Figure 6. Imp and Syp antagonistically regulate the growth and heterogeneity of pros^RNAi tumors.

(A) Syp knockdown in poxⁿ > pros^RNAi tumors triggers a dramatic increase of Chinmo⁺Imp⁺ tNBs. tNBs are marked with an anti-Mira antibody. (B) Proportion of Chinmo⁺Imp⁺ tNBs in poxⁿ > pros^RNAi (n = 6 VNC) and poxⁿ > pros^RNAi, Syp^RNAi (n = 6 VNC) tumors in 4-day-old adults. p=0.0022. (C) Simulation of tumor growth (number of cells) in pros^KD and pros^KD, Syp^KD (red) tumors. Quiescence probabilities are those set by the error minimization. (D) Volume of poxⁿ > pros^RNAi (n = 6 VNC) and poxⁿ > pros^RNAi, Syp^RNAi (n = 6 VNC) tumors in 1-day-old adults. p=0.002. Volume of poxⁿ > pros^RNAi (n = 7 VNC) and poxⁿ > pros^RNAi, Syp^RNAi (n = 4 VNC) tumors in 4-day-old adults. p=0.0061. (E) Volume of poxⁿ > pros^RNAi tumors in 3-day-old (n = 10 VNC) and 8-day-old (n = 6 VNC) adults. p=0.00025. Volume of poxⁿ > pros^RNAi (n = 10 VNC) and poxⁿ > pros^RNAi, UAS-Syp (n = 8 VNC) tumors in 3-day-old adults. p=0.0005. Volume of poxⁿ > pros^RNAi (n = 6 VNC) and poxⁿ > pros^RNAi, UAS-Syp (n = 8 VNC) tumors in 8-day-old adults. p=0.00067. Volume of poxⁿ > pros^RNAi, Syp^OE tumors in 3-day-old (n = 8 VNC) and 8-day-old (n = 8 VNC) adults. p=0.7209. (F) Volume of poxⁿ > pros^RNAi, Syp^RNAi (n = 6 VNC) and poxⁿ > pros^RNAi, Imp^RNAi, Syp^RNAi (n = 9 VNC) tumors in 4-day-old adults. p=0.0004.

Figure 6—figure supplement 1. Syp silences Chinmo and Imp to limit NB self-renewal during development.

(A) RNA affinity pull-down assay using biotinylated RNAs incubated with wild-type adult brain extracts. Proteins recovered in the bound fractions were visualized by western blot analysis. The 3’ UTR of chickadee (chic) and the coding sequence of y14 were used as positive and negative controls respectively. (B) Cartoons represent a ventral view of the adult Drosophila CNS. The nab-GAL4 driver is active in all NBs located in the VNC. In nab-Gal4, UAS-dicer2, UAS-GFP (nab >GFP) GFP labels NBs and their recent progeny. NBs, marked with GFP and Miranda (Mira) are not present in the VNC of wt pharate adults. (C) Syp knock-down (nab >Syp^RNAi) from early larval stages induces the persistence of active Chinmo⁺Imp⁺ NBs in the VNC of pharate adults. (D) Most of the VNC NBs lacking both Syp and Imp (nab >Syp^RNAi, Imp^RNAi) are eliminated before the pharate adult stage. The few persisting NBs exhibit a smaller size and cytoplasmic extensions. They don’t express Chinmo and Imp. (E) Most of the VNC NBs lacking both Syp and Chinmo (nab >Syp^RNAi, Chinmo^RNAi) are eliminated before the pharate adult stage. The few persisting NBs exhibit a smaller size and cytoplasmic extensions. They don’t express Chinmo and Imp. (F) Quantification of the number of NBs per VNC in nab >Syp^RNAi (n = 6 VNC), nab >Syp^RNAi, Imp^RNAi (n = 6 VNC) and nab >Syp^RNAi, chinmo^RNAi (n = 6 VNC). P_SypKD/_SypKD,ImpKD = 0.002. P_SypKD/_{SypKD,chinmoKD} = 0.002. P_SypKD,ImpKD/_{SypKD,chinmoKD} = 0.039. (G) Quantification of proliferating NBs, marked with PH3, in nab >Syp^RNAi (n = 6 VNC), nab >Syp^RNAi, Imp^RNAi (n = 6 VNC) and nab >Syp^RNAi, chinmo^RNAi (n = 6 VNC). P_SypKD/_SypKD,ImpKD = 0.002. P_SypKD/_{SypKD,chinmoKD} = 0.004. (H) Quantification of the NB area in nab > Syp^RNAi (n = 14 NBs), nab > Syp^RNAi, Imp^RNAi (n = 10 NBs) and nab > Syp^RNAi, chinmo^RNAi (n = 10 NBs). P_SypKD/_SypKD,ImpKD=<0.0001. P_SypKD/_{SypKD,chinmoKD}=<0.0001. (I) Scheme depicting the asymmetric divisions of NBs throughout larval and pupal stages and recapitulating the above experiments. Scale bars, 20 µm.

Figure 6—figure supplement 2. Imp and Syp antagonistically regulate tumor growth and chinmo expression in poxⁿ > pros^RNAi NB tumors.

(A) poxⁿ > pros^RNAi, Syp^RNAi  tumors overgrow compared to control poxⁿ > pros^RNAi tumors (Cntrl) in 1-day-old adults (1d).Tumors fail to grow in poxⁿ > pros^RNAi, Syp^RNAi, Imp^RNAitumors (4d). Overexpression of Syp in poxⁿ > pros^RNAi, Syp^OE tumors arrests tumor growth compared to control poxⁿ > pros^RNAi tumors (Cntrl) in 8-day-old adults (8d). Tumors are delimited by dashed lines. The UAS-mCherry^chinmoUTRs is expressed in tumors where it is post-transcriptionally regulated, reflecting chinmo expression (Figure 1—figure supplement 4). (B) poxⁿ > pros^RNAi, Syp^RNAi tumors are mostly composed of Chinmo⁺ tNBs. Chinmo is absent in poxⁿ > pros^RNAi, Syp^RNAi, Imp^RNAi tumors indicating that Imp is necessary to promote chinmo expression in poxⁿ > pros^RNAi, Syp^RNAi tumors. tNBs are marked with Mira. Scale bars, 20 µm.