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Dpp spreading is required for medial but not for lateral wing disc growth

Nature volume 527, pages 317–322 (2015)Cite this article

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Abstract

Drosophila Decapentaplegic (Dpp) has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the ‘temporal rule’ model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the ‘growth equalization model’ suggests that Dpp is only essential for proliferation control of the central cells. Here, to discriminate between these two models, we generated and used morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody, which enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. We find that in the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc.

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Figure 1: Morphotrap can block eGFP::Dpp spreading.
Figure 2: Blocking Dpp spreading results in a sharp p-Mad/Brk transition.
Figure 3: The uniform proliferation pattern is independent of Dpp spreading.
Figure 4: Block of Dpp spreading does not affect clonal proliferation rates.
Figure 5: The development of the medial but not the lateral wing disc requires Dpp spreading.

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Acknowledgements

We would like to acknowledge the work of the late William (Bill) Gelbart, who initiated the work on Dpp. We thank S. Matsuda, I. Alborelli and H. Belting for discussions; T. Schaffter for help and support with WingJ; the Biozentrum Imaging Core Facility for maintenance of microscopes and support. We are grateful to G. Struhl, K. Basler and G. Pyrowolakis for their input and discussion on the project. We thank K. Basler, S. Cohen, G. Morata, R. Bario and E. Laufer for flies and reagents. S.H. was supported by the ‘Fellowships for Excellence’ International PhD Program in Molecular Life Sciences of the Biozentrum, University of Basel. Funding is also acknowledged from the SystemsX.ch initiative within the framework of the WingX (E.C. and F.H.) and the MorphogenetiX projects (E.C.). F.H. is now supported by a Swiss National Science Foundation (SNSF) Professorship grant (PP00P3_150682). The work in the laboratory was supported by grants from Cantons Basel-Stadt and Basel-Land, from the SNSF and from SystemsX.ch (M.A.).

Author information

Author notes

  1. Markus Affolter and Emmanuel Caussinus: These authors contributed equally to this work.

Authors and Affiliations

  1. Growth & Development, Biozentrum, Klingelbergstrasse 50/70, University of Basel, Basel, 4056, Switzerland

    Stefan Harmansa, Markus Affolter & Emmanuel Caussinus

  2. Center for Integrative Genomics, University of Lausanne, Lausanne, 1015, Switzerland

    Fisun Hamaratoglu

  3. Institute of Molecular Life Sciences (IMLS), University of Zurich, Zurich, 8057, Switzerland

    Emmanuel Caussinus

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  1. Stefan Harmansa
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Contributions

S.H., E.C., F.H. and M.A. conceived and designed the study. S.H. performed the experiments. S.H. analysed the data. S.H., E.C. and M.A. wrote the paper.

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Correspondence to Markus Affolter.

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Extended data figures and tables

Extended Data Figure 1 eGFP::Dpp can compensate for endogenous Dpp during wing disc development.

a, Part of the protein sequence of the Dpp protein. The two different eGFP insertion sites9,10, and the two furin cleavage sites58 located in this region are marked. Furin cleavage of the inactive pro-form yields the active carboxy-terminal mature ligand. However, potential processing at cleavage site II may result in uncoupling of the eGFP from the mature ligand in the construct described previously10. We therefore inserted the EGFP C-terminal to the second furin cleavage site as was done previously9. b–d, Immunostainings for p-Mad and Brk in wild-type (b), dppd8/d12 mutant (c) and dppd8/d12 mutant wing discs rescued with eGFP::Dpp expressed under control of the dpp::LG48 line (d). In the dppd8/d12 mutant wing discs expressing eGFP::Dpp, the p-Mad and Brk profiles are rescued to a control-like pattern (d, bottom). The pouch outline and the A/P boundary (assessed by Wg/Ptc pattern, data not shown) are marked by dotted lines. e, The eGFP::Dpp gradient visualized by eGFP fluorescence or by an immunostaining for the extracellular fraction of eGFP (bottom). f, Quantification of wing pouch area assessed by the inner Wg ring of 98–100 h old wing discs (wild type n = 6, dppd8/d12 mutant n = 10, rescue n = 10; red crosses are outliers). eGFP::Dpp expression in dppd8/d12 mutants rescues pouch area close to wild-type size. g–i, Wing discs of 98–100 h old larvae stained for Drosophila Serum response factor (DSRF; also known as blistered). DSRF is expressed in the future intervein tissue of the wing disc. Positions of prospective wing veins 3, 4 and 5 are marked by arrowheads. The vein pattern is largely restored in mutant discs rescued by eGFP::Dpp expression (i)). j–l, Adult wings of a wild-type fly (j), a dppd8/d12 mutant (k) and a dppd8/d12 mutant expressing eGFP::Dpp (l) (W, wing). Rescued wings have a slightly elongated shape but their sizes are comparable to that of control wings. However, they show some additional vein tissue at the anterior cross-vein and wing vein 4 is absent in the distal part of the wing (marked by arrowhead). We speculate that this is due to lower eGFP::Dpp expression in the ventral compartment, which also manifests itself in lower ventral p-Mad levels (see d) and less well defined ventral vein patterns in the dSRF staining (i, arrow). Apart from these drawbacks, LexA-driven eGFP::Dpp can compensate for endogenous Dpp during wing disc development.

