Introduction

Certain families of master regulatory transcription factor control major developmental decisions in metazoa. This includes the bHLH proteins encoded by proneural genes, which were discovered in Drosophila and control neural fate specification and differentiation from sponges to humans(Weinberger et al., 2017; Baker & Brown, 2018; Dennis, Han, & Schuurmans, 2019; Johnson, 2020). One of the ways proneural gene activities are regulated, thereby modulating the location and timing of neurogenesis, is through Inhibitor of DNA binding proteins (Id proteins), which are HLH proteins lacking basic DNA-binding sequences. Using their HLH domains, Id proteins form inactive heterodimers with proneural bHLH proteins, preventing DNA binding and transcriptional regulation of target genes by the resulting heterodimer (Benezra, Davis, Lockshon, Turner, & Weintraub, 1990; Ellis, Spann, & Posakony, 1990; Garrell & Modolell, 1990; Cabrera, Alonso, & Huikeshoven, 1994; Ellis, 1994; Norton, 2000). Accordingly, mutations in Id protein genes, of which there are 4 in mammals and one in Drosophila, permit enhanced proneural bHLH function, deregulating neurogenesis at many developmental stages and in many tissues. Mammalian Id gene expression is particularly associated with proliferative and stem cell states, and is frequently affected in cancer(Ling, Kang, & Sun, 2014; Wang & Baker, 2015a; Roschger & Cabrele, 2017; Oproescu, Han, & Schuurmans, 2021).

An outstanding question has been whether heterodimerization with proneural bHLH proteins represents the only mechanism of Id protein function. This question arises because not all phenotypes of Id gene mutations obviously represent gain of proneural gene function.

Interpretation is complicated in mammals by the number of proneural bHLH genes and of Id protein genes, both functionally redundant so that single mutant phenotypes reflect only subsets of protein function(Ling et al., 2014; Wang & Baker, 2015a). In Drosophila, it is clear that mutating the sole Id protein gene extra macrochaetae (emc) affects some tissues where no proneural gene is known to function. The first example noted was the requirement for emc for normal growth of imaginal discs(Garcia-Alonso & Garcia-Bellido, 1988). Imaginal discs are larval progenitors of adult tissues and remain proliferative and undifferentiated until late in the final larval instar; no proneural gene is active in these proliferating cells. A second example is that emc is required for ovarian follicle cell development (Adam & Montell, 2004), which does not depend on proneural bHLH genes. Further examples are noted in Drosophila eye development. The emc gene is required to restrain the rate at which the morphogenetic furrow, a wave of fate specification that traverses across the eye imaginal disc as retinal patterning and differentiation begin(Brown, Sattler, Paddock, & Carroll, 1995). emc is also required for the specification of the R7 photoreceptor cells and the non-neuronal cone cells, neither of which depends on any known proneural bHLH gene (Bhattacharya & Baker, 2009). Most recently, emc was also found to be required for left-right asymmetry of the Drosophila gut(Ishibashi et al., 2019). Thus, Drosophila provides opportunities to investigate how at least one Id protein functions independently of proneural bHLH proteins, and potentially reveal new mechanisms of developmental regulation.

Insights into how Emc acts independently of proneural genes have progressively emerged from several studies. The first concerned the proper proliferation of imaginal disc cells(Garcia- Alonso & Garcia-Bellido, 1988). Independence of imaginal disc growth from proneural bHLH genes is confirmed by the lack of requirement for da, the only E protein in Drosophila(Bhattacharya & Baker, 2011; Andrade-Zapata & Baonza, 2014). E proteins are ubiquitously-expressed bHLH proteins that are heterodimer partners for proneural bHLH proteins. In Drosophila, proneural bHLH proteins cannot function without Da, so normal growth of da mutant cells implies independence from all Da heterodimer partners. Remarkably, it is actually ectopic Da activity that causes poor growth in emc mutant cells, normal growth being restored in emc da doubly mutant cells (Bhattacharya & Baker, 2011). Without emc, Da levels are elevated and by themselves activate transcription of expanded (ex), a component of the Salvador-Hippo-Warts (SHW) tumor suppressor pathway. This increased SHW pathway activity is what inhibits growth of imaginal disc cells(Wang & Baker, 2015b). Thus, one role of emc is to restrain Da(Reddy, Bhattacharya, & Baker; Bhattacharya & Baker, 2011; Wang & Baker, 2015a; Li & Baker, 2018).

