Centriole elimination during C. elegans oogenesis initiates with loss of the central tube protein SAS-1

  1. Marie Pierron
  2. Alexander Woglar
  3. Coralie Busso
  4. Keshav Jha
  5. Tamara Mikeladze-Dvali
  6. Marie Croisier
  7. Pierre Gönczy

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Abstract

Centrioles are lost during oogenesis in most metazoans, ensuring that the zygote is endowed with the correct number of two centrioles, which are paternally contributed. How centriole architecture is dismantled during oogenesis is not understood. Here, we analyze with unprecedent detail the ultrastructural and molecular changes during oogenesis centriole elimination in C. elegans . Centriole elimination begins with loss of the so-called central tube and organelle widening, followed by microtubule disassembly. The resulting cluster of centriolar proteins then disappears gradually, usually moving in a microtubule- and dynein-dependent manner to the plasma membrane. Moreover, we find that neither Polo-like kinases nor the PCM, which modulate oogenesis centriole elimination in Drosophila , do so in C. elegans . Furthermore, we demonstrate that the central tube protein SAS-1 normally departs first from the organelle, which loses integrity earlier in sas-1 mutants. Overall, our work provides novel mechanistic insights regarding the fundamental process of oogenesis centriole elimination.

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    Reply to the reviewers

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

    Centrioles are small cylindrical structures with roles in cell division, motility, and signaling. Typically, centrioles are highly stable structures which can persist for many cell generations. However, in some cells, such as the female germ line of many species, centrioles are programmed for elimination. This process is essential for maintaining centriole number from one generation to the next in sexually reproducing organisms, yet in nearly all species the molecular mechanisms underlying how centrioles are eliminated is unknown. The current study utilizes the nematode C. elegans to explore how centriole architecture changes during the elimination program in the female germ line. Using a suite of light microscopy techniques, the authors provide a stunning visual perspective of how centrioles are disassembled during oogenesis and show that removal of the central tube component SAS-1, a key regulator of centriole stability, is an early event in elimination. I have no major objections to the work and enthusiastically endorse its publication with the following minor revisions.

    Page 9 line 200: In the pcmd-1 mutant, the authors state that centriolar foci devoid of nuclei are present in rachis, but they do not mention in the text that there are also nuclei that lack centriole foci in early pachytene. This is mentioned in the figure legend, but I felt it was important enough to mention in the text.

    As per the reviewer’s suggestion, we will provide this information in the main text as well.

    Page 9 line 211. The authors found that in the absence of dynein heavy or light chain that centrioles remain associated with the nuclear envelope (rather than moving to the periphery). To me this was striking as dynein depletion in the embryo results in the opposite phenotype with centrioles losing attachment to the nuclear envelope and moving to the cell periphery (Gonczy et al. 1999 JCB 147:135). It might be worth pointing this out somewhere in the manuscript and speculating about the reasons for this difference.

    We will expand the Discussion section to better explain the difference of dynein’s involvement in the oocyte versus the embryo.

    Page 11 line 277: The authors state that elimination timing is not affected by the loss of SPD-5. This is a small but important point. It really is the absence of PCMD-1 and not SPD-5, as SPD-5 is still present in the cell. An alternative would be to say "in the absence of PCM" or "in absence of a pericentriolar accumulation of SAS-5".

    Fully agreed, we will modify the text accordingly.

    Figure 4D: Why does loss of PCMD-1 result in a delay in oocyte maturation as judged by RME-2 accumulation? This is not mentioned in the paper. Is this a general response to a loss of PCM or is this specific to a loss of PCMD-1?

    We realize that we were not sufficiently clear in explaining that RME-2 accumulation reflects the maturation state of oocytes. In the revised manuscript, we will clarify this point further and mention that a mild developmental delay (such as in pcmd-1(t3421ts) mutant animals) can impact the number of maturing oocytes present in the proximal gonad, and thereby lead to a slight shift in RME‑2::GFP distribution. See also related minor comment 2 of reviewer 2, and major comment 1 of reviewer 3.

    Figure 7 E and F. The authors measure the tubulin and SAS-4 intensity in wild-type and sas-1(t1521) embryos and conclude that microtubules and SAS-4 signals decay faster in the sas-1 mutant than in the control. To me, this is convinceingly the case with microtubules in panel E but I am not so sure this is the case with SAS-4 as shown in panel F. The differences in SAS-4 levels are much smaller between mutant and control. Could the authors provide statistical analysis to show how significant the differences are?

    We will provide the requested statistical analysis (which indeed shows significance).

    Page 15 line 363. I think this sentence should be reworded to: "Finally, we demonstrate that the central tube protein SAS-1 is the first of the factors analyzed here to leave centrioles..."

    In response to this suggestion and to the related comment of reviewer 2 (see below), we will rephrase this sentence to read “among the centriolar components analyzed to date, SAS-1 is the first to depart”.

