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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Dev Cell. Author manuscript; available in PMC 2013 May 15.
Published in final edited form as:
PMCID: PMC3366633
NIHMSID: NIHMS374784

IFT25 Links the Signal-Dependent Movement of Hedgehog Components to Intraflagellar Transport

Abstract

The intraflagellar transport (IFT) system is required for building primary cilia, sensory organelles that cells use to respond to their environment. IFT particles are composed of about 20 proteins and these proteins are highly conserved across ciliated species. IFT25, however, is absent from some ciliated organisms suggesting it may have a unique role distinct from ciliogenesis. Here, we generate an Ift25 null mouse and show that IFT25 is not required for ciliary assembly but is required for proper Hedgehog signaling, which in mammals occurs within cilia. Mutant mice die at birth with multiple phenotypes indicative of Hedgehog signaling dysfunction. Cilia lacking IFT25 have defects in the signal-dependent transport of multiple Hedgehog components including Patched-1, Smoothened, and Gli2 and fail to activate the pathway upon stimulation. Thus, IFT function is not restricted to building cilia where signaling occurs, but also plays a separable role in signal transduction events.

Keywords: intraflagellar transport, Hedgehog signaling, cilia

Introduction

Primary cilia extend from the surface of most types of vertebrate cells and function as sensory organelles. In vertebrates, but curiously not in invertebrates, the cilium is the organizing center for Hedgehog signaling, an essential pathway required for development (Corbit et al., 2005; Huangfu and Anderson, 2005). In the absence of Sonic Hedgehog (Shh) ligand, the Patched-1 (Ptch1) receptor resides in the ciliary membrane. Upon stimulation by Shh ligand, Ptch1 exits the cilium and consequently triggers the ciliary accumulation of the Smoothened (Smo) receptor (Rohatgi et al., 2007). Gli transcription factors, which are the downstream effectors, localize to cilia, accumulate at ciliary tips after pathway activation, and translocate to the nucleus to regulate gene expression (Haycraft et al., 2005; Kim et al., 2009; Tukachinsky et al., 2010). During development, Shh signaling plays key roles in the development of the limbs, brain, heart and most other organ systems and it is likely that alteration of the Shh pathway by ciliary defects underlies many of the pathologies seen in the ciliary diseases or ciliopathies. However, in spite of its importance to mammalian development, the mechanism that vertebrates use to compartmentalize Shh signaling within the cilium remains unknown.

Cilia are assembled by intraflagellar transport (IFT), a process that moves large complexes called IFT particles from the cell body into and along the ciliary microtubules. The particles are carried to the flagellar tip using kinesin-2 motors and then back to the cell body using cytoplasmic dynein 2 (Rosenbaum and Witman, 2002). The IFT particles are composed of more than twenty subunits organized into two subcomplexes called A and B (Cole et al., 1998; Follit et al., 2009). In mouse, mutations in complex B subunits and in the kinesin motor typically result in a loss of cilia and an attenuated Shh pathway (Houde et al., 2006; Huangfu and Anderson, 2005; Liu et al., 2005; Ocbina and Anderson, 2008). Most mutations in the retrograde IFT motor dynein 2 produce short stumpy cilia and also yield an attenuated Shh pathway (Huangfu and Anderson, 2005; May et al., 2005). Mutations in complex A have produced varying results with either stumpy cilia and enhanced Shh signaling (Ift139, Ift122sopb) or failed cilia assembly and weakened Shh signaling (Ift122Med1Δ1–3) (Cortellino et al., 2009; Qin et al., 2011; Tran et al., 2008).

IFT proteins are highly conserved throughout ciliated species. Two intriguing exceptions to this conservation are the complex B proteins IFT27 (NM_025931) and IFT25 (NM_028394), which are conserved in vertebrates but not found in C. elegans and Drosophila. IFT27 is a member of the small G protein family and is most closely related to the Rab subfamily (Qin et al., 2007). IFT25 was originally identified as a small heat shock protein (Bellyei et al., 2007) but its membership in the heat shock family was recently called into question (Bhogaraju et al., 2011). Work in mouse (Follit et al., 2009) and Chlamydomonas (Lechtreck et al., 2009; Wang et al., 2009) strongly support its role as an IFT complex B subunit. Furthermore, recent structural analysis revealed that IFT25 and IFT27 reside in a binary complex (Bhogaraju et al., 2011).