In the course of these studies, we noticed that ex mutations affected the number of sensory organ precursor (SOP) cells in the thorax(Wang & Baker, 2015b). We showed that this was due to activity of Yorkie (Yki), the transcription factor target of SHW signals, in the ex mutant cells. This in turn increased transcription of a direct Yki target gene, Death-Associated Inhibitor of Apoptosis 1 (diap1)(Wang & Baker, 2019). Diap1 is a ubiquitin ligase that prevents accumulation of a processed form of Dronc, an apical caspase in Drosophila(Muro, Hay, & Clem, 2002; Wilson et al., 2002; Yan, Wu, Chai, Li, & Shi, 2004; Li, Wang, & Shi, 2011). Caspases are proteases that are well known for driving apoptosis by cleaving cellular substrates. They are expressed as inactive zymogens that are then activated by signals (initiator caspases) or by cleavage by caspases (effector caspases) in a feed-forward process that is expected to kill cells rapidly once a threshold of caspase activity has been crossed(Fuchs & Steller, 2011). Many studies show that caspases can also mediate non-apoptotic processes, although what differentiates apoptotic from non-apoptotic outcomes is uncertain, as the same caspase cascade of initiator and effector caspases seems to be involved (Yamaguchi & Miura, 2015; Aram, Yacobi-Sharon, & Arama, 2017; Nakajima & Kuranaga, 2017; Baena-Lopez, Arthurton, Xu, & Galasso, 2018; Colon-Plaza & Su, 2022). Non-apoptotic caspase activities are of particular interest because of their contributions to multiple diseases(Su, 2020). In ex mutants, the increased diap1 transcription does not affect cell death much, but causes an increase in wg signaling activity in the thorax(Wang & Baker, 2019). Wg signaling promotes SOP cell determination in the thorax, and it has been shown previously that Wg signaling is antagonized by caspases, through a mechanism that is non-apoptotic but whose direct target is not yet certain(Baker, 1988; Kanuka et al., 2005; Wang & Baker, 2019).

Since emc mutations elevate ex expression(Wang & Baker, 2015b), we wondered whether emc also changes Diap1 levels and affects caspase activities. Increased ex expression in emc mutants would be expected to reduce transcription of the diap1 gene and potentially increase caspase activity, the opposite of what occurs in ex mutants. This predicts that caspases could contribute to aspects of the emc mutant phenotype, especially those that do not depend on proneural bHLH genes. Remarkably, we found that caspase activity was responsible for all the aspects of the emc phenotype that we tested. This included reduced growth of imaginal disc cells, accelerated morphogenetic furrow progression, and loss of R7 and cone cells fates in the eye. The effects on eye development all reflected caspase-dependent expression of the Notch ligand Delta, which is known to contribute to morphogenetic furrow progression, and R7 and cone cell fate specification. Thus, caspase-dependent non-apoptotic signaling underlies multiple roles of emc that are independent of proneural bHLH proteins.

Materials and Methods

Drosophila Strains

The following stocks were employed in this study and were maintained at 25°C unless otherwise stated - hsflp;;emc^AP6 FRT80/TM6B, hsflp;;droncⁱ²⁹FRT80/TM6B, hsflp;;droncⁱ²⁹emc^AP6, hsflp;;Df(3L)H99/TM3, hsflp;;Df(3L)H99 emc ^AP6/TM3, Ubi-GFP M(3)67C FRT80, FRT42 M(2)56F Ubi-GFP, FRT82 M(3)95A Ubi-GFP, hsflp;; Ubi-GFPFRT80, hsflp; Su(H)^Δ47FRT40, pie^eB3 FRT40 (Baker et al., 1992), psn[v1]FRT80/TM6B, Fz3-RFP;D/TM6B (kind gift from Yu Kimata, University of Cambridge). We obtained genomic Ci construct from Kalderon lab and used BestGene Inc. to target the transgene on the third chromosome and then recombined these flies to generate hsflp;;ci+M(3)67Calz FRT80/TM6B;ci94/y+spa.

Mosaic Analysis

Mosaic clones were obtained using FLP/FRT mediated mitotic recombination (Golic, 1991; Xu & Rubin, 1993). For non-Minute genotypes, larvae were subjected to heat shock for 30 minutes at 37℃, 60 ± 12 hours after egg laying. For Minute genotypes, heat shock was performed 84 ± 12 hours after egg laying for 50 minutes. Larvae were dissected 72 hours after heat shock. All flies were maintained at 25℃ unless otherwise stated.

