• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of hhmipaAbout Author manuscriptsSubmit a manuscriptHHMI Howard Hughes Medical Institute; Author Manuscript; Accepted for publication in peer reviewed journal
Dev Biol. Author manuscript; available in PMC Jul 1, 2010.
Published in final edited form as:
PMCID: PMC2799895

The Zn Finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis


Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and that utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian S. mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.


The Hedgehog (Hh) signaling pathway is of broad importance for development and disease (Varjosalo and Taipale, 2008). In vertebrates, Hh signaling can regulate limb and digit patterning (Tabin and McMahon, 2008), patterning of neurons in the neural tube (Dessaud et al., 2008), and has been implicated in diseases including cancer (Jiang and Hui, 2008). In recent years, primary cilia have been demonstrated to be required for Hh signaling in vertebrates (Eggenschwiler and Anderson, 2007).

Cilia are cellular projections that grow from a basal body and that involve nine doublet microtubules arranged in a ring with two singlet central pair microtubules (a 9+2 microtubule arrangement) in the case of motile cilia or no central microtubules (a 9+0 microtubule arrangement) in the case of primary cilia (Eggenschwiler and Anderson, 2007; Gerdes et al., 2009; Huangfu et al., 2003). Cilia dysfunction in humans can result in chronic bronchitis, situs invertis, immotile sperm, polydactyly, mental retardation, kidney cysts, and other disorders (Badano et al., 2006). Mutations in genes required for ciliogenesis in vertebrates can cause defects in Hh signaling (Huangfu et al., 2003; Liu et al., 2005). Several Hh signaling components can localize to cilia in vertebrates, including the Smoothened transmembrane protein (Corbit et al., 2005) and Gli transcription factors (Haycraft et al., 2005). The requirement of cilia for Hh signaling, however, may be an innovation that occurred during the evolution of the deuterostomes because no evidence exists to link cilia and Hh signaling in Drosophila. Given the significance of both Hh signaling and cilia for development and disease, understanding the identity and roles of proteins that impact Hh signaling and cilia is of great importance.

Iguana is a novel, C2H2-type, zinc finger protein (Sekimizu et al., 2004; Wolff et al., 2004), also referred to as DZIP1 (Moore et al., 2004). The zebrafish iguana gene is required for normal Hedgehog signaling (Odenthal et al., 2000; Sekimizu et al., 2004; Wolff et al., 2004), but the reason it is required for Hedgehog signaling is unknown. The planarian S. mediterranea is an emerging model system for studies of metazoan gene function in regeneration and tissue turnover (Reddien and Sánchez Alvarado, 2004). The essentially complete planarian genome sequence combined with the ability to perform RNAi screens has opened the door to molecular genetic studies of the robust biological attributes these animals display (Reddien et al., 2005). Because there are numerous ciliated cell types in planarians, including the ventral epidermis for accomplishing animal locomotion (Hyman, 1951), ciliogenesis genes can readily be studied. We found that an iguana-like gene is required for ciliogenesis in the planarian S. mediterranea. This observation suggested the possibility that it is a role in ciliogenesis that explains the requirement for Iguana in Hedgehog signaling. We therefore also studied the role of Iguana/DZIP1 proteins in zebrafish and in human cells and found that these proteins are required for vertebrate ciliogenesis and are components of the basal bodies of cilia. We conclude that Iguana proteins are novel, and broadly conserved, components of basal bodies with a critical role in ciliogenesis.

Materials and methods

planarian assays

Time to move across a defined distance in a light gradient was measured to determine speed. Head rim cilia length and density at the lateral animal edge were determined using differential interference contrast (DIC) microscopy and still images (Zeiss Axiovision).

planarian in situ hybridizations and antibody labeling

Fixations and hybridization methods were largely based upon Pearson et. al (Pearson et al., 2009). For antibody labeling, planarians were first placed in 5% N-acetyl cysteine in PBS for 5 min, followed by fixation with Carnoy's fixative, and labeling as previously described (Reddien et al., 2005) using an anti-acetylated tubulin antibody (Sigma).


