Gene Regulation during Carapacial Ridge Development of Mauremys reevesii: The Development of Carapacial Ridge, Ribs and Scutes
Round 1
Reviewer 1 Report
Kind regards,
Ms. Zinnia Liu
Assistant Editor
In consideration of the paper that I was entrusted to review entitled “Gene regulation during carapacial ridge development of Mauremys reevesii: the development of carapacial ridge, ribs and scutes” by Jiayu Yang , Yingying Xia , Shaohu Li , Tingting Chen , Jilong Zhang , Zhiyuan Weng , Huiwei Zheng , Minxuan Jin , Chuanhe Bao , Shiping Su , Yangyang Liang , Jun Zhang, I tell you that it is a current, detailed, novel and important paper for the readers of the journal and potentially for the experts in the area of study of turtles.
The paper shows us several aspects, the first was to establish an opportunity in the field of embryonic development to show that more studies were needed on the understanding of the metabolic processes in the generation of hard (bone) and soft (skin) shells.
The second aspect was to know the different stages in which the carapacial rigid can be studied and how to establish the collection of the sample in these 3 different stages of development of the shell.
The third thing is that we can use tools of molecular biology, bioinformatics and global databases of both proteins and nucleic acids to compare the results obtained with those previously obtained or to know those missing data and propose novel results at the time of conclusion.
Therefore, in my opinion, even when there is strong and constant evidence on the participation of various genes that turn on and off on what is called canonical and non-canonical Wnt signaling, it is essential to broaden the discussion on the fact of which are the factors that initiate the signaling in these routes to finally obtain a rigid or soft shell, apart from the genes already described in this work. Please extend the discussion of mechanism of stage 16 CR samples of 560 P. sinensis contained Col1a1, Col1a2, and Itga8. Could this mechanism also be inferred for Dermochelys coriacea?
Any idea if any of the pathways described in this paper are affected in those hatchling turtles whose plastron is not fully developed, that therefore the plastron does not "close" completely and the internal organs are seen? Explain briefly.
Linea 189: Get a better resolution of the formula used
Figue 3: Get a better resolution
Line 569: verify the word “reguating”
Line 577: verify the word “to15”
Author Response
Response to Reviewer 1 Comments
Point 1: It is essential to broaden the discussion on the fact of which are the factors that initiate the signaling in these routes to finally obtain a rigid or soft shell, apart from the genes already described in this work.
Response 1: Thank you for your recommendation. Most of factors that initiate the differentiation of hard shell turtle and soft shell turtle appear at stage 15. Different signaling pathways may lead to such differentiation, or different expression sites determine the CR development. For example, Moustakas demonstrated that the continuant and segmental expression of Fgf signal in soft-shell turtle and hard-shell turtle, respectively. Therefore, the analysis need more evidence, we can only make a small assumption for now. The additional explanation has been added in manuscript and highlighted in red. Such as,
Line 524-526: They also found the expression of Shh segmented pattern were lost in CR with inhibition of Bmp and Hedgehog signaling. Besides, this Shh segmented expression were also absent in embryo of P. sinensis [16].
Line 550-552: Experiments disturbed Shh, Bmp and Fgf signaling, leading to the destruction of segmental pattern expression and scutes development [16].
Line 598-599: In addition, the different expression location of key genes expressed in soft shell turtle and hard shell turtle also contribute to this differentiation.
Point 2: Please extend the discussion of mechanism of stage 16 CR samples of 560 P. sinensis contained Col1a1, Col1a2, and Itga8.
Response 2: Thank you for your recommendation. This has been added to lines 586-594 and is highlighted in red.
Point 3: Any idea if any of the pathways described in this paper are affected in those hatchling turtles whose plastron is not fully developed, that therefore the plastron does not "close" completely and the internal organs are seen? Explain briefly.
Response 3: Thank you for your recommendation. In this study, the transcriptome of carapacial ridge was analyzed. It only appears in embryonic stage 14 and stage 16, and may be involved in the development of ribs and scutes, but no clue was found to regulate in plastron closing. Thus, even in the absence of CR, internal organs may remain invisible.
Point 4: Linea 189: Get a better resolution of the formula used
Figue 3: Get a better resolution
Line 569: verify the word “reguating”
Line 577: verify the word “to 15”
Response 4: Thank you for your recommendation. We changed the following points.
- It has been revised in the original manuscript.
- It has been revised in the original manuscript.
- It has been revised in the original manuscript.
- It has been revised in the original manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Gene regulation during carapacial ridge development of Mauremys reevesii: the development of carapacial ridge, ribs and scutes by Yang et al presents the original study on the carapace development of turtles.
It is generally well designed and and it can be improved in below points.
There are some typho mistakes such as double dot in Figure 4 legand and there is no space between profile2 in some cases. These needs to be corrected.
One more thing is, the manuscript is so long and there are many analyses but in the results and discussion section, there is a need to stress on the differences among the stages and different genes. This would be very useful.
The manuscript can be shortened in general.
