Figures and data in Flamingo participates in multiple models of cell competition

Figures
Tables
Additional files

6 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements

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Fmi is required in clonal tumors to outcompete wildtype tissue.

(A) Schematic of the whole eye disc Ras^V12, scrib RNAi, RFP tumors. (**B–H**) Pupal RFP tumors imaged through the cuticle. All pupae are ey-Flp; act5C>CD2>Gal4, UAS-RFP with the indicated RNAi. N=5 (no tumor), 10 (control RNAi), 13 (*fmi* RNAi), 17 (fz RNAi), 19 (*dsh* RNAi), 17 (*vang* RNAi), and 8 (pk RNAi). (C) Control UAS-Ras^V12, UAS-scrib RNAi, UAS-w RNAi tumors. (**D–H**) UAS-RasV12, UAS-scrib RNAi tumors co-expressing UAS-RNAi against the planar cell polarity (PCP) genes indicated above each pupa. (**B’–H’**) Representative third instar larval brain and eye discs for each of the experimental groups from above. Arrowheads in B’ point to the eye-antenna imaginal disc (orange) and the brain lobes (green). N=3 (no tumor), 10 (control RNAi), 13 (*fmi* RNAi), 12 (fz RNAi), 15 (*dsh* RNAi), 20 (*vang* RNAi), and 10 (pk RNAi). (I) Schematic of eye disc Ras^V12, scrib RNAi, RFP clonal tumors. (**J–P**) Third instar larval eye discs RFP tumor clones generated via ey-Flp; FRT42D Gal80/FRT42D; act5C>CD2>Gal4, UAS-RFP. (J) Eye discs with non-tumor, control RFP clones. (K) Control Ras^V12, UAS-scrib RNAi, RFP clonal tumors. (**L–P**) Ras^V12, scrib RNAi, RFP clonal tumors carrying the PCP allele indicated above each panel. Scale bars: 100 μm.

Figure 1—figure supplement 1

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The ey-Flp recombination system causes almost every cell in the eye disc to undergo chromosomal recombination.

(A) GFP-marked clonal cells. Clones generated with ey-Flp; FRT42D tub-Gal⁸⁰/FRT42D tub-RFP; ac>Gal4 UAS-GFP, express GFP in one of two daughter cells undergoing recombination to create homozygous clones without tub-Gal⁸⁰. (B) RFP-marked cells. Upon recombination, the same cells that express GFP also express two copies of tub-RFP, whereas cells that do not recombine express one copy of tub-RFP and lack GFP expression. We see few heterozygous RFP cells lacking GFP, indicating that few cells fail to undergo recombination in this system.

Figure 1—figure supplement 2

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*fmi* RNAi co-expression decreases the size of clonal Ras^V12, *scrib* RNAi tumors.

(A) Ras^V12, *scrib* RNAi clonal tumors generated by ey-Flp co-expressing w RNAi, imaged in wandering third instar larvae. N=5 larvae. (B) Ras^V12, *scrib* RNAi clonal tumors generated by ey-Flp co-expressing *fmi* RNAi, imaged in wandering third instar larvae. N=6 larvae. (C) Ras^V12, *scrib* RNAi whole eye third instar larva tumor generated from ey-Flp clones, where the wildtype (WT) cells were eliminated using GMR-Hid, l(2)CL. N=8 larvae. (D) Ras^V12, *scrib* RNAi, *fmi^E59* whole eye tumor generated from ey-Flp clones, where the WT cells were eliminated using GMR-Hid, l(2)CL. N=7 larvae. Scale bars: 200 μM.

Figure 2 with 1 supplement

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Winners require Fmi in developmental supercompetition.