RNAi involved feeding a mixture of dsRNA-containing bacteria and liver, as previously described (Reddien et al., 2005). 10mL of pelleted culture was resuspended in either 100uL or 33uL of a 66% blended liver solution in planarian water, with similar results.

molecular biology

Smed-iguana sequence was obtained using 5’RACE (RLM-RACE, Ambion) and from the NB.10.6h cDNA. The NBE.7.10a RNAi construct was used for RNAi (Reddien et al., 2005). Riboprobes to BBS2 and dnah1 were obtained using RT-PCR and other riboprobes were generated from cDNAs (BBS1, saah-aac61a11.g1; BBS9,saah-aab84c07; IFT88, saah-aaa85e05). The rootletin riboprobe was generated from the H.2.8b cDNA. Zebrafish cmlc2 (AF425743) ribroprobe template was obtained using RT-PCR.

zebrafish lines and morpholinos

Zebrafish lines used included wild type AB, igutm79a (Brand et al., 1996; Karlstrom et al., 1996) and smohi229Tg (Chen et al., 2001). Control embryos for experiments involving igutm79a were phenotypically wild type and from a cross of two igutm79a/+ animals. Wild-type embryos were injected with 1nL of 1-2 mM igu splice targeted morpholinos (MOs) (Gene Tools; Splice MO1, 5’-GTACAGACCTTGTGGTAATTGGCAC-3’; Splice MO2, 5’-CAGATTGAACTCACTCATGTCGAAT-3’). 5 base-pair mismatched MOs were used as controls. 50 picograms of mRNA encoding CAAX-eGFP (membrane GFP; kindly provided by J.B. Green, Dana-Farber Cancer Institute Boston, MA) served as an injection control.

zebrafish immunohistochemistry

Embryos were fixed in 2% trichloroacetic acid (TCA) for 3 hours, blocked (phosphate buffer with 0.5% Triton-X100, 10% goat serum, 0.1% BSA), and labeled with anti-acetylated tubulin (1:1000) overnight at 4°C. in situ hybridizations utilized standard procedures.

Cell culture and siRNA transfection

cDNAs for the human Iguana (dZIP1) and Iguana-like (dZIP1L) protein were obtained as IMAGE clones (5271595 and 4940443, respectively). GFP-tagged proteins were transiently transfected using effectene (Qiagen) into HeLa or hTERT-RPE1 cells and processed 30 to 72 hours later. For experiments in hTERT-RPE1 cells, primary cilia formation was induced by serum starvation for 48 hours. RNAi experiments were conducted as described previously (Kline et al., 2006). All cells were transfected with Oligofectamine (Invitrogen). 6 hours post-transfection, OPTI-MEM media was replaced with DMEM and cells processed 48-72 hours later. siRNAs were obtained from Dharmacon as a pool of 4 sequences; DZIP1 (CCGCAGGAGUCGCCGUUAA, GGAGUGAGAUCGUAUU, ACAACAACUUUGCGAUGUA, and GUGACUGAUUGGAGCGACA) and dZIPl1L (AGAGGGAAAUAGAAGCUAA, CAAAGGACCACACGUGUAA, CUGACGAGCUCAAGGGUGU, and GGAACAAGCUAGGAUCAUU). Control RNAi was performed with siCONTROL Non-targeting siRNA Pool #1 (Dharmacon).

Immunofluorescence and microscopy with human cells

Cells were fixed with methanol for 5 minutes in −-20°C. Cells were stained with anti-rabbit centrin, and acetylated-tubulin (6-11B-1, Sigma-Aldrich). Transiently transfected HeLa and hTERT-RPE1 cells were also stained with anti-goat GFP. Cy2, Cy3, and Cy5-conjugated secondary antibodies (Jackson Laboratories) were used at 1:100. DNA was visualized using 10 μg/ml Hoechst. Images were acquired using a DeltaVision Core deconvolution microscope (Applied Precision) equipped with a CoolSnap HQ2 CCD camera. 40 Z-sections were acquired at 0.2-micron steps using a 100×, 1.3 NA Olympus U-PlanApo objective with 1×1 binning. Images were deconvolved using the DeltaVision software. Equivalent exposure conditions and scaling was used between controls and RNAi-depleted cells.


Smed-iguana is required for ciliogenesis in planarians

The Smed-iguana gene (abbreviated as “iguana”) encodes an Iguana-like Zn finger protein (Fig. S1) and is required for normal planarian locomotion (Reddien and Sánchez Alvarado, 2004). Inhibition of iguana with RNAi in adult planarians results in loss of the ability of animals to glide (Reddien et al., 2005); resultant animals display an inching behavior with slower than normal locomotion (Fig. 1A) accomplished by muscular contractions (Supplementary movies 1, 2). Regenerating planarians produce an epidermis-covered mesenchymal bud at the wound site called a blastema, within which some missing tissues are produced (Reddien and Sánchez Alvarado, 2004). iguana(RNAi) animals were capable of normal blastema formation and regeneration (Fig. 1B), but these regenerating animals also showed inching behavior (n=274/289 inching with 15 not moving versus 286/288 gliding and 2 not moving for the control). Regenerating iguana(RNAi) animals ultimately bloat with fluid and blister (Reddien et al., 2005), indicating a candidate defect in water homeostasis (Fig. 1B). Planarian locomotion is accomplished with the use of ventral motile cilia and water balance is maintained by a ciliated excretory/osmoregulatory system referred to as protonephridia (Hyman, 1951). Together, the locomotory and osmoregulatory defects of iguana(RNAi) animals suggested a possible defect in cilia function.

