teiresias, a Fruitless target gene encoding an immunoglobulin-superfamily transmembrane protein, is required for neuronal feminization in Drosophila

This study aims at identifying transcriptional targets of FruitlessBM (FruBM), which represents the major isoform of male-specific FruM transcription factors that induce neural sexual dimorphisms. A promoter of the axon-guidance factor gene robo1 carries the 16-bp palindrome motif Pal1, to which FruM binds. Our genome-wide search for Pal1-homologous sequences yielded ~200 candidate genes. Among these, CG17716 potentially encodes a transmembrane protein with extracellular immunoglobulin (Ig)-like domains similar to Robo1. Indeed, FruBM overexpression reduced CG17716 mRNA and protein expression. In the fru-expressing mAL neuron cluster exhibiting sexual dimorphism, we found that CG17716 knockdown in female neurons completely transformed all neurites to the male-type. Conversely, CG17716 overexpression suppressed male-specific midline crossing of fru-expressing sensory axons. We renamed CG17716 teiresias (tei) based on this feminizing function. We hypothesize that Tei interacts with other Ig superfamily transmembrane proteins, including Robo1, to feminize the neurite patterns in females, whereas FruBM represses tei transcription in males.

midline crossing in females, it is therefore important to see if simultaneously knocking down Tei and Robo1 would have a stronger effect on midline crossing. This at least could test whether Tei and Robo1 function together or in parallel.
2. I am wondering why the authors keep changing the developmental stages of flies that were used for immunostaining, some with adult, and some with larvae or pupae. I do not have problem with using either developmental stage for certain experimental purpose, e.g., whether overexpression of FruBM would inhibit Tei expression in larvae or other stage, but the authors are at least should mentioned the logic between using flies with different developmental stages for different experiments.
1 Brief summary of the manuscript Understanding the molecular mechanism of how behavioural neural circuits are developed is the holy grail of neuroscience. The Fruitless protein (Fru), which functions to establish sexually dimorphic courtship behavior in flies, provides an excellent model to unravel this puzzle. Fru is a Zn-finger containing transcription factor with other structural domains that may form protein-protein interactions with other protein partners. FruBM is the male-specific isoform of Fru. This manuscript describes the identification of the second FruBM gene target known to date, Teiresias (Tei). Robo1 is another FruBM target, also discovered by the same group.
The authors identified Tei as a possible gene target of FruBM through a genome-wide bioinformatic analysis of promoter sequences that contain the previously established Pal1 sequence as the essential binding site for FruBM. The authors proved that Tei is a target of FruBM by using different genetic manipulations to overexpress and knockdown Tei, FruBM, Robo, and then evaluating the phenotypic consequences of the morphology of several well-characterized sexually dimorphic regions of the Fru circuit. The authors concluded that 1) FruBM represses the expression level of Tei, both at the RNA and protein level; and 2) Tei has a feminizing function, responsible in developing feminized neurite patterns when it is expressed; in contrast, Tei has no effect on the cell number in clusters that show sexually dimorphic neuronal population. Finally, the authors hypothesized that the mechanism of Tei function may be to interact with other IgG domain containing proteins, like Robo1, which may lead to a change in intracellular signaling that alters neurite morphology.

Overall impression of the work
This manuscript contains a body of meticulously designed experiments that establishes Tei as the second gene target of FruBM. The authors presented a thought-provoking hypothesis that Tei may exert its function by forming a heteromeric complex with Robo, though the data presented may suggest otherwise -see my comments below. The hypothesis is interesting as the use of the immunoglobulin-like structural domain to generate functional variability is a recurrent theme observed in other fields. I think the authors can develop this hypothesis further by discussing more about how Slit/Robo1 signaling works. For example, can Robo1 work on its own? What is the stoichiometry of a functional Robo1  P12 lines 180-181: "... additional … tei + in these neurons counteracted … to increase midline crossing.." but you're actually looking for a decreased midline crossing in the cited Figure. I understand the meaning, but the sentence is a bit convoluted. Rewording or rephrasing may improve readability.
P12 lines 178-182, P16 lines 252-260 FruBM represses both Robo1 and Tei activity. Both Robo1 and Tei KD promote midline crossing. Results show that overexpression of Tei can counteract Robo1 KD effect. Wouldn't this observation disprove the hypothesis stated in the abstract (p2 lines 29-30) that "Tei interacts with other IgG superfamily transmembrane proteins, including Robo1, to feminize the neurite patterns in females…"? If Tei/Robo1 functions as a heterodimer, then KD of one gene should not be rescued by the OE of another? Can these receptors function as homodimers? Tei lacks a substantial C-terminal domain (Fig 1d). What does it mean when it interacts with other IgG transmembrane proteins with a larger C-terminal domain? Will it inhibit the intracellular effect completely or just attenuate it?!?
P14 lines 210-219: The observation that the intensity of Tei signals in FruM-expressing cells is strong is very interesting. The authors offer two explanations: 1) FruM only represses Tei expression in a subset of mAL neurons; and 2) There's a temporal lag between FruM expression and Tei expression. Can't possibility 2 be tested by fine-tuning the developmental time points for the co-staining analysis in the experiments for Fig 6? If possibility 1 is true, then there must be an unidentified factor that stops FruM from repressing Tei expression in a subset of mAL neurons? Could this factor be Fru/Bon/HP1a?!? For example, FruM is present but it's in complex with the unknown factor that prevents it from binding to the promoter of Tei? P14 lines 216-219: The Dsx experiment doesn't seem to belong in this section. Instead, the first paragraph of the next section (p15 lines 222-226) should go after line 216 on p14? P20 Fly strains: explain somewhere what poxn-Gal4 is. P23 line 348: missing a bracket P24 line 377: "so that the circle was centered …" how do you ensure this is done consistently from sample to sample?

Reviewer #1
Major points: Q1. Perhaps I missed this but somewhere, the figure legend, the text or a table, the entire genotype for each experiment/panel must be given. Q4. The authors should include copulation success in Fig. 7 even with a low courtship index, the key is whether this reduces copulation. It would be best to redo the experiment using tsh-Gal80 especially as there looks to be widespread VNC expression (in the larva at least). This would be possible as tsh-Gal80 is on the second chromosome.
A4. We reconducted our mating behavior assays to quantify the effect of fru[+] overexpression in males on mating success over a 1-hour-observation period. The reduction in courtship activities induced by tei[+] overexpression was accompanied by a decrease in mating success, as described in If Tei were to interact with Robo, how will this affect Slit/Robo1 signaling? A10. We added the following sentence to discuss the possible role of the cytoplasmic C-terminus of Robo1, which is absent from Tei. "The lack of the cytoplasmic portion in Tei might suggest that Tei contributes to the ligand specificity whereas the specificity of intracellular signal transduction following heteromeric receptor activation is determined by Robo1 or another Tei partner that carries the C-terminal cytoplasmic domain" (lines 267-271). A12. Three tei RNAi lines were used in this study to assure that the observed effects were truly due to tei knockdown, and this fact is now described in lines 140-144 of the original manuscript (corresponding to lines 143-146 of the revised manuscript). We also added text to the Methods subsection on fly lines in order to clarify that we made the two tei RNAi lines, UAS-tei RNAi ex6-7 and UAS-teiRNA ex4 , as well as tei-GAL4 and UAS-tei + (lines 342-343).
Q13. P11-12 lines 170-182 and Figures 5d-g: Why did you use a different Gal4 line for the overexpression experiment? I assume poxn-Gal4 is EY11779? Why not use PPk23-Gal4 with fru2/frusat? I assume for practical reasons, but some explanation is needed.
A13. To clarify why we used different combinations of GAL4 and UAS constructs to overexpress tei+ from one experiment to another, we modified the relevant passage as follows: "In this series of experiment, tei+ was overexpressed with EY11779, a Gene-Search fly line with paired UAS sequences inserted near the tei locus on chromosome-2 (UAS-tei[+]), so as to make chromosome 3 available for other transgenes to combine. Also, Poxn-GAL4, a pan-chemosensory driver, replaced ppk23-GAL4 in another set of experiment, because the chromosome-3 carrying both a fru mutant allele and Poxn-GAL4 was already available with no need to generate a new recombinant chromosome-3" (lines 173-179).
Q14. P12 lines 180-181: "... additional … tei+ in these neurons counteracted … to increase midline crossing.." but you're actually looking for a decreased midline crossing in the cited Figure. I understand the meaning, but the sentence is a bit convoluted. Rewording or rephrasing may improve readability.
A14. We rephrased the relevant passage as "Importantly, additional expression of tei+ in these neurons counteracted the effect of robo1 RNAi, resulting in a trace of midline crossing ( Fig. 4o-q)." (lines 188- A16. We expanded this part of the Results section based on our additional analysis of FruM and Tei expression in the adult brain by adding the following passage to the text (lines 228-230): "In the adult brain, most of FruM-expressing mAL neurons (~20 cells) were also positive for tei-GAL4 presumably due to perdurance of GAL4 protein ( Supplementary Fig. 6), an observation that appears to favor the second possibility". The newly added Supplementary Fig. 6 shows a male-adult brain that was doublestained with the anti-Tei and anti-FruM antibodies in an immunostaining-enhancing solution Q18. P20 Fly strains: explain somewhere what poxn-Gal4 is.
Q19. P23 line 348: missing a bracket A19. We changed the description as follows: "(residues 56-78 of Tei; GenBank accession number NP_523731)." Q20. P24 line 377: "so that the circle was centered …" how do you ensure this is done consistently from sample to sample? A20. The following description was added to the Methods section to explain how to position circle-a (lines 421-424): "A few sensory neurons originating from prothoracic legs send ascending axons directly to the brain, which turn perpendicularly within the prothoracic ganglion. The midpoint of bilateral ascending fibers at their turning points was used to center the circle". Q21. P35 lines 534-537: More description of the arboplot would be nice. For example, is there any consistent landmark you use to draw the yellow dotted box for optical slicing for each sample? Does it need a special plugin in Image J? Maybe useful to write a method section to describe how this is done. It's a very nice quantification procedure that other researchers will find useful to implement. A22. In the revised manuscript, we explicitly stated that EY11779 is a Gene-Search fly line that functions as a UAS-tei[+] to overexpress tei[+], which is activated by poxn-GAL4, a panchemosensory GAL4 driver (lines 173-176). Please see also A13 above.
Q23. P38 Figure 6: How do you know the single cluster that expresses Tei in female is the same cluster that expresses Fru in male (Figure a-f), since their expression is supposedly mutually exclusive?
A23. This was almost the only cluster that expressed fru[FLP] as monitored with UAS>STOP>mCD8::GFP at this developmental stage in the entire CNS in both females and males, and therefore, this cluster was inferred to represent a homologous cluster in the two sexes.
A24. The box-and-whisker plot shows the first quartile (25th percentile), median, third quartile (75th percentile) and minimum and maximum of each set of data. Thank you for pointing out this fundamental error. We corrected it in the revised manuscript. This is a revised manuscript by Sato et al., identifying a new Fruitless target gene, teiresias, that functions to feminize neuron structure in the Drosophila adult nervous system. The revisions address my concerns raised in the initial submission except one.
As a major point, it was asked that the authors provide specific optical sections for confocal images that could be influenced by the stacking of z-projections. As an example, I suggested SFig. 4. However, I was not initially clear and it isn't enough to add a line stating in the figure legend that a z-projection is shown. Rather it is critical to specify how many optical sections were used especially for Fig. 4 and Fig. 2b',d' where the midline crossing and outgrowth phenotype can be influenced by the number of optical sections. This needs to be done in the figure legends in the manuscript not in any supplementary information.

Point-by-point replies to the reviewers' comments
Reviewer #1 (Remarks to the Author): Q1. As a major point, it was asked that the authors provide specific optical sections for confocal images that could be influenced by the stacking of z-projections. As an example, I suggested SFig. 4. However, I was not initially clear and it isn&#x2019t enough to add a line stating in the figure legend that a z-projection is shown.
Rather it is critical to specify how many optical sections were used especially for Fig. 4 and Fig. 2b&#x2019,   d&#x2019; where the midline crossing and outgrowth phenotype can be influenced by the number of optical sections. This needs to be done in the figure legends in the manuscript not in any supplementary information.
A1. In the re-revised manuscript (R2), we described, in respective figure legends, the number of confocal slices used in z-stack reconstitution for every image subjected to quantification of neural structures. It ranged between 29 and 100 slices, which, we believe, were sufficient to yield reliable estimates for the midline crossing score or fluorescent intensity score.
Q2. The authors did include copulation success as requested although tsh-Gal80 was not included so the reader is left to wonder if this is a VNC or brain neuron phenotype.
A2. We attempted to repress GAL4 expression in the VNC with tsh-GAL80, but it turned out that tsh-GAL80 on its own reduced the male copulation success rate down to ~75% (cf. 85% in fru-GAL4/+ control flies) without tei + overexpression, which was statistically indistinguishable from the value upon brain-restricted tei + overexpression as shown in the figure pasted below. This made it difficult for us to judge which of the brain or VNC was the primary site of action of tei in modulating mating success.
NS: Not significant in the Fisher's exact probability test.
Reviewer #3 (Remarks to the Author): Q3. All my comments have been addressed adequately except for Q11. I don't quite understand how Seqs 1-16 in Supplementary Table 1 is related to the 7 downregulated genes described in the main text. Are these DNA sequences that were used to search for genes that have the Pal1 sequence motif in the genome? Maybe just take out the reference to this table on page 7 line 103 and also edit the title of this table? A3. We used Seqs 1-16 as query nucleotide sequences in our in silico searches for Pal1 homologous sequences in the genome. Seqs 1-16 were based on the FruBM binding motif (Pal1) identified in the robo1 promoter (Ito et al., 2016), while allowed to carry two mismatches in the entire 16-bp palindrome sequence. Also, according to the reviewer's suggestion, we removed the reference to Supplementary Data 1 (former Supplementary