Centriole Loss in Embryonic Development Disrupts Axonal Pathfinding and Muscle Integrity

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   Dec 12, 2025

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   Reply to the reviewers

   Dear editor and reviewers,

   We sincerely thank you for your thoughtful comments and constructive suggestions, which have greatly improved the quality and clarity of our manuscript. In response, we have implemented all requested changes, which are highlighted in yellow throughout the revised text, and updated several figures accordingly. Furthermore, we have performed all additional experiments recommended by the reviewers and incorporated the new data into the manuscript. To enhance clarity, we have also included a schematic representation of our proposed model in an additional figure, providing a concise visual summary of our findings.

   We hope that …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   Dear editor and reviewers,

   We sincerely thank you for your thoughtful comments and constructive suggestions, which have greatly improved the quality and clarity of our manuscript. In response, we have implemented all requested changes, which are highlighted in yellow throughout the revised text, and updated several figures accordingly. Furthermore, we have performed all additional experiments recommended by the reviewers and incorporated the new data into the manuscript. To enhance clarity, we have also included a schematic representation of our proposed model in an additional figure, providing a concise visual summary of our findings.

   We hope that these revisions fully address all concerns raised by the reviewers and meet all the expectations for publication.

   Below, we answer the reviewers point by point (in blue).

   ---

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   In this paper, the authors address the important question of the role of centrosomes during neuronal development. They use Drosophila as an in vivo model. The field is somewhat unclear on the role and importance of centrosomes during neuronal development, although the current data would suggest they are dispensable for axon specification and growth. Early studies in cultured mammalian neurons showed that centrosomes are active and that their microtubules can be cut and transported into the neurites. But a study then showed that centrosomes in these cultured neurons are deactivated relatively early during neuronal development in vitro and that ablating centrosomes even when they are active had no obvious effect on axon specification and growth. Consistent with this, a study in Drosophila provided evidence that centrosomes were not active or necessary in different types of neurons. More recently, a study showed that centrosomal microtubules are dispensable for axon specification and growth in mice in vivo but are required for neuronal migration in the cerebral cortex. However, another study has linked the generation of acetylated microtubules at centrosomes with axon development. In this current study, the authors examine the effect of centrosome loss on various motor and sensory neurons and muscles mainly by examining mutants in essential centriole duplication genes. They associate axonal routing and morphology defects with centrosome loss and provide some evidence that centrosomes could still be active in the developing neurons. Overall, they conclude that centrosomes are active during at least early neuronal development and that this activity is important for proper axonal morphology and routing.

   While I think this study addressing a very interesting and important question, I think as it stands the data is not sufficient to be conclusive on a role for centrosomes during neuronal development. My biggest concern is that most phenotypes have not yet been shown to be cell autonomous, as whole animal mutants have been analysed rather than analysing the effect of cell-specific depletion, and the evidence for active centrosomes needs to be strengthened. If the authors can provide stronger evidence for a role of centrosomes in axonal development then the paper will certainly be of interest to a broad readership.

   We thank the reviewer for the clear and concise summary and fully agree that our study addresses a critical gap in understanding. Centrosomes have long been implicated in morphogenesis, yet their precise contribution to nervous system development has remained unclear. Our findings provide compelling evidence that centrosomes are indispensable for proper nervous system formation and that their absence also triggers muscular defects, highlighting their broader role in tissue organization.

   We acknowledge that the original manuscript lacked some key details; therefore, we have now strengthened our conclusions with additional experiments. Specifically, we demonstrate that these effects are cell-autonomous by using two independent RNAi lines targeted to a subset of motor neurons. Furthermore, we present new data showing that neuronal centrosomes remain active during the early stages of axonal development, emphasising their functional relevance in morphogenesis. All new experiments, figures, and corresponding text revisions are detailed below.

   Major comments

   1. The sas-6 transallelic combination shows only 17% embryonic lethality compared to 50% embryonic lethality with sas-4 mutants. Given that both mutants should result in the same degree of centrosome loss (this should be quantified in sas-6 mutants) it would suggest that either sas-4 has other roles away from centrosomes or that the sas-4 mutant chromosome used in the experiment has other mutations that affect viability. The effect of picking up "second-site lethal" mutations on mutant chromosomes is common and so I would not be surprised if this is the reason for the difference in phenotypes. This can be addressed either by "cleaning up" the sas-4 mutant chromosome by backcrossing to wild-type lines, allowing recombination to occur and replace the potential second site mutations, or by using transallelic combinations of sas-4, as they did for sas-6. The "easier" option may just be to analyse all the phenotypes with the sas-6 transallelic combination.

   We appreciate this comment, as it brought to light an issue with the CRISPR line Sas-6-Δa. Upon reanalysing all the data, we determined that this line is embryonic lethal both in homozygosis and when combined with the deficiency uncovering the genomic region, Df(3R)BSC794. In contrast, Sas-6-Δb homozygotes are viable. The inconsistency between these results raised concerns about whether the Δa and Δb Sas-6 mutants carry deletions confined to the Sas-6 coding region. Although this would not hinder our cell biology analysis, it could represent a problem in viability tests. To address this, we repeated all analyses using Sas-6-Δb homozygotes and Sas-6-Δb combined with Df(3R)BSC794. These new results are more consistent and indicate that approximately 50% of Sas-6/Def individuals hatch as adults. Fig. 3 was redone and the manuscript text changed in view of these results.

