A mutation in the mouse ttc26 gene leads to impaired hedgehog signaling - PubMed (original) (raw)

A mutation in the mouse ttc26 gene leads to impaired hedgehog signaling

Ruth E Swiderski et al. PLoS Genet. 2014.

Abstract

The phenotype of the spontaneous mutant mouse hop-sterile (hop) is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the hop mutation remained unexplored. Here we show that the hop mutation is located in the Ttc26 gene and impairs Hedgehog (Hh) signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT) complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, hop did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The Ttc26 gene of the hop mouse contains a nonsense mutation.

(A) Schematic representation of genomic positions of genes that both fall within the 16-mega base pair (Mbp) interval to which the hop mutation was mapped and had a known association with the ciliome. (B) Comparison of the 15th exon of the Ttc26 gene in wild-type and hop/hop mice. Horizontal lines represent introns, and black and white rectangles represent the coding and non-coding regions of exons, respectively. A deoxycytidine nucleotide (C) of wild-type Ttc26 (upper chromatogram) is replaced with a deoxyadenosine (A) in the hop mouse, as indicated by an arrow in the lower chromatogram. The point mutation changes the tyrosine (Tyr) at position 430 of Ttc26 to a stop codon (Stop), as shown in the amino-acid sequence lines. (C) Schematic representation of the Ttc26 protein. Blue boxes indicate the predicted TPR motifs. The bracket indicates the C-terminal 125-amino acid region of the protein that is predicted to be missing in hop/hop cells. The asterisk indicates the position of the epitope that is recognized by the anti-Ttc26 antibody. (D) Immunoblot analysis of Ttc26 expression in transfected HEK293 cells, airway epithelial cells (AEC) and testis of wild type (wt) and hop/hop (hop) mice. HEK293 cells were transfected with the indicated Ttc26-encoding construct or an empty expression vector. Arrowheads indicate the positions of the 64, 49, and 37 kDa standards. The antibodies used for immunoblotting are indicated next to the upper and lower panels.

Figure 2

Figure 2. The hop mouse exhibits patterning defects and hearing impairment.

(A) Representative images of hop/+ and hop/hop mice at postnatal day 4. (B) Comparison of Alcian Blue-stained fore and hind limbs of hop/+ and hop/hop mice. Extra digits are indicated by arrows. (C) Statistical analysis of FoxA2-positive cells in the lumbar neural tubes of hop/+ and hop/hop mice (E10.5). Each symbol represents the average number of FoxA2+ cells per focal plane in a single embryo (for each embryo, 12 focal planes in 4 sections were analyzed, Mann-Whitney test: *P = 0.01). (D) Immunostaining of the lumbar neural tube of hop/+ and hop/hop mice (E10.5) with antibodies against the V3 progenitor marker Nkx2.2 (white contrast), the floor plate marker FoxA2 (red), and the motor neuron protein HB9 (white contrast). The hop/hop genotype is associated with reduced FoxA2 expression and ventralization of the Nkx2.2- and HB9-expressing cells. Scale bar: 50 µm. (E) Representative ABR waveforms for 3–4 week-old hop/+ and hop/hop mice. Broadband click stimuli were applied at the indicated sound pressure levels (in dB). (F) Statistical analysis of ABR thresholds measured in 3–4 week-old hop/+ and hop/hop mice. Broadband click stimuli between 30 and 90 dB sound pressure level (SPL) were used. Each symbol represents the value for a single mouse (Mann-Whitney test: ***P<0.0001).

Figure 3

Figure 3. The hop mutation does not impair ciliogenesis or ciliary localization of the Ttc26-interacting protein Ift46.

(A) Color test of protein-protein interactions in yeast transformed with the indicated combination of Ift46 and Ttc26wt or Ttc26hop. Blue staining of the yeast colony (upper patch) is indicative of a protein-protein interaction; a lack thereof indicates the absence of a protein-protein interaction (lower patch). (B) Immunoprecipitation analysis of the Ttc26wt-Ift46 interaction. HEK293 cells were transfected with the indicated combinations of HA-tagged Ift46 and flag (FL)-tagged Ttc26wt or Ttc26hop. The two upper panels show immunoblot analysis of tagged proteins pulled down with an anti-flag antibody, and the two lower panels show immunoblot analysis of input controls. (C) Percentages of ciliated hop/+ and hop/hop MEFs following 2 days of serum starvation. MEFs were isolated from 4 embryos per genotype, and 150 MEFs per embryo were analyzed. (D) Statistical analysis of cilium length in the primary cultures of serum-starved hop/+ and hop/hop MEFs (mean ± SEM, n = 200 cilia per genotype, unpaired _t_-test with Welch correction, ***P<0.0001). (E,F) Immunofluorescence analysis of (E) Ttc26 and (F) Ift46 expression in the cilia of hop/+ and hop/hop MEFs. The axoneme was visualized by immunolabeling of acetylated-α-tubulin (AαT).

