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Dpp from the anterior stripe of cells is crucial for the growth of the Drosophila wing disc

  1. Shinya Matsuda  Is a corresponding author
  2. Markus Affolter  Is a corresponding author
  1. Biozentrum der Universität Basel, Switzerland
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Cite as: eLife 2017;6:e22319 doi: 10.7554/eLife.22319

Abstract

The Dpp morphogen gradient derived from the anterior stripe of cells is thought to control growth and patterning of the Drosophila wing disc. However, the spatial-temporal requirement of dpp for growth and patterning remained largely unknown. Recently, two studies re-addressed this question. By generating a conditional null allele, one study proposed that the dpp stripe is critical for patterning but not for growth (Akiyama and Gibson, 2015). In contrast, using a membrane-anchored nanobody to trap Dpp, the other study proposed that Dpp dispersal from the stripe is required for patterning and also for medial wing disc growth, at least in the posterior compartment (Harmansa et al., 2015). Thus, growth control by the Dpp morphogen gradient remains under debate. Here, by removing dpp from the stripe at different time points, we show that the dpp stripe source is indeed required for wing disc growth, also during third instar larval stages.

https://doi.org/10.7554/eLife.22319.001

eLife digest

From the wings of a butterfly to the fingers of a human hand, living tissues often have complex and intricate patterns. Developmental biologists have long been fascinated by the signals – called morphogens – that guide how these kinds of pattern develop. Morphogens are substances that are produced by groups of cells and spread to the rest of the tissue to form a gradient. Depending on where they sit along this gradient, cells in the tissue activate different sets of genes, and the resulting pattern of gene activity ultimately defines the position of the different parts of the tissue.

Decades worth of studies into how limbs develop in animals from mice to fruit flies have revealed common principles of morphogen gradients that regulate the development of tissue patterns. Morphogens have been shown to help regulate the growth of tissues in a number of different animals as well. However, how the morphogens regulate tissue size and what role their gradients play in this process remain topics of intense debate in the field of developmental biology.

In the developing wing of a fruit fly, a morphogen called Dpp is expressed in a thin stripe located in the center and spreads to the rest of the tissue to form a gradient. Matsuda and Affolter have now characterized where and when the Dpp morphogen must be produced to regulate both the final size of the fly’s wing and the number of cells the wing eventually contains. The experiments involved preventing the production of Dpp in the developing wing in specific cells and at specific stages of development. This approach confirmed that Dpp must be produced in the central stripe for the wing to grow. Bosch, Ziukaite, Alexandre et al. and, independently, Barrio and Milán report the same findings in two related studies, and also conclude that the gradient of Dpp throughout the wing is not required for growth.

Further work will be needed to explain how the Dpp signal regulates the growth of the wing. The answer to this question will contribute to a better understanding of the role of morphogens in regulating the size of human organs and how a failure to do so might cause developmental disorders.

https://doi.org/10.7554/eLife.22319.002

Introduction

Morphogens are thought to disperse and form concentration gradients to control tissue patterning and growth (Rogers and Schier, 2011). The Drosophila wing imaginal disc has served as an excellent model to study how morphogens control patterning and growth. It has been shown that decapentagplegic (dpp), a homologue of vertebrate bone morphogenetic protein 2/4 (BMP2/4), is expressed in a stripe of cells in the anterior compartment along the anterior-posterior compartmental boundary of the wing imaginal disc. From this source, Dpp protein is thought to spread and form a concentration gradient to control patterning and growth of the wing imaginal disc (Lecuit et al., 1996; Nellen et al., 1996; Matsuda et al., 2016; Affolter and Basler, 2007; Restrepo et al., 2014). However, the precise spatial-temporal requirement of dpp remained elusive, mainly because it was not possible to generate an inducible null allele of dpp due to the haploinsufficiency of the locus and the lack of appropriate methods. Recently, two papers addressed the role of the dpp source at the compartment boundary using independent strategies. Akiyama and Gibson used CRISPR-Cas9-mediated genome engineering techniques to insert a FRT (Flippase Recognition Target) cassette into the dpp locus, and successfully generated a conditional null allele of dpp using the expression of Flippase (FLP) in a spatial-temporal controlled manner (Akiyama and Gibson, 2015). By genetically removing dpp from the anterior stripe, they showed that the Dpp morphogen gradient derived from this stripe is indeed critical for patterning. However, and rather surprisingly, they also found that dpp from the stripe is largely dispensable for wing disc growth during the third instar larval stage. Instead, growth of the wing disc was compromised by genetically removing dpp from the entire anterior compartment. Based on the constant requirement of dpp derived from the anterior compartment for growth of the wing disc, Akiyama and Gibson proposed that a not-yet identified anterior dpp source outside the stripe of cells is required for wing disc growth (Akiyama and Gibson, 2015).

Harmansa et al. used a membrane-anchored anti-GFP nanobody (morphotrap) to trap GFP-Dpp and manipulate GFP-Dpp dispersal (Harmansa et al., 2015). Since an endogenously tagged GFP-Dpp strain was not available, dpp disc mutants were rescued by expressing GFP-Dpp in the stripe (Entchev et al., 2000; Teleman and Cohen, 2000) and morphotrap was concomitantly expressed in the stripe in order to trap GFP-Dpp and block its dispersal. In this setup, the authors confirmed that dpp is required for wing disc patterning, and also found that Dpp morphogen dispersal from the stripe of cells is required for medial but not for lateral wing disc growth in the posterior compartment (the region they analyzed). However, since these experiments were done under rescue conditions, other sources of dpp important for growth in wild type individuals could have been missed. Thus, while both studies confirmed a role of dpp on wing disc patterning, these studies propose different scenarios for the spatial requirement of dpp on wing disc growth, and it remains debated whether the Dpp morphogen gradient derived from the anterior stripe of cells is required for wing disc growth (Vincent et al., 2016; Strzyz, 2016). In this study, we first show that the dpp-Gal4 driver line used to remove dpp from the anterior stripe in the previous study (Akiyama and Gibson, 2015) does not faithfully reflect the endogenous dpp expression pattern during third instar larval stages. We therefore genetically removed dpp at different time points using a different Gal4 line (ptc-Gal4 line), which covers the anterior stripe of cells from the early larval stages onward. Using this setup, we demonstrate that dpp from the stripe of cells is indeed critical for growth of the wing disc, even during third instar larval stages. Furthermore, this result indicates that an anterior dpp source outside the stripe of cells, even if it would exist, would not be sufficient to drive growth of the wing disc.