   1. Using "whole animal" mutants for assessing neuronal morphology is risky due to non-cell-autonomous effects. The authors have carried out some phenotypic analysis of neurons depleted of Sas-4 by cell-specific RNAi, but I feel they need to do this for all of their analysis. This includes embryonic lethality measures, quantification of centrosome numbers, and all axonal phenotypes in Sas-4 RNAi neurons. It would also be prudent to use 2 distinct RNAi lines to help ensure any phenotypes are not off-target effects (and this may help clarify why the authors see some additional phenotypes with RNAi). Indeed, there are relatively weak phenotypes in muscles when using RNAi compared to the mutants and these potential non-cell-autonomous effects could then have a knock-on effect on neuronal morphology. If the authors were concerned that RNAi is not very efficient (explaining any potential weaker phenotypes than in mutants) the authors could examine the effectiveness of RNAi lines by analysing protein depletion by western blotting or mRNA depletion by rt-qPCR (although this has to be done in a different cell type due to the difficulty in obtaining a neuronal extract).

   We have now added a new panel to supplementary Figure 1, showing how the expression of a different Sas-4 RNAi line (2) induces similar nervous system phenotypes when expressed only in aCC, pCC and RP2 pioneer neurons (Sup. Fig. 1 M-O).

   1. When analysing centriole presence or absence it is a good idea to stain with two different centriole markers e.g. Asl and Plp. This helps rule out unspecific staining. It is clear from the images that similar sized foci can be observed outside of the cells (see Figure 5A for example), so clearly some of the foci that appear to be within the cells may also be unspecific staining.

   In a new supplementary figure, we now show that Asl and Plp colocalize and quantify the number of times we find this colocalization in neurons (Supl. Fig 3). In addition, and we apologise for the confusion, but the reason why there are foci outside the marked cells is because these are wholemount embryonic stainings and the anti-Plp antibody marks all centrosomes in all cells in the embryo.

   1. The evidence for active centrosomes is not that convincing. Acetylated tubulin is associated with stable MTs, which are not normally organised by "active" centrosomes that nucleate dynamic microtubules. Moreover, it is plausible that centriole foci happen to overlap with the acetylated tubulin staining by chance. This would explain why not all centrosomes colocalise with acetylated tubulin signal. The authors could better test centrosome activity by performing live imaging with EB1-GFP. If centrosomes are active, it is very easy to observe the many comets produced by the centrosomes.

   We appreciate the reviewer’s comment and agree that acetylated tubulin alone is not an ideal marker for centrosome activity. To address this, we performed live imaging of aCC neurons expressing EB1-GFP together with Asl-Tomato. This was technically challenging because we were imaging only two neurons per segment in live embryos, under significant limitations in fluorescence detection and timing. Despite these constraints, we were able to clearly observe EB1 comets emerging from the centrosome and moving toward the cell periphery, providing direct evidence of microtubule nucleation from centrosomes in neurons.

   Importantly, we complemented this with a microtubule depolymerization/polymerization assay, which provides unequivocal evidence that polymerization initiates at the centrosome. After depolymerization, we observed microtubule regrowth from the centrosome, confirming its role as an active microtubule-organizing centre in these neurons. Together, we hope that these results are enough to demonstrate that neuronal centrosomes are functionally active during early axonal development. These experiments are presented in Figure 6 and corresponding text in the manuscript.

   1. If the authors believe that centrosomes have a role in axon pathfinding in sensory neurons, they should show that these centrosomes are active, at least during early stages (again using EB1-GFP imaging).

   We appreciate the reviewer’s suggestion and agree that EB1-GFP imaging would be the most direct way to assess centrosome activity in sensory neurons. However, performing time-lapse imaging in these neurons is technically very demanding due to their location and accessibility in live embryos, and we did not attempt this approach. Instead, we now provide new evidence showing that sensory neuron centrosomes colocalize with both α-tubulin and γ-tubulin. This strongly supports that these centrosomes are associated with microtubule nucleation machinery and are as likely as motor neuron centrosomes to be active during early stages of axon development. These new data have been included in the revised manuscript (see Figure 5 and corresponding text).

   1. The authors mention in the discussion that "increased JNK activity, can result in axonal wiggliness (Karkali et al, 2023)". I therefore wonder whether centrosome loss may induce JNK activation (the stress response), as this would then indicate an indirect effect of centrosome loss on axonal structure rather than a direct influence of centrosome-generated microtubules. The authors could assess whether the DNK-JNK pathway is activated in neurons lacking centrosomes by expression UAS-Puc-GFP and quantifying the nuclear signal.

   In a new supplementary figure, we now show by using a reporter for JNK signalling, as requested, that Sas-4 neurons do not activate the JNK pathway (Supl. Fig 4).

   1. In Figure 5, the authors claim that they find "a correlation between axonal guidance phenotypes and the numbers of centrioles per embryo". I don't think this is a strong correlation. The difference in centriole number between embryos with no defects and those with defects is very small. In contrast, the difference between centriole numbers in control (no defects) and mutant (no defects) is very large. So, there does not appear to be a strong correlation between centrosome number and phenotype.

   We agree and we have corrected this sentence to better explain the results.

   Minor comments

   1. I don't understand Figure 3C - why do the % of surviving homozygotes and heterozygotes add up to 100%? Should the grey boxes not relate to dead and the white to surviving?

   Thank you for pointing this out. Figures 1B and 3C represent only the surviving individuals. The grey boxes correspond to surviving homozygotes, and the white boxes correspond to surviving heterozygotes. The percentages add up to 100% only at embryonic stages because all embryos reach late embryonic stages. The grey and white boxes reflect the proportion of these two genotypes among the survivors, not the total number of embryos including those that died. We have changed the text to convey this.

