ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Reviewer #1 (Public Review):

In their revised manuscript Hijaze et al. adequately addressed the majority of my previous concerns in a satisfactory manner. In particular, they validated their morpholino knock-down experiments by explaining how they determined the optimal concentrations and provided an immunohistological evidence for the reduction in ROCK protein abundance. The authors also added new antibody stainings providing evidence that ROCK and F-actin do not interact directly but likely through other kinases that modulate f-actin, and that the localization of f-actin at the spicule tips remains unaffected by the knock-down. In addition, the authors revised their discussion to not overstate their observations, and by focusing on the potential mechanisms by which ROCK may affect biomineralization (i.e. mechano sensing and exocytosis of vesicles). Here I would like to add, that f-actin mediated exocytosis does not necessarily target mineral baring vesicles but may also promote the exocytosis of matrix proteins that are essential for the normal formation of the spicules and that are an integral component of other biominerals, as well. I strongly encourage the authors to continue on this exciting research, including the development of methods to analyze the molecular mechanisms that control vesicular trafficking in mineralizing systems.

Reviewer #2 (Public Review):

This project is on the role of ROCK in skeletogenesis during sea urchin development. That skeleton is produced by a small number of cells in the embryo with signaling inputs from the ectoderm providing patterning cues. The skeleton is built from secretion of CaCO3 by the skeletogenic cells. The authors conclude that ROCK is involved in the regulation of skeletogenesis with a role both in regulating actomyosin in the process, and in the gene regulatory network (GRN) underlying the entire sequence of events.

The strength of the paper is that they show in detail how perturbations of ROCK results in abnormal actomyosin activity in the skeletogenic cells, and they show alterations both in expression of transcription factors of the GRN, and expression of genes involved in assembly of the skeletal matrix. Two different approaches lead to this conclusion: morpholino perturbations and the actions of a selective inhibitor of the kinase activity. Thus, they achieved their goal which was to test the hypothesis that ROCK is involved in the process of skeletogenesis. Those tests support the hypothesis with data that was quantitatively significant.

The discussion was transparent regarding where the analysis ended and where the next phase of work should begin. While actomyosin involvement was altered when ROCK was perturbed, it isn't known how direct or indirect the role of ROCK might be. Also, while the regulatory input to spicule initiation and growth is affected when ROCK is inhibited, it isn't clear exactly where ROCK is involved.

Author Response

The following is the authors’ response to the original reviews.

Thank you and the two reviewers for the thorough review of our manuscript. We found the reviewer’s comments highly valuable and addressed them by the following additional experiments and changes in the text and the figures:

(1) We measured the effect of ROCK MASO’s on the ROCK expression by immunostaining and observed a reduction in ROCK signal, supporting the downregulation of ROCK protein level under ROCK MASO’s (new Fig. S3).

(2) We measured the effect of lower concertation of ROCK inhibitor, Y27632 (10µM), and observe the same phenotypes of skeletal loss, skeletal reduction and ectopic branching in this concentration (Fig. 2, S4). Importantly, these phenotypes were not observed when directly inhibiting PKA and PKC, in whole sea urchin embryos (1) and in skeletogenic cell cultures (2), further supporting the specificity of ROCK inhibitor.

(3) We added a time course of Pl-ROCK expression and immunostaining of ROCK in the fertilized egg, that show that this gene is maternal and the protein is present in the egg Fig. 2SA-C.

(4) We recorded F-actin in ROCK MASO’s and demonstrate that it is still detected around the spicules and their tips, similarly to ROCK inhibited embryos (new Fig.S3).

(5) We revised the paper text and figures to provide a better description of our results, distinguish clearly between our data and our interpretations and emphasize the novelty of our findings.

This paper demonstrates that ROCK, F-actin polymerization and actomyosin contractility play critical roles in biomineral growth and in shaping biomineral morphology in the sea urchin embryo, and that ROCK activity affects skeletogenic gene expression. Our findings together with previous reports of the role of actomyosin in Eukaryotes biomineralization, suggest that this molecular machinery is a part of the common molecular tool-kit used in biomineralization. The identification of a common molecular mechanism within the diverse gene regulatory networks, organic scaffolds and minerals that Eukaryote use to build their biominerals will be of high interest to the field of biomineralization and evolutionary biology. Furthermore, our paper portrays the interplay between the cellular and the genetic machinery that drives morphogenesis. We believe it would be of great interest to the broad readership of eLife and particularly to the fields of biomineralization, cell, developmental and evolutionary biology.

