In situ mutational screening and CRISPR interference reveal that the apterous Early enhancer is required for developmental boundary positioning

Article activity feed

1. 

   eLife

   Jan 8, 2026

   eLife Assessment

   This important paper presents the discovery of the molecular basis of differential apterous expression during early Drosophila wing disc development. The evidence supporting these conclusions is compelling, ranging from classical genetic approaches to state-of-the-art genetic engineering techniques. By opening new questions, this paper is expected to be of broad interest to developmental biologists and geneticists working on transcriptional regulation.

   Read the original source
2. 

   eLife

   Jan 8, 2026

   Reviewer #1 (Public review):

   Summary:

   The Drosophila wing disc is an epithelial tissue which study has provided many insights into the genetic regulation of organ patterning and growth. One fundamental aspect of wing development is the positioning of the wing primordia, which occurs at the confluence of two developmental boundaries, the anterior-posterior and the dorsal-ventral. The dorsal-ventral boundary is determined by the domain of expression of the gene apterous, which is set early in the development of the wing disc. For this reason, the regulation of apterous expression is a fundamental aspect of wing formation.

   In this manuscript the authors used state of the art genomic engineering and a bottom-up approach to analyze the contribution of a 463 base pair fragment of apterous regulatory DNA. They find compelling evidence about …

   Reviewer #1 (Public review):

   Summary:

   The Drosophila wing disc is an epithelial tissue which study has provided many insights into the genetic regulation of organ patterning and growth. One fundamental aspect of wing development is the positioning of the wing primordia, which occurs at the confluence of two developmental boundaries, the anterior-posterior and the dorsal-ventral. The dorsal-ventral boundary is determined by the domain of expression of the gene apterous, which is set early in the development of the wing disc. For this reason, the regulation of apterous expression is a fundamental aspect of wing formation.

   In this manuscript the authors used state of the art genomic engineering and a bottom-up approach to analyze the contribution of a 463 base pair fragment of apterous regulatory DNA. They find compelling evidence about the inner structure of this regulatory DNA and the upstream transcription factors that likely bind to this DNA to regulate apterous early expression in the Drosophila wing disc.

   Strengths:

   This manuscript has several strengths concerning both the experimental techniques used to address a problem of gene regulation and the relevance of the subject. To identify the mode of operation of the 463 bp enhancer, the authors use a balanced combination of different experimental approaches. First, they use bioinformatic analysis (sequence conservation and identification of transcription factors binding sites) to identify individual modules within the 463 bp enhancer. Second, they identify the functional modules through genetic analysis by generating Drosophila strains with individual deletions. Each deletion is characterized by looking at the resulting adult phenotype and also by monitoring apterous expression in the mutant wing discs. They then use a clever method to interfere in a more dynamic manner with the function of the enhancer, by directing the expression of catalytically inactive Cas9 to specific regions of this DNA. Finally, they recur to a more classical genetic approach to uncover the relevance of candidate transcription factors, some of them previously know and other suggested by the bioinformatic analysis of the 463 bp sequence. This workflow is clearly reflected in the manuscript, and constitute a great example of how to proceed experimentally in the analysis of regulatory DNA.

   Weaknesses:

   The previously pointed weakness (vg expression, P compartment specific effects, early vs late analysis of ap expression in mutants) have been throughly and satisfactorily addressed by the authors.

   Read the original source
3. 

   eLife

   Jan 8, 2026

   Reviewer #3 (Public review):

   In this manuscript, authors use the Drosophila wing as model system and combine state-of-the-arte genetic engineering to identify and validate the molecular players mediating the activity of one of the cis-regulatory enhancers of the apterous gene involved in the regulation of its expression domain in the dorsal compartment of the wing primordium during larval development. The paper is subdivided into the following chapters/figures:

   (1) In the first couple of figures, authors describe the methodology to genetically manipulate the apE enhancer (a cartoon summarizing all the previous work with this enhancer might help) and identify two well-conserved domains in the OR463 enhancer required for wing development (the m3 region whose deletion phenocopies OR463 deletion: loss of wing, and the m1 region, whose …

   Reviewer #3 (Public review):

   In this manuscript, authors use the Drosophila wing as model system and combine state-of-the-arte genetic engineering to identify and validate the molecular players mediating the activity of one of the cis-regulatory enhancers of the apterous gene involved in the regulation of its expression domain in the dorsal compartment of the wing primordium during larval development. The paper is subdivided into the following chapters/figures:

   (1) In the first couple of figures, authors describe the methodology to genetically manipulate the apE enhancer (a cartoon summarizing all the previous work with this enhancer might help) and identify two well-conserved domains in the OR463 enhancer required for wing development (the m3 region whose deletion phenocopies OR463 deletion: loss of wing, and the m1 region, whose deletion gives rise to AP identify changes in the P compartment).

