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A role for Flower and cell death in controlling morphogen gradient scaling

Abstract

During development, morphogen gradients encode positional information to pattern morphological structures during organogenesis1. Some gradients, like that of Dpp in the fly wing, remain proportional to the size of growing organs—that is, they scale. Gradient scaling keeps morphological patterns proportioned in organs of different sizes2,3. Here we show a mechanism of scaling that ensures that, when the gradient is smaller than the organ, cell death trims the developing tissue to match the size of the gradient. Scaling is controlled by molecular associations between Dally and Pentagone, known factors involved in scaling, and a key factor that mediates cell death, Flower4,5,6. We show that Flower activity in gradient expansion is not dominated by cell death, but by the activity of Dally/Pentagone on scaling. Here we show a potential connection between scaling and cell death that may uncover a molecular toolbox hijacked by tumours.

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Fig. 1: Cell death correlates with scaling.
Fig. 2: Extracellular Flower, association with Dally/Pentagone and cell competition.
Fig. 3: Flower in scaling and cell death.
Fig. 4: Role of Flower on Dally/Pentagone scaling and scaling of Flower itself.

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Data availability

The data that support the findings of this study are available within the paper and its Supplementary Information files. Data supporting the findings of this study are available from the corresponding author on reasonable request. Publicly available datasets used in this work are available at https://flybase.org/ (FB2021_06)71 and https://m.ensembl.org/index.html. Source data are provided with this paper.

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Acknowledgements

We thank WellGenetics and Y. Chen for the generation of Drosophila lines including flower mutants and eGFP fusion proteins, Bloomington Drosophila Stock Center, FlyORF, Vienna Drosophila Resource Center and the Developmental Studies Hybridoma Bank for reagents and G. Morata, H. D. Ryoo, J. Felix de Celis, K. Basler, N. Tapon, R. Levayer, S. B. Selleck, S. Casas-Tinto, S. Eaton and T. B. Kornberg for providing Drosophila fly lines and antibodies. We thank B. Habermann, O. Schaad and A. Daeden for helpful discussions and A. C. Oates, J. E. Castelli-Gair, D. Basagiannis, I. Castanon, R. Rashpa, R. Mateus and Z. Hadjivasiliou for critical reading of the manuscript. We thank T. Wagner for technical assistance. M.M.M. was supported by the Swiss National Science Foundation (SNSF) (SystemsX.ch, Transition Postdoc Fellowship) and Novartis Foundation Fellowships. This work was supported by grants from the SNSF, by the ERC (Sara and Morphogen), the NCCR Chemical Biology programme, the DIP of the Canton of Geneva and the SystemsX EpiPhysX (SNSF) granted to M. Gonzalez-Gaitan.

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Authors and Affiliations

Authors

Contributions

M.M.M. performed most of the experiments, quantifications and analysis. C.S. performed immunoprecipitation experiments, generated Drosophila lines and characterized dallygem mutant. M.D. generated Drosophila lines and performed sequencing work. M.M.M. prepared figures. M.M.M. and M.G.-G. designed the project and wrote the manuscript.

Corresponding authors

Correspondence to Marisa M. Merino or Marcos Gonzalez-Gaitan.

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Extended data

Extended Data Fig. 1 Cell death in the Scaling mutants.

