Effects of X-ray irradiation of DNA damage, apoptosis and cell cycle progression

(A) Cartoon overlay of wing disc showing PD regions. (B, C) IF of p-H2Av at 0 rad (B) and 4000 rad of irradiation, 30 min after exposure (C). (D, E) IF of cleaved Dcp-1 at 0 rad (D) and 4000 rad 4 h after exposure (E). Magenta arrowhead points to the DV boundary and white arrowheads point to regions of the hinge with less Dcp-1 signal. (F– I) IF of PHH3 at 0 rad (F) and 4000 rad 15 min (G), 2 h (H), and 4 h (I) after exposure. (J–M) EdU incorporation at 0 rad (J) and 4000 rad 15 min (K), 2 h (L) and (M) 4 h after exposure. Magenta arrowheads point to regions with high EdU signal at 4 h after irradiation. All scale bars are 100 μm.

Territories of the wing disc shown by immunofluorescence and UMAP plots

IF of Zfh2 (A, B), GFP expression in w-,nub-gal4,uas-gfp; fly wing discs (A’, B’), and merged images of both (A’’, B’’) at 0 rad (A–A’’) or 4 h after exposure to 4000 rad (B– B’’). All scale bars are 100 μm. (C) UMAP showing 35 cluster annotations with cluster annotations based on PD region. (D) UMAP showing broad epithelial region annotations. (E–H) UMAP plots showing the expression of markers used to annotate specific epithelial regions at 0 rad (top row) and 4000 rad (bottom row); Ubx for PE (E), tup for notum (F), zfh2 for hinge (G), nub for pouch (B). Combinations were used to determine hinge-notum and hinge-pouch regions. Plots for eyg (also used for the notum) and twi (used for the myoblasts) are shown in Figure 2—figure supplement 1.

Heterogeneity of gene expression across the wing disc following radiation exposure

(A) 521 genes across 9 functional categories plotted as points by their HHI score at 4000 rad. Point color represents the log2FC between conditions in the cluster that has the highest log2FC. The horizontal red bar represents the mean HHI score for the genes of that category. Genes with max log2FC > 2 or HHI > 0.075 were labeled where space permitted. The equation used to calculate the Herfindahl-Hirschman Index (HHI) is shown above the panel. (B) Heat map of 4 highest HHI scoring genes in apoptosis, DDR, ROS, cell cycle categories (low HHI scoring categories), and TF and ligand categories (categories with high HHI scoring genes). Box colors represent the proportion of mRNA found in each subregion relative to the sum of all mRNA found in all clusters (the values used to calculate HHI). (C) X-ray induced genes plotted by their HHI score (calculated on 7 broad regions, not the 35 clusters) and the Euclidean distance in gene expression in seven-dimensional space at 0 rad vs 4000 rad (calculated using the 7 broad regions as described in the text). Point color represents the log2FC of each gene when comparing all cells from the 4000 rad condition to all cells of the 0 rad condition. The dotted lines are drawn at the 95th percentile for each of the two parameters. (D–G) Gene expression UMAPs of example genes with <95th percentile Euclidean distance score belonging to the DDR (D), apoptosis (E), ROS (F), and ligand (G) groups. (H–I) Gene expression UMAPs of two genes with large differences in expression pattern between 0 rad and 4000 rad identified by a top 5% Euclidean distance. Genes in (D–I) are arranged from high (top) to low (bottom) HHI score.

X-ray-induced expression of genes encoding ligands

(A) Heatmaps showing log2FC between conditions for each cluster (left) and proportion of total mRNA in each cluster at 4000 rad (right) of the top 14 ligands ranked by max log2FC in any cluster. A Left: Boxes are colored based on the average log2FC from 0 rad to 4000 rad for each cluster. The left number in each box is the percentage of cells in the cluster expressing at least one transcript at 0 rad; the right number is the same at 4000 rad. Dark black numbers indicate that the change is statistically significant in that cluster (adjusted p value < 0.05, Wilcoxon Rank Sum Test, Bonferroni correction), while light gray numbers indicate that it is not (adjusted p value ≥0.05). Genes are sorted from left to right in descending order of the mean log2FC when comparing all cells of 0 rad to all cells of 4000 rad (“overall mean log2FC”); The overall mean log2FC value is in parentheses next to gene names (all significant). A Right: Boxes are colored based on the proportion of mRNA expressed in that cluster vs the total amount of mRNA expressed in all clusters. Genes are sorted from left to right in ascending order of HHI score. HHI score is noted in parentheses next to gene name. Genes shown in panels (B–G) are underlined with gray bars in (A). (B–M) In each panel: HCR signal of upd1, upd2, upd3 are in yellow and DAPI in blue at 0 rad (B, D, F, H, J, L) or 4000 rad (C, E, G, I, K, M). Wild-type discs (B, C, F, G, J, K) are compared to p53 mutant discs (D, E, H, I, L, M). To the right of each wild-type HCR image is an expression UMAP of each gene in each condition. The myoblast cluster was cropped into UMAP images for space, indicated by the dotted box around them.

