The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking

Abstract

Two intraflagellar transport (IFT) complexes, IFT-A and IFT-B, build and maintain primary cilia and are required for activity of the Sonic hedgehog (Shh) pathway. A weak allele of the IFT-A gene, Ift144, caused subtle defects in cilia structure and ectopic activation of the Shh pathway. In contrast, strong loss of IFT-A, caused by either absence of Ift144 or mutations in two IFT-A genes, blocked normal ciliogenesis and decreased Shh signaling. In strong IFT-A mutants, the Shh pathway proteins Gli2, Sufu, and Kif7 localized correctly to cilia tips, suggesting that these pathway components were trafficked by IFT-B. In contrast, the membrane proteins Arl13b, ACIII, and Smo failed to localize to primary cilia in the absence of IFT-A. We propose that the increased Shh activity seen in partial loss-of-function IFT-A mutants may be a result of decreased ciliary ACIII and that the loss of Shh activity in the absence of IFT-A is a result of severe disruptions of cilia structure and membrane protein trafficking.

Figure 1.

Mutations in Ift144 alter Shh-dependent neural patterning. (A) The motor neuron marker HB9-GFP (green) is not expressed in the rostral neural tube of Ift144^dmhd embryos (arrow) but is strongly expressed in the caudal spinal cord. WT, wild type. (B) Ift144^twt mutants show increased HB9-GFP expression compared with wild-type littermates. (C) The two Ift144 alleles cause contrasting changes in dorsal–ventral neural patterning, shown in transverse sections at three rostral–caudal levels of the E10.5 neural tube. (D) Expression of the Shh target gene Ptch1-lacZ in E10.5 Ift144 mutant and littermate control embryos at lumbar levels. Ift144^dmhd mutants lack strong expression Ptch1-lacZ at the ventral midline at all levels. Ptch1-lacZ is ectopically expressed in the mesoderm adjacent to the neural tube in Ift144^dmhd mutants (arrows). Ift144^twt mutants show dorsally expanded Ptch1-LacZ expression. Bars: (A and B) 1 mm; (C and D) 200 µm.

Figure 2.

Two mutations in mouse Ift144 affect cilia structure. (A) Schematic drawing of how the mutations affect protein structure. Two isoforms of the 1,341–amino acid WDR19/IFT144 protein have been described; conserved protein–protein interaction motifs are shown in colored boxes. Depending on the isoform, the dmhd splice site mutation would truncate the protein at amino acid 592 (ENSMUSP00000038098) or amino acid 543 (ENSMUSP00000109414). The twt mutation causes a leucine-to-proline substitution in the first tetratrico peptide repeat (TPR; Ashe et al., 2012). Asterisks indicate the position of the sequence changes in the mutant proteins. CHCR, clathrin heavy chain repeat; wt, wild type. (B) Altered cilia protein expression and structure in the mutants. Cilia in twt MEFs are of approximately normal length and width but accumulate higher than normal levels of IFT88 (an IFT-B protein) at cilia tips and lack detectable IFT140 (an IFT-A protein; 0/9 twt cilia were positive for IFT140 compared with 13/14 wild-type cilia). Cilia in dmhd MEFs are short and wide; they accumulate IFT88 but lack any detectable IFT144 protein, consistent with the hypothesis that dmhd is a null allele (0/18 were positive for IFT144). ac, acetylated. Bar, 2 µm.

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.

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.

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.

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.

Figure 7.

Localization of Shh pathway proteins in IFT-A mutant MEF cilia. (A and B) γ-Tubulin marks the basal body; IFT88 marks the ciliary axoneme. (A) Smo is present in the cilia of wild-type (wt), Ift144^twt, and Ift122^sopb MEFs in cells where the Shh pathway is activated by SAG (>90% of cilia are Smo+; n > 10). Smo movement into the cilium in response to activation of the Shh pathway by SAG is abolished in Ift144^dmhd (0/9 cilia were Smo+) and greatly reduced Ift144^twt Ift122^sopb (1/17). (B) Gli2 is two- to threefold enriched at wild-type MEF cilia tips in response to SAG (n > 14 for each condition). The level of Gli2 is slightly elevated at the tips of the Ift144^twt and Ift122^sopb mutant cilia in the absence of SAG and is further enriched in the presence SAG (n > 16 for each condition). The level of Gli2 is elevated at the tips of the Ift144^dmhd or Ift144^twt Ift122^sopb mutant cilia in the absence of SAG and does not increase further in the presence SAG (n > 9 for each condition). Bar, 2 µm.

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.