Immunohistochemistry and histology

Unless otherwise noted, preparation of eye and wing imaginal discs for immunostaining and confocal imaging were performed as described previously (Baker, Li, Quiquand, Ruggiero, & Wang, 2014). Antibodies from Developmental Studies Hybridoma Bank (DSHB): anti-Ptc (mouse, 1:40), anti-Elav (mouse, 1:100), anti-Elav (rat, 1:50, anti-Cut (mouse, 1:50), anti-Delta C594.9B (mouse, 1:2000), anti-βGal (mouse, 1:100), mouse anti-βGal (1:100, DSHB 40-1a) and anti-Ci (rat, 1:10). Other antibodies: anti-phospho-Smad1/5 (rabbit, 1:100, Cell Signaling), anti- DIAP1 (mouse, 1:50) (gift from Hyun Don Ryoo, NYU), anti-Dcp1 (rabbit, 1:100, Cell Signaling), anti-Sens (guinea pig, a gift from Hugo Bellen used at 1:50), anti-Da (mouse, 1:200), rabbit anti-Emc (1:40), anti-GFP (rat, 1:50 from Nacalai Tesque # GF090R), rabbit anti-GFP (1:500), rabbit anti-ß-Galactosidase (1:100, Cappel), E(spl)bHLH (1:50,mAb323) and guinea pig anti-runt (1:500). Secondary antibodies conjugated with Cy2, Cy3 and Cy5 dyes (1:200) were from Jackson ImmunoResearch Laboratories and Alexa 555 (1:500). Multi-labelling images were sequentially scanned with Leica SP8 confocal microscopes and were projected and processed with ImageJ. All images were assembled into figure format using Adobe Illustrator 2020.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

For labeling dead cells with TUNEL assay, ApopTag® Red In Situ Apoptosis Detection Kit (Cat #S7165) was used according to manufacturer’s instruction. Briefly, dissected eye discs were fixed for 20 minutes at room temperature followed by three washes with 1X PBS. Then the samples were incubated in equilibration buffer for 1 minute followed by incubation in reaction buffer (TdT enzyme; ratio 7:3) at 37 °C for 1 hour. TdT reaction mix was replaced with stop buffer (diluted 1:34 in dH2O) and incubated for 10 min at room temperature. Samples were washed three times with 1X PBS, 5 min per wash; and incubated with anti-digoxigenin antibody solution (diluted 31:34 in blocking solution) for 30 min at room temperature. The samples were then washed three times in 1X PBS, 5 min per wash. For the subsequent antibody staining, the samples were blocked in PBST (1X PBS + 0.5% Triton-X) for 30 min, and incubated with primary antibodies in PBST overnight at 4 °C. The samples were next washed with PBST and incubated for 2 h with secondary antibodies in PBST, and then again washed with PBST, followed by PBS wash and samples were mounted in mounting media.

Statistical analysis

Statistical analysis of clone size data reported in Figur 1N is described in the figure legend.

Prediction of caspase cleavage sites

Caspase cleavage sites were predicted for Delta using Cascleave 2.0(Wang et al., 2014). Cascleave 2.0 was set to a medium stringency threshold for prediction of cleavage sites.

Results

Caspase activity causes growth defects in emc mutants

To determine the contribution of caspases to growth inhibition in emc mutant cells, we used the FLP/FRT system (Xu & Rubin, 1993) to generate clones of emc mutant cells that were unable to activate caspases normally. We achieved this by homozygously deleting the linked reaper (rpr), head involution defective (hid) and grim genes using Df(3L)H99. Rpr, Hid and Grim proteins promote Diap1 degradation in response to apoptotic stimuli and allow activation of initiator caspases such as dronc to start apoptosis(Yoo et al., 2002). By removing all three pro-apoptotic proteins, Df(3L)H99 affects both apoptotic and non-apoptotic caspase functions in Drosophila (White et al., 1994; Tapadia & Gautam, 2011). Thus, clones of emc Df(3L)H99 mutant cells should be defective for caspase activation. In addition, we also made clones of emc mutant cells further mutated for dronc, which encodes the main initiator caspase in Drosophila that is necessary for most developmental apoptosis (C. J. Hawkins et al., 2000; Meier, Silke, Leevers, & Evan, 2000; Quinn et al., 2000). Dronc contributes to non-apoptotic caspase functions as well, so emc dronc mutant cells should also show reduced non-apoptotic and well as apoptotic caspase functions.

In comparison to neutral clones, which grew equivalently to their twin-spot controls, emc mutant clones showed greatly reduced growth in eye or wing imaginal discs, as described previously (Figures 1A-D) (Garcia-Alonso & Garcia-Bellido, 1988; Bhattacharya & Baker, 2011; Andrade-Zapata & Baonza, 2014). By contrast, emc mutant clones that were also mutant for dronc, or emc Df(3L)H99 clones that lacked the rpr, grim and hid genes, grew more like their twin spot controls in both eye and wing (Figures 1E-H). Deletion of the rpr, hid and grim genes restored growth indistinguishable from the wild type to emc clones, although mutation of dronc rescued less completely (see Figure 1N for quantification). Neither homozygosity for dronc, nor deletion of rpr, grim and hid, affected growth of otherwise wild type imaginal disc clones (Figure 1I-L).

