Zebrafish fin regeneration involves generic and regeneration-specific osteoblast injury responses

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   Version published to 10.7554/elife.77614 on eLife

   Jun 24, 2022
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   eLife

   Jun 6, 2022

   Author Response

   Reviewer #1 (Public Review):

   Weaknesses:

   The data presented in the first part of the study are convincing. However, it is unclear whether each step of cell elongation and alignment, cell migration, cell dedifferentiation and regenerative response, is required for fin regeneration following amputation. As indicated in the discussion, the authors cannot provide evidence for the requirement of migration or dedifferentiation for the overall success of fin regeneration. Such limitations should be more clearly stated.

   We have modified the title and abstract to avoid overstating the requirement of the particular responses to successful regeneration. Furthermore, we have stated the limitations of our study more clearly in the discussion.

   We have removed the word “requires” from the title, it now reads: Zebrafish fin …

   Author Response

   Reviewer #1 (Public Review):

   Weaknesses:

   The data presented in the first part of the study are convincing. However, it is unclear whether each step of cell elongation and alignment, cell migration, cell dedifferentiation and regenerative response, is required for fin regeneration following amputation. As indicated in the discussion, the authors cannot provide evidence for the requirement of migration or dedifferentiation for the overall success of fin regeneration. Such limitations should be more clearly stated.

   We have modified the title and abstract to avoid overstating the requirement of the particular responses to successful regeneration. Furthermore, we have stated the limitations of our study more clearly in the discussion.

   We have removed the word “requires” from the title, it now reads: Zebrafish fin regeneration involves generic and regeneration-specific osteoblast injury responses

   In the discussion we state the limitations on page 21 as follows:

   “Unfortunately, currently existing tools to block dedifferentiation are either mosaic (activation of NF- κB signalling using the Cre-lox system) or cannot be targeted to osteoblasts alone (treatment with retinoic acid). Due to these limitations in our assays, we can currently not test what consequences specific, unmitigated perturbation of osteoblast dedifferentiation has for overall fin / bone regeneration. Conversely, the interventions presented here that specifically perturb osteoblast migration are limited as they act only transiently, that is they can severely delay, but not fully block migration. Furthermore, while interference with actomyosin dynamics reduces regenerative growth, we cannot distinguish whether this is caused by the inhibition of osteoblast migration or due to other more direct effects on cell proliferation and tissue growth. Thus, an unequivocal test of the importance of osteoblast migration for bone regeneration requires different tools.”

   In the second part of the study, the term trauma needs to be clarified or reconsidered. A trauma model would imply that healing is impaired. Evidence for a non-healing phenotype is lacking and is expected in support of a trauma model.

   We apologize if our use of the term trauma has caused confusion. We have simply used it interchangeably with “injury”. We have now removed all references to “trauma” in the text.

   The authors describe the process of fin regeneration that may share common features with bone regeneration in other species. In the absence of direct evidence of common mechanisms between fin regeneration and bone regeneration in other systems, the authors should remain focused on "fin regeneration" in their conclusions rather than referring to "bone regeneration" and "bone formation" in more general terms.

   We have rephrased the conclusion to have it more centred on bone regeneration in the fin. The relevant parts of the discussion now read on page 25 as follows:

   In conclusion, our findings support a model in which zebrafish fin bone regeneration involves both generic and regeneration-specific injury responses of osteoblasts. Morphology changes and directed migration towards the injury site as well as dedifferentiation represent generic responses that occur at all injuries even if they are not followed by regenerative bone formation. While migration and dedifferentiation can be uncoupled and are (at least partially) independently regulated, they appear to be triggered by signals that emanate from all bone injuries. In contrast, migration off the bone matrix into the bone defect, formation of a population of (pre-) osteoblasts and regenerative bone formation represent regeneration-specific responses that require additional signals that are only present at distal-facing injuries. The identification of molecular determinants of the generic vs regenerative responses will be an interesting avenue for future research.

   Reviewer #2 (Public Review):

   The study by Sehring et al. depends on an extensive and thoroughly acquired collection of data points in combination with a robust and rigorous statistical analysis. I see that the authors have spent a lot of effort into this and I am overwhelmed by the number of analyzed data points that again depend on careful measurements at the cellular level in a more or less intact tissue. However, since just a fraction of cells has been chosen to be incorporated into the statistical analysis, there is a certain risk of a biased selection. I think the reader of the paper would appreciate a somewhat clearer picture of how the authors get to their final numbers, starting from the original image data. This appears of particular importance when it comes to determining the elongation of cells and the angular deviations from the proximo-distal axis. In many cases (e.g. Fig.2 A, B, D and E), the reader has to take those numbers without seeing any primary image data. A practicable solution to that issue would be to complement the accompanying Excel sheets of raw data with corresponding image material. This should show an overview of a representative sample for the dedicated experiment, together with some appropriate magnifications of analyzed cells including the axes along which those measurements have been performed. Also, it would be important to state within the methods section of the paper whether the measurements have been done manually using Fiji or whether a certain automated Fiji plug-in has been used for this part of the analysis.