X-ray induced expression of transcription factors.

(A) Heatmaps showing log2FC between conditions for each cluster (left) and proportion of total mRNA in each cluster at 4000 rad (right) of the top 14 TFs ranked by max log2FC in any cluster. Generated in the same way as figure 4A. Genes shown in panels (B–T) are underlined with gray bars in (A). (B, G, L, Q, S) HCR of p53, Ets21C, dysf, Xrp1, and Dif at 0 rad in Oregon R wing discs. (C, H, M, R, T) HCR of the same genes at 4000 rad in Oregon R wing discs. HCR signal is in yellow and DAPI in blue. To the right of 0 rad and left of 4000 rad HCR image is an expression UMAP of each gene in each condition. (D, I, N) Alternative irradiated wing disc showing non-induction of dysf after X-ray exposure. The myoblasts were cropped into the UMAP images for space, indicated by the dotted box around them. (E, J, O) HCR of p53, Ets21C and dysf in p53[5A-1-4] mutant wing discs at 0 rad. (F, K, P) HCR signal of the same genes at 4000 rad in p53[5A-1-4] mutant wing discs. HCR signal is in yellow and DAPI in blue.

Clustering of cells on 175 cell cycle genes

(A–A’) Cluster UMAPs of data processed and clustered on cell cycle genes at 0 rad (A) and 4000 rad (A’). (B–C’) Expression of trbl and PCNA in this UMAP object at 0 rad (B, C) and 4000 rad (B’, C’). (D) Heatmap showing average scaled expression of cell cycle marker genes in each cell cycle cluster considering both integrated conditions. Numbers are the percent of cells expressing each gene in each cluster. (E) Bar plot showing the proportion of cells each cluster contributes to total cells at 0 rad (cream) and total cells at 4000 rad (teal) conditions. (F–H’) High-trbl cluster (in orange) and both high-PCNA clusters (in purple) from panels A-A’ mapped onto the standard UMAP at 0 rad (F) and 4000 rad (F’). (G–H’) Standard UMAPs showing expression of PCNA and trbl at 0 rad (G, H) and 4000 rad (G’, H’).

Differences in X-ray induced gene expression between cell-cycle-based clusters

(A) Mean scaled expression of X-ray induced genes with avg log2FC ≥ 1 between conditions. Mean scaled expression values were calculated from cell-cycle-based clusters using cells from the 4000 rad condition only. Each row contains the same genes. (B) Heatmap showing the average scaled expression of the top 24 X-ray-induced genes with highest expression in the High-trbl Cluster. (C) 24 of the genes with maximum expression in a cell-cycle-based cluster other than the High-trbl Cluster. Numbers are the percentage of cells expressing the gene in each cluster. Only genes with ≥ 5% expression in any cluster at 4000 rad were selected from the initial 359.

HHI scoring of categorical genes in standard clusters vs cell-cycle-based clusters

(A–I) Transformed HHI scores calculated using the 35 standard clusters (y-axis) and the 6 cell-cycle-based clusters (x-axis). Genes used were the 521 categorical genes shown previously in Figure 3A. Scores were calculated using cells from the 4000 rad condition only. Genes displayed encode for proteins involved in apoptosis (A), DDR (B), ROS (C), cell cycle (D), TFs (E), phosphatases (F), kinases (G), ligands (H), and receptors (I). Genes are labeled where space permits but were otherwise chosen for labeling arbitrarily.

Two types of heterogeneity following X-ray irradiation

(A) Inter-regional heterogeneity. Many genes are expressed uniformly while some are expressed in specific territories following irradiation.

(B) Intra-regional heterogeneity. In the example shown, a gene is expressed at high levels in some cells, intermediate levels in some, and no expression in others. Cells with different levels of expression are interspersed.

UMAP plots of top cluster markers by region

(A) Expression UMAPs of eyg at 0 rad (left) and 4000 rad (right). (B) Expression UMAPs of twi at 0 rad (left) and 4000 rad (right). (C) Cropped and zoomed in UMAP plots of clusters belonging to each of the major PD regions. Each cluster is labeled by its cluster number and the genes that are most highly expressed in it when compared to all clusters (left of the slash) and when compared to other clusters within the same PD region (right of the slash).

