Tyrosine kinase activity in the developing olfactory system of Manduca sexta

Abstract

To begin to determine the role tyrosine kinases play in the development of the antennal lobe in Manduca sexta, phosphotyrosine levels were examined immunocytochemically during critical periods of development. Tyrosine phosphorylation was found to be highest in the region of the antennal lobe known to contain the growing tips of antennal-lobe neurons and forming synapses. To explore the relationship between synapses and tyrosine kinase activity a marker for synapses was developed for light level microscopy. Manduca synaptotagmin was cloned and sequenced and a polyclonal antibody (SYNT76) was produced against the amino terminal of the predicted peptide sequence. SYNT76 labeled known synaptic neuropils in the brain of Manduca and recognized a single 61 kd protein on immunoblots. Immunocytochemical double labeling of antennal lobes for synaptotagmin and phosphotyrosine showed that although the patterns were very similar there was surprisingly little direct signal overlap. The similarity of immunolabeling suggests that tyrosine kinases may affect synapses in the developing antennal lobe but the lack of dramatic signal overlap shows that this association does not occur at the active zone. A trk-like protein was identified immunocytochemically, using an antibody against mammalian neurotrophin receptors, in the same regions of the developing lobe that contained high levels of phosphotyrosine. Trk-like proteins and phosphotyrosine were also found in the growing processes of cultured antennal lobe neurons. To examine the role of trk-like kinases in process outgrowth of antennal-lobe neurons, a tyrosine kinase inhibitor specific for trk-like kinases (Tyrphostin AG879) was applied to cultured neurons and was found to inhibit process outgrowth from antennal-lobe neurons significantly when compared to cells grown under control conditions. To determine the identity of the antennal-lobe trk-like proteins outside of their kinase domains the process of cloning trk-like messenger RNA was begun with reverse transcriptase polymerase chain reactions (RT-PCR). Northern blot analysis using RT-PCR fragments with significant sequence similarity to mammalian trk receptors identified a single 4.5 kb trk-like transcript in the brain and specifically in the antennal lobe during development. These data suggest that a trk-like kinase may mediate a developmental signal that causes antennal-lobe neurons to grow processes.