   1. "In mouse fibroblasts, myoblasts and endothelial cells, centrosome orientation is important for nuclear positioning and cell migration(Chang et al, 2015; Gomes et al, 2005; Kushner et al, 2014)." Do you mean "centrosome position"?

   Yes, text changed, thank you for spotting it.

   1. In the introduction, the authors mention Meka et al. when saying the centrosomal microtubules are important for axonal development, but they should also discuss the counter argument from Vinopal et al., 2023 (Neuron) that showed how centrosomes were required for neuronal migration but not axon growth, which was instead mediated by Golgi-derived microtubules.

   Done, thank you very much.

   1. Lines 228-230 - repeated sentence

   Corrected, thank you very much.

   1. Additionally, we did not detect centrioles in the quadrant opposite the axon exit point (Fig. 2B n=75) - this data is not in Fig 2B

   Correct, it is in figure 4B, thank you very much.

   1. "This significant decrease in the humber of centrioles further supports the critical role of Sas-4 in pioneer neurons of the ventral nerve cord (VNC) during Drosophila embryogenesis". It rather highlights that Sas-4 is required for centriole formation in these neurons. Also, humber = number.

   We agree, and have changed the text, thank you very much.

   1. Result title: Non-ciliated sensory neurons have centrioles. This is kind of obvious. A better title may be "axon phenotypes correlate with centriole numbers in sensory neurons" but unfortunately i don't think there is good evidence for this (See major point above).

   We agree and we have changed. We now believe we have strong evidence to support it. We hope the additional data presented in the revision convincingly demonstrate this point.

   Reviewer #1 (Significance (Required)):

   As mentioned above, the advance will be important if more evidence is provided. In this case, the paper will be interesting to a broad readership. But currently the paper is limited by the lack of evidence for centrosome function and activity in the neurons.

   We hope that reviewer 1, now considers that the manuscript is not limited anymore and that it shows convincing evidence for centrosome function and activity in embryonic neurons.

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   Summary: In this manuscript, Gonzalez et al. examine the potential function of centrosomes in the neurons and muscle cells of Drosophila embryos. By studying various mutant and RNAi lines in which centriole duplication has been disrupted, they conclude that the loss of centrioles disrupts axonal pathfinding and muscle integrity.

   Major points:

   1. Throughout the manuscript, the phenotypes presented are often quite subtle. For this reason, I would really recommend that these experiments are scored blind. Perhaps the authors did this, but I didn't see any mention of this.

   All our phenotypic analyses are performed blind. We apologize for not having originally included this information in the Methods section; it has now been added. Embryos are stained using colorimetric methods (DAB) to label the nervous system, while balancer chromosomes are marked with a fluorescent antibody. This approach allows us to assess and quantify phenotypes using white light without knowing whether the embryos are homozygous mutants or heterozygous, which can only be detected by changing the channels to fluorescence.

   1. The authors conclude that neurons have active centrioles that function as centrosomes (Figure 6), but the data here is confusing. The authors state that in these cells they observe acetylated MTs extending from the centrosomes and these colocalised with g-tubulin. But the authors don't show the overlap between centrosomes, g-tubulin and MTs, as they stain for these separately. This is problematic, as it was not clear from these images that the majority of the MTs really are extending from the centrosome: the centrosome may just associate or be close by to these MT cables (Figure 6A,B). Moreover, the authors show that only a fraction of the centrosomes in these cells associate with g-tubulin, so presumably in cells where the centrosomes lack g-tubulin they would not expect the centrosomes to be associated with the MTs-but they do not show that this is the case. Perhaps the authors can't test this, but an alternative would be to show that these MT arrays are absent in Sas-4 mutants. This would give more confidence that these MTs arise from the centrosomes.

   We agree that the initial data based on acetylated microtubules and γ-tubulin colocalization were not sufficient to conclude that microtubules originate from the centrosome, as these markers can only suggest association. To address this, we have now included additional experiments that provide direct evidence of centrosome activity.

   First, we performed live imaging of aCC neurons expressing EB1-GFP together with Asl-Tomato. Despite the technical challenges of imaging only two neurons per segment in live embryos under strict fluorescence and timing constraints, we were able to clearly observe EB1 comets emerging from the centrosome and moving toward the cell periphery. This demonstrates active microtubule nucleation from centrosomes rather than mere proximity to microtubule bundles.

   Second, we carried out a microtubule depolymerization/polymerization assay, which provides unequivocal evidence that polymerization initiates at the centrosome. After depolymerization, microtubules regrew from the centrosome, confirming its role as an active microtubule-organizing center. These experiments go beyond colocalization and directly address the concern that centrosomes might simply be adjacent to microtubule cables.

   Regarding the suggestion to use Sas-4 mutants, while we did not perform this experiment, the regrowth assay combined with EB1 imaging strongly supports that these microtubules originate from the centrosome. All new data are presented in Figure 6 and the corresponding text in the revised manuscript.

   1. The authors show that muscle cell integrity is compromised by centriole-loss (Figure 2). This is very surprising as it is widely believed that centrosomes are non-functional in muscle cells, and the MTs are instead organised around the nuclear envelope. I'm not aware of the situation in Drosophila muscle cells, but the authors should ideally try to examine if the centrioles are functioning as centrosomes in these cells. At the very least they should discuss how they think centriole-loss is influencing the muscle integrity when it is widely believed they are inactive in these cells.

