Weak and strong alleles of Ift144 have opposing effects on specification of Shh-dependent ventral neural cell types

To define how IFT144 affects Shh signaling, we examined neural tube patterning in the mutants. Neural patterning in Ift144^dmhd embryos was unlike that of other previously characterized IFT mutants. Null mutations in two other IFT-A genes, Ift122 and Ift139a, cause a ventralization of the neural tube, in which the most ventral cell types that require the highest level of Shh (the floor plate and V3 interneuron progenitors) are expanded as a result of elevated ectopic activity of the Shh pathway (Tran et al., 2008; Qin et al., 2011). In contrast, Ift144^dmhd mutants lacked the cell types that require high levels of Shh activity, the FoxA2+ floor plate and Nkx2.2+ V3 progenitors, throughout the spinal cord (Figs. 1 C and S1 A). In anterior (cervical) regions, Ift144^dmhd embryo mutants lacked all Shh-dependent cell types, but motor neurons, which require intermediate levels of Shh activity, were present in the posterior (lumbar) neural tube, where they expanded to more dorsal positions than seen in wild type (Figs. 1 C and S1 A).

Whereas the ventral neural cell types that require high levels of Shh activity were absent in Ift144^dmhd at all rostrocaudal positions, Ift144^twt showed an opposing phenotype: the floor plate, V3 progenitor, and motor neuron domains were dorsally expanded in the caudal neural tube (Fig. 1 C). This ectopic specification of Shh-dependent ventral neural cell types in the Ift144^twt neural tube was similar to that caused by null mutations in two IFT-A genes, Ift139a^alien and Ift122^sopb (Tran et al., 2008; Cortellino et al., 2009; Qin et al., 2011).

To test whether the changes in neural patterning were a result of changes in activity of the Shh pathway, we examined expression of Ptch1, a direct transcriptional target of the Shh pathway, using a Ptch1-lacZ reporter (Goodrich et al., 1997). As predicted by the loss of ventral neural cell types, Ptch1-lacZ was reduced in the Ift144^dmhd neural tube anterior to the forelimbs (Fig. S1, C and D). In the caudal neural tube, Ptch1-lacZ was expressed ectopically in dorsal regions, although the strong Ptch1-lacZ expression at the ventral midline was absent (Figs. 1 D and S1 D). Ptch1-lacZ was ectopically expressed in the dorsal neural tube of Ift144^twt embryos, consistent with the dorsal expansion of ventral neural cell types (Figs. 1 D and S1 E). Thus, the two alleles of Ift144 had contrasting effects on activity of the Shh pathway in the neural tube: Ift144^twt increased pathway activity, and Ift144^dmhd decreased pathway activity but led to an increased number of cells with an intermediate level of activity in the caudal neural tube.

Other IFT proteins have been shown to act at a step in the Shh pathway between the membrane proteins Patched and Smoothened and the Gli transcription factors. Because the Ift144^dmhd phenotype was different than that of other IFT mutants, we used double-mutant analysis to identify the step in the Shh pathway affected by the mutation. Analysis of neural patterning in double mutants showed that Ift144^dmhd acted genetically downstream of the two membrane proteins in the pathway, Patched and Smoothened (Fig. S2, A and B), similar to other IFT-B and IFT-A genes (Huangfu et al., 2003: Huangfu and Anderson, 2005; Qin et al., 2011). Double-mutant analysis showed that the neural patterning phenotype of Ift144^dmhd was the result of changes in the activity of the Gli2 and Gli3 transcription factors (Fig. S2, C–L), as in other IFT mutants.

The expansion of the motor neuron domain in Ift144^dmhd depends on the presence of cilia

IFT144 could, in principle, exert its effect on Shh signaling through either a ciliary or nonciliary role of the protein. To test whether cilia were required for the specification of motor neurons in Ift144^dmhd embryos, we generated double mutants that lacked both IFT144 and the IFT-B complex protein IFT172. Embryos homozygous for the Ift172^wim mutation lack cilia and fail to specify Shh-dependent cell types in the neural tube, including motor neurons (Huangfu et al., 2003). The Ift144^dmhd Ift172^wim double mutants showed the same loss of ventral neural cell types seen in Ift172^wim single mutants (Fig. S3, A–D). Thus, the changes in the activity of the Shh pathway in Ift144^dmhd embryos must be a result of changes in cilia structure or ciliary trafficking caused by loss of this IFT-A protein.