Results and discussion

Akiyama and Gibson used a dpp-Gal4 driver line to genetically remove dpp from the anterior stripe. Based on the results they obtained, they proposed that the dpp stripe is not required for wing disc growth (Akiyama and Gibson, 2015). Although it appears straightforward to use dpp-Gal4 to remove dpp, this setup has intrinsic problems in removing dpp from the early onset of its expression. Since this setup requires the dpp disc enhancer to be activated, endogenous dpp is initially expressed before it can be removed by FLP/FRT recombination. Furthermore, since it takes about 18–24 hr to remove the engineered cassette in the dpp locus from the majority of cells in the anterior stripe and the wing disc grows dramatically during this time (Akiyama and Gibson, 2015) (Figure 1a), this delay may fail to reveal a potential early function of dpp on growth. In addition to this intrinsic problem, we found that dpp-Gal4 expression does not faithfully reflect the spatial-temporal endogenous dpp expression pattern until relatively late third instar stages (Figure 1a). Although dpp has been shown to be expressed in the entire anterior stripe of cells from the early third instar larval stages (Akiyama and Gibson, 2015), NLS-mCherry expressed under the control of dpp-Gal4 marked only the dorsal stripe of cells at the beginning of third instar larval stages (60 hr after egg laying (AEL) at 26°C) (Figure 1a). dpp-Gal4 was expressed in the entire stripe of cells only in relatively late third instar stages (Figure 1a). Most probably, the fragment of the dpp disc enhancer used to drive Gal4 does not cover all the cis-regulatory regions important for proper stripe expression.

Figure 1 with 1 supplement see all
Comparison of dpp-Gal4 and ptc-Gal4 expression pattern with Dpp expression in the Drosophila wing disc.

(a) Temporal expression pattern of dpp-Gal4 (dppFO/+; dpp-Gal4/UAS-NLS-mCherry) (b) temporal expression pattern of ptc-Gal4 (dppFO, ptc-Gal4/+; UAS-NLS-mCherry). Single confocal images except 50.5–52.5 hr AEL by maximum intensity projection. (c, d) Comparison of anti-Dpp staining and dpp-Gal4 expression (NLS-mCherry) in the early (c) and late (d) third instar wing disc of a dppFO/+; dpp-Gal4/UAS-NLS-mCherry larva. (e, f) Comparison of anti-Dpp staining and ptc-Gal4 expression (NLS-mCherry) in the early (e) and late (f) third instar wing disc of a dppFO/+; ptc-Gal4/UAS-NLS-mCherry larva. Average intensity projection from 5 sequential confocal images. Scale bars 50 μm. Anterior is to the left in all figures.

https://doi.org/10.7554/eLife.22319.003

Therefore, to reinvestigate the role of the dpp stripe on growth, we decided to remove dpp by FLP/FRT recombination using a different Gal4 line expressed in this anterior stripe of cells. Since ptc, a Hedgehog target gene, is expressed in a stripe of cells similar to dpp, we first analyzed the spatial-temporal expression pattern of ptc-Gal4. We found that NLS-mCherry expressed under the control of ptc-Gal4 marked the entire stripe of cells as early as late second instar (50 hr AEL at 26°C) and that stripe expression continued throughout third instar larval stages (Figure 1b). We then compared the expression pattern of these two Gal4 lines with endogenous Dpp expression using a Dpp antibody that recognizes the prodomain of Dpp (Akiyama and Gibson, 2015) (Figure 1c–f). Consistent with the above observations, dpp-Gal4 expression did not cover the ventral Dpp stripe in the early third instar larval stages and covered the entire stripe only in late third instar larval stages (Figure 1c,d). In contrast, ptc-Gal4 expression continuously covered the Dpp stripe from the early third instar larval stages onward (Figure 1e,f). To ascertain the validity of using NLS-mCherry to mark ptc-Gal4 or dpp-Gal4 expression, which might be somewhat compromised due to the lengthy maturation time for the mCherry protein, we marked Gal4 expression using α-mCherry antibody and found that fluorescent signal and antibody signal overlap well (Figure 1—figure supplement 1). Taken together, these results show that spatial-temporal ptc-Gal4 expression reflects endogenous dpp expression pattern in the wing disc more precisely than dpp-Gal4.

We then removed dpp using dpp-Gal4 or ptc-Gal4 and compared their effects on wing disc growth (CRISPR-Cas9-modified flies (dppFO) were generously provided by Akiyama and Gibson). When dpp was removed using dpp-Gal4, pMad was not detectable in the wing pouch but the wing disc grew normally, as reported in Akiyama and Gibson (Figure 2a,b) (Akiyama and Gibson, 2015). In sharp contrast, when dpp was removed using ptc-Gal4, the pMad signal was also lost from the wing pouch (except for the future alula region), and wing disc growth was severely affected (Figure 2c). Wing pouches were very small and often hardly visible, as shown by the lack of internal ring expression of Wg (Figure 2c). We confirmed that Dpp expression was lost from the stripe of anterior cells but remained detectable in the future alular region (Figure 2d), consistent with the pMad signal in this region (Figure 2c). Accordingly, the growth repressor Brk, normally repressed by pMad, was uniformly expressed in the wing pouch (Figure 2e).

Defects in wing disc growth by removing dpp using ptc-Gal4.