   We do not claim that centrosomes are active in muscle cells at these developmental stages. The observed muscle defects could result from earlier processes such as cell division, migration, or muscle fusion. We agree that this is an intriguing observation; however, pursuing this question further would go beyond the scope of the current manuscript. As requested by the reviewer, we have now expanded the discussion to consider how centriole loss might impact muscle integrity.

   Regardless of the strength of the supporting data, I think the authors should tone down their conclusions. The title and abstract led me to believe that centriole loss would cause significant problems in axonal pathfinding and muscle integrity. In all the mutant specimens examined (and certainly the low magnification views shown in Figure 1D'-F', Figure 1I'-K' and Figure 2D'-F') the mutants look very similar to the WT. Many readers may not get past the title and abstract, so the authors should make it clearer that these defects are very subtle.

   We have changed the text to convey this idea.

   Minor points:

   1. In Figures 4 and 5, CP309 staining is relied on to identify centrioles, but there is quite a background of non-specific dots, making it hard to be certain what is a centriole and what isn't. For example, in Figure 5D' there are lots of dots within some of the cells - are any of these centrioles? How can the authors be certain which dot is a centriole in some of the cells shown in Figure 5C'? Is it possible to use a second marker and only count as centrioles dots that are recognised by both antibodies?

   We thank the reviewer for this suggestion and agree that using a second marker improves confidence in centriole identification. In a new supplementary figure (Supplementary Fig. 3), we now show that Asl and Plp colocalize in neurons and provide a quantification of the frequency of this colocalization. This dual labelling confirms the identity of centrioles and addresses the concern about non-specific background.

   We also apologize for any confusion regarding the presence of foci outside the marked cells. These images are whole-mount embryonic stainings, and the anti-Plp antibody labels all centrosomes in all cells of the embryo, which explains the additional foci observed.

   In the abstract that authors state that traditionally centrosomes have been considered to be non-essential in terminally differentiated cells. I don't think this is correct. In the standard "textbook" view of a cell, the centrosome is normally positioned in the centre of the cell organising an extensive array of MTs that are thought play an important role in organising intracellular transport, the positioning and movement of organelles and the maintenance and establishment of cell polarity. I don't think it is only recent evidence that suggests they play vital roles in terminally differentiated cells.

   We thank the reviewer for this correction and we have changed the text accordingly.

   1. Line 162 the authors state that in the RNAi knockdown lines they observe several additional phenotypes, but then in the same sentence (Line 164) they say that these defects were also observed in the original mutant and mutant/Df lines.

   We apologise for this confusion, we have rearranged the sentence for clearance.

   The sentences in Line281-287 don't reference any of the Figures, so it seems the authors are just stating these results without presenting any data (e.g. "Significantly, we also found a correlation between axonal guidance phenotypes and the numbers of centrioles per embryo". If they've tested this correlation, they should show it.

   We have rearranged the sentences for better understanding.

   In Figure 7 I did not understand how the authors measured tortuosity (wiggliness) and could see no description in the methods. This is important as, again the defect seems quite subtle, but perhaps I am not understanding which bits of the axon are being measures. Is it just the small bit of the axons close to the asterixis that is being measured, or the whole FasII track?

   We have now added another quantification and additional descriptions in the methods section.

   Reviewer #2 (Significance (Required)):

   The potential function of centrosomes in axonal outgrowth is quite controversial, so this study is potentially of considerable interest.

   However, several aspects of the data presented here were confusing or not terribly convincing. In its present state, I don't think the main conclusions are strongly enough supported by the data.

   We hope that reviewer 2, now considers that the manuscript is not confusing anymore and that it shows convincing evidence for centrosome function and activity in embryonic neurons.

   Reviewer #3 (Evidence, reproducibility and clarity (Required)):

   The manuscript of González et al. entitled "Centriole Loss in Embryonic Development Disrupts Axonal Pathfinding and Muscle Integrity" deals with the role of centrosomes in shaping axonal morphology. To this aim the AA analysed Drosophila Sas-4 mutants that are reported to develop until adult stage without centrioles. Remarkably, the AA observe that 50% of the homozygous mutant embryos fail to hatch as larvae. The present observations suggest that centrosome loss results in axonemal shaping defects and muscle developmental abnormalities. Finally, the AA show the presence of functional centrosomes in neurons. In my opinion, the manuscript is interesting because shows unexpected findings. However, to justify these new findings the AA are required to improve some experimental observations.

   We thank the reviewer for his summary of our work and for considering it interesting. We have taken into account all the comments and believe that these have helped improve our manuscript.

   Major: Abstract- It is unclear in which phenotypic condition the observations of centrosome loss or centrosome presence have been found. Please better explain. l.36. embryos, larvae, adult, from Sas4 or controls? If mutants, the observations are very interesting since Sas4 would be without centrioles. Indeed, Basto et al., show that chemosensory neurons do not develop an axoneme in the absence of centrioles, but extend dendrites toward the sensory bristle.

   We have made clear which refer to wild-type and which are Centriole Loss (CL) conditions. CL conditions refer to mutant and downregulation conditions, whereas targeted downregulation refers to RNAi downregulation only in neurons.

   I do not think appropriate the use of "centriole" in the main title since the centrioles would be localized by true centriolar antigens rather than by centrosomal antigens. This problem occurs throughout the text and some figures where the AA image centrioles by centrosomal material. In Gig. 5A only the AA properly look at Asl localization. The other pictures of presumptive centrioles or centriole quantification report CP309 dots. This localization does not unequivocally reveal centrioles, since CP309 is essentially required for centrosome-mediated Mt nucleation. There are differentiated Drosophila tissues in which centrioles are present, but inactivated, and unable to recruit pericentriolar material. Mt are nucleated by ncMTOCs that contain centrosomal material and gamma-tubulin. Thus, the centrosomal antigens do not colocalize with centrioles.

