longitudinals lacking (lola) is among the most complex genes in Drosophila melanogaster, encoding up to twenty protein isoforms and acting as a key transcription factor in axonal pathfinding and neural reprogramming. Most of previous studies employed loss-of-function alleles disrupting common exons of lola, making it difficult to delineate its functions. To address this issue we have generated specific mutations in each isoform using the CRISPR/Cas9 system. Our targeted screen allows us to revisit the previously demonstrated roles for few isoforms and to demonstrate a specific function for one variant in axon guidance via activation of the microtubule-associated factor Futsch. Importantly, we also reveal a critical role for a second variant in preventing neurodegeneration via the control of the octopaminergic pathway. This variant is expressed almost exclusively in the octopaminergic cells and is involved in the transcriptional activation of a key enzyme of the pathway. Thus, our comprehensive study greatly expands the functional repertoire of Lola functions, and adds novel insights into the transcriptional regulatory control of neurotransmitter expression in vivo.