(A) Eye imaginal disc clones overexpressing UAS-Myc (>>Myc) and UAS-nGFP under control of the tub >CD2>GAL4 driver. Clones were generated with ey-Flp, so half of the cells are clones and the other half are wildtype twins. (B) Eye imaginal disc >>Myc, UAS-nGFP, *fmi^E59* clones, competing against wildtype twins. (C) Eye imaginal disc >>Myc, UAS-nGFP clones, competing against *fmi^E59* twins. (D) Ratio of RFP-labeled clone area vs unlabeled wildtype area. The unlabeled wildtype area was obtained by subtracting the RFP-labeled area from the total eye disc area. A ratio over 1 implies RFP-positive clones are over-represented, likely behaving as supercompetitors, while a ratio below 1 means the RFP cells are under-represented, likely losers. N=9 discs (>>Myc), 5 discs (*fmi^E59*,>>Myc), 4 discs (*fmi^E59* vs >>Myc), groups were analyzed using multiple unpaired, two-tailed t-test; p-values:<0.0001 (****), 0.6340 (ns). (E) Representative wing imaginal disc overexpressing >>Myc and UAS-nGFP under control of the tub-Gal4 driver. Clones were generated using hsp70-Flp, with a 15 min 37°C heat-shock. Wildtype cells are labeled with tub-nRFP. Non-recombinant cells are heterozygous for nRFP, while twin spots are homozygous nRFP. n=14 discs. (F) Representative wing imaginal disc with >>Myc, UAS-nGFP, *fmi^E59* clones, over a wildtype background. Clones were generated in the same fashion as D. n=19 discs. (G) Representative wing imaginal disc with >>Myc, UAS-nGFP clones, competing against *fmi^E59* twin spots. n=13 discs. (H) log10 of the >>Myc/Twin spot cell ratio. nGFP total cells were divided by the number of twin spot homozygous nRFP cells, and then log10-transformed. A ratio over 0 indicates over-representation (likely supercompetition) of nGFP+ clones, and below 0 indicates nGFP+ cells are under-represented (likely behaving as losers). The difference between groups was analyzed using a one-way ANOVA, with Dunnett correction for multiple comparisons; p-value: >>Myc vs *fmi^E59* >>Myc < 0.0001 (****); >>Myc vs *fmi^E59*=0.9705 (ns). Scale bars: 50 μm.

Figure 2—figure supplement 1

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Fmi by itself does not trigger cell competition.

(A) Eye disc GFP+ *fmiE^E59* vs an unlabeled wildtype background. Clones were generated with ey-Flp as in Figure 2A. (B) Eye disc GFP+, RFP control clones vs a wildtype background. (C) log of the GFP/WT clone ratio measured in either *fmi^E59* clones vs wildtype (n=11 discs) or RFP+ clones vs wildtype (n=15 discs). Differences were analyzed by a two-tailed, unpaired t-test, rendering a p-value of 0.7655. (D) Representative wing imaginal disc showing the GFP+ *fmiE^E59* clone cells. Clones were generated with hsp70-Flp. (E) Twin spot RFP+ cells for the disc shown in D. Homozygous RFP+ twin spots can be observed adjacent to the RFP- *fmi^E59* clones. (F) *fmi^E59*, GFP cells vs wildtype homozygous RFP twin spot cells counted in wing imaginal discs (n=10 discs). The differences were analyzed with a two-tailed, ratio-paired t-test, p-value = 0.4815. Scale bar: 50 μm.

Figure 3 with 1 supplement

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Winners require fmi in *scribble* cell competition.