The observation that IFT25 and IFT27 are not conserved in some ciliated organisms such as C. elegans and Drosophila but are conserved in mammals suggests that these two proteins may have distinct roles outside of the conventional IFT-B role in ciliary assembly. To further elucidate the function of IFT25, we generated an Ift25 null mutant mouse. All other mouse IFT complex B null mutants fail to assemble cilia properly and die mid-gestation. In contrast, Ift25 null mutants retained the ability to ciliate and survived through gestation. The mutants die at birth with severe heart defects and also display phenotypes indicative of aberrant Shh signaling. Cell culture experiments and in vivo analysis indicate that IFT25 is required for the export of Smo and Ptch1 receptors out of the cilium and the accumulation of Gli2 at the ciliary tip in response to activation of the Shh pathway. We propose that IFT25 is not required for ciliary assembly but rather for the dynamic transport of Shh signaling molecules within the cilium.

Experimental Procedures

Additional methods can be found in Supplemental Experimental Procedures

Mouse Breeding

Ift25-targeted ES cell line 2900042B11Rik was obtained from the EUCOMM project and injected into C57Bl/6J albino blastocysts to generate chimeric animals. Chimeric mice were mated to C57Bl/6J albino mice (B6(Cg)-Tyrc-2J/J, Jax 000058) or to C57Bl/6J mice (Jax 000664). Germline deletion was carried out using a C57Bl/6 congenic PrmCre (O’Gorman et al., 1997). Genotyping was carried out as described in Figure S1. All mouse work was carried out at UMMS and was approved by the UMMS IACUC.

Cell Culture

Primary mouse embryonic fibroblasts (MEF) from e14 embryos were cultured in 90% DMEM (4.5 g/L glucose), 10% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM NEAA, 10 mM HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco-Invitrogen). Early passage cells (<4 passages) were used for analysis. For Shh and SAG experiments, MEFs were plated at near confluent densities and serum starved (same culture medium described above but with 0.25% FBS) for 48 hr prior to treatment to allow ciliation. SAG (Calbiochem) was used at 400nM and Shh (R&D Systems) was used at 2μg/ml.

Immunofluorescence Microscopy, Histology, and Electron Microscopy

Cells for immunofluorescence microscopy were grown, fixed, and stained as described (Follit et al., 2006) except that the paraformaldehyde fixation time was reduced to 15 min. For some antibodies, an antigen retrieval step of 0.05% SDS in PBS for 5 minutes was used prior to the blocking step (Brown et al., 1996). For H&E staining and trachea immunofluorescence, tissues were fixed overnight at 4°C with 4% paraformaldehyde in PBS, paraffin embedded, and processed as previously described (Jonassen et al., 2008). For neural tube immunostaining, embryos were fixed in 4% paraformaldehyde in PBS for 1 hr at 4°C, rinsed in PBS, equilibrated overnight at 4°C in PBS with 30% sucrose, and embedded in OCT Tissue Freezing Medium (Triangle Biomedical Sciences). Cryosections (10 μm) were blocked for one hour at room temperature in TBS containing 5% goat serum and 0.1% Triton and then incubated in primary antibody overnight at 4°C.

Primary antibodies used included acetylated tubulin (611-B1, Sigma), beta-tubulin (B-5-1-2, Sigma), MmIFT20, MmIFT52, MmIFT57, MmIFT88 (Pazour et al., 2002), MmIFT25 (Proteintech), MmIFT140 (Jonassen et al., 2012), Gli1 (V812, Cell Signaling), Gli3 (AF3690, R&D), beta actin (13E5; Cell Signaling), Gli2 (gift of J. Eggenschwiler, Princeton Univ), Smo, and Ptch1 (gifts of R. Rohatgi, Stanford Univ.). Nkx2.2, FoxA2, Pax7, and Pax6 antibodies were from DSHB (University of Iowa). Anti-MmIFT27 was made by expressing the mouse protein in bacteria as a maltose binding protein fusion and injecting into rabbits (Figure S3). IFT antibodies were affinity purified against the same fragment expressed as a glutathione S-transferase fusion. Further microscopy details are provided in Supplemental Experimental Procedures.

