Author response:
The following is the authors’ response to the original reviews.
We thank the Editors for the positive assessment on our manuscript. We also thank the Reviewers for their positive remarks and constructive comments. Based on the Reviewers’ feedback, we have conducted additional experiments and provided supporting data to address Reviewers’ comments. Particularly, we provided quantitative measurement for rotational polarity of ependymal cells in Agbl5M1/M1 mutants and assessed the microtubule polarization. We quantified the intensity of apical actin network in ependymal cells to strength the role of CCP5 in organizing actin network. Using scanning electron microscopy, we demonstrated the affected polarity of trachea multicilia in Agbl5M1/M1. We co-immunostained ependymal cilia with GT335 and acetylated tubulin to address the effects on their length in cilia in the mutant. We assessed the presence and length of primary cilia in ependymal cell progenitors to identify their potential contribution to the defective polarity in Agbl5M1/M1 ependymal cells. We feel that these revisions have much strengthened this MS.
Public Reviews:
Reviewer #1 (Public review):
Summary:
Dad et al. explored the roles of cytosolic carboxypeptidase 5（CCP5）in the development of ependymal multicilia in the brain. CCP family are erasers of polyglutamylation of ciliary-axoneme microtubules. The authors generated a new mutant mouse of Agbl5 gene, which encodes CCP5, with deletion of its N-terminus and partial carboxypeptidase (CP) domain (named AGBL5M1/M1).
Strengths:
The mutant mice revealed lethal hydrocephalus due to degeneration of ependymal multicilia. Interestingly, this is in contrast with the phenotype of Agbl5 mutants with disruption solely in the CP domain of CCP5 (named AGBL5M2/M2) that did not develop hydrocephalus despite increased glutamylation levels in ependymal cilia as observed for AGBL5M1/M1 mutants. The study has been well-performed and the findings suggest a unique function of the N-domain of CCP5 in ependymal multicilia stability.
Weaknesses:
The content of this article is relatively descriptive and lacks molecular insights.
We thank the Reviewer’s positive comments. To address the molecular insights of the dysregulated planar cell polarity (PCP) in Agbl5M1/M1 ependyma, we have conducted additional experiments to assess the microtubule polarization in ependymal cells (Figure 7O-P). We quantified the intensity of actin networks around BB patches to better understand how it is affected in the ependyma of the mutants and contributes to the dispersion of BBs (Figure 4M-N), (Please see Recommendations for the authors).
We also assessed trachea multicilia in Agbl5M1/M1 mutants using SEM and found that the polarity of trachea multicilia was affected as well (Figure S2).
Reviewer #2 (Public review):
Summary:
This study analyzed the consequences of Agbl5 mutation on ependymal cell development and function. The authors first characterize their mutant mouse line reporting a reduced lifespand and severe hydrocephalus. Next, they report a defect in ependymal cell cilia number and motility. They provide evidence for impaired basal body organisation and cilia glutamylation.
Strengths:
Description of a mutant mouse which implicates Cytosolic Carboxypeptidase 5 (the product of Agbl5 gene) for proper ependymal cells.
Weaknesses:
Description of phenotype is incomplete:
We thank the Reviewer’s constructive comments. We have performed additional quantitative analysis of the phenotypes in Agbl5M1/M1 that we feel strengthen this study.
Figure 3G - the sequence from the movie is not really informative. Providing beating frequencies as quantification of the data would be more informative.
We have provided the beating frequency as well as the mean vector length of cilia beating directions (that reflects the coordination of cilia) in Figure 3H and 3I respectively in the revised manuscript.
Figure 3 - the quantification of actin network would strengthen the message.
We agree with the Reviewers. We have quantified the total intensity of actin around BBs and the actin intensity normalized to signals of the BB marker (CEP164). The data have been provided in Figure 4M and 4N respectively. The quantitative analysis showed that both the total intensity of apical actin network and the intensity of F-actin per BB are reduced in Agbl5M1/M1 ependymal cells compared to that in wild-type mice, suggesting that CCP5 is involved in organizing actin network around BB. This analysis certainly improves the clarity of this message.
Lines 219 -220 - the authors conclude «Taken together, in Agbl5M1/M1 ependymal cells, the expression of genes promoting multiciliogenesis were not impaired but certain proteins associated with differentiated ependymal cells are not properly expressed». However, they do not assess gene but protein expression (IF). In addition, their quantification shows differences in the number of FoxJ1 positive cells which indeed is an impaired expression.
We will clarify this statement and emphasize the number of FoxJ1-positive cells.