   (2) In the following three figures, authors characterize the m1 regulatory region, identify HOX and ETS binding sites, functionally validate their role in wing development and the activity of the genes/proteins regulating their activity (eg-. Hth and Pointed) by their ability to phenocopy (when depleted) the m1 loss of function wing phenotype. Authors conclude that Hth and Pointed regulate apterous expression through the m1 region.

   (3) In the last few figures, authors perform similar experiments with the m3 regulatory region to conclude that the Grn and Antennapedia regulate apterous expression through the m3 enhancer.

   My comments:

   Technically sound: As stated in my previous review, the work is technically excellent (authors use state-of-the-art genetic engineering to manipulate the enhancer and combine it with genetic analysis through RNAi and CRISPR/Cas9 and phenotypic characterization to functionally validate their findings), figures are nicely done and cartoons are self-explanatory.

   Poor paper writing: The paper is too long and difficult to read/understand, many grammatical mistakes are found, and formatting is in some cases heterodox.

   Science:

   (1) The question of "who is locating the relative position of the AP and DV boundaries in the developing wing?" is not resolved. I would then change the intro or reduce the tone of this question. Having said that, I agree that these results shed light on the wing phenotypes of some apterous alleles related to AP identify and growth and, as such, I congratulate the authors.

   (2) Identification of two TFs (Grain and Antp) mediating the regulation of apterous expression is interesting but some contextualization might be required. Data on Antp is not as convincing as data on Grn. I wonder whether Antp data can be removed at all.

   (3) I am not sure whether the term hemizygous is used properly

   Read the original source
4. 

   eLife

   Jan 8, 2026

   Author response:

   The following is the authors’ response to the original reviews.

   Public Reviews:

   Reviewer #1 (Public Review):

   Summary:

   The Drosophila wing disc is an epithelial tissue, the study of which has provided many insights into the genetic regulation of organ patterning and growth. One fundamental aspect of wing development is the positioning of the wing primordia, which occurs at the confluence of two developmental boundaries, the anterior-posterior and the dorsal-ventral. The dorsal-ventral boundary is determined by the domain of expression of the gene apterous, which is set early in the development of the wing disc. For this reason, the regulation of apterous expression is a fundamental aspect of wing formation.

   In this manuscript, the authors used state-of-the-art genomic engineering and a bottom-up approach to analyze the …

   Author response:

   The following is the authors’ response to the original reviews.

   Public Reviews:

   Reviewer #1 (Public Review):

   Summary:

   The Drosophila wing disc is an epithelial tissue, the study of which has provided many insights into the genetic regulation of organ patterning and growth. One fundamental aspect of wing development is the positioning of the wing primordia, which occurs at the confluence of two developmental boundaries, the anterior-posterior and the dorsal-ventral. The dorsal-ventral boundary is determined by the domain of expression of the gene apterous, which is set early in the development of the wing disc. For this reason, the regulation of apterous expression is a fundamental aspect of wing formation.

   In this manuscript, the authors used state-of-the-art genomic engineering and a bottom-up approach to analyze the contribution of a 463 base pair fragment of apterous regulatory DNA. They find compelling evidence about the inner structure of this regulatory DNA and the upstream transcription factors that likely bind to this DNA to regulate apterous early expression in the Drosophila wing disc.

   Strengths:

   This manuscript has several strengths concerning both the experimental techniques used to address the problem of gene regulation and the relevance of the subject. To identify the mode of operation of the 463 bp enhancer, the authors use a balanced combination of different experimental approaches. First, they use bioinformatic analysis (sequence conservation and identification of transcription factors binding sites) to identify individual modules within the 463 bp enhancer. Second, they identify the functional modules through genetic analysis by generating Drosophila strains with individual deletions. Each deletion is characterized by looking at the resulting adult phenotype and also by monitoring apterous expression in the mutant wing discs. They then use a clever method to interfere in a more dynamic manner with the function of the enhancer, by directing the expression of catalytically inactive Cas9 to specific regions of this DNA. Finally, they recur to a more classical genetic approach to uncover the relevance of candidate transcription factors, some of them previously known and others suggested by the bioinformatic analysis of the 463 bp sequence. This workflow is clearly reflected in the manuscript, and constitutes a great example of how to proceed experimentally in the analysis of regulatory DNA.

   We thank the reviewer for these positive comments on the manuscript.

   Weaknesses:

   There are several caveats with the data that might be constructed as weaknesses, some of them are intrinsic to this detailed analysis or to the experimental difficulties of dealing with the wing disc in its earliest stages, and others are more conceptual and are offered here in case the authors may wish to consider them.