(**A–L’**) scrib RNAi clone analysis in the prospective eye. UAS-scrib RNAi was expressed with the act5C>CD2>Gal4 driver and clones were generated by ey-Flp. Clones are marked both with UAS-nGFP and tub-nRFP. (A) Representative early third instar disc with control UAS-nGFP clones. (B) Representative late third instar disc with control UAS-nGFP clones. (**C, C’**) Representative adult eye with control UAS-nGFP clones. C’ shows fluorescently labeled control clone cells. (D) Representative early third instar disc with UAS-scrib RNAi clones vs wildtype twin clones. (E) Representative late third instar disc with UAS-scrib RNAi clones vs wildtype twin clones. (**F, F’**) Representative adult eye with UAS-scrib RNAi clones vs wildtype twin clones. F’ shows fluorescently labeled surviving UAS-scrib RNAi cells. (G) Representative early third instar disc with UAS-scrib RNAi clones vs *fmi^E59* twin clones. (H) Representative late third instar disc with UAS-scrib RNAi clones vs *fmi^E59* twin clones. (**I, I’**) Representative adult eye with UAS-scrib RNAi clones vs *fmi^E59* twin clones. This phenotype was lethal. The adult eye shown was from an escaper. Escapers had trouble eclosing and died within hours. I’ shows fluorescently labeled surviving scrib RNAi cells. tub-nRFP fluorescence was barely visible, so the stronger UAS-nGFP is shown. (J) Representative early third instar disc with *fmi^E59*, UAS-scrib RNAi clones vs wildtype twin clones. (K) Representative late third instar disc with *fmi^E59*, UAS-scrib RNAi clones vs wildtype twin clones. (**L–L’**) Representative adult eye with *fmi^E59*, UAS-scrib RNAi clones vs wildtype twin clones. L’ shows fluorescently labeled surviving *fmi^E59*, UAS-scrib RNAi cells. (M) Quantification of fluorescently tagged clone area for the phenotypes mentioned above. Each dot in the violin plot displays the ratio of RFP+ area vs total disc area. Of special significance is the fact that scrib RNAi clones competing with wildtype clones survive similarly to scrib RNAi clones competing with *fmi^E59*. p-Values are as follows: ****<0.0001; ***<0.0005; ns = 0.2634. Scale bars: 50 μm.

Figure 3—figure supplement 1

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Clone size during early third instar *scrib* competition.

(A) Percentage of RFP clone area in wildtype third instar discs. The X axes indicate the disc size measured as the total nuclear count in the eye imaginal disc. The line represents the best linear fit to the samples plotted. (B) Percentage of RFP, *scrib* RNAi clone area competing with wildtype twins in third instar discs. (C) Percentage of RFP, *scrib* RNAi clone area competing with *fmiE59* twins in third instar discs. (D) Percentage of RFP, *fmi^E59*, *scrib* RNAi clone area competing with wildtype twins in third instar discs.

Figure 4 with 2 supplements

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Lack of Fmi increases cell death and reduces proliferation in would-be winners.

(**A–C**) Ras^V12, scrib RNAi tumors stained for DAPI, Dcp1+ (A), and puc-lacZ (B). (C) Merged channels. (D) Representation of how Dcp1+ staining localizes in the wildtype (WT) cells at the boundary with the tumor. (**E–G**) Ras^V12, scrib RNAi tumors mutant for Fmi, stained with DAPI, Dcp1+ (E), and puc-LacZ (F). (G) Merged channels. (H) Representation of how Dcp1+ staining localizes in the tumor cells in contact with the surrounding WT tissue. Scale bar for A–G: 25 μm. (**I–K**) Dcp1+ staining in >>Myc clones in eye discs. >>Myc clones are marked by GFP (I) and were stained against Dcp1+ (J). The arrows show apoptotic WT (red arrow) and >>Myc (white arrow) cells at the clone boundary. (K) Merged channels, showing apoptotic cells evenly distributed between WT and >>Myc cells. (**L–N**) Dcp1+ staining in >>Myc clones lacking Fmi in the eye disc. Eye disc >>Myc clones are marked by GFP (L) and were stained for Dcp1+ (M, N) Merged channels, showing apoptotic cells localized mainly in the >>Myc, *fmi^E59* clones. Scale bar: 50 μm. (O) Quantification of apoptotic cells in WT vs >>Myc clones in eye discs. Apoptosis occurs similarly in WT and >>Myc cells (two-tailed paired t-test; p-value = 0.6049). The left side of the graph shows the number of apoptotic WT and >>Myc cells. Each imaginal disc is displayed as a pair of dots, linked by a line, to easily visualize the Dcp1+ apoptotic cells in WT vs >Myc cells. Dots represent the number of apoptotic WT (left) or >>Myc (right) cells per disc. The right side of the graph displays the difference (>>Myc minus WT apoptotic cells). The dashed line indicates the mean difference between those values for all samples. N=14 discs. (P) Quantification of apoptotic cells in WT vs >>Myc, *fmi^E59* clones in eye discs. Apoptosis is found mainly in >>Myc, *fmi^E59* cells (two-tailed paired t-test; p-value = 0.0006). The left side of the graph shows the number of apoptotic WT and >>Myc, *fmi^E59* cells, side by side. Dots represent the number of apoptotic >>WT (left) or >>Myc, *fmi^E59* (right) cells per disc. The right side of the graph displays the difference (>>Myc, *fmi^E59* minus WT apoptotic cells). The dashed line indicates the mean difference between those values for all samples. N=14 discs. (**Q–R**) Proliferation analysis performed by pHis3 staining in wing discs with either >>Myc clones (Q) or >>Myc, *fmi^E59* clones (R). Scale bar: 20 μm. (S) Proliferative ratio of GFP cells in a non-competition Control (n=9 discs), >>Myc (n=9 discs), or >>Myc, *fmi^E59* (n=13 discs) clones. The proliferative ratio for each group was calculated as the ratio of pHis3 cells within the GFP+ clone vs the non-GFP WT tissue and the differences were analyzed as an ordinary ANOVA with a Tukey’s test for multiple comparisons, with all p-values<0.0001 (****).