(a–c) Anti-pMad and anti-ptc/wg staining in a dppFO/+; UAS-FLP/+ (control) late third instar wing disc (a), in a dppFO/dppFO; dpp-Gal4/UAS-FLP late third instar wing disc (b), and in a dppFO, ptc-Gal4/dppFO; UAS-FLP/+ late third instar wing disc (c). (d) anti-Dpp staining in a dppFO, ptc-Gal4/+; UAS-FLP/+ (control) late third instar wing disc (left), and in a dppFO, ptc-Gal4/dppFO; UAS-FLP/+ late third instar wing disc (right). (e) anti-Brk staining in a dppFO, ptc-Gal4/+; UAS-FLP/+ (control) late third instar wing disc (left), and in a dppFO, ptc-Gal4/dppFO; UAS-FLP/+ late third instar wing disc (right). (*) marks the future alula region. (a–e) Average intensity projection from 5 sequential confocal images. (f, h) an experimental setup to test the efficiency of FLP/FRT mediated recombination. (g) anti-β-gal staining in a dppFO, ptc-Ga4/+; UAS-FLP/act5C(-FRT)lacZ late third instar wing disc (control). (i) anti β-gal staining in a dppFO, ptc-Gal4/dppFO;UAS-FLP/act5C(-FRT)lacZ late third instar wing disc. (g, i) A single confocal image. Scale bars 50 μm. Anterior is to the left in all figures.

https://doi.org/10.7554/eLife.22319.005

The above results show that wing disc growth is severely affected by removing dpp using ptc-gal4. To test where FLP/FRT recombination was driven by ptc-Gal4, we then marked cell lineages of ptc-Gal4 (Figure 2f–i). Since there is no lineage separation within the anterior compartment, the temporal Gal4 expression pattern does not necessarily reflect the actual domain, in which FLP/FRT-mediated recombination occurred. For example, cells that leave the stripe in the early stages might not express NLS-mCherry anymore at later stages, but could have excised dpp when they were located in the stripe in earlier stages. Alternatively, Gal4 may be transiently expressed outside the stripe in the early stages and be sufficient to excise dpp there. We found that when one copy of dpp was removed using ptc-Gal4 (control), the entire anterior wing pouch was marked in some wing discs, and only a stripe of anterior cells was marked in other wing discs (Figure 2f,g). Similar cell lineages were also observed when two copies of dpp were removed using ptc-Gal4 (Figure 2h,i). The variations in these ptc-Gal4 lineages observed may be due to slight differences in Gal4 or FLP expression or a degree of randomness in the excision events in each wing disc. Importantly, the wing discs where the ptc-Gal4 lineages were restricted to a stripe of cells showed drastic growth defects (Figure 2i), raising a possibility that dpp derived from the anterior stripe is critical for wing disc growth.

However, since ptc-Gal4 is expressed earlier than dpp-Gal4 (Figure 1), the severe growth defects by ptc-Gal4 may simply reflect an early role of dpp stripe on wing disc growth. Furthermore, since different FRT cassettes can have different sensitivities to FLPase, the FRT cassette in dpp locus and the FRT cassette to follow the ptc-Gal4 lineage could be excised in different regions. For example, the FRT cassette in the dpp locus may be excised outside the anterior stripe, although the excisions of the FRT cassette to follow the ptc-Gal4 lineages are restricted to the anterior stripe. Thus, the growth defect by ptc-Gal4 may be due to elimination of dpp in the early stages, and/or elimination of potential dpp source outside the anterior stripe that may drives wing disc growth.

To investigate the temporal requirement of the dpp stripe for wing disc growth, we therefore used the Gal80ts system (Figure 3a). Gal80ts represses Gal4 activity at 17°C and can be inactivated at 29°C. Thus, ptc-Gal4 can be conditionally activated upon a temperature shift. Indeed, we found that at the permissive temperature dppFO, ptc-Gal4 / dppFO; tub-Gal80ts / UAS-FLP adult wing had no obvious wing phenotype, suggesting that Gal80ts effectively suppresses ptc-Gal4 activity at 17°C (Figure 3—figure supplement 1).

Figure 3 with 4 supplements see all
dpp stripe is required for wing disc growth during second and third instar larval stages.

(a) A scheme to genetically remove dpp from the second or third instar larval stages using Gal80ts system. At 17°C, Gal4 activity is blocked by Gal80ts. At 29°C, Gal80ts is inactivated and Gal4 starts to induce FLP expression in the anterior stripe of cells. After embryo collection for 2–4 hr at room temperature, the embryos were incubated at 17°C until temperature shift. Temperature was shifted after 4 days (second instar) or 6 days (third instar) at 17°C, and late third instar wing discs were dissected after 53 hr or 43 hr later respectively. (b–e) Removal of dpp using ptc-Gal4 during second instar larval stages (b, c) or third instar larval stages (d, e). (b, d) anti-pMad staining and anti-β-gal staining (lineage tracing) in a control male wing disc (dppFO, ptc-Gal4/CyO; tub-Gal80ts/UAS-FLP, act5C(-FRT)lacZ) (left), and a male wing disc removing dpp during the specified time point (dppFO, ptc-Gal4/dppFO; tub-Gal80ts/UAS-FLP, act5C(-FRT)lacZ) (right). (c, e) anti-Dpp staining in a control male wing disc (dppFO, ptc-Gal4/+; tub-Gal80ts/ +) (left), and a male wing disc removing dpp during the specified time point (dppFO, ptc-Gal4/dppFO; tub-Gal80ts/UAS-FLP) (right). (b’–e’) Quantification of the wing disc size of (b–e). Mean ± s.d. p<0.001 by two sided Student’s t-test. Scale bars 100 μm.

https://doi.org/10.7554/eLife.22319.006

We first marked lineages of ptc-Gal4 when FLP/FRT-mediated recombination was temporally activated at the beginning of the second or third instar larval stage using Gal80ts. We found that ptc-Gal4 lineages were strictly restricted to the anterior stripe in control and mutant wing disc (Figure 3b,d). Thus, the more random lineages shown in Figure 2g,i appear to be derived from early expression of the ptc-Gal4 driver. To directly monitor where dpp is removed by conditionally activating ptc-Gal4, we utilized the dppFO-GFP allele originally generated as an intermediate allele to generate the final dppFO allele (Akiyama and Gibson, 2015). dppFO-GFP contains the same FRT cassette as dppFO and a ubiquitously expressed GFP construct (ubi-GFP) within the cassette (Figure 3—figure supplement 2). Thus, the regions in which dpp is excised will lack the GFP signal. Using this setup, we found that FLP/FRT-mediated excision in the dpp locus was indeed restricted to the anterior stripe from second and from third instar larval stages although the excision varied within the stripe (21/21 and 14/14 wing discs, respectively) (Figure 3—figure supplement 2). This result also strongly suggests that the temporal lineages of ptc-Gal4 indeed reflect the actual region where dpp is removed in this setup (Figure 3b,d).