   (1) The primordium of the wing region of the wing imaginal disc is defined by the expression of the gen vestigial, which is regulated by inputs coming from the dorsal-ventral boundary (Notch and wg) and from the anterior-posterior boundary (Dpp). Having such a principal role in wing primordium specification and expansion, I am surprised that this manuscript does not mention this gene in the main text and only contains indirect references to it. I consider that the manuscript would have benefited a lot by including vestigial in the analysis, at least as a marker of early wing primordium. This might allow us to visualize directly the positioning of the primordium in the apterous mutants generated in this study, adding more verisimilitude to the interpretations that place this domain based on indirect evidence.

   Vg does indeed play a critical role on the formation of the wing disc, and it is an ideal marker for the identification of the wing pouch. In the updated version of the article, we have now followed the expression of vg in some of the OR463 mutants via immunostaining of the Vg protein (Supplementary Figure 6). Cells within posterior wing outgrowths in Δm1flies were invariably positive for Vg. This result further supports our previous identification of these cells as pouch cells. In those mutants in which no cross-over between DV and AP was observed, vg expression was severely reduced or absent, indicating that the wing pouch had not been specified. We thank the reviewer for this experimental idea, which we believe strengthens the final manuscript.

   We have added to the text:

   “To identify the nature of the posterior outgrowths, we performed anti-Vestigal (Vg) antibody staining of Δm1 mutants (Supplementary Figure 6). Vg is a key regulator of wing specifications and also participates in wing growth and patterning (Baena-Lopez & García-Bellido, 2006; Kim et al., 1996; Zecca & Struhl, 2007a). In those discs, in which the stripe was extended and the P compartment was enlarged, Vg was detected throughout the outgrowth, supporting the wing pouch identity of this region (Supplementary Figure 6B). Hemizygous Δm3 mutants presented a highly reduced anti-Vg signal, which suggests that no wing pouch is specified in these mutants (Supplementary Figure 6C).”

   (2) The authors place some emphasis on the idea that their work addresses possible coordination between setting the D/V boundary and the A/P boundary:

   Abstract: "Thus, the correct establishment of ap expression pattern with respect to en must be tightly controlled", "...challenging the mechanism by which apE miss-regulation leads to AP defects." "Detailed mutational analyses using CRISPR/Cas revealed a role of apE in positioning the DV boundary with respect to the AP boundary"

   Introduction: "However, little is known about how the expression pattern of ap is set up with respect that of en. In other words, how is the DV boundary positioned with respect to the AP boundary?"

   "How such interaction between ap and the AP specification program arises is unknown."

   Results: "Some of these phenotypes are reminiscent of those reported for apBlot (Whittle, 1979) and point towards a yet undescribed crosstalk between ap early expression and the AP specification program."

   At the same time, they express the notion, with which this reviewer agrees, that all defects observed in A/P patterning arising as a result of apterous miss-regulation are due to the fact that in their mutants, apterous expression is lost mainly in the posterior dorsal compartment, bringing novel confrontations between the A/P and the D/V boundaries.

   To me, the key point is why the expression of apterous in different mutants of the OR463 enhancer affects only the posterior compartment. This should be discussed because it is far from obvious that apterous expression has different regulatory requirements in the anterior and posterior compartments.

   We agree with the reviewer that the differential effect of the mutations on the expression of ap in the A and P compartment is a key factor underlying our explanation of how the phenotypes arise. To clarify this point, we have now extended our first discussion point. Moreover, we have included some other references of differential enhancer regulation in different wing disc compartments. In addition, we have discussed whether this effect has to do with the different regulation of the enhancer in the A and P compartment or due to regulation of downstream effectors.

   Added paragraph:

   “Although apE is active throughout the dorsal compartment, its disruption leads to a preferential loss of ap expression in posterior cells. The asymmetric effect of apE perturbation on the anterior and posterior compartments suggests that apE transcriptional control is not equivalent across the A/P axis. Compartment-dependent differences in enhancer regulation have also been documented in other developmental contexts; for example, the Distal-less DMX-R element is interpreted through distinct cofactor combinations (Sloppy paired anteriorly and Engrailed posteriorly) (Gebelein et al., 2004), and specific mutations within DMX-R preferentially disrupt enhancer function in anterior versus posterior cells. It is possible that apE is more sensitive to misregulation due to differential transcriptional regulation across compartments. Nevertheless, we cannot exclude the possibility that the posterior bias we observe arises not from enhancer logic per se, but from intrinsic differences in tissue architecture or the dynamics of boundary positioning during wing disc development.”

   (3) The description of gene expression in the wing disc of novel apterous mutants is only carried out in late third instar discs (Figs. 2, 3, 5, and 7). This is understandable given the technical difficulties of dealing with early discs, as those shown in the analysis of candidate apterous regulatory transcription factors (Fig. 4F, Fig. 6 C-D). However, because the effects of the mutants on apterous expression are expected to occur much earlier than the time of expression analysis, this fact should be discussed.