Microtubules are involved in the local organization of ciliary basal bodies (see Werner et al., Vladar et al.,2011; Boutin et al., 2014). It would be interesting for the authors to check whether the subapical network of microtubules is glutamylated or not during ependymal cell differentiation and how this network is affected in their mutants.
We thank the Reviewer’s constructive comments. We conducted an immunostaining on whole-mount lateral walls of lateral ventricles for GT335 and Centrin1, the position of the latter being used to localize the subapical layer. While the GT335 signal in multicilia is increased in Agbl5M1/M1 ependyma (Figure S8E), its signals underneath BBs are not much different between the mutant and wild-type (Please see Figure S8C, D, G, H).
Showing the data mentioned in the discussion on Cep110 would be a nice addition to the paper.
These data have been provided in Supplementary Figure S9.
Line 354: "The latter serves as a component of tissue polarity that is required for asymmetric PCP protein localization in each cell (Boutin et al., 2014; Vladar et al., 2012)." The cited reference did not demonstrate that this microtubule network is required for asymmetric PCP localization.
We thank the Reviewer for critical reading. The cited reference (Bountin et al., 2014) has been removed.
Reviewer #3 (Public review):
Summary:
The authors developed a new Agbl5 KO allele, extending the deletion to the N-terminus of CCP5 to explore its function in mouse ependymal cells.
Strengths:
They show that the KO mice exhibit severe hydrocephalus due to disorganized and mislocated basal bodies. Additionally, they present evidence of both impaired beating coordination and a reduction in ciliary beating.
Weaknesses:
The manuscript is well-written but lacks specific interpretations of the results presented. Further experiments are needed to be fully convincing.
We thank the Reviewer’s comments. We have performed further analysis and conducted additional experiments to strengthen this study.
(1) We have quantified the intensity of actin staining around BB patches and its intensity relative to the number of BBs to assess to which extent the actin networks in Agbl5M1/M1 ependymal cells are affected (please refer to the above response to the comments of Reviewer 2#). The results were shown in Figure 4M-N.
(2) We Co-stained tdTomato with an ependymal cell-specific markers to strengthen the expression of Agbl5 in ependymal cells (please see Figure 6C-E).
(3) We have conducted co-immunostaining of GT335 and Ac-Tub and compared the length of their signals in ependymal multicilia between WT and Agbl5M1/M1 mice (please see Figure 6O, P, R, S).
(4) We quantified the area of ependymal cells in the wild-type and Agbl5M1/M1 mice. Indeed, the area of ependymal cells is increased in the mutants. However, the primary cilia are present in the ependymal cell progenitors of Agbl5M1/M1 mice and have similar length with that in the wild-type (Please see Figure 7M, N and our response to this point below).
(5) We performed additional analysis to address the affected rotational polarity in the Agbl5M1/M1 mutant mice (please see Figure 3I, Figure 7E).
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) The authors showed that the actin networks were severely affected, leading to impaired stability of basal bodies and that the intensity and length of acetylated tubulin signal in the multicilia were dramatically reduced in AGBL5M1/M1mutant mice (Figures 3 and 5). Data also suggested the dysregulation of planar cell polarity. Are expression and localization of other planar cell polarity proteins such as tyrosinated tubulin and Fzd6 affected in mutant mice?
We thank the Reviewer’s recommendations. We have assessed the expression of tyrosinated tubulins and found they are similarly polarized in ependymal cells from wild-type and Agbl5M1/M1 mice. The results are presented in Figure 7O, P in the revised MS. We also tried to assess the expression of Fzd6. However, with the antibody we tested, Fzd6 signals were not convincing. Therefore, we prefer to not showing the results and drawing a conclusion on it.
(2) The phenotype of multiciliated cells in tracheas should also be examined in mutant mice. It is important to elucidate whether AGBL5 commonly functions in multiciliated cells of other organs.
We thank the Reviewer’s suggestion. We have assessed the multicilia in the tracheas of P30 mice using scanning electron microscopy. Indeed, unlike the multicilia in wild-type mice that orientate to the same direction, those in the tracheas of Agbl5M1/M1 mice often radiate to different directions in individual cells (Figure S2). Therefore, Agbl5 appears commonly involved in the alignment of multicilia.
(3) According to Figure 1B, AGBL5 is highly expressed in the brain. Which cells in the brain express it besides ependymal cells?
