Abstract
The interactions that cells in Drosophila imaginal discs have with their neighbors are known to regulate their ability to survive. In a screen of genes encoding cell surface proteins for gene knockdowns that affect the size or shape of mutant clones, we found that clones of cells with reduced levels of echinoid (ed) are fewer, smaller, and can be eliminated during development. In contrast, discs composed mostly of ed mutant tissue are overgrown. We find that ed mutant tissue has lower levels of the anti-apoptotic protein Diap1 and has increased levels of apoptosis which is consistent with the observed underrepresentation of ed mutant clones and the slow growth of ed mutant tissue. The eventual overgrowth of ed mutant tissue results not from accelerated growth, but from prolonged growth resulting from a failure to arrest growth at the appropriate final size. Ed has previously been shown to physically interact with multiple Hippo-pathway components and it has been proposed to promote Hippo pathway signaling, to exclude Yorkie (Yki) from the nucleus, and restrain the expression of Yki-target genes. We did not observe changes in Yki localization in ed mutant tissue and found decreased levels of expression of several Yorkie-target genes, findings inconsistent with the proposed effect of Ed on Yki. We did, however, observe increased expression of several Yki-target genes in wild-type cells neighboring ed mutant cells, which may contribute to elimination of ed mutant clones. Thus, ed has two distinct functions: an anti-apoptotic function by maintaining Diap1 levels, and a function to arrest growth at the appropriate final size. Both of these are unlikely to be explained by a simple effect on the Hippo pathway.
Introduction
Nowhere is the influence of neighbors more important than for cells in developing epithelia. In unicellular organisms, the availability of nutrients is the primary regulator of cell proliferation. In addition to nutrient availability, the survival and proliferation of epithelial cells in multicellular organisms is regulated by a variety of signals emanating from other cells. Such signals include diffusible factors that could be secreted locally or circulate systemically. The packing of cells into tissue layers generate mechanical forces that can influence cell proliferation and survival. Additionally, especially in epithelial tissues, contacts between cells and their immediate neighbors, likely mediated by cellular adhesion molecules, can determine whether cells remain within the layer or get extruded; extrusion often accompanies or promotes cell death.
The importance of a cell’s immediate neighbors in regulating its survival and proliferation is best illustrated by the phenomenon of cell competition (Morata and Ripoll 1975) (reviewed by Amoyel and Bach 2014; Nagata and Igaki 2018; Baker 2020; Cumming and Levayer 2023). Cell competition does not refer simply to the ability of fast-growing cells to outpace slow-growing cells. Rather, it is a situation where cells that are less fit for a variety of reasons are underrepresented because they are eliminated only in the presence of faster-growing cells (i.e., in genetic mosaics). In their original discovery of this phenomenon, Morata and Ripoll found that cells heterozygous for a class of mutations known as Minute, that mostly disrupt ribosomal proteins, are eliminated in the presence of wild-type cells. However, as a homogeneous population, they can generate animals of relatively normal size and shape albeit much more slowly. Subsequently, small patches of wild-type cells were shown to be eliminated when they are surrounded by cells having even modest increases in the level of the Myc protein (Moreno and Basler 2004; de la Cova et al. 2004). Cells capable of eliminating wild-type cells were dubbed “supercompetitors”. Additionally, cells with mutations in the Hippo pathway that have increased activity of the transcriptional co-activator Yorkie (Tyler et al. 2007; Neto-Silva et al. 2010), cells with increased Wnt signaling (Vincent et al. 2011), cells with increased Jak/Stat signaling (Rodrigues et al. 2012) and cells with reduced crumbs function (Hafezi et al. 2012) were also shown to be supercompetitors. Collectively these findings show that the designation of “winners’’ and “losers’’ is not determined by the genotype of the cell itself but by how it compares itself to its neighbors. How this comparison is achieved is still not well understood since many of the mutations that alter competitive ability affect intracellular proteins and any kind of comparison must occur at the cell surface. Once the comparison has happened, specific isoforms of the cell surface protein Flower are upregulated in “loser” cells (Rhiner et al. 2010). Also, winner cells seem capable of expressing higher levels of the Toll ligand Spätzle which might promote the loser cell fate in adjacent cells (Alpar et al. 2018).
There are several other phenomena that resemble classical cell competition, some of which appear to be mechanistically distinct. Clones of cells with mutations in genes encoding proteins that regulate apicobasal polarity such as scribble (scrib) and discs large (dlg) are eliminated during development (Brumby and Richardson 2003). However imaginal discs or entire compartments of discs that are entirely composed of mutant cells overgrow and the epithelia are often multilayered (Bilder et al. 2000). The elimination of clones of scrib or dlg cells requires the TNF-ortholog Eiger (Igaki et al. 2009). The loss of apicobasal polarity re-localizes cell surface proteins including the Eiger receptor, Grindelwald, rendering it accessible to circulating Eiger with the result that cells undergo apoptosis (de Vreede et al. 2022). Also, a signaling event at the clone interface involving the ligand Stranded at second (Sas) and the receptor PTP10D that promotes the elimination of polarity-deficient cells has been proposed (Yamamoto et al. 2017) but a requirement for PTP10D has been questioned by others (Gerlach et al. 2022).
In addition to these phenomena, mis-specified cells can be eliminated by a JNK-dependent pathway (Adachi-Yamada and O’Connor 2002) that involves the transmembrane protein Fish-lips (Adachi-Yamada et al. 2005). More recently, there has been an increasing appreciation of the importance of mechanical forces in eliminating cells that proliferate more slowly (Shraiman 2005; Marinari et al. 2012; Mao et al. 2013; Levayer et al. 2016). Such mechanisms might make some cells more susceptible to elimination but this kind of mechanism cannot, on its own, easily account for observations that most, if not all cells, of specific genotypes are selectively eliminated.
One class of proteins that are likely to play an important role in mediating heterotypic interactions at clone interfaces are cell-surface proteins, particularly cell-cell adhesion proteins. The Drosophila genome encodes over one hundred proteins containing cadherin motifs or Ig-loops, which are commonly used for cell-cell adhesion (Hynes and Zhao 2000; Vogel et al. 2003). Although many of these genes have been studied extensively in neuronal contexts, relatively few have been examined for a role in cell survival or cell proliferation in epithelia (Finegan and Bergstralh 2020), and fewer yet in a mosaic context. Here we describe a genetic screen where we have reduced the function of individual cell-surface proteins in clones of cells in the wing imaginal disc and identified those that affect the size or shape of mutant clones. Of these, we focus on the properties of the Echinoid (Ed) protein because its depletion in clones results in clone elimination while reducing ed function in the entire disc results in overgrowth. We demonstrate a role of Ed in maintaining levels of the anti-apoptotic protein Diap1 in cells, and also a separate role in arresting growth when a tissue reaches its final size.
