Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

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.

References

  1. Hamaratoglu, F., Affolter, M. & Pyrowolakis, G. Dpp/BMP signaling in flies: from molecules to biology. Semin. Cell Dev. Biol. 32, 128–136 (2014).

    CAS  PubMed  Article  Google Scholar 

  2. Wartlick, O. et al. Dynamics of Dpp signaling and proliferation control. Science 331, 1154–1159 (2011).

    CAS  PubMed  Article  Google Scholar 

  3. Hamaratoglu, F., de Lachapelle, A. M., Pyrowolakis, G., Bergmann, S. & Affolter, M. Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc. PLoS Biol. 9, e1001182 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Vuilleumier, R. et al. Control of Dpp morphogen signalling by a secreted feedback regulator. Nat. Cell Biol. 12, 611–617 (2010).

    CAS  PubMed  Article  Google Scholar 

  5. Akiyama, T. et al. Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface. Dev. Biol. 313, 408–419 (2008).

    CAS  PubMed  Article  Google Scholar 

  6. Rhiner, C. et al. Flower forms an extracellular code that reveals the fitness of a cell to its neighbors in Drosophila. Dev. Cell 18, 985–998 (2010).

    CAS  PubMed  Article  Google Scholar 

  7. Barkai, N. & Ben-Zvi, D. ‘Big frog, small frog’—maintaining proportions in embryonic development: delivered on 2 July 2008 at the 33rd FEBS Congress in Athens, Greece. FEBS J. 276, 1196–1207 (2009).

    CAS  PubMed  Article  Google Scholar 

  8. Norman, M., Vuilleumier, R., Springhorn, A., Gawlik, J. & Pyrowolakis, G. Pentagone internalises glypicans to fine-tune multiple signalling pathways. eLife https://doi.org/10.7554/eLife.13301 (2016).

  9. Mateus, R. et al. BMP signaling gradient scaling in the Zebrafish pectoral fin. Cell Rep. 30, 4292–4302 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Chorsky, R. & Belote, J. M. Genitalia missing (gem): an autosomal recessive mutant that affects development of the genital disc derivatives. D. I. S. 75, 33 (1994).

    Google Scholar 

  11. Cen, H., Mao, F., Aronchik, I., Fuentes, R. J. & Firestone, G. L. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. FASEB J. 22, 2243–2252 (2008).

    CAS  PubMed  Article  Google Scholar 

  12. Gudipaty, S. A., Conner, C. M., Rosenblatt, J. & Montell, D. J. Unconventional ways to live and die: cell death and survival in development, homeostasis and disease. Annu. Rev. Cell Dev. Biol. 34, 311–332 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Moreno, E., Yan, M. & Basler, K. Evolution of TNF signaling mechanisms: JNK-dependent apoptosis triggered by Eiger, the Drosophila homolog of the TNF superfamily. Curr. Biol. 12, 1263–1268 (2002).

    CAS  PubMed  Article  Google Scholar 

  14. Igaki, T. et al. Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway. EMBO J. 21, 3009–3018 (2002).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Andersen, D. S. et al. The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature 522, 482–486 (2015).

    CAS  PubMed  Article  Google Scholar 

  16. Levayer, R., Dupont, C. & Moreno, E. Tissue crowding induces caspase-dependent competition for space. Curr. Biol. 26, 670–677 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Monier, B. et al. Apico-basal forces exerted by apoptotic cells drive epithelium folding. Nature 518, 245–248 (2015).

    CAS  PubMed  Article  Google Scholar 

  18. Tsuneizumi, K. et al. Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 389, 627–631 (1997).

    CAS  PubMed  Article  Google Scholar 

  19. Morin, X., Daneman, R., Zavortink, M. & Chia, W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl Acad. Sci. USA 98, 15050–15055 (2001).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Ben-Zvi, D., Pyrowolakis, G., Barkai, N. & Shilo, B. Z. Expansion-repression mechanism for scaling the Dpp activation gradient in Drosophila wing imaginal discs. Curr. Biol. 21, 1391–1396 (2011).

    CAS  PubMed  Article  Google Scholar 

  21. Crossman, S. H., Streichan, S. J. & Vincent, J. P. EGFR signaling coordinates patterning with cell survival during Drosophila epidermal development. PLoS Biol. 16, e3000027 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  22. Ohtsubo, T., Kamada, S. & Tsujimoto, Y. Inhibition of apoptosis by a baculovirus p35 gene.Nihon Rinsho J. J. Clin. Med. 54, 1907–1911 (1996).

    CAS  Google Scholar 

  23. Hay, B. A., Wassarman, D. A. & Rubin, G. M. Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83, 1253–1262 (1995).

    CAS  PubMed  Article  Google Scholar 

  24. Zhou, L., Hashimi, H., Schwartz, L. M. & Nambu, J. R. Programmed cell death in the Drosophila central nervous system midline. Curr. Biol. 5, 784–790 (1995).

