Cis-interactions between Notch and its ligands block ligand-independent Notch activity

The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity. DOI: http://dx.doi.org/10.7554/eLife.04415.001

Dl-/Dl-follicle cell clones (retaining a cis-ligand but without a trans-ligand) are comparable to wild-type cells before entry to endocycle ( Figure 1F,G). Removal of both cis-and trans-Dl through knockdown of Dl by RNA interference (RNAi) simultaneously in the germline and soma confirmed this finding ( Figure 2-figure supplement 1A,B). Together, these observations provide evidence that follicle cells without both cis-and trans-ligand sources can still enter the endocycle stages of oogenesis. This back-up route to the endocycle is not a co-option of Ser in place of Dl, as Dl RevF10 Ser Rx82 double clones recapitulated the Dl-/Dl-phenotype ( Figure 1E, Figure 1-figure supplement 2A).
To determine whether the entry into the endocycle in Dl-/Dl-follicle cells still requires the function of Notch, we implemented the mosaic analysis with a repressible cell marker (MARCM) system (Lee and Luo, 2001). The MARCM system enables us to create mutant clones while driving expression of a UAS transgene specifically in those clonal cells. Dl-/Dl-clones driving expression of Notch RNAi show a significantly higher proportion (p < 0.0001) of late Cut-expressing cells than the Dl-/Dl-clones alone, indicating that Notch is still required for the mitotic-to-endocycle switch ( Figures 1D and 2A (Stempfle et al., 2010) was also upregulated in Dl-/Dl-clones as early as stage 2, and this expression persisted beyond stage 6 ( Figure 2C,D), suggesting that NRE-GFP is probably more sensitive to Notch activation than Hnt in follicle cells. Together, these results suggest that Notch activity occurs independently of canonical ligands when both cis-and trans-ligands are removed, resulting in normal downstream developmental events in the follicle cells. Consistently, Dl RevF10 Ser Rx82 double mutant clones in the wing and eye discs show a slight cell-autonomous upregulation of NRE-GFP in the clone center, which would only occur if cis-inhibition blocked a DSL-independent mode of Notch activity, as interior cells have no access to trans-ligand ( Figure 2E,F). This NRE-GFP eLife digest Many biological processes require cells to send messages to one another. Typically, this is achieved when molecules are released from one cell and make contact with companion molecules on another cell. This triggers a chemical or biological reaction in the receiving cell.
One of the most common examples of this is the Notch pathway, which is used throughout the animal kingdom and plays an important role in helping cells and embryos to develop. The Notch protein itself is a 'receptor' protein that is embedded in the surface of a cell, and relays signals from outside the cell to activate certain genes inside the cell. In fruit flies, two proteins called Serrate and Delta act as 'ligands' for Notch-by binding to Notch, they can change how this receptor works.
If Serrate or Delta are present on the outside of one cell, they can activate Notch (and hence the Notch signaling pathway) in an adjacent cell. However, if the Serrate or Delta ligands are present on the surface of the same cell as Notch they turn the receptor off, rather than activate it. Notch can also work without being activated by Serrate or Delta, but whether the ligands can inhibit this 'ligandindependent' Notch activation if they are on the surface of the same cell as the Notch receptor was unknown.
Palmer et al. study Notch signaling in the fruit fly equivalent of the ovary, in cells that are naturally deficient in Serrate and from which Delta was artificially removed. The Notch protein was activated when these ligands were not present. Furthermore, the developmental processes that are activated by Notch were able to proceed as normal when triggered by ligand-independent Notch signaling. In total, Palmer et al. investigated three different types of fruit fly cell, and found that ligandindependent Notch signaling can occur in all of them.