By removing dpp during either second or third instar larval stages, we found that wing disc growth was severely affected in both cases, as measured in the late third instar larval stages (53 hr or 43 hr later after temperature shift, respectively) (Figure 3b’–e’). The majority of Dpp was eliminated around 20 ~ 24 hr after temperature shift as also reported by Akiyama and Gibson (Akiyama and Gibson, 2015) (Figure 3—figure supplement 3) and wing disc appeared to grow at least until 24 hr after temperature shift. Thus, the growth defects resulting from temperature shifting at the beginning of third instar larval stages likely reflect the effects seen from the absence of dpp around mid-third instar larval stages. These results show that the dpp stripe is required for wing disc growth during second and even third instar larval stages.

Since we used the same dpp allele (dppFO) and UAS-FLP line as Akiyama and Gibson, the different growth defects observed should be due to the differences between the dpp-Gal4 and ptc-Gal4 driver lines. We showed that while ptc-Gal4 is constantly expressed at the anterior stripe during third instar larval stages, dpp-Gal4 is initially expressed only at the dorsal stripe and only later, during third instar larval stages, in the entire stripe (Figure 1). Thus, the differences in the spatial-temporal expression may be responsible for the different phenotype observed when using dpp-Gal4 or ptc-Gal4. Interestingly, although both dpp-Gal4 and ptc-Gal4 are expressed in the dorsal stripe in the early third instar larval stages, the dorsal wing disc pouch still grew using dpp-Gal4 with minor growth defects (Figure 2b) (Akiyama and Gibson, 2015). To investigate why the dorsal compartment still grows when using dpp-Gal4, we analyzed Brk expression, the critical growth repressor repressed by Dpp signaling. (Figure 3—figure supplement 4). We found that at the mid third instar larval stage (80 hrAEL at 26°C), Brk was repressed in the ventral compartment where dpp was still expressed but was slightly upregulated in the dorsal compartment where the majority of dpp was removed (Figure 3—figure supplement 4). Interestingly, Brk upregulation in the dorsal compartment was not uniform but graded; lower in ventral and higher in dorsal regions within the dorsal compartment (Figure 3—figure supplement 4). At the late third instar larval stage, the majority of dpp was eliminated and Brk was upregulated in both dorsal and ventral compartment but again, Brk was not uniformly upregulated (Figure 3—figure supplement 4). Consistent with our findings, Omb has been shown to be weakly expressed in this setup (Akiyama and Gibson, 2015). These results suggest that the Dpp signal is not completely removed from the wing imaginal disc when using dpp-Gal4. The graded Brk expression in the dorsal compartment is consistent with weak Dpp signal derived from the ventral dpp stripe, and this lasting signal can explain the sustained growth there with minor growth defects. In contrast, when dpp was removed by ptc-Gal4 during the third instar larval stages, majority of dpp was removed from the entire anterior stripe (Figure 3—figure supplement 3) and wing disc growth was severely affected (Figure 3b–d).

Together, these results suggest that the critical role of the dpp stripe on wing disc growth was missed by Akiyama and Gibson due to imprecise spatial removal of dpp when using dpp-Gal4 during third instar larval stages (Akiyama and Gibson, 2015). Based on the constant requirement of dpp from the anterior compartment during third instar larval stages for proper wing disc growth, Akiyama and Gibson further proposed that a potential anterior dpp source outside the stripe was critical for wing disc growth (Akiyama and Gibson, 2015). However, our data show that wing disc growth was severely affected when dpp was removed only from the anterior stripe during third instar larval stages. Thus, the strong growth defects resulting from removing dpp from the entire anterior compartment are most likely due to excision of dpp from the anterior stripe and not due to the excision of the potential dpp outside the stripe.

In conclusion, our results establish that the anterior dpp stripe is critical for growth as well as patterning of the wing imaginal disc. Given the slow process of removing dpp by FLP/FRT mediated recombination (about 20–24 hr) compared to wing disc growth, it remains an open question whether the requirement of the dpp stripe on wing disc growth changes over time. It would be important to acutely manipulate the endogenous morphogen gradient at the protein level to address the precise temporal requirement of the dpp stripe on wing disc growth (Matsuda et al., 2016; Bieli et al., 2016).

Materials and methods

Fly stocks

Flies were kept in standard fly vials (containing polenta and yeast) in a 26°C incubator. The following fly lines were used: dppFO, dppFO-GFP, dpp-Gal4, and UAS-FLP (Matthew Gibson), UAS-NLS-mCherry (Caussinus et al., 2008), ptc-Gal4 (w*; P{GawB}ptc559.1), P{act5C(FRT.polyA)lacZ.nls1}3, ry506, tub-Gal80ts (Bloomington stock center).

Immunostainings and antibodies

Protocol was described previously (Harmansa et al., 2015). Each fly cross was set together with control and >10 wing imaginal discs from each genotype were processed in parallel. If the genotype could be distinguished, experimental and control samples were processed in the same tube. A representative wing disc was shown for all the experiments. Following primary antibodies were used; anti-Dpp (1:100; Matthew Gibson), anti-phospho-Smad1/5 (1:200; Cell Signaling, 9516S), anti-Brk (1:1000; Gines Morata), anti-Wg (1:120; DSHB, University of Iowa), anti-Ptc (1:40; DSHB, University of Iowa), anti-β-Galactosidase (1:1000; Promega Z378A), anti-mCherry (1:5000; Nigg lab, University of Basel). All the primary antibodies except anti-Dpp antibody were diluted in 5% normal goat serum (NGS) (Sigma) in PBT (0.03% Triton X-100/PBS). Anti-Dpp antibody was diluted in 5% NGS in Can Get Signal Immunostain Solution B (TOYOBO). All secondary antibodies from the AlexaFluor series were used at 1:500 dilutions. Wing discs were mounted in Vectashield (H-1000, Vector Laboratories). Images of wing discs were obtained using a Leica TCS SP5 confocal microscope (section thickness 1 μm).