Skeletal preparations were performed with Alcian Blue (Sigma) and Alizarin red (Sigma) as previously described (Tsumaki et al., 1999) with slight modifications (0.03% Alizarin red in 1% KOH).

For quantification of Gli2 at ciliary tips, the average Gli2 fluorescence intensity within a defined circular area of 320 pixels at the distal end of each axoneme was measured using the measurement tools of Openlab. Gli2 quantitation data was compiled from measurements of 50 cilia from each of 3 wild type cell lines and 3 mutant cell lines.

Protein and mRNA Analysis

For determining IFT protein levels, MEFs were lysed directly into denaturing loading buffer. Western blots were developed by chemiluminescence (Super Signal West Dura, Pierce Thermo) and imaged using a LAS-3000 imaging system (Fujifilm).

Isolation of mRNA and quantitative mRNA analysis was performed as previously described (Jonassen et al., 2008). Primers are listed in Supplemental Experimental Procedures.

Episcopic Fluorescence Image Capture (EFIC) Histology and MicroCT Imaging

EFIC and MicroCT imaging were performed as previously described (Rosenthal et al., 2004; Tobita et al., 2010). Detailed information is included in Supplemental Experimental Procedures.

Data analysis

All quantitations are presented as means ± SD and were compared between mutant and wild type mice with unpaired t tests using GraphPad software. Differences between groups were considered statistically significant if p < 0.05. Statistical significance is denoted with asterisks (*p=0.01–0.05; **p=0.001-0.01;***p<0.001).

Results

Ift25 mutants die at birth with multi-organ structural birth defects

To characterize the function of IFT25, we generated a mutant mouse using EUCOMM ES cells (Skarnes et al., 2011). The original ES cell line contained a splice acceptor, a promoterless β-galactosidase-neomycin resistance gene fusion and a LoxP recombination site inserted into intron 2 and an additional LoxP site inserted into intron 3 (Figure S1). This cell line was injected into blastocysts and used to create the Ift25neo allele. The Ift25neo allele was converted to the Ift25null1 allele by a germ line Cre. The null1 allele retained the splice acceptor and β-galactosidase-neomycin resistance gene fusion insertion but lacked exon 3. In both cases, the splice acceptor and fusion gene are expected to capture the upstream exons and disrupt splicing and or translation of the downstream exons. We examined both Ift25null1 and Ift25neo mutant animals but found no differences between the two genotypes (Figure 1, ,2).2). We were unable to detect IFT25 protein by western blot from either allele (Figure 3E and data not shown) and the mRNA levels of the 3′ exons were at background levels by quantitative PCR (Figure 1B) strongly suggesting that these are null alleles.

Figure 1
Ift25 null mutants display multiple developmental defects
Figure 2
Ift25 mutants have structural heart and lung disease
Figure 3
Unlike other complex B null mutants, Ift25 null mutant cells are ciliated

In stark contrast to the mid gestational lethality observed when other IFT B genes are deleted in the mouse, Ift25null1 and Ift25neo mutants are born live but none survive past the day of birth (Figure 1A). At birth the Ift25 mutant animals are smaller than littermates and are cyanotic (Figure 1C). To understand the pathology leading to the neonatal lethality, we examined embryos the day prior to birth (e18). At e18 the mutant embryos were alive but had a number of abnormalities. In addition to a slight growth restriction (Figure 2D), they frequently showed omphaloceles (23/29 Ift25neo/neo mutants and 21/40 Ift25null1/null1) (Figure 1D) and often had polydactyly (6/29 for Ift25neo/neo mutants and 6/40 for Ift25null1/null1) with a preaxial digit duplication (Figure 1Ec). Mutants also displayed other skeletal malformations such as micrognathia, cleft palate (Figure 1Ea, 1Ed), and malalignment of the sternal vertebrae (Figure 1Eb). The kidneys did not show any sign of cyst formation at e18 (data not shown).

