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), and d07478 (Lck), and one suppressor, d09869 (Cad99C), are shown. (B) Analysis of enrichment of GO terms among the 360 Drosophila genes identified in the screen. Only enriched terms with corrected p-value < 0.05 (using Benjamini–Hochberg correction) are shown. For numerical p-values, please see Supplementary file 2. (C) Gene association analysis among cell cycle genes identified in the genetic screen. Genetic interactions, physical interactions, predicted interactions, and shared protein domains were mapped using GeneMania (www.genemania.org) between the 31 cell cycle genes from our screen (black circles) and Notch (yellow). Genes labeled with grey circles are part of the network but were not identified in our screen.
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 coexpressed (A), the Nact large eye phenotype (C) is enhanced; in addition, occasional outgrowths of eye tissue can be seen (arrow). Note that d10338 alone (B) results in decreased eye size, whereas Nact alone (C) results in increased eye size compared to the control (D). Src42ACA and Src64B both cause a similar phenotype (E, G) when coexpressed with Nact under E1Gal4, and both also result in decreased eye size in the absence of Nact (F, H). (I–L) UAS-Nact and UAS-Src42ACA were driven in the developing wing using the vgGal4 driver. When Nact and Src42ACA are co-expressed (I), wing discs are overgrown compared to either Src42ACA (J) or Nact (K) alone and display a characteristic ‘crumpled ball’ phenotype indicative of tissue disorganization and cell migration. Note that Src42ACA alone (J) causes disorganization but not overgrowth. (M–P) Puc-LacZ reporter assay for JNK signal activation in wing discs expressing UAS constructs as indicated under the vgGal4 driver in a pucE69/+ background. Coexpression of Nact and Src42ACA (M) causes strong, global activation of the pucLacZ reporter. In contrast, expression of either gene alone (N, O) causes weaker activation that is limited in scope. Scale bars: 100 μm.
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 discs (blue bar) or MS1096Gal4/+ controls (red bar). Mean values are shown for two independent biological replicates.
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) compared to control (D). Scale bar: 100 μm.
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) and cl-casp3 (G), which is strongly reduced by BskDN (E, K). The combination of Nact and Mef2 results in an increase in MMP1 (F) but little effect on cc3 (L). (M) qPCR for egr and wgn in wing discs overexpressing genes as indicated under the vgGal4 driver reveals that both transcripts are strongly downregulated when Nact and Src42ACA are coexpressed. Scale bar: 100 μm.
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-Src42ACA (two UAS transgenes total) with vgGal4. Discs were stained for MMP1 (A, B) or cleaved caspase 3 (C, D). Scale bar: 100 μM.
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 independent experiments) than their FM7C/+;vgGal4/UAS-Src64B siblings (A, n = 16), and show a rescued phenotype similar to that of N55e11/+;vgGal4/+ controls (C). FM7C/+;vgGal4/UAS-Src64B (A) wings were indistinguishable from vgGal4/UAS-Src64B (D) wings. (E–H) Immunostaining for MMP1 (E, G) or cleaved caspase 3 (F, H) in wing discs with genotypes (D, E) FM7iGFP/+;vgGal4/UAS-Src64B or (F, G) N55e11/+;vgGal4/UAS-Src64B. Scale bar: 100 μm. (I–K) d10338 (Exelixis Src42A allele) lethality and phenotype at 25°C can be partially rescued by Notch RNAi. vgGal4/d10338 flies (I) are largely pupal lethal (n = 3 viable adults compared to 62 vgGal4/CyO-Tb siblings from the same cross) and the few escapers have no wings. In contrast, vgGal4/d10338, UAS-NRNAi flies (J) have narrow, short, and shriveled wings and much lower lethality (n = 67, compared to 108 vgGal4/CyO siblings from the same cross.) The wing phenotype appears to be a more severe version of the phenotype of vgGal4/UAS-NRNAi flies (K).
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 (cl-casp3, M–X). The combination of Nact and Src42ACA induces both MMP1 (A, G) and cl-casp3 (M, S), and Src42ACA alone does the same to a lesser extent (B, H, N, T). Green arrows: GFP positive cells that do not express MMP1 (G, H) or cl-casp3 (S, T). Red arrows: cl-casp3-positive cells that do not express GFP, indicating a potentially non-cell-autonomous effect. This effect can be largely rescued with BskDN (E, K, Q, W). Similarly, the combination of Src64B and Nact also induces both MMP1 (F, L) and cl-casp3 (R, X). Scale bar: 100 μM.