   We agree with the reviewer regarding the limitations of our analysis whenever we analyzed third instar larvae to assess the expression of the OE463 enhancer. We have included a statement in which this is mentioned in the discussion:

   “It is important to acknowledge that all expression analyses were conducted in third-instar discs, a stage that follows the initial establishment of ap expression. Earlier effects are therefore inferred rather than directly observed, as imaging and staging of early discs present significant technical challenges due to their small size and fragility. A direct observation of the early wing disc across mutant conditions would likely help to clarify the role of the discovered factors during early ap expression.”

   Reviewer #2 (Public Review):

   In their manuscript, "Transcriptional control of compartmental boundary positioning during Drosophila wing development," Aguilar and colleagues do an exceptional job of exploring how tissue axes are established across Drosophila development. The authors perform a series of functional perturbations using mutational analyses at the native locus of apterous (ap), and perform tissue-specific enhancer disruption via dCas9 expression. This innovative approach allowed them to explore the spatio-temporal requirements of an apterous enhancer. Combining these techniques allowed the authors to explore the molecular basis of apterous expression, connecting the genotypes to the phenotypical effects of enhancer perturbations. To me, this paper was a beautiful example of what can be done using modern drosophila genetics to understand classic questions in developmental biology and transcriptional regulation.

   In sum, this was a rigorous paper bridging scales from the molecular to phenotypes, with new insight into how enhancers control compartmental boundary positioning during Drosophila wing development.

   We would like to thank the reviewer for its positive and encouraging comments, as well as for the careful review of the manuscript and figures. We have adapted most of the suggestions in the new manuscript.

   Reviewer #3 (Public Review):

   In this manuscript, authors use the Drosophila wing as a model system and combine state-ofthe-art genetic engineering to identify and validate the molecular players mediating the activity of one of the cis-regulatory enhancers of the apterous gene involved in the regulation of its expression domain in the dorsal compartment of the wing primordium during larval development.

   (1) The authors raise two very important questions in the Introduction: (1) who is locating the relative position of the AP and DV boundaries in the developing wing, and (2) who is responsible for the maintenance of the apterous expression domain late in larval development. None of these two questions have been responded to and, indeed, the summary of the work (as stated in the conclusions of the last paragraph of the Introduction) does not resolve any of these questions.

   We believe the results presented, together with those added during the revision, shed some on the positioning of the boundary. We proposed that the combined integration of four TFs by the OR463 enhancer is fundamental for the correct positioning. Additionally, we proposed a model on how these positioning problems result in the phenotypes observed (Supplementary figure 7, now also shown in Figure 2D). Our results indicate that ap expression in the PD quadrant is particularly sensitive to mutations in the enhancer, which we have now further elaborated on in the first part of the discussion. Together, we believe that our results do tackle the first problem posed in the introduction, while not completely solving them. As for the second question, we have tried to remove any suggestions that this article tries to explain later regulation of apterous. Probably this misunderstanding arises from a sentence in the introduction which has now been deleted. The means of the maintenance of ap expression in later stages has been partially explored previously (See Bieli et al 2015) and it is subject of our current studies.

   (2) The authors have identified two different regions whose deletions give very interesting phenotypes in the adult wing (AP identify change & outgrowths, and loss of wing), and have bioinformatically identified and functionally verified 4 TFs that mediate the activity of these regions by their capacity to phenocopy the wing phenotype. While identification of the 2 TFs acting on the m1 is incremental with respect to previous work on the identification of the enhancer responsible for the early expression of Ap, identification of Antp and Grn does not explain the loss of function phenotype of the m3 enhancer. Does any of these results shed any light on the first two Qs? Do these results explain the compartment boundary position in the wing as stated in the title? Expression of lacZ reporter assays is fundamental to demonstrate their model of Figure 8. The reduction of the PD compartment is difficult to understand by the sole reduction in ap expression in this region (which has not been demonstrated).

   We agree that the identification of Antp and Grn does not by itself explain the loss-of-function phenotype of the m3 enhancer. However, these transcription factors represent the best current candidates for direct regulators for this enhancer. We have clarified in the text that Antp and Grn may not act as instructive inputs but rather play a permissive role in enabling ap expression through m3. Importantly, the dCas9-mediated perturbation experiments directly demonstrate that targeted manipulation of apE in this region is sufficient to produce the characteristic duplications, providing functional evidence that apE activity underlies the observed phenotypes. In addition, lacZ reporter assays confirm that apE expression is indeed affected in all cases where the experimental setup permitted detection. Together, these results validate that the observed morphological phenotypes stem from perturbation of apE activity and support the proposed model for enhancer regulation and its role in compartment boundary maintenance.