Weak and strong alleles of Ift144 have distinct effects on cilia structure

Each of the neural progenitor cells that respond to Shh has a single primary cilium that projects from its apical surface into the central lumen of the neural tube. When cells of the neural tube were examined en face by scanning electron microscopy, wild-type cilia on E10.5 neural progenitors were ~1 µm in length, with shorter cilia emanating from ciliary pockets (Fig. 3 A), consistent with the appearance of primary cilia in other cell types (Sorokin, 1962; Molla-Herman et al., 2010; Rohatgi and Snell, 2010). In contrast, the primary cilia on Ift144^dmhd neural epithelial cells were short and bulbous (Fig. 3 B and Table S1). The neural cilia in Ift144^twt were approximately normal in length (Fig. 3 C and Table S1), whereas Ift144^dmhd/twt compound heterozygote neural cilia were shorter and wider than wild type (Fig. 3 D and Table S1). The vast majority of Ift144^twt and Ift144^dmhd/twt cilia lacked the bulges near the tips that have been described in other IFT-A mutants.

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Figure 3.

Abnormal morphology of primary cilia in Ift144 mutants. (A–F) Scanning electron micrographs show cilia (arrows) on the lumenal face of the neural tube at E10.5. (A) Most wild-type (wt) cells have a single cilium that projects from the apical surface. (B) Ift144^dmhd mutant cilia are shorter, rounder, and more bulged than wild type. (C and D) Ift144^twt cilia (C) have normal morphology, whereas Ift144^dmhd/twt (D) cilia are shorter than wild type, but few cilia are bulged. (E) Neural tube cilia in embryos that carry a null allele of Ift122 (Ift122^sopb) are of approximately normal length but have bulges near the tip. (F) Ift144^twt Ift122^sopb double-mutant cilia are very short, similar to those of the Ift144^dmhd. Bars, 400 nm.

To characterize the aspects of ciliogenesis that require IFT144, we analyzed cilia and basal bodies in neuroepithelial cells by transmission EM (TEM). Cross-sections showed that wild-type basal bodies had the typical triplet microtubule structure, and cilia had the characteristic 9 + 0 doublet microtubule organization (Fig. 4 A). Basal bodies of Ift144^dmhd, Ift144^twt, and Ift144^dmhd/twt cilia appeared to be of normal size and structure and were present close to the apical surface (Fig. 4, B–D). Ift144^twt and Ift144^dmhd/twt cilia were similar in structure to wild type in both longitudinal sections and cross-sections, although there was a slightly greater distance between the plasma membrane and the microtubule axoneme, which could account for the greater cilia width seen by scanning electron microscopy (Fig. 4 [B and C] and Table S1). Despite the normal structure of the basal body, the majority of Ift144^dmhd cilia analyzed lacked any visible microtubules in longitudinal or transverse sections (Figs. 4 D and S4). Instead, the cilia compartment contained disorganized electron-dense material. This phenotype suggests that IFT144 is required for building the cilium rather than functioning exclusively in retrograde trafficking.

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Figure 4.

Ultrastructure of wild-type and mutant neural tube cilia. (A–F) Longitudinal sections (left) and cross-sections of cilia (top right) and basal bodies (bb; bottom right) from the neural tube in wild type (WT) and mutants. (A) Wild-type cilia have a 9 + 0 doublet microtubule organization in the axoneme and triplet microtubules in the basal body. (B and C) The microtubules in Ift144^twt (B) and Ift144^dmhd/twt (C) mutant cilia appear normal, whereas the cilia are slightly wider than wild type. (D) Ift144^dmhd mutant cilia lack axonemal microtubules and contain vesicle-like structures and have a normal-appearing basal body. (E) Longitudinal sections show that neural tube Dync2h1^mmi cilia are swollen and filled with arrays of electron-dense particles that resemble trains of IFT particles. A few singlet microtubules can be seen in cross-sections of Dync2h1^mmi cilia (arrow). (F) The cilia of Ift144^twt Dync2h1^mmi double mutants are less swollen than the Dync2h1^mmi single mutants and do not accumulate IFT trains but show a more severe phenotype than Ift144^twt cilia. Cilia are from E9.5 (wild type and Ift144^dmhd) and E10.5 (Ift144^twt, Ift144^dmhd/twt, Dync2h1^mmi, and Ift144^twt Dync2h1^mmi) embryos. Bars, 200 nm.