Results
The overall goal of our screen was to identify cell adhesion molecules that regulated the survival, proliferation, or arrangement of epithelial cells in imaginal discs. To that end we generated clonal patches of cells in wing imaginal discs, that had reduced levels of individual adhesion molecules, in the midst of wild-type cells, and assessed the number, size and shapes of the mutant clones. We used the FLP-out Gal4 system (Pignoni and Zipursky 1997) to activate the expression of Gal4 in random isolated cells and their progeny, and concurrently expressed an RNAi transgene under the control of Gal4-responsive UAS elements (Fischer et al. 1988; Brand and Perrimon 1993). The cells expressing the RNAi transgene were marked by concurrent expression of a fluorescent protein (UAS-GFP or UAS-RFP). The size and shape of clones were compared to those expressing an RNAi transgene directed against the white (w) gene (UAS-w-RNAi).
For our screen, we initially compiled a list of 151 genes that encoded known or putative cell-adhesion molecules, based on the assumption that they had cadherin or immunoglobulin domains (Figure 1A) (Supplementary Table 1). These included those identified by Hynes & Zhao (2000) or Vogel et al. (2003) as well as others that we included based on reports in the literature. Since we chose to conduct our screen in the wing imaginal disc, we excluded 43 genes that were not expressed in wing discs using the single-cell RNAseq data of Everetts et al. (2021) and 9 genes whose localization and function precluded a role in cell-cell adhesion (e.g. sarcomere components) (Supplementary Table 2). Of the remaining 99 genes, we screened 74 genes using 90 different RNAi lines (Supplementary Table 3).
(A) Summary of the screen where genes encoding cell-surface proteins were individually knocked down using RNAi transgenes
(B-L) Phenotypes of imaginal discs containing clones generated with a FLP-out Gal4 and UAS-RNAi transgenes, Clones are marked by the inclusion of a UAS-GFP or UAS-RFP transgene as indicated. A cyst-like clone in (K) is shown at higher magnification in (L) and an orthogonal view in (L’). (B-K) are shown at the same magnification; the scale bar, 50 μm is shown in panel (B). (L, L’) are shown at a higher magnification: scale bar is 25 μm.
While expression of most of the RNAi lines did not cause an obvious change in the number, size, or shape of the clones when compared to the expression of w-RNAi (Figure 1B), five lines caused a reduction in clone size, two affected clone shape without reducing clone size, and two reduced clone size and also generated rounder clones (Figure 1A). Those that caused a marked reduction in the number and size of clones were amalgam (ama), beaten path Vc (beat-Vc), sidestep VII (side-VII), Contactin (Cont), and shotgun (shg) (Figure 1C-G). Amalgam is a secreted protein whose best characterized function is in axonal fasciculation where it promotes the adhesion of cells and axons that express Neurotactin to each other (Frémion et al. 2000). Beat and Side family proteins interact with each other to facilitate cell adhesion. A comprehensive analysis of physical interactions between Beat and Side proteins did not predict an interaction between Beat-Vc and Side-VII; Side-VII was predicted to bind to Beat-IV and Beat-Vc was predicted to bind to Side-VI (Li et al. 2017). Contactin is a GPI-anchored protein needed to organize septate junctions of epithelial cells (Faivre-Sarrailh et al. 2004). shg encodes the Drosophila ortholog of E-cadherin that functions as a homophilic adhesion molecule that is a key component of adherens junctions (Oda et al. 1994; Tepass et al. 1996). Clones expressing RNAi constructs targeting fat (ft) (Figure 1H) and dachsous (ds) (Figure 1I) are clearly rounder and likely larger than w-RNAi clones. Fat and Dachsous are large atypical cadherins whose extracellular domains bind to each other and their role in regulating cell proliferation and the orientation of cell division has been studied extensively (reviewed by Fulford & McNeill, 2020; Thomas & Strutt, 2012). Knockdown of two genes, echinoid (ed) (Figure 1J) and off-track2 (otk2) (Figure 1K, L) generated fewer clones that were typically smaller and also appeared more rounded when compared to the irregularly-shaped elongated clones that are normally found in the wing pouch (Figure 1B). Otk2 clones were sometimes extruded as cysts (Figure 1L, L’). The function of Otk2 is not well understood other than it likely functions as a co-receptor for the Wnt2 ligand (Linnemannstöns et al. 2014).
Clones of cells with reduced ed function were infrequent, small, and had smooth outlines. The reduced growth of ed clones in imaginal discs has been noted previously (Escudero et al. 2003; Wei et al. 2005) but is not easily reconciled with some of the other functions of Ed (see below). We therefore chose to characterize the function of Echinoid in growth regulation in more detail. The Echinoid protein has an extracellular domain that includes seven Ig repeats, three FN-III repeats and an intracellular domain of 315 amino acids that includes a C-terminal PDZ-binding protein that can interact either with Bazooka (Baz) or Canoe (Canoe) (Bai et al. 2001; Wei et al. 2005). ed mutant clones are rounded, in contrast to the irregular (“wiggly”) boundaries of wild-type clones (Wei et al. 2005). ed mutant cells fail to assemble proper adherens junctions (AJs) at the interface wild-type cells (Wei et al. 2005; Laplante and Nilson 2006; Chang et al. 2011). This differential adhesiveness might result in the tendency for these cells to remain together and sort away from wild-type cells (Townes and Holtfreter 1955; Steinberg 1963). Additionally, an actomyosin cable that forms at Ed expression boundaries in the wild-type cells is likely to act as a “mechanical fence” at the clone perimeter (Wei et al. 2005; Laplante and Nilson 2006, 2011; Lin et al. 2007; Chang et al. 2011). Together, these properties explain the roundness and smoothness of ed clones at apical levels.
Ed is also thought to function as a signaling molecule. ed mutations were originally identified as dominant enhancers of a hypermorphic allele of the EGF receptor, Ellipse (Elp) (Bai et al. 2001). Ed has subsequently been shown to be a negative regulator of EGF receptor (EGFR) signaling (Bai et al. 2001; Islam et al. 2003; Rawlins et al. 2003a; Spencer and Cagan 2003; Fetting et al. 2009; Ho et al. 2010) and a positive regulator of Notch signaling (Ahmed et al. 2003; Escudero et al. 2003; Rawlins et al. 2003b), possibly by promoting endocytosis of EGFR and Delta respectively. More recently, reduced ed function has also been shown to cause tissue overgrowth by decreasing signaling via the Hippo pathway and increasing the levels of nuclear Yorkie (Yki) (Yue et al. 2012). Ed interacts with multiple Hippo -pathway components, promotes the stability of Salvador (Sav) and curtails the expression of Yki-target genes that normally promote growth and cell survival. Since both the EGFR pathway and Yki promote cell survival and cell proliferation (Díaz-Benjumea and García-Bellido 1990; Bergmann et al. 1998; Kurada and White 1998; Huang et al. 2005), and both EGFR signaling and Yki-target gene expression would be expected to be elevated in ed mutant tissue, the underrepresentation of ed mutant tissue is not easily explained. We therefore decided to examine the properties of ed mutant tissue in greater detail.
