Reviewer #1

Below one anonymous reviewer's comments.

In humans, the transcription factor FoxP2 gene is necessary for normal development of speech and language. In flies, the only ortholog FoxP has been recently analyzed in several studies. In a report by the Miesenb6ck lab published last year in Science, a transposon induced mutant affecting one of the two (or three) isoforms of the FoxP gene was used to show a requirement of FoxP in decision making processes. In a later report by the Brembs lab published in PLOSone the very same insertion mutant was used to demonstrate a requirement of FoxP in operant self learning. One of the problems in all these studies is that no "clean" FoxP mutants are available and that the expression pattern of FoxP is still elusive as no antibodies work and several FoxP Gal4 lines all show different expression patterns.

The present proposal of B. Brembs aims to clarify these issues by
a) the generation of additional strains allowing the expression of shRNA,
b) generation of an Anti-FoxP monoclonal antibody
c) generation of specific mutants and Gal4 strains by RMCE complemented
d) by a behavioral analysis of the generated lines.

In principle, this proposal addresses important and highly relevant questions but unfortunately there are many (!) problems with this application which make it rather weak and in no case fundable. The preliminary work mentioned in this proposal is odd. Basically we learn that there are many RNAi lines available in the stock centers, which have a phenotype when used to silence FoxP expression but strangely do not affect FoxP expression. What does this mean? I have seen no arguments why the generation of additional RNAi strains is now all the sudden expected to yield a breakthrough result.

Quite similar we learn in the preliminary result section that many attempts to generate specific antibodies failed and yet the generation of mAbs is proposed. Again, it is unclear what we will learn and alternative strategies are not even discussed. The authors could consider the generation of HA-tagged Fosmids /I minigenes or could use homologous recombination to manipulate the gene locus accordingly.

One page 2 of the application it is stated that "It is a technical hurdle for further mechanistic study of operant self-learning that the currently available FoxP mutant lines are insertion lines, which only affect the expression level of some of the isoforms. " This is not true! and the applicant himself states on page 11:

"However, as the Mi{MIC} insertion is contained within a coding exon which is spliced into all FoxP isoforms, it is likely that this insertion alone already leads to a null mutation at the FoxP locus."

Yes, by all means the insertion of a large transposon into the open reading frame of a gene causes a mutation!!!! Why this allele, which is available in the stock centers, has not yet been analyzed so far remains mysterious. Moreover, reading the entire third section of this application "genome editing using MiMIC" reveals that the applicant has not understood the rational behind the MiMIC technique at all. Venken et al clearly published that "Insertions (of the Minos-based MiMIC transposon) in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments." Importantly, insertions have to be in an intron!!!! The entire paragraph demonstrates the careless generation of this application. "we will characterize the expression of eGFP in the MiMIC transposen". Again, a short look into the Venken et aI., paper demonstrates the uselessness of this approach. The application requests two students. Although the entire application is far from being fundable, this request adds the dot on the i. The student is planned for the characterization of lines that are not available, characterization of antibodies that likely will not be on hand in the next two years and so on.

In summary, this is a not well prepared application, full of mistakes and lacking some necessary preliminary data. Not at all fundable.

My reply to reviewer #1:

http://bjoern.brembs.net/2015/12/why-cutting-down-on-peer-review-will-improve-it/

Thanks for sharing Bjoern. I really hope it was cathartic writing that response! ;) Moving ahead I'd just like to make sure everyone is on a positive, constructive footing. As I see it there's two things we can help you with:

1. We can help you improve the research plan to create the most impact in the field
2. We can help you improve the proposal to help you get funded!

So I'd like to invite everyone to join in that effort. To respond to an issues raised by reviewer #1 you can start a new discussion or simply reply in this thread. I've started a discussion posing the following question: How much expertise is it realistic to expect grant reviewers to have?

For anyone who wants to be extra helpful, I invite you to start a full review of Bjoern's proposal.