, 2005). We failed to find a role for Hhip1 in the AP guidance of commissural axons through genetic analysis in the mouse, with postcrossing commissural axons turning anteriorly in Hhip1−/− mice ( Figures S3A–S3D). Although it is possible that

Shh-mediated AP axon guidance in the chick and mammals uses different molecular mechanisms, this would be somewhat surprising given that all of the other guidance effects described so far for Shh are Smo dependent ( Charron et al., 2003; Fabre et al., 2010; Sánchez-Camacho and Bovolenta, 2008; Yam et al., 2009). The ability of axons to change responsiveness to guidance cues is critical as axons navigate PFI-2 through complex environments. We show that the switch in Shh response from attraction to repulsion depends on 14-3-3 proteins, which are highly expressed in nervous tissue. In Drosophila motor neurons, correct axon pathfinding requires 14-3-3ε, which antagonizes Semaphorin-1a/PlexinA-mediated axon repulsion and allows axons to become more responsive to integrin-mediated adhesion ( Yang and Terman, 2012). In postnatal rat DRG neurons, 14-3-3 proteins are important for conferring repulsive responses

to NGF, and antagonism of 14-3-3 proteins converts this NGF-mediated selleck repulsion to attraction ( Kent et al., 2010), a process that could be harnessed to promote neuronal repair after injury. We now demonstrate a role for 14-3-3 proteins in a developmental switch in response to a guidance cue. 14-3-3 proteins function as homodimers and heterodimers to control the spatial and temporal activity of substrate proteins (Bridges and Moorhead, 2004). One way that 14-3-3 proteins modulate growth cone turning is by inhibiting PKA activity, through binding and stabilizing

the PKA holoenzyme (Kent et al., 2010). Consistent with this, the increase in 14-3-3 levels in 3–4 DIV commissural neurons was accompanied by a decrease in active PKA levels, and PKA inhibition of could rescue the effect of 14-3-3 inhibition on commissural axon turning. According to our model, 14-3-3 levels regulate the global state of the neuron, changing the way the growth cone responds to Shh gradients. Our experiments showed that modulating 14-3-3 protein levels are sufficient to change the polarity of the turning response of commissural axons to Shh gradients. Thus, we hypothesize that 14-3-3 proteins regulate the turning response to Shh downstream of Shh reception, and we do not expect that Shh signaling itself regulates 14-3-3 levels. Consistent with this, neither treatment of commissural neurons with Shh nor a Smo antagonist affected 14-3-3 protein levels (Figure S3E). In vitro, changes in the relative levels of other intracellular molecules, such as cyclic nucleotides and Ca2+, can switch responses to guidance cues (e.g., Song et al., 1997, 1998; Wen et al., 2004).