Figure 1
Smed-iguana(RNAi) planarians display defects in locomotion and osmoregulation

We examined planarian cilia using differential interference contrast (DIC) microscopy at the head rims of iguana(RNAi) and control animals. Cilia number was grossly abnormal in iguana(RNAi) animals (Fig. 2A, B). Cilia began to disappear from the animals between five and seven days following RNAi, and reached a low point following approximately 14 days of RNAi (Fig. 2B). During the period of cilia loss, remaining cilia maintained normal length (Fig. 2B) and were capable of beating (Supplementary movies 3, 4). These data indicate a requirement for iguana in maintenance of the ciliated state of cells at the head rim.

Figure 2
Smed-iguana(RNAi) planarians lack head rim cilia

To test for a general requirement for iguana in maintenance of cilia and in the genesis of new cilia, we utilized an anti-acetylated tubulin antibody that can label planarian cilia. Prominent ciliated features labeled with this antibody include the entire ventral surface and a dorsal, midline stripe of cilia (Reddien et al., 2007; Robb and Sánchez Alvarado, 2002). iguana(RNAi) animals displayed a near complete loss of ventral and dorsal cilia (Fig. 3A). We also failed to observe cilia on iguana(RNAi) regeneration blastemas (Fig. 3B, Fig. S2). Because no cilia were observed on iguana(RNAi) blastemas during timepoints of regeneration at which cilia normally are forming (Fig. S2), iguana is likely required for ciliogenesis as opposed to the maintenance of cilia on blastemas following a normal phase of formation. The ciliated cells (flame cells) of the planarian excretory/osmoregulatory system can also be visualized with the anti-acetylated tubulin antibody. The ‘flame bulb’ of the flame cells in this system is a cluster of cilia that creates fluid flow. Bloating and blistering in planarians can be caused by disruption of microtubule function (Reddien et al., 2005), consistent with a possible defect in motile cilia explaining the bloating and blistering observed in iguana(RNAi) animals. We found that the regeneration blastemas of iguana(RNAi) animals had fewer flame bulbs than did the blastemas of control animals (Fig. 3C). Therefore, iguana is required for the regeneration of ciliated protonephridia. Together these data indicate that iguana is required for maintenance of a ciliated epidermis and for ciliogenesis in newly regenerated tissues.

Figure 3
Smed-iguana(RNAi) planarians cannot maintain or regenerate cilia

Smed-iguana is expressed with other ciliogenesis genes in ciliated planarian cell types

If iguana primarily functions in planarian ciliogenesis, it should be expressed predominantly in ciliated cell types. iguana expression was detected using whole-mount in situ hybridizations and was observed within the ciliated epidermis, including along a stripe on the dorsal epidermis (Fig. 4A). Expression was also observed within the pharynx, the highly ciliated feeding organ of planarians (Fig. S3). An additional site of iguana expression was within the region peripheral of the brain in the anterior, where sensory neurons containing sensory cilia reside (Fig. 4A). We compared the iguana expression pattern to that of numerous planarian genes predicted to encode proteins associated with cilia formation or function (Gerdes et al., 2009). Specifically, we identified and cloned the planarian genes BBS1 (Smed-BBS1; BBS, Bardet-Biedl Syndrome), BBS2 (Smed-BBS2), BBS9 (Smed-BBS9), IFT88 (Smed-IFT88; IFT, intraflagellar transport), and dynein Dnah1 (Smed-dnah1, Dnah1 is required for ciliary motility). All of these genes were expressed in a pattern similar to that of the Smediguana gene (Fig. 4A). We also found that the planarian rootletin gene (Smed-rootletin) was expressed in the ciliated epidermis (Fig. 4A); Rootletin is a component of ciliary rootlets (Yang et al., 2002). The expression pattern of iguana in planarians, and the similarity of this pattern to that of genes involved in cilia function, is consistent with a primary role for planarian iguana being in ciliogenesis and maintenance of a ciliated state.