Extended Data Figure 2 Morphotrap expression does not affect growth or patterning of the wing disc.

a, Wing disc expressing morphotrap in the posterior compartment controlled by hh-Gal4 (morphotraphh). The Wg/Ptc pattern is used as a coordinate system to assess pouch size (anterior (A) pouch, left two quadrants; posterior (P) pouch, right two quadrants). Gradient profiles are measured parallel to the dorso-ventral (D/V) boundary (for example, 15% ventral offset). b, Wild-type wing disc stained for p-Mad. c, Wings of a male wild-type fly and a fly expressing morphotrap in the posterior compartment under the control of hedgehog::Gal4 (morphotraphh). d, Morphotraphh wing discs show no significant change in anterior or posterior pouch size (t-test two-sided, unequal variance: anterior compartment P > 0.05, posterior compartment P > 0.05). e, Posterior expression of morphotrap does not cause obvious changes to the p-Mad profile. f, p-Mad pattern of a wild-type wing disc expressing eGFP::Dpp in the endogenous Dpp source area. g, Lateral morphotrap clones show elevated p-Mad signal at the clone boundary facing the Dpp source due to eGFP::Dpp accumulation. The region marked by a white rectangle is enlarged to the right. d, e, Control n = 11, morphotraphh n = 9, error bars in e show s.d.

Extended Data Figure 3 Domain width of Dpp targets depends on Dpp spreading.

a, Discs of a dppd8/d12 mutant rescued with eGFP::Dpp stained for Dpp targets Sal and Omb. Omb shows a wider distribution than Sal. b, dppd8/d12 mutant wing discs co-expressing eGFP::Dpp and morphotrap. The regions marked by a dotted rectangle are enlarged to the right of the respective image. The dotted red line marks the A/P compartment boundary. In the absence of Dpp spreading, target domains collapse onto a single cell row in the posterior compartment. In the anterior compartment domain borders are less sharp. We hypothesize that this is due to morphotrap-bound eGFP::Dpp that is dragged into the anterior compartment by dividing cells (see also e). Intensity profiles of the enlarged regions are plotted to the right. c, Wing disc of a dppd8/d12 mutant rescued with eGFP::Dpp stained for the proliferation marked BrdU. Uniform BrdU signal is obtained along the entire disc tissue. d, Rescued wing disc with blocked Dpp spreading stained for BrdU. Also in the absence of Dpp spreading the uniform BrdU signal is not lost. e, Expression of mCherry–CAAX under the control of the dpp::LexA driver line used for the rescue. mCherry–CAAX is a protein with a long half-life that localizes to the membrane. The graph to the right shows intensity plot of the region marked on the left. No posterior expression is observed; however, the protein profile is graded into the anterior compartment. Analogous to morphotrap-bound eGFP::Dpp, the stable mCherry–CAAX protein forms a concentration gradient into the anterior compartment due to dividing cells that are pushed further laterally into the anterior compartment. f, Wing of a rescued fly with blocked Dpp spreading. The hinge region, arising from the lateral wing disc region, is present and well patterned. In contrast, the wing field, arising from the medial wing disc region, is strongly reduced in size and patterning is lost.