a, Stainings showing dying cells by Cleaved Caspase-3 immunostaining (green), in representative wildtype (wt), dallygem/+, pent2/+, dallygem and pent2 wing discs. b, Corresponding Cleaved Caspase-3 Index in wt (n=15 discs), dallygem/+ (n=20), pent2/+ (n=20), dallygem (n=16) and pent2 (n=9). c, Stainings showing Active JNK immunostaining (green) in representative wt, dallygem/+, pent2/+, dallygem and pent2 wing discs. d, Corresponding Active JNK Index in: wt (n=13 discs), dallygem/+ (n=15), pent2/+ (n=13), dallygem (n=12) and pent2 (n=13). For details on Cleaved Caspase-3 Index and Active JNK Index measurement see Supplementary Information. e, Cell death (anti-Cleaved Caspase-3) in wt, dallygem and pent2 as a function of position in the wing in a region of interest (ROI) in a rectangular stripe 50 µm wide at the position of maximal width of the disc. Normalized positions along the anterior-posterior (x) and dorso–ventral axes (y) are indicated. In x, position is normalized to the maximal anterior-posterior length of the wing disc; in y, position is normalized to the constant width of the ROI (50µm). f, Average frequency of dying cells (*; in discs where apoptosis was observed) along the anterior-posterior axis in wt, dallygem and pent2. Note that the average frequency of dying cells (anti-Cleaved Caspase-3) is homogenous in space for these three conditions. For statistical significance see Supplementary information; Statistical analyses chapter. Error bars, SEM. g, Quantifications of the amplitudes of the anti-Pmad gradients of wt and dallygem wing imaginal discs. The difference is not statistically significant. h, P35-expressing Flip-out clones in wing imaginal discs expressing Dally::GFP, Pentagone::GFP and Thickveins::GFP at endogenous levels (MiMICs). Wing discs were fixed 48h after clone induction. RFP (Red; clones), Dally::GFP (green), Pentagone::GFP (green) and Thickveins::GFP (green). Note that the expression of Dally::GFP, Pentagone::GFP or Thickveins::GFP is not affected in the P35 overexpressing clones. i, Phosphorylated Mad scaling plots for the indicated genetic conditions. j, Representative images of blk-GFP (yellow) wing discs stained with nuclear marker (DAPI, blue) in the following conditions: UASlacZ, UASp35, UASeiger::mCherry and wt UV-irradiated. k, Quantification of Dpp expression levels (Dpp promoter driving GFP: blk-GFP) in the following conditions: UASlacZ, UASp35, UASeiger::mCherry and wt UV-irradiated. l, Representative images from wing imaginal discs 96h after egg laying (AEL) in control discs (UASlacZ) and in disc overexpressing P35 (UASp35), stained with nuclear marker DAPI (grey). m,n, Quantifications of the total wing disc area (m) and length of the posterior compartment (n) at 72h and 96h AEL of the following genotypes: UASlacZ and UASp35. Data are presented as mean values +/− SEM. * indicates ≤ 0.05 significance; **, ≤ 0.01 and ***, ≤ 0.001. Two-sided test of significance were used for all the comparisons. Scale bars in a, c, h, j 50 μm; 30 μm in l.

Source data

Extended Data Fig. 2 dallygem mutant characterization. NucView as a reporter of dying cells and Scaling plots in the scaling mutants.

a, Genomic region of dallygem mutant from exon 4 to exon 9. Orange arrows show the different exons from the region of interest, green arrows correspond to the gene CG43169. dallygem mutant has a hobo108 transposable element (brown box) landed into exon 4. This insertion leads to a frameshift generating two consecutive STOP codons (red triangle), leading to a truncated protein. Two PCR products containing the hobo108 insertion were amplified from cDNA (yellow and blue lines). These two PCR products were sequenced and aligned, covering the full hobo108 insertion (yellow arrows show the sequencing coverage from the ‘yellow’ PCR product and blue arrows show the sequencing coverage from the ‘blue’ PCR product). Primers are shown with small triangles. Numbers refer to base pair (bp). b,c Comparison of the different protein domains found in Dally and and Dallygem truncated protein. b, Dallygem contains the signal peptide and a truncated protein sequence (to aa289), including the N-linked glycosylation (c, green rectangle) sites but missing the O-linked glycosylation sites needed for Heparan sulfate addition (c, blue rectangle) and the GPI anchor (c, blue rectangle) present in wildtype Dally protein. Numbers refer to amino acids (aa). d, (top) Wildtype wing disc stained with NucView (Red) and anti-Cleaved Caspase-3 (grey). (bottom) Flip-out clones in a wing overexpressing eiger (see Methods). Wing discs were fixed 24h after clone induction. Stainings show NucView (Red), GFP (green), anti-Cleaved Caspase-3 (grey) and nuclear marker DAPI (blue). e, Scaling plots of the same data-set and genotypes as shown in Fig. 1h–j. Sample sizes (discs); wt (n=28, discs), dallygem/+ (n=26), pent/+ (n=42), dallygem (n=37), pent2 (n=18), C765>UASpentagone (n=18). Lines, linear fits. Error bars, SEM. Scale bars in d, 10 μm.

