Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila

SUMMARY

Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. Here, we show that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, we define a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP.

In brief

Shields et al. define a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as apoptosis-induced proliferation. This work identifies conceptual similarities between cell competition and apoptosis-induced proliferation.

Graphical Abstract

INTRODUCTION

Since the discovery of the original Toll gene in Drosophila (Toll-1 hereafter), a large number of Toll-related genes have been identified in both insects and mammals (reviewed in Anthoney et al., 2018; Bilak et al., 2003). The Drosophila genome encodes a total of 9 Toll-related genes, including Toll-1 (Ooi et al., 2002; Tauszig et al., 2000), while mammalian genomes encode between 10 and 13 Toll-like receptors (TLRs). TLRs are single-pass transmembrane proteins that upon ligand stimulation usually trigger a conserved intracellular signaling pathway, culminating in the activation of NF-κB transcription factors (Anthoney et al., 2018).

Initially identified as an essential gene for dorsoventral patterning in the early Drosophila embryo (Anderson et al., 1985a, 1985b) (reviewed by Nusslein-Volhard, 2021), Toll-1 was later also found to be a critical component for innate immunity (Lemaitre, 2004; Lemaitre et al., 1996). In this function, Toll-1 signaling via the NF-κB transcription factors Dorsal and Dorsal-related immunity factor (Dif) induces the expression of anti-microbial peptides (AMPs) that mediate innate immunity (Meng et al., 1999; Rutschmann et al., 2000; Tauszig et al., 2000). A role in innate immunity has also been demonstrated for all mammalian TLRs (Lemaitre, 2004). Likewise, Drosophila Toll-8 (aka Tollo) regulates immunity in the trachea (Akhouayri et al., 2011). Toll-7 may regulate anti-viral responses, albeit through an NF-κB-independent mechanism (Benton et al., 2016; Lamiable et al., 2016; Nakamoto et al., 2012; Pare et al., 2014). However, for the remaining Toll-related proteins in Drosophila, a function in innate immunity has not been clearly demonstrated (Anthoney et al., 2018; Tauszig et al., 2000).

Of particular interest is Toll-9 in Drosophila because it behaves genetically most similar to Toll-1 (Bettencourt et al., 2004; Ooi et al., 2002), and its intracellular TIR domain is most closely related to those of the mammalian TLRs (Khush and Lemaitre, 2000; Ooi et al., 2002; Wasserman, 2000). Overexpression of Toll-9 results in the production of AMPs, which led to the proposal that Toll-9 might be involved in innate immunity (Ooi et al., 2002). However, a loss-of-function analysis with a defined Toll-9 null allele did not confirm this prediction (Narbonne-Reveau et al., 2011). Nevertheless, although Drosophila Toll-9 does not appear to be directly involved in regulating expression of AMPs, it has been implicated together with a few other Toll-related proteins in cell competition, an organismal surveillance program that monitors cellular fitness and eliminates cells of reduced fitness (losers) (Alpar et al., 2018; Meyer et al., 2014). Depending on the type of cell competition, Toll-9 participates in the expression of the pro-apoptotic genes reaper and hid in loser cells, triggering their elimination (Alpar et al., 2018; Meyer et al., 2014). Therefore, it has been proposed that the function of Toll signaling for elimination of bacterial pathogens by AMPs and for elimination of unfit cells by pro-apoptotic genes bears a conceptual resemblance between innate immunity and cell competition (Alpar et al., 2018; Meyer et al., 2014).

Apoptosis is an evolutionarily well-conserved process of cellular suicide mediated by a highly specialized class of Cysproteases, termed caspases (reviewed by Fuchs and Steller, 2011; Salvesen et al., 2016; Shalini et al., 2015). In Drosophila, the pro-apoptotic genes reaper and hid promote the activation of the initiator caspase Dronc (caspase-9 homolog in Drosophila). Dronc activates the effector caspases DrICE and Dcp-1 (caspase-3 and caspase-7 homologs), which trigger the death of the cell (reviewed in Salvesen et al., 2016; Shalini et al., 2015). However, caspases not only induce apoptosis but can also have non-apoptotic functions (reviewed by Aram et al., 2017; Baena-Lopez, 2018) such as apoptosis-induced proliferation (AiP), during which caspases in apoptotic cells promote the proliferation of surviving cells independently of their role in apoptosis (Diwanji and Bergmann, 2019; Fogarty and Bergmann, 2017).

Early work has shown that AiP requires the initiator caspase Dronc (Fan et al., 2014; Huh et al., 2004; Kondo et al., 2006; Wells et al., 2006). To reveal the mechanism of AiP, we are expressing the pro-apoptotic gene hid and the apoptosis inhibitor p35 simultaneously using the ey-Gal4 driver (referred to as ey > hid,p35) which drives expression in the larval eye disc anterior to the morphogenetic furrow (MF) (Fan et al., 2014). p35 encodes a specific inhibitor of the effector caspases DrICE and Dcp-1 but does not block the activity of Dronc (Hawkins et al., 2000; Meier et al., 2000; Yoo et al., 2002). In ey > hid,p35-expressing discs, the apoptotic pathway is activated by hid expression but blocked downstream because of DrICE and Dcp-1 inhibition by P35, rendering cells in an “undead” condition. Although apoptosis is inhibited in undead tissue, AiP still occurs because of non-apoptotic signaling by the initiator caspase Dronc, triggering hyperplasia of the anterior portion of the larval eye imaginal discs at the expense of the posterior eye field, which is reduced in size (Fan et al., 2014). Combined, these effects result in overgrowth of the adult head capsule (Figure 1B), but a reduction or even absence of the eyes in the adult animal (Fan et al., 2014).

Figure 1. Toll-9 is required for ey > hid,p35-induced overgrowth, ROS generation, and hemocyte activation.

(A–D) Reduction of Toll-9 activity by RNAi (C) or the heterozygous Toll-9^rv17 mutant (D) suppressed ey > hid,p35-induced overgrowth (B). Arrow in (B) points to additional bristles and ocelli. Expression of p35 (ey > p35) (A) does not trigger overgrowth. Scale bar: 200 μm.

(E) Quantification of the data in (A)–(D). On the basis of qualitative screening criteria, overgrowth of progeny is scored by the presence of additional bristles and ocelli, expansion of the head capsule, and amorphic overgrowth. In severe cases, eye tissue is lost. At least 100 flies were scored for each genotype.

(F) ey > hid,p35 induces Toll-9 expression in a JNK (basket [bsk])-dependent manner. Relative mRNA levels of Toll-9 in ey-Gal4, ey > hid,p35 and ey > hid,p35,bsk^DN genetic backgrounds. The average of three independent experiments is plotted using one-way ANOVA with the Holm-Sidak test for multiple comparisons. *p < 0.05. Plotted is the mean intensity ± SEM. ns, not significant.