Figure 1:

Previous studies of emc phenotypes have often used Minute genetic backgrounds (ie heterozygosity for mutations in Rp genes) to retard growth and enhance the size of the emc mutant clones(Bhattacharya & Baker, 2009). When emc Df(3L)H99 clones were induced in the M(3)67C background, the clones took over almost the entire disc, leaving only a few M(3)67C heterozygous cells remaining (Figure 1M).

These results indicate that the growth disadvantage of emc mutant imaginal disc clones is mostly attributable to cell death genes. Preventing caspase activation, either by mutating the main initiator caspase, or by preventing Diap1 turnover, partially or completed restored normal growth. Our data did not support a previous suggestion that dronc was required for normal wing disc growth(Verghese, Bedi, & Kango-Singh, 2012).

Emc regulates furrow progression through non-apoptotic caspase activity

Emc mutations have multiple effects on the eye imaginal disc, although only a few aspects of retinal differentiation depend on proneural bHLH genes. The proneural gene atonal is required, along with da, for the specification of R8 photoreceptor precursors in the morphogenetic furrow that found each ommatidial cluster in the larval eye disc(Jarman, Grell, Ackerman, Jan, & Jan, 1994; Brown et al., 1996). Later, during pupal development, proneural genes of the Achaete-Scute gene Complex (AS-C), along with da, are required for the specification of the interommatidial bristles(Cadigan, Jou, & Nusse, 2002). All the other cell types develop independently of proneural bHLH genes, and most of them develop independently of da (Jimenez & Campos-Ortega, 1987; Brown et al., 1996).

In emc mutant clones, retinal differentiation begins precociously, associated with more rapid transit of the morphogenetic furrow across the disc (Figure 2A, B)(Brown et al., 1995; Bhattacharya & Baker, 2011, 2012). By contrast, we found that 75% of the time, the morphogenetic furrow progressed normally through emc dronc double mutant clones (Figure 2C). Eye discs containing emc Df(3L)H99 double mutant clones always appeared completely normal (Figure 2D). Both dronc and Df(3L)H99 mutant clones also showed normal furrow progression in the absence of emc mutations (Figure 2 Figure Supplement 1A,B).

Figure 2:

Not only the position of the morphogenetic furrow, but also the overall pattern of retinal differentiation revealed by labeling for Senseless, which is specific for R8 photoreceptor cells, and Elav, which labels all neuronal photoreceptor cells, appeared so normal in these genotypes that we deemed it necessary to confirm that the supposedly emc dronc mutant and emc Df(3L)H99 mutant clones were indeed mutated for emc. This was confirmed by lack of staining by an antibody against Emc protein, similar to plain emc mutant clones that did affect the morphogenetic furrow (Figure 2 Figure supplement 1C-E). We also found that emc dronc and emc Df(3L)H99 clones upregulated Da protein expression to a comparable degree to plain emc mutant clones (Figure 2 Figure Supplement 1C-E)(Bhattacharya & Baker, 2011). This further confirmed the absence of emc function from emc dronc mutant and emc Df(3L)H99 genotypes, and also showed that cell death pathways were not required for the regulation of Da protein levels by emc.

We have previously shown that diap1 transcription is reduced in emc mutant clones, and in Da-overexpressing cells, due to Yki inhibition downstream of ex (Wang & Baker, 2015b). In eye imaginal discs, Diap1 protein was normally present uniformly (Figure 3A). Diap1 protein levels were cell-autonomously reduced in emc mutant clones (Figure 3B). A comparable reduction was also seen in emc Df(3L)H99 clones, (Figure 3C). Because emc Df(3L)H99 clones developed normally, this suggested Diap1 protein levels were upstream of the cell death pathway in affecting morphogenetic furrow speed, and that rpr, grim, and hid activities were required downstream of diap1. To test the role of Diap1 directly, diap1 was over-expressed in the posterior eye by transcription under GMR-Gal4 control. This rescued morphogenetic furrow speed to normal in emc clones (Figure 3D). We also saw restoration of morphogenetic furrow speed in emc clones that expressed Baculovirus P35 under GMR-Gal4 control (Figure 3E).

Figure 3:

Baculovirus P35 encodes a caspase pseudo-substrate that inhibits all Drosophila caspases except Dronc(Hay, Wolff, & Rubin, 1994; Xue & Horvitz, 1995; C.J. Hawkins et al., 2000; Meier et al., 2000). The emc clones expressing p35 also lacked cell death (Supplemental Figure 2D). The restoration of morphogenetic furrow speed by Diap1, a dronc antagonist, as well as by the caspase inhibitor P35, suggest that the effect of morphogenetic furrow acceleration in emc mutant clones is due to the caspase cascade.