   We have changed centrioles to centrosomes in the title and most sections in the manuscript. We have also included an extra control, showing that Asl and Plp colocalize and quantify the number of times we find this colocalization in neurons (Supl. Fig 3). Asl is a reliable and widely used marker for centrioles, as it localizes specifically to the centriole structure (Varmark H, Llamazares S, Rebollo E, Lange B, Reina J, Schwarz H, Gonzalez C. Asterless is a centriolar protein required for centrosome function and embryo development in Drosophila. Curr Biol. 2007 Oct 23;17(20):1735-45. doi: 10.1016/j.cub.2007.09.031. PMID: 17935995.)

   Minor: l. 58. The early arrest is mainly due to a checkpoint control. In double mutant for Sas4 and P53 the embryos survive longer, even if their further development is asrrested.

   We thank the reviewer for this comment, and we have changed the text accordingly.

   1. Previous works, also quoted by the AA, reported that in mature neurons the centrosome are inactivated, whereas the present manuscript describes functional centrosomes in Drosophila motor and peripheral nervous system. This is an intriguing observations that needs a better explanation in Discussion section.

   We thank the reviewer for this comment, and we have changed the discussion accordingly.

   l.143-145. I understand that 50% of the Sas4 embryos that reach the adult stage have centrioles. Is it correct? But if it is so, how the AA explain the absence of centrioles in sensory neurons of adult flies as reported by Basto et al. ?

   According to our results they have less centrioles than controls already at embryonic stages. In addition, as reported in Basto et al. they continue losing centrioles during larval stages and metamorphosis, which explains why centrioles are not detected at adult stages.

   l.215. It is unclear for me why the AA analyse Sas6 flies, unless explain the mutant phenotype.

   To strengthen our conclusions with Sas-4 and exclude the possibility that the observed phenotypes arise from a centrosome-independent function of Sas-4. For this reason, we have taken additional steps to confirm that the effects are specifically due to centrosome loss and we used Sas-6 mutants as one of these.

   l.221. How the centrioles have been quantified? What antibody, the AA used.

   We have quantified centrosomes using antibodies agains Plp (CP309) and Asl-YFP expression.

   l.244. and Fig 4C,D. I see high background with CP309. As reported previously I think better to use antibodies against centriolar proteins, such as Sas6, Ana1, Asl, or Sas4 ( if centrioles are present in 50% of mutants as the AA claim, the antibody could be also useful). In addition, I can see some CP309 spots in Fig 4E,F. Are they centrioles?

   Indeed, as we report, Sas-4 mutant embryos are not totally devoid of centrosomes. In addition, and we apologise for the confusion, but the reason why there are foci outside the marked cells in control embryos is because these are wholemount embryonic stainings and the anti-Plp antibody marks all centrosomes in all cells in the embryo, not just in the neurons.

   l.270 and Fig. 5A and Fig.5 C-E. Why the AA localize Cp309 and not Asl (Fig. 5A) to detect centrioles?

   In a new supplementary figure, we now show that Asl and Plp colocalize and quantify the number of times we find this colocalization in neurons (Supl. Fig 3). So, we can use CP309 in neurons to the same effect as Asl-

   L295-296. I cannot see Mts, but only a diffuse staining. I am expecting to see distinct Mt bundles.

   In figure 5 it is now easier to see the MT bundles in the new experiment in Fig. 5F-I , where we performed MT depolymerisation/repolymerisation: Nevertheless, we need to stress out that we are doing these analyses in wholemount embryonic stainings.

   326-327. How the AA explain this different lethality, even if both the proteins are involved in centriole assembly?

   We have now redone all the viability and mutant phenotype analysis using Sas-6 CRISPR mutant over the Deficiency, which is a better way to access the phenotype.

   335-337. In my opinion the quoted publications are not relevant.

   We believe that these references back up our hypothesis because:

   • Metzger et al 2012 stress the importance of nuclear position in muscle development in Drosophila
   • Loh et al 2023, relate centrosomes with nuclear migration in Drosophila
   • Tillery et al 2018, is a review describing MTs in muscle development in Drosophila.

   358-359. Does maternal contribution persist after gastrulation?

   While bulk degradation occurs by midblastula transition, some stable maternal products persist beyond gastrulation. In our case, if centrioles are formed due to the maternal contribution, they will only be diluted by cell division, which explains why we can detect centrioles at late embryonic stages.

   l.366. This is an intriguing point, but as previously observed I have some problem with centriole localization. References. Please uniform Journal abbreviations and control page numbers.

   I hope we have clarified this problem with the new experiments showing MT repolarization from the centrosomes in neurons.

   Reviewer #3 (Significance (Required)):

   The manuscript is potentially interesting for peoples working of cell and molecular biology, and development. However, the paper needs an additional working to be suitable for publication.

   We hope that reviewer 3, considers that the additional work and revision make this manuscript suitable for publication.

