Figure 1. Single-cell RNA-seq identifies a subset of temporal patterning genes that are redeployed in NB tumors defining heterogeneity in NB states.

(A) PCA was performed on single-cell tNB transcriptomes to reduce the dimensions of the data for further analysis. Genes (rows) and cells (columns) are ordered by their PCA scores, and the 500 most extreme cells and 30 most extreme genes on both sides of the distribution are shown in the heatmap. PC4 reveals that tNBs can be discriminated by the expression of early (light red asterisks) vs late (light blue asterisks) larval NB temporal patterning genes. Other larval temporal genes are found in PC3 and PC7 (Figure 1—figure supplement 1C). (B) The UMAP representation of all single cells included in our analysis shows the separation of different clusters. We used a k-nearest neighbor algorithm to call seven clusters, which are shown in different colors on the UMAP plot. (C) Expression of Mira, early and late temporal markers on the UMAP map shown in B. (D) Cartoon representing a ventral view of an adult CNS containing a NB tumor induced during larval stages in the VNC. Green circles are tNBs. Green circles colored in red represent Chinmo⁺Imp⁺ tNBs. Immunostainings with anti-Chinmo, anti-Imp and anti-Syp, indicating that Imp/Chinmo and Syp are expressed in a complementary pattern in poxⁿ > pros^RNAi tumors found in 4-day-old adults. tNBs are labeled with Mira. Scale bar 20 µm. (E) Immunostainings with anti-Chinmo and anti-E93 indicating that these two transcription factors are expressed in a complementary pattern in poxⁿ > pros^RNAi tumors found in 4-day-old adults. tNBs are marked with anti-Mira. Scale bar 20 µm. (F) The light red and blue colors respectively designate ‘early’ and ‘late’ larval NB genes as determined by Liu et al. (2015), Syed et al. (2017) and Ren et al. (2017). A subset of these genes are redeployed in tumors to define distinct tNB states. Asterisks mark temporal patterning genes regulated at the post-transcriptional level in tNBs. Note that cas and svp transcription is associated with early Imp⁺ NBs, while br transcription is associated with late E93⁺ NBs during larval stages. In contrast, cas, svp and br transcription do not distinguish Imp⁺ and E93⁺ tNBs.

Figure 1—figure supplement 1. Seurat analysis of the single-cell transcriptomic data, with regression of cell-cycle genes.

(A) Plots depicting the number of genes detected in each cell (nFeature_RNA), the number of unique RNA molecules detected in each cells (nCount-RNA), and the percentage of reads mapping to the mitochondrial genome (percent.mt). (B) Plot of the standard deviations of PCs used to determine the number of PCs to include in downstream analyses of the RNA-seq analysis. (C) Heatmaps depicting the composition of the first 12 PCs. Light red asterisks mark early larval temporal patterning genes. Blue asterisks mark late larval temporal patterning genes.

Figure 1—figure supplement 2. Characterizing tNBs by their transcriptome.

(A) Expression levels of various marker genes for NBs (dpn); cholinergic, glutamatergic and GABAergic neurons (VAChT, VGlut and Gad1 respectively); and glia (repo); on the UMAP map. (B) Expression levels of genes enriched in the small clusters 5 and 6. (C) Heatmap depicting the top 10 most highly expressed genes in each cluster compared to all other clusters. Cluster three is highly enriched in early larval NB markers (red asterisks). (D) Upper Venn diagram depicts overlaps between genes, the expression of which is enriched in early NBs of various lineages (Mushroom Body (MB), Antennal Lobe (LB), Type II)) and in cluster 2 of tNBs. Lower Venn diagram depicts overlaps between genes enriched in late NBs of various lineages (MB, AL, Type II) and in cluster 0,1,3,4 of tNBs. Early or late temporal NB genes overlapping with tumor genes are indicated.

Figure 1—figure supplement 3. Comparison of genes that are temporally regulated genes in various larval NBs with genes defining clusters in the UMAP representation of the NB tumor.

Lists of genes enriched in NBs at various timepoints during larval development (24 hr after larval hatching (ALH), 36 hr, 50 hr, 84 hr). Lists have been extracted for Mushroom body (MB) NBs, antennal lobe (AL) NBs and type Liu et al. (2015) and Ren et al. (2017). Darks backgrounds encompass early genes, while light backgrounds encompass late larval genes. Expression of the gene br, a well-known marker for late NBs, has been chosen as the transition time from an early to late stage. Dark and light orange backgrounds represent genes that distinguish the Imp⁺ Cluster two from the E93⁺ Clusters 0,1,3,4 in the UMAP representation of tumor cells. A subset of temporal patterning genes highlighted in red distinguish Imp⁺ or E93⁺ clusters in the tumors.

Figure 1—figure supplement 4. Post-transcriptional regulation of chinmo in poxⁿ > pros^RNAi NB tumors.

The UAS-mCherry^chinmoUTRs transgene (Dillard et al., 2018) is transcribed in all tNBs using the poxⁿ-GAL4 driver. However, the mCherry protein (anti-RFP immunostaining in red) is only present in tNBs that express endogenous Chinmo (anti-Chinmo immunostaining in grey). This demonstrates post-transcriptional regulation via the chinmo UTRs in tNBs. This is consistent with the ubiquitous presence of chinmo mRNA in all tNBs as detected with the single-cell RNA-seq protocol.

Figure 1—figure supplement 5. Grh is expressed in all tNBs.

(A) grh mRNA is detected in most if not all tNBs throughout the UMAP representation. (B) All tNBs in 4 day-old adult express grh as shown by immunostainings.