RESUMO
Members of the Hedgehog (Hh) family of secreted proteins function as morphogens to pattern developing tissues and control cell proliferation. The seven-transmembrane domain (7TM) protein Smoothened (Smo) is essential for the activation of all levels of Hh signaling. However, the mechanisms by which Smo differentially activates low- or high-level Hh signaling are not known. Here we show that a newly identified mutation in the extracellular domain (ECD) of zebrafish Smo attenuates Smo signaling. The Smo agonist purmorphamine induces the stabilization, ciliary translocation, and high-level signaling of wild-type Smo. In contrast, purmorphamine induces the stabilization but not the ciliary translocation or high-level signaling of the Smo ECD mutant protein. Surprisingly, a truncated form of Smo that lacks the cysteine-rich domain of the ECD localizes to the cilium but is unable to activate high-level Hh signaling. We also present evidence that cilia may be required for Hh signaling in early zebrafish embryos. These data indicate that the ECD, previously thought to be dispensable for vertebrate Smo function, both regulates Smo ciliary localization and is essential for high-level Hh signaling.
Assuntos
Cílios , Proteínas Hedgehog/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Cílios/fisiologia , Cílios/ultraestrutura , Proteínas Hedgehog/genética , Dados de Sequência Molecular , Morfolinas/metabolismo , Estrutura Terciária de Proteína , Purinas/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Characterization of previously described intraflagellar transport (IFT) mouse mutants has led to the proposition that normal primary cilia are required for mammalian cells to respond to the sonic hedgehog (SHH) signal. Here we describe an N-ethyl-N-nitrosourea-induced mutant mouse, alien (aln), which has abnormal primary cilia and shows overactivation of the SHH pathway. The aln locus encodes a novel protein, THM1 (tetratricopeptide repeat-containing hedgehog modulator-1), which localizes to cilia. aln-mutant cilia have bulb-like structures at their tips in which IFT proteins (such as IFT88) are sequestered, characteristic of Chlamydomonas reinhardtii and Caenorhabditis elegans retrograde IFT mutants. RNA-interference knockdown of Ttc21b (which we call Thm1 and which encodes THM1) in mouse inner medullary collecting duct cells expressing an IFT88-enhanced yellow fluorescent protein fusion recapitulated the aln-mutant cilial phenotype, and live imaging of these cells revealed impaired retrograde IFT. In contrast to previously described IFT mutants, Smoothened and full-length glioblastoma (GLI) proteins localize to aln-mutant cilia. We hypothesize that the aln retrograde IFT defect causes sequestration of IFT proteins in aln-mutant cilia and leads to the overactivated SHH signaling phenotype. Specifically, the aln mutation uncouples the roles of anterograde and retrograde transport in SHH signaling, suggesting that anterograde IFT is required for GLI activation and that retrograde IFT modulates this event.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Alquilantes/toxicidade , Sequência de Aminoácidos , Animais , Transporte Biológico , Western Blotting , Células Cultivadas , Clonagem Molecular , Etilnitrosoureia/toxicidade , Feminino , Fibroblastos/metabolismo , Genes Recessivos , Hibridização In Situ , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Homologia de Sequência de Aminoácidos , Medula Espinal/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
The zone of polarizing activity (ZPA) in the posterior limb bud produces Sonic Hedgehog (Shh) protein, which plays a critical role in establishing distinct fates along the anterior-posterior axis. This activity has been modeled as a concentration-dependent response to a diffusible morphogen. Using recombinase base mapping in the mouse, we determine the ultimate fate of the Shh-producing cells. Strikingly, the descendants of the Shh-producing cells encompass all cells in the two most posterior digits and also contribute to the middle digit. Our analysis suggests that, while specification of the anterior digits depends upon differential concentrations of Shh, the length of time of exposure to Shh is critical in the specification of the differences between the most posterior digits. Genetic studies of the effects of limiting accessibility of Shh within the limb support this model, in which the effect of the Shh morphogen is dictated by a temporal as well as a spatial gradient.
Assuntos
Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Transativadores/fisiologia , Alelos , Animais , Western Blotting , Padronização Corporal , Linhagem da Célula , Proteínas de Ligação a DNA/fisiologia , Difusão , Genótipo , Proteínas Hedgehog , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Proteínas do Tecido Nervoso/fisiologia , Fenótipo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/metabolismo , Transdução de Sinais , Temperatura , Fatores de Tempo , Transativadores/metabolismo , Fatores de Transcrição/fisiologia , Proteína Gli3 com Dedos de Zinco , beta-Galactosidase/metabolismoRESUMO
Vertebrate limb outgrowth is driven by a positive feedback loop involving Sonic Hedgehog (Shh), Gremlin, and Fgf4. By overexpressing individual components of the loop at a time after these genes are normally down-regulated in chicken embryos, we found that Shh no longer maintains Gremlin in the posterior limb. Shh-expressing cells and their descendants cannot express Gremlin. The proliferation of these descendants forms a barrier separating the Shh signal from Gremlin-expressing cells, which breaks down the Shh-Fgf4 loop and thereby affects limb size and provides a mechanism explaining regulative properties of the limb bud.