Akhirin Functions as an Innate Immune Barrier to Preserve Neurogenic Niche Homeostasis During Mouse Brain Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper by Kudo el al. entitled “Akhirin functions as an innate immune barrier to preserve neurogenic niche homeostasis during mouse brain development” investigates the akhirin (AKH), a soluble molecule expressed during embryonal development that disappears by postnatal day 30 in mice. In this study, the authors demonstrate that AKH maintains the integrity of the neural stem cells (NSC) niche during mouse brain development. The results showed that AKH deficient mice had increased expression of inflammatory cytokines in the cerebrospinal fluid (CSF). This suggests that AKH acts as a barrier molecule that preserves NSC homeostasis.

The first set of findings indicated that AKH is localized in the ventricular zone, adjacent to the ventricular surface and the choroid plexus (ChP) at E14.5 mice brain. At this stage, mice brains were also immunostained for PHH3, a marker for dividing cells and Iba1, a marker for both microglia and macrophages. The data revealed a reduction in proliferative cells in the subventricular zone of the lateral ventricle of AKH⁻/⁻ mice brains compared with those of AKH⁺/⁺ mice. However, immunostaining for Iba1 showed an increase in Iba1⁺ cells in the VZ of AKH⁻/⁻ mice. Using immunostaining for a macrophage marker (CD206) and a microglia marker (P2RY12), AKH⁻/⁻ mice brains were found to contain CD206⁺/P2RY12⁻cells, suggesting that a macrophage-dominant population capable to destabilize the NSCs/NPCs microenvironment and reducing proliferation. The data presented are solid and appropriately interpreted, but I have serious reservations about the next series of experiments, particularly those aimed to examine neural migration and differentiation.

Specifically, to study cell migration, 16.5-day-old pregnant mice and postnatal day P1 mice were injected with BrdU, and brains from the embryos and P1 pups were collected 30 minutes later. E16.5 brains were immunostained for BrdU and Tbr1, and the cortical layer lengths containing BrdU+ and Tbr1+ cells were measured, which the authors interpreted as representing the SVZ and cortical plate, respectively. However, this interpretation is not accurate. BrdU marks cells in S-phase, which lasts only a portion of the neural progenitor cell cycle (approximately 4-6 hours of a 12-18-hour cycle), and therefore BrdU labels only a fraction of cells in the SVZ. To accurately distinguish the SVZ, intermediate zone would also need to be marked. Additionally, more reliable quantitative data would be obtained by counting BrdU+ and Tbr1+ cells and estimating their densities rather than relying solely on cortical layer length measurements. Furthermore, the results showed that at P1, the number of Sox2+ and BrdU+ double-labeled cells was reduced in the SVZ in AKH⁻/⁻, supporting the conclusion that “it was thought that cell migration was delayed in AKH⁻/⁻”. At E16.5, intense neurogenesis continues to occur, with newly born neurons forming the upper layers (layers 2/3). Most proliferating cells differentiate into neurons and migrate through the deep layers before reaching their final positions in layer 2/3. In mice, the formation of the six-layered cortex begins around at E12 and continues until E18. Therefore, by P1 mice the cortical layers are already established. Although proliferation is still observed at this developmental stage, it is primarily due to ongoing gliogenesis, which does not contribute to further cortical layer formation.

To assess cell migration I propose the following experimental design: BrdU should be injected into 16-day-old pregnant mice, and embryonic brains should be collected at different post-injection points, for example, 24, 48 and 96 h post injection, and the number of BrdU+ cells in each cortical layer should be determined. Cell differentiation should also be examined in these brains using BrdU with cell type markers.

Overall, the development of brain structures in AKH⁻/⁻ mice needs to be examined in greater depth, as the current findings suggest that the neocortex, and potentially other brain regions, may be structurally altered.

Minor

- The Materials and Methods section includes a description of the "Mouse Model of Ischemic Stroke," but the main text does not provide any results describing this method. Only the Appendix Figure 4, presents some data.

 

Author Response

Reviewer1

Although intrinsic proliferative capacity of NSCs appeared intact under defined culture conditions, the marked reduction in proliferation observed in vivo at E14.5 suggested the involvement of inhibitory extrinsic factors within the AKH⁻/⁻ neurogenic niche. Given that at E14.5 the CSF is in direct contact with the apical surface of NSCs during early brain development, we hypothesized that alterations in CSF composition may contribute to the observed NSCs dysfunction.

The paper by Kudo el al. entitled “Akhirin functions as an innate immune barrier to preserve neurogenic niche homeostasis during mouse brain development” investigates the akhirin (AKH), a soluble molecule expressed during embryonal development that disappears by postnatal day 30 in mice. In this study, the authors demonstrate that AKH maintains the integrity of the neural stem cells (NSC) niche during mouse brain development. The results showed that AKH deficient mice had increased expression of inflammatory cytokines in the cerebrospinal fluid (CSF). This suggests that AKH acts as a barrier molecule that preserves NSC homeostasis.