References

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Decision letter

  1. Utpal Banerjee
    Reviewing Editor; University of California, Los Angeles, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Dpp from the anterior stripe of cells is crucial for the growth of the Drosophila wing disc" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. We hope you will be able to submit the revised version within two months.

Summary:

A contentious item that continues to raise interest concerns the relationship between the gradient of the BMP4-like signaling protein Dpp produced in Drosophila wing discs and cell proliferation in the disc. Dpp is both necessary and sufficient for disc growth, and the problem basically boils down to why regions with different levels of Dpp and BMP signaling do not cause different amounts of growth. Evidence and arguments on this point remain of high current interest.

The present manuscript is a partial rebuttal to the 2015 Nature paper from Akiyama and Gibson, which used various types of dpp loss-of-function clones to argue that the BMP Dpp produced by the stripe of cells anterior to the A/P boundary in wing discs was not necessary for the growth of the disc. This argued that models based on reading a gradient of Dpp were likely wrong, and that levels could be greatly reduced without greatly affecting growth, and thus models based on a temporal gradient of increasing BMP signaling were probably wrong as well. Nonetheless, that study showed that the Dpp produced by the entire anterior compartment was necessary for growth, at least up until 36 hours before wandering third instar, presumably this was supplied by low-level Dpp produced outside the normal stripe of high level Dpp expression.

The present manuscript argues that stripe Dpp is necessary for growth, using the same conditional dpp allele used by Akiyama, but a different (ptc) Gal4 driver that the authors show covers more of the stripe at earlier stages, especially in the ventral pouch.

Essential revisions:

Reviewer 1:

1) Since Akiyama already showed that removal from the entire anterior compartment reduces growth, the claim in the present study rests entirely on whether ptc-gal4 drives the excision of the conditional dpp allele in the stripe, or whether it drives excision more widely in the disc. This is a real worry, because the endogenous ptc gene is expressed at low levels throughout the anterior compartment. And as the authors (and others previously) show, ptc-gal4 can drive excision of a G-TRACE maker throughout the anterior as well.

Unfortunately, there is no direct way of telling where the dpp allele has been recombined. The conditional allele contains no marker of excision, and loss of dpp itself cannot be detected except where dpp expression is very strong, as Akiyama did.

The authors argue that there are some discs where ptc-gal4-driven G-TRACE is not excised throughout the anterior, and that therefore these discs must also be those where the conditional dpp allele is not recombined throughout the anterior. However, this makes the unwarranted assumption that the dppFO and G-TRACE are identically sensitive to FLPase. In my experience, different flpout constructs show different sensitivities. The authors also seem to be implying that these are disc to disc differences in cell "lineage", but I doubt greatly whether stripe cells ever give rise to far anterior cells. Rather, the variation likely to be due to slight differences in Gal4 or FLPase expression, or simply a level of randomness in the excision events. In my hands, G-TRACE from Bloomington and ptc-Gal4 can even give rise to expression in the far posterior compartment, cells that have certainly not descended from anterior compartment stripe cells.

I cannot think of a way around this problem, short of building a new excision allele with a marker in the excised DNA. I am open to counter-arguments, but without something I cannot accept the authors' interpretation.

2) One difficulty is that Akiyama show that their dpp-gal4 technique removes most or all stripe Dpp from the dorsal wing pouch and hinge, and also greatly reduces pMad there, as early as 72 hours AEL (their Figure 3). Nonetheless, the dorsal pouch reaches a pretty normal-looking size by late third (although they did not measure pouch size alone, so it is possible there was a slight defect). If this is correct, then loss of the gradient and stripe do not affect growth from 72 hours on.

Either the authors need to disprove this, or they have to incorporate it into their discussion. Does the Akiyama allele version of the experiment lead to loss of Dpp and the pMad gradient in parts of the disc at 72 hours, and is growth in those regions affected or unaffected?

If Akiyama is correct, this should be mentioned. One possible explanation is that the authors might investigate is that the early pMad loss was not enough to increase brinker expression at early time points, as Akiyama only examined brinker at late third. Since the authors have Akiyama's allele, could they look? My thinking here is that the different results might not be due to whether stripe Dpp is lost, per se, but how much residual Dpp signaling is left from Dpp elsewhere in the disc, and whether that residual signaling is enough to suppress brinker expression during the growth phase.

3) In the Gal80ts experiment, the authors also need to show a control wing that is homozygous for the dppFO allele, but reared continuously at 18°C.

Reviewer 2:

1) Figure 2 is a single addition to the GAL4 drivers explored in Extended Data 6 by Akiyama & Gibson, 2015; Figure 3 is an extension of Extended Data 4e of Akiyama & Gibson, 2015; Figure 4 is an extension of Extended Data 7 and lacks the temporal resolution for the Gal80ts experiment as well. There are no experiments to independently substantiate their claim of requirement of central stripe of Dpp for growth.

2) Usage of mCherry: NLS as a probe to study the activity of GAL4 domains: It has been demonstrated that GFP matures in about 5-20 mins, while mCherry matures in about 40-80 mins at 37°C. Slow maturation of mCherry is a property used in design of fluorescent timers (Khmelinskii et al., 2012). Maturation of mcherry is further slower at lower temperatures (~20-25°C). The kinetics of the marker (production/degradation) is crucial for marking the boundaries of GAL4s. Therefore, another probe should be used to confidently determine the boundaries of dpp-GAL4/ ptc-GAL4 domain. Ideally as Dpp is a secretory protein, the domain comparison should be studied with respect to Dpp mRNA and not protein.