Figure 4—figure supplement 1

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Fmi-/- tumor cell debris is found in vesicles inside wildtype cells.

(A) Closeup of RFP-tagged *fmi^E59* RasV12, scrib RNAi tumors competing against unlabeled wildtype cells in eye discs. RFP+ tumor cells can be observed at the periphery, and RFP+ debris is detected in the unlabeled wildtype cells inside the annotated area. (B) Lysotracker staining marking acidic vesicles. Large, stained vesicles can be observed in the annotated area. (C) The merged channels show colocalization of the RFP+ cell debris with the lysosomal vesicles inside wildtype cells. (D) Closeup of the annotated region in A–C. Scale bar: 25 μm.

Figure 4—figure supplement 2

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Lack of fmi does not affect the activation of JNK signaling.

(**A–C**) Closeup of late third instar eye discs showing *scrib* RNAi clones marked with RFP (A), *puckered* transcriptional activation using the puc-LacZ reporter (B) and a merge image showing both channels (C). (**D–F**) Closeup of late third instar eye discs showing *scrib* RNAi, fmi^E59 clones marked with RFP (D), *puckered* transcriptional activation (E), and a merge image showing both channels (F). Scale bar: 25 μm.

Figure 5 with 1 supplement

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Vang is not required for >>Myc supercompetition.

(A) Representative wing imaginal disc showing the GFP-tagged, *vang^A3* >>Myc clones. Clones were generated using hsp70-Flp. (B) RFP-labeled twin spots for the clones shown in A. Twin spot homozygous for RFP can be observed adjacent to the supercompetitor clones. (C) Graph showing the total GFP and homozygous RFP counts per disc, with each disc counts linked by a line and the log10 ratio of GFP/hRFP cells per disc (n=12 discs). Differences between GFP/hRFP clone size were analyzed using a two-tailed, ratio-paired t-test. p-Value<0.0001. When the ratio was compared with a two-tailed t-test to >>Myc, the difference was not significant, with a p-value of 0.078. Scale bar: 50 μm.

Figure 5—figure supplement 1

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Fmi levels are not affected by cell competition.

(**A–D**) Overview of representative imaginal discs with >>Myc clones labeled with nGFP (green) and Fmi immunostaining (white), obtained from different larvae. (B) >>Myc clone (green), from the annotated box shown in (A). The clone boundary is delimited with a dashed line. (C) Fmi staining (white) from the annotated box shown in (A). The clone boundary is delimited with a dashed line. (E) >>Myc clone (green), from the annotated box shown in (D). The clone boundary is delimited with a dashed line. (F) Fmi staining (white) from the annotated box shown in (D). The clone boundary is delimited with a dashed line. Scale bars: 50 μm.

Figure 6

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The Fmi cadherin repeats are not required for cell competition.