Cyanosis is often caused by heart and lung defects. Histological analysis indicated that cellular development of the Ift25 mutant lungs was not greatly affected and at most, only slight hypoplasia was detected. However, a subset of the animals (2/6 for Ift25neo/neo mutants and 6/15 for Ift25null1/null1) had a left pulmonary isomerism where a single lobe was present on both sides rather than the normal arrangement of four lobes on the right and one on the left (Figure 2B). Necropsy indicated that in many cases, the rib cage was small and impinged on the lungs, deforming their surface, findings consistent with a short-rib phenotype. Quantitative analysis using microCT imaging showed a reduction in the chest cross sectional area. However, mutant animals were smaller and the reduction in chest size was proportional to the crown to rump length. In contrast, the heart size was not reduced proportionally, and it is likely that a normal size heart in a smaller chest cavity caused the compression of the lungs (Figure 2C). Besides the pulmonary isomerism, no other evidence of left-right asymmetry defects was observed (0/6 Ift25neo/neo mutants and 0/14 Ift25null1/null1embryos showed alterations in orientation of the heart or position of stomach and spleen).

The Ift25 mutant animals also displayed severe structural heart defects including ventricular septal defects (VSD) and outflow tract malalignment defects comprising of overriding aorta or double outlet right ventricle (Figure 2Ab). In addition, Ift25 mutants had severe endocardial cushion defects, also referred to as atrioventricular septal defects (AVSD) (Figure 2Aa). Instead of two AV orifices with mitral and tricuspid valves separating the left atrium and ventricles versus right atrium and ventricles, Ift25 mutants have a single orifice with common AV valves. In some mutants, the AV orifice is located more in favor of the right ventricle, referred to as an unbalanced AVSD. These defects are associated with a common atrium due to complete failure in formation of the septum primum and septum secondum (Figure 2Aa). The severity of these congenital heart defects, particularly the common atrium and AVSD are not compatible with postnatal survival and this is likely the cause of the neonatal lethality seen in the mutant animals.

IFT25 is not required for cilia assembly

The ability of the Ift25 mutants to survive past the point at which most IFT complex B mutants die suggests that they would not completely lack cilia. Indeed, examination of e18 lung sections showed normal numbers and lengths of cilia on the epithelial cells of the bronchi (Figure 3A) and TEM analysis of trachea cilia revealed no major ultrastructural differences (Figure 3B). Furthermore, no significant differences were seen in either the percentage of ciliated cells or the ciliary lengths in mouse embryonic fibroblast (MEF) cultures (Figure 3D). Strikingly, the assembled cilia on Ift25 mutant cells contained normal distributions of IFT88 and IFT140 but were devoid of IFT27 (Figure 3C, S3) suggesting that IFT27 may be specifically affected by the Ift25 mutation. To examine how the lack of IFT25 affected IFT27 and other IFT proteins, extracts derived from MEF cell lines were examined by western blotting. As expected, IFT25 was undetectable in homozygous Ift25 mutant cells. Although most of the IFT subunits we tested (IFT88, 57, 20, 140) were not significantly affected by the loss of IFT25, IFT27 was strongly depleted (Figure 3E). These data indicate that the stability of IFT27 is dependent on IFT25 and that both proteins are likely dispensable for cilia assembly. We also did not detect significant mitotic defects in Ift25 mutant cells (data not shown), suggesting that mammalian IFT25 is likely not required for cell cycle control.

Ift25 mutants have Hedgehog signaling defects

Mouse mutants that fail to assemble cilia typically exhibit aberrant Shh signaling. Interestingly, although the Ift25 mutant embryos form cilia normally, they still display phenotypes of Shh signaling defects, including polydactyly, cleft palate, lung isomerisms and ventricular septal defects (Goddeeris et al., 2008; Johnson, 1967; McMahon et al., 2003; Mo et al., 1997; Tsukui et al., 1999) (Figure 1, ,2).2). This observation suggests that the cilia on Ift25 mutants may have defects in Shh signaling. However, these phenotypes may be caused by defects in other signaling pathways. To more directly test the Shh pathway, we used an in vitro assay to measure the upregulation of Gli1 and Ptch1 in response to pathway activation by the Smo agonist SAG. Treatment of wild type cells with SAG activates the pathway at Smo, stimulating the downstream events to elevate Gli1 and Ptch1 expression (Humke et al., 2010). As previously reported, cells from ciliary assembly mutants like Ift88Tg737Rpw assemble reduced numbers of cilia (Figure 4C) and display weakened Shh signaling (indicated by decreased upregulation of Gli1 and Ptch1 in response to SAG compared to controls) (Figure 4B) (Haycraft et al., 2005). In contrast, the Ift25 mutant cells assembled normal numbers of cilia (Figure 4C) yet still showed a weakened Shh signaling response (Figure 4A). Therefore, although both Ift25 and Ift88 mutants have a weakened Shh signaling response, the underlying causes are likely quite different.