Source data

Extended Data Fig. 3 Co-immunoprecipitations and Western blots of Flower-Dally.

Immunoprecipitations with GFP-Trap beads. a, Co-immunoprecipitation of Dally::mCherry and Pentagone::GFP and control immunoprecipitation assays (Dally::mCherry and GFP). Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. Approximate sizes (kDa) are indicated for BenchMark protein ladder. b, Co-immunoprecipitation of Dpp::mCherry and Pentagone::GFP, Dpp::mCherry and Dally::GFP and control immunoprecipitation (Dpp::mCherry and GFP). Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. Sizes (kDa) are indicated for Novex Sharp protein ladder. c, Co-immunoprecipitation of Dally::mCherry and Fwe-Ubi::CFP, Dally::mCherry and Fwe reporter (YFP-GFP-RFP), Fwe reporter (YFP-GFP-RFP) and control immunoprecipitation assays (Dally::mCherry and GFP). Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. Note the presence of Flower dimers in the immunoprecipitacion. Protein ladder, BenchMark. d, Co-immunoprecipitation of Dally::mCherry and Fwe-LoseA::CFP, Dally::mCherry and Fwe-Ubi::CFP, Dally::mCherry and Fwe-LoseB::CFP and control immunoprecipitation assays (Dally::mCherry and GFP). Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. Protein ladder, BenchMark. e, Co-immunoprecipitations of GFP::Dally and Fwe-Ubi::mCherry, GFP::Dally and Fwe-LoseA::mCherry, GFP::Dally and Fwe-LoseB::mCherry, GFP::Dally and Fwe-ΔExtra::mCherry and control immunoprecipitations assays: GFP and Fwe-Ubi::mCherry, GFP and Fwe-LoseA::mCherry, GFP and Fwe-LoseB::mCherry, GFP and Fwe-ΔExtra::mCherry. Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. Protein ladder, BenchMark.

Source data

Extended Data Fig. 4 Co-immunoprecipitations and Western blots of Fwe-Pentagone, Fwe-Tkv, Fwe-Dpp and Fwe-Dally mutants.

Immunoprecipitations with GFP-Trap beads. a, Co-immunoprecipitations Pentagone::GFP and Fwe-Ubi::mCherry, Pentagone::GFP and Fwe-LoseA::mCherry, Pentagone::GFP and Fwe-LoseB::mCherry, Pentagone::GFP and Fwe-ΔExtra::mCherry and control immunoprecipitation assays: GFP and Fwe-Ubi::mCherry, GFP and Fwe-LoseA::mCherry, GFP and Fwe-LoseB::mCherry, GFP and Fwe-ΔExtra::mCherry. Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. b, Co-immunoprecipitations Tkv::mCherry and Flower reporter (YFP-GFP-RFP) and control immunoprecipitation assays: Tkv::mCherry and GFP. Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. c, Co-immunoprecipitations HRP::Dpp and Flower reporter (YFP-GFP-RFP) and control immunoprecipitation assays: HRP::Dpp and GFP. Western blots show inputs and immunoprecipitations with anti-GFP and anti-HRP. d, Co-immunoprecipitations GFP::Dally and Fwe-Ubi::mCherry, GFP::DallyΔHS and Fwe-Ubi::mCherry, GFP::DallyΔGPI and Fwe-Ubi::mCherry and control immunoprecipitation assays: Fwe-Ubi::mCherry and GFP. Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. e, Co-immunoprecipitations GFP::Dally and Fwe-LoseA::mCherry, GFP::DallyΔHS and Fwe-LoseA::mCherry, GFP::DallyΔGPI and Fwe-LoseA::mCherry and control immunoprecipitation assays: Fwe-LoseA::mCherry and GFP. Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. Protein ladder, BenchMark.