The phenotype of IFT-A double mutants confirms that the IFT-A complex is required for ciliogenesis and for high-level responses to Shh

The Ift144^dmhd mutation caused a much more severe disruption of cilia architecture than null mutations in the mouse IFT-A genes Ift122 or Ift139 (Tran et al., 2008; Qin et al., 2011). To test whether this represented a unique property of the Ift144^dmhd allele or was a result of a more crucial role of IFT144 in the IFT-A complex, we analyzed double mutants that were homozygous for both the weak allele Ift144^twt and the null allele Ift122^sopb. Whereas neural cilia in both Ift144^twt and Ift122^sopb embryos were of nearly normal length (Fig. 3, C and E), the Ift144^twt Ift122^sopb double-mutant cilia were less than half the length of wild type and more bulbous than either single mutant (Fig. 3 F and Table S1). The Ift144^twt Ift122^sopb double-mutant cilia were similar in size and shape to those present in Ift144^dmhd embryos.

Whereas Ift144^twt and Ift122^sopb single mutants showed expansion of Shh-dependent ventral neural cell types, neural patterning in Ift144^twt Ift122^sopb double mutants was similar to that seen in Ift144^dmhd embryos. Like Ift144^dmhd, the Ift144^twt Ift122^sopb double mutants lacked all ventral cell types in spinal cord regions anterior to the forelimbs and lacked floor plate and most Nkx2.2+ V3 progenitors cells at more posterior positions (Figs. 5 [A and B] and S1 A). Also like Ift144^dmhd, motor neurons in the double mutant spanned the midline and were expanded dorsally in the caudal neural tube (Fig. 5 B). We conclude that the Ift144^twt Ift122^sopb double-mutant and Ift144^dmhd single-mutant phenotypes in cilia formation and neural patterning are the result of stronger disruption of the IFT-A complex than seen in Ift122-null single mutants.

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Figure 5.

Neural patterning in Ift144^twt Ift122^sopb double mutants resembles that in Ift144^dmhd. (A) Based on expression of HB9-GFP, Ift144^twt Ift122^sopb double-mutant embryos lack motor neurons in the anterior spinal cord (arrow) but retain them in caudal regions. The double mutants are exencephalic and have laterality defects, similar to Ift144^dmhd. WT, wild type. (B) Cross-sections through Ift144^twt, Ift122^sopb, and Ift144^twt Ift122^sopb double-mutant embryos at E10.5, marked by the expression of HB9-GFP and FoxA2. Ift144^twt/Ift122^sopb double mutants lack FoxA2+ floor plate cells at all levels, lack cervical motor neurons, and show dorsally expanded motor neurons at lumbar levels, like Ift144^dmhd. Bars: (A) 1 mm; (B) 200 µm.

IFT-A and cytoplasmic dynein-2 have distinct roles in cilia structure

Because previous studies have shown that both IFT-A proteins and cytoplasmic dynein-2 are required for normal retrograde ciliary trafficking, we compared the structure of Ift144 mutant cilia with that of mutants homozygous for a strong allele of the gene encoding the heavy chain of the retrograde dynein motor Dync2h1^mmi (Liem et al., 2009; Ocbina et al., 2011). TEM sections showed that Dync2h1^mmi cilia were filled with electron-dense particles between the axoneme and the ciliary membrane (Fig. 4 E), similar to the accumulation of particles in Chlamydomonas flagella that lack cytoplasmic dynein-2 (Pazour et al., 1999). In cross-sections, it was apparent that Dync2h1^mmi cilia lacked most axonemal microtubules, and most of the remaining microtubules were singlets rather than doublets. The particles in the Dync2h1^mmi cilia appeared to be highly organized and were regularly spaced at ~40-nm intervals, very different than the unstructured content of the Ift144^dmhd cilium.

As both IFT-A and cytoplasmic dynein-2 are important for retrograde ciliary trafficking, we analyzed the structure of cilia of Ift144^twt Dync2h1^mmi double mutants. The double-mutant cilia lacked the regular repeating electron-dense particles and were less swollen than Dync2h1 single mutants, and, in the best cases, a reasonably normal-appearing axoneme was restored (Fig. 4 F). Thus, the accumulation of trains of IFT particles in Dync2h1^mmi cilia was relieved by the Ift144^twt mutation. The partial rescue of cilia structure was paralleled by a partial rescue of Shh signaling, as Ift144^twt Dync2h1^mmi double mutants specified the most ventral neural cell type, the floor plate, which was never seen in Dync2h1^mmi single mutants (Fig. S3, E–J).