(G–N) ROS generation (G–J) and hemocyte recruitment (K–N) in ey > hid,p35 eye imaginal discs are strongly reduced by Toll-9 RNAi and the heterozygous Toll-9^rv17 mutant. Dihydroethidium (DHE) was used as ROS indicator. Arrow in (H) highlights DHE labeling. Anti-NimC antibody was used as hemocyte marker. Scale bars: 50 μm.

(O and P) Quantification of the data in (G)–(J) and (K)–(N), respectively. DHE counts and number of hemocyte clusters were counted across entire discs. Data were analyzed using one-way ANOVA with the Holm-Sidak test for multiple comparisons. Plotted is the mean ± SEM. ***p < 0.001 and ****p < 0.0001.

The following numbers of discs were analyzed: 4 (G), 8 (H), 4 (I), 7 (J), 5 (K), 6 (L), 6 (M), and 5 (N). The Toll-9^RNAi(BL) line in (B), (I), and (M) is Bloomington stock #BL35035. See also Figures S1–S4.

Using the undead model, we showed that AiP is mediated by extracellular reactive oxygen species (ROS) generated by the NADPH oxidase Duox (Fogarty et al., 2016). ROS trigger the recruitment of hemocytes, Drosophila immune cells most similar to mammalian macrophages, to undead imaginal discs (Diwanji and Bergmann, 2020). Hemocytes in turn release signals that stimulate JNK activity in undead cells, which then promotes AiP (Fogarty et al., 2016). In addition, JNK can also induce the expression of hid (Moreno et al., 2002), thus setting up an amplification loop in undead cells which continuously signals for AiP (Fogarty et al., 2016).

Given that undead cells are abnormal cells (Martin et al., 2009) with potentially altered cellular fitness and that signaling by Toll-related proteins surveilles cellular fitness (Alpar et al., 2018; Meyer et al., 2014), we examined if signaling by Toll-9 has an important function for undead AiP. Here, we show that Toll-9 is strongly up-regulated in undead cells and is necessary for the overgrowth of undead tissue. Overexpression of Toll-9 with p35 induces all hallmarks of undead AiP signaling, including Duox-dependent ROS generation, hemocyte recruitment and JNK signaling. Mechanistically, we provide genetic evidence for a heterologous interaction between Toll-9 and Toll-1, which engages the canonical Toll-1 signaling pathway to promote nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces the expression of reaper and hid. This activity contributes to the establishment of the feedback amplification loop that signals continuously for AiP. In conclusion, although Toll-9 does not appear to have an important function in innate immunity, it appears to be involved in the expression of pro-apoptotic genes such as reaper and hid in stress situations such as cell competition and undead AiP.

RESULTS

Toll-9 is required for overgrowth of undead tissue

We examined if Drosophila Toll-9 is involved in undead AiP. Indeed, depletion of Toll-9 by RNAi suppressed ey > hid,p35-induced overgrowth (Figures 1A–1C; quantified in Figure 1E). We also generated a Toll-9 deletion mutant by imprecise excision of an existing P element insertion, referred to as Toll-9^rv17 (Figure S1A). Heterozygosity of Toll-9^rv17 suppressed ey > hid,p35-induced overgrowth of undead tissue (Figures 1D and 1E). These results suggest that Toll-9 is required for ey > hid,p35-induced AiP. Furthermore, by qRT-PCR, we found that Toll-9 mRNA levels increased 8- to 10-fold in ey > hid,p35 undead tissue compared with control tissue (Figure 1F). This upregulation of Toll-9 expression is dependent on JNK encoded by the gene basket (bsk) (Figure 1F).

Toll-9 is required for ROS generation and hemocyte recruitment

To place Toll-9 into the AiP network, we performed a number of genetic tests in the ey > hid,p35 genetic background. Toll-9 RNAi suppressed the ectopic JNK and Wg activity in ey > hid,p35 background, suggesting that it acts upstream of these genes (Figure S2). Furthermore, Toll-9 RNAi or Toll-9^rv17 heterozygosity strongly suppressed the generation of ROS (Figures 1G–1J; quantified in Figure 1O). The same set of experiments with a different ROS indicator, H₂DCF-DA, gave similar results (Figure S3). In addition, recruitment of hemocytes to undead discs is reduced to levels seen at control discs in response to Toll-9 RNAi or Toll-9^rv17 heterozygosity (Figures 1K–1N; quantified in Figure 1P). We conclude that Toll-9 is required for ROS generation and the subsequent recruitment of hemocytes to ey > hid,p35-expressing discs.

Toll-9 overexpression in ey > p35 background engages a similar AiP pathway as ey > hid,p35

Although ey > Gal4-induced expression of Toll-9 does not induce any visible phenotype (Figures S1B and S1C), co-expression of Toll-9 with p35 (ey > p35,Toll-9) is sufficient to cause overgrowth of the resulting head cuticle (Figures 2A and 2B; quantified in Figure 2M). It is unknown why overexpression of Toll-9 can induce overgrowth only in the presence of p35 (see Discussion), but we have made a similar observation when overexpressing another factor involved in AiP, called Myo1D (Amcheslavsky et al., 2018). Nevertheless, the ey > p35,Toll-9-induced overgrowth phenotype gives us an assay to examine the mechanism by which Toll-9 works in the AiP pathway.

Figure 2. Toll-9 promotes overgrowth of ey > p35 head capsules and hemocyte recruitment via Duox-generated ROS.

(A and B) Transgenic expression of Toll-9 causes overgrowth of the head capsule of ey > p35-animals. Quantified in (M). The arrow in (B) highlights additional bristles, the arrowhead overgrown cuticle at the expense of eye tissue. Scale bar: 200 μm.

(C–F) Transgenic expression of Toll-9 in ey > p35 eye imaginal discs triggers ROS generation (D) and hemocyte recruitment (F). DHE and NimC were used as ROS indicator and hemocyte marker, respectively. Quantified in (N) and (O). Scale bars: 100 μm.

(G and H) Duox RNAi (H), but not Nox RNAi (G), can suppress Toll-9-induced overgrowth of ey > p35 head capsules. Quantified in (M). The arrow in (G) highlights overgrown tissue with additional bristles.

(I–L) ROS generation and hemocyte clustering of ey > p35,Toll-9 eye discs are suppressed by Duox RNAi (J–L) but not by Nox RNAi (I and K). DHE and NimC were used as ROS indicator and hemocyte marker, respectively. Quantified in (N) and (O).

