The transcriptional response to tumorigenic polarity loss in Drosophila
- University of California, Berkeley, United States
- Department of Biological Sciences, United States
Figures
Figure 1 with 1 supplement
Transcriptome analysis of neoplastic tumors.
(A–C) F-actin staining reveals dramatic overgrowth and architecture defects of neoplastic dlg and scrib wing discs relative to WT. (D) Overlap of genes upregulated (left) or downregulated (right) in …
Figure 1—figure supplement 1
Decreasing oxidative stress or reexpressing eyelesss does not suppress neoplasia.
(A) 19 genes activated in response to oxidative stress are significantly upregulated upon polarity loss. (B–C) Loss of dlg leads to higher superoxide levels, as evidenced by increased DHE staining, …
Figure 2 with 1 supplement
JAK/STAT activation drives overgrowth upon polarity loss.
(A and B) A JAK/STAT pathway reporter (green) is highly elevated throughout dlg as compared to WT discs, indicating strong pathway activation. (C) The ligand-encoding upd genes, but not other …
Figure 2—figure supplement 1
upd3 knockdown is not sufficient to prevent neoplastic tumors.
Eye imaginal discs expressing upd3 RNAi alone (A), dlg RNAi alone (B), and dlg RNAi + upd3 RNAi (C). Scale bar: 50 μm.
Figure 3 with 2 supplements
Identification of a polarity-responsive enhancer in upd3.
(A) Schematic of upd3 reporter constructs in relation to the corresponding genomic region. (B and C) 3 kb upd3LacZ is not expressed in WT, but is upregulated in dlg discs. (D and E) upd3.3LacZ …
Figure 3—figure supplement 1
Imaginal expression of polarity-responsive target genes in neoplasia.
(A–B) The upd3LacZ and upd3.3LacZ reporters are expressed primarily in the disc proper, and not the hemocytes or the peripodial membrane. (C–H) The JNK pathway reporter AP-1-GFP, and transcriptional …
Figure 3—figure supplement 2
Conserved AP-1 and Sd binding sites in genes upregulated in neoplasia.
(A) The upd3.3 enhancer contains two evolutionarily conserved (between D. melanogaster, D. yakuba and D. erecta) AP-1 binding sites (green boxes), and one semi-conserved Sd binding site (red box). …
Figure 4 with 3 supplements
JNK-Dependent transcription is necessary for overgrowth and upd3.3 activation upon polarity loss.
WT wing discs (A) do not express either the JNK target Mmp1 or upd3.3LacZ (A′). Expression of dlgRNAi promotes overgrowth and disorganization (B), as well as Mmp1 and upd3.3LacZ upregulation (B′). …
Figure 4—figure supplement 1
Inhibitor constructs do not significantly affect WT tissue growth and viability.
(A–B) Blocking JNK activity with JNKDN or FosDN (C) has no effect on normal growth or tissue architecture, relative to wild-type. Expression of miRGH does not affect normal tissue architecture or …
Figure 4—figure supplement 2
Quantification of upd3.3LacZ staining.
(A) Expression of dlgRNAi increases upd3.3LacZ fluorescence, which is suppressed by blocking JNK or Trx activity. (B) Expression of aPKCact stimulates upd3.3LacZ in a JNK-independent, but …
Figure 4—figure supplement 3
Neoplasia induced by scrib loss is also dependent on JAK-STAT, JNK, and Yki pathway activity.
(A–C) Reducing JAK-STAT activity with DomeDN or Socs36E attenuates scribIR-mediated overgrowth. (D–H) Blocking JNK pathway activation by depletion of the JNK kinase hep or overexpression of JNKDN …
Figure 5 with 3 supplements
aPKC activity drives upd3.3LacZ activation in a yki-dependent manner.
(A) Expression of constitutively active aPKC (aPKCact) induces upd3.3LacZ and Mmp1 upregulation and neoplasia. (B) Expressing JNKDN suppresses Mmp1, but does not prevent aPKCact-mediated upd3.3LacZ …
Figure 5—figure supplement 1
Ectopic aPKC activity drives upd3.3LacZ in a JNK-independent manner.
(A) Expression of aPKCmild and its partner Par6 drives upd3.3LacZ as well as strong overgrowth and Mmp1 expression in the wing pouch. (B) Co-expression of JNKDN does not suppress upd3.3LacZ …
Figure 5—figure supplement 2
Scrib module and wts mutant expression profiles display limited overlap.
