ABSTRACT: The analysis presented here aims at exposing single-cell transcriptome profiles of Drosophila larval brain type II neural stem cells (NSCII) and of their intermediate neural progenitor (INP) cell progeny during normal development and upon brain tumour initiation using the brain tumour (brat) mutant model. We have used brains 24 hours after larval hatching (ALH), a developmental stage when brat INPs (b_INP) have transformed into brain tumour initiating cells, expressing aberrantly the self-renewal transcription factor Deadpan unlike their iINP control counterparts (c_iINP), but have not yet started to overproliferate. iINP will acquire expression of proneural Asense (Ase) becoming mature INPs (m_INPs), yet brat INPs never express Ase and will lead to massive tumour growth (Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008). NSCIIs, adjacent smaller iINPs and one mature INP were individually harvested from live freshly dissected brains expressing membrane tagged GFP specifically in NSCII lineages, and single cell transcriptomes obtained as previously described (Bossing et al., 2012; Barros & Bossing, 2022). PCRs confirmed iINPs and INP samples to lack or show ase expression, respectively. Single cell cDNAs were hybridised on whole-genome microarrays in pairs and data processed as indicated below. References: Bello, B.C., Izergina, N., Caussinus, E., and Reichert, H. (2008). Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development. Neural Dev 3, 5. Bowman, R.L., Wang, Q., Carro, A., Verhaak, R.G., and Squatrito, M. (2017). GlioVis data portal for visualization and analysis of brain tumor expression datasets. Neuro Oncol 19, 139-141. Boone, J.Q., and Doe, C.Q. (2008). Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells. Dev Neurobiol 68, 1185-1195. Bossing, T., Barros, C.S., Fischer, B., Russell, S., and Shepherd, D. (2012). Disruption of microtubule integrity initiates mitosis during CNS repair. Dev Cell 23, 433-440. Barros C.S. & Bossing, T. (2022). Direct isolation of single cells from living brains of Drosophila melanogaster without dissociation for transcriptome analysis. STAR Protoc. Dec 16;3(4):101735.