(M) Quantification of the data in (A), (B), (G), and (H). Scoring criteria as in Figure 1E. At least 100 flies were scored for each genotype.

(N and O) Quantification of the DHE counts in (C), (D), (I), and (J) and hemocyte clusters in (E), (F), (K), (L). DHE counts and hemocyte clusters were counted across entire discs. Data were analyzed using one-way ANOVA with the Holm-Sidak test for multiple comparisons. Plotted is the mean ± SEM. **p < 0.01, **p < 0.001, and ****p < 0.0001. ns, not significant.

The genotypes of heads and eye discs are indicated above the panels. n = 4 (C), n = 4 (D), n = 7 (E), n = 12 (F), n = 6 (I), n = 6 (J), n = 7 (K), and n = 6 (L).

The expression of Toll-9 in ey > p35 discs is associated with the generation of ROS as well as recruitment of hemocytes (Figures 2C–2F; quantified in Figures 2N and 2O), consistent with the loss of ROS and hemocyte recruitment by Toll-9 depletion in ey > hid,p35 background (Figures 1I, 1J, 1M, and 1N). We showed previously that the membrane-bound NADPH oxidase Duox is required for ROS generation in ey > hid,p35 imaginal discs, while the second NADPH oxidase, Nox, is not (Fogarty et al., 2016). Consistently, Duox RNAi, but not Nox RNAi, suppressed the head overgrowth, the generation of ROS, and the recruitment of hemocytes in eye imaginal discs of ey > p35, Toll-9 animals (Figures 2G–2L; quantified in Figures 2M–2O), suggesting that Toll-9’s overgrowth-promoting effect through ROS generation by Duox, but not Nox, is required for the recruitment of hemocytes at ey > p35,Toll-9 imaginal discs.

JNK signaling is known to mediate signaling and overgrowth in undead tissue (Fan et al., 2014; Martin et al., 2009; Ryoo et al., 2004). Consistently, expression of a RNAi transgene targeting JNK (bsk^RNAi) suppressed the head overgrowth of ey > p35,Toll-9 adult animals (Figures S4A–S4C). We also observed an increase of JNK activity in ey > p35,Toll-9 imaginal discs based on the JNK reporter TRE-RFP (Figures S4D–S4G), suggesting that JNK is induced and required for Toll-9-induced overgrowth in the ey > 35 background.

These similarities between ey > hid,p35 and ey > p35,Toll-9 imply that overexpression of Toll-9 in ey > 35 background engages a similar hyperplasia pathway compared with ey > hid,p35 animals. However, in ey > hid,p35 discs, hid induces caspase (i.e., Dronc) activity, which is required for AiP (Fan et al., 2014; Huh et al., 2004; Kondo et al., 2006; Wells et al., 2006). Therefore, we examined if ey > p35,Toll-9 induces caspase activity. For that purpose, we used the cleaved Dcp-1 (cDcp-1) antibody, which is commonly used as a marker of caspase activity in Drosophila tissue (Li et al., 2019). Indeed, although there is no significant caspase activity in ey > Toll-9 discs, there is increased cDcp-1 labeling evident in ey > p35,Toll-9 imaginal eye discs (Figures 3C–3E; quantified in Figure 3F). Furthermore, depletion of dronc by RNAi resulted in strong suppression of ey > p35,Toll-9-induced overgrowth (Figures 3A and 3B), suggesting that Toll-9 overexpression can induce Dronc activity in the presence of p35. These data raise the question of how Toll-9 can achieve this activity.

Figure 3. dronc is required and the intracellular domain of Toll-9 is sufficient for overgrowth of ey > p35-expressing head capsules.

(A) RNAi knockdown of dronc suppressed ey > p35,Toll-9-induced overgrowth. Quantified in (B). Scale bar: 200 μm.

(B) Quantification of the data in (A), (G), and (H). Scoring criteria as in Figure 1E. At least 100 flies were scored for each genotype.

(C–E) cDcp1 labeling of imaginal eye discs of ey-Gal4 (C), ey > Toll-9 (D), and ey > p35,Toll-9 (E) third-instar larvae. Quantified in (F). Scale bar: 100 μm.

(F) Quantification of the data in (C)–(E). cDcp1 labeling was measured across entire discs and normalized to disc size. Data were analyzed using one-way ANOVA with the Holm-Sidak test for multiple comparisons. Relative signal intensity is plotted ±SEM. **p < 0.01. ns, not significant. The following numbers of discs were analyzed: 7 (C), 9 (D), and 12 (E).

(G and H) Transgenic expression of the intracellular domain of Toll-9 (Toll-9^INTRA) (G), but not the extracellular domain (Toll9^EXTRA) (H), causes overgrowth of the head capsule of ey > p35-expressing animals. Quantified in (M). The arrow in (G) highlights overgrown amorphic tissue. Scale bar: 200 μm.

(I–L) Transgenic expression of Toll-9^INTRA, but not Toll-9^EXTRA, triggers ROS generation (I and J) and hemocyte recruitment (K and L) in ey > p35-expressing eye imaginal discs. DHE and NimC were used as ROS indicator and hemocyte marker, respectively. Quantified in (M and N). Scale bar: 100 μm.

(M and N) Quantification of the data in (I) and (J) and in (K) and (L). DHE counts and hemocyte clusters were obtained across entire discs. Data were analyzed using one-way ANOVA with the Holm-Sidak test for multiple comparisons. Plotted is the mean ± SEM. ***p < 0.001 and ****p < 0.0001. ns, not significant.

The following numbers of discs were analyzed: 4 (ey > p35 in M), 4 (ey > Toll-9,p35 in M) 6 (I), 7 (J), 5 (ey > p35 in N), 11 (ey > Toll-9,p35 in N), 10 (K), and 4 (L).

Toll-9 requires the intracellular domain to induce overgrowth

To address this question, we determined first whether Toll-9 mediates this activity in the AiP network through an intracellular signaling pathway or through cell-cell adhesion mediated by the extracellular domain (Anthoney et al., 2018; Keith and Gay, 1990; Ward et al., 2015). We separately expressed intra- and extracellular domains of Toll-9 (Toll-9^INTRA and Toll-9^EXTRA, respectively) linked to the signal peptide and transmembrane domain, in ey > p35 background. Expression of Toll-9^INTRA is sufficient to induce overgrowth of ey > p35 head capsules, accompanied by ROS generation and hemocyte activation at imaginal discs (Figures 3G, 3I, and 3K; quantified in Figures 3B, 3M, and 3N). In contrast, expression of Toll-9^EXTRA did not trigger these phenotypes (Figures 3H, 3J, and 3L; quantified in Figures 3B, 3M, and 3N). These data suggest that the intracellular but not the extracellular domain of Toll-9 is sufficient for the generation of ROS, hemocyte activation, and overgrowth of ey > p35,Toll-9-expressing tissue, most likely by activation of an intracellular signaling pathway.