Thank you very much for the helpful review of our paper.

Reviewer #1 (Public Review):

We thank the reviewer for the appreciation of our work the helpful comments that guided us to strengthen the experimental evidence for our conclusions and increase the paper’s clarity. Below are our responses to the specific comments:

Major comments

One MASO led to reduced skeleton formation while the other one additionally induced ectopic branching. How was the optimum concentration for the MASOs determined? Did the authors perform a dose-response curve? What is the reason for this difference? Which of the two MASOs can be validated by reduced ROCK protein abundance? Since the ROCK antibody works, I would like to see a control experiment on Rock protein abundance in control and ROCK MO injected larvae which is the gold-standard for validating the knock-down.

We tested several MASO concentrations to identify a concentration where the control embryos injected with Random MASO were overall healthy and ROCK MASO’s showed clear phenotypes.

To test the effect of ROCK MASO’s on ROCK protein levels we did immunostaining experiments that are now presented in new Fig. S3. We could not do Western blot for injected embryos since ROCK antibody requires thousands of embryos for Western blot, which is not feasible for injected embryos. Therefore, we tested the effect of the two translation ROCK MASO’s on ROCK abundance compared to uninjected and Random MASO injected embryos using immunostaining. We observed a reduction of ROCK signal, supporting the downregulation of ROCK protein level in these genetic perturbations (new Fig. S3).

L212 "Together, these measurements show that ROCK is not required for the uptake of calcium into cells." But what about trafficking and exocytosis? As mentioned earlier, I think this is a really important point that needs to be confirmed to understand the function of ROCK in controlling calcification. In their previous study (reference 45) the authors demonstrated that they have superior techniques in measuring vesicle dynamics in vivo. Here an acute treatment with the ROCK inhibitor would be sufficient to test if calcein-positive vesicle motion, including the observed reduction in velocity close to the tissue skeleton interface, is affected by the inhibitor.

We thank the reviewer for the appreciation of our previous work where we studied calcium vesicle dynamics in whole embryos (Winter et al, Plos Com Biol 2021). We agree with the reviewer that the best way to test directly the effect of ROCK on mineral deposition and vesicle kinetics is to observe it in live skeletogenic cells. However, in Winter et al 2021, we found that the skeleton (spicules) doesn’t grow when the embryos are immobilized in either control or treated embryos. We have to immobilize the embryos to record live timelapses of whole embryos. Hence, this means that we can not determine the role of ROCK or any other perturbation in vesicle trafficking and exocytosis based on experiments conducted in immobilized whole embryos, since skeletogenesis is arrested. We believe that we can do it in skeletogenic cell cultures and we are currently developing this assay for vesicle tracking, but this is beyond the scope of this current work.

Is there a colocalization of ROCK and f-actin in the tips of the spicules? This would support the mechano-sensing-hypothesis by ROCK.

Our studies show that F-actin is localized around the spicule cavity and in the cortex of the cells (Figs. 5 and 6) while ROCK is enriched in the skeletogenic cell bodies, with some localization near the skeletogenic cell membranes (Fig. 1). To directly address the reviewer question we immune-stained ROCK and F-actin in the same embryos, and showed that their sub-cellular localizations does not show a strong overlap (Fig. S3 Q-T). However, ROCK does not bind F-actin directly: ROCK activates another kinase, LimK that phosphorylates Cofilin that interacts with F-actin. Therefore, the fact that ROCK is not colocalized with F-actin does not support nor contradicts the possible role of ROCK in mechano-sensing.

L 283. "F-actin is enriched at the tips of the spicules independently of ROCK activity" The results of this paragraph clearly demonstrate that ROCK inhibition has no effect on the localization of f-actin at the tips of the growing spicules. In addition, the new cell culture experiments underline this observation. Still, the central question that remains is, what is the interaction between ROCK, f-actin, and the mineralization process, that leads to the observed deformations? What does the f-actin signal look like in a branched phenotype or in larvae that failed to develop a skeleton (inhibition from Y20)?

