Contrasting developmental axon regrowth and neurite sprouting of Drosophila mushroom body neurons reveals shared and unique molecular mechanisms.

The molecular mechanisms regulating intrinsic axon growth potential during development or following injury remain largely unknown despite their vast importance. Here, we have established a neurite sprouting assay of primary cultured mushroom body (MB) neurons. We used the MARCM technique to both mark and manipulate MB neurons, enabling us to quantify the sprouting abilities of single WT and mutant neurons originating from flies at different developmental stages. Sprouting of dissociated MB neurons was dependent on wnd, the DLK ortholog, a conserved gene that is required for axon regeneration. Next, and as expected, we found that the sprouting ability of adult MB neurons was significantly decreased. In contrast, and to our surprise, we found that pupal-derived neurons exhibit increased sprouting compared with neurons derived from larvae, suggesting the existence of an elevated growth potential state. We then contrasted the molecular requirements of neurite sprouting to developmental axon regrowth of MB ɣ neurons, a process that we have previously shown requires the nuclear receptor UNF acting via the target of rapamycin (TOR) pathway. Strikingly, we found that while TOR was required for neurite sprouting, UNF was not. In contrast, we found that PTEN inhibits sprouting in adult neurons, suggesting that TOR is regulated by the PI3K/PTEN pathway during sprouting and by UNF during developmental regrowth. Interestingly, the PI3K pathway as well as Wnd were not required for developmental regrowth nor for initial axon outgrowth suggesting that axon growth during circuit formation, remodeling, and regeneration share some molecular components but differ in others.