Comments1: The first set of findings indicated that AKH is localized in the ventricular zone, adjacent to the ventricular surface and the choroid plexus (ChP) at E14.5 mice brain. At this stage, mice brains were also immunostained for PHH3, a marker for dividing cells and Iba1, a marker for both microglia and macrophages. The data revealed a reduction in proliferative cells in the subventricular zone of the lateral ventricle of AKH⁻/⁻ mice brains compared with those of AKH⁺/⁺ mice. However, immunostaining for Iba1 showed an increase in Iba1⁺ cells in the VZ of AKH⁻/⁻ mice. Using immunostaining for a macrophage marker (CD206) and a microglia marker (P2RY12), AKH⁻/⁻ mice brains were found to contain CD206⁺/P2RY12⁻cells, suggesting that a macrophage-dominant population capable to destabilize the NSCs/NPCs microenvironment and reducing proliferation. The data presented are solid and appropriately interpreted, but I have serious reservations about the next series of experiments, particularly those aimed to examine neural migration and differentiation.

Response 1: We sincerely appreciate the reviewer's insightful comments and valuable suggestions. In response to this comment, we have revised our analysis and added new quantitative data.

Specifically, we have now counted the number of BrdU⁺ cells and Tbr1⁺ cells present within each cortical layer, rather than relying solely on cortical layer length measurements. These quantitative results have been incorporated into Figures 3B and 3C, and the corresponding descriptions in the Results section have been revised accordingly (Lines: 507-511).

Comments2: Specifically, to study cell migration, 16.5-day-old pregnant mice and postnatal day P1 mice were injected with BrdU, and brains from the embryos and P1 pups were collected 30 minutes later. E16.5 brains were immunostained for BrdU and Tbr1, and the cortical layer lengths containing BrdU+ and Tbr1+ cells were measured, which the authors interpreted as representing the SVZ and cortical plate, respectively. However, this interpretation is not accurate. BrdU marks cells in S-phase, which lasts only a portion of the neural progenitor cell cycle (approximately 4-6 hours of a 12-18-hour cycle), and therefore BrdU labels only a fraction of cells in the SVZ. To accurately distinguish the SVZ, intermediate zone would also need to be marked. Additionally, more reliable quantitative data would be obtained by counting BrdU+ and Tbr1+ cells and estimating their densities rather than relying solely on cortical layer length measurements. Furthermore, the results showed that at P1, the number of Sox2+ and BrdU+ double-labeled cells was reduced in the SVZ in AKH⁻/⁻, supporting the conclusion that “it was thought that cell migration was delayed in AKH⁻/⁻”. At E16.5, intense neurogenesis continues to occur, with newly born neurons forming the upper layers (layers 2/3). Most proliferating cells differentiate into neurons and migrate through the deep layers before reaching their final positions in layer 2/3. In mice, the formation of the six-layered cortex begins around at E12 and continues until E18. Therefore, by P1 mice the cortical layers are already established. Although proliferation is still observed at this developmental stage, it is primarily due to ongoing gliogenesis, which does not contribute to further cortical layer formation.

To assess cell migration I propose the following experimental design: BrdU should be injected into 16-day-old pregnant mice, and embryonic brains should be collected at different post-injection points, for example, 24, 48 and 96 h post injection, and the number of BrdU+ cells in each cortical layer should be determined. Cell differentiation should also be examined in these brains using BrdU with cell type markers.

Overall, the development of brain structures in AKH⁻/⁻ mice needs to be examined in greater depth, as the current findings suggest that the neocortex, and potentially other brain regions, may be structurally altered.

Response 2: We thank the reviewer for the careful and thoughtful evaluation of our manuscript and for the constructive comments, which have helped us to improve the clarity and rigor of the study. In light of this, and to avoid overinterpretation, we have revised the manuscript to refrain from using the term “cell migration.” Instead, we now describe our findings more conservatively as abnormalities in cortical development and neurogenic progression in AKH⁻/⁻ mice. While the data indicate altered spatial distribution of BrdU⁺ and lineage-marker–positive cells, we agree that definitive conclusions regarding migratory dynamics would require pulse–chase experiments across multiple time points, as suggested by the reviewer.

Importantly, we note that a more detailed investigation of cortical layer formation and neuronal positioning is currently ongoing in a separate study, and therefore such in-depth analyses are beyond the scope of the present manuscript.

Minor

- The Materials and Methods section includes a description of the "Mouse Model of Ischemic Stroke," but the main text does not provide any results describing this method. Only the Appendix Figure 4, presents some data.