To explore the possibility that Toll-9 engages an NF-κB-like transcription factor for this overgrowth, we tested the two NF-κB factors involved in Toll-1 signaling in Drosophila, Dorsal and Dif (Ip et al., 1993; Meng et al., 1999; Rutschmann et al., 2000; Steward, 1987). Consistently, RNAi targeting either dorsal or Dif suppressed the overgrowth of ey > p35,Toll-9^INTRA-expressing animals (Figures 4A–4C; quantified in Figure 4I). Further support for the notion of an intracellular pathway comes from the observation that knockdown of Myd88 and a mutant of tube, known intracellular components acting upstream of Dorsal and Dif in the Toll-1 pathway (Horng and Medzhitov, 2001; Letsou et al., 1991; Tauszig-Delamasure et al., 2002), also suppressed ey > p35,Toll-9 ^INTRA-induced overgrowth strongly and moderately, respectively (Figures 4D and 4E; quantified in Figure 4I). RNAi against another gene involved downstream in Toll-1 signaling, pelle (Shelton and Wasserman, 1993; Towb et al., 2001), weakly suppressed ey > p35,Toll-9 ^INTRA (Figures 4F–4I). Consistently, RNAi targeting the components of the intracellular Toll-1 signaling pathway also suppressed the ey > hid,p35 overgrowth phenotype (Figure S5A) although Myd88 and pelle are quite weak suppressors.

Figure 4. Canonical, intracellular Toll-1 signaling contributes to ey > p35,Toll-9-induced overgrowth.

(A–C) ey > p35,Toll-9-induced overgrowth (A) is strongly suppressed by RNAi-mediated knockdown of dorsal (B) and Dif (C). Scale bar: 200 μm.

(D–H) RNAi-mediated knockdown of Myd88 (D), pelle (F), Toll-1 (G), and SPE (H) as well as a tube mutant (E) suppressed ey > p35,Toll-9-induced overgrowth.

(I) Quantification of the data in (A)–(H). Scoring criteria as in Figure 1E. At least 100 flies were scored for each genotype.

See also Figure S5.

One function of the intracellular Myd88/Tube/Pelle pathway is to mediate the nuclear translocation of the NF-κB transcription factor Dorsal (Roth et al., 1989; Rushlow et al., 1989; Steward, 1989). Therefore, we examined if misexpression of Toll-9 can trigger nuclear localization of Dorsal in wing imaginal discs using ptc-Gal4 as a driver. We chose ptc-Gal4 in these experiments, because the striped expression of Toll-9 in the ptc domain allows side-by-side comparisons between ptc-expressing and ptc-non-expressing areas. Consistent with the expectation, there is strong nuclear accumulation of Dorsal in the ptc domain upon Toll-9 (ptc > Toll-9) misexpression compared with control (Figures 5A and 5B; see also orthogonal sections on top of the panels). Interestingly, while ptc > Toll-9 is sufficient to trigger nuclear translocation of Dorsal (Figure 5B2), in the presence of p35 (ptc > p35,Toll-9), the expression domain of ptc and the domain where Dorsal is nuclear are strongly enlarged (compare Figures 5C2 and 5C4 with Figures 5B2 and 5B4) further supporting the notion that expression of Toll-9 induces hyperplasia only in the presence of p35 (see Discussion). Consistently, we also find nuclear Dorsal in ey > hid,p35-expressing eye imaginal discs in a Toll-9-dependent manner (Figure S6).

Figure 5. Toll-9-induced nuclear localization of Dorsal is dependent on canonical Toll-1 signaling.

All wing imaginal discs were labeled with anti-Dorsal antibody (red). GFP is pseudo-colored in blue and highlights the ptc domain. Nuclei (green) were stained with Hoechst. Scale bar: 100 μm. The yellow lines in merged panels indicate the regions from which orthogonal sections (shown above panels) were derived. At least 20 wing imaginal discs were analyzed per genotype.

(A) A control (ptc > GFP) wing imaginal disc.

(B and C) Misexpression of Toll-9 results in nuclear localization of Dorsal in ptc > GFP,Toll-9^INTRA (B) and ptc > GFP,p35,Toll-9^INTRA (C) wing imaginal discs. White arrows point to nuclear Dorsal in the ptc expression domain. Notably, the ptc expression domain was significantly expanded in ptc > GFP,p35,Toll-9^INTRA (C) wing imaginal discs, confirming that in the presence of p35, expression of Toll-9 induces overgrowth.

(D and E) RNAi knockdown of Myd88 (D) and Toll-1 (E) blocked Toll-9-induced nuclear localization of Dorsal.

See also Figure S6.

To determine if the intracellular Toll-1 pathway is required for the nuclear translocation of Dorsal, we examined if knockdown of Myd88 can block the nuclear localization of Dorsal. Indeed, depletion of Myd88 by RNAi resulted in strong loss of nuclear localization of Dorsal (Figure 5D). Combined, these data suggest that Toll-9 overexpression engages the intracellular Toll-1 pathway involving Myd88, Tube, and Pelle for nuclear localization of Dorsal.

Toll-9 laterally interacts with Toll-1 to stimulate nuclear localization of Dorsal

These observations raise the question whether Toll-9 can promote the nuclear localization of Dorsal by directly engaging the Myd88/Tube/Pelle intracellular pathway or whether it laterally interacts with the Toll-1 receptor, which then activates the intracellular pathway for nuclear translocation of Dorsal. To address this question, we examined the effect of Toll-1 RNAi on the ey > p35,Toll-9 overgrowth phenotype. As shown in Figure 4G (quantified in Figure 4I), Toll-1 RNAi is a strong suppressor of ey > p35,Toll-9-induced overgrowth. We also observed that the nuclear localization of Dorsal in the ptc > p35,Toll-9 expression domain is abolished by Toll-1 RNAi (Figure 5E). These data imply that Toll-9 can directly or indirectly interact with Toll-1 to engage the known intracellular signaling pathway of Toll-1 for nuclear localization of Dorsal. Consistently, overexpression of Toll-1 in ey > p35 background triggers strong overgrowth of the head capsule (Figures S5B and S5C).

