(a), Mutations in Dync2h1 lead to the absence of Shh-dependent cell types in the E10.5 neural tube. In Dync2h1^lln/lln mutants, floor plate (FoxA2, green) and V3 progenitor (Nkx2.2, red) domains are not specified, and motor neurons (HB9, green) are present only in the caudal neural tube (shown here); dorsal up. Scale bars represent 100 μm. (b), Scanning electron micrographs show that neural tube primary cilia in Dync2h1^lln/lln mutants are bloated; dimensions are given in Supplementary Table1. Scale bars represent 500 nm. (c), IFT88 (green) in cilia of serum-starved wild-type MEFs is enriched at the base and the tip of the cilium, marked with acetylated α-tubulin (red). In Dync2h1^lln/lln mutant MEFs, the amount of IFT88 in the cilium is increased and is found all along the axoneme. Quantitation is in Supplementary Table 2. Scale bars represent 1 μm (c).

Localization of Smo (a, b), Gli2 (c, d) and Ptch1 (e, f) to the primary cilium in wild-type and Dync2h1^lln/lln MEFs (a, c, e) and E10.5 neural tube (b, d, f). (a) Smo (green) was enriched in cilia of wild-type MEFs only after exposure to Shh. Smo was enriched in cilia of Dync2h1^lln/lln mutant cells even in the absence of Shh. (b) Smo was enriched in cilia of ventral neural progenitors in wild-type. Smo was strongly enriched in primary cilia of Dync2h1^lln/lln neural progenitors at all dorsal-ventral levels. (c) Gli2 (green) localized to the tips of cilia in wild-type MEFs and accumulated further after Shh treatment. Gli2 levels were elevated along the axoneme of Dync2h1^lln/lln mutant MEF cilia. (d) Gli2 was elevated in the cilia of Dync2h1^lln/lln neural progenitors. (e) Low amounts of endogenous Ptch1 (green) were detected near the base and along the length of primary cilia in wild-type MEFs only in the absence of Shh, whereas Ptch1 was strongly enriched along the axoneme of Dync2h1^lln/lln cilia in unstimulated cells; strong Ptch1 immunofluorescence remained near the base of the cilium after stimulation with Shh. (f) Ptch1 appeared localized to the cytoplasm of wild-type neural progenitors, and was strongly enriched in cilia throughout the neural tube in Dync2h1^lln/lln mutants. Acetylated α-tubulin (red) marks cilia in (a, c, e); (b, d, f) are ventral views of transverse sections through the ventral half of the neural tube at the level of the forelimb. Scale bars represent 500 nm (a, c, e), 25 μm (b, d, f) and 10 μm (insets b, d, f).

(a), E10.5 embryos and transverse sections through the caudal neural tube of wild-type, Dync2h1^lln/lln and Dync2h1^lln/lln Ift172^avc1/+ embryos. Specification of floor plate (FoxA2, green), V3 progenitors (Nkx2.2, red) and motor neurons (HB9, green) were rescued in Dync2h1^lln/lln Ift172^avc1/+ embryos. Scale bars represent 100 μm. (b), Dync2h1^lln/lln Ift172^avc1/+ mutants survive to at least E16.5 (n=5). Scale bar is 5 mm. (c), Right forelimbs and digits of embryos in (b) stained with Alcian blue (cartilage) and Alizarin red (bone) staining shows incomplete penetrance of polydactyly in Dync2h1^lln/lln Ift172^avc1/+ embryos at E16.5. (d), Scanning electron micrographs of cilia from the neural tube at E10.5 showing near-normal morphology of Dync2h1^lln/lln Ift172^avc1/+ mutant cilia (quantitation in Supplementary Table 1). Scale bar is 500 nm. IFT88 (green, e) and Smo (green, f) and Gli2 (red, g) localize normally in primary cilia of MEFs derived from Dync2h1^lln/lln Ift172^avc1/+ embryos. Acetylated α-tubulin (red) marks cilia in (e-g). Scale bars represent 1 μm in (e-g).