Source data

Extended Data Fig. 5 Co-immunoprecipitations and Western blots of Fwe-Dally mutants, Fwe-Pentagone in dallygem background, Fwe-Dally in pentagone2 background and Dally-Pentagone in Flower silencing.

Immunoprecipitations with GFP-Trap beads. a, Co-immunoprecipitations GFP::Dally and Fwe-LoseB::mCherry, GFP::DallyΔHS and Fwe-LoseB::mCherry, GFP::DallyΔGPI and Fwe-LoseB::mCherry and control immunoprecipitation assays: Fwe-LoseB::mCherry and GFP. Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. Protein ladder, BenchMark. b, Co-immunoprecipitations Pentagone::GFP and Fwe-Ubi::mCherry in dallygem mutant background, Pentagone::GFP and Fwe-LoseA::mCherry in dallygem mutant background, Pentagone::GFP and Fwe-LoseB::mCherry in dallygem mutant background and control immunoprecipitation assays: GFP and Fwe-Ubi::mCherry, GFP and Fwe-LoseA::mCherry, GFP and Fwe-LoseB::mCherry, Pentagone::GFP and Fwe-Ubi::mCherry, Pentagone::GFP and Fwe-LoseA::mCherry, Pentagone::GFP and Fwe-LoseB::mCherry. Protein ladder, Novex Sharp. c, Co-immunoprecipitations Dally::mCherry and Fwe reporter (YFP-GFP-RFP) in pent2 mutant background and control immunoprecipitation assays: Dally::mCherry and GFP, Dally::mCherry and Fwe reporter (YFP-GFP-RFP). Protein ladder, BenchMark. d, Co-immunoprecipitations in wing imaginal discs Dally::mCherry and Pentagone::GFP downregulating fwe (C765>fweRNAi) and control immunoprecipitation assays: Dally::mCherry and GFP, Dally::mCherry and Pentagone::GFP. Protein ladder, Novex Sharp. e, Co-immunoprecipitations GFP::Dally and Fwe-Ubi::mCherry, Pentagone::GFP and Fwe-Ubi::mCherry using increasing detergent concentrations (0.2%, 0.5% and 1%) and control immunoprecipitation assays using increasing detergent concentrations (0.2%, 0.5% and 1%): GFP and Fwe-Ubi::mCherry. Western blots show inputs and immunoprecipitations with anti-GFP and anti-mCherry. Protein ladder, Novex Sharp.

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Extended Data Fig. 6 Dally, Pentagone and Flower localization, twin clone analysis of pentagone and Dally/Pentagone expression in cell death and Super-competition.

a, Extracellular staining of wildtype Fwe-Ubi::mcherry isoform expressed in the posterior compartment by the UAS/Gal4 system (hhGal4 driver) in the background of Dally::GFP (top) and Pentagone::GFP (bottom). Dally::GFP (green), Pentagone::GFP (green), Anti-mCherry nanobody immunostaining (anti-mCherry-Atto647N), grey; mCherry fluorescent signal, red. Only cells from the posterior compartment are shown. b, Out-competition assay. Twin clone analysis of control and pentagone2 null mutant clones in wing discs fixed 96 (n=22 clones), 72 (n=82) and 48h (n=78) after clone induction (ACI). Left, pent2 mutant clone and control (pent+) twins; right, percentage of control clones without pent2 mutant twins. c, Representative examples of the twin clone analysis. Dark areas correspond to pent2 homozygous mutant clones and brighter green areas, to pent+ control twins in pent2/+ wing discs (lighter green). d, Triple staining showing dying cells labelled by NucView (NV, green) as well as Patched (Ptc, red) and Wingless (Wg, red) immunostaining in representative wildtype (wt) and dally80 wing discs. e, Representative image of the twin clone analysis of control and dally80 null mutant clones stained for NV (red); GFP clones in green. Wing discs were fixed at 72h ACI. f, Corresponding Death Index in the following conditions: wt (n=12 discs), dally80 homotypic (n=12 discs) and dally80 heterotypic (dally80 mutant clones in dally80/+ background, n=33 clones) wing discs. g, Death Index (NV, red) in dally80 clones (n=33) in the center or clone border. h, Flip-out clones in wing imaginal discs overexpressing eiger in the background of Dally::GFP and Pentagone::GFP. Wing discs were fixed 24h after clone induction. RFP (Red; clones), Dally::GFP (green) and Pentagone::GFP (green). Note that the expression of Dally::GFP and Pentagone::GFP is not affected in the eiger overexpressing clones. i, Super-competition assay in the background of Dally::GFP and Pentagone::GFP. Flip-out clones without a dmyc overexpression cassette (‘loser clones’). Clones (Red), Dally::GFP (green) and Pentagone::GFP (green). Note that the expression of Dally::GFP and Pentagone::GFP is not affected in the clones without dmyc overexpression cassette. Data are presented as mean values +/− SEM. ** indicates ≤ 0.01 and ***, ≤ 0.001 significance. Two-sided test of significance were used for all the comparisons. Sample sizes (discs/clones) are indicated. Scale bars: in a, 10µm; c-e,h,i, 50 µm.