Interestingly, RNAi targeting Spätzle-processing enzyme (SPE), which processes and activates the Toll-1 ligand Spätzle (Spz), strongly suppressed both the ey > p35,Toll-9^INTRA and ey > hid,p35 overgrowth phenotypes (Figures 4H, 4I, and S5A). Although we do not observe a suppression of the ey > p35,Toll-9 overgrowth by spz RNAi, overexpression of an active form of spz, spz^Act, in ey > p35 animals induces strong overgrowth of the head capsules (Figures S5D and S5E). These data imply that the Toll-1 ligand Spz may also be involved in undead overgrowth.

Toll-9 induces expression of reaper and hid in p35-expressing tissue

In the models of cell competition in imaginal discs, evidence was provided that Toll-9 contributes to the expression of the pro-apoptotic genes reaper (rpr) and hid (Meyer et al., 2014). Consistently, RNAi targeting rpr strongly suppressed ey > p35,Toll-9-induced overgrowth (Figures 6A and 6B; quantified in Figure 6D). Furthermore, depletion of hid by RNAi also significantly suppressed ey > p35,Toll-9-induced overgrowth (Figures 6C and 6D). These observations suggest that rpr and hid are required for p35,Toll-9-induced overgrowth. Furthermore, although hid RNAi suppressed the ey > hid,p35-induced overgrowth phenotype as expected, rpr RNAi as well as an RNAi line targeting the three pro-apoptotic genes rpr, hid, and grim (miR[RHG]) also suppressed ey > hid,p35 (Figure S7A), implying that rpr also plays a critical role for ey > hid,p35-induced overgrowth. This notion is further supported by examining the expression of rpr and hid in ey > p35,Toll-9 background using lacZ reporter genes (Figures 6E–6H; quantified in Figure 6I). Interestingly, despite the nuclear localization of Dorsal in ptc > Toll-9 imaginal discs (Figure 5B), it is able to induce the expression of the rpr and hid reporter transgenes only in the presence of p35 (Figures 6F, 6H, and 6I). To explain this discrepancy, we reasoned that because Dorsal acts as a concentration-dependent transcription factor during embryonic dorsoventral patterning (Roth et al., 1989; Rushlow et al., 1989; Steward, 1989), rpr and hid expression requires a certain nuclear concentration of Dorsal, and therefore we quantified the amount of Toll-9-induced nuclear Dorsal in the presence and absence of p35. This analysis indeed demonstrates that the amount of nuclear Dorsal is increased in the presence of p35 (Figure S7B) which would be consistent with a concentration-dependent control of rpr and hid expression by Dorsal. However, it is also possible that another cofactor is required for Dorsal’s ability to induce rpr and hid expression. This cofactor may not be active or nuclear in the absence of p35.

Figure 6. Toll-9 induces expression of reaper and hid in ey > p35 tissue.

(A–C) Toll-9-induced overgrowth of ey > p35 head capsules is suppressed by RNAi knockdown of rpr (B) and hid (C). Scale bar: 200 μm.

(D) Quantification of the data in (A)–(C). Scoring criteria as in Figure 1E. At least 100 flies per genotype were analyzed.

(E–H) Toll-9-induced expression of transcriptional reporters of reaper (rpr-lacZ) (E and F) and hid (hid-lacZ) (G and H) in ey > p35 eye discs. Anti-beta-galactosidase was used to detect lacZ expression. Elav labels photoreceptor neurons. Scale bars: 100 μm.

(I) Quantification of the data in (E)–(H). An unpaired t test, two-tailed distribution, was performed to test the significance. Plotted is the mean intensity ± SEM. *p < 0.05 and ***p < 0.001.

The following numbers of discs were analyzed: 7 (E), 15 (F), 4 (G), and 17 (H). See also Figure S7.

Nevertheless, the lack of rpr and hid induction in ey > Toll-9 discs also explains why there is little caspase activity in these discs, while ey > p35,Toll-9 discs have increased caspase activity (Figures 3C–3F) and are dependent on Dronc for overgrowth (Figures 3A and 3B). Furthermore, given that expression of hid and reaper in the presence of p35 induces overgrowth (see, e.g., Figure 1B), these observations explain why ey > p35,Toll-9 animals have overgrown heads.

DISCUSSION

Toll-9 is the most closely related Drosophila TLR compared with mammalian TLRs, but a biological function of Toll-9 has not been clearly defined. All mammalian TLRs are involved in innate immunity; therefore, the close homology has led to the prediction that Drosophila Toll-9 also participates in innate immunity (Ooi et al., 2002). However, in Toll-9 mutants, the basal as well as bacterially induced AMP production is not affected, leading to the conclusion that Toll-9 is not involved in innate immunity (Narbonne-Reveau et al., 2011). Nevertheless, instead of eliminating foreign pathogens, previous work has shown that Toll-9 together with several other Toll-related receptors participates in elimination of unfit cells during cell competition (Alpar et al., 2018; Meyer et al., 2014). This is achieved through the expression of the pro-apoptotic gene rpr. Here, we demonstrate that Toll-9 has a similar rpr- and hid-inducing function during AiP, thereby adding to the database of Toll-9 function.

To identify the mechanism by which Toll-9 participates in AiP, we took advantage of the observation that misexpression of Toll-9 is sufficient to induce overgrowth of ey > p35 animals. There are multiple aspects of this phenotype that are worth being discussed. First, key for many of the observations presented in this paper is the presence of P35, a very efficient inhibitor of the effector caspases DrICE and Dcp-1. In the absence of P35, overexpression of Toll-9 does not induce any obvious phenotypes in eye discs or adult heads (see, e.g., Figure S1C). The exact reason for this P35 dependence is currently unknown, but it has also been observed upon misexpression of other genes involved in AiP, such as Myo1D (Amcheslavsky et al., 2018), Toll-1, and Spz^Act (this study). The only known function of P35 is to inhibit DrICE and Dcp-1. Therefore, one possible explanation for the P35 dependence is that these caspases cleave and inactivate an as yet unidentified component of the AiP network, possibly to block inappropriate AiP under normal conditions. In the presence of P35, the AiP-blocking activity of DrICE is inhibited and with the addition of an AiP-inducing stimulus such as misexpression of Toll-9, AiP is engaged and can trigger tissue overgrowth.

Second, our data show that ectopic p35,Toll-9 co-expression triggers overgrowth through a similar mechanism as hid,p35 co-expression. This includes Dronc activation, Duox-generated ROS, hemocyte recruitment, and JNK activation. These similarities allow us to place the function of Toll-9 into the AiP network.