Figure 4
Smed-iguana is expressed in ciliated cell types

We sought to determine whether iguana acts by promoting the expression of a ciliogenesis program or by promoting the process of ciliogenesis. If iguana acts to specify a ciliated cell fate, a gross general defect in expression of genes involved in cilia biology would be anticipated in iguana(RNAi) planarians. However, we failed to detect any defect in expression of cilia genes in intact and regenerating iguana(RNAi) planarians that lacked cilia (Fig. 4B, C). Although it is possible that the IGUANA protein has a role in the expression of one or more critical cilia proteins, these data are most consistent with a role for IGUANA in the physical process of ciliogenesis as opposed to a role in the specification of a cilia program as part of a cell fate.

Smed-iguana is not required for planarian Hedgehog signaling

There is no evidence that cilia are required for Hedgehog signaling in protostomes, nor is there evidence that Hedgehog signaling is required for ciliogenesis. Because the defects associated with RNAi of iguana in planarians are in ciliogenesis, the possibility therefore exists that iguana is not required for Hedgehog signaling in protostomes. We identified a single hedgehog gene in the S. mediterranea genome (Smed-hedgehog), and inhibited the gene with RNAi. hedgehog(RNAi) animals did not have detectable defects in ciliogenesis, but did display defects in tail regeneration (Fig. 5A, B). Similar results were obtained following inhibition of a gene encoding a Gli2-like protein, Smed-gli2-1 (Fig. 5A, B). The similar phenotypes caused by RNAi of hedgehog and gli2-1 indicates that tail formation is promoted by a Hedgehog signaling pathway similar to that of other organisms. By contrast, iguana(RNAi) animals did not display tail regeneration defects (Fig. 5A, B). These data suggest that Iguana is not a component of planarian Hedgehog signaling.

Figure 5
hedgehog(RNAi) and iguana(RNAi) planarians have different phenotypes

iguana is required for ciliogenesis and left/right asymmetry in zebrafish

Because cilia have recently been demonstrated to be required for Hedgehog signaling in vertebrates the implication of iguana in cilia biology in planarians raised the intriguing possibility that it is a cilia role for Iguana that explains its requirement for Hedgehog signaling in zebrafish. Zebrafish Smoothened protein can localize to primary cilia (Aanstad et al., 2009), and genetic manipulations that disrupt cilia can cause Hedgehog signaling defects (Aanstad et al., 2009; Beales et al., 2007; Huang and Schier, 2009). These observations suggest the role for cilia in Hedgehog signaling is conserved in zebrafish.

The Hh pathway works, in brief, as follows. The Hh receptor, Patched, can inhibit translocation of the Smoothened protein to cilia (Varjosalo and Taipale, 2008). Hh signals by binding to and inhibiting Patched. Within cilia, Smoothened acts with other factors to regulate the activity state of Gli transcription factors. In the presence of Hh, formation of repressive Gli proteins is inhibited, and formation of activating Gli proteins is promoted (Varjosalo and Taipale, 2008). The genetic attributes of the zebrafish iguana gene (igu), with respect to action of the Hh pathway, share similarities to the attributes of ciliogenesis genes in mice. Aspects of the igu mutant phenotype are similar to mutants for the Sonic hedgehog-like sonic-you (syu) gene, a smoothened (smo or smu, smooth muscle omitted) gene, the Gli1-like detour (dtr) gene, and the Gli2-like you-too (yot) gene (Brand et al., 1996; Chen et al., 2001; Karlstrom et al., 1999; Karlstrom et al., 2003; Schauerte et al., 1998; Varga et al., 2001). However, the igu phenotype is consistent with defects in both activating and repressive roles of Gli proteins. igu mutants show defects in Hh-dependent marker expression in the ventral neural tube (Sekimizu et al., 2004), a decrease in somite cell populations promoted by low and high Hh levels, and an increase in somite cell populations promoted by intermediate Hh levels (Wolff et al., 2004). Multiple experiments indicate that igu acts at a step in the Hh pathway downstream of smoothened and that igu may affect the relative activities of repressive and activating Gli proteins (Sekimizu et al., 2004; Wolff et al., 2004). Loss of function of cilia genes in mice can also affect the Hh pathway downstream of smoothened and have impacts on the activity states of Gli proteins (Eggenschwiler and Anderson, 2007; Huangfu et al., 2003; Liu et al., 2005; May et al., 2005). The similarities between genetic data for igu in zebrafish and for cilia genes in mouse, with respect to Hh signaling, are consistent with a hypothesis of a cilia-related role for Iguana in vertebrates.