Clones of echinoid mutant cells in epithelia are eliminated by a process that resembles cell competition
To avoid the possibility of off-target effects generated with the ed-RNAi transgene used in our screen (V104279), we also tested three other ed-RNAi lines. In each case, clones were induced 72 h after egg lay (AEL) and imaginal discs were dissected 48 h later at 120 h AEL. Both V104279 (Figure 2A) and V3087 (Figure 2B) generated fewer and smaller clones and Ed protein was almost undetectable in those clones. The BL38243 line generated clones that were not smaller and had wiggly outlines, much like wild-type clones (Figure 2D); the knockdown was least effective in this line (Figure 2D’, 2D’’). The V938 line had an intermediate effect (Figure 2C, C’, C’’). We also used mitotic recombination with the MARCM method where mutant clones are positively marked with GFP (Figure 2E-H). Using this approach, homozygous mutant clones of two different null alleles of ed (edIF20 and ed1×5) (de Belle et al. 1993; Bai et al. 2001), that have early stop codons and would be predicted to encode proteins with small portions of the extracellular domain (Escudero et al. 2003), were completely absent from the disc epithelium (Figure 2F-G). Clones that are homozygous for the hypomorphic allele, edsIH8 (de Belle et al. 1993), which has a missense mutations that changes a conserved cysteine residue in the sixth Ig domain of the extracellular portion to serine (Escudero et al. 2003), are observed but are also far less frequent and smaller than wild-type clones (Figure 2H). Notably, in all of these experiments, large clones of myoblasts are often observed beneath the epithelium in the notum region of the disc (Figure 2F’). Although Ed is expressed in wing disc-associated myoblasts based on single cell RNA-seq data (Everetts et al. 2021) and is required for the targeting and morphogenesis of some embryonic and larval body wall muscles (Swan et al. 2006), unlike ed-depleted epithelial cells, ed-depleted myoblasts do not have a growth or survival disadvantage.
(A-D) Imaginal discs containing GFP-marked clones of cells that express an RNAi directed against ed. Four different UAS-RNAi transgenes were used. Panels (A’-D’) and (A’’-D’’) show GFP-marked clones at higher magnification that are stained with an anti-Ed antibody. The magnified regions are indicated with dashed lines in (A-D).
(E-H) GFP-marked clones of cells that are homozygous for the chromosome arm bearing FRT40A alone (E) or FRT40A and an allele of ed (F-H). Note the near absence of clones in the epithelium with the two null alleles of ed (F, G) and the presence of myoblast clones underlying the notum (indicated by an arrowheads). An orthogonal view is shown in (F’) with the disc epithelium outlined; the GFP-marked myoblasts are located basal to the epithelium. Both epithelial and myoblast clones are observed with the hypomorphic allele (H).
(I-K) Clones of cells homozygous for either a wild-type chromosome arm distal to FTR40A (I) or edIF20, FRT40A (J) marked white, generated using eyFLP. The tester chromosome carries a recessive cell lethal resulting in the absence of wild-type twin spots. An overlay of the overgrown eye containing ed clones and the normally sized eye containing wild-type clones is shown in (K).
(L, M) Clones generated using eyFLP using a wild-type tester chromosome that does not carry a recessive cell lethal mutation. FRT40A clones (L) or ed FRT40A clones (M) are white while the wild-type twin spots appear red in both panels. Note the almost complete absence of homozygous ed/ed tissue in eyes that contain wild-type twin spots.
Yue et al. (2012) had reported that ed/ed clones generated during eye development were overgrown. An important difference between that experiment and our experiments is that their clones were generated in a stock where the wild-type chromosome carried a recessive cell-lethal mutation. Thus, following eyFLP-driven mitotic recombination, the eye disc would contain mostly ed/ed tissue as well as a small amount of ed/+ tissue that had not undergone mitotic recombination. Consistent with their observations, we find that eyes composed almost entirely of edIF20/edIF20tissue are overgrown (Figure 2I-K). When the wild-type twin clones are not eliminated with a recessive cell-lethal, we observed almost no homozygous ed/ed tissue (Figure 2L-M). Thus ed/ed tissue is able to survive, and even overgrow, when the disc is mostly composed of ed/ed tissue. In contrast, ed/ed tissue is extremely underrepresented in the presence of wild-type tissue.
To further explore this phenomenon, we generated discs where an increasing proportion of tissue had decreased ed function by increasing the density of clones expressing ed-RNAi. This was achieved by expressing hs-FLP for 12 min, 15 min, or 30 min (Figure 3A-F). At low clone densities, ed-RNAi-expressing clones were far fewer than w-RNAi (control) clones (Figure 3A, B). As clone density increased, until the clones collectively accounted for the majority of tissue in the wing disc, the representation of ed-RNAi tissue and w-RNAi tissue was similar (Figure 3C-F). Thus, the loss of ed mutant tissue is most likely to occur when those clones are completely surrounded by wild-type cells.
(A-F) Wing discs containing clones expressing either UAS-w-RNAi (A, C, E) or UAS-ed-RNAi (B, D, F). Clones also express GFP. The clone density was varied by increasing the duration of the heat shock at 37° C during which hs-FLP is expressed. Thus, heat shocks of 12 min (A, B), 15 min (C, D) and 30 min (E, F) generate clones at progressively higher density.
(G-K) An experiment to examine ed/ed clones 24 h and 72 h after generation by mitotic recombination. The design of the experiment is shown in (G). Discs were dissected 120 h AED and clones were induced either 24 h (H, I) or 72 h (J, K) prior to dissection. Clones generated using the FRT40A chromosome (H, J) are compared to clones homozygous for edIF20 (I, K).
In their original description of cell competition, Morata & Ripoll (1975) observed that Minute clones were eliminated when they were generated early in development but were still observable when they were generated much later. To determine if this was also the case with clones of ed/ed tissue, we generated clones either 48 h or 96 h AEL using mitotic recombination and discs were dissected at 120 h AEL (Figure 3G). Thus, the discs were allowed to develop for 72 h or 24 h after clone induction respectively (Figure 3H-K). With marked wild-type clones (Figure 3H, J), clones were much larger 72 after clone generation (Figure 3J) than they were 24 h after clone generation (Figure 3H). In marked contrast ed/ed clones were observed readily 24 h after clone generation (Figure 3I) but mostly absent 72 h after clone generation (Figure 3K). At 72 h after clone generation their wild-type sister clones were similar in size to wild-type clones generated at similar times (Figure 3J-K). Thus ed/ed clones proliferate for a short time and are then eliminated. The propensity of ed/ed tissue to be eliminated when surrounded by wild-type tissue and to survive and grow when it accounts for most of the tissue is very similar to other instances of cell competition.
