Requirement of RNA synthesis for pathfinding by growing axons

The effects of actinomycin D were studied in cultured grasshopper embryos at different stages of development by following the outgrowth patterns of identified neurones known as aCC, pCC, and Q1. When administered at stages occurring before 31% of embryonic development, actinomycin D (0.05-0.10 microM for 24-48 hours) prevented axon extension, whereas it did not affect the development of the nervous system in embryos older than 34% of development. At 31-34% of development, actinomycin D perturbed pathfinding of aCC without blocking axon extension. Thus, only 22% of the aCCs (n = 271) in embryos treated with actinomycin D extended an axon along the intersegmental nerve as in control embryos. In the remaining embryos, aCC failed to turn into the intersegmental nerve root; its growth cone remained in the longitudinal connective, above or below the turning point. Neurones of the group caudal to the intersegmental nerve root could extend along either the anterior or posterior commissure of the next posterior segment. In contrast to the observations made with aCC, only 1.2% of pCC (n = 166) and 0.0% of Q1 (n = 45) in embryos treated with actinomycin D showed axon growth along aberrant pathways. The position of the growth cones of most pCCs and all Q1s observed were in various points along their normal pathway. Both pCC and Q1, as a population, showed an extension rate significantly lower than that of their control counterparts. The effect of actinomycin D on aCC pathway choice was probably mediated by inhibition of RNA synthesis, because incorporation of uridine into RNA was reduced by 40%. The labelling of several monoclonal antibodies (1C10, 3B11, 7F7) that recognise surface glycoproteins (lachesin, fasciclin I, and REGA-1) involved in nervous system development of grasshopper embryos was suppressed. Our results suggest that the navigation of some axons along different pathways requires the synthesis of new mRNA.