Figure 2. Dynamics of cell heterogeneity in poxⁿ > pros^RNAi NB tumors.

(A,B) Cartoons represent a ventral view of the Drosophila CNS at late L2 and late L3. Gray circles represent normal NBs. Green circles are pros^RNAi tNBs. Red tNBs express chinmo. Tumors were induced by knocking down pros in six NBs located in the VNC, throughout larval development, using the poxⁿ-Gal4, UAS-pros^RNAi, UAS-GFP, UAS-dicer2 system (poxⁿ > pros^RNAi). Because chinmo is always co-expressed with Imp in NBs, we use either anti-Chinmo or anti-Imp to label Chinmo⁺Imp⁺ tNBs. We also used Syp to label Syp⁺E93⁺ tNBs. (A) poxⁿ > pros^RNAi initially induces pools of tNBs all expressing Chinmo in early larvae (L2). tNBs are marked with anti-Mira and anti-GFP. (B) In late larvae (L3), poxⁿ > pros^RNAi tumors are composed by two distinct populations of tNBs respectively expressing Chinmo and Syp. Tumors are marked with anti-GFP and delineated by dashed lines. (C) Mitotic Imp⁺ tNBs and Syp⁺tNBs are marked with anti-PH3 in poxⁿ > pros^RNAi tumors persisting in 4-day-old adults. (D) Quantification of the mitotic index of Chinmo⁺Imp⁺ tNBs (n = 7 VNCs) and Syp⁺E93⁺ tNBs (n = 7 VNCs) in poxⁿ > pros^RNAi tumors of 4-day-old adults. p=0.0006. (E) Proportion of Chinmo⁺Imp⁺ tNBs over all tNBs composing tumors (volumes of each population are measured) at 8 days (8d) (n = 5), 10d (n = 6), 11d (n = 6) and 15d (n = 6) after tumor induction. Each dot represents the % for one tumor. (F) Scheme depicting the dynamics of tumor composition: from a homogeneous pool of Chinmo⁺Imp⁺ tNBs in early larvae to a heterogeneous tumor with a minor population of Chinmo⁺Imp⁺ and a majority of Syp⁺E93⁺ tNBs. Scale bars, 20 µm.

Figure 2—figure supplement 1. poxⁿ > pros^RNAi tumors exhibit low levels of neuronal differentiation in adults.

The G-TRACE system allows the permanent GFP labeling of neurons generated by poxⁿ > pros^RNAi tNBs. Tumor neurons, marked with anti-GFP and anti-Elav, are usually grouped and represent a minor population of cells poorly contributing to tumor mass in 4-day-old poxⁿ > pros^RNAi adult tumors. Tumor neurons localize outside the Chinmo⁺Imp⁺ tNB clusters marked with anti-GFP and anti-Imp.