    Reviewer #1 (Significance (Required)):

    The work contained in this manuscript represents a fundemental step forward in understanding the process of centriole elimination. The authors have carefully described the stepwise disassembly of the centriole including changes in the architechure during oogenesis. They have identified loss of the centriole stability factor SAS-1, as an early event in the elimination program and have found that in a sas-1 mutant, the centriole disassembles prematurely. They have also shown that loss of SAS-1 is followed by expansion of the centriole and ultimately loss of structural integrity. This work should be of interest to a broad range of scientists including those interested in centrosome dynamics, germ line development, and more generally cell biologists.

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

    Summary In this manuscript Pierron et al. explore the mechanisms of centriole elimination during oogenesis in C. elegans. Centriole elimination is a common feature of oogenesis in many species, but it is relatively poorly understood and understudied. Here, the authors characterise the kinetics with which several key centriole and centrosome proteins are lost during this process in living worms, and they correlate this with an EM and expansion microscopy (U-Ex-STED) analyses of fixed tissues. They conclude that centriole elimination begins with the loss of SAS-1 from the central region of the centrioles, which correlates with the widening of the structure and the loss of the centriole MTs. A remnant structure containing several core centriole proteins remains, however, and this often ultimately detaches from the nuclear envelope and moves towards the plasma membrane in a MT-motor-dependent fashion before it dissipates (although detachment from the nucleus does not seem to be required for the eventual elimination of this residual structure). Intriguingly, centriole loss in this system does not appear to require the down-regulation of PLK activity, which is in contrast to the situation in Drosophila oogenesis.

    The manuscript is generally well written and the data is of a high quality and is logically and clearly presented. Although the ultimate mechanisms regulating centriole elimination remain obscure (i.e. what triggers the loss of SAS-1, and how is this regulated?), the data presented here will be of significant interest to the centriole/centrosome field and I am supportive of publication. I have a few points that the authors should consider prior to publication.

    Major comment:

    In the EM shown in Figure 5F the authors claim that the central tube of the centriole is disrupted, but the other elements (inner tube, MTs and paddlewheel) are not. I don't think this is as clear cut as the authors claim-at least from comparing the images of the one normal centriole (5E) and one centriole that is starting to be eliminated (5F). It seems much harder to distinguish the MTs and the inner tube in the image in 5F. Perhaps this is obvious to the authors as they have compared many more images, but I think they need to find some way of showing this more convincingly (a montage of multiple centrioles)?

    We understand that Figure 5F alone may have left the reviewer wondering whether the central tube is truly the first element to be disrupted during centriole elimination. We plan on strengthening this point by providing additional EM images as a Supplemental Figure.

    This same issue is compounded in Figure 6D where, using a different technique (U-Ex-STED), the authors claim that the centriolar distribution of SAS-1 is gradually disrupted as centriole elimination proceeds. It does look like the amount of SAS-1 has decreased from early prophase to late pachytene, but the central tube it stains doesn't look particularly disrupted and, if anything, the MTs look more disrupted (and also possibly of lower intensity, perhaps explaining why the ratio of SAS-1/tubulin doesn't change very much over these stages, as shown in Figure 6G).

    As the reviewer correctly noticed, there is some variability in central tube removal during oogenesis. In some cases, such as in the centriole on the right of the late pachytene panel in Fig. 6D, SAS-1 signal intensity diminishes uniformly, without apparent holes in the central tube. By contrast, in other cases, such as in the centriole on the left of the late pachytene panel, SAS-1 signal intensity diminution is accompanied by a loss of central tube continuity. We will clarify the writing and qualify our findings on this important point in the revised manuscript.

    These points are important, as throughout the manuscript the authors assume it as a fact that SAS-1 leaves the centriole early (which is clear), and that this leads to the specific loss of the central tube (which, at least on the basis of this data, is not so clear).

    As mentioned above, we will make certain that the results linking SAS-1 departure and central tube loss are explained in a clear and balanced manner in the revised manuscript.

    Minor comments:

    1. The authors state that the kinetics of GFP-SAS-7 or SAS-4 loss were not altered in pcmd-1 mutants (Figure 4A-C; Figure S3E,F). This doesn't look correct to me, as both proteins seem to stay brighter for longer in the mutant embryos (and this is quite easy to see on the quantification graph for SAS-7 in Figure 4C). It looks similar for SAS-4 from the pictures shown in Figure S3E,F, although this data is not quantified (and is there any reason why this data is not quantified?).

    As mentioned in response to reviewers 1 and 3, we will mention in the revised manuscript that a mild developmental delay can impact the number of maturing oocytes present in the proximal gonad, thereby leading to this slight shift in GFP::SAS-7 and GFP::SAS-4 persistence.

    1. The authors state that they demonstrate that SAS-1 is the first component to leave the disassembling centrioles. I would rephrase as they can't know this for sure (i.e. there could be some untested component that leaves earlier).