(a), In contrast to the lack of ventral neural cell types in Dync2h1^lln/lln mutants, both Ift122^sopb/sopb single and Dync2h1^lln/lln Ift122^sopb/sopb double mutants specify floor plate (FoxA2, green), V3 progenitors (Nkx2.2, red) and motor neurons (HB9, green) in the lumbar neural tube. Scale bars represent 100 μm. (b), Scanning electron micrographs of neural tube cilia from the neural tube of E10.5 Ift122^sopb/sopb and Dync2h1^lln/lln Ift122^sopb/sopb embryos. The distal ends of Ift122^sopb/sopb mutant cilia appeared swollen. Dync2h1^lln/lln Ift122^sopb/sopb mutant cilia were similar in diameter to Ift122^sopb/sopb but were shorter than either Dync2h1^lln/lln or Ift122^sopb/sopb single mutants (See Supplementary Table 1). Scale bars represent 500 nm. (c), IFT88 (green) accumulates specifically at the distal tips of both Ift122^sopb/sopb and Dync2h1^lln/lln Ift122^sopb/sopb mutant MEF cilia. Acetylated α-tubulin staining (red) marks primary cilia. Localization of Smo (d, green) and Gli2 (e, green) in the cilia of Ift122^sopb/sopb and Dync2h1^lln/lln Ift122^sopb/sopb mutant MEFs. Acetylated α-tubulin (red) marks cilia. (f), Dync2h1 protein is present at the base of the cilium and along the ciliary axoneme in wild-type cells. In Ift122^sopb/sopb mutant cilia, Dync2h1 localization accumulates mainly at the base of the cilium. Orientation for (b-f) is distal tip up. Scale bars are 1 μm (d-f).

Whole embryos and patterning in the lumbar neural tube of E10.5 wild type, Dync2h1^lln/lln, Shh^-/-, Dync2h1^lln/lln Ift122^sopb/+ and Dync2h1^lln/lln Ift122^sopb/+ Shh^-/- compound mutants. Specification of floor plate (FoxA2, red, middle panels), V3 progenitors (Nkx2.2, red, bottom panels) and motor neurons (HB9, green, middle panels) were partially rescued in Dync2h1^lln/lln Ift122^sopb/+ mutant embryos but all these cell types were absent in Dync2h1^lln/lln Ift122^sopb/+ Shh^-/- embryos. The Pax6 domain (green, bottom panels), which is restricted by low levels of Shh signaling, was ventrally expanded in Shh^-/- and Dync2h1^lln/lln Ift122^sopb/+ Shh^-/- embryos. Scale bars represent 100 μm.

(a), SEM analysis of neural tube primary cilia show the more normal length and width of Dync2h1^lln/lln Ift122^sopb/+ mutants compared to Dync2h1^lln/lln. Quantitation in Supplementary Table 1. Scale bars are 500 nm. (b-d), Localization of IFT88 (b, green), Smo (c, green) and Gli2 (d, green) in cilia (acetylated α-tubulin, red) appear normal in Dync2h1^lln/lln Ift122^sopb/+ mutants. Scale bars are 500 nm (b-d). (e) Model of the trafficking of mammalian IFT and Hh pathway proteins in the primary cilium, shown in the absence of Hh ligand. In wild-type cells, IFT directs the formation of cilia, which accumulate a basal level of Gli2 at cilia tips, while Smo traffics through the cilium at a low basal rate. Loss of retrograde motor in Dync2h1^lln/lln mutant cilia leads to the accumulation of IFT particles and blocks the movement of both Smo and Gli2 out of the cilium. In Ift122^sopb/sopb mutants, Dync2h1 protein fails to enter the cilium, leading to the accumulation of IFT-B particles. Loss of IFT122 also results in the accumulation of Gli2 but does not affect Smo trafficking. Decreased anterograde ciliary trafficking in Dync2h1^lln/lln Ift122^sopb/+ suppresses the Dync2h1^lln/lln phenotype and permits normal transport of both Smo and Gli2 through the cilium.