Figure 4
IFT25 is required for normal Hedgehog signaling response

The expression pattern of transcription factors within the developing neural tube is often used as a readout of Shh signaling. The notochord resides ventral to the neural tube and produces high levels of Shh. This establishes a gradient across the neural tube with high Shh on the ventral side and low Shh on the dorsal side. In response to high levels of Shh, the most ventral cells differentiate into the floor plate and become a secondary source of Shh that reinforces the gradient. The Shh gradient within the neural tube effects which target transcription factor genes will be expressed (Goetz et al., 2009). For example, Pax6 is normally expressed in the dorsal region of the neural tube but is repressed at the ventral pole due to the high level of Shh signaling in this region. Similarly, Olig2 is repressed by high levels of Shh signaling, but requires moderate levels of Shh signaling for expression so this transcription factor is found in a zone at the middle of the neural tube. Mouse mutants that fail to properly assemble cilia (e.g. mice with mutations in most complex B genes) exhibit aberrant expansion of dorsal markers into ventral regions of the neural tube (Ko et al., 2009). This is interpreted as weakened Shh signaling. Similar to what is seen in cilia-defective embryos, Ift25 mutants had an aberrant expansion of the Pax6 and Olig2 domains towards the ventral pole of the caudal neural tube (Figure 5A,B) suggesting that the Ift25 mutation also causes weakened Shh signaling. However, this is complicated in both our Ift25 mutant and in other cilia defective mutants (Ko et al., 2009) as the floor plate cells in these lines fail to become secondary sources of Shh (Figure 5B) and so it is likely that the gradient is less established. Despite these defects at e9.5, the neural tube patterning appeared to be largely corrected by e10.5 (Figure S5), suggesting a significant diminution but not a complete block of Shh signaling in the Ift25 mutant neural tube at e.9.5.

Figure 5
Hedgehog signaling is altered in the neural tube and limb buds of Ift25 mutants

Patterning of limb is also dependent on Shh signaling and the finding of polydactyly in the hindlimbs of Ift25 mutant animals suggested that Shh signaling might be altered. During limb development, the Shh gradient affects the processing of Gli3 into the cleaved repressor form and cilia-defective mutants typically show increased amounts of Gli3 full length protein. Likewise, the Ift25 mutants also displayed significantly increased levels of full length Gli3 in the hindlimbs (Figure 5C) suggesting that Ift25 is required for the processing of this transcription factor.

Ift25 mutants are defective in signal-dependent redistribution of Hedgehog components

The weakened Shh signaling seen in other loss of cilia mutants could be due to the cell’s inability to concentrate Shh signaling components into its signaling compartment (the cilium). However, the Ift25 mutants also have weakened Shh signaling despite the presence of normal cilia. This suggests that the dynamic localizations of Shh signaling components within cilia might be perturbed in Ift25 mutants. To test this, we monitored the redistribution of Gli2, Smo and Ptch1 in MEFs in response to activation of the pathway. In the basal state, Gli2 is normally found in the cytoplasm with a small enrichment at the ciliary tip. Upon stimulation, Gli2 rapidly accumulates at the ciliary tip so that by 30 min a detectable increase is observed; after 24 hrs there is a large pool at this site and Gli2 is now observed in the nucleus (Figure 6A, B). In Ift25 mutant cells, the small pool is present at the ciliary tip in the basal state, but there is greatly diminished accumulation of Gli2 at both the ciliary tips and the nucleus after stimulation of the pathway (Figure 6A, B). A similar phenotype was found in the embryonic neural tube. Normally, Gli2 is highly enriched at the tip of cilia in the neural tube. In the Ift25 mutant embryos, the ciliary level was reduced and the remaining Gli2 was more evenly distributed along the ciliary shaft (Figure 6C). In wild type cells under basal conditions, Ptch1 is found in the cilium but Smo is not, and upon activation of the pathway, ciliary Ptch1 decreases and Smo increases (Figure 7A). In Ift25 mutant MEFs, Ptch1 is localized to the cilia under unstimulated conditions as expected, but is not exported from the cilium after pathway activation. Although ciliary Smo levels increase after activation of the pathway, Smo is abnormally present in the cilia of unstimulated Ift25 mutant MEFs (Figure 7A). This accumulation of ciliary Smo and Ptch1 was even more striking in vivo. Embryonic tissues that are normally Shh responsive, such as the paraxial mesoderm, have broad cell body distributions of Smo and Ptch1 and relatively low levels within cilia. In contrast, Smo and Ptch1 are highly enriched in the cilia of Ift25 mutants (Figure 7B, C, S7). Since low levels of Smo and Ptch1 are thought to dynamically enter and exit the cilium under unstimulated conditions (Kim et al., 2009; Ocbina et al., 2011) their abnormal ciliary accumulation in Ift25 mutants suggest defects in the ability of these cilia to export Smo and Ptch1 in response to extracellular cues.