3) Evans et al., 2009 using GTRACE show that the ptc-GAL4 marks the entire anterior compartment while dpp-GAL4 only marks a portion of anterior compartment although both show a similar real-time expression profile along the A/P boundary. It is important to "quantify" the variation observed in GTRACE to confidently negate the following possibility: Is dpp from the set of cells excluded by dpp-GAL4 lineage but included in ptc-GAL4 lineage (and ci GAL4) in the anterior compartment important for growth?

Reviewer 3:

The requirement of Dpp signaling for growth of the Drosophila wing has been a subject of much debate recently. Despite a large amount of evidence showing that Dpp is required for wing growth, and that it acts by repressing the growth-repressor Brinker, recent work has suggested that Dpp is only required for growth early in wing development, and not later on (as of mid-third instar). The manuscript here by Matsuda and Affolter revisits this issue. They show that Dpp is indeed required for wing disc growth, providing a technical explanation for the lack of a growth effect seen in Akiyama and Gibson 2015. In terms of temporal requirements, they show that Dpp is required as of early L3 for disc growth (although this was not really debated). In terms of spatial requirement, they find that discs where Dpp was removed only from the medial expression stripe display growth defects, indicating that the medial expression domain of Dpp is needed to support wing growth. Overall, the data quality are good. The manuscript analyzes the issue less in depth than the two other manuscripts that were co-submitted.

From Akiyama and Gibson, there is little debate whether Dpp is needed for growth at early 3rd instar. The debate is whether it is needed later on, as of 96h AEL (mid 3rd instar) (see Figure 2m of Akiyama and Gibson, where the reduction in size at 72h is by 50% and statistically significant, whereas the reduction in size at 96h is not significant). Hence Figure 4 presented here is not very novel, but instead should be repeated at 96h AEL.

https://doi.org/10.7554/eLife.22319.012

Author response

Essential revisions:

Reviewer 1:

1) Since Akiyama already showed that removal from the entire anterior compartment reduces growth, the claim in the present study rests entirely on whether ptc-gal4 drives the excision of the conditional dpp allele in the stripe, or whether it drives excision more widely in the disc. This is a real worry, because the endogenous ptc gene is expressed at low levels throughout the anterior compartment. And as the authors (and others previously) show, ptc-gal4 can drive excision of a G-TRACE maker throughout the anterior as well.

Unfortunately, there is no direct way of telling where the dpp allele has been recombined. The conditional allele contains no marker of excision, and loss of dpp itself cannot be detected except where dpp expression is very strong, as Akiyama did.

The authors argue that there are some discs where ptc-gal4-driven G-TRACE is not excised throughout the anterior, and that therefore these discs must also be those where the conditional dpp allele is not recombined throughout the anterior. However, this makes the unwarranted assumption that the dppFO and G-TRACE are identically sensitive to FLPase. In my experience, different flpout constructs show different sensitivities. The authors also seem to be implying that these are disc to disc differences in cell "lineage", but I doubt greatly whether stripe cells ever give rise to far anterior cells. Rather, the variation likely to be due to slight differences in Gal4 or FLPase expression, or simply a level of randomness in the excision events. In my hands, G-TRACE from Bloomington and ptc-Gal4 can even give rise to expression in the far posterior compartment, cells that have certainly not descended from anterior compartment stripe cells.

I cannot think of a way around this problem, short of building a new excision allele with a marker in the excised DNA. I am open to counter-arguments, but without something I cannot accept the authors' interpretation.

We agree with the reviewer that our setup (and also Akiyama and Gibson’s setup) is based on the unwarranted assumption that the dppFO allele and G-TRACE are identically sensitive to FLPase, and that random lineage of ptc-gal4-expressing cells (Figure 3 in old version, Figure 2 in new version) outside the stripe raises a serious worry on whether dpp is removed only from the stripe.

Considering the randomness of the ptc-gal4 lineages, we performed temporal ptc-Gal4 lineage experiments from second or from third instar larval stages using Gal80ts. We found that in both cases, the ptc-Gal4 lineage was strictly confined to a stripe of cells abutting the anterior-posterior compartment boundary, and that wing disc growth was severely affected (Figure 3). Thus, we focus on the role of dpp on wing disc growth from second or from third instar larval stages using such temporally staged larvae.

Nevertheless, and as the reviewer suggested, the ideal allele to directly monitor where dpp is removed would be a dpp excision allele with a marker in the excised DNA. We found such an allele (dppFO-GFP) originally generated as an intermediate allele to generate the final dppFO allele (Akiyama and Gibson, 2015). dppFO-GFP contains the same FRT cassette as dppFO and a ubiquitously expressed GFP construct (ubi-GFP) within the cassette (Figure 3—figure supplement 2). Thus, the regions in which dpp is excised will lack the GFP signal. Using this setup, we found that FLP/FRT mediated excision in the dpp locus was indeed restricted to the anterior stripe from second and from third instar larval stages (Figure 3—figure supplement 2). This result also strongly suggests that the temporal lineages of ptc-Gal4 indeed reflect the actual region where dpp is removed in this setup (Figure 3B, D).

“Rather, the variation likely to be due to slight differences in Gal4 or FLPase expression, or simply a level of randomness in the excision events.”

We agree that the variation may be due to slight differences in Gal4 or FLPase expression, or simply a level of randomness in the excision events between different wing discs. Based on the temporal lineage experiments (Figure 3) (Figure 3—figure supplement 2), the variation is clearly derived from activation earlier than second instar larval stages. We now discuss these possibilities in our manuscript and the new temporal lineage results we obtained (Figure 3) (Figure 3—figure supplement 2).

2) One difficulty is that Akiyama show that their dpp-gal4 technique removes most or all stripe Dpp from the dorsal wing pouch and hinge, and also greatly reduces pMad there, as early as 72 hours AEL (their Figure 3). Nonetheless, the dorsal pouch reaches a pretty normal-looking size by late third (although they did not measure pouch size alone, so it is possible there was a slight defect). If this is correct, then loss of the gradient and stripe do not affect growth from 72 hours on.

Either the authors need to disprove this, or they have to incorporate it into their discussion. Does the Akiyama allele version of the experiment lead to loss of Dpp and the pMad gradient in parts of the disc at 72 hours, and is growth in those regions affected or unaffected?