    In response to this suggestion and to the related comment of reviewer 1 (see above), we will rephrase this sentence to read “among the centriolar components analyzed to date, SAS-1 is the first to depart”.

    In the latter part of the Discussion the authors state that SAS-1 is critical for centriole elimination. I would rephrase, as this seems to suggest it is required for centriole elimination, which is not the case. It might also be worth discussing that the elimination machinery clearly seems to target SAS-1 early on, but we don't yet know what this machinery is or how it is regulated.

    We thank the reviewer for raising this important point, which we will implement in the Discussion accordingly.

    Reviewer #2 (Significance (Required)):

    The manuscript is generally well written and the data is of a high quality and is logically and clearly presented. Although the ultimate mechanisms regulating centriole elimination remain obscure (i.e. what triggers the loss of SAS-1, and how is this regulated?), the data presented here will be of significant interest to the centriole/centrosome field and I am supportive of publication. I have a few points that the authors should consider prior to publication.

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

    Pierron et al. uses C. elegans oocytes to tackle a fundamental, yet heavily under-studied question in developmental biology: how are centrioles are eliminated during gamete formation/maturation? The paper's main conclusion is that SAS-1 (a key protein that make up the central tube in C. elegans centrioles) plays a critical part to regulate the timing of centriole elimination. I congratulate the authors on all the experiments related to SAS-1 part of their story, as they are done meticulously and in unprecedented detail (particularly all the fascinating EM and expansion microscopy data!).

    The paper also concludes that the Polo-like kinase family does not have a central role in this process, in stark contrast to a previous report demonstrating their importance for centriole elimination in Drosophila oogenesis (Pimenta-Marques et al. 2016 Science). Unfortunately, I am less convinced about this part of the paper, and half of my major comments below relate to the experiments/analyses in this regard. I was similarly not very enthusiastic about a part of story that I didn't find very relevant to the main point of the paper: half of the centrioles detach from the nucleus and translocate to plasma membrane prior to their elimination. I find the observations here quite epiphenomenal and lacking a direct/mechanistic relevance to either the PLK or SAS-1 part of the story. In my view, the authors should consider taking this part out.

    Regarding this last suggestion: we think that even if the movement of centrioles remnant is not essential for final removal, an account of this process provides important information about cellular dynamics during oocyte maturation. We note also that the two other reviewers did not raise this point, but leave the final decision to the editor.

    Overall, the piece is well written and organized, however it suffers from several shortcomings that preclude it from publication in its current form. I list my criticisms and suggestions below.

    Major comments:

    1. The authors state firmly at several places in the text that PCM components do not contribute to the timing of centriole elimination (e.g., lines 420-421), particularly given their experiments with Polo kinase paralogs. In my view, the data speaks otherwise. The centriole elimination process appears strikingly premature when SPD5__1__ (another PCM component) is overexpressed with the fluorescent transgene (Figure 1I). The opposite is also true - when another PCM component, PCMD-1, is knockdown by a temperature sensitive allele, the centriole elimination process is severely delayed 2 (Figure 4C). Even more extremely in the epistatic Polo mutant conditions (Fig. S3B), the centrioles do not appear to be eliminated at all__3__ (though the authors prefer to interpret this result differently in line 260-263, which could be flawed per my second comment below). How do the authors explain all these intriguing results? (underlining and numbering added above to clarify our responses point by point hereafter)

    1 > We respectfully disagree, since our quantifications show clearly that the SAS-7 signal disappears with an analogous timing in the line expressing RFP::SPD-5 (Fig. 1J) when compared to the other lines (Fig. 1D, 1F and 1H). The image shown currently for RFP::SPD-5 (Fig. 1I) is somewhat of an outlier compared to the others (Fig. 1C, 1E and 1G), and we will therefore provide a more representative specimen in the revised manuscript to avoid confusion.

    2 > As mentioned also in response to reviewers 1 and 2, we realize that we were not sufficiently clear in explaining that RME-2 accumulation reflects the maturation state of oocytes. In the revised manuscript, we will clarify this point and mention that a mild developmental delay (such as in pcmd-1(t3421ts) mutant animals) can impact the number of maturing oocytes present in the proximal gonad, and thereby lead to a slight shift in RME‑2::GFP distribution (as opposed to representing a delay in centriole elimination in pcmd-1(t3421ts) mutant animals).

    3 > We used *plk-1(or683ts); plk-2(ok1936) *double mutants to further test whether there might be premature elimination in this strong reduction-of-function condition compared to RNAi-mediated depletion. Although centriolar foci appear to remain for a longer time, these gonads are extremely disorganized, so that our conclusion regarding PLK-1 and PLK-2 are based primarily on the combined data shown in Fig. 3 and Fig. S3, which do not exhibit premature centriole elimination. We will rectify the writing to clarify these points.