Increased apoptosis and reduced levels of Diap1 are observed in echinoid mutant tissue
In many situations where clones of cells of a certain genotype (e.g. Minute heterozygotes) are eliminated by cell competition, their elimination occurs by caspase-mediated apoptotic cell death and their death can be rescued by expression of the baculovirus p35 protein (Martín et al. 2009) which is an inhibitor of effector caspases (Hay et al. 1994). We therefore generated clones expressing either w-RNAi or ed-RNAi either in the presence or absence of p35 (Figure 4A-D). While expression of p35 modestly increases the recovery of wild-type clones (Figure 4A, B), there was a dramatic increase in the recovery of clones expressing ed-RNAi (Figure 4C, D).
(A-D) Wing discs containing GFP-marked clones of cells that express either UAS-w-RNAi (A, B) or UAS-ed-RNAi (C, D). Clones that also express UAS-p35 are shown in (B, D).
(E, F) Imaginal discs containing GFP-marked clones expressing UAS-w-RNAi (E) or UAS-ed-RNAi
(F). Anti Dcp-1 antibody staining of the same discs is shown in (E’, F’). Images were taken at a basal Z-plane where Dcp-1 staining was most prominent.
(G, H) Effect of reducing ed function in the entire wing pouch on levels of apoptosis. nub-Gal4 drives expression of UAS-GFP in (G, G’) or UAS-ed-RNAi in (H, H’). The pouch is visualized by GFP expression in (G) and with anti-Nubbin in (H). Apoptotic cells are visualized with anti-Dcp1. Note increased levels of apoptosis at the periphery of the pouch where nb-Gal4 expression is higher.
(I, J) Discs expressing ed-RNAi in clones (I) or in the entire posterior compartment (J) stained with an anti-Diap1. The insets in (I, I’) show a clone at higher magnification.
(K) Discs with GFP-marked clones that express both UAS-ed-RNAi and UAS-diap1 which results in an increase in both the number and size of clones. Staining with anti-Diap1 is shown in (K’). Note that the “rescued” clones still have smooth borders consistent with the propensity of cells with decreased ed to sort away from wild-type cells.
These observations indicate that the activity of effector caspases is necessary for the elimination of ed mutant tissue. Apoptotic cells in Drosophila express Death Caspase-1 (Dcp-1) (Song et al. 1997). To visualize cells undergoing apoptosis, we used the anti-Dcp-1 antibody. In discs containing clones expressing w-RNAi, occasional Dcp-1 staining was observed (Figure 4E, E’). In contrast, in discs containing ed-RNAi clones, punctate Dcp-1 staining was observed within ed-RNAi clones, especially in basal focal planes (Figure 4F, F’). These puncta were often at the edge of the clone where it abuts wild-type tissue, suggesting that competition with neighbors may be involved. We also examined Dcp-1 staining when using the nub-Gal4 driver. Very little apoptosis was seen when nub-Gal4 drove the expression of GFP (Figure 4G, G’), but very high levels of apoptosis were seen in the pouch of nub-Gal4, UAS-ed-RNAi discs (Figure 4H, H’). Taken together, these observations indicate that cells with reduced ed function die by apoptosis at an increased rate, and this contributes, in significant part, to the elimination of ed clones.
An important regulator of apoptosis is the IAP protein Diap1 (Hay et al. 1995) which inhibits caspase activation. When discs containing ed-RNAi clones were stained with an anti-Diap1 antibody, a clear reduction in Diap1 levels was observed within the clones (Figure 4I, I’). Intriguingly, the wild-type cells immediately adjacent to the clones had elevated levels of Diap1. The elevation of Diap1 in neighboring wild-type cells was inconsistent in that it was not always observed throughout the entire border of the clone, or around every clone. Importantly, a reduction in Diap1 levels was observed not just in ed mutant clones, but also when ed function was decreased in an entire region of the disc such as the posterior compartment (Figure 4J, J’).
If the reduced level of Diap1 had a role in promoting the death of ed mutant cells, then restoring Diap1 levels should be able to prevent clone elimination. To examine this possibility, we expressed a UAS-diap1 transgene concurrently with UAS-ed-RNAi. This resulted in the recovery of many clones that expressed UAS-ed-RNAi, an effect that was comparable to that obtained by expressing p35 (Figure 4K, K’). This indicated that the reduced Diap1 protein level in ed mutant clones has a role in their elimination.
The finding that Diap1 levels were consistently reduced in ed clones was surprising because a previous study reported that there is decreased activity of the Hippo signaling pathway and increased expression of Yorkie target genes including diap1 (Yue et al. 2012). Clones mutant for components of the Hippo pathway have increased levels of Diap1 protein (Tapon et al. 2002) and increased expression of transcriptional reporters of diap1 (Wu et al. 2003). Our finding of decreased Diap1 protein levels in ed mutant tissue is inconsistent with the proposed role of Ed promoting Hippo pathway activity; there needs to be another mechanism by which Ed positively regulates Diap1 protein levels. Importantly, our observation of decreased Diap1 in ed mutant clones is consistent with their elimination by apoptosis.
Imaginal discs with reduced echinoid function grow more slowly but fail to arrest their growth at the appropriate size
Previous work has indicated that reducing ed function in most, or all, cells of the imaginal disc results in overgrowth (Bai et al. 2001; Yue et al. 2012). This, at first glance, seems inconsistent with the increased levels of cell death observed in ed mutant tissue. To analyze the phenomenon further in the wing disc, we first examined the effect of reducing ed function on the size of adult wings (Figure 5A-I). Adult flies that were homozygous for the null alleles of ed were not obtained. We therefore examined the size of adult wings in a heteroallelic combination, edIF20/edsIH8. edIF20 is a null allele, while edsIH8 is a hypomorphic allele. Wings of these flies were 9% larger than the wings of wild-type (Oregon-R) flies. We also reduced ed function in the wing pouch using the nub-Gal4 driver line and found that this increased wing area by 15% compared to flies with nub-Gal4 driving w-RNAi. Because the nub-Gal4 chromosome itself appears to reduce wing area (Supplementary Figure 1), all wing-area experiments using nub-Gal4 were compared to controls that also contained nub-Gal4, rather than to Oregon-R. Thus, as described previously (Bai et al. 2001; Yue et al. 2012), reducing ed function results in tissue overgrowth.