(A) Representative disc with nGFP-labeled, >>Myc (A), >>Myc, *fmi^E59* (B) or >>Myc, *fmi^E59*, *arm-fmiΔcad* (C) clones competing against wildtype twin spots in the wing disc. Twin spot clones are labeled with homozygous nRFP and clones were generated with hsp70-Flp. (D) Ratio of GFP vs RFP cells in the three groups, represented as the log10(GFP/hRFP) cell ratios. To evaluate the effect of the *arm-fmiΔcad* rescue (n=14 discs), the GFP/hRFP cell ratio was directly compared against the two other groups, already quantified and shown in Figure 2F. Differences between the groups were analyzed using an unpaired, ordinary one-way ANOVA, which found a p-value<0.0001. Inter-group differences were analyzed with a Tukey’s multiple comparisons test, which found no differences between >>Myc and >>Myc, *fmi^E59*, *arm-fmiΔcad* clones, whereas both groups strongly differed from >>Myc, *fmi^E59* clones, both returning a p-value<0.0001 (****) when compared directly against >>Myc, *fmi^E59*. Scale bar: 50 μm.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Drosophila melanogaster)	fmi	FlyBase	FBgn0024836	Also known as stan
Gene (D. melanogaster)	fz	FlyBase	FBgn0001085
Gene (D. melanogaster)	dsh	FlyBase	FBgn0000499
Gene (D. melanogaster)	vang	FlyBase	FBgn0015838	Also known as stbm
Gene (D. melanogaster)	pk	FlyBase	FBgn0003090
Gene (D. melanogaster)	dgo	FlyBase	FBgn0086898
Gene (D. melanogaster)	scrib	FlyBase	FBgn0263289
Gene (D. melanogaster)	myc	FlyBase	FBgn0262656
Gene (D. melanogaster)	Ras85D	FlyBase	FBgn0003205
Strain, strain background (Escherichia coli)	5-alpha High Efficiency	NEB	C2987H
Genetic reagent (D. melanogaster)	W RNAi	BDSC	33623
Genetic reagent (D. melanogaster)	Fmi RNAi	BDSC	26022
Genetic reagent (D. melanogaster)	Fz RNAi	BDSC	31311
Genetic reagent (D. melanogaster)	Dsh RNAi	BDSC	31306
Genetic reagent (D. melanogaster)	Vang RNAi	BDSC	34354
Genetic reagent (D. melanogaster)	Pk RNAi	BDSC	32413
Genetic reagent (D. melanogaster)	Scrib RNAi	BDSC	39073
Transfected construct (D. melanogaster)	Arm-fmiΔCad	This paper		Located in chromosome 2R
Antibody	Rabbit polyclonal α-pHis3	Millipore		1:100
Antibody	Rabbit monoclonal α-Dcp1	Cell Signaling	RRID:AB_2721060	1:100
Antibody	Mouse monoclonal α-LacZ	Promega		1:500
Antibody	Rabbit polyclonal α-Cas3	AbCam		1:200
Antibody	488-Goat polyclonal α-rabbit	Thermo Scientific	RRID:AB_3251385	1:500
Antibody	546-Goat polyclonal α-mouse	Thermo Scientific	RRID:AB_2535765	1:500
Antibody	546-Goat polyclonal α-rabbit	Thermo Scientific	RRID:AB_2534077	1:500
Antibody	647-Donkey polyclonal α-mouse	Thermo Scientific	RRID:AB_162542	1:500
Commercial assay or kit	HiFi DNA assembly kit	New England Biolabs
Chemical compound, drug	Alexa 350 phalloidin	Thermo Scientific		1:500
Chemical compound, drug	Alexa 635 phalloidin	Thermo Scientific		1:500
Chemical compound, drug	DAPI	Invitrogen		1 μg/mL
Chemical compound, drug	Vectashield	Vector Labs
Software, algorithm	Fiji	https://fiji.sc	RRID:SCR_002285
Software, algorithm	Counting macros	https://github.com/iPabloSB/Nuclear-counts; Shcherbina and Sanchez Bosch, 2023
Software, algorithm	Prism 10	GraphPad	RRID:SCR_002798