(A) Comparison of the Scrib module and wts mutant transcriptomes (see ‘Materials and methods’) reveals a limited degree of overlap. (B) Most canonical Yki growth targets are not upregulated in …
Figure 5—figure supplement 3
Co-activation of JNK and Yki are not sufficient to drive neoplasia.
(A–B) Ectopic expression of wild-type JNKK causes only slight morphological defects and upregulates Mmp1, but cannot activate upd3.3LacZ. (C) Co-expression of JNKK and Ykiact activates both upd3.3Lac…
Figure 6 with 1 supplement
The Scrib module and PcGs regulate common targets.
(A and B) Loss of the paralogous PcGs Psc and Su(z)2 leads to activation of upd3.3lacZ, along with dramatic overgrowth and architecture defects. Activation is observed in areas of epithelial …
Figure 6—figure supplement 1
PcG depletion does not cause widespread loss of polarity.
(A) Depletion of the paralogous PcGs ph-p and ph-d leads to overgrowth and upd3.3LacZ activation, including in areas with mild architecture defects. Arrows show areas of upd3.3LacZ expression in …
Figure 7
PcGs cooperate with Scrib module proteins to regulate growth.
(A–D) Knockdown of ph-p has little effect on WT growth but increases the growth of dlghypo tissue. Quantification is in E (***p < 0.0001). (F–G) BrmDN expression in dlgRNAi tissue decreases both upd3…
Additional files
-
Supplementary file 1
Transcriptome Analysis of scrib Tissue. Differential expression analysis of scrib versus white RNA-Seq data by DESeq. Each column contains the following information: Flybase ID- Flybase Gene Identifier; Gene Name- Name of each gene; baseMean_allconditions- Average normalized read count for that gene, across all samples, baseMean_white- Normalized read count for that gene in white tissue; baseMean_scribble- Normalized read count for that gene in scrib tissue; foldChange- Change of the gene in scribble, relative to white tissue; foldChangelog2- Logarithm to base 2 of the fold change; pval- p-value for the statistical significance of the fold change; padj- p-value adjusted for multiple testing with the Benjamini-Hochberg procedure, which controls for false discovery rate (FDR).
- https://doi.org/10.7554/eLife.03189.021
-
Supplementary file 2
Transcriptome Analysis of dlg Tissue. Differential expression analysis of dlg versus white RNA-Seq data by DESeq. Each column contains the following information: Flybase ID- Flybase Gene Identifier; Gene Name- Name of each gene; baseMean_allconditions- Average normalized read count for that gene, across all samples, baseMean_white- Normalized read count for that gene in white tissue; baseMean_discslarge- Normalized read count for that gene in dlg tissue; foldChange- Change of the gene in dlg, relative to white tissue; foldChangelog2- Logarithm to base 2 of the fold change; pval- p-value for the statistical significance of the fold change; padj- p-value adjusted for multiple testing with the Benjamini-Hochberg procedure, which controls for false discovery rate (FDR).
- https://doi.org/10.7554/eLife.03189.022
-
Supplementary file 3
Transcriptome Analysis of Psc/Su(Z)2 Tissue. Differential expression analysis of scrib versus white RNA-Seq data by DESeq. Each column contains the following information: Flybase ID- Flybase Gene Identifier; Gene Name- Name of each gene; baseMean_allconditions- Average normalized read count for that gene, across all samples, baseMean_iso- Normalized read count for that gene in iso42 tissue; baseMean_PscSuZ2- Normalized read count for that gene in Psc/Su(Z)2 tissue; foldChange- Change of the gene in scribble, relative to white tissue; foldChangelog2- Logarithm to base 2 of the fold change; pval- p-value for the statistical significance of the fold change; padj- p-value adjusted for multiple testing with the Benjamini-Hochberg procedure, which controls for false discovery rate (FDR).
- https://doi.org/10.7554/eLife.03189.023
-
Supplementary file 4
RNA-Seq alignment statistics. Table of combined number of 50-bp single-end sequencing reads for each sequencing replicate. Reads were considered ‘non-aligned’ if they had >2 mismatches relative to the reference genome, and ‘low complexity’ reads had multiple matches within the genome, reflecting sequencing reads from repeated DNA elements. Percentages listed refer to the number of reads for each category relative to the total number of reads.
- https://doi.org/10.7554/eLife.03189.024
-
Supplementary file 5
Contains the number of differentially expressed genes for each genotype.
- https://doi.org/10.7554/eLife.03189.025
-
Supplementary file 6
Contains the primer sequences used for quantitative PCR in Figure 7.
- https://doi.org/10.7554/eLife.03189.026