    Also, I believe these claims (on the PCM components and their role in centriole elimination) will benefit from more nuanced statements. For instance, although Plk paralogs may not be necessary for the centriole elimination process, some other centrosome components clearly are. Paradoxically, the effects observed here (when disrupting or promoting PCM formation) has the totally opposite effects observed in Pimenta-Marques et al. 2016 Science. The 2016 piece claimed that the loss of PCM renders centrioles more vulnerable to losing their stability (which makes sense). How do the authors interpret their own results (i.e. that a disturbed PCM leads to slower centriole elimination, and vice versa)?

    As suggested by the reviewer, we will consider toning down claims regarding the role of PCM components in centriole elimination. Moreover, we will expand the section in the Discussion comparing our results with the published work of Pimenta-Marques et al. in Drosophila. This being written, as mentioned above, our findings do not suggest that removing the PCM (in pcmd-1(t3421ts) mutant animals) alters centriole elimination timing in C. elegans.

    I invite the authors to more carefully tread these nuances throughout their manuscript, which otherwise may cast major doubt on their claims.

    See point above.

    1. When investigating the role of Polo-like kinases, the authors assume that centriole elimination must follow (or correlate with) the dynamics of RME-2 (as a proxy for oocyte maturation). What guarantees that the centriole elimination process has to follow oocyte maturation? As far as I could tell, there is no direct evidence presented in the paper about this point. Do the authors have direct data (or reference to another work) that this trend must hold true at all times? I can readily see several places in the paper where this correlation doesn't appear to hold (e.g., in Fig. 4D the centriole elimination precedes the oocyte maturation under pcmd-1 condition).

    We will provide further data supporting the view that oocyte maturation and centriole elimination are correlated, whereby premature oocyte maturation mutants, such as* let-60(ga89ts)* and kin-18(ok395), exhibit precocious elimination.

    To correctly interpret their results on the epistatic Polo mutants, the authors could examine centriole elimination timing with mutants that can pre-maturely trigger or delay oocyte maturation (and do so without affecting the centriole biology itself).

    See above point.

    1. Lines 155-159 on the dimness of the SAS-6 signal make me worried about how successfully the transgenes were generated. Could the authors comment on, or perhaps extend in detail in the Methods section, through what assays the transgenes were validated? For example, did the authors try to rescue a SAS-6-/- with a SAS-6::GFP transgene? I would like to see further support for their validities.

    We will explicitly explain in the Material and Methods section that the SAS-6::GFP transgene indeed rescues the sas-6 null phenotype.

    If the authors can demonstrate the validity of their transgenes more reliably, could they possibly comment on the bunch of seemingly random SAS-6::GFP foci in Fig. 1G?

    We will comment on the presence of small SAS-6::GFP foci in the most mature oocytes, which correspond to potential precursors of centriolar elements later assembled in the embryo.

    1. Starting from line 204, the authors use the percentage of oocytes with detached centrioles (from the nucleus) as a proxy for movement to plasma membrane. This can be very confounding in my view (due to erroneous detachments etc.). As the authors explicitly state that the detachment is a process followed by a directed movement (with a defined velocity) towards the plasma membrane, this calls for a much better measurement in general. The authors should directly measure how far the centrioles are from the closest plasma membrane region in each condition they are examining (and should do this as a function of the "time progression" in different oocytes as they get closer to fertilization).

    As mentioned above, we think that an account of the movement of centriole remnants provides important information about cellular dynamics during oocyte maturation. However, given that this movement is not essential for the elimination of such remnants, it appears that providing additional complex 3D analysis as suggested by the reviewer will not benefit the present manuscript.

    Do the authors observe any propensity in sas1(t1521ts) oocytes as to where the centrioles are being degraded more prominently in the cytoplasm (i.e., when attached to the nucleus vs. when near the plasma membrane)? They could perform analyses à la their assessments in Fig. S2 and see whether they can extract some more information about this. In other words, I am wondering whether SAS-1 regulates the centriole elimination process more prominently at near the nucleus or near the plasma membrane.

    Centriole elimination occurs during pachytene in sas-1(t1521) mutant animals, when nuclei are packed in the gonad and surrounded by little cytoplasm. Therefore, even if foci were to detach from nuclei at this stage, we would not be able to quantify it with certainty. We will discuss these points in the revised manuscript.

    I ask this because the section about "centrioles moving to plasma membrane" appears epiphenomenal and rather random (i.e., the chances of a centriole moving to plasma membrane appears 50-50 under some control conditions - see control RNAi in Fig. 2G for example). Could the authors explore their existing data more closely (like suggested above), to see whether they could find intriguing correlations that tells us a little more about whether the centriole elimination at these two places are achieved differently? Otherwise, I frankly do not think this section contributes significantly to the essence of the story.