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   Referee #3

   Evidence, reproducibility and clarity

   The manuscript of González et al. entitled "Centriole Loss in Embryonic Development Disrupts Axonal Pathfinding and Muscle Integrity" deals with the role of centrosomes in shaping axonal morphology. To this aim the AA analysed Drosophila Sas-4 mutants that are reported to develop until adult stage without centrioles. Remarkably, the AA observe that 50% of the homozygous mutant embryos fail to hatch as larvae. The present observations suggest that centrosome loss results in axonemal shaping defects and muscle developmental abnormalities. Finally, the AA show the presence of functional centrosomes in neurons. In my opinion, the …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   The manuscript of González et al. entitled "Centriole Loss in Embryonic Development Disrupts Axonal Pathfinding and Muscle Integrity" deals with the role of centrosomes in shaping axonal morphology. To this aim the AA analysed Drosophila Sas-4 mutants that are reported to develop until adult stage without centrioles. Remarkably, the AA observe that 50% of the homozygous mutant embryos fail to hatch as larvae. The present observations suggest that centrosome loss results in axonemal shaping defects and muscle developmental abnormalities. Finally, the AA show the presence of functional centrosomes in neurons. In my opinion, the manuscript is interesting because shows unexpected findings. However, to justify these new findings the AA are required to improve some experimental observations.

   Major:

   Abstract- It is unclear in which phenotypic condition the observations of centrosome loss or centrosome presence have been found. Please better explain. l.36. embryos, larvae, adult, from Sas4 or controls? If mutants, the observations are very interesting since Sas4 would be without centrioles. Indeed, Basto et al., show that chemosensory neurons do not develop an axoneme in the absence of centrioles, but extend dendrites toward the sensory bristle.

   I do not think appropriate the use of "centriole" in the main title since the centrioles would be localized by true centriolar antigens rather than by centrosomal antigens. This problem occurs throughout the text and some figures where the AA image centrioles by centrosomal material. In Gig. 5A only the AA properly look at Asl localization. The other pictures of presumptive centrioles or centriole quantification report CP309 dots. This localization does not unequivocally reveal centrioles, since CP309 is essentially required for centrosome-mediated Mt nucleation. There are differentiated Drosophila tissues in which centrioles are present, but inactivated, and unable to recruit pericentriolar material. Mt are nucleated by ncMTOCs that contain centrosomal material and gamma-tubulin. Thus, the centrosomal antigens do not colocalize with centrioles.

   Minor:

   l. 58. The early arrest is mainly due to a checkpoint control. In double mutant for Sas4 and P53 the embryos survive longer, even if their further development is asrrested.

   l. 102. Previous works, also quoted by the AA, reported that in mature neurons the centrosome are inactivated, whereas the present manuscript describes functional centrosomes in Drosophila motor and peripheral nervous system. This is an intriguing observations that needs a better explanation in Discussion section.

   l.143-145. I understand that 50% of the Sas4 embryos that reach the adult stage have centrioles. Is it correct? But if it is so, how the AA explain the absence of centrioles in sensory neurons of adult flies as reported by Basto et al. ?

   l.215. It is unclear for me why the AA analyse Sas6 flies, unless explain the mutant phenotype.

   l.221. How the centrioles have been quantified? What antibody, the AA used.

   l.244. and Fig 4C,D. I see high background with CP309. As reported previously I think better to use antibodies against centriolar proteins, such as Sas6, Ana1, Asl, or Sas4 ( if centrioles are present in 50% of mutants as the AA claim, the antibody could be also useful). In addition, I can see some CP309 spots in Fig 4E,F. Are they centrioles?

   l.270 and Fig. 5A and Fig.5 C-E. Why the AA localize Cp309 and not Asl (Fig. 5A) to detect centrioles?

   L295-296. I cannot see Mts, but only a diffuse staining. I am expecting to see distinct Mt bundles.

   L. 326-327. How the AA explain this different lethality, even if both the proteins are involved in centriole assembly?

   l. 335-337. In my opinion the quoted publications are not relevant.

   l. 358-359. Does maternal contribution persist after gastrulation?

   l.366. This is an intriguing point, but as previously observed I have some problem with centriole localization.

   References. Please uniform Journal abbreviations and control page numbers.

   Significance

   The manuscript is potentially interesting for peoples working of cell and molecular biology, and development. However, the paper needs an additional working to be suitable for publication.

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   Referee #2

   Evidence, reproducibility and clarity

   Summary: In this manuscript, Gonzalez et al. examine the potential function of centrosomes in the neurons and muscle cells of Drosophila embryos. By studying various mutant and RNAi lines in which centriole duplication has been disrupted, they conclude that the loss of centrioles disrupts axonal pathfinding and muscle integrity.

   Major points:

   1. Throughout the manuscript, the phenotypes presented are often quite subtle. For this reason, I would really recommend that these experiments are scored blind. Perhaps the authors did this, but I didn't see any mention of this.
   2. The authors conclude that neurons have active centrioles that …

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   Referee #2

   Evidence, reproducibility and clarity

   Summary: In this manuscript, Gonzalez et al. examine the potential function of centrosomes in the neurons and muscle cells of Drosophila embryos. By studying various mutant and RNAi lines in which centriole duplication has been disrupted, they conclude that the loss of centrioles disrupts axonal pathfinding and muscle integrity.