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), WT control (black), UAS-BskDN;UAS-Src64B;UAS-Nact (light green), UAS-Nact;UAS-Src42ACA (purple), UAS-Nact;d10338 (dark blue) or UAS- Nact;UAS-Mef2 (orange). Comparative histograms show relative frequencies on the y-axis, normalized to total number of counts for each sample. (B–E) EdU incorporation assay in dppGal4;UAS-GFP wing discs expressing d10338;UAS-Nact (B), d10338 (C), UAS-Nact (D), or UAS-GFP (E) at 22°C. A closeup of the areas denoted by boxes is shown below each image, and the GFP-positive area is marked with dotted yellow lines. Whereas UAS-Nact alone expands the ZNC (zone of non-proliferating cells) and also non-cell-autonomously induces proliferation in the dorsal-posterior region of the disc, thus increasing the size of the dorsal compartment (D), the combination of d10338 and UAS-Nact eliminates the expansion of the non-proliferative zone and causes cells within the ZNC proper to begin incorporating EdU; furthermore, the area of increased proliferation in the dorsal compartment appears to be expanded (B). (F–J) Nact and Src42ACA together cause a reduction in dacapo (dap) levels. (F) qPCR for dap expression in wing discs expressing Nact and/or Src42ACA or Mef2 under the vgGal4 driver. (G–J) A dap-LacZ reporter assay was used to visualize dap expression in vgGal4 wing discs in a dapk07309/+ background. Both Nact and Src42ACA together (G) and Src42ACA alone (H) show a reduction in dap-LacZ compared to both Nact alone (I) and vgGal4 controls (J). Scale bars: 100 μM.
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 (grey), or UAS-BskDN;UAS-Src64B;UAS- Nact (yellow-green). Comparative histograms show relative frequencies on the y-axis, normalized to total number of counts for each sample.
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 bars), and this upregulation is dependent upon JNK signaling as BskDN rescues it (lavender bars). Coexpression of Nact and Mef2 (orange bars) induces a much lower level of the upd ligands. Note that the y-axis is on a logarithmic scale. (B–E) An upd-LacZ reporter assay in vgGal4 wing discs validates the qPCR data and demonstrates that Nact+Src42ACA causes strong, widespread activation of upd transcription (B); in contrast, either gene alone (C, D) causes lower, more restricted levels of upd upregulation. (F–J) The 10XStatGFP reporter was used to assess JAK/STAT signal activation in vgGal4 discs grown at 18°C. Nact+Src42ACA strongly upregulates 10XStatGFP (G), whereas either gene alone (H, I) only weakly upregulates the reporter. The addition of BskDN (F) reduces the 10XStatGFP induced by Nact+Src42ACA (G) to levels similar to those of Nact alone (I). Note that since the upd-LacZ discs were grown at 25°C and the 10XSTATGFP discs were grown at 18°C, the latter displays a somewhat weaker phenotype, hence the difference in disc size between B/D and G/I. Scale bar: 100 μm.
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 (D) induces cut expression, which is suppressed in Nact+Src42ACA discs (B). Note that both ectopic and endogenous cut appear to be suppressed. (F) NRE-GFP expression in wing discs expressing Nact+Src42ACA under vgGal4. Scale bar: 100 μm.
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 reporter (Nellesen et al., 1999). Note that E(spl)mγ induced by Nact (C) is not suppressed by the addition of Src42ACA (A). Src42ACA alone (B) seems to suppress the endogenous E(spl)mγ staining (D) in the proneural cells.
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 bypass of G1 phase of the cell cycle via dap downregulation. Other downstream outputs, such as apoptosis, level of JAK/STAT activation, and regulation of Notch target genes, diverge between N/Src and N/Mef2 synergy.
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
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The Notch-mediated hyperplasia circuitry in Drosophila reveals a Src-JNK signaling axis
eLife 4:e05996.
https://doi.org/10.7554/eLife.05996