Third, mis-expressed Toll-9 can genetically interact with Toll-1. This interaction results in nuclear translocation of Dorsal and is dependent on Myd88, Tube, and Pelle, all canonical components of the intracellular Toll-1 signaling pathway (Horng and Medzhitov, 2001; Letsou et al., 1991; Shelton and Wasserman, 1993; Tauszig-Delamasure et al., 2002; Towb et al., 2001). Importantly, the nuclear translocation of Dorsal and the p35,Toll-9-induced overgrowth is also dependent on Toll-1, suggesting that the activation of the Myd88/Tube/Pelle pathway is directly triggered by Toll-1 and not by Toll-9. Mechanistically, Toll-9 may activate Toll-1 either directly through hetero-dimerization or mediated by additional factors. Future work will be necessary to identify the molecular mechanism of the Toll-9/Toll-1 interaction.

Fourth, the outcome of the Toll-9/Toll-1 interaction is the expression of the pro-apoptotic genes reaper and hid. Because Toll-9 expression is strongly up-regulated in undead cells (Figure 1F), these data suggest that Toll-9-induced expression of reaper and hid in undead cells is setting up an amplification loop (Figure 7). The cause of the strong transcriptional upregulation of Toll-9 in undead cells is unknown, but it requires JNK activity (Figures 1F and 7). The Toll-9 amplification loop contributes to the strength of undead signaling during AiP and propels the overgrowth of the tissue.

Figure 7. Model of Toll-9 function during undead AiP.

Shown is an undead epithelial cell of a larval eye-antennal imaginal disc and an attached hemocyte. The interactions revealed in this paper are highlighted in yellow. Toll-9 engages a Toll-1-dependent intracellular pathway, culminating in the nuclear translocation of the NF-κB factor Dorsal, which directly or indirectly induces expression of reaper and hid. Previously shown interactions including the activation of Duox, recruitment of hemocytes, and activation of JNK (Amcheslavsky et al., 2018; Fogarty et al., 2016) are also shown. Wg, Dpp, and Spi are mitogens produced by undead cells for AiP.

Fifth, another important question is how Toll signaling becomes activated during AiP. Toll-1 is activated by the ligand Spätzle during embryogenesis and the immune response (Morisato and Anderson, 1994; Weber et al., 2003). Spz requires proteolytic processing for activation, which during the immune response is mediated by the Ser-protease Spätzle-processing enzyme (Jang et al., 2006; Kambris et al., 2006). Consistently, SPE RNAi can suppress both the ey > p35,Toll-9^INTRA and ey > hid,p35-induced overgrowth phenotypes (Figures 4H and S5A). In the experiment in Figure 4H, SPE RNAi suppressed ey > p35,Toll-9^INTRA, which lacks the extracellular domain and should be insensitive to a ligand. Thus, the suppression of ey > p35,Toll-9^INTRA suggests that SPE does not act through Toll-9 but instead on Toll-1. This result is consistent with a recent finding that Toll-9 RNAi cannot suppress apoptosis induced by a dominant active SPE (SPE^Act) transgene (Alpar et al., 2018). Although we cannot rule out that there is an unknown Toll-9 ligand, Toll-9 may not need to be activated by a ligand. Toll-9 naturally carries an amino acid substitution in the cysteine-rich extracellular domain similar to the gain-of-function Toll-1¹ mutant (Ooi et al., 2002; Schneider et al., 1991). Indeed, Toll-9 behaves as a constitutively active receptor in cell culture assays (Ooi et al., 2002). As TLRs can form homo- and heterodimers (Hu et al., 2004; Zhang et al., 2002), it is possible that the constitutive activity of Toll-9 and the strong transcriptional upregulation of Toll-9 (Figure 1F) together with ligand stimulation of Toll-1 by Spz is sufficient for the activation of the Toll-1/Toll-9 complex. However, it remains unknown how SPE becomes activated during AiP.

With these considerations in mind, the following model for Toll-9 function during undead AiP emerges (Figure 7). The initial stimulus for AiP is the Gal4-induced expression of hid and p35, which leads to the activation of Dronc. Because of P35, Dronc cannot induce apoptosis but instead activates Duox for generation of ROS (Figure 7). ROS attract hemocytes which release signals for JNK activation in undead cells. JNK signaling directly or indirectly induces Toll-9 transcription. Up-regulated Toll-9 interacts with Toll-1, and after Spz ligation the Myd88/Tube/Pelle pathway triggers the nuclear accumulation of Dorsal and potentially Dif (Figure 7). These factors transcriptionally induce reaper and hid expression setting up the feedback amplification loop, which maintains and propels AiP and overgrowth.

One other interesting question to examine in the future will be how the intracellular pathway of Toll-1 signaling including Dorsal and Dif can induce different target genes under different conditions. For dorsoventral patterning of the Drosophila embryo, Dorsal induces the expression of twist and snail (Ip et al., 1992; Jiang et al., 1991; Pan et al., 1991; Thisse et al., 1991). During immune responses in the fat body, it promotes the expression of AMP genes as well as Kennedy pathway genes for the synthesis of phospholipids (Buchon et al., 2014; Martinez et al., 2020), while during cell competition and AiP which occur in larval imaginal discs, pro-apoptotic genes hid and rpr are induced. One potential answer to this question is that the specificity of Toll-1 signaling may be modified by the interaction with Toll-9. Although this interaction occurs at the plasma membrane, it also might influence the activity in the nucleus. It will also be interesting to examine if Toll-1 can interact with some or all of the other Toll-related receptors in Drosophila and how this interaction might influence the specificity of the transcriptional outcome.

Although Toll-9 in Drosophila does not appear to be required for innate immunity, on the basis of its non-essential function to induce AMP production during bacterial infection (Narbonne-Reveau et al., 2011), our work and work by others (Meyer et al., 2014) reveals that Toll-9 may have a function during stress responses which involves expression of pro-apoptotic genes such as rpr and hid. That was demonstrated previously for cell competition (Meyer et al., 2014) and now for undead AiP, indicating potential similarities between cell competition and undead AiP. At first, such similarities appear to be at odds with the common dogmas that proliferating winner cells trigger apoptosis of loser cells, while during AiP, apoptotic cells induce proliferation of surviving cells. However, it has also been reported that loser cells have a much more active role during cell competition and can promote the winner status of cells with increased fitness (Kucinski et al., 2017). Therefore, there appear to be significant similarities between cell competition and undead AiP. The common denominator for both systems is the expression of pro-apoptotic genes. These responses have different outcomes in both systems. During cell competition, this response involves the death of the loser cells (Alpar et al., 2018; Meyer et al., 2014). During undead AiP, it sets up the amplification loop known to operate in undead cells (Fogarty et al., 2016), which propels hyperplasia and tissue overgrowth.