    CAS  PubMed  Article  Google Scholar 

  25. Luo, X., Puig, O., Hyun, J., Bohmann, D. & Jasper, H. Foxo and Fos regulate the decision between cell death and survival in response to UV irradiation. EMBO J. 26, 380–390 (2007).

    CAS  PubMed  Article  Google Scholar 

  26. Merino, M. M. et al. Elimination of unfit cells maintains tissue health and prolongs lifespan. Cell 160, 461–476 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Wartlick, O., Jülicher, F. A. & Gonzalez-Gaitan, M. Growth control by a moving morphogen gradient during Drosophila eye development. Development 141, 1884–1893 (2014).

  28. Baillon, L. & Basler, K. Reflections on cell competition. Semin. Cell Dev. Biol. 32, 137–144 (2014).

    PubMed  Article  Google Scholar 

  29. Bowling, S., Lawlor, K. & Rodriguez, T. A. Cell competition: the winners and losers of fitness selection. Development https://doi.org/10.1242/dev.167486 (2019).

  30. Fahey-Lozano, N., La Marca, J. E., Portela, M. & Richardson, H. E. Drosophila models of cell polarity and cell competition in tumourigenesis. Adv. Exp. Med. Biol. 1167, 37–64 (2019).

    CAS  PubMed  Article  Google Scholar 

  31. Merino, M. M., Levayer, R. & Moreno, E. Survival of the fittest: essential roles of cell competition in development, aging and cancer. Trends Cell Biol. 26, 776–788 (2016).

    PubMed  Article  Google Scholar 

  32. Moreno, E., Basler, K. & Morata, G. Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development. Nature 416, 755–759 (2002).

    CAS  PubMed  Article  Google Scholar 

  33. Adachi-Yamada, T., Fujimura-Kamada, K., Nishida, Y. & Matsumoto, K. Distortion of proximodistal information causes JNK-dependent apoptosis in Drosophila wing. Nature 400, 166–169 (1999).

    CAS  PubMed  Article  Google Scholar 

  34. Adachi-Yamada, T. & O’Connor, M. B. Mechanisms for removal of developmentally abnormal cells: cell competition and morphogenetic apoptosis. J. Biochem. 136, 13–17 (2004).

    PubMed  Article  CAS  Google Scholar 

  35. Adachi-Yamada, T. & O’Connor, M. B. Morphogenetic apoptosis: a mechanism for correcting discontinuities in morphogen gradients. Dev. Biol. 251, 74–90 (2002).

    CAS  PubMed  Article  Google Scholar 

  36. Coelho, D. S. et al. Culling less fit neurons protects against amyloid-β-induced brain damage and cognitive and motor decline. Cell Rep. 25, 3661–3673 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Madan, E. et al. Flower isoforms promote competitive growth in cancer. Nature 572, 260–264 (2019).

    CAS  PubMed  Article  Google Scholar 

  38. Merino, M. M., Rhiner, C., Portela, M. & Moreno, E. ‘Fitness fingerprints’ mediate physiological culling of unwanted neurons in Drosophila. Curr. Biol. 23, 1300–1309 (2013).

    CAS  PubMed  Article  Google Scholar 

  39. Nagata, R., Nakamura, M., Sanaki, Y. & Igaki, T. Cell competition is driven by autophagy. Dev. Cell 51, 99–112 (2019).

    CAS  PubMed  Article  Google Scholar 

  40. Casas-Tinto, S., Maraver, A., Serrano, M. & Ferrus, A. Troponin-I enhances and is required for oncogenic overgrowth. Oncotarget 7, 52631–52642 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  41. Basagiannis, D. et al. VEGF induces signalling and angiogenesis by directing VEGFR2 internalisation through macropinocytosis. J. Cell Sci. 129, 4091–4104 (2016).

    CAS  PubMed  Google Scholar 

  42. Hemalatha, A., Prabhakara, C. & Mayor, S. Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs. Proc. Natl Acad. Sci. USA 113, E6993–E7002 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Basagiannis, D., Zografou, S., Galanopoulou, K. & Christoforidis, S. Dynasore impairs VEGFR2 signalling in an endocytosis-independent manner. Sci. Rep. 7, 45035 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Lamb, J. E., Ray, F., Ward, J. H., Kushner, J. P. & Kaplan, J. Internalization and subcellular localization of transferrin and transferrin receptors in HeLa cells. J. Biol. Chem. 258, 8751–8758 (1983).

    CAS  PubMed  Article  Google Scholar 

  45. Portela, M. et al. Drosophila SPARC is a self-protective signal expressed by loser cells during cell competition. Dev. Cell 19, 562–573 (2010).