Based on the localization of tdTomato tracer engineered in Agbl5 mutant alleles (Figure 5B), Agbl5 is broadly expressed in the brain, including most if not all neurons, but its expression is much weaker in the subventricular zone (Please see Figure 5B). We clarified this in the revised MS.
(4) From a mechanistic point of view, it is necessary to identify binding proteins with the N-domain of AGBL5 and perform functional analyses.
We agree with the Reviewer. We feel that identification of the binding partners of CCP5 N-domain and functional analysis may be more suitable to go along with other mechanistic analysis on the function of CCP5 in ependymal cell polarities in our future study.
Reviewer #2 (Recommendations for the authors):
(1) Movie 3: The authors could comment on beating direction that seems impaired at the cell scale here, analysis of rotational polarity would be a plus.
We thank the reviewer’s recommendation. We have analyzed the beating directions of cilia in individual cells and presented their consistency in each cell using mean vector length. These results indeed demonstrated defective rotational polarity in the cell level in Agbl5M1/M1 mice (please refer to Figure 3I). We also analyzed the beating directions of ependymal multicilia in earlier stage in tissue level (Figure 7E). The mean vector length of cilia beating direction in Agbl5M1/M1 mice is significantly reduced compared to that in wild-type, suggesting an aberrant rotational polarity in the tissue level in the mutant (Figure 7E).
(2) Line 166 : ref to Werner et al., 2011 is not correct (no ependymal cells in that paper).
We thank the reviewer’s critical reading. This reference has been removed.
(3) Figure S4: B and D look similar picture to me same for C and F.
We apologize for using the wrong images in this Figure. It has been corrected (Revised Figure S5).
(4) Line 328: "Therefore, CCP5 apparently contributes to the establishment of both translational and tissue polarities in ependymal cells." Should be rephrased since translational polarity is also a tissue-level parameter which is the coordinated positioning of the ciliary patch. Cf Mirzadeh et al., 2010; Boutin et al., 2014.
We thank the Reviewer’s comments. The sentence has been rephrased. This concept has been clarified where else needed in the revised manuscript.
(5) Line 348: "Planar cell polarity (PCP) pathway is essential for the establishment of rotational and tissue polarities in ependymal cells" Rotational polarity also has a tissular component (ie coordination of beating direction across tissue which is reflected by coordination of basal body polarities across tissue).
We thank the Reviewer’s comments. We have clarified this point in the revised MS.
(6) Incomplete bibliography citation (ie Walentek et al. without date).
We thank the Reviewer’s critical reading. This bibliography citation has been fixed.
Reviewer #3 (Recommendations for the authors):
(1) Figure 3: The authors assert that the mutant's apical actin networks are significantly disrupted. However, the cell shown in Figure 3Q-R exhibits less compact centrioles than the controls, which could account for the reduction in phalloidin staining. Because centriole dispersion is variable in the mutant, quantifying actin staining in representative cells would be necessary to support such a statement.
We thank the Reviewer’s comments. To address this concern, we have quantified the total intensity of actin network around BBs as well as the intensity of F-actin signals normalized to the level of immunosignals of BBs ((revised Figure 4M, N) please also refer to our response to Reviewer 1#). The results indicated the intensity of actin signal per BB is reduced in the mutant compared to that of wild-type mice. We feel that this analysis strengthened our statement.
(2) Figures S3 and 4A-B show that the authors examine tdT expression to show that Agbl5 is expressed in ependymal cells but not in the SVZ. However, the tdT signal intensity is very low, and cells are very dense in this brain region. Double staining with specific markers of ependymal and/or SVZ cells would help convince readers that tdT is not expressed in SVZ cells.
We agree with the Reviewer that the intensity of tdT signal is low, but broadly detectable in brain. Compared with its expression in ependymal cells, that in SVZ is much lower if any (Figure 4B’). To further confirm the identity of tdT-positive cells along the surface of ventricles, we have co-stained the brain sections of Agbl5WT/M1 mice for tdT and S100b, a marker of mature ependymal cells (Figure 5C-E). The signal of tdt is colocalized with that of S100b and is much lower in cell layers next to S100b-positive cells.
(3) Figure 4C-D and S4: The authors demonstrate that the number of FoxJ1+ cells per section increases at P7 (4C-E), while the number of S100β+ cells per mm decreases. Quantifications should be carried out in a similar manner to ensure comparability (number of positive cells per mm). Additionally, it remains unclear how to interpret these results, as S100β and FoxJ1 are two markers of differentiated cells, yet they exhibit opposite trends compared to controls. Is this a direct or indirect effect of Agbl5 mutation? The increase in the number of FoxJ1+ cells is particularly surprising given that the number of GT335 multicilia per mm remains unchanged (Figure 5).