Effect of nub-Gal4 on size of adult wings. Quantification of wing areas of “wild type” (Oregon-R or nub-Gal4 driving UAS constructs [GFP, w-RNAi] that should not affect sing size) and ed-depleted wings, with and without nub-Gal4. Wing blades are smaller in flies carrying the nub-Gal4 chromosome. n=10 wings (+/+ [Oregon-R]; edsIH8/edIF20; nub-Gal4, >GFP, >w-RNAi; same as shown in Figure 5I, 6D), 11 wings (nub-Gal4, >GFP [homozygotes]), 4 wings (edsIH8/edIF20, nub-Gal4; same as Figure 6D). “ns” indicates p> 0.05, ** indicates p< 0.01, calculated using ANOVA with post-hoc Tukey’s HSD test. Error bars indicate standard deviation.
(A-I) Effect of reducing ed function on size of adult wings. (A-F) show adult wings of the indicated genotypes. An overlay of a wild-type wing and an overgrown wing of a heteroallelic combination of ed mutations is shown in (G). An overlay of nub-Gal4, UAS-w-RNAi and nub-Gal4, UAS-ed-RNAi is shown in (H). Quantification of wing areas is shown in (I). n=10 wings (+/+ [Oregon-R]; edsIH8/+; edIF20/+; edsIH8/edIF20; nub-Gal4, >GFP, >w-RNAi), 9 wings (nub-Gal4, >GFP, >ed-RNAi). “ns” indicates p> 0.05, ** indicates p< 0.01, calculated using ANOVA with post-hoc Tukey’s HSD test. Error bars indicate standard deviation.
(J-P) A time course of growth of imaginal discs expressing either w-RNAi (J, K) or ed-RNAi (L-P) in the posterior compartment. All larvae expressing w-RNAi have pupariated soon after 120 h. However, much older larvae are observed in the population expressing ed-RNAi. Examples of discs from larvae that have delayed their pupariation are shown. All images are shown at the same scale (scale bar in panel J).
We then examined the growth of wing discs at different time points during development using the hh-Gal4 driver which is expressed in the posterior compartment (Figure 5J-P). At 120 h AEL, when control larvae (hh-Gal4, UAS-w-RNAi) reached the late third instar, the posterior compartments of discs of mutant larvae (hh-Gal4, UAS-ed-RNAi) were much smaller (Figure 5K, M). However, the discs of mutant larvae continued to grow through an extended larval stage and could reach a size much larger than ever observed in wild-type discs (Figure 5P). Interestingly, although the ed-RNAi is only expressed in the posterior compartment, increased growth was also observed in the anterior compartment, possibly as a secondary effect. From these experiments we conclude that, at least under the conditions of this experiment, ed mutant tissue grows more slowly than wild-type tissue, likely due to the increased cell death. However, ed mutant tissue can continue to grow beyond the appropriate size suggesting a defect in the mechanism that normally arrests growth.
Mechanisms that function downstream of Echinoid that regulate growth arrest and clone survival
Ed could function in cell-cell adhesion and also as a signaling molecule. To determine which of these properties of Ed are necessary for growth arrest, we examined the ability of either full length Ed protein (encoded by UAS-edFull) or one that contains the extracellular domain and the transmembrane domain but lacks the intracellular domain (encoded by UAS-edΔC-GFP) (Laplante and Nilson 2011) to rescue the overgrowth phenotype (Figure 6A-D). We used the heteroallelic combination edsIH8/edIF20, since flies of this genotype were viable and, as shown previously (Figure 5A-D, I), their wings were larger when compared to edsIH8/+ or edIF20/+ flies. Inclusion of nub-Gal4 itself resulted in a reduction of wing sizes back to a size similar to that of the wild-type and heterozygous flies (Figure 6A, D; Supplemental Figure 1). Wing size was further reduced by including the UAS-edFulltransgene and even more so by including the UAS-edΔC-GFP. That nub-Gal4 on its own caused a reduction in wing size which was exacerbated by either transgene makes it difficult to know whether this is a true rescue of the overgrowth phenotype or whether it is due to a non-specific growth inhibitory effect that counteracts the overgrowth.
(A-E) Effect of expressing full length ed (edFull) and a version where the cytoplasmic domain has been replaced by GFP (edΔC-GFP) on wing size and wing shape. (A) Wings of a heteroallelic combination of ed alleles that generate viable adults together with nub-Gal4. Inclusion of UAS-edFull (B) and UAS-edΔC-GFP(C) reduces wing size and brings the aspect ratio closer to wild-type. (B’) and (C’) show overlays of (B) and (C) over (A) respectively. For (D), n=10 wings (edsIH8/+; edIF20/+; edsIH8/edIF20; same as shown in Fig 5I), 4 wings (edsIH8/edIF20, nub-Gal4; edsIH8/edIF20, nub-Gal4, UAS-edFull), 6 wings (edsIH8/edIF20, nub-Gal4, UAS-edΔC-GFP). For (E), n=10 wings (edsIH8/+; edIF20/+), 9 wings (edsIH8/edIF20, edsIH8/edIF20, nub-Gal4, UAS-edΔC-GFP), 4 wings (edsIH8/edIF20, nub-Gal4; edsIH8/edIF20, nub-Gal4, UAS-edFull). The same wings were used in (D) and (E); number of wings differ if damage or mounting artifacts prevented measurement of both wing area and aspect ratio. “ns” indicates p> 0.05, ** indicates p< 0.01, calculated using ANOVA with post-hoc Tukey’s HSD test. Error bars indicate standard deviation.
(F, F’) GFP-marked clones expressing ed-RNAi stained with anti-Diap1 (F’). Yellow arrowhead indicates wild type cells immediately adjacent to the clone.
(G, G’) RFP marked clone expressing ed-RNAi shows decreased expression of a diap1 transcriptional reporter generated using 8 copies of the Hippo-response element (HRE) from the diap1 promoter (G’).
(H, H’) RFP-labeled clones expressing ed-RNAi show no obvious alteration in the localization of a GFP-tagged Yki. The region of the disc enclosed by the dashed line is shown at higher magnification in the insets.
(I, I’) GFP-labeled clone expressing ed-RNAi shows decreased expression of a ban-lacZ reporter (I’). The yellow arrowhead indicates wild-type cells adjacent to the clone that show increased expression of ban-lacZ.
(J, J’) RFP-labeled clone expressing ed-RNAi shows decreased expression of a fj-lacZ reporter (J’). The yellow arrowhead indicates wild-type cells adjacent to the clone that show increased expression of ban-lacZ.
(K, K’) Expression of ed-RNAi in the posterior compartment of the wing disc results in increased ex-lacZ expression (K’). The posterior compartment is identifiable because it does not express Ci (K).