   Major points:

   1. Throughout the manuscript, the phenotypes presented are often quite subtle. For this reason, I would really recommend that these experiments are scored blind. Perhaps the authors did this, but I didn't see any mention of this.
   2. The authors conclude that neurons have active centrioles that function as centrosomes (Figure 6), but the data here is confusing. The authors state that in these cells they observe acetylated MTs extending from the centrosomes and these colocalised with g-tubulin. But the authors don't show the overlap between centrosomes, g-tubulin and MTs, as they stain for these separately. This is problematic, as it was not clear from these images that the majority of the MTs really are extending from the centrosome: the centrosome may just associate or be close by to these MT cables (Figure 6A,B). Moreover, the authors show that only a fraction of the centrosomes in these cells associate with g-tubulin, so presumably in cells where the centrosomes lack g-tubulin they would not expect the centrosomes to be associated with the MTs-but they do not show that this is the case. Perhaps the authors can't test this, but an alternative would be to show that these MT arrays are absent in Sas-4 mutants. This would give more confidence that these MTs arise from the centrosomes.
   3. The authors show that muscle cell integrity is compromised by centriole-loss (Figure 2). This is very surprising as it is widely believed that centrosomes are non-functional in muscle cells, and the MTs are instead organised around the nuclear envelope. I'm not aware of the situation in Drosophila muscle cells, but the authors should ideally try to examine if the centrioles are functioning as centrosomes in these cells. At the very least they should discuss how they think centriole-loss is influencing the muscle integrity when it is widely believed they are inactive in these cells.
   4. Regardless of the strength of the supporting data, I think the authors should tone down their conclusions. The title and abstract led me to believe that centriole loss would cause significant problems in axonal pathfinding and muscle integrity. In all the mutant specimens examined (and certainly the low magnification views shown in Figure 1D'-F', Figure 1I'-K' and Figure 2D'-F') the mutants look very similar to the WT. Many readers may not get past the title and abstract, so the authors should make it clearer that these defects are very subtle.

   Minor points:

   1. In Figures 4 and 5, CP309 staining is relied on to identify centrioles, but there is quite a background of non-specific dots, making it hard to be certain what is a centriole and what isn't. For example, in Figure 5D' there are lots of dots within some of the cells - are any of these centrioles? How can the authors be certain which dot is a centriole in some of the cells shown in Figure 5C'? Is it possible to use a second marker and only count as centrioles dots that are recognised by both antibodies?
   2. In the abstract that authors state that traditionally centrosomes have been considered to be non-essential in terminally differentiated cells. I don't think this is correct. In the standard "textbook" view of a cell, the centrosome is normally positioned in the centre of the cell organising an extensive array of MTs that are thought play an important role in organising intracellular transport, the positioning and movement of organelles and the maintenance and establishment of cell polarity. I don't think it is only recent evidence that suggests they play vital roles in terminally differentiated cells.
   3. Line 162 the authors state that in the RNAi knockdown lines they observe several additional phenotypes, but then in the same sentence (Line 164) they say that these defects were also observed in the original mutant and mutant/Df lines.
   4. The sentences in Line281-287 don't reference any of the Figures, so it seems the authors are just stating these results without presenting any data (e.g. "Significantly, we also found a correlation between axonal guidance phenotypes and the numbers of centrioles per embryo". If they've tested this correlation, they should show it.
   5. In Figure 7 I did not understand how the authors measured tortuosity (wiggliness) and could see no description in the methods. This is important as, again the defect seems quite subtle, but perhaps I am not understanding which bits of the axon are being measures. Is it just the small bit of the axons close to the asterixis that is being measured, or the whole FasII track?

   Significance

   The potential function of centrosomes in axonal outgrowth is quite controversial, so this study is potentially of considerable interest.

   However, several aspects of the data presented here were confusing or not terribly convincing. In its present state, I don't think the main conclusions are strongly enough supported by the data.

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   Referee #1

   Evidence, reproducibility and clarity

   In this paper, the authors address the important question of the role of centrosomes during neuronal development. They use Drosophila as an in vivo model. The field is somewhat unclear on the role and importance of centrosomes during neuronal development, although the current data would suggest they are dispensable for axon specification and growth. Early studies in cultured mammalian neurons showed that centrosomes are active and that their microtubules can be cut and transported into the neurites. But a study then showed that centrosomes in these cultured neurons are deactivated relatively early during neuronal development in …

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   Referee #1

   Evidence, reproducibility and clarity

   In this paper, the authors address the important question of the role of centrosomes during neuronal development. They use Drosophila as an in vivo model. The field is somewhat unclear on the role and importance of centrosomes during neuronal development, although the current data would suggest they are dispensable for axon specification and growth. Early studies in cultured mammalian neurons showed that centrosomes are active and that their microtubules can be cut and transported into the neurites. But a study then showed that centrosomes in these cultured neurons are deactivated relatively early during neuronal development in vitro and that ablating centrosomes even when they are active had no obvious effect on axon specification and growth. Consistent with this, a study in Drosophila provided evidence that centrosomes were not active or necessary in different types of neurons. More recently, a study showed that centrosomal microtubules are dispensable for axon specification and growth in mice in vivo but are required for neuronal migration in the cerebral cortex. However, another study has linked the generation of acetylated microtubules at centrosomes with axon development. In this current study, the authors examine the effect of centrosome loss on various motor and sensory neurons and muscles mainly by examining mutants in essential centriole duplication genes. They associate axonal routing and morphology defects with centrosome loss and provide some evidence that centrosomes could still be active in the developing neurons. Overall, they conclude that centrosomes are active during at least early neuronal development and that this activity is important for proper axonal morphology and routing.

   While I think this study addressing a very interesting and important question, I think as it stands the data is not sufficient to be conclusive on a role for centrosomes during neuronal development. My biggest concern is that most phenotypes have not yet been shown to be cell autonomous, as whole animal mutants have been analysed rather than analysing the effect of cell-specific depletion, and the evidence for active centrosomes needs to be strengthened. If the authors can provide stronger evidence for a role of centrosomes in axonal development then the paper will certainly be of interest to a broad readership.