To test whether reduced Diap1 expression promoted apoptosis and thereby accelerated the morphogenetic furrow, we assessed apoptosis levels by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of eye discs containing mutant clones. We found almost no cell death posterior to the furrow in emc clones (Supplemental Figure 2B), and cell death in emc clones anterior to the furrow was comparable to controls and less than that of cells surrounding the clones (Supplemental Figure 2A). Some cell death is expected outside emc or control clones, as these cells have the M background that is itself associated with an increase in apoptosis(Coelho et al., 2005; Li & Baker, 2007; Kale, Li, Lee, & Baker, 2015). To test whether apoptosis in the eye disc would be sufficient to promote morphogenetic furrow progression, we generated mosaic clones for a pineapple eye (pie) mutation. These pie mutant cells have an elevated rate of apoptosis in imaginal discs, but not sufficient to prevent pie homozygous clones surviving late into larval and even adult life (Shi, Stampas, Zapata, & Baker, 2003). The rate of morphogenetic furrow progression was unaffected in eye discs containing pie clones (Supplemental Figure 2C). Because no excess cell death was detected in emc clones, and cell death was insufficient to accelerate the morphogenetic furrow in otherwise wild type eye discs, emc clones might be affected by a non-apoptotic caspase activity.

Wingless and Dpp signaling are unaffected by emc

To understand how caspases could affect the speed of the morphogenetic furrow, we analyzed pathways known to contribute. Hedgehog (Hh) and Decapentaplegic (Dpp) signaling drive this differentiation wave, along with a contribution from Notch signaling (U. Heberlein, T. Wolff, & G. M. Rubin, 1993; Ma, Zhou, Beachy, & Moses, 1993; Borod & Heberlein, 1998; Baonza & Freeman, 2001b; Fu & Baker, 2003). A negative regulator of morphogenetic furrow progression is Wingless (Wg), which is expressed at the dorsal and ventral eye disc margins (Maurel-Zaffran & Treisman, 2000; Lee & Treisman, 2002).

Because we found that ex mutations affected thoracic bristle patterning through a caspase-dependent non-apoptotic effect on Wg signaling (Wang & Baker, 2019), we looked first to see whether emc mutations reduced Wg signaling in the eye. We used Frizzled-3 RFP (Fz3- RFP) as a reporter (Sato, Kojima, Ui-Tei, Miyata, & Saigo, 1999). In control eye discs, the Fz3- RFP recapitulates the pattern of endogenous Wg signaling activity at the wing margins (Figure 4 figure supplement 1) (Treisman & Rubin, 1995). Frizzled-3 RFP expression was not changed in emc clones (Figure 4A). We then used a mutation in naked cuticle (nkd), encoding a negative feedback regulator of Wg signaling, to modulate Wg signaling(Zeng et al., 2000; Chang, Chang, Barolo, & Cadigan, 2008). If the morphogenetic furrow was accelerated in emc mutant clones due to reduced Wg signaling, more normal development should occur in emc nkd clones. The morphogenetic furrow was still accelerated in emc nkd clones, however (Figure 4B). These results provided no evidence that Wg signaling was the relevant emc target in the eye.

Figure 4:

We also examined Dpp signaling, since ectopic Dpp signaling is sufficient to accelerate the morphogenetic furrow(Pignoni & Zipursky, 1997). The pattern of pMad, a readout of Dpp signaling, was identical in emc mutant and control clones spanning the morphogenetic furrow (Figure 4C, D). Thus, Dpp signaling did not seem altered by emc mutants either.

Hedgehog pathway

Hedgehog signaling is a key mover of the morphogenetic furrow(U. Heberlein, T. Wolff, & G.M. Rubin, 1993; Ma et al., 1993; Treisman, 2013). Elevated Hh signaling is sufficient to accelerate the morphogenetic furrow(Heberlein, Singh, Luk, & Donohoe, 1995; Ma & Moses, 1995). Notably, it has been suggested previously that emc mutations affect the morphogenetic furrow by activating Hedgehog signaling, because emc mutant cells accumulate Ci protein (Spratford & Kumar, 2013). Full-length Ci protein (Ci155) is targeted to the proteosome by Cul1 for processing into a transcriptional repressor protein Ci75(Aza-Blanc, Ramirez-Weber, Laget, Schwartz, & Kornberg, 1997). By inhibiting this processing, Hh prevents repression of target genes by Ci75 and promotes transcriptional activation downstream of Ci155.

Accordingly, Ci155 accumulation is a feature of cells receiving Hh signals(Motzny & Holmgren, 1995).

We confirmed that emc mutant cells contain higher levels of Ci155, as reported previously (Figure 5A)(Spratford & Kumar, 2013). Ci155 was elevated in emc dronc clones (Figure 5B) but reduced to wild-type levels in emc Df(3L)H99 clones (Figure 5C, Figure 5 figure supplement 1). Thus, Ci155 levels did not correlate perfectly with behavior of the morphogenetic furrow.