Author Response
Thanks for editor and reviewers’ comments. Please find our point-by-point response after reviewers’ comments
Manuscript “Transcriptomic Analysis …… Setaria viridis” deals with the RNA-Seq based analysis of EOD-FR light conditions dynamics. Authors identified several differentially expressed genes related to EOD-FR and chilling stress. They also try to study the regulatory network involved in EOD-FR signaling and chilling tolerance in C4 plants.
The study is good, but I have few concerns as follows:–
- Setaria viridis is commonly considered weed in the temperate region, authors have to justify the choice of plant its usage and why they selected for the chilling and EOD-FR stress?
Response: Thanks for the comments. Setaria viridis belongs to Poaceae and monocotyledonous plant, closely related to important crops such as maize, foxtail millet and sorghum. Compared with its relatives, Setaria viridis has many advantages, such as small stature, fast life cycle, prolific seed production, simple growth requirements, small genome and high efficiency by Agrobacterium-mediated transformation. Hence, it is very suitable for performing experiments in greenhouse, such as the chilling and EOD-FRtreatment.
- For the study of networking regulation and other mechanism, a model plant can also be used for such study?
Response: Thanks for the comments. In the evolution, Setaria viridis is closely related to maize and foxtail millet. Partial genes may be performed highly conserved function or involved in similar biological processes. As a model plant of C4, using Setaria viridis to study networking regulation and other mechanisms makes it easier to found the genes related to the traits we are interested in, and thereby laying a theoretical foundation for improving crops such as maize and foxtail millet. In our study, we found the DEGs, and then constructed the regulatory network under chilling stress and far red treatment. Key genes that may be participate in these two stresses have been discovered. For instance, the gene function of BBX2 orthologous gene from maize have been validated. The overexpression of BBX2 could enhance the cold tolerance in Setaria viridis.
- Section, 2.2: Why RT-qPCR Assays were performed?
Response: Thanks for the comments. In the previous version, we performed RT-qPCR assays to determine the relative expression level of ZmBBX2 in Setaria viridis after chilling and FR treatment. The result was exhibited in Fig. 8E.
However, considering the integrity of the story, ZmBBX2 acting as the homologous genes of BBX2 in maize is incongruity. Thus, we have decided to remove the analysis related to ZmBBX2, which will be reported in another paper.
- Are authors performing before RNASeq? Why?
Response: Thanks for the comments. In the previous version, we performed RT-qPCR assays after RNA-seq. Firstly, acting as a key candidate gene, BBX2 was founded through RNA-seq. Subsequently, we performed RT-qPCR assay to detect the expression pattern of BBX2 under cold stress and far red treatment.
However, we have removed the results about ZmBBX2 overexpression lines. Thus, no content about RT-qPCR assay in this version.
- Section 2.3: How many plants were used for RNASeq?
Response: Thanks for the comments. Totally 78 samples from three biological replicates under different conditions were performed RNA-seq. Each sample contains 10 individual seedlings above ground.
- What were different stress conditions? Not clear
Response: We designed three stress conditions to treat Setaria viridi. The three stress conditions were cold, EOD-FR, cold and EOD-FR.
- What was reference? Control?
Response: Thanks for the comments. The control is the RNA-Seq data coming from 25°C growth condition.
- Why they selected and overexpressed ZmBBX2 gene in Setaria viridi?
Response: Thanks for the comments. Based on transcriptome data analysis, we demonstrated that the expression of SvBBX2 is induced by low temperature and EOD-FR in Setaria viridi. In the evolution, Setaria viridis is closely related to maize. SvBBX2 orthologous gene from maize were identified and named ZmBBX2. To clarify whether the function of BBX2 performed conservation in different species, we detect the function of ZmBBX2 in Setaria viridis in order to apply to improve the performance of maize in the future.we cloned homologous gene in maize and generated overexpression lines.
- A gene responsible for chilling and EOD-FR signaling/stress may selected and overexpressed? Why a gene from Zea mays
Response: Thanks for the comments. The expression of SvBBX2 is elevated under chilling and EOD-FR conditions (Fig. 7C). Then, we selected SvBBX2 orthologous gene from maize to do further study. On the one hand, we would like to pay more attention to the evolutionary conservation between Setaria viridis and maize. On the other hand, we expect to discover genes that involved in maize trait improvement.
- Discussion is too short.
Response: Thanks for the comments. We have added more content in the section of discussion in lines 469-475, 481-484, 494-498 and 503-507.
- No conclusion?
Response: Thanks for the comments. The conclusion was added in lines 520-526.
A large number of independent and interactive genes in response to chilling stress and EOD-FR signaling were identified. Subsequently, regulatory network was constructed using well-designed transcriptomics study. BBX2 was identified as responsive to chilling and EOD-FR through the coexpressiong network analysis. In addition, this dataset will be useful for unravelling the signaling network related to these two environmental factors.
- In Results there is no information about RT-qPCR Assays, which was done in section 2.2
Response: Thanks for the comments. The part of RT-qPCR assays have been removed.
In the previous version, the result of RT-qPCR was exhibited in Fig. 8E.
Author Response File: Author Response.docx