Figure 4

Figure 4. The Ttc26 mutation in the hop mouse impairs Shh signaling.

(A) Induction of an adenovirus-delivered Glix8-luciferase reporter gene in wild-type (+/+) and hop/hop MEFs following 2-day incubation with DMSO (0.02%), Shh-conditioned medium, or SAG (400 nM) as indicated (mean ± SEM, n = 3–7). The increase in luciferase expression is shown relative to that in the DMSO control. Constitutive GFP expression from the Glix8-luciferase-encoding viral vector was used for normalization. (B) SAG-dependent induction of the Glix8-luciferase gene in hop/hop MEFs following adenoviral delivery of Ttc26hop or Ttc26wt (mean ± SEM, n = 3–7). The control group of MEFs was not transduced with a Ttc26-encoding virus (no Ttc26). The luciferase signal was normalized to GFP expression as described in panel A. (C) Immunoblot analyses of expression of Gli3-F and Gli3-R in wild-type and hop/hop MEFs, following SAG treatment (400 nM) for the indicated times. ß-actin serves as a loading control (lower panel). (D) Statistical analysis of Gli3-F band intensities in the immunoblot experiments described in panel C (mean ± SEM, n = 3; two-way ANOVA, P<0.006 for the genotype variable; post-hoc Bonferroni test, **P<0.01). (E) Statistical analysis of the ratio of the Gli3-F and Gli3-R bands at the 0 time point in the experiments described in panel C (mean ± SEM, n = 3; unpaired _t_-test, *P = 0.014).

Figure 5

Figure 5. The hop mutation inhibits Gli-Sufu dissociation without altering Gli accumulation at the ciliary tip.

(A) Immunofluorescence analysis of Smo (red) localization to primary cilium (AαT-labeled, green) in wild-type (+/+) and hop/hop MEFs following 4-h treatment with DMSO (0.02%) or SAG (400 nM) as indicated. (B) Immunofluorescence analysis of Gli3 (red) localization to primary cilium tip in wild-type (+/+) and hop/hop MEFs following 2-h treatment with DMSO (0.02%) or SAG (400 nM). (C) Quantitative analysis of Gli3 immunofluorescence signal intensities at the primary cilium tip in +/+ and hop/hop MEFs following SAG treatment (400 nM) for the indicated times (mean ± SEM, n = 40–50). AU: arbitrary units. (D) Immunofluorescence analysis of Gli2 (red) localization to primary cilium tip in +/+ and hop/hop MEFs following 2-day treatment with DMSO (0.02%) or SAG (400 nM). (E) Quantitative analysis of Gli2 immunofluorescence signal intensities at the primary cilium tip in +/+ and hop/hop MEFs following 2-day treatment with the indicated concentrations of SAG (mean ± SEM, n = 45–55). AU: arbitrary units. (F) Gli-Sufu dissociation in +/+ and hop/hop MEFs following 5-h incubation with SAG (400 nM) and the proteasome inhibitor bortezomib (2 µM) or with DMSO (0.02% negative control). Left panels show the relative amounts of Gli3-F, Gli2-F, and Sufu pulled down from the cell lysates using an anti-Sufu antibody. Right panels show the relative amounts of Gli3-F, Gli3-R, Gli2-F, Sufu and ß-actin (loading control) in the total cell lysates. (G) Ratios of band intensities of Gli-F and Sufu were calculated based on the immunoprecipitation results in the left column of panel F, and the Gli-F/Sufu ratios in the immunoprecipitated fractions of SAG-treated +/+ (wt) and hop/hop (hop) cells are shown relative to the Gli-F/Sufu ratios in the immunoprecipitated fractions of non-treated cells.

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