Aberrant Sonic Hedgehog signaling in bbs morphants. (A) Expression of ptc1, a marker of Sonic Hedgehog pathway activity, is down-regulated in bbs morphants, most strikingly so in bbs8 morphants (6D). (E and F) Conversely, pax6, a gene negatively regulated by Shh signaling, is up-regulated in the neural tube of bbs8 morphants. (I) Injection of ptc1 MO alone up-regulates expression of ptc1 by inducing the Shh pathway. (J) Coinjection of ptc1 MO with bbs8 MO partially rescues this induction, implicating Bbs8 downstream of Ptc1. (K) Injection of dominant-negative PKA causes ectopic activation of the Shh pathway. (L) Coinjection with bbs8 MO rescues this induction, suggesting that it could act downstream of PKA, likely in Gli3 transport. (M) Addition of Purmorphamine to control transfected cells induces the Gli activity by 2.5-fold. Bbs8 knockdown cells show attenuated response to Purmorphamine stimulation. (N and O) Western blot showing reduction of Gli3R in response to stimulation with Purmorphamine, resulting in a higher Gli3A;Gli3R ratio. This ratio remains unchanged when Bbs8 knockdown cells are stimulated. [Scale bars: 500 μm (A–D and I–L) and 200 μm (E–H).]
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