We thank the reviewer for pointing this out. In the revised manuscript. We have added a description of the tMCAO experiments to the adult section of the Results (Figure 5, Lines:622-634), so that the ischemic stroke model described in the Materials and Methods is now directly accompanied by corresponding results in the main text. Data that were previously shown only in Appendix Figure 4 are now explicitly addressed in the Results section.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of this manuscript show that Akhirin (AKH) secretion supports NSCs/NPCs during mid-stage development. The origin of AKH resides on the apical surface of both the choroid plexus and the ventricular wall. Ablation of AKH increases CSF levels of inflammatory cytokines. Furthermore, a loss of coordination between NSC proliferation and their differentiation. In addition, AKH binds to and inhibits bacterial spread through its LCCL domain. The manuscript fills a gap in neurodevelopmental research by presenting original data showing that AKH maintains immune balance at the developing brain-CSF interface. Overall, this manuscript adds to the field by showing that AKH plays a role in regulating the immune balance to control neurostem cell/progenitor cell proliferation and differentiation.

The proper controls have been carried out. As noted by the authors, the main improvement would be to use a mouse model in which AKH expression is controlled spatially and temporally in contextually in specific neural cells.

The authors' findings are consistent with their data and their interpretations addressing their main question. Overall, their findings suggest that AKH serves as a "molecular barrier" in the brain during development, attenuating the innate immune response to support neurogenesis. Finally, the authors impose limitations on their study by stating that a whole-body knockout could trigger systemic compensatory mechanisms. Their recommendations for future studies involve the use of cell- or tissue-specific spatial knockouts of AKH in the choroid plexus and astrocytes to examine the contextual cell-specific function of AKH.

The references are relevant and not excessive, giving the manuscript a strong focus on AKH.

Overall, the figures are appropriate, except that the text does not refer to Figures 5H and 5I or Appendix Figure 4A, which need to be corrected for the revision.

Author Response

Reviewer2

The authors of this manuscript show that Akhirin (AKH) secretion supports NSCs/NPCs during mid-stage development. The origin of AKH resides on the apical surface of both the choroid plexus and the ventricular wall. Ablation of AKH increases CSF levels of inflammatory cytokines. Furthermore, a loss of coordination between NSC proliferation and their differentiation. In addition, AKH binds to and inhibits bacterial spread through its LCCL domain. The manuscript fills a gap in neurodevelopmental research by presenting original data showing that AKH maintains immune balance at the developing brain-CSF interface. Overall, this manuscript adds to the field by showing that AKH plays a role in regulating the immune balance to control neurostem cell/progenitor cell proliferation and differentiation.

We sincerely appreciate the reviewer's insightful comments.

The proper controls have been carried out. As noted by the authors, the main improvement would be to use a mouse model in which AKH expression is controlled spatially and temporally in contextually in specific neural cells.

Thank you very much.

The authors' findings are consistent with their data and their interpretations addressing their main question. Overall, their findings suggest that AKH serves as a "molecular barrier" in the brain during development, attenuating the innate immune response to support neurogenesis. Finally, the authors impose limitations on their study by stating that a whole-body knockout could trigger systemic compensatory mechanisms. Their recommendations for future studies involve the use of cell- or tissue-specific spatial knockouts of AKH in the choroid plexus and astrocytes to examine the contextual cell-specific function of AKH.

The references are relevant and not excessive, giving the manuscript a strong focus on AKH.

We thank the reviewer for the kind comments.

Overall, the figures are appropriate, except that the text does not refer to Figures 5H and 5I or Appendix Figure 4A, which need to be corrected for the revision.

We are sorry about it. References to Figure 5H and 5I have been corrected in the revised manuscript (Line:615). In addition, we note that the figure lettering in the original submission contained an error, which has now been fixed; the corresponding panels are currently labeled as Figure 5G and 5H, and all in-text citations have been updated accordingly (Lines:613-615). Regarding Appendix Figure 4A, these data have now been incorporated into the main text (Lines:622-634) and are presented as Supplementary Figure 5.

