Abstract
As a central model for morphogen action during animal development, the bone morphogenetic protein 2/4 (BMP2/4)-like ligand Decapentaplegic (Dpp) is proposed to form a long-range signalling gradient that directs both growth and pattern formation during Drosophila wing disc development1,2,3,4,5,6. While the patterning role of Dpp secreted from a stripe of cells along the anterior–posterior compartmental boundary is well established1,2,6, the mechanism by which a Dpp gradient directs uniform cell proliferation remains controversial and poorly understood7,8,9,10,11,12,13. Here, to determine the precise spatiotemporal requirements for Dpp during wing disc development, we use CRISPR–Cas9-mediated genome editing to generate a flippase recognition target (FRT)-dependent conditional null allele. By genetically removing Dpp from its endogenous stripe domain, we confirm the requirement of Dpp for the activation of a downstream phospho-Mothers against dpp (p-Mad) gradient and the regulation of the patterning targets spalt (sal), optomotor blind (omb; also known as bifid) and brinker (brk). Surprisingly, however, third-instar wing blade primordia devoid of compartmental dpp expression maintain relatively normal rates of cell proliferation and exhibit only mild defects in growth. These results indicate that during the latter half of larval development, the Dpp morphogen gradient emanating from the anterior–posterior compartment boundary is not directly required for wing disc growth.
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Acknowledgements
We thank G. Struhl and K. Wharton for extensive discussion and suggestions, S. Kondo for technical advice with CRISPR, K. Irvine, W. Deng and the Bloomington Stock Center for fly stocks, and R. Barrio, G. Pflugfelder, A. Teleman and the Developmental Studies Hybridoma Bank for antibodies. We thank K. Marr and L. Ellington for a critical reading of the manuscript, L. Gutchewsky for administrative support, and members of the Gibson laboratory for discussions and advice. This work was supported by funding from the Stowers Institute for Medical Research.
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T.A. and M.C.G. conceived the project, designed the experiments and wrote the manuscript. T.A. performed the experiments and analysed the data.
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Extended data figures and tables
Extended Data Figure 1 Endogenous Dpp expression in imaginal discs.
a–f, Wing (a, b), eye–antenna (c, d), and leg (e, f) imaginal discs from UAS-GFP/+; dpp-GAL4/+ larvae are dissected and stained with anti-Dpp antibody. GFP (green) indicates dpp-GAL4-expressing cells. Note that dpp-GAL4 is not expressed in the morphogenetic furrow of the third instar eye–antenna disc (arrow in d). Dotted lines show outlines of imaginal discs. Blue: DNA. Scale bars, 100 μm. Anterior is left.
Extended Data Figure 2 Dpp and p-Mad expression in dppd12 clones.
a–h, Control (a, b, e, f) and dppd12 mutant (c, d, g, h) clones are stained with anti-Dpp (a–d) and anti-p-Mad (e–h) antibodies. Clones are marked by the absence of GFP (green). Disc boundaries are indicated by dotted lines. Scale bars, 50 μm. Anterior is to the left.
Extended Data Figure 3 FLP/FRT-mediated conditional dpp-null allele.
a, A flowchart describing the establishment of an FLP/FRT-mediated conditional dpp-null allele. Grey and white boxes indicate untranslated region (UTR) and dpp coding sequences, respectively. FRT sequences flank the first coding exon (exon 5). Since this exon contains the Dpp start codon and almost half of its coding sequence (the first 288/588 amino acids), FLP/FRT mediated recombination is predicted to yield a null allele. b, dppFO-GFP heterozygous, dppFO heterozygous, and dppFO homozygous adult flies. Importantly, dppFO homozygous animals have normal adult morphology.