IFT-A is required for recruitment of specific cilia components, but not IFT88, to cilia

The TEM analysis showed that Ift144^dmhd cells have short, abnormal cilia. However, standard molecular markers for cilia were not detectable in the mutant cilia. Arl13b, a palmitoylated membrane-associated protein of the ARL family of small GTPases (Caspary et al., 2007; Cevik et al., 2010; Larkins et al., 2011), is localized to all cilia in the wild-type embryos and mouse embryo fibroblasts (MEFs). Arl13b was present in primary cilia of Ift144^twt and Ift122^sopb embryos and MEFs (Fig. 6, A and B). In contrast, Arl13b was not detectable in cilia in the neural tube of Ift144^dmhd single mutants or in Ift144^twt Ift122^sopb double mutants (Fig. 6 A) or in primary MEFs derived from Ift144^dmhd or Ift144^twt Ift122^sopb embryos (Fig. 6 B). Acetylated α-tubulin, another standard marker for cilia, was easily detected in MEF cilia in wild-type, Ift144^twt, and Ift122^sopb embryos but was not detected in most Ift144^dmhd mutant or Ift144^twt Ift122^sopb double-mutant MEF cilia (Fig. 6 C). A similar pattern of expression of acetylated α-tubulin was observed in cilia on mesenchymal cells near the notochord in wild-type and mutant embryos (Fig. S5). Another ciliary marker, the membrane protein adenylyl cyclase III (ACIII), was present in neural cilia in wild type but appeared to be present in far fewer cilia in the IFT-A mutants (Fig. 6 A). Because of the density of neural cilia, it was difficult to quantitate the effect on ACIII localization in the neural tube, but the decreased localization of ACIII to mutant cilia could be confirmed in MEFs. ACIII localized to wild-type MEF cilia but was not detected in Ift144^dmhd mutant or Ift144^twt Ift122^sopb double-mutant MEFs (Fig. 6 D). ACIII was detected in Ift144^twt and Ift122^sopb MEFs but at significantly lower levels than in wild-type cilia (Fig. 6 D). A stronger effect on ACIII localization was seen in mesenchymal cells in the embryo, where ACIII was present in wild-type mesenchymal cells, but ACIII was reduced in mesenchymal cells of the weak IFT-A mutants, and little or no ACIII was detected in strong IFT-A mutants (Fig. S5 and Table S2).

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Figure 6.

A subset of cilia markers is not detected in IFT-A mutant cilia. (A) Neural tube cilia. Arl13b is expressed in the cilia that project into the lumen of the E10.5 neural tube in wild-type (wt), Ift144^twt, and Ift122^sopb embryos. Arl13b cannot be detected in Ift144^dmhd or Ift144^twt Ift122^sopb double-mutant cilia. IFT88 localizes to cilia in all genotypes analyzed. ACIII is present in neural cilia in wild type but is present in fewer neural cilia in the IFT-A mutants. Bars: (top row) 200 µm; (bottom rows) 10 µm. (B–D) MEF cilia. The basal body is marked by γ-tubulin and the axoneme by IFT88. (B) Arl13b marks the ciliary axoneme in wild-type (35/35), Ift144^twt (27/32), and Ift122^sopb (12/13) mutant MEFs but is not detectable in Ift144^dmhd (0/9) and greatly reduced or Ift144^twt Ift122^sopb (1/12) mutant cilia. IFT88 accumulates in the cilia of Ift144^dmhd or Ift144^twt Ift122^sopb mutants. (C) Like Arl13b, acetylated α-tubulin (ac α-tub) is detected in the primary cilia of wild-type (12/12), Ift144^twt (17/17 cilia scored were positive for Arl13b), and Ift122^sopb (8/9) mutant MEFs but is detected in only a small fraction of Ift144^dmhd (1/13) or Ift144^twt Ift122^sopb (4/14) mutant cilia. (D) ACIII is strongly expressed in wild-type MEF cilia (12/13) but is not detectable in either Ift144^dmhd (0/8) or Ift144^twt Ift122^sopb cilia (0/12). Ift144^twt MEF cilia showed ACIII staining (13/13), but the intensity of staining was reduced to 62 ± 14% of wild type. Only 30% of Ift122^sopb (6/21) showed ACIII staining, and, in the ACIII+ cilia, the intensity was 22 ±10% of wild type. Bars, 2 µm.