    We apologize for the confusion our writing seems to have generated. The chances of moving to the plasma membrane are not 50-50. The actual figure is 78.7% (reported as ~80% in the manuscript, line 187), and stems from the live imaging experiments where every travelling event can be monitored. By contrast, the analysis of fixed specimens is an underestimate as it provides only a snapshot of a dynamic process. We will expand the writing in the revised manuscript to clarify this point.

    Finally, the statements about a deterministic function for the plasma membrane re-localization should be toned down, because unlike what the authors claim in the paper (that ~80% of the centrioles move to plasma membrane), the control data (in Fig. 2B) clearly demonstrates that this number is more like ~60% (hence close to its chances being 50-50).

    Please see response just above.

    The paper carefully quantifies most of the data (for which I sincerely congratulate the authors!), however the experiments in Fig. S3 fall short of this. It would be nice if the authors could do the same here for completion.

    We will provide quantifications for Fig. S3E and S3F. However, due to the high disorganization of *plk-1(or683ts); plk-2(ok1936) *gonads, the presence of centriolar foci relative to oocyte position cannot be quantified accurately in this case.

    Minor comments:

    1. Sentence in lines 110-113 is too long and perturbs the flow. This should be shortened or be broken into better clauses. Perhaps the following way? "Prior analysis of centriole elimination in C. elegans oogenesis uncovered that this process takes place during diplotene..."

    The text will be modified accordingly.

    What are the orange arrowheads in the figure panels? They are not stated explicitly in the figure legends. My prediction was that they point to regions where centrioles are in another plane (though the overview is depicted from a different slice in the stack). Is this right? Either way, it will be useful to over-guide the reader on these orange arrowheads.

    The meaning of the orange arrowheads is explained in lines 520-521.

    If I am not wrong, the data/graph in Figures S2G and 2E are essentially the same (i.e., the data are duplicated). I couldn't find any statement in the figure legends indicating this. This should be added.

    Apologies about this oversight -the reviewer is correct and we will make a mention of this redundancy in the legend of Fig. S2.

    Some may consider the discussion on C2CD3 a little far-fetched, as this protein localizes to the distal end of centrioles (completely unlike SAS-1). Also, unlike the C. elegans centrioles, mammal centrioles do not contain a discernible central tube, casting doubt on the possibility of speculations made in the Discussion section. I suggest to remove out this paragraph, and instead to explicitly state whether the SAS-1 dependent mechanism could be applicable to other species is unclear.

    We will nuance these thoughts, further stressing their speculative nature, but intend to maintain them in some form as they provide a potential parallel that will be of interest to the human cell biology community.

    Could the authors add in their Discussion section some comment/thought on what the remaining GFP::SAS-7 pool (line 300-302) might possibly be? Curiously, there doesn't seem to be any structure associated with it in their EM tomograms, so it would be helpful to guide the reader further on this interesting finding.

    Although we would love to comment on this further, the remaining GFP::SAS-7 foci lack ultrastructural organization and do not exhibit recognizable electron densities. That this is the case will be stated explicitly in the revised manuscript.

    Reviewer #3 (Significance (Required)):

    General Assessment: This paper's strength is in its rigorous cell biology approaches to tackle a fundamental developmental biology problem. However, some of their conclusions are too firm while not being well-supported by the data, so the paper requires major revision before its publication.

    Advance: Discovery of a new molecular player in the centriole elimination process in worm oocytes, which can pave the way for future discoveries of centriole elimination mechanisms in other species. It is not yet clear whether the results will be broadly applicable, as some of the findings presented are in stark contrast to previous studies published on centriole elimination processes in Drosophila oocytes (e.g., Pimenta-Marques et al. 2016 Science). However, as summarized in the above section, these conclusions require further experimental evidence/support.

    Audience: Centriole elimination mechanisms are not widely studied, so I am not entirely sure whether this piece will be of immediate interest to the broad cell biology community. It will certainly be of general interest to several groups studying centriole elimination mechanisms, as well as developmental biologists trying to understand the oocyte maturation process.

    My expertise: Molecular and cellular mechanisms of cytoplasmic organization in development

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    Referee #3

    Evidence, reproducibility and clarity

    Pierron et al. uses C. elegans oocytes to tackle a fundamental, yet heavily under-studied question in developmental biology: how are centrioles are eliminated during gamete formation/maturation? The paper's main conclusion is that SAS-1 (a key protein that make up the central tube in C. elegans centrioles) plays a critical part to regulate the timing of centriole elimination. I congratulate the authors on all the experiments related to SAS-1 part of their story, as they are done meticulously and in unprecedented detail (particularly all the fascinating EM and expansion microscopy data!).

    The paper also concludes that the Polo-like kinase family does not have a central role in this process, in stark contrast to a previous report demonstrating their importance for centriole elimination in Drosophila oogenesis (Pimenta-Marques et al. 2016 Science). Unfortunately, I am less convinced about this part of the paper, and half of my major comments below relate to the experiments/analyses in this regard. I was similarly not very enthusiastic about a part of story that I didn't find very relevant to the main point of the paper: half of the centrioles detach from the nucleus and translocate to plasma membrane prior to their elimination. I find the observations here quite epiphenomenal and lacking a direct/mechanistic relevance to either the PLK or SAS-1 part of the story. In my view, the authors should consider taking this part out.

