Therefore, we next considered BDNF, which exhibits a highly specific ophthalmic and maxillary epidermal expression pattern, with no detectable levels in the mandibular region of the face (Arumäe et al., 1993 and O’Connor and Tessier-Lavigne, 1999). Application of BDNF to severed axons caused a 1.7-fold increase in axonal SMAD levels in 30 min (Figures 7A and 7B). This increase was blocked by coapplication of anisomycin, indicating

that axonal SMAD induction by BDNF is local synthesis dependent (Figures 7A and 7B). As a control, Tau1 and TrkB receptor levels were not affected by these treatments (Figures S7A–S7C). These data indicate that neurotrophins, especially BDNF, induce SMAD expression in axons via local protein synthesis. Our data indicate that retrograde induction of nuclear pSMAD1/5/8 and Tbx3 requires target-derived BDNF-dependent intra-axonal synthesis of SMADs. Previous studies Regorafenib in vivo mTOR inhibitor suggested that BMP4 was the primary regulator of pSMAD1/5/8 induction (Hodge et al., 2007). However, these experiments utilized bath application of BMP4, which results in activation of BMP4 receptors primarily on cell bodies. Because cell bodies express SMAD1/5/8 even in the absence of neurotrophins (Figure 3B), these studies do not address signaling in axons, which express

SMAD1/5/8 only in the presence of neurotrophins. To test the idea that both BMP4 and neurotrophins are required for retrograde signaling, we cultured E13.5 trigeminal ganglia neurons in microfluidic chambers, and after 2 days switched the media to neurotrophin-free media for 4 hr. Under these modified culture conditions, axonal application of BMP4 for 1 hr failed to elicit retrograde induction of nuclear pSMAD1/5/8 (Figure 7C). Similarly, axonal application of BDNF for 1 hr was unable to induce nuclear pSMAD1/5/8. However, coapplication of BDNF and BMP4 resulted in retrograde induction of nuclear pSMAD1/5/8 (Figure 7C). In each of these treatments, total cell body SMAD1/5/8 levels were not significantly affected (Figure S7D). These data indicate

that BMP4 is not sufficient for retrograde signaling, but requires collaboration with neurotrophins. To Fossariinae determine whether the induction of pSMAD1/5/8 and Tbx3 by axonally applied BDNF also requires a retrogradely trafficked TrkB signaling endosome, we used the Trk inhibitor K252a (Tapley et al., 1992). Application of K252a to axons of E13.5 trigeminal ganglia neurons in microfluidic chambers blocked the ability of axonally applied BDNF and BMP4 to induce Tbx3, without affecting retrograde transport of BMP4 signaling endosomes (Figures 7D and S7E). These data indicate that the effects of BDNF in mediating retrograde BMP4 signaling reflect local, intra-axonal actions of BDNF and do not require the activity of Trk receptors in the cell body. Together, these data indicate that BDNF and BMP4 receptors have roles in distinct compartments within the neuron to mediate retrograde signaling.