Flies with nub-driven ed-RNAi have rounder wings due to a hexagonal packing defect during pupal wing elongation (Chan et al. 2021). We also observed rounder wings in edsIH8/edIF20 flies (Figure 6E). While addition of nub-Gal4 alone did not change the aspect ratio of edsIH8/edIF20flies, nub-Gal4 driving expression of UAS-edFull reduced the roundness of edsIH8/edIF20 wings. nub-Gal4 driving expression of UAS-edΔC-GFP gave a significant, though less pronounced, reduction in roundness. This result suggests that Ed is functioning in cell-cell adhesion in regulating organ size and shape. However, since the hypomorphic allele edsIH8 encodes a protein with a disruption in the extracellular domain, we cannot exclude the possibility that this protein might form a heterodimer with the EdΔC-GFP protein, and that this heterodimer could potentially function in some way as a signaling molecule (since the heterodimer would contain one wild-type extracellular domain and a wild-type cytoplasmic domain, albeit on different molecules).
We further examined the reduction of Diap1 protein in cells with reduced ed function. This finding is contrary to a previous study that reported that ed tissue had increased expression of Yki-target genes including diap1 (Yue et al. 2012). In addition to finding that clones of cells with reduced ed function had reduced Diap1 protein levels (Figure 6F, F’), we also found that wild-type cells immediately adjacent to these clones sometimes had increased Diap1 protein levels - a “border effect” (Figure 4G, G’; Figure 6F, F’). We did not observe a change in diap1 RNA either in the clone or in the adjacent cells by RNA in-situ hybridization using the hybridization chain reaction (data not shown). However, when we used a transcriptional reporter of diap1 which contains 8 copies of a Hippo-responsive element (HRE) (Wu et al. 2008), we observed decreased expression in clones expressing diap1-RNAi and sometimes observed increased expression in cells just outside the clone (Figure 6G, G’). Also, contrary to that same study, we did not find elevated levels of nuclear Yki. Using a GFP-tagged Yki, which has proven to be a reliable indicator of Yki localization (Fletcher et al., 2018), we observed no change in the localization of Yki in ed-RNAi clones (Figure 6H, H’). Additionally, two other reporter of Yki-driven expression, bantam-lacZ (Herranz et al. 2012) (a direct reporter of bantam transcription, not the bantam sponge which is inversely correlated to ban levels) and fj-lacZ (Brodsky and Steller 1996), are expressed at lower levels in the clone and at higher levels in some neighboring cells (Figure 6I, I’, J, J’). In contrast to all of these reporters, another Yki-dependent reporter, ex-lacZ (Boedigheimer and Laughon 1993), was expressed at higher levels in cells expressing ed-RNAi (Figure 6K, K’). In summary, our results are inconsistent with the previous conclusion that ed mutant tissue has increased levels of nuclear Yki and increased expression of Yki-target genes. We observed no change in Yki localization and lower levels of expression of three Yki-dependent reporter genes. One, ex-lacZ, was indeed expressed at higher levels in ed mutant cells. When ed was overexpressed using nub-Gal4, there was a strong reduction in wing size (Supplementary Figure 2A, B). Once again, inconsistent effects on Yki-targets were observed. Diap1 protein levels were unaffected in ed-overexpressing clones (Supplementary Figure 2C, C’) but reduced when ed was overexpressed in the entire posterior compartment (Supplementary Figure 2D, D’). ban-lacZ expression and ex-lacZ expression were both increased (Supplementary Figure 2E, E’, F, F’). Taken together, these results do not support a simple relationship between Ed levels and Yki-target gene expression.
(A, B) Effect of ed overexpression on wing size. Panels show adult wings of the indicated genotype. Wings overexpressing ed (B) are severely reduced in size, especially in the proximal regions.
(C, D) Effect of ed overexpression on Diap1 levels. GFP-marked clone that expresses UAS-ed (C) showing no obvious effect on Diap1 levels (C’). Overexpression of ed in the entire posterior compartment (D) results in a decrease in Diap1 (D’).
(E) Overexpression of ed in GFP-marked clones (E) results in an increase in ban-lacZ expression (E’).
(F) Overexpression of ed in the posterior compartment results in increased ex-lacZ expression (F’). The autonomous increase is less obvious in the pouch, except in the ed-overexpressing cells at the border with wild-type cells (inset, arrowhead).
Border effects such as that observed in ed clones have been described previously with manipulations of the Fat/Dachsous pathway (Willecke et al. 2008; Matakatsu and Blair 2012). To investigate a possible role for this pathway, we stained discs containing ed-RNAi clones with anti-Fat. We observed increased staining in the clones (Figure 7A) which might be due to a combination of increased apical Fat levels as well as the reduced apical profiles of cells in the clone which has been noticed previously (Wei et al. 2005; Laplante and Nilson 2006; Chang et al. 2011). Importantly, we observed decreased anti-Fat staining in the cells immediately adjacent to the clone. A reduction in Fat levels would be expected to result in reduced Hippo pathway activity (Cho et al. 2006; Bennett and Harvey 2006; Silva et al. 2006; Willecke et al. 2006) and increased expression of Yki-target genes as we have observed.
(A, A’) GFP-marked clones expressing ed-RNAi (A) stained with an anti-Fat antibody. The inset shows a close-up of the boundary of the clone.
(B) A model figure showing the effect of ed loss in ed cells and wild-type neighbors. Within ed clones, Fat levels are increased and Diap1 levels are decreased; the opposite is true in the wild-type neighbors. These differences may confer a competitive advantage to the wild-type neighbors, which could facilitate the elimination of ed clones from mosaic tissue.
Discussion
In order to systematically assess the requirement of individual cell-surface proteins in regulating cell survival or proliferation in clones, we examined the effect of reducing the expression of 75 such genes that have detectable levels of expression in the wing disc. Although we did not verify the efficacy of the knockdown by RNAi in each case, we found that for 66 of these, there was no obvious decrease in clone size or alteration in clone shape. Thus, most cell surface proteins, at least individually, do not function to sustain cell survival or proliferation. We found that both reducing levels of either ft or ds resulted in rounder clones that appeared larger. Since these genes have been studied in extensive detail, we did not pursue them further.
Of the remaining genes identified in the screen, we chose to focus on ed for two reasons. First, reducing ed levels has very different effects in clones and in entire tissues. Clones with reduced ed function are eliminated, while imaginal discs that lack ed function in large regions are overgrown. In some ways this phenotype is reminiscent of cells that have disruptions in apicobasal polarity such as scrib (Bilder et al. 2000; Brumby and Richardson 2003; Hariharan and Bilder 2006) with the important difference being that cells lacking ed still seem to have preserved most aspects of their apicobasal polarity, and tissue architecture appears relatively normal.