    Overall, the piece is well written and organized, however it suffers from several shortcomings that preclude it from publication in its current form. I list my criticisms and suggestions below.

    Major comments:

    1. The authors state firmly at several places in the text that PCM components do not contribute to the timing of centriole elimination (e.g., lines 420-421), particularly given their experiments with Polo kinase paralogs. In my view, the data speaks otherwise. The centriole elimination process appears strikingly premature when SPD5 (another PCM component) is overexpressed with the fluorescent transgene (Figure 1I). The opposite is also true - when another PCM component, PCMD-1, is knockdown by a temperature sensitive allele, the centriole elimination process is severely delayed (Figure 4C). Even more extremely in the epistatic Polo mutant conditions (Fig. S3B), the centrioles do not appear to be eliminated at all (though the authors prefer to interpret this result differently in line 260-263, which could be flawed per my second comment below). How do the authors explain all these intriguing results?

    Also, I believe these claims (on the PCM components and their role in centriole elimination) will benefit from more nuanced statements. For instance, although Plk paralogs may not be necessary for the centriole elimination process, some other centrosome components clearly are. Paradoxically, the effects observed here (when disrupting or promoting PCM formation) has the totally opposite effects observed in Pimenta-Marques et al. 2016 Science. The 2016 piece claimed that the loss of PCM renders centrioles more vulnerable to losing their stability (which makes sense). How do the authors interpret their own results (i.e. that a disturbed PCM leads to slower centriole elimination, and vice versa)?

    I invite the authors to more carefully tread these nuances throughout their manuscript, which otherwise may cast major doubt on their claims.

    1. When investigating the role of Polo-like kinases, the authors assume that centriole elimination must follow (or correlate with) the dynamics of RME-2 (as a proxy for oocyte maturation). What guarantees that the centriole elimination process has to follow oocyte maturation? As far as I could tell, there is no direct evidence presented in the paper about this point. Do the authors have direct data (or reference to another work) that this trend must hold true at all times? I can readily see several places in the paper where this correlation doesn't appear to hold (e.g., in Fig. 4D the centriole elimination precedes the oocyte maturation under pcmd-1 condition).

    To correctly interpret their results on the epistatic Polo mutants, the authors could examine centriole elimination timing with mutants that can pre-maturely trigger or delay oocyte maturation (and do so without affecting the centriole biology itself).

    1. Lines 155-159 on the dimness of the SAS-6 signal make me worried about how successfully the transgenes were generated. Could the authors comment on, or perhaps extend in detail in the Methods section, through what assays the transgenes were validated? For example, did the authors try to rescue a SAS-6-/- with a SAS-6::GFP transgene? I would like to see further support for their validities.

    If the authors can demonstrate the validity of their transgenes more reliably, could they possibly comment on the bunch of seemingly random SAS-6::GFP foci in Fig. 1G?

    1. Starting from line 204, the authors use the percentage of oocytes with detached centrioles (from the nucleus) as a proxy for movement to plasma membrane. This can be very confounding in my view (due to erroneous detachments etc.). As the authors explicitly state that the detachment is a process followed by a directed movement (with a defined velocity) towards the plasma membrane, this calls for a much better measurement in general. The authors should directly measure how far the centrioles are from the closest plasma membrane region in each condition they are examining (and should do this as a function of the "time progression" in different oocytes as they get closer to fertilization).
    2. Do the authors observe any propensity in sas1(t1521ts) oocytes as to where the centrioles are being degraded more prominently in the cytoplasm (i.e., when attached to the nucleus vs. when near the plasma membrane)? They could perform analyses à la their assessments in Fig. S2 and see whether they can extract some more information about this. In other words, I am wondering whether SAS-1 regulates the centriole elimination process more prominently at near the nucleus or near the plasma membrane.

    I ask this because the section about "centrioles moving to plasma membrane" appears epiphenomenal and rather random (i.e., the chances of a centriole moving to plasma membrane appears 50-50 under some control conditions - see control RNAi in Fig. 2G for example). Could the authors explore their existing data more closely (like suggested above), to see whether they could find intriguing correlations that tells us a little more about whether the centriole elimination at these two places are achieved differently? Otherwise, I frankly do not think this section contributes significantly to the essence of the story.

    Finally, the statements about a deterministic function for the plasma membrane re-localization should be toned down, because unlike what the authors claim in the paper (that ~80% of the centrioles move to plasma membrane), the control data (in Fig. 2B) clearly demonstrates that this number is more like ~60% (hence close to its chances being 50-50).

    1. The paper carefully quantifies most of the data (for which I sincerely congratulate the authors!), however the experiments in Fig. S3 fall short of this. It would be nice if the authors could do the same here for completion.

