The Notch-mediated hyperplasia circuitry in Drosophila reveals a Src-JNK signaling axis
- Harvard Medical School, United States
- Translational Health Science and Technology Institute, India
- Biogen Idec, United States
Figures
Figure 1
A genetic screen for modifiers of Notch-induced proliferation in the Drosophila eye.
(A) Examples of screen phenotypes. E1>Nact results in larger eyes (second panel), compared to wild-type (E1Gal4 alone) controls (top panel). Examples of three enhancers, c01597 (fng), c03191 (Mef2), …
Figure 2 with 2 supplements
Synergy between Notch and Src in the eye and wing causes hyperplastic phenotypes and activates JNK.
(A–H) Various UAS-Src constructs were driven by E1Gal4 along with UAS-Nact in the developing eye. When d10338, an Exelixis allele that causes Gal4-dependent overexpression of Src42A, and Nact are …
Figure 2—figure supplement 1
d10338 is a UAS allele of Src42A.
(A, B) UAS-GFP/d10338; dppGal4/+ wing discs were stained for anti-phosphoY418-Src (p-Src, red), which labels activated Src. Scale bar: 100 μm. (C) qPCR for Src42A in MS1096Gal4/+; d10338/+ wing …
Figure 2—figure supplement 2
Src64B also synergizes with Nact in the wing disc.
When driven with vgGal4, Src64B and Nact synergize to produce an overgrown, disorganized disc (A), whereas Src64B alone causes disorganization (B) and Nact alone causes large but organized discs (C) …
Figure 3 with 2 supplements
N/Src synergy induces both MMP1 and apoptosis.
(A–L) Immunofluorescence for MMP1 (A–F) and cleaved caspase 3 (cl-casp3, G–L) in wing discs expressing UAS constructs under vgGal4. Together, Nact and Src42ACA cause robust activation of both MMP1 (A…
Figure 3—figure supplement 1
Gal4/UAS titration does not affect the N/Src phenotype.
(A, C) One copy each of UAS-Nact, UAS-Src42ACA, and UAS-GFP (three UAS transgenes total) were driven with vgGal4 in the wing disc, and compared to (B, D) wing discs expressing only UAS-Nact and UAS-S…
Figure 3—figure supplement 2
A heterozygous null mutation of Notch can rescue lethality and phenotype of Src alone.
N55e11/FM7C;UAS-Src64B virgins were crossed to vgGal4 males at 18°C (A, B) and the resultant female progeny were scored. N55e11/+;vgGal4/UAS-Src64B flies were more viable (B, n = 126 over four …
Figure 4
dpp-Gal4 driven expression of Nact and Src42ACA also upregulates MMP1 and induces apoptosis.
UAS transgenes as indicated were driven with dppGal4 along with UAS-GFP at 18°C. Controls express an extra copy of UAS-GFP. Wing discs were stained with anti-MMP1 (A–L) or anti-cleaved caspase 3 …
Figure 5 with 1 supplement
N/Src synergy disrupts the cell cycle.
(A) DNA content analysis was performed on Hoechst-labeled dissociated cells from vgGal4;UAS-GFP wing discs expressing UAS-Src64B;UAS-Nact (dark green trace), UAS-Src64B (light blue), UAS-Nact (red), …
Figure 5—figure supplement 1
Elimination of G1 phase of the cell cycle also occurs in dppGal4 wing discs expressing Nact and Src64B.
DNA content analysis was performed on Hoechst-labeled dissociated cells from dppGal4;UAS-GFP wing discs expressing UAS-Src64B;UAS-Nact (green trace), UAS-Src64B (blue), UAS-Nact (red), WT control …
Figure 6
N/Src synergy activates the JAK/STAT signaling pathway.
(A) qPCR for unpaired family ligands in vgGal4 discs expressing UAS constructs as indicated. All three upd family genes are highly upregulated by the combination of Nact and Src42ACA (dark purple …
Figure 7 with 1 supplement
Notch targets are differentially affected by N/Src synergy.
(A) qPCR assay for expression levels of E(spl) complex members in vgGal4 wing discs expressing UAS constructs as indicated. (B–E) Immunostaining with anti-cut (red) in vgGal4 wing discs. Nact alone …
Figure 7—figure supplement 1
E(spl)mγ reporter staining in N/Src wing discs.
VgGal4 wing discs expressing UAS-Nact and/or UAS-Src42ACA in an E(spl)mγ-LacZ/+ background were stained for anti-β-gal. E(spl)mγ-LacZ consists of the 234-bp mγ enhancer region fused to a LacZ …
Figure 8
Model of convergence and divergence of the Notch/Mef2/JNK and Notch/Src/JNK signaling axes.
N/Mef2 and N/Src synergies converge on JNK, through eiger-dependent and eiger-independent means respectively. Some downstream processes are common to both synergies, such as MMP1 activation and …
Additional files
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Supplementary file 1
List of Exelixis mutant lines that enhance or suppress the E1Gal4; UAS-Nact -induced large eye phenotype. Lines in bold appear more than once in this table as they may affect more than one target gene. 26 of these lines were retested using qPCR for puc and MMP1 in vgGal4;UAS-Nact wing discs. These genes were determined to be upregulated if the fold change by qPCR vs isogenic wild-type controls was at least 1.5. MMP1 upregulation was further verified by immunostaining.
- https://doi.org/10.7554/eLife.05996.017
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Supplementary file 2
GO term enrichment analysis for Notch interactors. Enrichment analysis was performed with DAVID (http://david.abcc.ncifcrf.gov), using the total Exelixis gene list as background; corrected p-values are shown using both the Benjamini–Hochberg and Bonferroni corrections.
- https://doi.org/10.7554/eLife.05996.018
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Supplementary file 3
List of genes synergistically regulated by Nact and Src42ACA as determined by RNA-seq analysis (P-adj < 0.05 for NS vs N, S, and WT). Genotypes: NS: vgGal4/UAS-Nact; UAS-Src42ACA/+, S: vgGal4/+; UAS-Src42ACA/+, N: vgGal4/UASNact, WT: vgGal4/+, BNS: UAS-BskDN/+, vgGal4/UAS-Nact, UAS-Src42ACA/+.
- https://doi.org/10.7554/eLife.05996.019
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Supplementary file 4
RNA-seq data for known N/Src targets. Genotypes: NS: vgGal4/UAS-Nact; UAS-Src42ACA/+, S: vgGal4/+; UAS-Src42ACA/+, N: vgGal4/UASNact, WT: vgGal4/+, BNS: UAS-BskDN/+, vgGal4/UAS-Nact, UAS-Src42ACA/+.
- https://doi.org/10.7554/eLife.05996.020