Figure 5:

We noticed that Ci155 levels were elevated in emc mutant clones in the posterior, differentiating eye disc, as well as in and ahead of the morphogenetic furrow (Figure 5A). This is significant, because Ci155 is not affected by Hh-dependent Cul1 processing posterior to the morphogenetic furrow (Ou, Lin, Chen, & Chien, 2002; Baker, Bhattacharya, & Firth, 2009).

Ci155 accumulation posterior to the furrow suggests a Hh-independent mechanism.

To test whether Ci155 accumulation in emc clones indicates elevated Hh target signaling, we looked at the transmembrane receptor Patched (Ptc) which is a transcriptional target of Hh signaling, acting in a negative feedback loop(Hepker, Wang, Motzny, Holmgren, & Orenic, 1997). We saw no changes in Ptc protein levels in either emc or emc Df(3L)H99 clones compared to controls, questioning the notion that Hh signaling was altered by emc (Figure 5 figure supplement 2).

To test definitively whether Ci155 is responsible for accelerating the furrow in emc clones, we generated emc ci double mutant clones. To achieve this, a genomic transgene that rescues ci⁹⁴ flies to adulthood (Little, Garcia-Garcia, Sul, & Kalderon, 2020), was introduced into chromosome arm 3L where it is linked to the wild type emc locus, so that mitotic recombination in the ci⁹⁴null background leads to emc ci double mutant clones. For unknown reasons, emc ci double mutant clones were small and difficult to obtain, even in the Minute background. In all the emc ci double mutant clones we found that spanned the morphogenetic furrow; eye differentiation was still accelerated (Figure 5D). These data might not exclude a quantitative contribution of Ci to more rapid morphogenetic furrow progression in the absence of emc but are sufficient to show that emc regulates the speed of the morphogenetic furrow through at least one other caspase target.

Delta expression is a target of caspases

The remaining signaling pathway that contributes to morphogenetic furrow movement is Notch. Specifically, only cells where Notch signaling is active are competent to initiate retinal differentiation in response to Dpp (Baonza & Freeman, 2001a; Fu & Baker, 2003). Accordingly, ectopic expression of Delta, the transmembrane ligand for Notch, is sufficient to accelerate retinal differentiation anterior to the morphogenetic furrow by expanding the effective range of Dpp signaling (Baonza & Freeman, 2001a; Li & Baker, 2001).

To test whether emc restrains Notch, we examined the bHLH proteins of the E(spl)-C, widely characterized targets of the Notch pathway (Bray, 2006). We used mAb323 to detect up to five E(spl) bHLH proteins with Notch-dependent expression (Jennings, Preiss, Delidakis, & Bray, 1994). E(spl) protein expression, and hence Notch signaling, was higher in the morphogenetic furrow in emc clones than in wild type cells (Figure 6A). By contrast, E(spl) protein expression was normal in emc dronc clones (Figure 6B).

Figure 6:

To test whether elevated Notch signaling was required to accelerate the morphogenetic furrow in emc mutants, we examined emc psn double mutant clones. Presenilin (Psn) is the enzymatic component of γ-secretase that releases the intracellular domain of Notch during active Notch signaling, and the psn gene is linked to emc. Loss of psn function leads to a Notch loss of function phenotype (Struhl & Greenwald, 1999; Ye, Lukinova, & Fortini, 1999). Accordingly, psn clones lead to a neurogenic phenotype in the eye, without affecting the progression of the morphogenetic furrow (Figure 6C)(Li & Baker, 2001). The position of the morphogenetic furrow was also not affected in emc psn clones (Figure 6D). Thus, the furrow was not accelerated in emc mutant clones also defective for Notch signaling.

These two results together indicated that emc mutants promoted Notch signaling in the morphogenetic furrow, acting through caspase signaling on a step prior to γ-secretase cleavage of the intracellular domain of Notch. Accordingly, we decided to check Delta (Dl) protein levels.

We found that Dl protein levels were clearly elevated cell-autonomously throughout emc clones, both posterior and anterior to the furrow (Figure 7B). Importantly, this includes the region just ahead of the morphogenetic furrow that lacks Delta expression in normal development(Parks, Turner, & Muskavitch, 1995; Baker & Yu, 1998)(Figure 7B). By contrast, levels of Dl protein in emc dronc clones and emc Df(3L)H99 clones were similar to those of wild type controls (Figure 7A,C,D). This indicated that Dl protein is a target of caspases, directly or indirectly.

