Western blot analysis HEK293 or other cells were cultured in 6-well plates

nclusion that PD173074 does not block EGFR activation in M. sexta. We lack an antibody for the activated form of the only other Manduca receptor tyrosine kinase characterized to date, the Eph receptor, so we could not check for its possible inactivation. However, PD173074 was designed to competitively bind to the Glial FGFRs in Glia-Neuron Signaling ATP-binding pocket of the FGF receptor, and amino acid alignments show that the Eph receptor lacks 8 of the 18 amino acids at specific locations needed to form the binding pocket for PD173074. Thus PD173074 appears an unlikely candidate for binding to and blocking activation of the Eph receptor. Because it was important to determine whether the altered fasciculation of ORNs traversing the sorting zone in PD173074treated animals was a direct result of blocking ORN FGFR activation, we also looked for evidence of expression of FGFRs by olfactory receptor neurons. We found no evidence for pFGFRs in ORN cell bodies, axons, or dendrites within antennal sensilla, suggesting that the altered behavior of ORN axons in the sorting zone is the consequence of interrupting an interaction between the ORNs and glial cells that depends on FGFR activation in the glial cells. Glial FGFRs in Glia-Neuron Signaling Blocking glial FGFR activation: effects on glia During development of the olfactory pathway, glial migration occurs in response to the arrival of ORN axons and leads to the formation of the sorting zone and formation of the glial envelopes that stabilize developing glomeruli. We have observed previously that NP glia fail to buy Debio1347 migrate but do extend processes following blockade of neuron-to-glial cell signaling via nitric oxide or disruption of sterol-rich membrane subdomains with methyl-b-cyclodextrin. We have shown here the same phenotype in PD173074-treated animals. Together, these several observations indicate that glial cell migration in response to ORN axon ingrowth and coupling of cell-body movement to process extension depends on several signaling systems, including FGFR activation.At stage 12, apoptotic nuclei were found 24291101 in the sorting zone and antennal nerve. “n”= number of frozen sections examined. Alternatively, pathways downstream of calcium influx and FGFR activation could intersect to produce glial cell migration via, for example, activation of doublecortin, src-family kinases, and focal adhesion kinases. In contrast to the effect on NP glial cells, pharmacologic blockade of FGFR activation did not prevent the migration of SZ or AN glial cells. Blockade of ORN-mediated nitric oxide signaling or disruption of sterol-rich membrane subdomains with methyl-b-cyclodextrin also failed to block SZ glial cell migration. Our inability to block SZ glial migration by these various methods may be due to the fact that the initial contact between ORN growth cones and the glial cells that become SZ glia occurs late in stage 3, and thus the signaling necessary for SZ glial migration 8199874 may have occurred before the various drug treatments could take effect. Injecting drugs at earlier stages generally results in developmental arrest a short distance into the sorting zone. B: PD173074-treated animals exhibited unchanged fasciculation in traveling through the sorting zone, although they did show increased fasciculation on exiting the sorting zone. Projection depths = 35 mm in A, 45 mm in B. doi:10.1371/journal.pone.0033828.g011 lished). Another possibility is that redundancy in the signaling pathways that elicit SZ

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