Limitations of the study

This work was performed largely under undead conditions (i.e., in the presence of the effector caspase inhibitor p35, which is not an endogenous gene in Drosophila). In reality, however, in the absence of p35, effector caspases are also activated in apoptotic cells, which will eventually lead to the death of the cell. Therefore, the question arises as to how apoptosis and AiP are linked to allow compensatory proliferation under normal conditions. Recently, we presented evidence that certain apoptotic cells (dying enterocytes in the adult intestine) can adopt a transiently undead-like state that enables them to signal for AiP before they are dying and removed (Amcheslavsky et al., 2020). The transiently undead-like state is achieved by transient localization of Dronc to the plasma membrane, which might serve as a non-apoptotic compartment of the cell (Amcheslavsky et al., 2018; Bergmann, 2018). In that way, apoptotic cells, before they die, can trigger AiP in a p35-independent manner. Therefore, in future research, it will be important to examine if the Toll-9/Toll-1 interaction sets up a similar amplification loop in transiently undead enterocytes.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Andreas Bergmann (andreas.bergmann@umassmed.edu).

Materials availability

All unique/stable materials generated in this study is available from the lead contact without restriction.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request. This study does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

The experimental model organism is Drosophila melanogaster. Details of genotypes used in this study and their sources are described in the key resources table (KRT). All crosses were performed on standard cornmeal-molasses medium (60g/L cornmeal, 60mL/L molasses, 23.5g/L bakers yeast, 6.5g/L agar, 4mL/L acid mix and 0.13% Tegosept). For expression of RNAi transgenes and mis-expressing transgenes in eye and wing imaginal discs the Gal4/UAS system was used. Crosses not involving conditional expression of transgenes were incubated at 25°C.

KEY RESOURCES TABLE.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-NimC antibody	Dr. Istvan Ando	RRID: AB_2568423
Dorsal	Developmental Studies Hybridoma Bank (DSHB)	DSHB Cat# anti-dorsal 7A4; RRID:AB_528204
Mmp1	DSHB	DSHB Cat# 3A6B4; RRID:AB_579780
Wingless	DSHB	DSHB Cat# 4d4; RRID:AB_528512
Elav	DSHB	DSHB Cat# Rat-Elav-7E8A10 anti-elav; RRID:AB_528218
Mouse monoclonal anti-β-Galactosidase	DSHB	DSHB Cat# 40–1a; RRID:AB_528100
Cleaved Dcp-1 (Asp176)	Cell Signaling Technology	Cat# 9578; RRID:AB_2721060
Chemicals, peptides, and recombinant proteins
Dihydroethidium (DHE)	Invitrogen	Cat#D23107
H2DCF-DA	Molecular Probes	Cat#C6827
Vectorshield with DAPI	Vector Laboratories	Cat#H-1200; RRID: AB_2336790
Hoechst 33342	Invitrogene	Cat#H3570
Experimental models: Organisms/strains
Drosophila melanogaster: Toll-9rv17	this study	N/A
Drosophila melanogaster: UAS-Toll-9	this study	N/A
Drosophila melanogaster: UAS-Toll-9INTRA	this study	N/A
Drosophila melanogaster: UAS-Toll-9INTRA	this study	N/A
Drosophila melanogaster: UAS-Toll-9 RNAi	Bloomington Drosophila Stock Center (BDSC)	BDSC 30535 FlyBase: FBst0030535
Drosophila melanogaster: UAS-Toll-9 RNAi	Vienna Drosophila Resource Center (VDRC)	VDRC v36308 FlyBase: FBst0461624
Drosophila melanogaster: UAS-Toll-9 RNAi	VDRC	VDRC v109635 FlyBase: FBst0481299
Drosophila melanogaster: UAS-Dif RNAi	VDRC	VDRC v100537 FlyBase: FBst0472410
Drosophila melanogaster: UAS-dorsal RNAi	VDRC	VDRC v105491 FlyBase: FBst0477317
Drosophila melanogaster: UAS-dorsal RNAi	BDSC	BDSC 34938 FlyBase: FBst0034938
Drosophila melanogaster: UAS-MyD88 RNAi	VDRC	VDRC v106198 FlyBase: FBst0478023
Drosophila melanogaster: UAS-pelle RNAi	VDRC	VDRC v103774 FlyBase: FBst0475632
Drosophila melanogaster: UAS-Toll-1 RNAi	VDRC	VDRC v100078 FlyBase: FBst0471952
Drosophila melanogaster: UAS-reaper RNAi	BDSC	BDSC 51846 FlyBase: FBst0051846
Drosophila melanogaster: UAS-hid RNAi	VDRC	VDRC v7912 FlyBase: FBst0470883
Drosophila melanogaster: UAS-dronc RNAi	VDRC	FlyBase: FBst0472297; VDRC v100424
Drosophila melanogaster: UAS-bsk RNAi	VDRC	VDRC v34138 FlyBase: FBst0460476
Drosophila melanogaster: UAS-Luciferase RNAi	BDSC	BDSC 31603 FlyBase: FBst0031603
Drosophila melanogaster: 4kb rpr-lacZ	Hermann Steller	FlyBase: FBal0105175
Drosophila melanogaster: hid[10–1kb]-lacZ	this study	N/A
Drosophila melanogaster: UAS-Toll-1	Tony Ip	FlyBase : FBal0181209
Drosophila melanogaster: UAS-SPE RNAi	VDRC	VDRC v104906 FlyBase: FBst0476734
Drosophila melanogaster: UAS-spzAct	Laura Johnson	FlyBase: FBal0138129
Drosophila melanogaster: UAS-Duox RNAi (line #44)	Won-Jae Lee	FlyBase: FBal0190061
Drosophila melanogaster: UAS-Nox RNAi (line #4)	Won-Jae Lee	FlyBase: FBal0191562
Drosophila melanogaster: TRE-RFP	Dirk Bohmann	FlyBase: FBst0059011
Oligonucleotides
Toll-9, Forward: 5′CCATTACAAGCACTATAGG	this study	N/A
Toll-9, Reverse: 5′-GACCTCTTCGGCCTCTTC	this study	N/A
RP-49: Forward: 5′-CCAGTCGGATCGATATGCTAA	this study	N/A
RP-49: Reverse: 5′-ACGTTGTGCACCAGGAACTT	this study	N/A
Software and algorithms
Photoshop	Adobe	Version 2021
GraphPad Prism Software	GraphPad	Version 9
Zen imaging software	Carl Zeiss AG	Version 3.1

METHOD DETAILS

Fly stocks and genetics

The exact genotype of ey > hid,p35 is UAS-hid (on X); ey-Gal4 UAS-p35 (on 2^nd) and of ey > p35 it is ey-Gal4 UAS-p35 (on 2^nd).

