Nucleolar stress has been implicated in the pathogenesis of various human diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the cause of nucleolar stress and its effect in ALS are incompletely understood. G4C2 hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of both ALS and FTD (C9-ALS/FTD). In this study, we used fly C9-ALS/FTD models that express G4C2 repeats to characterize nucleolar stress and investigate its cause. We find that pan-neuronal expression of G4C2 in flies induces enlargement of nucleoli selectively in glutamatergic neurons. Some of the basic components of ribosomes, such as rRNA and ribosomal proteins RPS6 and RpL10Ab, are accumulated in these enlarged nucleoli. Myc is a proto-oncogene that regulates cell growth and proliferation primarily through induction of ribosome biogenesis. Interestingly, Myc is up-regulated in C9-ALS/FTD flies as are some of its downstream targets, pit and ppan. Pit is an RNA helicase and ppan is an rRNA processing factor, both of which have been linked to ribosome biogenesis. Notably, we find that pit is enriched in the enlarged nucleoli of C9-ALS/FTD flies; investigation of its potential involvement in the pathogenesis is ongoing. Overexpression of Myc in glutamatergic neurons increases nucleolar size, but these nucleoli are not enriched with rRNA, suggesting that this phenotype in C9-ALS/FTD flies is independent of Myc. Recently, Sirozh et al. showed that Myc depletion mitigates C9orf72-PR (proline-arginine) repeat-induced toxicity in U2OS and NSC34 cells. However, it remains unclear how Myc is upregulated, nor is it clear how its depletion is protective in C9-ALS/FTD. We are currently investigating these areas of uncertainty using our C9-ALS/FTD fly models. In sum, our current study suggests that G4C2 expression induces nucleolar expansion along with accumulation of rRNA and ribosomal proteins and that up-regulation of Myc may play an important role.