Second, previous studies have reported that ed mutant tissue has increased EGFR signaling (Bai et al. 2001; Islam et al. 2003; Rawlins et al. 2003a; Spencer and Cagan 2003; Fetting et al. 2009; Ho et al. 2010) and reduced activity of the Hippo pathway resulting in increased Yki-target gene expression (Yue et al. 2012). Both of these changes would be expected to promote cell survival and proliferation. Based on these observations, the underrepresentation and elimination of ed mutant clones in imaginal disc is unexpected. Previous work, however, has also shown that ed mutant cells appear to sort away from wild-type cells and that there is an actomyosin cable generated at the clone interface in wild-type cells that could potentially promote clone extrusion. We confirmed that EGFR signaling is likely elevated as assessed by reduced levels of nuclear Capicua (Supplementary Figure 3A-C) and also observed an actomyosin cable at the clone borders (Supplementary Figure 3D) like others have reported (Wei et al. 2005; Laplante and Nilson 2006, 2011; Lin et al. 2007; Chang et al. 2011). However, our observations are not consistent with the proposed effect on the Hippo pathway (Figure 6E-J, Supplementary Figure 2C-F).
(A-C) Effect of ed knockdown on Capicua (Cic) levels. (A) Cic levels are lower in some ed clones (arrowheads) than in wild-type neighbors, indicating increased EGFR activity within the clones. (B, C) and (B’, C’) show GFP-marked clones and Cic antibody staining at higher magnification. The location of these clones is indicated in (A, A’) by dashed lines.
(D, D’) An apical actomyosin cable is observed at the interface of ed mutant tissue and the wild type neighbors (edIF20/ edIF20 mitotic recombination clone is shown), consistent with previous reports (Wei et al. 2005; Laplante and Nilson 2006, 2011; Lin et al. 2007; Chang et al. 2011).
Why are ed clones eliminated?
We have found that ed mutant tissue has reduced levels of the anti-apoptotic protein Diap1. We observed this consistently in ed clones as well as in the entire posterior compartment of the wing disc when ed function was reduced using RNAi. Furthermore, the elimination of ed clones could be rescued by co-expressing diap1 with ed-RNAi. Together, these observations suggest the reduction in Diap1 levels underlies, in significant part, the elimination of ed clones.
Although we could not detect a reduction in diap1 RNA by in situ hybridization (data not shown), we were able to detect decreased expression of a commonly-used diap1 transcriptional reporter (Wu et al. 2008) which is a more sensitive reagent. Since this reporter was constructed using multiple copies of a Hippo-responsive element from the diap1 locus, these data indicate that the change in Diap1 protein levels is due, at least in part, to a decrease in Yki-dependent diap1 transcription. This finding differs from a previous study that reported increased Diap1 expression in ed clones that was attributed to increased expression of Yki target genes (Yue et al. 2012). In contrast to the conclusions of that study, we find decreased expression of at least three different transcriptional reporters of Yki in ed mutant clones: diap-lacZ, ban-lacZ and fj-lacZ and also no change in nuclear Yki localization. We do, however, find that a different reporter of Yki-dependent transcription, ex-lacZ is expressed at increased levels in ed mutant tissue indicating that the regulation of Yki-target genes by Ed is not easily explained by a simple effect on alterations in Hippo pathway activity.
Ed depleted tissues have increased apoptosis. We observed this in clones, especially at clone boundaries, and when ed was depleted in broader domains. Our results also explain why a reduction of ed function in the posterior compartment of the wing disc slows its growth; the affected posterior compartments are smaller than equivalently staged wild-type discs. This could simply be because the increased level of cell death in this compartment reduces tissue mass.
We have also identified a boundary effect at the border of ed clones: Some, but not all, wild-type cells immediately adjacent to the clones have elevated expression of Yki-target genes and elevated Diap1 protein. In this way, these cells resemble cells that have mutations in the Hippo pathway and behave as supercompetitors (Tyler et al. 2007). Thus, the reduction of Diap1 in ed mutant clones coupled with the generation of boundary cells that have some of the properties of supercompetitors could together contribute to the elimination of ed cells from the disc (Figure 7B).
Why are ed mutant tissues overgrown?
Although ed tissue grows more slowly than wild-type tissue, it appears to grow beyond an appropriate size. Thus, Ed has a second function in sensing tissue size and arresting growth at the appropriate time. Although not definitive, our experiments suggest that this function of Ed might primarily involve its role as an adhesion molecule. The levels of E-cadherin are known to be altered in ed mutant tissue and Ed and E-cadherin may function with some degree of redundancy to mediate cell-cell adhesion (Wei et al. 2005; Laplante and Nilson 2006; Chang et al. 2011). The mechanisms by which tissues sense their final size and arrest their growth accordingly are still poorly understood. While growth arrest is clearly disrupted in mutants that completely disrupt apicobasal polarity (Hariharan and Bilder 2006), it is possible that even subtle changes in junctional components could disrupt growth arrest.
Concluding remarks
Ed participates in, and regulates, at least two key cellular processes: adhesion at the adherens junction (Wei et al. 2005; Laplante and Nilson 2006, 2011; Chang et al. 2011), and endocytosis and endosomal trafficking (Rawlins et al. 2003b; Fetting et al. 2009; Ho et al. 2010; Li et al. 2015; Yang et al. 2018; Chan et al. 2021). Moreover, at least based on genetic interactions, ed influences the EGFR, Notch, and Hippo pathways. Ed may interact with more pathways or processes than we currently appreciate, either directly or indirectly via its effects on adhesion, endomembrane trafficking, or through signaling crosstalk. The collection of phenotypic abnormalities and alterations in gene expression that we observe in ed mutants are therefore likely to involve a summation of alterations in multiple pathways rather than a disruptive effect on any single pathway.
Methods
Fly Stocks & Husbandry
Unless otherwise noted, all experimental crosses were raised at 25°C on food prepared according to the recipe from the Bloomington Drosophila Stock Center.
Stocks used in this study include or were derived from the following: Oregon-R (“Ore-R,” used as wild type), y w hs-FLP; act<[y+]<Gal4 UAS-GFP/SM5-TM6B, hs-FLP;; act<stop<Gal4 UAS-RFP/SM5-TM6B, TIE-DYE (Worley et al. 2013), FRT40A and FRT40A, white+ ubi-GFP (Xu and Rubin 1993), FRT40A MARCM (Lee and Luo 1999), eyFLP; FRT40A CL white+/CyO (BL5622), UAS-ed (Bai et al. 2001), edIF20 FRT40A, ed1×5 FRT40A, and edsIH8 FRT40A (Bai et al. 2001), UAS-edFulland UAS-edΔC-GFP (Laplante and Nilson 2011), Yki:GFP (Fletcher et al., 2018), nub-Gal4 (AC-62, BL25754), hh-Gal4 (BL45169), ban-lacZ (BL10154, Herranz et al., 2012), fj-LacZ (fjP1, BL44253; Brodsky & Steller, 1996), ex-lacZ (Boedigheimer and Laughon 1993), diap1-GFP.HREx8 (Wu et al. 2008), UAS-w-RNAi (BL33644), UAS-ama-RNAi (BL33416), UAS-beat-Vc-RNAi (BL60067), UAS-Cont-RNAi (BL34867), UAS-shg-RNAi (BL32904), UAS-side-VII-RNAi (V10011), UAS-ft-RNAi (BL34970), UAS-ds-RNAi (BL32964), UAS-ed-RNAi (V104279, V3087, V938, BL38423 [“ed-RNAi” refers to V104279 unless otherwise indicated]), UAS-otk2-RNAi (BL55892), UAS-p35 (BL5073). “BL#” or “V#” indicates stocks obtained from the Bloomington Drosophila Stock Center (BDSC; Bloomington, IN, USA) or Vienna Drosophila Resource Center (VDRC; Vienna, Austria), respectively.