We sought to determine whether the zebrafish iguana gene is broadly required for zebrafish ciliogenesis by examining the cilia of multiple tissues in igu-defective zebrafish embryos. We utilized the tm79a allele (Brand et al., 1996; Karlstrom et al., 1996) of igu, which is a nonsense mutation (Sekimizu et al., 2004), and also used splice-site morpholinos to create igu morphants (morpholino antisense oligonucleotide-injected embryos). igu mutants were recently reported to contain fewer cilia in the zebrafish neural tube and floor plate (Huang and Schier, 2009). We also detected fewer motile cilia in the pronephric ducts of igu mutant embryos than were observed in control embryos (Fig. 6A). The similar phenotypes observed in igu mutants and morphants indicate that the observed reduction in cilia number was the result of loss of igu function. If Iguana has a primary function in ciliogenesis it should be required for the functioning of ciliated tissues. The cilia of Kupffer's vesicle generate a counter-clockwise flow of fluid that is required for the production of left-right asymmetry in zebrafish (Essner et al., 2005; Kramer-Zucker et al., 2005). iguana mutant zebrafish were previously described to affect cardiac asymmetry (Chen et al., 1997), consistent with a hypothesized general role in the functioning of ciliated tissues. We examined left-right asymmetric gene expression in the lateral plate mesoderm of control and igu morphant zebrafish embryos. The heart is formed on the left side of zebrafish embryos, and the developing heart can be visualized with in situ hybridizations using a riboprobe corresponding to the cardiac myosin light chain 2 (cmlc2) gene. igu-defective embryos displayed a highly penetrant left-right asymmetry abnormality, with the heart forming either on the left, centrally, or on the right side of embryos (Fig. 6B). By contrast, embryos mutant for the Hh pathway gene smoothened (smo) displayed normal left-right cmlc2-expression asymmetry (Wilson et al., 2009) and data not shown). igu morphants had a marked reduction in the number of cilia in Kupffer's vesicle (Fig. 6C). By contrast, Hedgehog signaling does not appear to be required for left-right asymmetry in zebrafish (Chen et al., 2001) and we failed to observe cilia number abnormalities in Kupffer's vesicle in smoothened (smo) morphants (Fig. 6C). The fact that many ciliated tissues displayed a reduction in cilia number in igu mutants and morphants supports the hypothesized primary role for iguana in ciliogenesis.Together these data demonstrate a Hedgehog-independent role for Iguana in vertebrate ciliogenesis.

Figure 6
iguana mutant zebrafish have defects in ciliogenesis and left-right asymmetry

The human Iguana-like proteins dZIP1 and dZIP1L localize to the basal bodies of primary cilia and are required for primary ciliogenesis

Because Iguana proteins are required for ciliogenesis, we sought to determine if Iguana proteins localize to cilia. Two Iguana-like genes are found in humans, dZIP1 and a second dZIP1-like gene, dZIP1L (Moore et al., 2004). The localization of GFP-tagged versions of the human Iguana-like proteins was examined in RPE1-hTERT cells, which allow induction and visualization of primary cilia (Vorobjev and Chentsov Yu, 1982). dZIP1 and dZIP1L localized to two discrete puncta. These puncta localized in close proximity to centrin, a centriolar component (Fig. 7A and S4). Furthermore, dZIP1, dZIP1L, and centrin localized near to the basal body of primary cilia in hTERT-RPE1 cells. In cells lacking cilia (HeLa cells), dZIP1 localized to the centrioles (Fig. S4). Potential centriolar and basal body localization of Iguana proteins was unexamined in prior localization studies (Sekimizu et al., 2004; Wolff et al., 2004). RNAi was utilized to determine if human Iguana proteins are required for human primary cilia formation. RNAi of dZIP1 and dZIP1L together resulted in a reduction in the percentage of hTERTRPE1 cells that were capable of primary cilia formation (Fig. 7B). Together, our observations indicate that dZIP1/Iguana proteins are previously unrecognized centriole and basal body-associated proteins required for normal ciliogenesis.