Extended Data Figure 4 Validation of an FLP/FRT-mediated conditional allele.
a, FRT 5′-loxP and FRT 3′ genomic regions are sequenced. b, Southern blot analysis of dppFO-GFP. Genomic DNAs from w1118 and dppFO-GFP are digested by ClaI and are subjected to Southern blot analysis using a GFP probe. c, Molecular confirmation of the FLP/FRT-mediated dpp FLP-OFF system. As expected, an FLP-OFF product (2,349 bp PCR fragment) is only detected in the dppFO/dppFO; dpp-GAL4/UAS-FLP lane. Asterisk indicates a non-specific PCR product. d, Biochemical evidence of the FLP/FRT-mediated dpp FLP-OFF system. y,w,hs-FLP; dppFO/dppFO larvae are incubated at 37 °C for 30 min at 96 h AEL to eliminate Dpp expression. After 24 h, wing discs are homogenized in SDS sample buffer and analysed by western blot analysis with anti-Dpp. Non-specific bands are indicated by an asterisk. Anti-α-tubulin is used as a loading control. e, A system to visualize the efficiency of FLP/FRT-mediated recombination. f–k, dppFO/+; dpp-GAL4/UAS-FLP,Act5c(-FRT)lacZ controls (f, g, h) and dppFO/dppFO; dpp-GAL4/UAS-FLP,Act5c(-FRT)lacZ (i, j, k) wing discs are stained with anti-Dpp and β-galactosidase antibodies. The lineage of dpp-GAL4-expressing cells is visualized by anti-β-galactosidase staining. Scale bar, 100 μm. Anterior is left.
Extended Data Figure 5 p-Mad staining of wing discs lacking dpp function in the dorsal compartment.
a–d, dppFO/ap-GAL4,UAS-GFP; UAS-FLP/+ (a, b) and dppFO/dppFO,ap-GAL4,UAS-GFP; UAS-FLP/+ (c, d) are dissected and stained with anti-p-Mad antibody. The dorso-ventral boundaries are indicated by green dotted lines. Yellow dotted lines show the disc areas. Scale bar, 50 μm.
Extended Data Figure 6 Elimination of Dpp from specific regions of wing discs.
a–f, Anti-Dpp antibody staining of wild-type (a), dppFO,ci-GAL4,en-GAL4/dppFO; UAS-FLP/+ (b), dppFO,en-GAL4/dppFO; UAS-FLP/+ (c), dppFO,ci-GAL4/dppFO; UAS-FLP/+ (d), dppFO,ap-GAL4/dppFO; UAS-FLP/+ (e), and dppFO,nub-GAL4/dppFO; UAS-FLP/+ (f). Gal4-expressing domains are highlighted in grey in each illustration. Wing disc boundaries are shown by dotted lines. Scale bar, 100 μm. Anterior is left.
Extended Data Figure 7 Spatiotemporal Dpp removal from the anterior region of wing discs.
a, A strategy for temporal Dpp elimination from the anterior compartment of wing discs using the GAL80ts system. At 18 °C, Gal4 activity is blocked by Gal80. When flies are kept at 29 °C (non-permissive temperature for GAL80ts), Gal4 induces expression of FLP and GFP. b, Timing of temperature shift. Larvae are reared at 18 °C, and are transferred to 29 °C at the indicated time points before dissection. c–f, dppFO/ci-GAL4,UAS-GFP; UAS-FLP,tub-GAL80ts/+ controls are stained with anti-Dpp. Gal4 activity is monitored by GFP expression. Scale bar, 100 μm. g, Size comparison between wing discs: dppFO/ci-GAL4,UAS-GFP; UAS-FLP,tub-GAL80ts/+ (0 (n = 21) and 72 h (n = 40) before dissection) and dppFO/dppFO,ci-GAL4,UAS-GFP; UAS-FLP,tub-GAL80ts/+ (0 (n = 36), 18 (n = 33), 24 (n = 40), 36 (n = 33), 48 (n = 80) and 72 h (n = 43) before dissection). Mean ± s.d. *P < 0.001, not significant (NS), two-sided Student’s t-test.
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Akiyama, T., Gibson, M. Decapentaplegic and growth control in the developing Drosophila wing. Nature 527, 375–378 (2015). https://doi.org/10.1038/nature15730
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DOI: https://doi.org/10.1038/nature15730
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