Taken together, these data support a model in which axonal application of BMP4 elicits a retrograde signal that is translocated by dynein, and involves the appearance of both BMP4 and active BMP4 receptors in the cell body. GDC-0941 solubility dmso During the development of the nervous system, intra-axonal mRNA translation is a component of several signaling pathways, notably those involving axon guidance cues (Lin and Holt, 2008 and Martin and Ephrussi, 2009). We therefore asked if local protein synthesis is required for retrograde BMP4 signaling. Coapplication of either of the translation

inhibitors cycloheximide or anisomycin with BMP4 to axons substantially blocked retrograde BMP4 signaling (Figures 2A and 2B). The effect of axonal anisomycin treatment is not due to inhibition of protein synthesis in the cell body, as a labile control protein ODC in cell bodies is not affected (Figure S2A). In addition to inducing Tbx3, retrograde BMP4 signaling also leads to the repression of OC1, OC2, and Hmx1 in the cell body ( Hodge et al., 2007). These effects were also blocked by axonal application of translation inhibitors ( Figures 2C–2E). We considered the possibility

Protease Inhibitor Library that inhibition of local translation could impair the retrograde translocation of BMP4 signaling endosomes. However, application of anisomycin to axons did not prevent the retrograde transport of biotinylated BMP4 (Figure S2B). Under these experimental conditions, the translation inhibitors did not elicit axonal or cell body toxicity compared to vehicle treatment (Figures S2C and S2D). Additionally, the effects of the protein synthesis inhibitors were not due to alterations in the levels of axonal BMPR1a, 1b, and 2 or cell body SMAD1/5/8, or due to diffusion of the inhibitors into the cell body compartment (Figures

Parvulin S2E–S2J, S1F, and S2K). These data indicate that translation of axonal mRNA(s) are required for retrograde BMP4 signaling. To identify axonal mRNAs that may mediate retrograde BMP4 signaling, we considered proteins that are present in axons and may be translated locally. Although transcription factors are typically localized in the nucleus, previous studies have detected prominent labeling of phospho-SMAD1/5/8 (pSMAD1/5/8) in certain axonal branches of the trigeminal ganglia (Hodge et al., 2007). The axonal localization of these transcription factors raises the possibility that they are synthesized locally and that this axonal pool of SMAD1/5/8 has a role in conveying retrograde patterning signals from target tissues. Although pSMAD1/5/8 is selectively localized to ophthalmic and maxillary axons (Hodge et al., 2007), the absence of pSMAD1/5/8 immunoreactivity in mandibular axons could reflect the absence of SMAD phosphorylation in these axons, or the absence of SMAD protein altogether.