Reviewer 3 Report

Comments and Suggestions for Authors

In the article "Akhirin functions as an innate immune barrier to preserve neurogenic niche homeostasis during mouse brain development," the authors show the regulatory role of achirin, which controls the anti-inflammatory status in the neurogenic niche. The article is devoted to an urgent area of research in the biology of neurogenic niches and is of great interest. The following comments were made during the review.
1. In the introduction section, the authors convincingly and consistently describe the features of neurogenesis in the neurogenic niche, justifying the need for further study of the regulation of neurogenesis. The authors convincingly describe the composition of neurogenic niches and substantiate the involvement of achirine in the regulation of homeostasis and protection against changes in the composition of cerebrospinal fluid. In general, this section does not cause any comments.
2. In the Materials and Methods section, the authors describe in detail the experimental procedures related to the design of the article, however, the model of ischemic stroke is not described in sufficient detail. It is necessary to provide a more complete description of the experimental research protocol in this section.
3. The section of experimental administration of bromodeoxyuredin also needs to be detailed and a more precise description of the research protocol. In the sections Obtaining antibodies to achirine, it is also necessary to specify the procedure for conjugation with hemocyanin.  This section is not described enough. 
4. The section profiling cytokines in cerebrospinal fluid also needs to be detailed and described in more detail about the use of the RayBio® C-Series Mouse Cytokine Antibody Array. The authors should explain the cytokine detection process step by step. 
5. It is necessary to describe how quantitative proteomic analysis of mouse cerebrospinal fluid samples was performed without using a label. This information is missing from the presented version of the article.
6. The authors should specify the group sizes for statistical analysis and provide a justification for choosing the t-criterion for quantitative assessment. 
7. In the results section, the authors provide references to previous studies in which they investigated the expression of AKH mRNA in neurogenic niches. This information should be moved to the Discussion section, and the results of this study should be clearly presented in the effective part. 
8. In section 3.1, the authors should quantify the expression of achirin in cells. For this purpose, the author should calculate the number of cells in the profile field and present the average values. Without quantification, IHC research data is largely descriptive. 
9. In section 3.2, the authors should also transfer previously established information to the discussion section in order not to overload the effective part and enable the reader to better understand what was new in this study. The results of the quantitative analysis are not clear, in the captions to Fig. 2B It is necessary to clarify which groups of proliferation were compared. 
10. In the Discussion section, the authors, on the contrary, need to add links to relevant literature sources after the statement "emphasizing that transient intrauterine inflammatory disorders in the womb can have long-term behavioral consequences." The authors also need to reinforce and discuss the further development of their ideas with well-known literature. Many of the statements presented in the Results section and supported by references (see the comments above) need to be moved to the Discussion section. In general, the Discussion section needs a more detailed discussion of the results obtained by the authors with the data from the available literature. In the presented version, it is not enough to discuss the numerous effects obtained during experimental studies.

Comments on the Quality of English Language

English can be improved

 

Author Response

Reviewer3

 

In the article "Akhirin functions as an innate immune barrier to preserve neurogenic niche homeostasis during mouse brain development," the authors show the regulatory role of achirin, which controls the anti-inflammatory status in the neurogenic niche. The article is devoted to an urgent area of research in the biology of neurogenic niches and is of great interest. The following comments were made during the review.

 

We sincerely appreciate the reviewer's insightful comment.

1. 
   In the introduction section, the authors convincingly and consistently describe the features of neurogenesis in the neurogenic niche, justifying the need for further study of the regulation of neurogenesis. The authors convincingly describe the composition of neurogenic niches and substantiate the involvement of achirine in the regulation of homeostasis and protection against changes in the composition of cerebrospinal fluid. In general, this section does not cause any comments.

 

We thank the reviewer for the positive and encouraging evaluation of the Introduction section.

2. 
   In the Materials and Methods section, the authors describe in detail the experimental procedures related to the design of the article, however, the model of ischemic stroke is not described in sufficient detail. It is necessary to provide a more complete description of the experimental research protocol in this section.

 

We added the description for the model of ischemic stroke more detail (Lines:113-120).

3. 
   The section of experimental administration of bromodeoxyuredin also needs to be detailed and a more precise description of the research protocol. In the sections Obtaining antibodies to achirine, it is also necessary to specify the procedure for conjugation with hemocyanin.  This section is not described enough. 

 

We added the precise description for the experimental administration of BrdU more detail (Lines: 141-149).

4. 
   The section profiling cytokines in cerebrospinal fluid also needs to be detailed and described in more detail about the use of the RayBio® C-Series Mouse Cytokine Antibody Array. The authors should explain the cytokine detection process step by step. 

 

We added the precise description for the use of the RayBio® C-Series Mouse Cytokine Antibody Array more detail (Lines: 200-219).

5. 
   It is necessary to describe how quantitative proteomic analysis of mouse cerebrospinal fluid samples was performed without using a label. This information is missing from the presented version of the article.

 

We added the precise description for the label free quantitative proteomic analysis more detail (Lines: 286-346).

6. 
   The authors should specify the group sizes for statistical analysis and provide a justification for choosing the t-criterion for quantitative assessment. 

 

We are sorry about it. We thank you for pointing this out. In the revised manuscript, we have now clearly specified the sample sizes (n values) for all quantitative analyses in the figure legends and in the Statistical Analysis subsection of the Materials and Methods (Lines: 393-399).

7. 
   In the results section, the authors provide references to previous studies in which they investigated the expression of AKH mRNA in neurogenic niches. This information should be moved to the Discussion section, and the results of this study should be clearly presented in the effective part. 

 

We appreciate this comment. The references to our previous studies have been relocated from the Results to the Discussion (Lines: 752-759), and the Results section now clearly presents only the data generated in the current study.

8. 
   In section 3.1, the authors should quantify the expression of achirin in cells. For this purpose, the author should calculate the number of cells in the profile field and present the average values. Without quantification, IHC research data is largely descriptive. 

 

We appreciate this suggestion. We have now performed quantitative analysis of AKH expression by counting AKH-positive cells per Hoechst signals in one neurosphere, and the averaged data are presented as a new graph in Figure 1E (Lines 418-420).

 

9. In section 3.2, the authors should also transfer previously established information to the discussion section in order not to overload the effective part and enable the reader to better understand what was new in this study. The results of the quantitative analysis are not clear, in the captions to Fig. 2B It is necessary to clarify which groups of proliferation were compared. 