The following fly stocks were used: ey > hid,p35 and ey > p35 (Fan et al., 2014); UAS-Nox RNAi (line#4) and UAS-Duox RNAi (#44) (Ha et al., 2005; Tanji et al., 2010); Toll-9^rv17, UAS-Toll-9, UAS-Toll-9^INTRA and UAS-Toll-9^EXTRA (this study); UAS-Toll-9 RNAi stocks: BL30535, v36308 and v109635; UAS-Dif RNAi: v100537; UAS-dl RNAi: BL34938 and v105491;UAS-Myd88 RNAi: v106198; tube³ mutant (Letsou et al., 1991); UAS-pelle RNAi: v103774; UAS-Toll-1 RNAi: v100078; UAS-rpr RNAi: BL51846; UAS-hid RNAi: v7912, UAS-dronc RNAi: v100424; UAS-bsk RNAi (v34138); UAS-Lucif-erase RNAi: BL31603 (used as the mock RNAi); 4kb rpr-lacZ (Jiang et al., 2000); hid[10–1kb]-lacZ (line1) (this work); TRE-RFP (Chatterjee and Bohmann, 2012); UAS-SPE RNAi: v104906; UAS-Toll-1 (Hu et al., 2004); UAS-spz^Act (Ligoxygakis et al., 2002).

Generation of the Toll-9^rv17 allele

The parental P element insertion strain EY14405 from Bloomington stock center was used for a jump out excision screen. The strain was crossed with a Δ2–3 strain and offspring with loss of red eye color were collected. Individual flies collected were crossed with balancer strain to establish individual stocks. Genomic DNA from these collected stocks were isolated and tested by PCR using primers surrounding the P element insertion site. The revertant 17 (rv17) stock showed absence of PCR product on primary screen and the homozygous strain were subject to further PCR and sequencing to confirm the deletion of the Toll-9 locus, from the insertion site in the second intron to the last coding exon as shown in Figure S1A.

Generation of UAS-Toll-9^INTRA and UAS-Toll-9^EXTRA

For UAS-Toll-9^EXTRA, the Toll-9 cDNA was used to PCR amplify the first part of the coding sequence, from 5′ UTR to the beginning of cytoplasmic domain including the transmembrane domain. The clone encodes a.a. 1 to 700. The PCR fragment was cloned into the XbaI and BglII sites of the pUAST vector. For UAS-Toll-9^INTRA, the Toll-9 cDNA was used to amplify a short fragment encoding the first 15 a.a., as signal peptide sequence, and a second fragment encoding a.a. 650, to the stop codon, including the transmembrane and the cytoplasmic domains. The two fragments were cloned head to tail into the XbaI and BglII sites of the pUAST vector. The resulting plasmids were used to generate transgenic flies (Rainbow Transgene, California) in the w¹¹¹⁸ background. The resulting transgenic flies were used for crosses into the ey > p35 background.

Generation of hid[10-1kb]-lacZ

The 10 kb genomic DNA upstream of the hid transcriptional start site was cloned and inserted into a Drosophila reporter plasmid, pCaSpeR-hs43 lacZ. Transgenic flies were then generated using standard procedures. The hid[10-1]-lacZ reporter was found to respond to Toll-9 signaling and was used in this study as the hid reporter.

Immunolabeling and ROS staining

For labelings with antibodies, fixed eye-antennal or wing imaginal discs from 3^rd instar larvae were used following standard protocols (Fogarty et al., 2016). The monoclonal NimC antibody is a kind gift of István Andó (Kurucz et al., 2007) and was used at a dilution of 1:300. Primary antibodies from the Developmental Studies Hybridoma Bank (DSHB) included anti-Dorsal (7A4) used at 1:20, anti-Mmp1 (3A6B4) used at 1:50, anti-Wingless (4D4) used at 1:50, anti-beta-galactosidase (40–1a) used at 1:20, and anti-Elav (7E8A10) used at 1:50. Anti-cDcp1 (Asp216) antibody from Cell Signaling Technology was used at 1:200. Hoechst 33342 solution was used at 1:1000 to stain nuclei. Fluorescent secondary antibodies were obtained from Jackson ImmunoResearch. ROS staining with DHE (Invitrogen #D23107) and H₂DCF-DA (Molecular Probes #C6827) were performed on unfixed imaginal discs using published protocols (Owusu-Ansah et al., 2008).

qRT-PCR for determination of Toll-9 transcript levels

Total mRNA was prepared from eye-imaginal discs using TRIzol reagent (Ambion, Cat#15596026). cDNA was prepared using QuantiTect Reverse Transcription Kit (Qiagen, Cat# 205311) and qPCR was performed with Power SYBR Green PCR master mix (Applied Biosystems, Cat#4367659). The following primer pairs were used:

Toll-9, Forward: 5′-CCATTACAAGCACTATAGG; Reverse: 5′-GACCTCTTCGGCCTCTTC.

RP-49: Forward: 5′-CCAGTCGGATCGATATGCTAA; Reverse: 5′-ACGTTGTGCACCAGGAACTT.

QUANTIFICATION AND STATISTICAL ANALYSIS

For quantification of confocal images, the ‘Histo’ function of Zen (2012) imaging software (Carl Zeiss) was used. Region of interest was outlined for each disc and mean fluorescence signal intensity was determined. Crosses were repeated at least three times. Analysis and graph generation was done using GraphPad Prism 9. The statistical method used was one-way ANOVA with Holm-Sidak test for multiple comparisons, unless otherwise indicated. Plotted is mean intensity ±SEM. Levels of significance are depicted by asterisks in the figures: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

N numbers are as follows:

Figure 1O: 4, 8, 4, 7 (from left to right)

Figure 1P: 5, 6, 6, 5 (from left to right)

Figure 2N: 4, 4, 6, 6 (from left to right)

Figure 2O: 7, 12, 7, 6 (from left to right)

Figure 3F: 7, 9, 12 (from left to right)

Figure 3M: 4, 4, 6, 7 (from left to right)

Figure 3N: 5, 11, 10, 4 (from left to right)

Figure 6I: 7, 15, 4, 17 (from left to right)

Figure S3F: 3, 6, 5, 6, 7 (from left to right)

Figure S4G: 8, 9, 11 (from left to right)

Figure S7B: 50, 50 (from left to right)

Highlights.

• Toll-9 is required for undead apoptosis-induced proliferation (AiP)

• Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway

• NF-κB-like transcription factors induce expression of apoptotic genes reaper and hid

• Toll-9/Toll-1 signaling establishes an amplification loop for AiP