Figure 6
IFT25 is required for accumulation of Gli2 at ciliary tips in response to pathway activation
Figure 7
Ciliary export of Smo and Ptch1 is defective in Ift25 mutants

Discussion

IFT25 is not required for ciliary assembly

The function of the IFT particle in the transport of ciliary components and the assembly of cilia is well established but little is known about the functions of individual subunits of the particle. The high degree of conservation across eukaryotes of the ~20 IFT particle proteins suggests that each subunit has important and unique functions. However, analysis of individual subunits has been hampered by the fact that strong alleles of most IFT genes disrupt the particle and cause ciliary assembly defects. A defective ciliary assembly phenotype precludes the ability to attribute specific functions to individual IFT proteins (as opposed to a function of IFT in general) and precludes the ability to study ciliary trafficking of signaling components.

In this work, we generated an Ift25 knockout mouse. In contrast to all previously characterized null alleles of IFT complex B genes in mouse, Ift25 is not required for ciliary assembly. Our analysis indicates that cilia are present in the mutants in normal numbers and with normal structure both in cultured fibroblasts and throughout the embryo. However, cilia of the Ift25 mutants contained no detectable IFT27. This finding is interesting in light of the observation that these two proteins are direct binding partners of each other and may exist as a heterodimer outside of the IFT particle (Bhogaraju et al., 2011). Thus, we cannot rule out the possibility that some of the phenotypes in the Ift25 mutant might be partially due to a combined loss of IFT25/IFT27. No Ift27 mouse mutants have been published so it is unclear whether Ift27 mutants would phenocopy the Ift25 mutants. The only known perturbations of Ift27 are Chlamydomonas RNAi knockdown lines, which suggested that IFT27 was critical for cytokinesis and cell cycle control (Qin et al., 2007). We did not observe cytokinesis defects in our Ift25 mutant fibroblasts and the mitotic index was the same in mutant and control MEFs suggesting that cell cycle is not greatly altered by the loss of mammalian IFT25/27.

Ift25 mutant mice have Hedgehog phenotypes

The finding that IFT25 is not required for ciliary assembly is consistent with the fact that Ift25 was lost from some invertebrates, like Caenorhabditis and Drosophila that still assemble cilia. However, IFT25 is not dispensable in organisms that retained the gene as Ift25 mutant mice died shortly after birth with a constellation of phenotypes reminiscent of Shh signaling defects. These phenotypes include polydactyly, cleft palate, lung isomerisms and structural heart defects (Figure 1, ,2)2) Although lung and heart defects are also found in mouse mutants with defects in left-right axis determination due to motile cilia defects, the phenotypes in the Ift25 mutant are more consistent with Shh signaling defects than motile ciliary defects. For example, motile cilia defects often result in situs inversus or reversal of the lung pattern (Tan et al., 2007), but the Ift25 mutants show duplication of the left lung, a phenotype nearly identical to that seen in the Shh mutant mouse (Tsukui et al., 1999). Likewise an AVSD similar to one in the Ift25 mutant heart is seen in the Shh mutant and when Smo is specifically deleted in the dorsal mesenchyme protrusion, an extracardiac tissue that migrates into the heart to form the atrial septum (Goddeeris et al., 2008; Hildreth et al., 2009). In contrast, motile cilia defects cause left-right phenotypes like heterotaxy and situs inversus (Tan et al., 2007) not seen in the Ift25 mutant hearts.