 

In accordance with the comment, we have removed the previously established background information in Section 3.2 and the information already included in the Discussion (Line:780-781. In addition, we have clarified the quantitative analysis of proliferating cells in the caption of Figure 2B (Lines:442-444).

10. 
    In the Discussion section, the authors, on the contrary, need to add links to relevant literature sources after the statement "emphasizing that transient intrauterine inflammatory disorders in the womb can have long-term behavioral consequences." The authors also need to reinforce and discuss the further development of their ideas with well-known literature. Many of the statements presented in the Results section and supported by references (see the comments above) need to be moved to the Discussion section. In general, the Discussion section needs a more detailed discussion of the results obtained by the authors with the data from the available literature. In the presented version, it is not enough to discuss the numerous effects obtained during experimental studies.

We thank you for this important and constructive comment. We have revised the Discussion extensively by adding relevant references to the statement regarding long-term behavioral consequences of intrauterine inflammation (Line:702-709) and by integrating additional well-established literature throughout the section (Lines:734, 736, 745). Moreover, reference-supported background information has been moved from the Results to the Discussion to better distinguish novel findings from contextual interpretation. As a result, the Discussion now provides a more comprehensive and literature-grounded interpretation of our experimental results.

Reviewer 4 Report

Comments and Suggestions for Authors

This interesting paper reports results from a study that identified akhirin as a key regulator in neurogenic niche in the developing murine CNS. If akhirin, being secreted from choroid plexus endothelial cells, is lost, neural stem cell homeostasis is disrupted. Akhirin can also provide innate immune response support after being cleaved. The LCCL domain is essential for this.

 

The paper is very interesting and timely, features a broad spectrum of relevant methods and reveals meaningful results plus some mechanistic insight. It is of clear interest for the journal’s audience.

 

However, a few aspects require the authors’ attention. Specifically:

 

Major aspects

(1) Much more information must be presented around animal experiments. Please report animal numbers, sex, strain, age, time of sacrifice and other relevant information. Please also report details about the applied perioperative pain management regimen. How was proper MCAO occlusion monitored? Were there animal exclusion and inclusion criteria? Please also report the number of excluded or dropped-out animals with reasons.

 

(2) It is not clear from the results which experiments were conducted in stroke animals. This must be clarified and made very clear.

 

(3) Please report the n in each experiment. Authors should also present individual data points, not such bar charts.

 

(4) The statistics must be improved. First, SD, not SEM is the proper error indicator (see doi: 10.2337/db13-0692 for details). Parametric tests were applied without confirming normal data distribution. This may result in false-positive findings. Please check for normal data distribution (and report procedure) and use parametric and non-parametric tests as appropriate. Please note that reliably assessing normal distribution requires a minimum n of 5-6; non-parametric tests must be used for lower n. Finally, some analyses contained multiple testing procedures but only t tests were used. Results must be corrected for multiple testing.

 

(5) Results of anti-akhirin antibody specificity tests should be shown in a supplement.

 

(6) Why was behavior assessed during the light phase, which is the animal’s resting phase? This may have concealed performance. Please explain. Please also provide rationale for the individual items of the behavioral test battery - which domains of cognitive or motor function were to be addressed?

 

Minor aspects

(7) The manuscript is well written, especially considering that the authors are not native speakers. Nevertheless, there are many minor language issues:

 

• “von willebrand factor” (line 78) should read “von Willebrand factor”
• “then centrifuge” (line 196) should read “centrifuged”
• “This processed as described[20].” (lines 198-199 and elsewhere); the sentence is incomplete and not understandable in this form. Please rephrase.
• “Signal” (line 209; potentially also elsewhere) should read “A signal…” etc.
• “were used” (line 252); should read “were used:”
• “Bacteria culture” (line 271); should read “Bacterial culture”
• “generated AKH-specific antibody” (line 383); should read “… a … antibody”
• “Appendix figure” (multiple occasions) may better read “Supplementary figure”
• “western blotting” (line 391) may read “Western blotting”
• “Blanching points” (A. Figure 1H) should read “Branching points”
• “Bar” should read “Scale bar” where appropriate (also applies to plural use)
• “(C’ )” in A. Figure 4B: to what does this refer / what does it indicate?
• “conditining” (A. Fig. 3B) should read “conditioning”)
• “All test shows no” (legend A. Figure 3) must read “No test revealed a statically significant difference”
• Line 397: redundant blank space after full stop
• “It is indicating” (line 480) may read “This indicates”
• “…this delay in layer-one formation was thin around P1..” (lines 481-482); a delay can be short or long, not thin. It may also read “layer I” (Roman numbers for all cortical layers)
• “NSCs exposed to … displayed a significant reduction in BrdU-positive cells” (lines 521-522); should better read “NSCs exposed to … were less frequently labelled with BrdU” etc.
• “nerusphere” (Fig. 2C, 3E) must read “neurosphere”. It must also read “secondary”, not “secondry”

 

Please note these are just some examples, not a complete list. Authors should perform a thorough spell check, ideally with the help of a native speaker or AI, before resubmission. Any human or AI assistance should be indicated. This includes all items, the main manuscript, the supplements as well as figures and their legends.