Reintroducing Delta to the same cell as Notch turns the receptor off, suggesting that ligands on the surface of the same cell as the receptor can inhibit ligand-independent Notch activity. Many genetic diseases and cancers have been linked to Notch being activated when it should not be; therefore, understanding how Notch is controlled could help guide the development of new treatments for these conditions.  These germline/follicle cell clones (D and E) show increased nuclear size comparable to wild-type (WT) follicle cells which have entered the endocycle (n = 8 for each stage/genotype) (F and G). For (G), Welch t-tests were done to assess significance between each condition. The only comparisons that were not significant were between WT stage 10B and Dl-/Dl-clones and between WT stage 6 and Dl germline clones, indicating nuclear size in germline clones alone is similar to that of cells before the endocycle, whereas Dl-/Dl-clonal nuclei are more similar in size to cells that have entered the endocycle. Scale bars represent 20 μm, except in F, where the scale bar represents 5 μm.   upregulation was spatially variable in the wing disc, having the highest prevalence in the notum region (25% incidence), a low incidence in the dorsal pouch (8%), whereas in the ventral pouch region it was never seen (n = 80) (Supplementary file 1), perhaps owing to the differential regulation of Notch degradation throughout the wing disc (Hori et al., 2011). As reported previously, most wing disc clones showed a higher NRE-GFP upregulation in the clone boundary where there is access to trans-ligand, indicating that the ligand-independent Notch activity observed occurs at a rather low level.
Drosophila S2 cells are reported to have no Dl expression and a very low level of Ser expression, which had no effect on Notch signaling (Fehon et al., 1990;Graveley et al., 2011) (Figure 3-figure supplement 1), and have been used as a model to study ligand-independent Notch activity (Hori et al., 2011). Upon transfection with pMT-N FL , a CuSO 4 -inducible full-length Notch construct, Notch activation was increased by a factor of 5.13 compared with the control cells, as indicated by a NRE-firefly luciferase reporter gene (p < 0.0001) ( Figure 3C). Notch activation in S2 cells is at least partially dependent on endosomal trafficking, as double-stranded (ds) RNA against early endosome component, Rab5, or multivesicular body sorting protein, hrs, reduced the levels of Notch activation ( Figure 3A,B). This is consistent with the in vivo studies indicating that ligand-independent Notch activation relies heavily on receptor trafficking (Hori et al., 2012) (Rab5 p = 0.00623, hrs p = 0.0159), and our observation that Notch accumulates in Dl-/Dl-clones (Figure 3-figure supplement 2). A requirement for trafficking is consistent with the results of others who have demonstrated aberrant Notch activation in follicle cell mutants for trafficking components (Wilkin et al., 2004;Vaccari et al., 2008;Schneider et al., 2013), such as tsg101 mutant clones, which show early Notch activation in the follicle cells ( Figure 3-figure  supplement 3). Furthermore, co-transfecting pMT-N FL with pMT-GAL4 and pUASt-Ser del3 , a form of Ser that cannot activate Notch, but only cis-inhibit, (Fleming et al., 2013) almost entirely abolished the Notch activation detected when N FL was transfected alone (p = 0.0048) ( Figure 3C). These results suggest that if Notch is expressed in a cell free of cis-and trans-ligands, DSL ligand-independent activity will occur and that cis-inhibition is extremely efficient in preventing this 'accidental' Notch activity as it travels through the endosomal pathway en route to degradation.