As we report in Fig. 6, and now on new Fig. S3, under ROCK late inhibition or in ROCK morphants, we still detect F-actin around the spicule and enriched at the tips. When ROCK is inhibited and the embryo fails to develop a skeleton, we observe Factin accumulation in the skeletogenic cells, but the F-actin is not organized (Fig. 5). As the spicule is absent in this condition, it is hard to conclude whether the effect on F-actin organization is direct or due to the absence of spicule in this condition. We stated that explicitly in the current version in the results, lines 324-326 and in the discussion, lines 405-408.

Immunohistochemical analyses on f-actin localization and abundance should be additionally performed with ROCK knock-down phenotypes to confirm the pharmacological inhibition.

We did that in our new Figure S3 and showed that ROCK morphant show the same F-actin localization at the tips like control and ROCK inhibited embryos.

L 365 "...supporting its role in mineral deposition..." "...Overall, our studies indicate that ROCK activity....is essential for the formation of the spicule cavity......which could be essential for mineral deposition..." I think the authors need to do a better job in clearly separating between the potential processes impacted by ROCK perturbation. Is it stabilization and mechano-sensing in the spicule tip or the intracellular trafficking and deposition of the ACC? If the dataset does not allow for a definite conclusion, I suggest clearly separating the different possibilities combined with thorough discussion-based findings from other mineralizing systems where the interaction between ROCK and F-actin has been described.

We thank the reviewer for this important comment. We believe that ROCK and the actomyosin are involved in both, mechano-sensing of the rigid biomineral and in the transport and exocytosis of mineral-bearing vesicles. In the current version we provide explicit explanations of these two hypotheses in the discussion section. The possible role in exocytosis and the experiments that are required to assess this role are described in lines 427-439, and the possible mechano-sensing role and effect on gene expression is described in lines 440-453.

Reviewer #1 (Recommendations For The Authors):

Minor comments

L185 "These SR-µCT measurements show that the rate of mineral deposition is significantly reduced under ROCK inhibition." To correctly support this statement I would suggest to calculate the real growth rates (µm3 time-1). For example, an increase in volume from 6,850 µm3 at 48 hpf to 14,673 µm3 at 72 hpf would result in a growth rate of 7823 µm3 24h-1.

We thank the reviewer for this suggestion. We calculated the rate of spicule growth as the reviewer suggested and we added this information in lines 218-221.

L343: "This implies that....within the skeletogenic lineage." This concluding sentence is very speculative and therefore misplaced in the results section.

We removed this sentence from the results section into the discussion, lines 443-445.

L382: "The participation of F-actin and ROCK in polarized tip-growth and vesicle exocytosis has been observed in both, animals and plants." L407-409: "...F-actin could be regulating the localized exocytosis of mineral-bearing vesicles...." I think this is exactly the core question that remains unresolved in this study. To reduce speculations I strongly recommend addressing the effect of ROCK inhibition on vesicle trafficking and exocytosis (Monitoring of calcein-positive Vesicles in PMCs).

We agree with the reviewer that this is a critical question that we would have address, but as we explained above, is beyond the scope of this study.

Figure 5: The values below the scale bars in the newly added figures U+V are extremely small. Also, the Legend for this figure sounds incorrect. Should read: "...and skeletogenic cell cultures that were treated with 30µM ROCK inhibitor that was added at 48hpf and recorded at 72hpf.

We increased the font near the scale bars and corrected the figure caption. Thanks for this and your other helpful comments!

Reviewer #2 (Public Review):

We thank the reviewer for raising the important issue of inhibitor concentration which led us to do additional experiments with lower concentration that were valuable and strengthen the manuscript. We also thank the reviewer for asking us to be clearer with the interpretation of the results. Below are our responses to the specific comments:

My concerns are the interpretation of the experiments. The main overriding concern is a possible over-interpretation of the role of ROCK. In the literature that ROCK participates in many biological processes with a major contribution to the actin cytoskeleton. And when a function is attributed to ROCK, it is usually based on the determination of a protein that is phosphorylated by this kinase. Here that is not the case. The observation here is in most cases stunted growth of the spicule skeleton and some mis-patterning occurs or there is an absence of skeleton if the inhibitor is added prior to initiation of skeletal growth. They state in the abstract that ROCK impairs the organization of F-actin around the spicules. The evidence for that as a direct role is absent.