 

Please also check for occasional case sensitivity issues, especially when referring to molecules (e.g., “Colchlin”). Please also make sure that all abbreviations are properly introduced at first use. I acknowledge there is a list of abbreviations in the supplements, but this could also be presented in the main manuscript. There are also many missing or redundant blank spaces and style inconsistencies (i.e., blank space use when indicating scale bar length).

 

(8) Authors may briefly discuss whether akhirin may also be important (or interesting as a tool or agent) for attempts to regenerate the adult CNS. This is trickier because many anatomical cues present during ontogenesis are not present there anymore (doi: 10.1016/j.neuroscience.2024.05.009)

 

(9) Please provide all full (i.e., uncropped) Western blots for the sake of transparency. This can be done as supplementary material.

 

(10) When referring to centrifugation, g and rpm are reported. Please exclusively report g.

 

(11) Please report any blinding or randomization procedures being applied.

 

(12) Some data, e.g. in Figure 2G, is not shown as mean and SEM. Please indicate what exactly is shown there.

 

(13) Figure 5A is generic and can be removed. Figure 6K is distorted (vertically stretched)

 

(14) Figures are of poor resolution in general. This is no drawback during the review process, but authors must make sure that high-res figures are available after potential article acceptance.

 

(15) All references come with double numbers.

 

(16) Please remove any unnecessary colored margins from bar charts.

Comments on the Quality of English Language

Please see comments to authors. There are many minor typos and unclear phrases.

Author Response

Reviewer4

This interesting paper reports results from a study that identified akhirin as a key regulator in neurogenic niche in the developing murine CNS. If akhirin, being secreted from choroid plexus endothelial cells, is lost, neural stem cell homeostasis is disrupted. Akhirin can also provide innate immune response support after being cleaved. The LCCL domain is essential for this.

The paper is very interesting and timely, features a broad spectrum of relevant methods and reveals meaningful results plus some mechanistic insight. It is of clear interest for the journal’s audience.

We sincerely appreciate the reviewer's insightful comments and valuable suggestions.

 

However, a few aspects require the authors’ attention. Specifically:

Major aspects

(1) Much more information must be presented around animal experiments. Please report animal numbers, sex, strain, age, time of sacrifice and other relevant information. Please also report details about the applied perioperative pain management regimen. How was proper MCAO occlusion monitored? Were there animal exclusion and inclusion criteria? Please also report the number of excluded or dropped-out animals with reasons.

 We have revised the Materials and Methods section to provide detailed information on animal numbers, sex, strain, age, time of sacrifice, perioperative care, and experimental design. For the tMCAO experiments, we now clearly describe anesthesia, temperature management, postoperative care, monitoring of occlusion success, inclusion and exclusion criteria, and the number and reasons for excluded animals. Randomization and blinding procedures have also been explicitly stated. These revisions address all concerns raised regarding the reporting of animal experiments.

(2) It is not clear from the results which experiments were conducted in stroke animals. This must be clarified and made very clear.

We thank the reviewer for pointing out this lack of clarity. We have revised the Results section to clearly specify which experiments were performed in tMCAO (stroke) animals, including the addition of a dedicated adult/tMCAO subsection and explicit clarification in the main text (Lines:622-634)

(3) Please report the n in each experiment. Authors should also present individual data points, not such bar charts.

We thank the reviewer for this important comment. We have updated all figure legends to report the n values for each experiment, and we now present individual data points in the graphs instead of bar charts alone.

(4) The statistics must be improved. First, SD, not SEM is the proper error indicator (see doi: 10.2337/db13-0692 for details). Parametric tests were applied without confirming normal data distribution. This may result in false-positive findings. Please check for normal data distribution (and report procedure) and use parametric and non-parametric tests as appropriate. Please note that reliably assessing normal distribution requires a minimum n of 5-6; non-parametric tests must be used for lower n. Finally, some analyses contained multiple testing procedures but only t tests were used. Results must be corrected for multiple testing.

We thank the reviewer for the detailed and constructive comments regarding the statistical analyses. We have carefully revised the manuscript to address all points raised. All data are now presented as mean ± SD instead of SEM. Next, for analyses involving multiple group comparisons, we have revised the statistical approach. Instead of using multiple pairwise t-tests, we now applied one-way ANOVA followed by appropriate post hoc tests for normally distributed data.These changes are now clearly indicated in the figure legends and Methods section (Lines:393-399). Importantly, while these revisions improved the rigor and transparency of the statistical analyses, they did not alter the overall conclusions of the study.

We believe that these comprehensive changes substantially strengthen the statistical validity of the manuscript and fully address the reviewer’s concerns.

 

(5) Results of anti-akhirin antibody specificity tests should be shown in a supplement.

