ABSTRACT
Primary cilia are required for proper Sonic Hedgehog (Shh) signaling in mammals. However, their role in the signal transduction process remains unclear. We have identified sister of open brain (sopb), a null allele of mouse Intraflagellar transport protein 122 (Ift122). IFT122 negatively regulates the Shh pathway in the cilium at a step downstream of the Shh ligand and the transmembrane protein Smoothened, but upstream of the Gli2 transcription factor. Ift122(sopb) mutants generate primary cilia, but they show features of defective retrograde intraflagellar transport. IFT122 controls the ciliary localization of Shh pathway regulators in different ways. Disruption of IFT122 leads to accumulation of Gli2 and Gli3 at cilia tips while blocking the ciliary localization of the antagonist TULP3. Suppressor of Fused and Smoothened localize to the cilium through an IFT122-independent mechanism. We propose that the balance between positive and negative regulators of the Shh pathway at the cilium tip controls the output of the pathway and that Shh signaling regulates this balance through intraflagellar transport.
Subject(s)
Cilia/metabolism , Hedgehog Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing , Animals , Blotting, Western , Cells, Cultured , Cilia/genetics , Cilia/ultrastructure , Cytoskeletal Proteins , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hedgehog Proteins/genetics , Immunohistochemistry , In Situ Hybridization , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins/genetics , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Mutant Strains , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Proteins/genetics , Proteins/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Smoothened Receptor , Zinc Finger Protein Gli2 , Zinc Finger Protein Gli3ABSTRACT
Recent findings indicate that mammalian Sonic hedgehog (Shh) signal transduction occurs within primary cilia, although the cell biological mechanisms underlying both Shh signaling and ciliogenesis have not been fully elucidated. We show that an uncharacterized TBC domain-containing protein, Broad-minded (Bromi), is required for high-level Shh responses in the mouse neural tube. We find that Bromi controls ciliary morphology and proper Gli2 localization within the cilium. By use of a zebrafish model, we further show that Bromi is required for proper association between the ciliary membrane and axoneme. Bromi physically interacts with cell cycle-related kinase (CCRK), whose Chlamydomonas homolog regulates flagellar length. Biochemical and genetic interaction data indicate that Bromi promotes CCRK stability and function. We propose that Bromi and CCRK control the structure of the primary cilium by coordinating assembly of the axoneme and ciliary membrane, allowing Gli proteins to be properly activated in response to Shh signaling.
Subject(s)
Carrier Proteins/physiology , Cilia/physiology , Cyclin-Dependent Kinases/physiology , Hedgehog Proteins/physiology , Adaptor Proteins, Signal Transducing , Animals , Base Sequence , Body Patterning , Carrier Proteins/genetics , Cilia/ultrastructure , Cloning, Molecular , DNA/genetics , Enzyme Stability , Epistasis, Genetic , Female , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Mutation , Neural Tube/embryology , Pregnancy , Signal Transduction , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/physiology , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Tubby-like protein 3 (TULP3) is required for proper embryonic development in mice. Disruption of mouse Tulp3 results in morphological defects in the embryonic craniofacial regions, the spinal neural tube and the limbs. Here, we show that TULP3 functions as a novel negative regulator of Sonic hedgehog (Shh) signaling in the mouse. In Tulp3 mutants, ventral cell types in the lumbar neural tube, which acquire their identities in response to Shh signaling, are ectopically specified at the expense of dorsal cell types. Genetic epistasis experiments show that this ventralized phenotype occurs independently of Shh and the transmembrane protein Smoothened, but it is dependent on the transcription factor Gli2. The ventralized phenotype is also dependent on the kinesin II subunit Kif3A, which is required for intraflagellar transport and ciliogenesis. In addition, TULP3 is required for proper Shh-dependent limb patterning and for maintaining the correct balance between differentiation and proliferation in the neural tube. Finally, the localization of TULP3 to the tips of primary cilia raises the possibility that it regulates the Hedgehog pathway within this structure.