We agree with the reviewer that since the spicule doesn’t form under ROCK continuous inhibition, it is unclear if the absence of F-actin around the spicule in this condition is a direct outcome of the lack of ROCK activation of F-actin polymerization, or an indirect outcome due to the lack of spicule to coat. We therefore deleted this line in the abstract and explicitly stated that we cannot conclude whether the impaired F-actin organization is directly due to ROCK effect on actin polymerization in the results, lines 324-326 and in the discussion, lines 405-408.

They use morpholino data and ROCK inhibitor data to draw their conclusion. My main concern is the concentration of the inhibitor used since at the high concentrations used, the inhibitor chosen is known to inhibit other kinases as well as ROCK (PKA and PKC). They indicate that this inhibition is specifically in the skeletogenic cells based on the isolation of skeletogenic cells in culture and spicule production either under control or ROCK inhibition and they observe the same - stunting and branching or absence of skeletons if treated before skeletogenesis commences. Again, however, the high concentrations are known to inhibit the other kinases.

In the previous version of the paper we used the range of 30-80µM Y-27632 to block ROCK activity. These concentrations are commonly used in mammalian systems and in Drosophila to block ROCK activity (3-8). The reviewer is correct stating that at high concentration, this inhibitor can block PKA and PKC. However, the affinity of the inhibitor for these kinases is more than 100 times lower than its affinity to ROCK as indicated by the biochemical Ki values reported in the manufactory datasheet: 0.14-0.22 μM for ROCK1, 0.3 μM for ROCK2, 25 μM for PKA and 26 μM for PKC.

Importantly, these Ki values are based on biochemistry assays where the activity of the inhibitor is tested in-vitro with the purified protein. Therefore, these concentrations are not relevant to cell or embryo cultures where the inhibitor has to penetrate the cells and affect ROCK activity in-vivo. Y-27632 activity was studied both in-vitro and in-vivo in Narumiya, Ishizaki and Ufhata, Methods in Enzymology 2000 (9). This paper reports similar concentrations to the ones indicated in the manufactory datasheet for the in-vitro experiments, but shows that 10µM concentration or higher are effective in cell cultures. We therefore tested the effect of 10µM Y-27632 added at 0hpf (continuous inhibition) and at 25hpf (late inhibition) and added this information to Figs. 2 and S3. Continuous inhibition at this concentration resulted with three major phenotypes: skeletal loss, spicule initiations and small spicules with ectopic branching. This result supports our conclusion that ROCK activity is necessary for spicule formation, elongation and prevention of branching. Late inhibition in this concentration resulted with the majority of the embryos developing branched spicules, which is very similar to the effect of MyoII inhibition with Blebbistatin. This result again, supports the inference that ROCK activity is required for normal skeletal growth and the prevention of ectopic branching. Importantly, there are two papers were PKA and PKC were directly inhibited in whole sea urchin embryos (1) and in skeletogenic cell cultures (2). In both assays, PKC inhibition resulted with mild reduction of spicule length while PKA inhibition did not affect skeletal formation. Neither skeletal loss nor ectopic branching were ever observed under PKC or PKA inhibition, supporting the specific inhibition of ROCK by Y-27362. Furthermore, both genetic and pharmacological perturbations of ROCK resulted with significant reduction of skeletal growth and with the enhancement of ectopic branching. Therefore, we believe we provide convincing evidence for the role of ROCK in spicule formation, growth and prevention of branching. We revised Fig. 2 and S3 to include the 10µM Y-27632 data and the text describing the inhibition to include the explanations and references we provided here.

They use blebbistatin and latrunculin and show that these known inhibitors of actin cytoskeleton lead to abnormal spiculogenesis, This coincidence is suggestive but is not proof that it is ROCK acts on the actomyosin cytoskeleton given the specificity concerns.