 We thank the reviewer for this suggestion. The results of the anti-akhirin antibody specificity tests have now been added as Supplementary Figure 1.

(6) Why was behavior assessed during the light phase, which is the animal’s resting phase? This may have concealed performance. Please explain. Please also provide rationale for the individual items of the behavioral test battery - which domains of cognitive or motor function were to be addressed?

We thank you for raising this important point regarding behavioral testing.  Behavioral tests were conducted during the light phase for experimental consistency across groups, a widely accepted approach in mouse behavioral studies, with prior handling to minimize stress (Lines:341-346). We have also clarified the rationale for each behavioral assay in the Results section, specifying the cognitive, motor, and emotional domains assessed by each test (Lines: 601-605).

Minor aspects

(7) The manuscript is well written, especially considering that the authors are not native speakers. Nevertheless, there are many minor language issues:

We thank the reviewer for the careful reading of the manuscript and for the detailed and helpful language-related comments. All corrections have been implemented, and the corresponding revised lines are indicated in the response document.

• “von willebrand factor” (line 78) should read “von Willebrand factor”

Line:74

• “then centrifuge” (line 196) should read “centrifuged”

Line:197

• “This processed as described[20].” (lines 198-199 and elsewhere); the sentence is incomplete and not understandable in this form. Please rephrase.

Line:199

• “Signal” (line 209; potentially also elsewhere) should read “A signal…” etc.

Line:212

• “were used” (line 252); should read “were used:”

Line:255

• “Bacteria culture” (line 271); should read “Bacterial culture”

Line:274

• “generated AKH-specific antibody” (line 383); should read “… a … antibody”

Line:405

• “Appendix figure” (multiple occasions) may better read “Supplementary figure”

We changed all “Appendix figure” to “Supplementary figure”.

• “western blotting” (line 391) may read “Western blotting”

Lines:414, 415.

• “Blanching points” (A. Figure 1H) should read “Branching points”

We collected “Branching points” in Supplementary figure 2H.

• “Bar” should read “Scale bar” where appropriate (also applies to plural use)

We collected all “Bar” to “Scale bar” in all Figure legend.

• “(C’ )” in A. Figure 4B: to what does this refer / what does it indicate?

We collected just AKH in Supplementary figure 5C.

• “conditining” (A. Fig. 3B) should read “conditioning”)

We collected “conditining” to “conditioning” in Supplementary figure 4B.

• “All test shows no” (legend A. Figure 3) must read “No test revealed a statically significant difference”

We collected “All test shows no” to “No test revealed a statically significant difference” in legend of Supplementary figure 4.

• Line 397: redundant blank space after full stop

We collected it.

• “It is indicating” (line 480) may read “This indicates”

We deleted this sentence.

• “…this delay in layer-one formation was thin around P1..” (lines 481-482); a delay can be short or long, not thin. It may also read “layer I” (Roman numbers for all cortical layers)

We collected “Layer I” in Line:515.

• “NSCs exposed to … displayed a significant reduction in BrdU-positive cells” (lines 521-522); should better read “NSCs exposed to … were less frequently labelled with BrdU” etc.

We collected “NSCs exposed to … displayed a significant reduction in BrdU-positive cells” to“NSCs exposed to … were less frequently labelled with BrdU” (Lines: 556-558)

• “nerusphere” (Fig. 2C, 3E) must read “neurosphere”. It must also read “secondary”, not “secondry”

We collected in Figure 2C and 3E.

• Please note these are just some examples, not a complete list. Authors should perform a thorough spell check, ideally with the help of a native speaker or AI, before resubmission. Any human or AI assistance should be indicated. This includes all items, the main manuscript, the supplements as well as figures and their legends.

 We have thoroughly proofread the entire manuscript, including all figures, legends, and supplementary materials, with the assistance of AI-based language editing tools, and this assistance is now indicated in the manuscript.

• Please also check for occasional case sensitivity issues, especially when referring to molecules (e.g., “Colchlin”). Please also make sure that all abbreviations are properly introduced at first use. I acknowledge there is a list of abbreviations in the supplements, but this could also be presented in the main manuscript. There are also many missing or redundant blank spaces and style inconsistencies (i.e., blank space use when indicating scale bar length).

We thank the reviewer for these detailed stylistic and formatting suggestions. We have corrected case sensitivity issues, ensured that all abbreviations are defined at first use in the main text, and standardized formatting throughout the manuscript, including scale bar notation and spacing consistency.

(8) Authors may briefly discuss whether akhirin may also be important (or interesting as a tool or agent) for attempts to regenerate the adult CNS. This is trickier because many anatomical cues present during ontogenesis are not present there anymore (doi: 10.1016/j.neuroscience.2024.05.009)

We thank the reviewer for this insightful suggestion. In response, we have added a brief discussion on the potential relevance of AKH in adult CNS regeneration to the Discussion section and incorporated the suggested reference {49}.

(9) Please provide all full (i.e., uncropped) Western blots for the sake of transparency. This can be done as supplementary material.

