Guidance of neuronal growth cones in the grasshopper embryo. IV. Temporal delay experiments.

In the previous 3 papers, we demonstrated that neuronal growth cones display selective affinities for both specific axonal and glial pathways in the grasshopper embryo; for example, the pCC growth cone selectively recognizes the MP1/dMP2 axons, while the aCC growth cone selectively recognizes the U axons and a specific glial cell (the segment boundary cell). We were interested in further testing the temporal specificity of these affinities. To address this issue, we performed specific temporal transplant experiments by using a laser microbeam in ovo to ablate the neuronal precursor cell, neuroblast (NB) 1-1, that generates the aCC and pCC neurons. Neighboring ectodermal cells regulate and replace the ablated NB 1-1; the new NB 1-1 then generates the aCC and pCC neurons with a temporal delay of 10-20 hr (2-4% of development), depending upon the experimental paradigm. The results of these temporal delay experiments further demonstrate the selective affinities of the aCC and pCC growth cones for specific axonal and glial surfaces and confirm that these specificities are absolute and not hierarchical. Furthermore, they suggest that precise timing is not important; both the pCC and aCC growth cones are able to selectively recognize their appropriate axonal and glial pathways after delays of 10-20 hr despite being confronted with the surfaces of many additional axons and pathways.