As stated above, we believe that in the current vesion we overcame the specificity concerns and provided solid evidence that ROCK activity is necessary for spicule formation, growth and prevention of branching. Furthermore, the skeletogenic phenotypes of late 10µM Y-27632 are highly similar to those of MyoII inhibition (Blebbistatin) while the phenotypes of higher concetrations resemble the inhibition of actin polymerization by Latrunculin. We agree with the reviewer that: “This coincidence is suggestive but is not proof that ROCK acts on the actomyosin cytoskeleton” and we revise the discussion paragraph to differentiate between our solid findings and our speculations (lines 421-426): “These correlative similarities between ROCK and the actomyosin perturbations lead us to the following speculations: the low dosage of late ROCK inhibition is perturbing mostly ROCK activation of MyoII contractility while the higher dosage affects factors that control actin polymerization (Fig. 8F). Further studies in higher temporal and spatial resolution of MyoIIP activity and F-actin structures in control and under ROCK inhibition will enable us to test this.”

Reviewer #2 (Recommendations For The Authors):

The following areas require attention:

(1) You begin and end the abstract with statements on evolution in which the actomyosin cytoskeleton is associated with skeletogenesis despite different GRNs, different contributing proteins, etc. You then move to ROCK and claim to reveal that ROCK is a central player in the process. As above, in the judgement of this reviewer, you fail to establish a direct role of ROCK to the actomyosin role in skeletogenesis. Sure, the ROCK inhibitors suggest that ROCK plays some kind of role in the process but you also indicate that ROCK could act on many processes, none of which you directly associate with the necessary activity of ROCK.

We agree that our paper provides correlative similarities between the phenotypes of ROCK and those of direct pertrubations of the actomyosin network, and lacks causal relationship. We made this point clear throughout the current version of the manuscript.

(2) In the abstract you report that ROCK inhibition impairs the actin cytoskeleton around the skeleton. In examining your images in Fig. 5 that is not the case. Based on Phalloidin staining, actin surrounds both the control and the ROCK-inhibited skeleton. The distribution of actin is the same in both cases. Myosin is also stained in this figure and it too shows similar staining both in experimental and control. So, to this reviewer, there is insufficient evidence to suggest that the actin cytoskeleton is impaired, and there is no evidence directly relating ROCK with that cytoskeleton. I'm not questioning the observation that inhibition of ROCK causes stunting and mispatterning of the skeleton. That you show and quantify well. The issue is the precise target of ROCK. Your data does not establish the specific cause. It could be the actin cytoskeleton but your experiments do not directly address that.

Fig. 5 shows a clear difference between F-actin in control and under ROCK inhibition. In control F-actin is enriched around the spicule and under ROCK inhibition the spicule doesn’t form and disorganized F-actin is accumulated in the skeletogenic cells. Yet, as we stated above – this is not a proof for the direct effect of ROCK on F-actin polymerization, and we explain it explicitly in the results, lines 324-326 and in the discussion, lines 405-408.

(3) In parts of the manuscript you use the term filopodia and in other parts I think you use pseudopodia to refer to the same structure. Since Ettensohn has provided the most evidence on the organization of the skeletogenic syncytia, I suggest you use the same term he used for those cellular extensions.

The filopodia and the pseudopodia are two distinct structures generated by the skeletogenic cells. The filopodia is the common cellular extension described in many cells, while the term “pseudopodia cable” describes the specific structure that forms between the skeletogenic cells in which the spicule cavity forms, in agreement with Prof. Ettensohn terminology.

(4) In trying to find relationships you cite a number of previous papers at the end of the introduction. I went back to those papers and they describe (from your work) calcium exocytosis, plus filopodia formation, plus planar cell polarity, plus CDC42, any one of which could involve an actin cytoskeleton. You even cite a paper saying that perturbations of ROCK prevent spicule formation. I went back to that paper and that isn't the case. You then summarize the Introduction by relating ROCK and the actin cytoskeleton, thereby raising reader expectation that the two will be connected. As above, in reality, your evidence here does not connect the two.

We thank the reviewer for giving us credit for all these works, but only the paper on vesicle kinetics is from our lab (winter et al 2021). As for Croce et al, 2006 that the reviewer refers to: in Fig. 9A, 75µM of Y-27632 is used to inhibit ROCK in the same sea urchin species that we use, and the phenotype is identical to what we observe – the skeletogenic cells are there, but the spicule is not formed. As mentioned above, in the current version we distinguished clearly between our solid findings and our interpretations.