Observations from numerous groups have led to the idea that the cilium functions in a seemingly paradoxical manner to either activate or repress the Shh pathway, depending on the cellular context (Goetz et al., 2009; May et al., 2005). In the neural tube and agonist stimulated MEFs, loss of a cilium typically impedes the upregulation of Gli activators (such as Gli1) thus causing a weakened signaling output. In the limb bud, however, loss of the cilium typically reduces the processing of Gli3 into Gli3-R and causes polydactyly, which is thought to be the result of increased pathway activity. This difference is probably related to the observation that the cilium is needed for the Gli transcription factors to be modified by proteolysis and phosphorylation to stabilize them and convert them from inactive to active activator or repressor forms (Tuson et al., 2011). Despite the presence of normal cilia, Ift25 mutants displayed Shh phenotypes that are similar to loss of cilia mutants (i.e. weakened upregulation of Gli1 activator in MEFs and increased levels of Gli3-FL in limb buds). These data suggest that IFT25 functions downstream of ciliary assembly, but like the cilium, is critical for production of the appropriate forms of Gli transcription factors in various tissues. There is much left to understand about the mechanics of the Shh pathway, but it appears that Ptch1 plays a dual role in transmitting a signal to Smo when the pathway is activated and a negative role in inhibiting Smo when the pathway is inactive. The mechanism by which Ptch1 activates Smo is not known but is proposed to be through small molecule intermediates (Wang et al., 2007) and is likely to involve the phosphorylation of Smo (Chen et al., 2011). Likewise the repressive role of Ptch1 is not clear, but Ptch1 may sequester ciliary Smo keeping it inactive. In Ift25 mutants, there is an increase in both Ptch1 and Smo in the cilium and the pathway is attenuated. This may simply be due to the level of Ptch1 in the cilium being adequate to sequester all of the Smo in the cilium or may be due to a more complicated mechanism. For example, Gli2 is not trafficked to the ciliary tip in response to pathway activation. If Gli transcription factors are actively transported by IFT25, the failure to transport them to the tip would cause an attenuation of the pathway regardless of what is happening to Ptch1 and Smo.

The proposed role of IFT25 in Shh signaling fits well with the conservation of the gene in vertebrates and its loss in Drosophila and Caenorhabditis. While Shh signaling is cilia-dependent in vertebrates, this pathway does not appear to be present in Caenorhabditis and is cilia-independent in Drosophila (Wilson and Stainier, 2010). However, the fact that IFT25 is conserved in Chlamydomonas, which does not appear to use Shh signaling since core components are missing from its genome, suggests that it also had an ancestral role in other signaling pathways prior to the evolution of Shh signaling. For example, the mating response in Chlamydomonas involves dynamic movement of signaling components in cilia that are reminiscent of Shh signaling (Wang et al., 2006) and perhaps these are dependent upon IFT25.

IFT25 is required for the dynamic movements of Hedgehog components in cilia

The role of the cilium and IFT in vertebrate Shh signaling is well established but the fundamental question of whether IFT plays a direct role in the signaling cascade or whether it functions only to assemble the cilium where the components are organized remains unanswered. Null or strong hypomorphic mutations in IFT complex B components and the anterograde motor Kif3a typically cause a complete failure of the cilium to assemble and result in attenuated Shh signaling (Ko et al., 2009). In these cases it is not possible to distinguish a direct role for IFT in Shh signaling from its role in ciliary assembly. IFT complex A mutations are more complicated. A null allele of Ift139 resulted in short and stumpy cilia with overactive Shh signaling (Tran et al., 2008). IFT complex A has often been described as being primarily involved in retrograde transport and it was proposed that the defects in retrograde transport caused an accumulation of Shh components in the cilia and this resulted in an overactive pathway. However, alleles of the retrograde dynein motor also accumulate Shh components in cilia but result in attenuation of the pathway rather than its activation (May et al., 2005; Ocbina and Anderson, 2008). The phenotype of Ift25 is distinct from all of the previous mouse mutations in IFT components. The Ift25 mutants have some phenotypes similar to IFT-A mutants (ciliary accumulation of Shh components) yet also have some phenotypes similar to IFT-B mutants (attenuated Shh pathway). These data indicate that the view that complex B plays predominantly a role in anterograde transport while complex A plays predominantly a role in retrograde transport is overly simplistic. It is more likely that individual subunits within the two complexes have specific functions in maintaining the particle structure, in connecting the particle to the motors and in coupling the cargos to the particle. For example, the lack of IFT25 in the ciliated organisms Drosophilaan d Caenorhabditis coupled with the lack of ciliary structural defects in Ift25mutant mice suggest that IFT25 is not integral for basic “housekeeping” functions of the IFT particle such as axoneme assembly. Instead, IFT25 is more likely to function in signaling dependent transport of specific cargo within the cilium. Our finding that Ptch1 and Smo accumulate in the Ift25 mutant cilia suggests that IFT25 functions to couple the dynamic movement of these proteins to the IFT system. Our work is consistent with a model whereby the ciliary export, but not import, of Ptch1 and Smo requires IFT25 (Figure 7D). In the absence of Shh ligand, Ptch1 does not get exported by IFT, but Smo is actively removed from the cilium. Upon stimulation of the pathway, the inhibitor (Ptch1) gets removed from the cilium and the activator (Smo) accumulates there. However, when IFT25 is missing, Ptch1 remains ectopically trapped in the cilium and thus keeps the pathway in an attenuated state (Figure 7D).