Extended Data Figure 4 Time course of eGFP::Dpp spreading, signalling and the mitotic index.

a–i, Time course of extracellular eGFP::Dpp (exGFP), Dpp signalling (p-Mad) and p-H3 from 64–112 h AEL of larval development. a, b, Representative discs of the six time points examined of control animals (a) and animals with blocked Dpp spreading (b) stained for exGFP. The region marked by a red rectangle is enlarged below each image. eGFP::Dpp spreading is tightly blocked by morphotrap at all time points. c, Average exGFP profiles for all time points (control in black/block in red: n = 43/29). d, e, Discs of control animals (d) and animals with blocked Dpp spreading (e) stained for p-Mad. When Dpp spreading is blocked, the p-Mad gradient also collapses onto the source region at all time points. f, Average p-Mad profiles (control/block: n = 50/35). g, h, Control discs (g) and discs with blocked Dpp spreading (h) stained for p-H3. i, Quantification of the mitotic index (p-H3 spot density). No significant differences were observed between control discs (black, n = 55) and discs with blocked Dpp spreading (red, n = 43) at any time point (n > 0.05 for all time points, two-sided t-test, unequal variance).

Extended Data Figure 5 Shortening of the Dpp gradient by posterior morphotrap expression.

a, Scheme of morphotrap expression in the posterior compartment (using hh::Gal4) in dppd8/d12 mutant wing discs rescued with eGFP::Dpp. b, Posterior morphotrap expression in the rescue background results in strong eGFP signal in the first three cell rows of the posterior compartment due to eGFP::Dpp accumulation; after three cell rows the eGFP fluorescence signal drops. c, p-Mad staining in a dppd8/d12 mutant wing disc rescued with eGFP::Dpp. d, p-Mad staining in a dppd8/d12 mutant wing disc rescued by eGFP::Dpp and expressing morphotrap in the posterior compartment. Note that the eGFP::Dpp accumulation (marked by a yellow line) directly overlaps with the observed p-Mad signal. e, The average p-Mad profiles show that the p-Mad gradient range directly depends on the range of Dpp spreading (error bars are s.d.). f, dppd8/d12 mutant wing discs rescued with eGFP::Dpp expressing morphotrap in the posterior compartment stained for Sal and Omb (for control discs see Extended Data Fig. 3a). The A/P boundary is marked by a dotted red line and the range of the eGFP::Dpp accumulation is marked by a dotted yellow line. In this condition the domain width of both targets is strongly reduced. The Sal domain directly collapses onto the eGFP::Dpp accumulation domain. However, Omb, which can be activated at lower Dpp signalling levels, shows a slightly wider distribution. We hypothesize that this is again due to morphotrap-stabilized eGFP::Dpp being dragged into the posterior compartment (as discussed in Extended Data Fig. 3). Intensity profiles of the enlarged regions are plotted to the right. g, Representative dppd8/d12 mutant wing discs rescued with eGFP::Dpp expressing morphotrap in the posterior compartment stained for Brk at the indicated time points (79–12 h AEL). In this condition the medial region shows strongly reduced growth (compare to Fig. 5a–f). However, the growth dynamics of the lateral domain are similar to the lateral growth observed in control wing discs (right).

Extended Data Figure 6 Clonal growth rates do not change in the absence of Dpp spreading.

a–h, Estimation of clonal proliferation rates as shown in Fig. 4, inducing Raeppli at different time points: either 20 h before dissection (a–d) or 41 h before dissection (e–h). Discs were dissected at 96–100 h AEL. a, e, Representative control discs. b, f, Representative discs with blocked Dpp spreading. c, g, Clone size (number of cells per clone) plotted against the relative position in the posterior compartment (0 corresponding to the A/P boundary and 1 to the posterior edge of the disc). Low numbers of small clones in proximity to the A/P boundary are found in discs with blocked Dpp spreading (red dots), while these small clones are not present in control discs (black dots; see also Extended Data Fig. 7). d, h, Boxplots showing the number of cells per clone. When the small clones are excluded (right boxplots) no significant differences are detected in clonal proliferation between control discs and discs with blocked Dpp spreading (P > 0.05).

Extended Data Figure 7 Small clones in discs with blocked Dpp spreading.

a–c, Wing discs with blocked Dpp spreading carrying small Raeppli clones in proximity of the A/P boundary. Raeppli was induced by heat shock (HS) at different time points during larval development: 20 h (a), 30 h (b) and 41 h (c) before dissection. The regions marked by a white rectangle in the left column are magnified to the right.