   Major comments

   1. The sas-6 transallelic combination shows only 17% embryonic lethality compared to 50% embryonic lethality with sas-4 mutants. Given that both mutants should result in the same degree of centrosome loss (this should be quantified in sas-6 mutants) it would suggest that either sas-4 has other roles away from centrosomes or that the sas-4 mutant chromosome used in the experiment has other mutations that affect viability. The effect of picking up "second-site lethal" mutations on mutant chromosomes is common and so I would not be surprised if this is the reason for the difference in phenotypes. This can be addressed either by "cleaning up" the sas-4 mutant chromosome by backcrossing to wild-type lines, allowing recombination to occur and replace the potential second site mutations, or by using transallelic combinations of sas-4, as they did for sas-6. The "easier" option may just be to analyse all the phenotypes with the sas-6 transallelic combination.
   2. Using "whole animal" mutants for assessing neuronal morphology is risky due to non-cell-autonomous effects. The authors have carried out some phenotypic analysis of neurons depleted of Sas-4 by cell-specific RNAi, but I feel they need to do this for all of their analysis. This includes embryonic lethality measures, quantification of centrosome numbers, and all axonal phenotypes in Sas-4 RNAi neurons. It would also be prudent to use 2 distinct RNAi lines to help ensure any phenotypes are not off-target effects (and this may help clarify why the authors see some additional phenotypes with RNAi). Indeed, there are relatively weak phenotypes in muscles when using RNAi compared to the mutants and these potential non-cell-autonomous effects could then have a knock-on effect on neuronal morphology. If the authors were concerned that RNAi is not very efficient (explaining any potential weaker phenotypes than in mutants) the authors could examine the effectiveness of RNAi lines by analysing protein depletion by western blotting or mRNA depletion by rt-qPCR (although this has to be done in a different cell type due to the difficulty in obtaining a neuronal extract).
   3. When analysing centriole presence or absence it is a good idea to stain with two different centriole markers e.g. Asl and Plp. This helps rule out unspecific staining. It is clear from the images that similar sized foci can be observed outside of the cells (see Figure 5A for example), so clearly some of the foci that appear to be within the cells may also be unspecific staining.
   4. The evidence for active centrosomes is not that convincing. Acetylated tubulin is associated with stable MTs, which are not normally organised by "active" centrosomes that nucleate dynamic microtubules. Moreover, it is plausible that centriole foci happen to overlap with the acetylated tubulin staining by chance. This would explain why not all centrosomes colocalise with acetylated tubulin signal. The authors could better test centrosome activity by performing live imaging with EB1-GFP. If centrosomes are active, it is very easy to observe the many comets produced by the centrosomes.
   5. If the authors believe that centrosomes have a role in axon pathfinding in sensory neurons, they should show that these centrosomes are active, at least during early stages (again using EB1-GFP imaging).
   6. The authors mention in the discussion that "increased JNK activity, can result in axonal wiggliness (Karkali et al, 2023)". I therefore wonder whether centrosome loss may induce JNK activation (the stress response), as this would then indicate an indirect effect of centrosome loss on axonal structure rather than a direct influence of centrosome-generated microtubules. The authors could assess whether the DNK-JNK pathway is activated in neurons lacking centrosomes by expression UAS-Puc-GFP and quantifying the nuclear signal.
   7. In Figure 5, the authors claim that they find "a correlation between axonal guidance phenotypes and the numbers of centrioles per embryo". I don't think this is a strong correlation. The difference in centriole number between embryos with no defects and those with defects is very small. In contrast, the difference between centriole numbers in control (no defects) and mutant (no defects) is very large. So, there does not appear to be a strong correlation between centrosome number and phenotype.

   Minor comments

   1. I don't understand Figure 3C - why do the % of surviving homozygotes and heterozygotes add up to 100%? Should the grey boxes not relate to dead and the white to surviving?
   2. "In mouse fibroblasts, myoblasts and endothelial cells, centrosome orientation is important for nuclear positioning and cell migration(Chang et al, 2015; Gomes et al, 2005; Kushner et al, 2014)." Do you mean "centrosome position"?
   3. In the introduction, the authors mention Meka et al. when saying the centrosomal microtubules are important for axonal development, but they should also discuss the counter argument from Vinopal et al., 2023 (Neuron) that showed how centrosomes were required for neuronal migration but not axon growth, which was instead mediated by Golgi-derived microtubules.
   4. Lines 228-230 - repeated sentence
   5. Additionally, we did not detect centrioles in the quadrant opposite the axon exit point (Fig. 2B n=75) - this data is not in Fig 2B
   6. "This significant decrease in the humber of centrioles further supports the critical role of Sas-4 in pioneer neurons of the ventral nerve cord (VNC) during Drosophila embryogenesis". It rather highlights that Sas-4 is required for centriole formation in these neurons. Also, humber = number.
   7. Result title: Non-ciliated sensory neurons have centrioles. This is kind of obvious. A better title may be "axon phenotypes correlate with centriole numbers in sensory neurons" but unfortunately i don't think there is good evidence for this (See major point above).

   Significance

   As mentioned above, the advance will be important if more evidence is provided. In this case, the paper will be interesting to a broad readership. But currently the paper is limited by the lack of evidence for centrosome function and activity in the neurons.

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   Version published to 10.1101/2025.01.26.634909 on bioRxiv

   Jan 27, 2025