Cis-interactions between Notch and its ligands block ligand-independent Notch activity
- Florida State University, United States
Figures
Figure 1 with 2 supplements
Follicle cells without DSL ligand bordering germline cells without DSL ligand show proper Notch activation and downstream differentiation.
Illustrations legend: active Notch = white cytoplasm, inactive Notch = red cytoplasm, WT cell = grey nuclei, mutant clone = white nuclei. (A–E). Follicle cells downregulate Cut at stage 7 of …
Figure 1—figure supplement 1
A schematic depiction of the early stages of Drosophila oogenesis.
Oogenesis begins in the germarium, where germline stem cells divide four times, producing a 16-cell germline cyst which is encapsulated by somatic follicle cells (FCs). When the FCs complete …
Figure 1—figure supplement 2
Z-stacked images of Dl-/Dl- clones and quantification of Cut staining in egg chamber clones.
Z series confocal images of DlrevF10SerRx82 (A) or DlrevF10 (B) germline/follicle cell clones from Figure 1D,E stained for Hnt (A) or Cut (B). Notice Hnt staining in the anterior end of (A) is owing …
Figure 2 with 1 supplement
Cis-ligand represses ligand-independent Notch activity in the follicle cells and imaginal discs.
DlrevF10 mutant MARCM germline/follicle cell clones co-expressing NotchRNAi show prolonged Cut expression (A). Su(H)47 MARCM mutant germline/follicle cell clones co-expressing DlRNAi show failure to …
Figure 2—figure supplement 1
Control experiments relating to Figure 2.
The Dl-/Dl- phenotype can also be recapitulated using DlRNAi, which knocks down Dl in both the germline and soma using the FLP-out method (A and B). See the arrowhead in (B) for wild-type (WT) Dl …
Figure 3 with 3 supplements
DSL-ligand-independent Notch activity in S2 cells is buffered by cis-ligand.
Trafficking is important for Notch activation in S2 cells, as treatment with Rab5 dsRNA (A) or hrs dsRNA (B) significantly decreases the amount of Notch activated in S2 cells as shown by …
Figure 3—figure supplement 1
Addition of Ser dsRNA had no effect on the Notch activation in S2 cells in comparison with cells treated with control green fluorescent protein (GFP) dsRNA, indicating that the small amount of Ser expression is either not translated or does not significantly contribute to Notch activation upon transfection with pMT-NFL.
This validates our assumption that the Notch activation which occurs in S2 cells is by a DSL-ligand-independent mechanism. Dl was not tested, as studies have already shown a lack of Dl mRNA and …
Figure 3—figure supplement 2
Notch accumulates in Dl-/Dl- clones.
Staining either Notch extracellular domain (A) or intracellular domain (B) showed increased Notch levels in DlrevF10 mutant germline/follicle cell clones. This could be seen as early as stage 2 …
Figure 3—figure supplement 3
Follicle cells mutant for ESCRT component tsg101 show early Notch activity in the follicle cells (Vaccari et al., 2008).
tsg101111019 clones show early Cut downregulation.
Figure 4 with 3 supplements
Notch ligand buffers against genetically induced DSL-independent activation.
Wing discs were stained with Wg antibody and illustrations are colored red where Wg is expressed (A–E). A wing disc with regions of interest is labeled and WT Wg staining shown (A). lgdd7/lgdd7 wing …
Figure 4—figure supplement 1
Co-expression of UAS-Dx and UAS-Serdel3 has a variable effect on DSL-independent Notch activation.
Wing discs were stained with Wg antibody (A–E). Illustrations show Wg staining in red, with lower intensities of Wg presence being shown in pink (A–E). ptcGAL4 driving green fluorescent protein …
Figure 4—figure supplement 2
Endogenous DSL-independent Notch activity in crystal cells is reduced by cis-inhibition.
Lz-GAL4-driven green fluorescent protein (GFP) expression is an efficient marker of crystal cells which show a low incidence of bursting (A and E). Misexpressing UAS-Serdel3 increased the frequency …
Figure 4—figure supplement 3
Reduced Notch reporter activity in crystal cells was not caused by indirect effects on early ligand-dependent Notch signaling in prohaemocytes.
Normal Hnt expression in crystal cells expressing green fluorescent protein (GFP) driven by lz-GAL4 (A) and in lz-GAL4 driving expression of UAS-SerWT (B). There was no noticeable effect on the …
Additional files
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Supplementary file 1
Supplementary clonal data file. Excel worksheet containing clonal data from the egg chamber and the wing disc.
- https://doi.org/10.7554/eLife.04415.016