    CAS  PubMed  Article  Google Scholar 

  46. Morata, G. & Ripoll, P. Minutes: mutants of Drosophila autonomously affecting cell division rate. Dev. Biol. 42, 211–221 (1975).

    CAS  PubMed  Article  Google Scholar 

  47. Meyer, S. N. et al. An ancient defense system eliminates unfit cells from developing tissues during cell competition. Science 346, 1258236 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Blanco, J., Cooper, J. C. & Baker, N. E. Roles of C/EBP class bZip proteins in the growth and cell competition of Rp (‘Minute’) mutants in Drosophila. eLife https://doi.org/10.7554/eLife.50535 (2020).

  49. Vincent, J. P., Kolahgar, G., Gagliardi, M. & Piddini, E. Steep differences in wingless signaling trigger Myc-independent competitive cell interactions. Dev. Cell 21, 366–374 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Froldi, F. et al. The lethal giant larvae tumour suppressor mutation requires dMyc oncoprotein to promote clonal malignancy. BMC Biol. 8, 33 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. Rodrigues, A. B. et al. Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis. Development 139, 4051–4061 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Chen, C. L., Schroeder, M. C., Kango-Singh, M., Tao, C. & Halder, G. Tumor suppression by cell competition through regulation of the Hippo pathway. Proc. Natl Acad. Sci. USA 109, 484–489 (2012).

    CAS  PubMed  Article  Google Scholar 

  53. Tamori, Y. et al. Involvement of Lgl and Mahjong/VprBP in cell competition. PLoS Biol. 8, e1000422 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  54. Moreno, E. & Basler, K. dMyc transforms cells into super-competitors. Cell 117, 117–129 (2004).

    CAS  PubMed  Article  Google Scholar 

  55. Ben-Zvi, D. & Barkai, N. Scaling of morphogen gradients by an expansion-repression integral feedback control. Proc. Natl Acad. Sci. USA 107, 6924–6929 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. Green, E. W., Fedele, G., Giorgini, F. & Kyriacou, C. P. A Drosophila RNAi collection is subject to dominant phenotypic effects. Nat. Methods 11, 222–223 (2014).

    Article  CAS  Google Scholar 

  57. Yao, C. K. et al. A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis. Cell 138, 947–960 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Beira, J. V. et al. The Dpp/TGFβ-dependent corepressor Schnurri protects epithelial cells from JNK-induced apoptosis in Drosophila embryos. Dev. Cell 31, 240–247 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Weiss, A. et al. A conserved activation element in BMP signaling during Drosophila development. Nat. Struct. Mol. Biol. 17, 69–76 (2010).

    CAS  PubMed  Article  Google Scholar 

  60. Levayer, R., Hauert, B. & Moreno, E. Cell mixing induced by myc is required for competitive tissue invasion and destruction. Nature 524, 476–480 (2015).

    CAS  PubMed  Article  Google Scholar 

  61. Massague, J. TGFβ in cancer. Cell 134, 215–230 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Jakowlew, S. B. Transforming growth factor-β in cancer and metastasis. Cancer Metastasis Rev. 25, 435–457 (2006).

    CAS  PubMed  Article  Google Scholar 

  63. Dahmann, C. Drosophila: Methods and Protocols (Springer, 2018).

  64. Ryoo, H. D., Gorenc, T. & Steller, H. Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways. Dev. Cell 7, 491–501 (2004).

    CAS  PubMed  Article  Google Scholar 

  65. Derivery, E. et al. Polarized endosome dynamics by spindle asymmetry during asymmetric cell division. Nature 528, 280–285 (2015).

    CAS  PubMed  Article  Google Scholar 

  66. Loubery, S. & Gonzalez-Gaitan, M. Monitoring notch/delta endosomal trafficking and signaling in Drosophila. Methods Enzymol. 534, 301–321 (2014).

    CAS  PubMed  Article  Google Scholar 

  67. Basagiannis, D. & Christoforidis, S. Constitutive endocytosis of VEGFR2 protects the receptor against shedding. J. Biol. Chem. 291, 16892–16903 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  68. Venken, K. J. et al. MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. Nat. Methods 8, 737–743 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. Entchev, E. V., Schwabedissen, A. & Gonzalez-Gaitan, M. Gradient formation of the TGF-β homolog Dpp. Cell 103, 981–991 (2000).

    CAS  PubMed  Article  Google Scholar 

  70. Kondo, S. & Ueda, R. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195, 715–721 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  71. Larkin, A. et al. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res. 49, D899–D907 (2021).

    CAS  PubMed  Article  Google Scholar 

Download references

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.

Author information

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

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.

Source data

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.

Source data

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.

Source data

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.

Source data

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.

Source data

Supplementary information

Supplementary Information

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

Reporting Summary

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 10

Statistical source data.

Source Data Extended Data Fig. 10

Unprocessed western blots.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-022-00858-3

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing