Key homeobox transcription factors regulate the development of the firefly’s adult light organ and bioluminescence

Adult fireflies exhibit unique flashing courtship signals, emitted by specialized light organs, which develop mostly independently from larval light organs during the pupal stage. The mechanisms of adult light organ development have not been thoroughly studied until now. Here we show that key homeobox transcription factors AlABD-B and AlUNC-4 regulate the development of adult light organs and bioluminescence in the firefly Aquatica leii. Interference with the expression of AlAbd-B and AlUnc-4 genes results in undeveloped or non-luminescent adult light organs. AlABD-B regulates AlUnc-4, and they interact with each other. AlABD-B and AlUNC-4 activate the expression of the luciferase gene AlLuc1 and some peroxins. Four peroxins are involved in the import of AlLUC1 into peroxisomes. Our study provides key insights into the development of adult light organs and flash signal control in fireflies.


REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author):

Summary
The manuscript entitled "Key homeobox transcription factors regulate Firefly adult light organ development and bioluminescence" by Xinhua Fu and Xinlei Zhu aims at providing a better understanding of the molecular control of the light organ bioluminescence in adult fireflies.To this end, they used as a model Aquatica leii.They first employed genome sequencing to assemble more indepth the genome.They next performed comparative analysis at the genome and transcriptome levels to characterize transcription factors (TFs) presence and expression level.Focusing on the homeodomain proteins, they used RNAi injection combined with expression and functional assays to assess their importance in pupae light organ formation and bioluminescence.They further focused on AlABD-B and AlUNC-4 and performed interaction and reporter assays in human cells and in vitro to propose a possible mechanism of AlLUC1 regulation and peroxisome transport.

Strength
From genome assembly, comparative analysis, transcriptome and gene-function relationship, this manuscript is an integrative study on an interesting topic.The field is specific but the evolutionary aspect studied holds the potential for broader interest in the evolution and gene regulation communities.

Weakness
The problem of syntax makes the manuscript often difficult to read.Moreover, there is a lack of essential information and the description of the figures in text and legends is too superficial.This makes the reading often confusing (for example, male VS female, n number, data mentioned but not provided like Alap2-RNAi and videos) The result part is in particular lacking description and importantly, conclusion and reasoning.The discussion is missing.

Overall
While the story is interesting, with a lot of data, the dissonance of these data and of the methods employed as well as the lack of discussion, clarity and description impact strongly on the potential of the story.In the reviewer's opinion, the present manuscript does not fulfil the requirement and accessibility necessary for publishing in Nature Communication.Thus, this reviewer cannot recommend it for publication.

Major issues Results:
-Fig1D and 1A are not mentioned in the text -page6 lines 141-142: how about the expression pattern in females?-extended data fig5: how about the analysis for other TFs?What does the distribution look like?-Interference efficiency: is it ubiquitous or tissue-specific?How was the qPCR performed?On whole individuals?Only the end of the abdomen?-Fig2: AlAP2-RNAi: there is a decrease of intensity of bioluminescence (fig2A) in male but not in female.It would be interesting to understand why and provide quantification and reasoning to explain this discrepancy between males and females.-page8: overall, what is the conclusion?What does it suggest?This seems missing -Fig2B: on which tissue was the qPCR experiments performed?-Fig2E is confusing and would gain visibility by changing the colour or pattern or size -page12, line256: a sentence seems missing between "verified36" and "Analysis…", it seems to weaken the following description.-page13, line 286: how about the protein level of luciferase?-is it possible to perform RNAi of peroxisomal transporter and analyse the effect on the luciferase localisation?-Fig4 and the following: Why using human cell lines as model?Why not insect cells?or staining of dissected abdominal segment?-Fig4F: a negative control may strengthen the figure (mutant DNA sequence of the Abd-B binding site that should not compete) -why doing YOH for UNC-4 and EMSA for ABD-B? this really confuses this reviewer.EMSA would have been sufficient for both proteins, + DNA mutant of each binding site for showing specificity.
-Is it possible to do a reporter assay in vivo within this specie?This would greatly enhance the quality of the study -Fig5E and extended data fig12: it is rather confusing to detect BiFC signal of AIPEX14 and AlLuc1 in the nucleus.How would you explain this result?-BiFC-related result: why not using tri-colour BiFC for showing the requirement of several subunits for AlLUC1 transport in the peroxisome?Related to this, this reviewer gets a bit lost as it seems that AlLUC1 expression was not revealed specifically in the peroxisome.May you clarify this please as it is major information for the manuscript?-it would be interesting to test the requirement of the DNA binding ability of Unc-4 to interact and synergize with Abd-B activity on the reporter.One experiment could be to assess the transactivation of the reporter by the combinatorial expression of Unc-5 and Abd-B when a mutation of the Unc-4 binding site is inserted.
Reasoning missing: page8: it sounds like something is missing after "axongenesis22" Data/figure missing -video and supplementary video missing -extended data fig6: no flash behaviour present -data AlShox2 knockdown is missing (page 8) -quantification of fig5A is missing Mix of figures: -among many of them, fig1B cited before Fig1A, fig2A cited after 2B, fig 2C after 2D, fig4B before 4A, fig4E before 4D -extended data fig7C not cited -fig5E and extended data fig12 left panel are exactly the same Legend lacking information: -extended fig5, Fig2, Fig3A, Fig4 (and AF in particular), extended data fig6, extended data fig10, extended data fig11, extended data fig12 Discussion: missing Minor issues Abstract: -present tense to uniformise -page1, line 20: "our results demonstrate that …" Introduction: -page2 line 39: "luciferaseS are" or "luciferase is" -a short sum-up of the major finding would have been appreciated Results: Page6, line 143: "only 6 homeobox genes were always.." Page7, line 150: quantitative PCR (not quantitation) Page7, line162 : corrective dash between Abd-B and specifies Page10, ine 200-202: the syntax of the sentence is confusing, same line 215-216, 217-218, 227-228 Same page10, Line 221: "were assessed" insteatd of "were detected" would provide better clarity line 222, 226, 230: citing the fig 3B here would help the reader page11, line 231: syntax is confusing page12 line23: "functional" instead of "function" page12: tense seems sometimes confusing (example line240: "AlLuc1 is located" instead of "was") page13, line 275: syntax "of AlLuc1 was down-regulated.." Reviewer #2 (Remarks to the Author): This is an interesting study that provides new insight in the regulation of firefly luminescence.Given the availability of 4 already published firefly genomes, it is not clear why they needed to sequence a different one.Is it a better model system?Does it provide a different perspective on the problem?The genome sequencing, quality control and gene annotation follow standard protocols and provide the information required for the current study.The work is presented in a linear fashion and the overall meaning is clear.However the word usage and grammar need additional attention.The follow represent only a few instances, where the clarity of the argument was obscured.
Line 39 peroxisomes where luciferase….. peroxisomes containing luciferase.Line 49 sifted should probably be filtered.Line 165 no significant difference line 215 and 217 is possibly Line 224 sifted should be filtered Line 267 in female abdomens among with female Line 270 AlLuc1 is needed for transport to must be transported to Line 273 was interfered by was knock down by Line 322 Need to included AlABDB in this sentence Line 331 AlABD-B activated Line 332 between in cells Line 352 AlLuc1 functions within in.and requires import by peroxins.Line 354 sifted filtered Line 355 were involved in import ……., RNAi was performed for each gene.Line 361 Verification by real-time……….. were down-regulated

Summary
The manuscript entitled "Key homeobox transcription factors regulate Firefly adult light organ development and bioluminescence" by Xinhua Fu and Xinlei Zhu aims at providing a better understanding of the molecular control of the light organ bioluminescence in adult fireflies.To this end, they used as a model Aquatica leii.They first employed genome sequencing to assemble more in-depth the genome.They next performed comparative analysis at the genome and transcriptome levels to characterize transcription factors (TFs) presence and expression level.Focusing on the homeodomain proteins, they used RNAi injection combined with expression and functional assays to assess their importance in pupae light organ formation and bioluminescence.They further focused on AlABD-B and AlUNC-4 and performed interaction and reporter assays in human cells and in vitro to propose a possible mechanism of AlLUC1 regulation and peroxisome transport.

Strength
From genome assembly, comparative analysis, transcriptome and gene-function relationship, this manuscript is an integrative study on an interesting topic.The field is specific but the evolutionary aspect studied holds the potential for broader interest in the evolution and gene regulation communities.

Weakness
The problem of syntax makes the manuscript often difficult to read.
Moreover, there is a lack of essential information and the description of the figures in text and legends is too superficial.This makes the reading often confusing (for example, male VS female, n number, data mentioned but not provided like Alap2-RNAi and videos) The result part is in particular lacking description and importantly, conclusion and reasoning.
The discussion is missing.

Overall
While the story is interesting, with a lot of data, the dissonance of these data and of the methods employed as well as the lack of discussion, clarity and description impact strongly on the potential of the story.
Response: Thank you for your summary.We really appreciate your efforts in reviewing our manuscript.
We apologize for the syntactic problems in the original manuscript and the problems they caused.The manuscript has been thoroughly revised, and parts of it rewritten where needed, by a native English speaker proficient in genetics, so we hope that the revised version can meet the journal's standard.We have also revised the manuscript according to other comments: (Male Vs Female), tests and analysis on female tissue were added; (n number), numbers of tested materail were added in Methods part; (data mentioned but not provided like Alap2-RNAi), data mentioned like AlAp2 RNAi were added; Three Supplementary videos were uploaded; The result parts were re-written; The discussion part was added.
Our point-by-point responses are detailed below.

:
-Fig1D and 1A are not mentioned in the text Response:Thank you for pointing out these problems: Fig1A is now mentioned in the Introduction, and Fig1D is mentioned in line 70.
-page6 lines 141-142: how about the expression pattern in females?
Response:During our sample collection at various developmental stages, our primary concern was securing a substantial number of individuals for dissection purposes.In addition, males also have a larger luminous organ.Therefore, we focused our sampling efforts and analyses solely on male specimens.To address your concerns, and analyze the expression patterns of these genes within the female luminous organ, we detected the relative expression level of 8 homeobox genes by qPCR in the light organof female individuals at different developmental stages (n=3).
The relative expression levels of AlAbd-A, AlAbd-B, AlUbx, AlAntp, AlUnc-4, AlShox2, AlRepo and AlAp2 were analysedin three developmental stages of female light organs using qPCR (Supplementary Fig. 5B).AlAbd-B and AlUnc-4 were continuously up-regulated during the female pupal development, while AlAbd-A, AlAntp and AlUbx were down-regulated.The expression pattern of AlAp2 had a peak expression in the mid-pupal stage, and the expression levels of AlRepo did not change during these three stages.Furthermore, the expression level of AlShox2 could not be detected (Cq>37) in female light organs.The results are shown in Supplementary Fig. 5b.
As fireflies undergo one reproductive cycle annually, we currently lack a sufficient female sample size for conducting more extensive RNA sequencing and analyses.
-extended data fig5: how about the analysis for other TFs?What does the distribution look like?
Response: In addition to the Homeobox transcription factor that we highlighted in yellow, the expression patterns of other transcription factors can be broadly categorized into two types: (1) down-regulated during the development of the light organ, and (2) peak expression in the mid-pupal stage.We hypothesise that these two types of transcription factors may be more important in the early stages of pupal development then in the late stages, or these genes may play an inhibitory role, but they don't affect the entire process of light organ development and they have no direct relationship with the control of bioluminescence and flashing.Therefore, they are not in the focus of this study.The results are shown in Supplementary Fig. 5A, and if the editor deems it necessary, we will add this explanation to the main manuscript.
-Interference efficiency: is it ubiquitous or tissue-specific?How was the qPCR performed?On whole individuals?Only the end of the abdomen?
Response: For each dsRNA treatment group, we used 2 males and 1 female.The total RNA was extracted from the whole individuals (ubiquitous).For the qPCR, we used three technical replicates.
We have added the missing content to the line 783-984 of the Methods section.
-Fig2: AlAP2-RNAi: there is a decrease of intensity of bioluminescence (fig2A) in male but not in female.It would be interesting to understand why and provide quantification and reasoning to explain this discrepancy between males and females.
Response:Thanks for pointing out a phenomenon that we had overlooked originally.Based on your feedback, we have re-treated nine specimens (4♀ and 5♂) with dsAlAP2: 2 died, 6 exhibited a continuous glow phenotype, and one female exhibited a continuous glow phenotype and a significant decrease in light intensity.These results indicated that the reduction of intensity of bioluminescence is not a universal phenomenon.Additionally, we assessed the video data, and failed to observe a significant decrease in the brightness.This indicates that the observed decrease might be an artefact caused by random fluctuations in brightness.Based on the latest experimental results, we have replaced the male photo of dsAlAP2 in Figure 2A.
-page8: overall, what is the conclusion?What does it suggest?This seems missing Response: Thank you for underlining this deficiency.We have added the missing content to the manuscript on page 8: "Overall, based on the fact that the knockdown of homeobox genes AlAbd-B and AlUnc-4 resulted in non-luminescence and empty peroxisomes, these two genes may be the key regulators required for normal light organ development.In addition, we found evidence that AlAntp, AlRepo and AlAp2 are involved in flash control.".
-Fig2B: on which tissue was the qPCR experiments performed?
Response: For each dsRNA treatment group, extracted total RNA from the whole body.We have added the missing content to the line 783-784 of the Methods section.
-Fig2E is confusing and would gain visibility by changing the colour or pattern or size Response: Thank you for your suggestion.According to the reviewer's comment, we have deleted Fig. 2D and Fig. 2E, and moved these data to Supplementary Fig. 6.
-page12, line256: a sentence seems missing between "verified36" and "Analysis…", it seems to weaken the following description.Response: The sentence (line 231) was changed to "Surprisingly, the expression level of AlUnc-4 decreased significantly in response to the knockdown of AlAbd-B (Fig. 3B).".page12 line23: "functional" instead of "function" Response: "function" was changed to "functional".page12: tense seems sometimes confusing (example line240: "AlLuc1 is located" instead of "was") Response: past tense was changed to present tense.page13, line 275: syntax "of AlLuc1 was down-regulated.." Response: Sentence was changed to "Verification of real-time qPCR confirmed that the expression level of AlLuc1 was down-regulated significantly in non-luminescent adults (Supplementary Fig. 8A).".

Response to reviewer #2:
This is an interesting study that provides new insight in the regulation of firefly luminescence.Given the availability of 4 already published firefly genomes, it is not clear why they needed to sequence a different one.Is it a better model system?Does it provide a different perspective on the problem?
Response: ①The genome of A.leii is the first sequenced aquatic firefly genome, while the other 4 already published firefly genomes are from terrestrial fireflies.②We have been able to raise A.leii in large numbers in the lab, which will provide us with sufficient experimental materials such as pupae to study development of adult light organs.Thus make A. leii as a better model system to study adult light organ development and flash control.The other 4 species of fireflies are difficult to be raised, i.e they can only be collected in the wild.③We used single-molecule Nanopore sequencing and Hi-C technologies for genome sequencing and assembly, with an N50 125Mb, resulting in a higher-quality genome compared to the other 4 genomes.
The genome sequencing, quality control and gene annotation follow standard protocols and provide the information required for the current study.
The work is presented in a linear fashion and the overall meaning is clear.However the word usage and grammar need additional attention.The follow represent only a few instances, where the clarity of the argument was obscured.
Response: Thank you for your careful review.We appreciate the reviewer's positive evaluation of our work.We are very sorry for the mistakes in this manuscript and for the inconvenience they caused.
According to your advice, we amended the relevant parts of manuscript.In addition, the manuscript has been thoroughly revised and rewritten by a native English speaker, so we hope it can meet the journal's high standards.
-Line 49 sifted should probably be filtered.
Response: changed to "In this study, we present a chromosome-level genome assembly for Aquatica leii Fu and Ballantyne, a rare aquatic firefly, using single-molecule Nanopore sequencing and high-throughput chromosome conformation capture sequencing technologies.We also present the functional studies and analyses of two key homeobox transcription factors regulating the adult light organ development in A. leii.".
-Line 165 no significant difference Response: Changed to " The depletion of AlUbx transcript resulted in pale yellow hindwings, but it did not produce any significant impacts on the development of adult light organs and flash behavior (Supplementary Fig. 6A-D and Supplementary Video 1).".
-line 215 and 217 is possibly Response: Sentences were rewritten to " Similarly, the expression of closely related genes, Alpex3, Alpex16 and Alpex19, which might be related to the transportation of Peroxisome Membrane Protein (PMP) 17, 18 , also decreased significantly in the dsAlAbd-B group.The expression of AlPxmp2, possibly related to the nonspecific transportation of small molecules 19 , decreased significantly in dsAlAbd-B and dsAlUnc-4 groups.".
Line 224 sifted should be filtered Response: "sifted" changed to "Candidate genes for more detailed analyses were selected using two criteria: high expression level (in the top 10% of genes) and the change in expression -log2FC (dsAlAbd-B/dsGFP) > 1.5.The selected genes comprised Alluc1, AlPx11c.1,AlPx11c.2,AlPx11c.2,AlPex5,AlPxmp2,AlPex13,AlPex14,AlPex16 and AlPex1.".Line 267 in female abdomens among with female Response: The sentence was changed to "Whereas AlLuc1 was specifically expressed in light organs (Supplementary Fig. 7A), AlLuc2 had the highest expression in female abdominal tissue (excluding the light organ) (Supplementary Fig. 7B).".Line 270 AlLuc1 is needed for transport to must be transported to Response: The sentence was changed to "To test our hypothesis that nonfunctional peroxisomes were directly caused by the lack of AlLUC1 protein, we applied RNAi to the AlLuc1 gene.".Line 273 was interfered by was knock down by Response: "was interfered by" changed to "we applied RNAi to the AlLuc1 gene.".Line 322 Need to included AlABDB in this sentence Response: The sentence was changed to "These results indicated that AlABD-B and AlUNC-4 are likely to be upstream regulators of AlLuc1.".

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): This reviewer thank the authors for addressing the issues step by step and improving the quality of the manuscript.The paper is in my opinion a new manuscript, so this reviewer has only evaluated the revised status.Some extra comments are provided below: Page 9: Why talking only about the qPCR in the female?How about the male?It is followed by the RNAi data, but it is not clear if it has been done in males or females.Same Fig2B, it is still not clear or written if it is data from male or female (legend, text).This needs further clarification in text and figure legend.
Fig4 YOH: mut1 & mut2 have different effects with alAbdB & AlUnc4 (fig4E-F) but this doesn't seem to correlate with the reporter assay (fig4G).Why?There are still several syntax problems that should be addressed in particular in the introduction and discussion.Just a few examples are listed but more are included.Line22 : syntax Line46 developmen Line 52-53 : fu ballantyne ?Line 483: organs Etc… Line527: The posterior Hox genes do not only suppress the function or more anterior one, but they also repressed their transcriptional expression Discussion: it would be interesting to discuss the decreased expression of unc4 with ds-abdB and how it would fit in the cartoon model.
Reviewer #2 (Remarks to the Author): The arguments in the manuscript are much clearer.The use of this model system is improved with the new genome, as demonstrated by the analysis of the adult lantern organ.