However, it was possible to visualize Ift144^dmhd cilia by immunofluorescent staining, as the mutant cilia showed strong expression of the IFT-B protein IFT88. IFT88 was localized to neural cilia that point into the lumen of the neural tube in all genotypes analyzed: wild type, Ift144^dmhd, Ift144^twt, Ift122^sopb, and Ift144^twt Ift122^sopb (Fig. 6 A). Thus, we infer that IFT-A is not required for targeting or entry of IFT88 into cilia. In wild-type MEFs, IFT88 was enriched at the transition zone at the base of the cilium and in a punctate pattern along the ciliary axoneme (Figs. 2 B and 6 [B–D]). In Ift144^dmhd and Ift144^twt Ift122^sopb MEF cilia, IFT88 extended distal to the basal bodies (marked by γ-tubulin) and often appeared to be continuous from the transition zone into the short mutant axoneme, in contrast to its more punctate localization in wild type, suggesting that this IFT-B protein accumulates in cilia in the absence of IFT-A.

Overlapping functions of mouse IFT-A genes

Our genetic analysis of mouse mutants provides a different perspective on the function of individual IFT-A proteins than seen in biochemical experiments. Those studies suggested that mammalian IFT144/Wdr19 and IFT122 were core components of the mammalian IFT-A complex, whereas IFT139/Ttc21b and IFT121/Wdr35 were peripheral proteins that were not required for the formation of the core complex (Mukhopadhyay et al., 2010). In contrast, the genetic data indicate that loss of IFT144 or IFT121/Wdr35 (Mill et al., 2011) has a stronger effect on IFT-A function than does loss of IFT139a or IFT122.

Double mutants that are homozygous for both a null allele of Ift122 and the weak allele of Ift144 (Ift144^twt Ift122^sopb) have stronger defects in ciliogenesis than either single mutant and are indistinguishable from Ift144^dmhd mutants in both cilia structure and protein localization. Therefore, we conclude that IFT144 and IFT122 partially overlap in function, and, in the absence of IFT122, decreased activity of IFT144 is sufficient to block the activity of the IFT-A complex. This conclusion is consistent with data from Chlamydomonas that some IFT proteins can substitute for each other (Iomini et al., 2009) and reveals functional differences among the IFT-A proteins.

Loss of Shh pathway activity in strong IFT-A mutants is associated with the disruption in cilia structure

Strong disruption of IFT-A, as in Ift144^dmhd mutants, blocks the formation of a normal ciliary axoneme and the specification of ventral neural cell types that require high levels of Shh activity (Fig. 8 C). Because of the important roles of cilia in promoting Shh activity, a simple model is that the decreased activity of the Shh pathway in Ift144^dmhd is a result of the collapse of cilia structure in the absence of the IFT-A complex and that normal cilia structure is required for cilia-associated Shh pathway proteins to respond correctly to ligand.

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Figure 8.

Model for the relationship between the composition of the primary cilium and Shh-dependent patterning of the neural tube in IFT144 mutants. (A) In wild type (wt), IFT is required to maintain the axonemal structure and to mediate trafficking of mammalian Shh pathway proteins in the primary cilium. When Shh ligand is present (illustrated here), Smo moves into cilia, and a complex of proteins required for activation of the pathway, including Kif7, Gli2, and Sufu, is present at cilia tips. Primary cilia are required for the production of the Gli2 transcription activator and therefore to respond to a gradient of Shh activity, which specifies ventral cell types in the developing neural tube, including floor plate, V3 progenitors, and motor neurons. Arl13b and ACIII are present in cilia both in the presence or absence of Shh and can modulate the activity of the pathway. (B) In Ift144^twt mutants, cilia appear to have nearly normal morphology but are slightly wider than wild type. IFT-A complex proteins are reduced in cilia, whereas IFT-B particles tend to accumulate in the cilia tips. Smo trafficking into cilia in response to ligand is not affected, but Gli2 is enriched in the cilia regardless of pathway activation, as seen in other IFT-A complex mutants such as Ift122^sopb. The level of ACIII in the ciliary membrane is reduced, which may lead to decreased cAMP and decreased activity of PKA, a negative regulator of the pathway. Shh-dependent ventral cell types are dorsally expanded in the neural tube. (C) Ift144^dmhd mutant cilia have only rare microtubules and accumulate IFT-B particles, but no IFT-A particles are present. Smo, Arl13b, and ACIII fail to localize to the ciliary membrane, but Kif7, Sufu, and Gli2 accumulate at the tip of the short cilium. In the caudal neural tube, floor plate and V3 progenitors are absent, but motor neurons are formed, demonstrating the presence of an intermediate level of Shh pathway activity. Ift144^twt Ift122^sopb double mutants are indistinguishable from Ift144^dmhd mutants in both cilia structure and neural patterning. D, dorsal; V, ventral.