(5) You emphasize in Fig. 1 the inhibition of ROCK in the presence of VEGFR inhibition. However, at no place in the manuscript do you say anything about how VEGFR is inhibited, when it is inhibited, or how you know it is inhibited. That oversight must be corrected. You mention axitinib but don't say anything about what it does. Some readers may know its activity but many will not.

We now indicate that we use Axitinib to block VEGFR in the results section (line 104) and in the methods section (lines 470-471).

(6) Fig. 2. The use of Y27632 as a selective inhibitor of ROCK. According to data sheets from the manufacturer, at the levels used in your experiments, 120 µm, 80 µm and 30 µm, those levels of inhibitor also inhibit the activity of PKA and PKC (both inhibited at around 25 µm). This is concerning because of the literature indicating that activation of the VEGFR operates through PKA. Inhibition of PKA, then, would inhibit the activity of VEGF signaling. Thus, the inhibitory effects of Y27632 may actually not be attributed specifically to ROCK. Furthermore, the heading of this section states that ROCK activity controls initiation, growth, and morphology of the spicule. Yet, even in high levels of inhibitor spicule production is initiated. Yes, the growth and the morphology are compromised, but the initiation doesn't seem to be.

The spicule fails to form under ROCK continuous inhibition in all concentrations (Fig. 2). Also, as we explained in details above, these Ki values are based on biochemical experiments with purified proteins and are not relevant to in-vivo use of the inhibitor. Yet, these Ki values demonstrate that the affinity of the inhibitor to ROCK is 100 higher than of its affinity to PKA and PKC. Specifically to the reviewer suggestion here: direct inhibition of PKA does not have skeletogenic phenotypes, not in whole embryos (1) and not in skeletogenic cell culture (2). Since we see the same skeletogenic phenotypes at low Y-27362 concentration and the genetic and pharmacological pertrubations of ROCK reconcile, we believe that these phenotypes can be atributed directly to ROCK.

(7) The synchrotron study is very nice with two points that should be addressed. Again, a high concentration of Y27632 was used giving a caveat on ROCK specificity. And second, the blue and green calcein pulses are very nice but the recent paper by the Bradham group should be cited.

We added a reference to Bradham recent paper on two calcein pulses (10).

(8) Fig. 5 is where an attempt is made to associate ROCK inhibition to alterations in actomyosin. Again, a high concentration of the inhibitor is used casting doubt on whether it specifically inhibits ROCK. However, even if the inhibition is specific to ROCK the images do not provide convincing evidence that ROCK activity normally is directed toward actomyosin. This is crucial to the manuscript.

As stated above, we addressed the specificity in this version and we modified the text to emphasize the correlation and not cuasation: Fig. 5 shows a clear difference between F-actin in control and under ROCK inhibition. In control F-actin is enriched around the spicule and under ROCK inhibition the spicule doesn’t form and disorganized F-actin is accumulated in the skeletogenic cells. Yet, as we stated above – this is not a proof for the direct effect of ROCK on F-actin polymerization, and we explain it explicitly in the results, lines 324-326 and in the discussion, lines 405-408.

(9) Again in Fig. 6 the inhibitor is used with the same concern about whether the effects noted are due to ROCK.

Fig. 6 is now Fig. 7 – the effect of ROCK on gene expression and as explained above, we addressed the specificity in this version.

(10) Lines 350-358. This interpretation falls apart without showing that the inhibitor is specific for ROCK as indicated above. Also, Fig. 5 is unconvincing in showing a difference in actin or myosin distribution in control vs ROCK inhibited embryos. Yes, the spicules are stunted, but whether actin or myosin have anything to do with that as a result of lack of ROCK activity is not demonstrated.

As stated above, we addressed the specificity in the revised version and we modified the text to emphasize the correlation and not cuasation: Fig. 5 shows a clear difference between F-actin in control and under ROCK inhibition. In control F-actin is enriched around the spicule and under ROCK inhibition the spicule doesn’t form and disorganized F-actin is accumulated in the skeletogenic cells. Yet, as we stated above – this is not a proof for the direct effect of ROCK on F-actin polymerization, and we explain it explicitly in the results, lines 324-326 and in the discussion, lines 405-408.

(11) Throughout, the manuscript spelling, grammar, and sentence structure will require extensive editing. The mistakes are numerous.

We did our best to correct the spelling and grammar. If we still missed some mistakes, we would be happy to further correct them.

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