We agree with the Reviewer that quantifications should be carried out in a similar manner. In the revised MS, the quantification of Foxj1-positive cells is presented in number per mm (Figure 5I). To be noted, the expression of Foxj1 was assessed at P7 when ependymal cells are differentiating. while the expression of S100β was assessed at P17 when ependymal cells are supposed to be fully mature. Although S100b is used as a marker of mature ependymal cells, given its unclear function, we removed the results of S100b-positiving cell counting to avoid confusion in the revised manuscript.
(4) Figure 5: In this figure, the authors analyze the labeling obtained with GT335, Acetylated Tubulin, and Arl13b antibodies. They show that the area of the cilium labeled by GT335 has increased, while the area labeled by the Acetylated Tubulin antibody has decreased in the knockout (KO) compared to the control. However, the length of the cilia observed through labeling with the Arl13b antibody remains unchanged. These observations are intriguing, but the low-magnification images in Figure 4 do not allow for the differences in ciliary axoneme labeling to be seen. Double GT335/AcTub labeling and higher magnifications are necessary for improved visualization of the differences in labeling along the axonemes.
We thank the Reviewer comments. We have co-stained the cilia with GT335 and Ac-Tub antibodies, re-quantified cilia length labeled with respective antibodies and provided high magnification images. Please see the revised Figure 6O,P,R,S.
(5) Figure 6: An analysis of ciliary beats using a high-speed camera shows no difference in ciliary beat frequency between the control and KO groups. At least, 3 animals should be analyzed. According to Figure 5, these findings indicate that the decrease in ciliary acetylation and the increase in ciliary glutamylation do not affect the beat frequency; instead, they disrupt the orientation of the beats. While these results are intriguing, they require further confirmation. Analyzing ciliary beats with a high-speed camera is informative, but at least three animals per genotype should be examined to ensure rigor. Furthermore, if the coordination of ciliary beats is impaired within the cells, this should be validated by double-labeling centrioles and basal feet to demonstrate that the orientation of cilia within the cells is abnormal.
We thank the Reviewer’s comments. Sections shown in Figure 5 (currently Figure 6) are from P7 mice, while the ciliary beating analysis shown in Figure 6 (currently Figure 7) is from P15 mice. As the PTM changes in cilia were also observed in Agbl5M2/M2, we don’t think this is the cause that disrupts the orientation of the beats. The rotational polarity of Agbl5M1/M1 ependymal cells is affected. Please refer to the analysis in Figure 3I and Figure 7E in the revised manuscript.
(6) Figure 6F-G: β-Catenin labeling reveals cells of varying sizes in the KO. This phenotype is typical of ciliary mutants that lack primary cilia (Mirzadeh et al., 2010). Hence, it is essential to examine the mutation's impact on the presence, length, and positioning of the primary cilium in ependymal cell progenitors.
We thank the Reviewer’s constructive comments. We assessed the area of ependymal cells labeled with β-Catenin. Indeed, the ependymal cells in the mutant showed larger area than that of wild-type. The ratio of the area of BB patch over that of cell surface is reduced (please see Figure 7O, P in the revised manuscript). However, primary cilia are present in ependymal cell progenitors in the mutant and exhibit comparable length with those in the wild-type (Figure S8). Due to some technique problems, we were unable to get convincing results from whole-mount ventricle walls for the primary cilium positioning at this time. We speculate that the localization of certain sensory proteins in primary cilia or the positioning of primary cilia might be affected in Agbl5M1/M1 mice. We discussed this possibility and will certainly systemically assess this intriguing aspect in our future investigation.
(7) Given the regular beating frequency in the KO at P15, how do the authors explain the complete absence of ciliary beating in the adult? How many animals were analyzed? One would expect ciliary beating to remain unaffected as it was at P15 unless the cilia structure was specifically altered at the adult stage. Is that the case?
We thank the Reviewer’s critical questions. We do think that the ciliary structure of Agbl5M1/M1 ependymal cells is likely altered during aging. Given that only Agbl5M1/M1 but not Agbl5M2/M2 mice develop hydrocephalus, we speculate the N-domain of CCP5 may contribute to the integrity of ependymal multicilia. We have added this in the Discussion section. For each genotype, 2 mice were analyzed.
(8) Line 264 of the manuscript: replace intercellular with intracellular.
It has been revised.
(9) Indicate the number of animals analyzed in each experiment
It has been included in figure legends.
