Cilia and Hedgehog responsiveness in the mouse

Abstract

The intraflagellar transport (IFT) proteins Ift172/Wimple and Polaris/Ift88 and the anterograde IFT motor kinesin-II are required for the production and maintenance of cilia. These proteins are also required for the activation of targets of the mouse Hedgehog (Hh) pathway by Gli transcription factors. The phenotypes of the IFT mutants, however, are not identical to mutants that lack Smoothened (Smo), an essential activator of the Hh pathway. We show here that mouse embryos that lack both Ift172 and Smo are identical to Ift172 single mutants, which indicates that Ift172 acts downstream of Smo. Ift172 mutants have a weaker neural patterning phenotype than Smo mutants, because Ift172, but not Smo, is required for proteolytic processing of Gli3 to its repressor form. Dnchc2 and Kif3a, essential subunits of the retrograde and anterograde IFT motors, are also required for both formation of Gli activator and proteolytic processing of Gli3. As a result, IFT mutants display a loss of Hh signaling phenotype in the neural tube, where Gli activators play the major role in pattern formation, and a gain of Hh signaling phenotype in the limb, where Gli3 repressor plays the major role. Because both anterograde and retrograde IFT are essential for positive and negative responses to Hh, and because cilia are present on Hh responsive cells, it is likely that cilia act as organelles that are required for all activity of the mouse Hh pathway.

Fig. 1.

Ift172 Smo double mutant analysis. (A) Phenotypes of wild-type (WT), Ift172, and Smo single mutants and Ift172 Smo double mutants at e10.5. Both the small body size and early lethality of Smo mutants were rescued in Ift172 Smo double mutants. Heart-looping reversal was observed in about one-half (five of nine) Ift172 Smo double mutants, similar to Ift172 single mutants (3). (B) Immunofluorescent staining of neural tube markers (Shh, Nkx2.2, Lhx3, Chx10, and Pax7) in e10.5 wild-type and Ift172 Smo double mutants.

Fig. 2.

Gli3 processing. (A) Both full length (FL) and repressor (R) forms of Gli3 were detected in wild-type embryos but absent in Gli3 mutants, confirming these bands correspond to Gli3 proteins. The repressor form was undetectable in Ptch1 mutants. (B) Gli3 processing was affected in both Smo and Ift172 mutants but in opposite directions: Smo mutants showed a relative increase in Gli3 repressor, whereas Ift172 mutants showed a relative decrease in Gli3 repressor. (C) Mutants lacking the anterograde and retrograde motors, Kif3a and Dnchc2, both showed decreased Gli3 repressor, as did Ift172 mutants. (D and E) Quantification of results in B and C.(D) The average repressor to full length Gli3 protein ratios (R/FL) in wild-type, Smo, and Ift172 mutants were 0.74 ± 0.10 (mean ± SD), 2.60 ± 0.07, and 0.089 ± 0.044, respectively. (E) In an independent experiment (C), the average R/FL ratios in wild-type, Kif3a, and Dnchc2^GT mutants were 1.11 ± 0.13, 0.34 ± 0.27, and 0.26 ± 0.05.

Fig. 3.

Dnchc2 phenotypes. (A) The motor domain of Dnchc2 protein has six AAA (ATPase associated with cellular activities) motifs (A1–A6) (35). The Dnchc2^lln mutation in A6 is likely to disrupt the motor activity of the protein. The Dnchc2^GT mutation produced a truncated protein lacking all six of the AAA motifs. (B) Exencephaly (arrows) and reversal of heart looping (arrowhead) in Dnchc2 mutants at e10.5. (C) Occasional Dnchc2^GT mutants that survived to e13.5 showed polydactyly in both hindlimbs (asterisk) and forelimbs (FL).

Fig. 4.

Dnchc2 is required for ciliogenesis. (A) Bilateral expression of nodal-lacZ in the lateral plate mesoderm of Dnchc2^lln mutants (8 of 10). (B) Lateral view of Dnchc2-lacZ staining in the node of head-fold-stage embryos (Dnchc2^GT/+) in whole-mount and section (rostral to the right). (C) Nodal cilia visualized by scanning electron microscopy (SEM). The average length of cilia in Dnchc2^lln mutants was ≈70% of those in wild types (n = 51 in wild types, n = 37 in Dnchc2^lln mutants). Cilia in Dnchc2^lln mutants showed an unusual bulging morphology (arrowheads). (Bar, 1 μm.)

Fig. 5.

Expression of neural tube markers (Shh, Nkx2.2, Lhx3, Chx10, and Pax7) in e10.5 wild-type and Dnchc2^lln mutant embryos.

Fig. 6.

Presence of cilia in the neural tube at e10.5. Punctate expression of γ-tubulin (A) and cilia-like expression of acetylated α-tubulin (B and C) in the neural tube using confocal microscopy. A and B are ventral neural tubes; arrowhead points to the notochord. (C) A high-magnification view of the boxed area in B, with the arrow pointing to cilia-like protrusions into the lumen of the neural tube. Acetylated α-tubulin is also expressed in the notochord and the surrounding mesenchymal cells.