RESUMO
Sonic hedgehog (Shh) signaling is critical in development and oncogenesis, but the mechanisms regulating this pathway remain unclear. Although protein phosphorylation clearly affects Shh signaling, little is known about phosphatases governing the pathway. Here, we conducted a small hairpin RNA (shRNA) screen of the phosphatome and identified Eya1 as a positive regulator of Shh signaling. We find that the catalytically active phosphatase Eya1 cooperates with the DNA-binding protein Six1 to promote gene induction in response to Shh and that Eya1/Six1 together regulate Gli transcriptional activators. We show that Eya1, which is mutated in a human deafness disorder, branchio-oto-renal syndrome, is critical for Shh-dependent hindbrain growth and development. Moreover, Eya1 drives the growth of medulloblastoma, a Shh-dependent hindbrain tumor. Together, these results identify Eya1 and Six1 as key components of the Shh transcriptional network in normal development and in oncogenesis.
Assuntos
Carcinogênese/patologia , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Meduloblastoma/patologia , Proteínas Nucleares/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Receptores de Superfície Celular/genética , Rombencéfalo/citologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinogênese/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Knockout , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Patched , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rombencéfalo/metabolismo , Transdução de Sinais , Proteína GLI1 em Dedos de ZincoRESUMO
The olfactory system relies on precise circuitry connecting olfactory sensory neurons (OSNs) and appropriate relay and processing neurons of the olfactory bulb (OB). In mammals, the exact correspondence between specific olfactory receptor types and individual glomeruli enables a spatially precise map of glomerular activation that corresponds to distinct odors. However, the mechanisms that govern the establishment and maintenance of the glomerular circuitry are largely unknown. Here we show that high levels of Sonic Hedgehog (Shh) signaling at multiple sites enable refinement and maintenance of olfactory glomerular circuitry. Mice expressing a mutant version of Shh (Shh(Ala/Ala)), with impaired binding to proteoglycan co-receptors, exhibit disproportionately small olfactory bulbs containing fewer glomeruli. Notably, in mutant animals the correspondence between individual glomeruli and specific olfactory receptors is lost, as olfactory sensory neurons expressing different olfactory receptors converge on the same glomeruli. These deficits arise at late stages in post-natal development and continue into adulthood, indicating impaired pruning of erroneous connections within the olfactory bulb. In addition, mature Shh(Ala/Ala) mice exhibit decreased proliferation in the subventricular zone (SVZ), with particular reduction in neurogenesis of calbindin-expressing periglomerular cells. Thus, Shh interactions with proteoglycan co-receptors function at multiple locations to regulate neurogenesis and precise olfactory connectivity, thereby promoting functional neuronal circuitry.