We next explored whether cis-inhibition can also block ligand-independent Notch activity induced in aberrant genetic backgrounds. The Notch target, Wingless (Wg) is normally expressed along the dorsoventral boundary of the wing disc ( Figure 4A). Lethal giant disc (lgd) homozygous mutant (lgd d7 ) larvae display overgrown imaginal discs and ubiquitous ligand-independent Notch activation in the wing pouch region, as shown by upregulation of Wg ( Figure 4B). Notch activation in lgd mutant cells is caused by a defect in Notch trafficking and degradation, as the receptor is aberrantly transported to the limiting membrane of the lysosome which facilitates production of N ICD (Childress et al., 2006;Gallagher and Knoblich, 2006;Jaekel and Klein, 2006;Schneider et al., 2013). Using dpp-GAL4 to misexpress UAS-Dl along the anterior-posterior axis of the wing disc in lgd d7 homozygous larvae, Wg expression was considerably reduced along the dpp expression domain, indicating that cis-inhibition can block the ligand-independent Notch activity observed in this situation ( Figure 4C). Overexpression of Deltex (Dx), an E3 ubiquitin ligase that stimulates Notch monoubiquitination and promotes its trafficking to the lysosomal limiting membrane, has also been shown to induce ligand-independent Notch activation specifically in the ventral wing pouch region (Matsuno et al., 2002;Hori et al., 2004;Wilkin et al., 2008;Schneider et al., 2013) (Figure 4D). We used patched (ptc)-GAL4 to drive expression of UAS-Dx with either UAS-Dl or UAS-Ser del3 , whose ectopic expression leads to a reduction of Wg staining along the dorsoventral boundary (Micchelli et al., 1997;Fleming et al., 2013) (controls in Figure 4figure supplement 1A,E). Co-expression of Dx and Dl led to a decrease in Wg expression in the ventral ptc domain as compared with expression of Dx alone ( Figure 4E). When UAS-Dx and UAS-Ser del3 were co-expressed, there was a small but noticeable, albeit variable, decrease in Dx-induced Notch activation (Figure 4-figure supplement 1B-D). This incomplete reduction was probably due to the previously noted, slightly compromised, cis-inhibitory potential of UAS-Ser del3 (Fleming et al., 2013) (Figure 4-figure supplement 1A). Taken together, these results provide evidence that cisligand has a negative effect on the raised levels of DSL-ligand independent Notch activation incurred in genetically abnormal cells.
To quantify this effect, we co-transfected pMT-Dx with pMT-N FL , causing an increase by a factor of 4.21 (p = 0.0021) in the Notch activation compared with transfecting pMT-N FL alone ( Figure 4F). Transfection of pMT-N FL , pMT-Dx, pMT-GAL4, and pUASt-Ser del3 significantly (p = 0.0194) reduced the level of Notch activation ( Figure 4F). We next treated cells with dsRNA for either lgd or shrub (a component of the ESCRT-III complex). Lgd dsRNA induced an increase in Notch activation by a factor of 1.73 compared with GFP dsRNA-treated cells (p = 0.00286) ( Figure 4G). Likewise, shrub dsRNA caused a 3.93-fold increase (p < 0.0001) in Notch activation in S2 cells ( Figure 4H) (Thompson et al., 2005). Expression of Ser del3 in both situations led to a significant decrease in the amount of Notch activated in comparison with Notch-expressing cells treated with control dsRNA (lgd p = 0.0093, shrub p = 0.0257) ( Figure 4G,H).
To explore whether cis-acting ligands might block endogenous raised levels of ligand-independent Notch activation, in addition to the raised levels induced by genetic defects, we examined the effect of increased ligand expression in crystal cells in the larval lymph gland, which have recently been shown to have ligand-independent Notch activation (Mukherjee et al., 2011). Notch activity in crystal cells promotes cell survival, and decreased Notch activity leads to a 'bursting' phenotype (Mukherjee et al., 2011) (Figure 4-figure supplement 2B,E). Evidence for this bursting phenotype is provided by the disorganization of membrane-associated GFP (Mukherjee et al., 2011). Using Lozenge (Lz)-GAL4, a crystal cell lineage-specific driver (Terriente-Felix et al., 2013) to misexpress UAS-Notch RNAi or UAS-Ser del3 led to a significantly higher proportion of cells showed the 'bursting' phenotype than wild-type crystal cells (Notch RNAi p = 0.0434, Ser del3 p = 0.0286) (Figure 4-figure supplement 2A,B,E). Furthermore, overexpression of UAS-Ser WT led to a significant decrease of the Notch reporter E(spl):mβ-CD2expression in mature crystal cells (Figure 4-figure supplement 2C,D,F). Reduced Notch reporter activity was not caused by indirect effects on early ligand-dependent Notch signaling in prohaemocytes, as Hnt, a Notch target in differentiating crystal cells, (Terriente-Felix et al., 2013) was unaffected by ligand misexpression (Figure 4-figure supplement 3A,B). These observations indicate that increased ligand expression in crystal cells decreases cell survival by blocking Notch ligand-independent activation, and therefore the buffering role of cis-expressed ligand can be extended to endogenous cases of DSLindependent Notch activity.
