CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity

Essential revisions:

1) All three reviewers have agreed that the paper is more appropriate as a "Tools and Resources" paper. While there are some clear biological insights, these are not followed up in sufficient detail to lead to a major advance. However, the technical aspects will be useful to others, and the work generated here will almost certainly allow these authors and others to advance this particular system and possibly create significant breakthroughs in our understanding of the mechanism at work to set the position of the L2 vein and how it has evolved. The authors should revise the paper with this in mind.

We have revised the manuscript to emphasize the technical aspects of the technology where possible.

2) The authors should provide some quantitation on the shift of the position and length of the L2 vein after the enhancer swaps (from other Dipteran species). In particular, the authors should quantify the shift in L2 position seen with the M. domestica CRM (distance of shift after normalizing for variation in wing size) and shortening of L2 seen with the C. capitata CRM (length of L2 relative to total wing length).

We have quantified the M. domestica vein displacement phenotype by three different means: 1) measuring the ratio of the length of the L2 versus L3 veins; 2) measuring the angle between the L2 and L3 veins; and 3) measuring the distance (counted in number of thick marginal bristles) distal to the point where the L2 vein intersects the wing margin. All three of these measures reveal highly significant anterior shifts of the L2 vein in the M. domestica replacement. With regard to the C. capitata replacement, we have measured the length of the rescued L2 vein segment normalized by the length the intact L3 vein. We also quantitated the anterior displacement of the rescued L2 segment relative to that of the wild-type L2 vein in a fixed proximo-distal position.

3) The authors should provide some data on the expression of kni and sal in the wing disks of M. domestica and C. capitata to assess if indeed the pattern of these genes suggests general similarities in the mechanisms of vein placement, and to assess if the pattern seen when the respective CRMs are placed in D. melanogaster is reflective of the normal pattern of expression for kni in the source species.

We have included as supplemental material a figure (Figure 6—figure supplement 1) showing expression of kni and salm in C. capitata wing imaginal disc. These results confirm that these two genes are expressed in a similar pattern in C. capitata and D. mel. In consultation with the editors, it was decided, in order to avoid a significant potential publication delay, that it would not be necessary to provide comparable data from M. domestica given that: 1) the M. dom. replacement expresses kni in a stripe abutting the salm domain in D. mel.; and 2) because kni is expressed in a similar pattern relative to spalt in a yet more diverged fly species (Megaselia abdita) as well as in C. capitata.

[Editors’ note: after resubmission, the authors sent the note below. The paper was then accepted for publication.]

Since submission of our revised manuscript on November 29, 2017, we have done a stain for Knirps protein expression in M. domestica that worked very well, revealing a pattern strikingly similar to what we observed in the D. mel. riCC-Mdom CRM replacement. We provide this result in comparison to the D.mel. wt and riCC-Mdom CRM to help readers appreciate that the broadened stripe we observe in the riCC-Mdom CRM replacement most likely reflects the endogenous Kni expression pattern in its species of origin. We have included this stain in the revised version of Figure 6—figure supplement 1.

With these newly added data, we have now addressed all of the reviewer requests.