   (3) The authors state in one of the sections "Spatio-temporal analysis of apE via dCas9 ". No temporal manipulation of gene activity is shown. The authors should combine GAL4/UAs with the Gal80ts to demonstrate the temporal requirements of Antp/Grn and Pnt/Hth as depicted in their model of Figure 8.

   We agree with the reviewer that the temporal dimension was not explored in the first version of the manuscript (aside of the temporal constrains of en-Gal4 driver). As suggested by the reviewer, we have now used a tub-Gal80ts allele to temporally control the enhancer perturbation and delimit its window of activity. The results are included in two new panels in the figure 3 (H and H’). The new data agrees with the notion that apE enhancer is important up to L2 stages but dispensable later in development. We have added the following paragraph to the text:

   “To define the developmental time window during which the apE enhancer remains sensitive to repression, we combined the temperature-sensitive tub-Gal80^ts system with temporally controlled expression of dCas9. Animals carrying the en-Gal4, tub-Gal80^ts, UAS-dCas9 and U6-OR463gRNA(4x) transgenes were maintained at 18 °C to suppress dCas9 expression. Independent sets of embryos were then shifted to 29 °C at successive developmental intervals ranging from 0 to 168 h after egg laying (AEL), so that dCas9 induction occurred at distinct time points in development (Figure 3H). Under these conditions, dCas9 transcription was induced only after the temperature shift, while the gRNAs were expressed constitutively. Wing phenotypes were quantified in adult progeny as a readout of apE enhancer perturbation. When dCas9 was expressed from embryonic or early larval stages (0–48 h AEL), nearly all wings (70–90%) displayed severe ap-like phenotypes, including posterior compartment duplication and loss of anterior–posterior boundary integrity. Shifting animals later (48–72 h AEL) still produced a majority (~66%) of abnormal wings, whereas induction after 72 h AEL resulted in progressively weaker effects and complete loss of phenotypes by 96 h AEL (Figure 3H’).

   These results delineate the developmental period during which apE activity is required for proper wing patterning. Perturbation during the first half of the second larval instar (≤ 96 h at 18 °C) was sufficient to elicit strong ap-like transformations, consistent with the enhancer being functionally required during early larval stages and becoming dispensable thereafter. The temporal decline in phenotype penetrance thus reflects the progressive loss of apE sensitivity to dCas9-mediated repression, providing a precise estimate of when its activity is no longer required for wing morphogenesis.”

   (4) The authors have not managed to explain the AP phenotype. Thus, this work opens many unresolved questions and does not resolve the title, which is a big overstatement. Thus, strengths (technically excellent), weakness (there is not much to learn about wing development and apterous regulation from these results besides the incremental identification of 4 additional TFs mediating the regulation of ap expression by their ability to phenocopy regulatory mutations of the apterous gene).

   As mentioned in response to reviewer 1, we have indeed no concrete explanation for why the P compartment seems more sensitive to mutations. We have now further discussed this point (see below paragraph, now included in the discussion). As for how the adult phenotypes arise from the mutant wing discs, we have a good idea (see Supplementary figure 7 and Figure 2).

   We are pleased to hear that the reviewer considers our article technically valuable. Therefore, we have reformulated the title such as the technical merits play a bigger role in it:

   ”in situ mutational screening and CRISPR interference demonstrate that the apterous Early enhancer is required for developmental boundary positioning”

   Paragraph added to the discussion:

   " Although apE is active throughout the dorsal compartment, its disruption leads to a preferential loss of ap expression in posterior cells. The asymmetric effect of apE perturbation on the anterior and posterior compartments suggests that apE transcriptional control is not equivalent across the A/P axis. Compartment-dependent differences in enhancer regulation have also been documented in other developmental contexts; for example, the Distal-less DMX-R element is interpreted through distinct cofactor combinations (Sloppy paired anteriorly and Engrailed posteriorly) (Gebelein et al., 2004), and specific mutations within DMX-R preferentially disrupt enhancer function in anterior versus posterior cells. It is possible that apE is more sensitive to misregulation due to differential transcriptional regulation across compartments. Nevertheless, we cannot exclude the possibility that the posterior bias we observe arises not from enhancer logic per se, but from intrinsic differences in tissue architecture or the dynamics of boundary positioning during wing disc development.”

   Recommendations for the authors:

   Reviewer #1 (Recommendations For The Authors):

   Formatting of references should be checked throughout the manuscript

   Reviewer #2 (Recommendations For The Authors):

   Here, I note a few points that would help clarify the manuscript and connect it with a broader community.

   Figure 1: it could help the reader to add the landing site genetic scheme to the main figure.

   In a first draft that was exactly the original configuration, but after comparing both versions we determined that the presence of the landing site removes a bit of the focus of the phenotypes.

   Figure 1: what species were used for the conservation alignment? Further details would be nice to add here.