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Extended Data Fig. 7 Flip-out clones in various genetic conditions.

a, Representative images of wing discs with flip-out clones fixed 24h ACI in various genetic conditions. GFP clones (green) and nuclear marker DAPI (blue). b, d Quantifications of clone areas (μm2) from discs fixed 24h (b) and 72h (d) ACI in various genetic conditions. c, Representative images of wing discs with flip-out clones fixed 72h ACI in various genetic conditions. GFP clones (green) and nuclear marker DAPI (blue). Sample sizes (clones) are shown in the corresponding quantification. Sample sizes in b; (UASlacZ=125), (UASGFP::dally=91), (UASpent=117), (dallyRNAi=93), (pentRNAi=99). Sample sizes in d; (UASlacZ=69), (UASGFP::dally=54), (UASpent=94), (dallyRNAi=106), (pentRNAi=106). e, Representative images of wing discs with flip-out clones fixed 48h ACI, overexpressing UASlacZ (control, left), UASpentagone (middle) and UASpentagone 2X (two UASpentagone transgenes, right). Wing discs are immunostained for Phosphorylated Mad (anti-PMad, green), GFP clones in red. f, Normalized average curve for Phosphorylated Mad gradient in the previous genetic conditions. λ shows the decay length of the average curve of each condition. g, Average decay length of the individual PMad exponential gradients of each condition. Sample sizes (discs); (UASlacZ=10), (UASpent=11), (UASpent 2X=10). For details on PMad exponential gradients measurement see Methods. Data are presented as mean values +/− SEM. * indicates ≤ 0.05 significance; **, ≤ 0.01 and ***, ≤ 0.001. Two-sided test of significance were used for all the comparisons. Sample sizes are indicated. Scale bars in a,c,e, 50 μm.

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Extended Data Fig. 8 Flower, Dally and Pentagone in scaling and cell death.