In this study, we show that cells devoid of DSL ligands activate Notch sufficiently to stimulate reporter activity, and in the ovarian follicle cells the level of activation is above the threshold required to mediate normal Notch-induced downstream developmental events. During development, this type of noncanonical Notch activity is normally prevented by cis-expressed DSL ligands in numerous tissues. Cis-inhibition can also attenuate DSL-ligand independent Notch activity both in endogenous and genetically induced situations. Mechanistically, this could be explained if DSL ligands sequestered Notch at the membrane, made Notch more sensitive to degradation, or increased the stability of the heterodimer as it travels through the endosomal pathway. As we and others (Fiuza et al., 2010) have shown that increasing or decreasing ligand has variable effects on receptor distribution among tissues, and given that we observe a consistent effect among tissues on Notch activation upon cis-ligand removal, we prefer the stability hypothesis. Fiuza et al. (2010) show that ligand affects Notch stability during Notch activation by EDTA, giving support to the stability hypothesis as the most parsimonious explanation (Fiuza et al., 2010). It is suggested that retaining a pool of translated Notch receptor keeps the pathway in a condition capable of almost instant activation (Sprinzak et al., 2010). Therefore, we propose that a role of cis-ligands might be to keep the Notch pathway in a state of readiness by buffering against unintentional stochastic Notch activity resulting from normal processing through the endosomes. Endogenously, this may aid the ability of a cell to mediate future Notch-dependent developmental events that have strict temporal regulation.

S2 cell transfection and RNA interference
S2 cells were grown under standard conditions and passaged once every three days in serum-free Gibco media (Invitrogen, Waltham, MA) supplemented with antibiotics. In preparation for transfection 10 6 cells per milliliter were seeded into either 24-well plates or 96-well plates for experiments with or without dsRNA treatment, respectively. Transfections were carried out with Qiagen Effectene (Qiagen, Netherlands) transfection reagent according to the manufacturer's instruction. Plasmids used for transfection were pMT-Notch FL (a gift from Renjie Jiao), pMT-GAL4 (DGRC #1042), pUASt-Ser del3 (a gift from Robert J Fleming), pMT-Deltex (a gift from Spyros Artavanis-Tsakonas), NRE-firefly luciferase (a gift from Sarah Bray), or Renilla luciferase (a gift from Sarah Bray). Aliquots (75 ng for 24-well plates or 50 ng for 96-well plates) of each non-luciferase plasmid were added and, where applicable, 10 ng of each luciferase plasmid. DNA concentration between transfections was kept constant with an empty vector. For experiments without dsRNA treatment, CuSO 4 was added to a concentration of 500 µM 24 hr after transfection, and cells were assayed 24 hr later. dsRNA was transcribed in vitro using the RiboMAX large-scale RNA production system-T7 kit (Promega, Madison, WI). The following primers were used to amplify genomic DNA taken from a single male fly from the NRE-GFP stock:

GFP
Forward: GAATTAATACGACTCACTATAGGGAGCTGGACGGCGACGTAAAC Reverse: GAATTAATACGACTCACTATAGGGATGGGGGTGTTCTGCTGGTAG Cells were treated with dsRNA at a concentration of 50 nM, and then transfected shortly after. CuSO 4 was added to a concentration of 500 µM later that day. Cells were incubatedfor five days, with an additional treatment of dsRNA on the fourth day.

Luciferase assay
Cells were transfected with plasmids of interest together with an NRE-driving firefly luciferase expression and a constitutively activated Renilla luciferase to control for transfection efficiency. Luciferase measures were inspected with the Dual-Luciferase Assay Kit (Promega) in 96-well luminometer plates. Each transfection was performed in duplicate and repeated several times. Student's t test was used to test for statistical significance.