Transcriptional control of compartmental boundary positioning during Drosophila wing development

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.

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.

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).