a, Triple staining showing dying cells labelled by NucView (NV, green) as well as Patched (Ptc, red) and Wingless (Wg, red) immunostaining in representative images of various genetic conditions. b, Death Index in the indicated genotypes. Sample sizes (discs); (white=14), (UASUbi::CFP=10), (UASLoseA::CFP=12), (UASLoseB::CFP=12), (UASΔExtra::mCherry=10), (ΔExtra::eGFP/+=12). c,d Comparison of the downregulation efficiency of yellowRNAi, fweRNAi (VDRC, KK) and fweRNAi 2 (Bloomington). c, Representative wing disc images of Fwe-LoseA::GFP (green) expression from Fwe reporter (Ubi::YFP, LoseA::GFP and LoseB::RFP). RNAis were ubiquitously expressed in the wing discs by means of the C765 Gal4 driver.d, Quantification of the downregulation efficiency of Fwe-LoseA::GFP of yellowRNAi, fweRNAi (VDRC, KK) and fweRNAi 2 (Bloomington). Note that fweRNAi line is considerably more efficient in silencing Flower than the line fweRNAi 2. Sample sizes (discs); (yellowRNAi=12), (fweRNAi 2=13), (fweRNAi=11). For details on these measurements see Methods. e, Scaling plots of various genetic conditions. Sample sizes (discs); (wt=34), (fweRNAi 2=53), (fweDB56/+=32). f, Representative images of blk-GFP (yellow) wing discs stained with nuclear marker (DAPI, blue) in the following conditions: UASlacZ and fweRNAi. g, Quantification of the Dpp expression levels (using a construct where the Dpp promoter region (blk) drives GFP expression (blk-GFP)) in the following conditions: UASlacZ and fweRNAi. Sample sizes (discs); (UASlacZ=10), (fweRNAi=13). h, Representative images of wing discs overexpressing the GCaMP5G Ca2+ reporter (cyan) in control (hhGal4>yellowRNAi) and fwe downregulation (hhGal4>fweRNAi). i, Quantification of the Ca2+ levels (GCaMP5G) in the following conditions: yellowRNAi and fweRNAi. For details on these measurements see Methods. Sample sizes (discs); (yellowRNAi=10), (fweRNAi=11). j, Active JNK Index (see methods) in: wt and ΔExtra::eGFP mutant. Sample sizes (discs); (wt=15), (ΔExtra::eGFP=13). k, Active JNK immunostaining (green) in representative wt and ΔExtra::eGFP mutant. l, Death Index in the indicated genotypes. Sample sizes (discs); (ΔExtra::eGFP =10), (ΔExtra::eGFP ; UASpuckered=10), (ΔExtra::eGFP ; UASschnurri=14). m, Triple staining showing dying cells labelled by NucView (NV, green) as well as Patched (Ptc,red) and Wingless (Wg, red) immunostaining in representative images of various genetic conditions. n, Hid Index (see methods) in various genetic conditions. Sample sizes (discs); (wt=13), (ΔExtra::eGFP=11), (ΔExtra::eGFP ; UASpuckered=15), (ΔExtra::eGFP ; UASschnurri=14). o, Triple staining showing Hid (green) as well as Patched (Ptc,red) and Wingless (Wg, red) immunostaining in representative images of various genetic conditions. p, q, and r Death Index in various genetic conditions. Sample sizes in p (discs); (UASlacZ=13), (dallygem=17), (dallygem ; UASpent::GFP=13), (pent2=16), (pent2 ; UASGFP::dally=15). Sample sizes in q (discs); (UASlacZ=13), (ΔExtra::eGFP=11), (ΔExtra::eGFP ; UASGFP::dally=13), (ΔExtra::eGFP ; UASpent=10), (ΔExtra::eGFP ; UASUbi::CFP=11), (ΔExtra::eGFP ; UASΔExtra::mCherry=10). Sample sizes in r (discs); (UASlacZ=13), (UASp35=10), (UASGFP::dally=13), (UASpent=12), (UASGFP::dally ; UASpent::GFP=12), (UASGFP::dally ; fweRNAi=8), (UASpent::GFP ; fweRNAi=11). s, Triple staining showing dying cells labelled by NucView (NV, green) as well as Patched (Ptc, red) and Wingless (Wg, red) immunostaining in representative images of the genetic conditions shown in q and r. Data are presented as mean values +/− SEM. * indicates ≤ 0.05 and ***, ≤ 0.001. Two-sided test of significance were used for all the comparisons. Sample sizes (discs) are indicated. Scale bars in a, c, f, h, k, m, o, s 50 μm.