   We have now added a section of bioinformatical analysis, which was missing in the original manuscript:

   Sequence conservation of the OR463 fragment within the ap upstream intergenic region was analysed across different dipteran species using the “Cons 124 Insects” multiple-alignment track of the D. melanogaster dm6 genome on the UCSC Genome Browser (Kent et al., 2002, https://genome.ucsc.edu). Conservation scores were obtained from the phastCons (Siepel et al., 2005) and used to delineate conserved and less conserved blocks within OR463. Conserved transcription factor binding sites were predicted with MotEvo (Arnold et al., 2011), which defined four conserved modules (m1–m4) and six inter-modules (N1–N6). Additional motif analysis was performed using the JASPAR CORE Insecta database and the Target Explorer tool to cross-validate conserved binding-site predictions and refine motif assignments within the enhancer.

   From Figure 2: I would consider moving the model or portions of it to a main figure. These models, while descriptive, really help make the manuscript more approachable. Note that eLife does not have forced figure requirements.

   We have adapted the reviewer’s suggestion and we are very grateful for it. We think the figure has greatly improved. The final figure now highlights a small part of the model, which is still included in the Supplementary Figure.

   Figure 5: This figure is fantastic, and the results are particularly important. I would recommend increasing the weight of the arrows from D to E, making it more obvious. Did the authors consider any temperature or other perturbations to look at robustness? They mention "robustness" a few times, and this could be an excellent system to explore a bit further. For panels F and G, it would be nice to have a bit of biochemistry here to test the spacing requirements' effects on the distances (but it's great phenotypical data, regardless).

   We have chosen a darker grey to highlight the lines.

   We appreciate the reviewer’s suggestions. With respect to robustness assays, such as temperature perturbations, we agree that the apE enhancer would be a suitable system for such experiments. However, these analyses would move the study beyond its current scope, which is focused on defining the regulatory logic of boundary positioning through mutational dissection and CRISPRi. We therefore prefer not to expand the work in this direction here, but we note that this would be an interesting avenue for future investigation.

   Similarly, biochemical assays probing spacing requirements would provide additional mechanistic insight but would represent a separate line of work. In this manuscript, we aimed to establish the functional consequences of motif spacing using in vivo genetic and phenotypic analyses, which we believe sufficiently support our conclusions.

   Thank you for the insight.

   Discussion: To the point "most point mutations or short deletions in enhancer regions have little effect on gene expression" I would push the authors to discuss their work in relation to Fuqua et al., (Nature 2020) and Kvon et al., (Cell 2020). Their work is consistent with enhancers being sensitive to mutations, and this warrants further discussion because it could be important for the transcription field.

   Hox genes as pioneer factors, I would recommend citing Loker et al., (Curr Biol 2021), as an example of Hox genes functioning as a pioneer factor.

   We thank the reviewer for this suggestion. We have now added a short paragraph in the Discussion noting how our observations may relate to the mutational patterns described in Fuqua et al. (2020) and Kvon et al. (2020), while keeping the interpretation tentative. The text now says:

   “Recent large-scale enhancer mutagenesis studies have shown that the mutational consequences within enhancers can vary widely. In some cases, many nucleotide positions appear tolerant to single-base changes and only a small subset of mutations produce clear functional effects (Kvon et al., 2020). In other enhancers, regulatory information is distributed more densely, and mutations at multiple positions can alter output (Fuqua et al., 2020). Together, these studies illustrate that enhancer sensitivity is not uniform but depends on enhancer-specific features such as motif organization, cooperativity, and redundancy. Within this broader landscape, the apE enhancer appears to represent a particularly sensitive case.”

   We also included a citation to Loker et al. (2021) in connection with the possible pioneer-like contribution of HOX input to apE.

   We would like to thank all reviewers for their effort.

   Read the original source
5. 

   Version published to 10.7554/elife.91713.2 on eLife

   Jan 8, 2026
6. 

   Version published to 10.7554/elife.91713 on eLife

   Jan 8, 2026
7. 

   Version published to 10.7554/elife.91713.1 on eLife

   Oct 11, 2023
8. 

   eLife

   Oct 10, 2023

   eLife assessment

   This paper presents an important discovery of the molecular basis of differential apterous expression during early Drosophila wing disc development. The evidence supporting these conclusions is compelling, ranging from classical genetic approaches to state-of-the-art genetic engineering techniques. By opening new questions, this paper is expected to be of broad interest to developmental biologists and geneticists working on transcriptional regulation.

   Read the original source
9. 

   eLife

   Oct 10, 2023

   Reviewer #1 (Public Review):

   Summary:
   The Drosophila wing disc is an epithelial tissue, the study of which has provided many insights into the genetic regulation of organ patterning and growth. One fundamental aspect of wing development is the positioning of the wing primordia, which occurs at the confluence of two developmental boundaries, the anterior-posterior and the dorsal-ventral. The dorsal-ventral boundary is determined by the domain of expression of the gene apterous, which is set early in the development of the wing disc. For this reason, the regulation of apterous expression is a fundamental aspect of wing formation.