Despite the loss of high-level responses to Shh, the Ift144^dmhd and Ift144^twt Ift122^sopb embryos show ectopic activation of the pathway in the caudal neural tube, albeit at a lower level than in Ift144^twt. As the ectopic motor neurons seen in Ift144^dmhd embryos are also present in Ift144^dmhd Dync2h1^mmi double-mutant embryos (Fig. S3 F), where retrograde trafficking should be completely disrupted, the data argue that the activation of the Shh pathway seen in IFT-A mutants is not the result of defects in retrograde trafficking. Instead, the results suggest that the ligand-independent activation of the Shh pathway is the result of a defect in IFT-A–dependent anterograde trafficking.

Cilia localization of soluble Shh pathway proteins does not depend on IFT-A

Given the absence of most microtubules in the ciliary axoneme in the null allele Ift144^dmhd, it is remarkable that the mutants can specify the ventral neural cell types that require intermediate levels of Shh activity and that this intermediate level of pathway activity depends on the presence of the Ift144^dmhd cilium. We observed that Gli2, Kif7, and Sufu, which are critical for activity of the Shh pathway, are all present at the tips of the small Ift144^dmhd and Ift144^twt Ift122^sopb double-mutant cilia, which suggests that the IFT-B complex is sufficient for trafficking Gli, Kif7, and Sufu to cilia tips and therefore for the activation of midlevel Shh activity. A critical role for IFT-B in cilia localization of Gli proteins is consistent with the reduction of Gli2 at cilia tips caused by a modest reduction in the level of the IFT-B protein IFT172 (Friedland-Little et al., 2011).

IFT-A–dependent trafficking of membrane proteins into cilia may account for the neural patterning phenotypes of IFT-A mutants

Our analysis indicates that a variety of proteins and protein complexes, including IFT88, Gli2, Sufu, and Kif7, can translocate into cilia that lack a functional IFT-A complex. In contrast, several membrane proteins, including Arl13b, ACIII, and Smo, are not detectable in cilia of either of the strong IFT-A mutants (Ift144^dmhd single mutants or Ift144^twt Ift122^sopb double mutants). A role for the IFT-A–associated protein Tulp3 in trafficking of specific G protein–coupled receptors into cilia was previously defined (Mukhopadhyay et al., 2010), and our findings suggest that IFT-A has a general role in the trafficking of membrane proteins into cilia. Recent work has shown that a membrane-associated protein complex at the transition zone that includes the protein Tectonic1 is important for entry of some membrane proteins (ACIII and Arl13b) but had less effect on others (Smo) into cilia (Garcia-Gonzalo et al., 2011). This raises the possibility that the Tectonic1 and IFT-A complexes act in concert to promote the transport of membrane proteins into cilia.

The membrane proteins affected by loss of IFT-A (Arl13b, Smo, and ACIII) all have documented or plausible roles in Shh signaling and are likely to contribute to the Shh phenotypes of the IFT-A mutants. Mouse mutants that lack Arl13b show strong defects in Shh signaling and neural patterning (Caspary et al., 2007), so the absence of detectable ciliary Arl13b in the strong IFT-A mutants (Ift144^dmhd and Ift144^twt Ift122^sopb) could be responsible for some aspects of the neural patterning phenotypes of these embryos. Indeed, cell types that require high levels of Shh activity (the floor plate and V3 interneurons) fail to be specified, and the motor neuron domain is expanded in the caudal neural tube of Arl13b mutants, as in Ift144^dmhd and Ift144^twt Ift122^sopb embryos. However, there must be additional targets of IFT-A that contribute to the neural patterning defects in the mutant embryos because the Arl13b and IFT-A neural patterning phenotypes are not identical. For example, neural patterning is normal in the rostral neural tube of Arl13b mutant embryos, but ventral neural cell types are not specified in this region of the neural tube in the strong IFT-A mutants; this could be explained if an unidentified membrane protein with an Arl13b-like function depends on IFT-A for its cilia localization in the rostral neural tube. However, even in the caudal neural tube, where the Arl13b and IFT-A phenotypes are similar, double mutants reveal differences between the genotypes. For example, motor neurons are specified in Arl13b Gli2 double mutants but not in Ift144^dmhd Gli2 double-mutant embryos. Therefore, we suggest that the IFT-A neural patterning phenotype could be the result of the absence of several different membrane proteins in the cilium, including Arl13b.