Figure 7:

Because Dl activates Notch signaling cell non-autonomously, we wondered whether the effect of emc mutant clones was cell-autonomous. We note that, in all the experiments reported here, and in all the previous studies of emc mutant clones affecting morphogenetic furrow movement, the morphogenetic furrow is maintained as a continuous groove across the eye disc (Brown et al., 1995; Bhattacharya & Baker, 2011, 2012; Spratford & Kumar, 2013). That is, the advanced front of retinal differentiation within emc clones is always smoothly continuous with the normal morphogenetic furrow outside the clones, implying a progressive gradual increase in morphogenetic furrow speed near the lateral edges of emc mutant clones (eg Figures 2B, 4B, 4D, 5A, 6A). Clones of Su(H) null mutants, which act cell-autonomously, provide a contrasting example. Complete loss of Su(H) accelerates the morphogenetic furrow due to loss of default Su(H) repression of Notch targets(Li & Baker, 2001). The boundaries of Su(H) null clones exhibit a clear discontinuity between the rates of differentiation within and outside the clones (Figure 6E). This difference between Su(H) and emc mutant clones is consistent with the idea that emc affects morphogenetic furrow progression non-autonomously.

Specific ommatidial cell fates are regulated by caspases in emc mutants

Because the general pattern of neurogenesis revealed by pan-neuronal anti-Elav staining appeared so normal in emc dronc and emc Df(3L)H99 clones (Figure 2C,D), we examined whether effects of emc on particular retinal cell fates was caspase dependent. Emc is also required for R7 differentiation and for timely onset of cone cell differentiation(Bhattacharya & Baker, 2009), two cell fate decisions that also depend on Notch signaling(Cooper & Bray, 2000; Flores et al., 2000; Tomlinson & Struhl, 2001; Treisman, 2013). These cell fates are normally independent of da, but like imaginal disc cell growth and morphogenetic furrow progression, ectopic da activity perturbs them in emc mutants(Reddy et al.; Brown et al., 1996).

Clones of emc mutant cells lack R7 photoreceptor cells (Figure 8A,B)(Bhattacharya & Baker, 2009). R7 differentiation was restored to 90% of ommatidia in emc Df(3L)H99 clones (Figure 8C). As shown before, emc mutant clones delayed Cone Cell differentiation by 2-3 columns (corresponding to a delay of 3-6 h)(Bhattacharya & Baker, 2009)(Figure 8D,E). We found no delay in cone cell differentiation in most emc Df(3L)H99 clones (Figure 8F). These results indicated that caspase activity contributes to the R7 and cone cell differentiation defects that are also characteristic of emc mutants.

Figure 8:

Discussion

The Inhibitor of DNA binding (Id) proteins are important regulators of differentiation(Massari & Murre, 2000; Ling et al., 2014; Wang & Baker, 2015a; Roschger & Cabrele, 2017; Singh et al., 2022). They are well known to antagonize proneural basic helix- loop-helix (bHLH) proteins (Benezra et al., 1990; Ellis et al., 1990; Garrell & Modolell, 1990; Cabrera et al., 1994; Ellis, 1994; Norton, 2000). It has been uncertain whether this is their only function, as in Drosophila it is clear that emc mutations affect processes that are independent of proneural genes. Here, we report that multiple effects of emc mutations that occur independently of proneural bHLH genes, and are due to the loss of restraint on da function in emc mutant cells, are caused by caspase-dependent non-apoptotic processes that result from reduced expression of Diap1 protein.

One known effect of emc is that it restrains the Drosophila E protein Da. Da is expressed ubiquitously and is the obligate heterodimer partner of proneural bHLH proteins in neurogenesis.

In undifferentiated progenitor cells, most or all Da is thought to be sequestered in inactive heterodimers with Emc(Li & Baker, 2018). In emc mutant cells, Da levels rise and become functional, potentially as homodimers(Bhattacharya & Baker, 2011).

In the context of imaginal disc cell growth, emc mutant cells experience da-dependent over-expression of the ex gene(Wang & Baker, 2015b, 2018). Although most study of ex has focused on its role in growth control through the SHW pathway, ex was recently shown to influence patterning through Yki-dependent transcription of diap1, an important regulator of cell death pathways. This was in the developing thorax, where ex mutations affect bristle patterning through diap1 and a non-apoptotic effect of caspases on Wg signaling(Wang & Baker, 2019).

This observation led us to explore whether aspects of the emc phenotype could reflect reduced diap1 expression and elevated caspase activity.