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Extended Data Fig. 9 Expression of Dally and Pentagone in fwe silencing and overexpression and Dally and Pentagone in scaling.

a, Scaling plots of various genetic conditions and Phospho-Mad immunostaining (PMad) in wing discs. Sample sizes (discs); (wt=34), (dallygem=33), (pent2=42), (dallygem ; UASpentagone::GFP=39), (pentagone2; UASGFP::dally=38). b, Scaling plots of various genetic conditions. Sample sizes (discs); (UASlacZ=36), (UASGFP::dally=29), (UASpentagone=26), (UASGFP::dally ; UASlacZ=37), (UASpent::GFP ; UASlacZ=32), (UASGFP::dally ; UASpent::GFP=25). c, Phospho-Mad immunostaining (PMad) in wing discs of the genotypes: UASlacZ, UASGFP::dally, UASpentagone and UASGFP::dally; UASpentagone::GFP. d, Scaling plots and Phospho-Mad immunostaining (PMad) in wing discs of various genetic conditions. Sample sizes (discs); (dallygem ; UASp35=18), (dallygem=33), (pent2; UASp35=30), (pent2=42), (UASGFP::dally ; UASp35=30), (UASGFP::dally ; UASlacZ=37), (UASpent::GFP ; UASp35=36), (UASpent::GFP ; UASlacZ=32). e, Flip-out clones in wing imaginal discs downregulating fwe and overexpressing fwe in the background of Dally::GFP, Pentagone::GFP, and Thickveins::GFP. Wing discs were fixed 48h after clone induction. RFP clones (Red), Dally::GFP (green), Pentagone::GFP (green) and Thickveins::GFP (green). Data are presented as mean values +/− SEM. Sample sizes (discs) are indicated. Scale bars in a, c, d, e, 10 μm, 10 μm, 10 μm, 50 μm respectively.

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Extended Data Fig. 10 Flower expression in wildtype and mutant conditions.

a, Extracellular staining of the Fwe eGFP-tagged endogenous isoforms. Anti-GFP nanobody immunostaining (anti-GFP-Atto647N), grey; eGFP fluorescent signal, green. b, Co-immunoprecipitation of Fwe-Ubi::eGFP, Fwe-LoseA::eGFP, Fwe-LoseB::eGFP and ΔExtra::eGFP immunoprecipitation assays. Immunoprecipitation with GFP-Trap beads. Western blot shows inputs and immunoprecipitations with anti-GFP antibodies. BenchMark protein ladder. c, Expression pattern of the Fwe eGFP-tagged endogenous isoforms along the A/P length of the wing imaginal discs. d, Flip-out clones in wing imaginal discs overexpressing dally and overexpressing pentagone in the background of Fwe-Ubi::eGFP. Wing discs were fixed 48h after clone induction. RFP clones (Red), Fwe-Ubi::eGFP (green). e, Representative wing imaginal discs showing endogenous expression of Fwe-Ubi::eGFP in dallygem and pentagone2 mutant background. f, Mean fluorescence intensity of Fwe-LoseA::eGFP, Fwe-LoseB::eGFP, Fwe-ΔExtra::eGFP, Fwe-Ubi::eGFP, Fwe-Ubi::eGFP in dallygem mutant background, Fwe-Ubi::eGFP in pentagone2 mutant background and ubi-GFP in a rectangular region of interest along the A/P axis of the wing (indicated in Fig. 4d) as a function of the A/P length of the disc. Line, linear fit to the data. β, slope of the linear fit, is indicated. Sample sizes (discs); (Fwe-LoseA::eGFP=38), (Fwe-LoseB::eGFP=35), (Fwe-ΔExtra::eGFP=39), (Fwe-Ubi::eGFP=38), (Dallygem ; Fwe-Ubi::eGFP=32), (Pent2 ; Fwe-Ubi::eGFP=34), (ubi-GFP=40). Data are presented as mean values +/− SEM. Sample sizes (n=discs) are indicated. Scale bars in a,d,e, 50 μm.

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Statistical analyses, alleles, detailed genotypes, parameters and sample sizes.

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Merino, M.M., Seum, C., Dubois, M. et al. A role for Flower and cell death in controlling morphogen gradient scaling. Nat Cell Biol 24, 424–433 (2022). https://doi.org/10.1038/s41556-022-00858-3

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