Histogram of cluster HHI scores, broad region HHI scores, and Euclidean expression distance scores.

(A) Histogram of cluster HHI scores for 3,767 X-ray induced genes that meet the following criteria: Adjusted p value < 0.05, average log2FC > 0.1, percent of cells expressing the gene in either condition ≥0.01. Inset is the same histogram cropped on the y-axis to better view the distribution. (B) Histogram of broad region HHI scores of 3,655 X-ray induced genes that meet the following criteria Adjusted p value <0.05, Average log2FC>0.1, Percent of cells expressing the gene in both conditions ≥ 1%. Inset is the same histogram cropped on the y-axis to better view the distribution. (C) Histogram of Euclidean expression distances for the same 3,655 genes as in (B). Inset is the same histogram cropped on the y-axis to better view the distribution.

Proportion mRNA heatmaps of ligands and transcription factors

Heatmaps showing the proportion of mRNA found in each of the 35 clusters in the 4000 rad condition for all genes in the ligand (A) and TF (B–B’) gene categories.

Proportion mRNA heatmaps of apoptosis, ROS, cell cycle, and DDR genes

Heatmaps showing the proportion of mRNA found in each cluster in the 4000 rad condition for all genes in the Apoptosis (A) ROS (B), cell cycle (C), and DDR (D) gene categories.

Proportion mRNA heatmaps of kinase, phosphatase, and receptor genes

Heatmaps showing the proportion of mRNA found in each cluster in the 4000 rad condition for all genes in the kinase (A–A’), phosphatase (B), and receptor (C) gene categories.

Examples of genes spanning range of Euclidean expression distance scores

(A) Heat map showing the difference in the proportion of gene expression in each PD region between 4000 and 0 rad. Numbers in the tiles are the values of this difference with positive values indicating a higher proportion at 4000 rad for a given region, and negative values indicating a lower proportion at 4000 rad. For example, a value of 0.4 means that the proportion of expression in a given region at 4000 rads was 0.4 greater than at 0 rads and a value of −0.4 means that it is 0.4 less. Euclidean distance was calculated on the absolute values of these differences. Genes were selected to span the range of Expression Distances. (B) Plot showing the distribution of Euclidean expression distances for the 3,655 genes plotted in Figure 3C. The dotted line is drawn at the 95th percentile of Euclidean expression distance scores. Genes from panel A are highlighted. (C) Expression UMAPs of genes spanning the range of Euclidean expression distance scores, ordered in ascending order of Euclidean expression distance (number above UMAPs). The dotted line separates genes below (left of the line) and above (right of the line) the 95th percentile.

Toll and PDGF/VEGF ligands and receptors

Expression UMAPs of Toll receptors (A), Toll ligands (B), PDGF/VEGF receptors (C), and PDGF/VEGF ligands (D).

JAK/STAT receptors and TNF ligands and receptors

Expression UMAPs of JAK/STAT receptors (A), TNF receptors (B), and TNF ligands (C).

Expression UMAP of Toll TF, high magnification images of dysf HCR, and increased intensity images of Xrp1 and p53

(A) Heatmap of the Toll TF Dl. (B) 63X magnification images of dysf HCR. White arrowheads point to the location of normal developmental expression of dysf in the posterior hinge (pouch is below this location). Magenta arrowheads point to X-ray induced dysf mRNA which is localized to the nucleus. Scale bars are 50 μm. (C) HCR of p53 and Xrp1 with increased gain. Images in each row have the same min/max display. Scale bars are 100 μm.

Cluster tree showing cluster stability at different clustering resolutions

Tree showing clusters generated using different Louvain clustering resolution parameters on the integrated principal component dimensions derived from the 175 cell cycle genes. Each node is a cluster (clusters are numbered in descending order of cluster size but the numbering is otherwise arbitrary). Each row of clusters from top to bottom are derived from increasing resolutions. Arrows represent cells moving from one cluster identity at a lower resolution to a different cluster identity at a higher resolution. Arrow color represents the number of cells making the transition, and arrow transparency represents the proportion of contribution of those cells to the new cluster.

Dendrogram of cell-cycle-based cluster relationships

Dendrogram showing the relationship of cell-cycle-based clusters to one another in integrated PC space generated from 175 cell cycle genes. Shorter lines represent a closer relationship; Longer lines a more distant relationship.