    Minor comments:

    1. Sentence in lines 110-113 is too long and perturbs the flow. This should be shortened or be broken into better clauses. Perhaps the following way? "Prior analysis of centriole elimination in C. elegans oogenesis uncovered that this process takes place during diplotene..."
    2. What are the orange arrowheads in the figure panels? They are not stated explicitly in the figure legends. My prediction was that they point to regions where centrioles are in another plane (though the overview is depicted from a different slice in the stack). Is this right? Either way, it will be useful to over-guide the reader on these orange arrowheads.
    3. If I am not wrong, the data/graph in Figures S2G and 2E are essentially the same (i.e., the data are duplicated). I couldn't find any statement in the figure legends indicating this. This should be added.
    4. Some may consider the discussion on C2CD3 a little far-fetched, as this protein localizes to the distal end of centrioles (completely unlike SAS-1). Also, unlike the C. elegans centrioles, mammal centrioles do not contain a discernible central tube, casting doubt on the possibility of speculations made in the Discussion section. I suggest to remove out this paragraph, and instead to explicitly state whether the SAS-1 dependent mechanism could be applicable to other species is unclear.
    5. Could the authors add in their Discussion section some comment/thought on what the remaining GFP::SAS-7 pool (line 300-302) might possibly be? Curiously, there doesn't seem to be any structure associated with it in their EM tomograms, so it would be helpful to guide the reader further on this interesting finding.

    Significance

    General Assessment: This paper's strength is in its rigorous cell biology approaches to tackle a fundamental developmental biology problem. However, some of their conclusions are too firm while not being well-supported by the data, so the paper requires major revision before its publication.

    Advance: Discovery of a new molecular player in the centriole elimination process in worm oocytes, which can pave the way for future discoveries of centriole elimination mechanisms in other species. It is not yet clear whether the results will be broadly applicable, as some of the findings presented are in stark contrast to previous studies published on centriole elimination processes in Drosophila oocytes (e.g., Pimenta-Marques et al. 2016 Science). However, as summarized in the above section, these conclusions require further experimental evidence/support.

    Audience: Centriole elimination mechanisms are not widely studied, so I am not entirely sure whether this piece will be of immediate interest to the broad cell biology community. It will certainly be of general interest to several groups studying centriole elimination mechanisms, as well as developmental biologists trying to understand the oocyte maturation process.

    My expertise: Molecular and cellular mechanisms of cytoplasmic organization in development

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In this manuscript Pierron et al. explore the mechanisms of centriole elimination during oogenesis in C. elegans. Centriole elimination is a common feature of oogenesis in many species, but it is relatively poorly understood and understudied. Here, the authors characterise the kinetics with which several key centriole and centrosome proteins are lost during this process in living worms, and they correlate this with an EM and expansion microscopy (U-Ex-STED) analyses of fixed tissues. They conclude that centriole elimination begins with the loss of SAS-1 from the central region of the centrioles, which correlates with the widening of the structure and the loss of the centriole MTs. A remnant structure containing several core centriole proteins remains, however, and this often ultimately detaches from the nuclear envelope and moves towards the plasma membrane in a MT-motor-dependent fashion before it dissipates (although detachment from the nucleus does not seem to be required for the eventual elimination of this residual structure). Intriguingly, centriole loss in this system does not appear to require the down-regulation of PLK activity, which is in contrast to the situation in Drosophila oogenesis.

    The manuscript is generally well written and the data is of a high quality and is logically and clearly presented. Although the ultimate mechanisms regulating centriole elimination remain obscure (i.e. what triggers the loss of SAS-1, and how is this regulated?), the data presented here will be of significant interest to the centriole/centrosome field and I am supportive of publication. I have a few points that the authors should consider prior to publication.

    Major comment:

    In the EM shown in Figure 5F the authors claim that the central tube of the centriole is disrupted, but the other elements (inner tube, MTs and paddlewheel) are not. I don't think this is as clear cut as the authors claim-at least from comparing the images of the one normal centriole (5E) and one centriole that is starting to be eliminated (5F). It seems much harder to distinguish the MTs and the inner tube in the image in 5F. Perhaps this is obvious to the authors as they have compared many more images, but I think they need to find some way of showing this more convincingly (a montage of multiple centrioles)?

    This same issue is compounded in Figure 6D where, using a different technique (U-Ex-STED), the authors claim that the centriolar distribution of SAS-1 is gradually disrupted as centriole elimination proceeds. It does look like the amount of SAS-1 has decreased from early prophase to late pachytene, but the central tube it stains doesn't look particularly disrupted and, if anything, the MTs look more disrupted (and also possibly of lower intensity, perhaps explaining why the ratio of SAS-1/tubulin doesn't change very much over these stages, as shown in Figure 6G).