The one core component of the Shh pathway that does not traffic into cilia of the strong IFT-A mutants (Ift144^dmhd and Ift144^twt Ift122^sopb) is Smo, a positive regulator of the pathway. However, the absence of Smo in the cilium cannot account for the neural patterning phenotypes in Ift144^dmhd embryos, as our double-mutant analysis shows that the Ift144^dmhd phenotype is independent of the presence or absence of Smo. Nevertheless, our data provide the first evidence that IFT proteins play a role for Smo movement into cilia and contrast with models that suggest Smo moves into cilia by lateral transport from the plasma membrane (Milenkovic et al., 2009). IFT-A could directly promote Smo movement from the transition zone into the cilium. Alternatively, the strong IFT-A mutations might disrupt axonemal structure in a way that other proteins cannot transport Smo past the transition zone.

The increased Shh pathway activity seen in the weak IFT-A mutants is a long-standing puzzle. The neural patterning phenotype of the hypomorphic mutant Ift144^twt is similar to those of null alleles of Ift122 and Ift139a (Fig. 8, A and B; Tran et al., 2008; Qin et al., 2011). All three mutations cause an expansion of Shh-dependent cell types in the neural tube, and the three mutants also cause polydactyly and craniofacial phenotypes (Tran et al., 2008; Qin et al., 2011; Ashe et al., 2012). Mutations in Tulp3, an IFT-A–associated protein, also cause ectopic activation of the Shh pathway in the neural tube, but the structure of Tulp3 cilia appears to be normal (Cameron et al., 2009; Norman et al., 2009; Patterson et al., 2009), and it has been suggested that the Tulp3 neural patterning phenotype reflects its role in the anterograde trafficking of a membrane-associated negative regulator of the pathway (Mukhopadhyay et al., 2010).

Our findings suggest a possible explanation for gain of Shh activity in the weak IFT-A mutants. Although ACIII is generally considered a cilia marker, ACIII and other adenylyl cyclases (Bishop et al., 2007; Choi et al., 2011) that are localized to cilia are likely to be important for Shh signaling. cAMP-dependent protein kinase is a strong negative regulator of Shh signaling (Tuson et al., 2011). It is reasonable to propose that the adenylyl cyclases in the cilium are the source of the cAMP that activates PKA (Barzi et al., 2010). In the absence of ciliary cAMP, we predict that PKA activity would be decreased, which would lead to ligand-independent activation of the Shh pathway. Therefore, we hypothesize that the gain of Shh activity seen in the weak IFT-A mutants may be the result of decreased levels of adenylyl cyclase in the cilia. The activation of intermediate, but not high, levels of Shh pathway activity in the strong IFT-A mutants (Fig. 8 C) could be a result of the combined loss of ACIII and Arl13b in the mutant cilia or a result of the absence of those proteins in combination with the disrupted cilia structure.

Cell culture and antibodies

MEFs were derived from E10.5 embryos using standard protocols, as previously described (Ocbina and Anderson, 2008). Confluent MEF cultures were serum starved for 48 h to induce ciliogenesis and treated with 100 nM SAG (EMD) for 24 h to activate the Shh pathway. Cultures were fixed for 10 min in 2% PFA followed by 5 min in 100% methanol. The Gli2 antibody (raised in guinea pig) was a gift from J.T. Eggenschwiler. The IFT88 and IFT140 antibodies (both raised in rabbits) were gifts from B. Yoder (University of Alabama, Birmingham, AL) and G. Pazour (University of Massachusetts Medical School, Worcester, MA), respectively. The Smo antibody (rabbit) was previously described (Ocbina et al., 2011). The rabbit Kif7 polyclonal antibody (Pocono Rabbit Farm and Laboratory, Inc.) was raised to a Kif7 peptide (amino acids 362–600) fused to a His tag expressed in bacteria. The following additional antibodies were used: rabbit anti-Arl13b (Caspary et al., 2007), mouse anti-Nkx2.2 (Developmental Studies Hybridoma Bank), rabbit anti-FoxA2 (Abcam), mouse anti-WDR19/IFT144 antibody (immunofluorescence; Abnova), rabbit anti-WDR19/IFT144 antibody (Western blot; GeneTex Inc.), mouse anti–γ-tubulin (Sigma-Aldrich), mouse antiacetylated α-tubulin (Sigma-Aldrich), rabbit anti-IFT88 (Proteintech Group, Inc.), and rabbit anti-ACIII (Santa Cruz Biotechnology, Inc.). Immunofluorescence staining was performed with Alexa Fluor (488, 594, and 633)–conjugated secondary antibodies (Invitrogen). Double antibody staining with rabbit antibodies was performed in series using Dylight 488-Fab fragment goat anti–rabbit H+L (Jackson ImmunoResearch Laboratories, Inc.).