Additional stocks which were included in the genetic screen but are not mentioned in the main text are listed in Supplementary Table 3 with BDSC or VDRC numbers indicated.
Mosaic tissue generation
Clones induced by heat shock were generated in a 37°C water bath 48 h before dissection, unless otherwise noted. FLP-out Gal4 clones were made using heat shocks of 12 minutes (to generate clones at low density), 15 minutes (for medium density), or 30 minutes (for high density). MARCM clones and mitotic recombination clones were generated using a 1-hr heat shock.
Mitotic recombination clones made in the eye were induced by expression of the eyFLP driver.
Screen
∼10 UAS-RNAi males were crossed to ∼20 y, w, hs-FLP; act<[y+]<Gal4, UAS-GFP/SM5-TM6B or TIE-DYE (Act<stop<lacZ.nls, Ubi <stop<GFP.nls; Act<stop<GAL4, UAS-his2A::RFP/SM5-TM6B) virgin females. Crosses were kept on Bloomington food supplemented with yeast and flipped once daily. Clones were induced by heat shock on day 3. Early rounds of screening used a 15-minute heat shock, although we later switched to a 12-minute heat shock for the majority of the screen since the low clone density made identifying deviations in either direction easier. Wing imaginal discs from ∼6 wandering L3 larvae per line were dissected ∼48h after heat shock, stained with DAPI, and imaged.
Immunohistochemistry and fluorescence microscopy
Imaginal discs were dissected in PBS, fixed in 4% paraformaldehyde in PBS, and permeabilized in 0.1% Triton in PBS. Primary antibody incubations were done overnight at 4°C. Secondary antibody incubations were done for 2-3h at room temperature, or overnight at 4°C. Discs were mounted in SlowFade Diamond Antifade Mountant (S36963, Invitrogen).
Primary antibodies used are: rabbit anti-Ed (1:500, J-C. Hsu, Wei et al., 2005), rabbit anti-cleaved Dcp-1 (1:250; Asp216, Cell Signaling Technology), mouse anti-Diap1 (1:200, B. Hay), mouse anti-β-Galactosidase (1:500, WH0051083M1 Sigma-Aldrich), mouse anti-β-Galactosidase (1:500, SAB4200805, Sigma-Aldrich), chicken anti-GFP (1:500, ab13970 Abcam); rat anti-Ci (1:500, #2A1; Developmental Studies Hybridoma Bank, DSHB), rat anti-Fat (1:400, K. Irvine, Feng & Irvine, 2009), and guinea pig anti-Cic (1:300, Tseng et al., 2007).
Secondary Alexa-Fluor antibodies from Invitrogen were used at 1:500. Nuclei were stained with DAPI (1:1,000, Cell Signaling).
Fluorescence images were taken on a Zeiss Axio Imager M2 equipped with a 20× objective (Plan-Apochromat, 20×/0.8, Zeiss), LED light source (Excelitas Technologies), AxioCam 506 mono camera (Zeiss), and ApoTome.2 slider for optical sectioning. Images and image stacks were acquired and optically sectioned in ZEN 2.3 software (Zeiss). Imaged were processed using FIJI software (Schindelin et al. 2012). Unless otherwise noted, images show a single Z-plane.
Adult wing imaging and quantification
Adult wings were dissected from female flies. One wing per fly was mounted in Gary’s Magic Mountant (Lawrence et al. 1986). Wings were imaged using a Keyence VHX-5000 digital microscope, using the 20-200× lens at 150×. Brightness, contrast, and color tone of wing images have been adjusted on some images for improved visibility of features relevant to this study (wing shape and size).
For qualitative comparisons of wing sizes, wing images or traced silhouettes were overlaid in PowerPoint. For quantitative comparisons of wing sizes, wing outlines were traced and area was quantified in Fiji. Charts were generated using the ggplot2 package in RStudio (Wickham 2016).
Wing aspect ratios were calculated by dividing the length of proximodistal (PD) axis (measured from the posterior junction of the wing and hinge to the tip of the L3 vein) by the length of the anteroposterior (AP) axis (measured as the shortest distance from the tip of the L5 vein to the L1 margin).
Statistical analysis
P values were obtained by one-way ANOVA with Tukey’s HSD test using Astatsa freeware (Vasavada 2016). P value significance <0.01: **; 0.01 to 0.05: *; >0.05: not significant. All error bars show standard deviation.
Hybridization Chain Reaction
In situ hybridization chain reaction (HCR) was performed on wing discs based on HCR v3.0 protocol (Choi et al. 2018; S Bruce et al. 2021) excluding methanol dehydration. Larvae were dissected, fixed in 4% paraformaldehyde in PBS, permeabilized, and incubated overnight with RNA probes at 37°C. Samples were then washed and incubated overnight with fluorescently-tagged RNA hairpins and DAPI at room temperature. Probe sequences were designed in an open-source probe design program (ÖzpolatLab-HCR 2021) and synthesized by Integrated DNA Technologies. Hairpins and buffers were from Molecular Instruments.
Author Contributions
DCS and IKH conceptualized and designed the study. DCS, WYS, and AR-V performed the experiments. DCS conducted data analysis and visualization. DCS and IKH wrote the manuscript. All authors read and approved the manuscript.
Acknowledgements
We thank current and former members of the Hariharan laboratory for helpful feedback and discussions, especially Luigi Viggiano, who conducted related experiments that influenced our thinking on this study, and Melanie Worley. We also thank Bruce Hay, Jui-Chou Hsu, Ken Irvine, Laura Nilson, Duojia Pan, Nic Tapon, the Bloomington Drosophila Stock Center, the Vienna Drosophila Resource Center, and the Developmental Studies Hybridoma Bank for reagents and fly stocks. This work was funded by a NIH grant R35 GM122490 (to I.K.H) and NSF GRFP DGE 1752814 (to D.C.S).
References