Figure 7
The human Iguana-like DZIP1 and DZIP1L proteins localize to basal bodies and are required for primary ciliogenesis


Data presented here demonstrate a broad requirement for Iguana-like proteins in ciliogenesis in planarians, zebrafish, and in human cells. A requirement was found for both motile and primary cilia formation and Iguana/dZIP1 proteins localized to the basal bodies of cilia in human cells. These observations explain two seemingly disparate pieces of initial information about Iguana proteins: requirements for planarian locomotion and for zebrafish Hedgehog signaling. The highly abundant and prominent usage of motile cilia in planarians for locomotion (Hyman, 1951), coupled with the efficient use of RNAi (Sánchez Alvarado and Newmark, 1999), can allow for ease of discovery of novel ciliogenesis genes by study of planarians. In this case, the identification of a ciliogenesis role for the planarian iguana gene generated several predictions. First, a ciliogenesis role could explain the similarity of the iguana loss-of-function phenotype to that caused by loss of function of other Hedgehog signaling genes in zebrafish because cilia can be required for vertebrate Hedgehog signaling. We found that iguana mutant zebrafish had fewer cilia in pronephric ducts and in Kupffer's vesicle. Furthermore, these embryos displayed left-right asymmetry defects that can be explained by a lack of cilia in Kupffer's vesicle. Second, iguana-like genes should be required for primary cilia formation, because primary cilia can be the site of events of Hedgehog signaling. We found that primary cilia induction in human cells was disrupted by RNAi of the human iguana-like dZIP1 and dZIP1L genes. Finally, Iguana proteins may localize to cilia if they are directly involved in the process of ciliogenesis. We found that both human Iguana-like proteins localized to the basal bodies of cilia and in cells lacking cilia, to the centrioles.

Cilia are not apparently required for Hedgehog signaling in Drosophila. However, it is possible that Drosophila have lost the use of cilia in many cell types in the course of evolution, leaving only sensory neurons and sperm as ciliated cells (Jiang and Hui, 2008). We observed a clear defect in tail formation following inhibition of the planarian hedgehog gene, but did not observe any defect in tail regeneration in iguana(RNAi) animals. Because we failed to see a Hedgehog-defective phenotype in iguana(RNAi) planarians, our data further support the idea that the use of cilia in Hedgehog signaling is an evolutionary innovation of the deuterostomes.

The linkage between ciliogenesis and Hh signaling in the course of evolution is poorly understood. For example, the gene fused (fu) in Drosophila is required for Hh signaling together with an atypical kinesin Costal2 (Robbins et al., 1997; Sisson et al., 1997). However, a Fused gene is required for normal ciliogenesis but not apparently for Hh signaling in the mouse (Chen et al., 2005; Merchant et al., 2005; Wilson et al., 2009). In zebrafish, fused is required for both Hedgehog signaling and ciliogenesis (Wilson et al., 2009). There may exist multiple additional genes with unidentified roles in ciliogenesis that have impacts on Hh signaling.

Ciliogenesis involves the modification of one of the centrioles of the cell to produce a basal body (Sorokin, 1962). The basal body becomes modified to display features such as a basal foot and ciliary rootlets, localizes near the cell surface, and is associated with growth of both primary and motile cilia (Hoyer-Fender, 2009). Because the human Iguana-like DZIP1 proteins co-localized with a marker of centrioles and basal bodies, Iguana proteins are newly identified basal body components. We conclude that Iguana is a novel regulator of ciliogenesis broadly in the Metazoa and that this activity in cilia biology has evolved to be critical for normal Hedgehog signaling in vertebrates.