    These points are important, as throughout the manuscript the authors assume it as a fact that SAS-1 leaves the centriole early (which is clear), and that this leads to the specific loss of the central tube (which, at least on the basis of this data, is not so clear).

    Minor comments:

    1. The authors state that the kinetics of GFP-SAS-7 or SAS-4 loss were not altered in pcmd-1 mutants (Figure 4A-C; Figure S3E,F). This doesn't look correct to me, as both proteins seem to stay brighter for longer in the mutant embryos (and this is quite easy to see on the quantification graph for SAS-7 in Figure 4C). It looks similar for SAS-4 from the pictures shown in Figure S3E,F, although this data is not quantified (and is there any reason why this data is not quantified?).
    2. The authors state that they demonstrate that SAS-1 is the first component to leave the disassembling centrioles. I would rephrase as they can't know this for sure (i.e. there could be some untested component that leaves earlier).
    3. In the latter part of the Discussion the authors state that SAS-1 is critical for centriole elimination. I would rephrase, as this seems to suggest it is required for centriole elimination, which is not the case. It might also be worth discussing that the elimination machinery clearly seems to target SAS-1 early on, but we don't yet know what this machinery is or how it is regulated.

    Significance

    The manuscript is generally well written and the data is of a high quality and is logically and clearly presented. Although the ultimate mechanisms regulating centriole elimination remain obscure (i.e. what triggers the loss of SAS-1, and how is this regulated?), the data presented here will be of significant interest to the centriole/centrosome field and I am supportive of publication. I have a few points that the authors should consider prior to publication.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Centrioles are small cylindrical structures with roles in cell division, motility, and signaling. Typically, centrioles are highly stable structures which can persist for many cell generations. However, in some cells, such as the female germ line of many species, centrioles are programmed for elimination. This process is essential for maintaining centriole number from one generation to the next in sexually reproducing organisms, yet in nearly all species the molecular mechanisms underlying how centrioles are eliminated is unknown. The current study utilizes the nematode C. elegans to explore how centriole architecture changes during the elimination program in the female germ line. Using a suite of light microscopy techniques, the authors provide a stunning visual perspective of how centrioles are disassembled during oogenesis and show that removal of the central tube component SAS-1, a key regulator of centriole stability, is an early event in elimination. I have no major objections to the work and enthusiastically endorse its publication with the following minor revisions.

    Page 9 line 200: In the pcmd-1 mutant, the authors state that centriolar foci devoid of nuclei are present in rachis, but they do not mention in the text that there are also nuclei that lack centriole foci in early pachytene. This is mentioned in the figure legend, but I felt it was important enough to mention in the text.

    Page 9 line 211. The authors found that in the absence of dynein heavy or light chain that centrioles remain associated with the nuclear envelope (rather than moving to the periphery). To me this was striking as dynein depletion in the embryo results in the opposite phenotype with centrioles losing attachment to the nuclear envelope and moving to the cell periphery (Gonczy et al. 1999 JCB 147:135). It might be worth pointing this out somewhere in the manuscript and speculating about the reasons for this difference.

    Page 11 line 277: The authors state that elimination timing is not affected by the loss of SPD-5. This is a small but important point. It really is the absence of PCMD-1 and not SPD-5, as SPD-5 is still present in the cell. An alternative would be to say "in the absence of PCM" or "in absence of a pericentriolar accumulation of SAS-5".

    Figure 4D: Why does loss of PCMD-1 result in a delay in oocyte maturation as judged by RME-2 accumulation? This is not mentioned in the paper. Is this a general response to a loss of PCM or is this specific to a loss of PCMD-1?

    Figure 7 E and F. The authors measure the tubulin and SAS-4 intensity in wild-type and sas-1(t1521) embryos and conclude that microtubules and SAS-4 signals decay faster in the sas-1 mutant than in the control. To me, this is convinceingly the case with microtubules in panel E but I am not so sure this is the case with SAS-4 as shown in panel F. The differences in SAS-4 levels are much smaller between mutant and control. Could the authors provide statistical analysis to show how significant the differences are?

    Page 15 line 363. I think this sentence should be reworded to: "Finally, we demonstrate that the central tube protein SAS-1 is the first of the factors analyzed here to leave centrioles..."

    Significance

    The work contained in this manuscript represents a fundemental step forward in understanding the process of centriole elimination. The authors have carefully described the stepwise disassembly of the centriole including changes in the architechure during oogenesis. They have identified loss of the centriole stability factor SAS-1, as an early event in the elimination program and have found that in a sas-1 mutant, the centriole disassembles prematurely. They have also shown that loss of SAS-1 is followed by expansion of the centriole and ultimately loss of structural integrity. This work should be of interest to a broad range of scientists including those interested in centrosome dynamics, germ line development, and more generally cell biologists.

  5. Version published to 10.1101/2023.06.19.545600v2 on bioRxiv
  6. Version published to 10.1101/2023.06.19.545600v1 on bioRxiv