Extended Data Figure 8 Temporal development and fitting procedure of Brk data set.

a, b, As can be seen in Fig. 2f, there is a gap in Brk expression in the lateral-most region of the posterior compartment, indicative of Dpp expression from another, laterally located source. Indeed, it has been shown that Dpp is expressed during the third instar larval stage in a posterior, lateral position and exerts a patterning role on the wing imaginal disc. However, this late Dpp expression does not affect the growth properties of wing disc cells59. Despite this, the additional Dpp source might complicate the interpretation of our growth analyses. To circumvent this problem, we also measured the growth properties in the anterior compartment in the presence (a) and in the absence of the eGFP::Dpp gradient (b; high uniform levels of Brk are indeed present in all cells outside the source). Indeed, we found that the lateral anterior region still grows despite the absence of the Dpp gradient and the lack of Dpp signalling. c, d, Width of the anterior and posterior compartment respectively in dppd8/d12 mutant wing discs rescued with eGFP::Dpp (black, n = 34) and dppd8/d12 mutant wing discs co-expressing eGFP::Dpp and morphotrap (red, n = 37, error bars show s.d.). e, The red dots mark the 15 points used as landmarks for the affine transformation. Using affine transformation allowed us to overlay discs of slightly different shapes and sizes when generating the mitotic density maps shown in Fig. 3c, e (see also Methods for details). f, Computation of Brk data set shown for the posterior compartment: (1) The compartment width LA or LP was defined as the distance from the A/P boundary to the anterior or posterior edge of the wing tissue, respectively. Brk profiles were measured along a straight line with 30%D offset. (2) Profiles were extracted using WingJ software. (3) The single gradients were fitted to the shown Hill function. The fitting procedure returns the parameter k, which corresponds to the position of half-maximum Brk levels and hence to the width of the medial domain. Therefore, the lateral domain equals L − k.

Extended Data Figure 9 Impact of Dpp spreading on wing pouch and adult wing size.

a, Dpp mutant wing disc rescued with eGFP::Dpp stained for Wg (outlining the wing pouch) and Ptc (marking the A/P boundary). In this background eGFP::Dpp spreading is not hindered and a normal gradient forms. The size of the posterior wing pouch is estimated by the area enclosed by the Wg ring and the A/P boundary (coloured orange) and plotted in g. b, Adult wing of a rescued fly. The border between the hinge region and the wing blade is marked by a dotted orange line; the alula is labelled with an A. (Wing is the same as shown in Extended Data Fig. 1l.) c, Rescued wing disc expressing morphotrap in the posterior compartment, reducing Dpp dispersal range in the posterior compartment. In this condition pouch size is significantly decreased (see g). d, Wing of a rescued fly expressing morphotrap in the posterior compartment. The wing blade area is strongly decreased and patterning in the posterior part of the wing is lost. e, Rescued wing disc expressing morphotrap in the Dpp stripe, completely blocking Dpp spreading, and hence gradient formation. Full block of Dpp spreading results in a further decrease of the Wg/Ptc-encircled posterior pouch area. f, Wing of a rescued fly co-expressing eGFP::Dpp and morphotrap. Full block of Dpp spreading results in a strong reduction of wing blade area. Only a small amount of unpatterned wing tissue is left, while the hinge region seems to be patterned normally (alula is present). g, Plot of the posterior pouch area, as accessed by the Wg/Ptc staining shown in (a, c, e, right) when Dpp spreads normally (black), Dpp spreading is reduced (blue) or when Dpp spreading is fully blocked (red). With decreasing Dpp dispersal range also the posterior pouch area decreases (n = 22).

Extended Data Figure 10 Linear range imaging conditions.

a−e, Linear range imaging for the quantitative data sets acquired (corresponding figure is labelled at top left in each plot). Dilutions of the secondary antibodies used (anti-rb-Alexa 405 (blue) and anti-gp-CF405S (green)) in Vectashield mounting medium yield fluorescent intensities proportional to their concentrations under the established imaging conditions. Mean intensities were extracted using the Histogram function in ImageJ on the whole imaging field of a mean projection. The background fluorescence was measured by imaging a slide only containing Vectashield and subtracted from the mean values. Dotted lines indicate linear fits.

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Harmansa, S., Hamaratoglu, F., Affolter, M. et al. Dpp spreading is required for medial but not for lateral wing disc growth. Nature 527, 317–322 (2015). https://doi.org/10.1038/nature15712

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