Supplementary Material






P.W.R. is an early career scientist of the Howard Hughes Medical Institute. We acknowledge support by the Keck, Smith, Searle, and Rita Allen Foundations. P.W.R. holds a Thomas D. and Virginia W. Cabot career development professorship. We thank Ellie Graeden, Jessica Chang, and members of the Reddien lab for comments and suggestions. We also greatly thank Dr. Hazel Sive for zebrafish support.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Aanstad P, Santos N, Corbit KC, Scherz PJ, Trinh LA, Salvenmoser W, Huisken J, Reiter JF, Stainier DY. The Extracellular Domain of Smoothened Regulates Ciliary Localization and Is Required for High-Level Hh Signaling. Curr Biol. 2009 [PMC free article] [PubMed]
  • Badano JL, Mitsuma N, Beales PL, Katsanis N. The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet. 2006;7:125–48. [PubMed]
  • Beales PL, Bland E, Tobin JL, Bacchelli C, Tuysuz B, Hill J, Rix S, Pearson CG, Kai M, Hartley J, Johnson C, Irving M, Elcioglu N, Winey M, Tada M, Scambler PJ. IFT80, which encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic dystrophy. Nat Genet. 2007;39:727–9. [PubMed]
  • Brand M, Heisenberg CP, Warga RM, Pelegri F, Karlstrom RO, Beuchle D, Picker A, Jiang YJ, Furutani-Seiki M, van Eeden FJ, Granato M, Haffter P, Hammerschmidt M, Kane DA, Kelsh RN, Mullins MC, Odenthal J, Nusslein-Volhard C. Mutations affecting development of the midline and general body shape during zebrafish embryogenesis. Development. 1996;123:129–42. [PubMed]
  • Chen JN, van Eeden FJ, Warren KS, Chin A, Nusslein-Volhard C, Haffter P, Fishman MC. Left-right pattern of cardiac BMP4 may drive asymmetry of the heart in zebrafish. Development. 1997;124:4373–82. [PubMed]
  • Chen MH, Gao N, Kawakami T, Chuang PT. Mice deficient in the fused homolog do not exhibit phenotypes indicative of perturbed hedgehog signaling during embryonic development. Mol Cell Biol. 2005;25:7042–53. [PMC free article] [PubMed]
  • Chen W, Burgess S, Hopkins N. Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity. Development. 2001;128:2385–96. [PubMed]
  • Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DY, Reiter JF. Vertebrate Smoothened functions at the primary cilium. Nature. 2005;437:1018–21. [PubMed]
  • Dessaud E, McMahon AP, Briscoe J. Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network. Development. 2008;135:2489–503. [PubMed]
  • Eggenschwiler JT, Anderson KV. Cilia and developmental signaling. Annu Rev Cell Dev Biol. 2007;23:345–73. [PMC free article] [PubMed]
  • Essner JJ, Amack JD, Nyholm MK, Harris EB, Yost HJ. Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut. Development. 2005;132:1247–60. [PubMed]
  • Gerdes JM, Davis EE, Katsanis N. The vertebrate primary cilium in development, homeostasis, and disease. Cell. 2009;137:32–45. [PMC free article] [PubMed]
  • Haycraft CJ, Banizs B, Aydin-Son Y, Zhang Q, Michaud EJ, Yoder BK. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet. 2005;1:e53. [PMC free article] [PubMed]
  • Hoyer-Fender S. Centriole maturation and transformation to basal body. Semin Cell Dev Biol. 2009 [PubMed]
  • Huang P, Schier AF. Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia. Development. 2009;136:3089–98. [PMC free article] [PubMed]
  • Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L, Anderson KV. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature. 2003;426:83–7. [PubMed]
  • Hyman LH. The Invertebrates: Platyhelminthes and Rhynchocoela The acoelomate bilateria. McGraw-Hill Book Company Inc.; New York: 1951.
  • Jiang J, Hui CC. Hedgehog signaling in development and cancer. Dev Cell. 2008;15:801–12. [PubMed]
  • Karlstrom RO, Talbot WS, Schier AF. Comparative synteny cloning of zebrafish you-too: mutations in the Hedgehog target gli2 affect ventral forebrain patterning. Genes Dev. 1999;13:388–93. [PMC free article] [PubMed]
  • Karlstrom RO, Trowe T, Klostermann S, Baier H, Brand M, Crawford AD, Grunewald B, Haffter P, Hoffmann H, Meyer SU, Muller BK, Richter S, van Eeden FJ, Nusslein-Volhard C, Bonhoeffer F. Zebrafish mutations affecting retinotectal axon pathfinding. Development. 1996;123:427–38. [PubMed]
  • Karlstrom RO, Tyurina OV, Kawakami A, Nishioka N, Talbot WS, Sasaki H, Schier AF. Genetic analysis of zebrafish gli1 and gli2 reveals divergent requirements for gli genes in vertebrate development. Development. 2003;130:1549–64. [PubMed]
  • Kline SL, Cheeseman IM, Hori T, Fukagawa T, Desai A. The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation. J Cell Biol. 2006;173:9–17. [PMC free article] [PubMed]
  • Kramer-Zucker AG, Olale F, Haycraft CJ, Yoder BK, Schier AF, Drummond IA. Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis. Development. 2005;132:1907–21. [PubMed]
