Figure 8. The Imp-to-Syp transition triggers down-regulation of glutamine and glucose metabolism genes along the differentiation trajectory to reduce tNB growth and proliferative potential.

(A) Glucose metabolism pathways (glycolysis, TCA cycle and respiratory electron transport chain). Genes whose expression is enriched in poxⁿ > pros^RNAi, Syp^RNAi tumors compared to poxⁿ > pros^RNAi tumors are highlighted in blue. Log2 fold change is indicated in brackets. (B) Expression dynamics of various glycolytic, respiratory electron transport chain and glutamine metabolism genes along the tumor pseudotime. p-values for differential expression along the pseudotime are indicated for each gene. (C) poxⁿ > pros^RNAi NB tumors are marked with anti-Mira (green). Silencing of glycolytic (Gapdh-1^RNAi, Pglym78^RNAi) or respiratory genes (Cyt-c-p^RNAi, Cyt-c1^RNAi) leads to smaller poxⁿ > pros^RNAi tumors in late L3 larvae. Silencing of glycolytic or respiratory genes arrests tumor growth in 4-day-old and 7-day-old adults respectively. (D) Volumes of poxⁿ > pros^RNAi tumors (n = 5 VNC), poxⁿ > pros^RNAi, Gapdh-1^RNAi tumors (n = 7 VNC); poxⁿ > pros^RNAi, Pglym78^RNAi tumors (n = 5 VNC); poxⁿ > pros^RNAi,Cyt-c-p^RNAi tumors (n = 5 VNC); and poxⁿ > pros^RNAi,Cyt-c1^RNAi tumors (n = 5 VNC) in late L3. P_{Cntrl/Gapdh-1RNAi} = 0,002. P_{Cntrl/Pglym78RNAi} = 0,007. P_{Cntrl/Cyt-c-pRNAi=} 0,007. P_{Cntrl/Cyt-c1RNAi} = 0,007. Volumes of poxⁿ > pros^RNAi tumors (n = 6 VNC); poxⁿ > pros^RNAi, Gapdh-1^RNAi tumors (n = 8 VNC); poxⁿ > pros^RNAi, Pglym78^RNAi tumors (n = 9 VNC); poxⁿ > pros^RNAi, Cyt-c-p^RNAi tumors (n = 10 VNC); and poxⁿ > pros^RNAi, Cyt-c1^RNAi tumors (n = 6 VNC) in 4-day-old adults. P_{Cntrl/Gapdh-1RNAi} = 0,0007. P_{Cntrl/Pglym78RNAi} = 0,0002. P_{Cntrl/Cyt-c-pRNAi} = 0,0002. P_{Cntrl/Cyt-c1RNAi} = 0,002. Scale bars, 50 µm. (E) Each dot represents the average area of Chinmo⁺Imp⁺ tNBs over the average area of Syp⁺E93⁺ tNBs for a single confocal section of a poxⁿ > pros^RNAi NB tumor in a 4-day-old adult. (F) Schematic representation of the tumor hierarchy. Chinmo⁺Imp⁺ tNBs are proliferative and tend to self-renew. However, they can also engage in a differentiation process triggered by the Imp-to-Syp transition and involving a subset of larval temporal genes. Syp⁺E93⁺ tNBs can self-renew but also tend to exit the cell-cycle as they decrease expression of glucose/glutamine metabolism genes. Syp⁺E93⁺ tNBs, that have exited the cell-cycle, express genes of the E(spl) family. The mechanisms controlling the Imp-to-Syp transition in NB tumors, to determine the CSC proportion are unknown.

Figure 8—figure supplement 1. nAChRalpha3 and nAChRalpha5 are expressed in tNBs positioned at the beginning of the pseudotime.

Figure 8—figure supplement 2. Branch analysis of the single-cell trajectory.

(A) Cell trajectory reconstructed from the population of tNBs using semi-supervised pseudotime ordering. Cells are colored by States. The three main branches of the tree are labeled as: Pre-branch, E93⁺stg⁺ branch and E93⁺E(spl)⁺ branch. (B) stg and E(spl)m7-HLH are enriched in distinct branches of the trajectory. (C) Kinetic heatmap depicting the expression of genes that are differentially expressed between the Pre-branch, the E93⁺stg⁺ branch and the E93⁺E(spl)⁺ branch (qval < 1e-20).

Figure 8—figure supplement 3. Segmentation of NB tumors Segmented confocal section of a tumor using Tissue Analyzer.

Anti-Mira staining was used as membrane reference. Accurate segmentation was manually double-checked using Dapi as nuclear marker. Imp-GFP⁺ cells represent Chinmo⁺Imp⁺ tNBs. GFP^- tNBs represent Syp⁺E93⁺ tNBs.