Clinical Implications

Despite the multitude of known ciliopathy genes, IFT80 is the only complex B gene found mutated in human patients thus far (Beales et al., 2007). This dearth of IFT complex B mutations in human diseases is likely explained by the fact that loss of cilia in mice causes mid-gestational lethality around the time of heartbeat initiation (~e10.5), which in humans would correlate with an early term miscarriage. The fact that Ift25 mutant mice survive gestation suggests that IFT25 is a good candidate for human ciliopathies. Interestingly, the IFT25 mouse phenotype is quite similar to the clinical manifestations of Jeune Asphyxiating Dystrophy which combines skeletal abnormalities such as polydactyly, with lung hypoplasia. Furthermore, Ellis-van Creveld syndrome and McKusick-Kaufman syndromes present with similar skeletal abnormalities in addition to cardiac disorders such as septal defects and a common atrium (Hills et al., 2011), which are all observed in the Ift25 mutant mouse.

Conclusions

Our finding that IFT25 is not required for ciliary assembly but does cause major defects in Shh signaling allows us, to demonstrate that IFT participates in Shh signaling and is not just involved in ciliary assembly. Since all previous IFT mutants had at minimum, moderate defects in ciliary assembly it has been argued that IFT does not play a direct role in Shh signaling but is only required for producing the proper ciliary architecture for signaling to occur. Our data demonstrates that IFT plays a larger role as mutations in Ift25 do not produce any changes in ciliary abundance nor cause any detectable structural defects or abnormal accumulations of IFT proteins yet the Shh components fail to undergo the dynamic movements that normally occur during Shh signaling. Thus, IFT25 couples Shh components to IFT to allow for the dynamic movements that are needed for signaling.

Highlights

  • IFT25 is not required for cilia assembly
  • Ift25 mutant mice die at birth with Hedgehog dysfunction phenotypes
  • Ift25 mutant cells are defective in dynamic movements of signaling components

Supplementary Material

02

03

04

Acknowledgments

We thank Drs. S. Jones (Transgenic Mouse Core) G. Hendricks (Electron Microscopy Core) and P. Furcinitti (Digital Imaging Core) for assistance during this work and Dr. R. Bloodgood for carefully reading the manuscript. We also thank Dr. P. Odgren for use of his bright field microscope, and Drs. J. Eggenschwiler (Princeton Univ) and R. Rohatgi (Stanford Univ) for reagents. This work was supported by the National Institutes of Health (GM060992 to GJP) and (5U01HL098180 to CWL). Core resources supported by the Diabetes Endocrinology Research Center grant DK32520 were also used.

Abbreviations

Shh
Sonic Hedgehog
Smo
Smoothened
Ptch1
Patched 1
H&E
hematoxylin and eosin
IFT
intraflagellar transport
SD
standard deviation
MEF
mouse embryonic fibroblast
TEM
transmission electron microscopy
e
embryonic
p
postnatal
EFIC
episcopic fluorescence image capture
AVSD
atrioventricular septal defect
AV
atrioventricular
VSD
ventricular septal defect

Footnotes

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