If Akiyama is correct, this should be mentioned. One possible explanation is that the authors might investigate is that the early pMad loss was not enough to increase brinker expression at early time points, as Akiyama only examined brinker at late third. Since the authors have Akiyama's allele, could they look? My thinking here is that the different results might not be due to whether stripe Dpp is lost, per se, but how much residual Dpp signaling is left from Dpp elsewhere in the disc, and whether that residual signaling is enough to suppress brinker expression during the growth phase.

We agree that by removing dpp using dpp-gal4, Dpp protein and pMad signal was greatly reduced from the dorsal compartment during third instar larval stages, but that dorsal wing disc pouch still grew with minor growth defects (Akiyama and Gibson, 2015) (Figure 2 in this study). We incorporated this point in the discussion to explain the different growth phenotype caused by using either dpp-Gal4 or ptc-Gal4.

To ask why the dorsal compartment can still grow when using dpp-Gal4 despite reduced pMad and Dpp levels there, we analyzed Brk expression. We found that Brk was repressed in the ventral compartment where dpp was still present but slightly upregulated in the dorsal compartment where the majority of dpp was removed at the mid third instar larval stage (80hrAEL at 26°C) (Figure 3—figure supplement 4). Interestingly, the Brk upregulation in the dorsal compartment is not uniform but graded; it is lower in the ventral and higher in the dorsal part within the dorsal compartment (Figure 3—figure supplement 4). At the late third instar larval stage, the majority of dpp was eliminated and Brk was upregulated in both dorsal and ventral compartment, but again, Brk was not uniformly upregulated (Figure 3—figure supplement 4). Consistent with this finding, Omb has been shown to be weakly expressed in this setup (Akiyama and Gibson, 2015). These results suggest that the Dpp signal is not completely removed from the wing imaginal disc using dpp-Gal4. The graded Brk expression in the dorsal compartment is consistent with weak Dpp signal mediated by the ventral dpp stripe, and the lasting signal can explain the sustained growth there. In contrast, when dpp was removed by ptc-Gal4 during the third instar larval stages using Gal80ts, the majority of dpp was removed from the entire anterior stripe (Figure 3—figure supplement 3), and we indeed found severe growth defects (Figure 3).

Together, these results suggest that Akiyama and Gibson missed the critical role of the dpp stripe on wing disc growth due to the imprecise spatial removal of dpp using dpp-Gal4 during third instar larval stages. Based on the finding that there is a constant requirement of dpp for proper wing disc growth from the anterior compartment during third instar larval stages, Akiyama and Gibson proposed that a potential anterior dpp source outside the stripe was critical for wing disc growth (Akiyama and Gibson, 2015). However, the growth defects they observed are most likely due to the excision of dpp from the stripe.

3) In the Gal80ts experiment, the authors also need to show a control wing that is homozygous for the dppFO allele, but reared continuously at 18°C.

We performed the control experiment and found that a control wing (dppFO, ptc-Gal4 / dppFO; tub-Gal80ts / UAS-FLP) has no obvious phenotype at 17 °C (Figure 3—figure supplement 1), suggesting that Gal80ts effectively suppresses Gal4 activity at 17 °C.

Reviewer 2:

1) Figure 2 is a single addition to the GAL4 drivers explored in Extended Data 6 by Akiyama & Gibson, 2015; Figure 3 is an extension of Extended Data 4e of Akiyama & Gibson, 2015; Figure 4 is an extension of Extended Data 7 and lacks the temporal resolution for the Gal80ts experiment as well. There are no experiments to independently substantiate their claim of requirement of central stripe of Dpp for growth.

Although both ptc-Gal4 and dpp-gal4 are intensively used in the field and known to be expressed in a stripe of anterior cells, Akiyama and Gibson used only dpp-Gal4 to propose that the dpp stripe is not required for wing disc growth during third instar larval stages.

We followed up this question by using the same setup but a different diver line, namely ptc-Gal4. Contrary to their results, we found that the dpp stripe was indeed required for wing disc growth during third instar larval stages (Figure 3 in new version). Since we used an experimental setup as close as possible to the one used by Akiyama and Gibson, we can conclude that the different results are not due to different experimental setups but due to different driver lines (dpp-Gal4 vs ptc-Gal4).

“lacks the temporal resolution for the Gal80ts experiment”

Following the reviewer’s comment, we performed the temporal Gal80ts experiments to ask when dpp is eliminated from the stripe after a temperature shift. We found that, consistent with Akiyama and Gibson, the majority of dpp is removed from the entire stripe around 20 hr after temperature shift (Figure 3—figure supplement 3).

2) Usage of mCherry: NLS as a probe to study the activity of GAL4 domains: It has been demonstrated that GFP matures in about 5-20 mins, while mCherry matures in about 40-80 mins at 37°C. Slow maturation of mCherry is a property used in design of fluorescent timers (Khmelinskii et al., 2012). Maturation of mcherry is further slower at lower temperatures (~20-25°C). The kinetics of the marker (production/degradation) is crucial for marking the boundaries of GAL4s. Therefore, another probe should be used to confidently determine the boundaries of dpp-GAL4/ ptc-GAL4 domain.

To avoid maturation problem of a fluorescent protein, we compared mCherry fluorescent signal with anti-mCherry antibody staining (since maturation is dependent on oxidation within the FP structure and epitope recognition by antibody is independent on the mCherry maturation). We found that the mCherry fluorescent signal nicely overlaps with antibody staining in the early and late third instar larval stages (Figure 1—figure supplement 1).

Nevertheless, the critical setup to determine the boundaries of ptc-Gal4 relevant to our study would be to determine where FLP/FRT recombination occurs under the control of ptc-Gal4. As we discussed above, using the dppFO-GFP allele, we also confirmed that dpp was indeed excised only from the anterior stripe when FLP/FRT recombination was induced in second or third instar larval stages (Figure 3—figure supplement 2).

Ideally as Dpp is a secretory protein, the domain comparison should be studied with respect to Dpp mRNA and not protein.

The Dpp antibody has been shown to recognize only Dpp prodomain but not mature ligands (Akiyama and Gibson, 2015). Thus, we used the Dpp prodomain antibody to compare the domains.