 We thank the reviewer for this comment. All full-length (uncropped) Western blot images have already been provided in the Supplementary Materials to ensure transparency.

(10) When referring to centrifugation, g and rpm are reported. Please exclusively report g.

All centrifugation parameters are now reported exclusively in ×g.

(11) Please report any blinding or randomization procedures being applied.

 We thank the reviewer for this important comment. Information regarding randomization and blinding procedures has now been added to the Materials and Methods section.

(12) Some data, e.g. in Figure 2G, is not shown as mean and SEM. Please indicate what exactly is shown there.

Thank you for the comment. The data presentation in Figure 2G has been corrected, and the figure and legend now clearly indicate what is shown.

(13) Figure 5A is generic and can be removed. Figure 6K is distorted (vertically stretched)

Thank you for the comment. Figure 5A has been removed as suggested. The distortion in Figure 6K has been corrected, and the figure is now presented with the proper aspect ratio.

(14) Figures are of poor resolution in general. This is no drawback during the review process, but authors must make sure that high-res figures are available after potential article acceptance.

We appreciate this comment. All figures have been generated and uploaded in high-resolution format following the journal guidelines, ensuring appropriate quality for publication after acceptance.

(15) All references come with double numbers.

Thank you for pointing this out. We carefully checked all citations and reference formatting throughout the manuscript and corrected any inconsistencies to ensure that references are cited and numbered appropriately.

 

(16) Please remove any unnecessary colored margins from bar charts.

Thank you for the suggestion. Unnecessary colored margins have been removed from all bar charts, and the figures have been updated accordingly.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Although the manuscript addresses most of the comments, additional revisions are still required.

In the materials and methods section, the description of the 2. SDS-PAGE, 3. In-gel Digestion and 4.1. NanoLC procedures require revision. These subsections should be rewritten to describe the methodology rather than listing step-by-step instructions clearly. Additionally, SDS-PAGE is conducted in volts (V), not kilovolts (kV).

I suggest removing the results from experiments in which mice were injected with BrdU at P1 (Lines 511-515). The authors use these data to explain the formation of cortical layer I. However, cortical layer I forms during early embryonic development, and by P1, it is already established. All data and conclusions related to these experiments should be removed from the manuscript, including the abstract, materials and methods, results and discussion sections.

Alternatively, if the authors intend to study the development of cortical layer I, they should conduct additional experiments assessing Cajal-Retzius cells and non-Cajal-Retzius cells in both AKH+/+ and AKH-/- mice.

Author Response

Although the manuscript addresses most of the comments, additional revisions are still required.

In the materials and methods section, the description of the 2. SDS-PAGE, 3. In-gel Digestion and 4.1. NanoLC procedures require revision. These subsections should be rewritten to describe the methodology rather than listing step-by-step instructions clearly. Additionally, SDS-PAGE is conducted in volts (V), not kilovolts (kV).

We thank the reviewer for the helpful suggestion. We have rewritten the SDS-PAGE, in-gel digestion, and Nano LC–MS/MS and Data analysis subsections to describe the methodologies in a narrative format rather than as step-by-step instructions. In addition, the electrophoresis conditions for SDS-PAGE have been corrected from kilovolts (kV) to volts (V). These revisions have been made in the Materials and Methods section accordingly (Line,299-337).

 

I suggest removing the results from experiments in which mice were injected with BrdU at P1 (Lines 511-515). The authors use these data to explain the formation of cortical layer I. However, cortical layer I forms during early embryonic development, and by P1, it is already established. All data and conclusions related to these experiments should be removed from the manuscript, including the abstract, materials and methods results and discussion sections.

Alternatively, if the authors intend to study the development of cortical layer I, they should conduct additional experiments assessing Cajal-Retzius cells and non-Cajal-Retzius cells in both AKH+/+ and AKH-/- mice.

We thank the reviewer for this important and insightful comment. We agree that cortical layer I is established during early embryonic development and that analysis at P1 is not appropriate for assessing its formation. Accordingly, we have removed all data, descriptions, and conclusions related to the P1 experiments from the manuscript, including the Materials and Methods, Results, and Discussion sections.

We retained the analysis at P10 to demonstrate that the developmental delay in cortical layer formation observed during embryogenesis was compensated by the postnatal stage when cortical lamination is generally completed. The text has been revised to clarify this point and to avoid any interpretation related to layer I formation (Line, 506-512).

Reviewer 3 Report

Comments and Suggestions for Authors

The article has been improved enough and can be recommended for publication.

Comments on the Quality of English Language

English can be improved

 

Author Response

Comments and Suggestions for Authors

The article has been improved enough and can be recommended for publication.

We are grateful for the reviewer’s constructive feedback throughout the review process

English can be improved.

Thank you very much for your comment regarding the English language.

In response, we have carefully revised the manuscript to improve clarity and readability, including language editing with the assistance of AI-based tools.

All newly revised sections are highlighted in yellow to clearly indicate the changes made in this revision.