembly and Hedgehog Signal Transduction" @default.
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