Image acquisition

Immunofluorescent imaging of cilia was performed using an image restoration system (DeltaVision; Applied Precision) with a wide-field microscope (IX70; Olympus). Imaging was performed using a 100× 1.4 NA objective at room temperature mounted in VECTASHIELD (Vector Laboratories) and captured with a cooled charge-coupled device camera (CoolSNAP QE; Photometrics). Images were acquired using SoftWorks, and datasets were processed using the Volocity software package to produce an extended-view snapshot (PerkinElmer). To improve clarity, some images were adjusted using the curves tool in Photoshop (Adobe); in each case, the entire image was adjusted identically, and all panels in a figure were adjusted with identical settings. Quantification of proteins within cilia was performed using MetaMorph software (Molecular Devices) and measuring the sum of intensities in DeltaVision z stacks. Neural tube sections were analyzed with a microscope (Axioplan2; Carl Zeiss) using a 10× 0.5 NA lens at room temperature in VECTASHIELD. Images were acquired with a wide-field monochrome camera (AxioCam MR) using AxioVision software (v4.6; Carl Zeiss).

Identification of the dmhd mutation

The dmhd mutation was mapped to a segment of chromosome 5 based on the identification of regions homozygous for C57BL6/J polymorphisms in DNA from mutant embryos on a genome-wide single-nucleotide polymorphism panel (Moran et al., 2006). The mutation mapped between two single-nucleotide polymorphisms, CEL-5_34263494 and rs6409508 (Ensembl build M37). A splice site mutation was identified in the Wdr19 gene: a T to C substitution at the second nucleotide of intron 16–17 (Ensembl transcript ENSMUST00000041892). No wild-type transcript was detected by PCR from mutant embryo cDNA, indicating that this mutation completely blocks normal splicing. The Ift144^dmhd mutation disrupts a Kpn1 restriction site present in the wild-type sequence, creating a restriction fragment–length polymorphism that was used for genotyping. PCR amplification from genomic DNA (primers 5′-TGATGCGACCTATGAGATTCC-3′ and 5′-CCAGCCAAAATAACCTTGGA-3′) generated a 287-bp PCR product containing the restriction fragment–length polymorphism. Kpn1 digestion cleaves the wild-type, but not the dmhd mutant, amplicon.

EM

E9.5 or 10.5 embryos were fixed with 2% PFA and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. For scanning electron microscopy, embryos were dissected to expose the lumen of the neural tube in 0.1 M cacodylate buffer and dehydrated in ethanol (Huangfu et al., 2003). Scanning electron microscopy was performed using a field emission microscope (Supra 25; Carl Zeiss), and images were acquired with SmartSEM (Carl Zeiss). For TEM, samples were postfixed with 1% osmium tetroxide followed by 1% uranyl acetate, dehydrated through a graded series of ethanol, and embedded in LX112 resin (Ladd Research Industries). Ultrathin sections were cut on an ultramicrotome (Reichert Ultracut; Leica), stained with uranyl acetate followed by lead citrate, and viewed on a transmission electron microscope (1200 EX; JEOL Ltd.) at 80 kV.

We thank Leslie Gunther-Cummins (Albert Einstein College of Medicine, Bronx, NY) for assistance with TEM, Carlo Iomini (Mount Sinai Hospital, New York, NY) for helpful discussions, Jonathan Eggenschwiler for antibodies and the Ift122^sopb mice, and Brad Yoder and Greg Pazour for antibodies. We thank Sarah Goetz for helpful comments on the manuscript. We thank Nina Lampen for help with scanning electron microscopy and the Memorial Sloan-Kettering Cancer Center core facilities and Tao Tong of the Rockefeller University Bio-Imaging Resource Center for help with imaging.

This work was supported by National Institutes of Health grant no. NS044385 to K.V. Anderson, National Health and Medical Research Council of Australia project grant no. 631493 to C. Wicking, and National Institutes of Health grant no. HD43430 to D. Beier.