   In this manuscript, the authors used state-of-the-art genomic engineering and a bottom-up approach to analyze the contribution of a 463 base pair fragment of apterous regulatory DNA. They find compelling evidence …

   Reviewer #1 (Public Review):

   Summary:
   The Drosophila wing disc is an epithelial tissue, the study of which has provided many insights into the genetic regulation of organ patterning and growth. One fundamental aspect of wing development is the positioning of the wing primordia, which occurs at the confluence of two developmental boundaries, the anterior-posterior and the dorsal-ventral. The dorsal-ventral boundary is determined by the domain of expression of the gene apterous, which is set early in the development of the wing disc. For this reason, the regulation of apterous expression is a fundamental aspect of wing formation.

   In this manuscript, the authors used state-of-the-art genomic engineering and a bottom-up approach to analyze the contribution of a 463 base pair fragment of apterous regulatory DNA. They find compelling evidence about the inner structure of this regulatory DNA and the upstream transcription factors that likely bind to this DNA to regulate apterous early expression in the Drosophila wing disc.

   Strengths:
   This manuscript has several strengths concerning both the experimental techniques used to address the problem of gene regulation and the relevance of the subject. To identify the mode of operation of the 463 bp enhancer, the authors use a balanced combination of different experimental approaches. First, they use bioinformatic analysis (sequence conservation and identification of transcription factors binding sites) to identify individual modules within the 463 bp enhancer. Second, they identify the functional modules through genetic analysis by generating Drosophila strains with individual deletions. Each deletion is characterized by looking at the resulting adult phenotype and also by monitoring apterous expression in the mutant wing discs. They then use a clever method to interfere in a more dynamic manner with the function of the enhancer, by directing the expression of catalytically inactive Cas9 to specific regions of this DNA. Finally, they recur to a more classical genetic approach to uncover the relevance of candidate transcription factors, some of them previously known and others suggested by the bioinformatic analysis of the 463 bp sequence. This workflow is clearly reflected in the manuscript, and constitutes a great example of how to proceed experimentally in the analysis of regulatory DNA.

   Weaknesses:
   There are several caveats with the data that might be constructed as weaknesses, some of them are intrinsic to this detailed analysis or to the experimental difficulties of dealing with the wing disc in its earliest stages, and others are more conceptual and are offered here in case the authors may wish to consider them.

   1. The primordium of the wing region of the wing imaginal disc is defined by the expression of the gen vestigial, which is regulated by inputs coming from the dorsal-ventral boundary (Notch and wg) and from the anterior-posterior boundary (Dpp). Having such a principal role in wing primordium specification and expansion, I am surprised that this manuscript does not mention this gene in the main text and only contains indirect references to it. I consider that the manuscript would have benefited a lot by including vestigial in the analysis, at least as a marker of early wing primordium. This might allow us to visualize directly the positioning of the primordium in the apterous mutants generated in this study, adding more verisimilitude to the interpretations that place this domain based on indirect evidence.

   2. The authors place some emphasis on the idea that their work addresses possible coordination between setting the D/V boundary and the A/P boundary:

   Abstract: "Thus, the correct establishment of ap expression pattern with respect to en must be tightly controlled", "...challenging the mechanism by which apE miss-regulation leads to AP defects." "Detailed mutational analyses using CRISPR/Cas revealed a role of apE in positioning the DV boundary with respect to the AP boundary"
   Introduction: "However, little is known about how the expression pattern of ap is set up with respect that of en. In other words, how is the DV boundary positioned with respect to the AP boundary?"
   "How such interaction between ap and the AP specification program arises is unknown."
   Results: "Some of these phenotypes are reminiscent of those reported for apBlot (Whittle, 1979) and point towards a yet undescribed crosstalk between ap early expression and the AP specification program."

   At the same time, they express the notion, with which this reviewer agrees, that all defects observed in A/P patterning arising as a result of apterous miss-regulation are due to the fact that in their mutants, apterous expression is lost mainly in the posterior dorsal compartment, bringing novel confrontations between the A/P and the D/V boundaries.

   To me, the key point is why the expression of apterous in different mutants of the OR463 enhancer affects only the posterior compartment. This should be discussed because it is far from obvious that apterous expression has different regulatory requirements in the anterior and posterior compartments.

   1. The description of gene expression in the wing disc of novel apterous mutants is only carried out in late third instar discs (Figs. 2, 3, 5, and 7). This is understandable given the technical difficulties of dealing with early discs, as those shown in the analysis of candidate apterous regulatory transcription factors (Fig. 4F, Fig. 6 C-D). However, because the effects of the mutants on apterous expression are expected to occur much earlier than the time of expression analysis, this fact should be discussed.

