Phosphatome RNAi Screen Identifies Eya1 as a Positive Regulator of Hedgehog Signal Transduction

The Hedgehog (Hh) signaling pathway is vital for vertebrate embryogenesis and aberrant activation of the pathway can cause tumorigenesis in humans. In this study, we used a phosphatome RNAi screen for regulators of Hh signaling to identify a member of the Eyes Absent protein family, Eya1, as a positive regulator of Hh signal transduction. Eya1 is both a phosphatase and transcriptional regulator. Eya family members have been implicated in tumor biology, and Eya1 is highly expressed in a particular subtype of medulloblastoma (MB). Here we show that RNAi-mediated knock-down of Eya1, as well as knock-down of its co-factor, Six1, blocks Hh signaling as assessed by induction of Hh response genes. Utilizing small molecule agonists, RNAi, and protein over-expression methods, we place the influence of Eya1 and Six1 within the Hh signaling pathway downstream of Smoothened (Smo) and at or above the level of Suppressor of Fused (Sufu). Interestingly, Eya1 appears to be specifically required for Hh-responsive gene activation mediated by Gli transcriptional activators but not for Hh-mediated transcriptional de-repression mediated by the inhibition of Gli transcriptional repressors. Furthermore, we find that Eya1 and Six1 regulate the expression of Neuropilin1 (Nrp1) and Neuropilin2 (Nrp2), known positive regulators of Hh signaling, providing a mechanism by which Eya1 and Six1 exert their effects. Based on these data, we investigated a role of Eya1 in Hh signaling in vivo. We obtained Eya1-/- mice and focused our attention on the developing cerebellum, where Sonic Hedgehog (Shh) is a major factor promoting neural precursor proliferation. In the Eya1-/- cerebellum, we find a striking reduction in neural precursor proliferation. In addition, we surveyed several other locations where Shh and/or Eya1 are known to be important for development. These include the embryonic otic vesicle, neural tube, and lung. In the developing inner ear we find Eya1-/- mice display reduced Hh signaling in vivo and a genetic interaction between Eya1 and Hh signaling. In lung tissue, Eya1-/- mice have reduced levels of Nrp expression. Together, these data further our understanding of the Hh signaling pathway and provide evidence for a role of Eya1 in Hh signal transduction.