3) Evans et al., 2009 using GTRACE show that the ptc-GAL4 marks the entire anterior compartment while dpp-GAL4 only marks a portion of anterior compartment although both show a similar real-time expression profile along the A/P boundary. It is important to "quantify" the variation observed in GTRACE to confidently negate the following possibility: Is dpp from the set of cells excluded by dpp-GAL4 lineage but included in ptc-GAL4 lineage (and ci GAL4) in the anterior compartment important for growth?

As we discussed above, we found that the temporal ptc-gal4 lineages induced during second or third instar larval stages was strictly restricted to the anterior stripe of cells (Figure 3). Using the dppFO-GFP allele, we also confirmed that dpp was indeed excised only from the anterior stripe when FLP/FRT recombination was induced in second or third instar larval stages (Figure 3—figure supplement 2). Thus, we focused on the role of the dpp stripe on growth from second or from third instar larval stages.

Reviewer 3:

The requirement of Dpp signaling for growth of the Drosophila wing has been a subject of much debate recently. Despite a large amount of evidence showing that Dpp is required for wing growth, and that it acts by repressing the growth-repressor Brinker, recent work has suggested that Dpp is only required for growth early in wing development, and not later on (as of mid-third instar). The manuscript here by Matsuda and Affolter revisits this issue. They show that Dpp is indeed required for wing disc growth, providing a technical explanation for the lack of a growth effect seen in Akiyama and Gibson 2015. In terms of temporal requirements, they show that Dpp is required as of early L3 for disc growth (although this was not really debated). In terms of spatial requirement, they find that discs where Dpp was removed only from the medial expression stripe display growth defects, indicating that the medial expression domain of Dpp is needed to support wing growth. Overall, the data quality are good. The manuscript analyzes the issue less in depth than the two other manuscripts that were co-submitted.

From Akiyama and Gibson, there is little debate whether Dpp is needed for growth at early 3rd instar. The debate is whether it is needed later on, as of 96h AEL (mid 3rd instar) (see Figure 2m of Akiyama and Gibson, where the reduction in size at 72h is by 50% and statistically significant, whereas the reduction in size at 96h is not significant). Hence Figure 4 presented here is not very novel, but instead should be repeated at 96h AEL.

In Figure 2M, Akiyama and Gibson claim that the reduction in wing disc size is significant when removing dpp using hs-Flp at 96 AEL (Akiyama and Gibson, 2015). Together with the temporal ci-Gal4 experiments, they concluded that dpp is constantly required for wing disc growth during third instar larval. However, based on the results of the dpp-Gal4 experiments, they claimed that the dpp stripe is not required for wing disc growth during third instar larval stages, and propose a role for a potential anterior dpp source outside of the stripe region.

Since we used the same setup (dppFO) as Akiyama and Gibson and temporal resolution (~20hr to remove dpp) is similar, we do not challenge the temporal requirement of dpp on growth during third instar larval stages. Thus, the debate here is whether the dpp stripe is indeed dispensable for wing disc growth, since Akiyama and Gibson used only dpp-gal4 to challenge the role of dpp stripe on wing disc growth. Furthermore, it remains unclear whether the dpp stripe is required for wing disc growth from these experiments. In our study, we used ptc-Gal4 to revisit the importance of the dpp stripe for growth. Using Gal80ts, we found that the majority of dpp was removed from the stripe around 20 hr after temperature shift (Figure 3—figure supplement 3), and that wing disc growth was severely affected at the late third instar larval stages (43hr later after temperature shift) (Figure 3). Since wing discs can grow for 24 hr until the majority of dpp is removed from the stripe (Figure 3—figure supplement 3), the growth defects that we observed likely reflect the absence of dpp from mid-third instar larval stage. Our result thus clearly demonstrates that the dpp stripe is indeed critical for wing disc growth during third instar larval stages.

Given the slow process of removing dpp by FLP/FRT mediated recombination compared to wing disc growth, it remains an open question whether the requirement of the dpp stripe on wing disc growth changes over time. It would be important in the future to acutely manipulate the endogenous morphogen gradient at the protein level to investigate the precise temporal requirement of dpp stripe. We are presently tempting such experiments using the previously published morphtrap approach as well as other newly designed synthetic receptors capable to trap Dpp fusion protein or Dpp protein itself.

https://doi.org/10.7554/eLife.22319.013

Article and author information

Author details

  1. Shinya Matsuda

    Biozentrum der Universität Basel, Basel, Switzerland
    Contribution
    SM, Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    For correspondence
    shinya.matsuda@unibas.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-7541-7914
  2. Markus Affolter

    Biozentrum der Universität Basel, Basel, Switzerland
    Contribution
    MA, Supervision, Funding acquisition, Writing—review and editing
    For correspondence
    markus.affolter@unibas.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-5171-0016

Funding

Basel-Stadt

  • Markus Affolter

Basel-Land

  • Markus Affolter

Japan Society for the Promotion of Science (Postdoctoral Fellowship for Research Abroad)

  • Shinya Matsuda

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Akiyama and Gibson for flies; the Biozentrum Imaging Core Facility for maintenance of microscopes and support; the Developmental Studies Hybridoma Bank at The University of Iowa for antibodies; Dimitri ‘Chin-Chin’ Bieli and Gustavo Aguilar for discussion, sharing reagents, and comments on the manuscript. We would like to thank Bernadette Bruno, Gina Evora and Karin Mauro for constant and reliable supply with world’s best fly food. SM is supported by a JSPS Postdoctoral Fellowship for Research Abroad. The work in the laboratory was supported by grants from Cantons Basel-Stadt and Basel-Land and from the SNSF (MA).

Reviewing Editor

  1. Utpal Banerjee, University of California, Los Angeles, United States

Publication history

  1. Received: October 12, 2016
  2. Accepted: June 4, 2017
  3. Accepted Manuscript published: July 4, 2017 (version 1)
  4. Accepted Manuscript updated: July 5, 2017 (version 2)
  5. Version of Record published: August 17, 2017 (version 3)

Copyright

© 2017, Matsuda et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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