   Read the original source
10. 

    eLife

    Oct 10, 2023

    Reviewer #2 (Public Review):

    In their manuscript, "Transcriptional control of compartmental boundary positioning during Drosophila wing development," Aguilar and colleagues do an exceptional job of exploring how tissue axes are established across Drosophila development. The authors perform a series of functional perturbations using mutational analyses at the native locus of apterous (ap), and perform tissue-specific enhancer disruption via dCas9 expression. This innovative approach allowed them to explore the spatio-temporal requirements of an apterous enhancer. Combining these techniques allowed the authors to explore the molecular basis of apterous expression, connecting the genotypes to the phenotypical effects of enhancer perturbations. To me, this paper was a beautiful example of what can be done using modern drosophila genetics to …

    Reviewer #2 (Public Review):

    In their manuscript, "Transcriptional control of compartmental boundary positioning during Drosophila wing development," Aguilar and colleagues do an exceptional job of exploring how tissue axes are established across Drosophila development. The authors perform a series of functional perturbations using mutational analyses at the native locus of apterous (ap), and perform tissue-specific enhancer disruption via dCas9 expression. This innovative approach allowed them to explore the spatio-temporal requirements of an apterous enhancer. Combining these techniques allowed the authors to explore the molecular basis of apterous expression, connecting the genotypes to the phenotypical effects of enhancer perturbations. To me, this paper was a beautiful example of what can be done using modern drosophila genetics to understand classic questions in developmental biology and transcriptional regulation.

    In sum, this was a rigorous paper bridging scales from the molecular to phenotypes, with new insight into how enhancers control compartmental boundary positioning during Drosophila wing development.

    Read the original source
11. 

    eLife

    Oct 10, 2023

    Reviewer #3 (Public Review):

    In this manuscript, authors use the Drosophila wing as a model system and combine state-of-the-art genetic engineering to identify and validate the molecular players mediating the activity of one of the cis-regulatory enhancers of the apterous gene involved in the regulation of its expression domain in the dorsal compartment of the wing primordium during larval development.

    (1) The authors raise two very important questions in the Introduction: (1) who is locating the relative position of the AP and DV boundaries in the developing wing, and (2) who is responsible for the maintenance of the apterous expression domain late in larval development. None of these two questions have been responded to and, indeed, the summary of the work (as stated in the conclusions of the last paragraph of the Introduction) does …

    Reviewer #3 (Public Review):

    In this manuscript, authors use the Drosophila wing as a model system and combine state-of-the-art genetic engineering to identify and validate the molecular players mediating the activity of one of the cis-regulatory enhancers of the apterous gene involved in the regulation of its expression domain in the dorsal compartment of the wing primordium during larval development.

    (1) The authors raise two very important questions in the Introduction: (1) who is locating the relative position of the AP and DV boundaries in the developing wing, and (2) who is responsible for the maintenance of the apterous expression domain late in larval development. None of these two questions have been responded to and, indeed, the summary of the work (as stated in the conclusions of the last paragraph of the Introduction) does not resolve any of these questions.

    (2) The authors have identified two different regions whose deletions give very interesting phenotypes in the adult wing (AP identify change & outgrowths, and loss of wing), and have bioinformatically identified and functionally verified 4 TFs that mediate the activity of these regions by their capacity to phenocopy the wing phenotype. While identification of the 2 TFs acting on the m1 is incremental with respect to previous work on the identification of the enhancer responsible for the early expression of Ap, identification of Antp and Grn does not explain the loss of function phenotype of the m3 enhancer. Does any of these results shed any light on the first two Qs? Do these results explain the compartment boundary position in the wing as stated in the title? Expression of lacZ reporter assays is fundamental to demonstrate their model of Figure 8. The reduction of the PD compartment is difficult to understand by the sole reduction in ap expression in this region (which has not been demonstrated).

    (3) The authors state in one of the sections "Spatio-temporal analysis of apE via dCas9 ". No temporal manipulation of gene activity is shown. The authors should combine GAL4/UAs with the Gal80ts to demonstrate the temporal requirements of Antp/Grn and Pnt/Hth as depicted in their model of Figure 8.

    (4) The authors have not managed to explain the AP phenotype. Thus, this work opens many unresolved questions and does not resolve the title, which is a big overstatement. Thus, strengths (technically excellent), weakness (there is not much to learn about wing development and apterous regulation from these results besides the incremental identification of 4 additional TFs mediating the regulation of ap expression by their ability to phenocopy regulatory mutations of the apterous gene).

    Read the original source
12. 

    Version published to 10.1101/2023.08.05.552106 on bioRxiv

    Aug 6, 2023