Remarkably, we found that all four features of the emc phenotype we examined depended on the caspase-mediated cell death pathway. The normal growth of imaginal disc cells, normal speed of the morphogenetic furrow, specification of R7 photoreceptor cells, and timing of non- neuronal cone cell development were all restored when caspase activation was prevented, which was achieved by inactivating the initiator caspase Dronc, deleting the proapoptotic rpr, grim, and hid genes, over-expressing diap1, or over-expressing baculovirus p35. These appeared to be non-apoptotic effects, because no elevation in apoptosis was apparent in emc mutant cells, because generating apoptosis by another means did not mimic the effects, and because the effects were mediated by elevated Dl protein expression in cells that were not apoptotic. They appeared to be effects of caspase activity, because they depended in part on dronc, an initiator caspase that cleaves and activates effector caspases, because they were suppressed by baculovirus p35, a caspase inhibitor that is a caspase pseudo-substrate, and because they were suppressed by Diap1, a ubiquitin ligase that inhibits caspases and targets them for degradation. The effects may depend on the full caspase cascade of initiator and effector caspases, because sensitivity to p35 indicates that caspases other than Dronc are necessary, and because mutating dronc alone gave lesser degrees of rescue than deleting rpr, grim, and hid. This is consistent with many other studies that describe the pathways for apoptotic and non-apoptotic caspase functions as similar(Kanuka et al., 2005; Kuranaga & Miura, 2007; Aram et al., 2017; Nakajima & Kuranaga, 2017; Baena-Lopez et al., 2018; Su, 2020; Colon-Plaza & Su, 2022).

Whereas non-apoptotic caspase activity promotes Wg signaling during specification of thoracic bristles(Kanuka et al., 2005), in the Drosophila eye the non-apoptotic caspase target was Notch signaling, due to elevated Delta protein levels. We do not know whether Delta is the direct caspase target. The Delta protein sequence contains multiple predicted caspase target sites, as do many proteins (Wang et al., 2014). Only one candidate site lies in the intracellular domain, where it would potentially be accessible to caspases (Figure 9B). Truncation of the Dl intracellular domain is usually associated with loss of Dl function, not stabilization and enhanced function, however (Daskalaki et al., 2011). Another possibility is that a regulator of Dl expression or stability and function is the direct caspase target.

Figure 9:

Notably, emc mutations accelerate the morphogenetic furrow by enhancing Notch signaling, apparently cell-autonomously, but inhibit R7 and cone cell differentiation through cell-autonomously reduced N signaling(Reddy et al.; Bhattacharya & Baker, 2009). These contrasts may be explained through the dual functions of the Notch ligand Delta in trans- activation and in cis-inhibition (Micchelli, Rulifson, & Blair, 1997; Jacobsen, Brennan,

Martinez-Arias, & Muskavitch, 1998; Li & Baker, 2004; Bray, 2016). The eye disc region ahead of the morphogenetic furrow lacks Delta expression (Parks et al., 1995; Baker & Yu, 1998).

Ectopic Dl expression here is sufficient to activate N non-autonomously and drive morphogenetic furrow progression (Baonza & Freeman, 2001a; Li & Baker, 2001). Within the R7 equivalence group, Delta is expressed early in R1 and R6 photoreceptor precursors, protecting them from N activation through cis-inhibition, and establishing the distinction between R1,6 and R7 precursor cells (Miller, Lyons, & Herman, 2009). Ectopic Dl expression posterior to the morphogenetic furrow has cis-inhibitory effect. Elevated Dl levels within emc mutant clones may render potential R7 and cone cell precursors cell-autonomously resistant to N activation, as normally seen in R1/6 precursor cells.

A previous study suggested that emc mutations might affect Hh signaling (Spratford & Kumar, 2013). Our data point much more clearly to an effect on Notch signaling. Although the Hh target Ci155 accumulates in emc mutant cells, increased Hh signaling is not the only possible cause of this. Ci155 is cytoplasmic, and thought to be only a precursor for a labile nuclear activator molecule(Ohlmeyer & Kalderon, 1998). Preventing activation and nuclear translocation of Ci155 by mutating the kinase fused also leads to Ci155 accumulation, without activating Hh signaling. Presumably Ci155 accumulates because it is more stable than its activated derivative(Ohlmeyer & Kalderon, 1998). Significantly, emc mutant cells have been reported to over-express the fused antagonist su(fu), providing a potential alternative explanation of Ci155 accumulation in emc mutant cells(Spratford & Kumar, 2013). Analyzing emc ci double mutant cells suggested that ci was not required to accelerate the morphogenetic furrow in emc mutant clones.

Da protein binds to and potentially regulates hundreds of genes throughout the Drosophila genome(Li et al., 2008). Accordingly, Da activity in emc mutant cells might be expected to lead to non-specific and pleiotropic effects. Contrary to this expectation, multiple aspects of proneural bHLH-independent emc mutant phenotypes have a simple common basis, ie reduced diap1 expression and elevated caspase activity. Remarkably, mutating emc may be the most general way to activate non-apoptotic caspase activities yet discovered.