  • Liu A, Wang B, Niswander LA. Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors. Development. 2005;132:3103–11. [PubMed]
  • May SR, Ashique AM, Karlen M, Wang B, Shen Y, Zarbalis K, Reiter J, Ericson J, Peterson AS. Loss of the retrograde motor for IFT disrupts localization of Smo to cilia and prevents the expression of both activator and repressor functions of Gli. Dev Biol. 2005;287:378–89. [PubMed]
  • Merchant M, Evangelista M, Luoh SM, Frantz GD, Chalasani S, Carano RA, van Hoy M, Ramirez J, Ogasawara AK, McFarland LM, Filvaroff EH, French DM, de Sauvage FJ. Loss of the serine/threonine kinase fused results in postnatal growth defects and lethality due to progressive hydrocephalus. Mol Cell Biol. 2005;25:7054–68. [PMC free article] [PubMed]
  • Moore FL, Jaruzelska J, Dorfman DM, Reijo-Pera RA. Identification of a novel gene, DZIP (DAZ-interacting protein), that encodes a protein that interacts with DAZ (deleted in azoospermia) and is expressed in embryonic stem cells and germ cells. Genomics. 2004;83:834–43. [PubMed]
  • Odenthal J, van Eeden FJ, Haffter P, Ingham PW, Nusslein-Volhard C. Two distinct cell populations in the floor plate of the zebrafish are induced by different pathways. Dev Biol. 2000;219:350–63. [PubMed]
  • Pearson BJ, Eisenhoffer GT, Gurley KA, Rink JC, Miller DE, Sanchez Alvarado A. Formaldehyde-based whole-mount in situ hybridization method for planarians. Dev Dyn. 2009;238:443–50. [PMC free article] [PubMed]
  • Reddien PW, Bermange AL, Kicza AM, Sánchez Alvarado A. BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration. Development. 2007;134:4043–51. [PubMed]
  • Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sánchez Alvarado A. Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Dev. Cell. 2005;8:635–49. [PMC free article] [PubMed]
  • Reddien PW, Sánchez Alvarado A. Fundamentals of planarian regeneration. Ann. Rev. Cell Dev. Bio. 2004;20:725–57. [PubMed]
  • Robb SMC, Sánchez Alvarado A. Identification of immunological reagents for use in the study of freshwater planarians by means of whole-mount immunofluorescence and confocal microscopy. Genesis. 2002;32:293–298. [PubMed]
  • Robbins DJ, Nybakken KE, Kobayashi R, Sisson JC, Bishop JM, Therond PP. Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell. 1997;90:225–34. [PubMed]
  • Sánchez Alvarado A, Newmark PA. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl. Acad. Sci. 1999;96:5049–5054. [PMC free article] [PubMed]
  • Schauerte HE, van Eeden FJ, Fricke C, Odenthal J, Strahle U, Haffter P. Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development. 1998;125:2983–93. [PubMed]
  • Sekimizu K, Nishioka N, Sasaki H, Takeda H, Karlstrom RO, Kawakami A. The zebrafish iguana locus encodes Dzip1, a novel zinc-finger protein required for proper regulation of Hedgehog signaling. Development. 2004;131:2521–32. [PubMed]
  • Sisson JC, Ho KS, Suyama K, Scott MP. Costal2, a novel kinesin-related protein in the Hedgehog signaling pathway. Cell. 1997;90:235–45. [PubMed]
  • Sorokin S. Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol. 1962;15:363–77. [PMC free article] [PubMed]
  • Tabin CJ, McMahon AP. Developmental biology. Grasping limb patterning. Science. 2008;321:350–2. [PubMed]
  • Varga ZM, Amores A, Lewis KE, Yan YL, Postlethwait JH, Eisen JS, Westerfield M. Zebrafish smoothened functions in ventral neural tube specification and axon tract formation. Development. 2001;128:3497–509. [PubMed]
  • Varjosalo M, Taipale J. Hedgehog: functions and mechanisms. Genes Dev. 2008;22:2454–72. [PubMed]
  • Vorobjev IA, Chentsov Yu S. Centrioles in the cell cycle. I. Epithelial cells. J Cell Biol. 1982;93:938–49. [PMC free article] [PubMed]
  • Wilson CW, Nguyen CT, Chen MH, Yang JH, Gacayan R, Huang J, Chen JN, Chuang PT. Fused has evolved divergent roles in vertebrate Hedgehog signalling and motile ciliogenesis. Nature. 2009;459:98–102. [PMC free article] [PubMed]
  • Wolff C, Roy S, Lewis KE, Schauerte H, Joerg-Rauch G, Kirn A, Weiler C, Geisler R, Haffter P, Ingham PW. iguana encodes a novel zinc-finger protein with coiled-coil domains essential for Hedgehog signal transduction in the zebrafish embryo. Genes Dev. 2004;18:1565–76. [PMC free article] [PubMed]
  • Yang J, Liu X, Yue G, Adamian M, Bulgakov O, Li T. Rootletin, a novel coiled-coil protein, is a structural component of the ciliary rootlet. J Cell Biol. 2002;159:431–40. [PMC free article] [PubMed]
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Gene
    Gene links
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene links
  • Nucleotide
    Published Nucleotide sequences
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Published protein sequences
  • PubMed
    PubMed citations for these articles
  • Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...