Response to reviewer #1:
This reviewer thank the authors for addressing the issues step by step and improving the quality of the manuscript.The paper is in my opinion a new manuscript, so this reviewer has only evaluated the revised status.Some extra comments are provided below: Page 9: Why talking only about the qPCR in the female?How about the male?It is followed by the RNAi data, but it is not clear if it has been done in males or females.
Answer: For this issue, we hope to explain it through four parts: experimental purpose, experimental design, experimental results, and supplementary experiments.
1. Experimental purpose: The experimental purpose was to identify key homeobox transcription factors among different light organ developmental stages by comparative transcriptome analysis, and apply them in subsequent functional validation studies.2. Experimental design: It is very difficult to get pupae directly in the wild, and it is also difficult to raise fireflies in the laboratory.It is necessary to retain sufficient female adults for reproduction.Therefore, in our experimental design, we only selected the material of male pupae as the research object.The larger area of the male light organ is another key factor that we took into account.

Experimental results:
From the final results of the experiments, we have screened several homeobox transcription factors related to the development of light organ, achieving our experimental purpose.4. Supplementary experiments: Following the reviewer's comment, we made an effort to conduct additional experiments.We used qPCR (instead of the transcriptional method in the experimental design) to explore the expression patterns of 8 candidate homeobox transcription factors at different developmental stages in the female light organ (page 9 lines 155-161, and Supplementary Fig. 5B). 5.In the RNAi experiments, in order to ensure the reproducibility of the experiment, both sexes were analyzed, and the same or similar stable phenotypes were obtained in both males and females.Finally, as suggested by the reviewer, we revised the text in this round of revision to specify the sex of specimens used for the experiments in the text and figure legends.In the discussion, we mentioned these problems briefly in the limitations paragraph: "Compared with other model insects, firefly breeding is relatively difficult, which limits our research.For example, transcriptomic data from different developmental stages are available for males, but not for females.In the follow-up study, we will strengthen the research on the differences between the two sexes in fireflies.".Same Fig2B, it is still not clear or written if it is data from male or female (legend, text).This needs further clarification in text and figure legend.
Response:Thank you for your comment.Due to the lack of depth of the PTS2-type protein in this research, we removed the discussion about this content completely.-page13,line 286: how about the protein level of luciferase?We deeply appreciate the suggestion.According to the reviewer's comment, we added a Western blot to detect the protein level of luciferase in different dsRNA-treated groups.The result was shown in Supplementary Fig. 8.The results indicate that treatment with dsAlPex13 did not affect the expression level of AlLUC1, but the expression level of AlLUC1 was significantly down regulated in the dsAlAbd-B and dsAlUNC-4 treatment groups.Page10, ine 200-202: the syntax of the sentence is confusing, same line 215-216, 217-218, 227-228 Response: The sentence (line 200-202) was changed to "To further study the regulation of the development of adult firefly light organs, we focused on AlABD-B and AlUNC-4 as putatively key transcription factors.".Sentences (line 215-218) were changed to " Similarly, the expression of closely related genes, Alpex3, Alpex16 and Alpex19, which might be related to the transportation of Peroxisome Membrane Protein (PMP)17, 18 , also decreased significantly in the dsAlAbd-B group.The expression of AlPxmp2, possibly related to the nonspecific transportation of small molecules 19 , decreased significantly in dsAlAbd-B and dsAlUnc-4 groups.".Same page10, Line 221: "were assessed" insteatd of "were detected" would provide better clarity Response: The word "detected" was changed to "assessed".line 222, 226, 230: citing the fig 3B here would help the reader Response: Fig. 3B was cited.page11, line 231: syntax is confusing