   Osteoblasts line the bony hemirays on the inner and outer surface (see Figure 1A), and for quantifications of osteoblast morphology, we analysed the osteoblasts of the outer layer of one hemiray (the hemiray facing the objective in whole mount imaging). While we have no direct evidence for this, we think it is reasonable to assume that osteoblasts in the other “sister” hemiray behave the same, and we have anecdotal evidence that osteoblasts on the inner surface of the hemirays also migrate and dedifferentiate. Thus, we don’t think that restriction of the analysis to one hemiray and the outer surface introduces bias.

   For measurement of morphology, we used a transgenic line expressing a fluorescent protein (FP) in osteoblasts in combination with Zns5 antibody labelling. Zns5 is a pan-osteoblastic marker which localizes to the cell membrane. Therefore, combination of a cytosolic FP labelling with the membrane labelling by Zns5 provides solid definition of single cell outlines. For general morphology studies and drug intervention studies, we used bglap:GFP transgenics. In the transgenic intervention studies (manipulation of NF-kB signalling), mCherry is expressed together with CreERT2 under the osterix promoter and used as cytosolic labelling of osteoblasts. Our analyses are always based on segments, e.g. we present data for segments 0, -1, 2. Within these segments all FP+ Zns5+ cells were included into the analysis, and cells along the whole proximodistal axis of a segment were measured. Measurements were performed manually, and the analysist was blinded. With these set-ups, not only a fraction but all FP+ Zns5+ osteoblasts present in those segments that we analysed were included into the analysis, and thus no selection was necessary that could have introduced bias. As suggested by Reviewer #2, we have added representative sample images to the accompanying Excel sheets of raw data for the dedicated experiments. Within these, the axes along which the measurements have been performed are indicated.

   We have expanded the description of the analysis in the method section. It now reads on page 36 as follows:

   “To quantify osteoblast cell shape and orientation, the transgenic line bglap:GFP in combination with Zns5 AB labelling was used. Osteoblasts of the outer layer of one hemiray (facing the objective in whole fin mounting) were imaged and analysed. As Zns5 localizes to the plasma membrane of all osteoblasts, the combination of both markers provides solid definition of single cell outlines. All GFP+ Zns5+ cells with such a defined outline within an analysed segment were included into the analysis, and cells along the whole proximodistal axis of a segment were measured. In the transgenic intervention studies, mCherry is expressed under the osx promoter and was used as cytosolic labelling of osteoblasts. Using Fiji (Schindelin et al., 2012), the longest axis of a FP+ Zns5+ cell was measured as maximum length, the short axis as maximum width, and the ratio calculated. Simultaneously, the angle of the maximum length towards the proximodistal ray axis was measured for angular deviation. All measurements were performed manually, with the analyst being blinded.”

   Along the same line, it would strengthen the statement provided by the statistical diagram in Fig.3A if the authors could show images of cells from segment -1 and -2 for all three experimental conditions. In particular, since the depicted segment -1 osteoblasts look rather roundish than elongated (compare with Fig.1 C and D, images and width/length ratio).

   As suggested by the reviewer, we have added representative sample images of cells in segment -1 to the figure, the images that were already there in the previous version of the figure were from segment -2 (new data in Figure 4A). As legible from the graphs, there is a certain range of morphology within each segment / assay with an obvious overlap between the segments. This can make it difficult to realize the difference between the segments by looking on the images alone, and we have therefore added arrowheads to highlight examples of roundish and elongated cells. Yet as mentioned above, all cells were included into the analysis.

   In regards to the biology itself, Sehring and colleagues claim that the complement system is required for injury-induced directed osteoblast migration. To strengthen this point it would be beneficial if the authors could show that the central complement components C3 and C5 are indeed expressed at the amputation site where the dedifferentiated pre-osteoblasts migrate to. It would be interesting to learn about the localization of C3 and C5 expression in the conventional amputation as well as the double-injury condition. Apparently, the RNAscope-based in situ hybridization seems to work quite well in the Weidinger lab.

   Complement precursor proteins are thought to be mainly expressed in the liver and distributed throughout the body via the circulation. Injury would then result in local production of the activated C3a and C5a peptides via a cascade of proteolytic processing. Unfortunately, we lack the tools to detect the C3 and C5 precursor proteins or the mature cleavage products of the complement factors, which mediate the biological function of the cascade (e.g. antibodies against the zebrafish proteins / peptides). We have also attempted RNAScope for c5a and c3a.1 in fins, but these turned out to not produce any specific stainings, thus the results of these experiments remained inconclusive and we have not included them in the manuscript.

   However, we analysed expression of the RNA coding for the precursors of the complement factors c5 and the six zebrafish paralogs of c3 using qRT-PCR on liver, non-injured fins and fins at 6 hpa (samples derived from segment -1 plus segment 0). These new data can be found in Figure 5B. Compared to the expression levels in the liver, expression in non-injured fins could hardly be detected. Interestingly, c5 and c3a.5 levels were upregulated in injured fins, but compared to the expression in the liver still only slightly, e.g. c5 is about 17 Ct values (2 to the power of 17 = 130000 times) more highly expressed in the liver than in the injured fin. These results are consistent with the idea that the majority of complement factors that are activated after injury is derived from precursors that are expressed in the liver and are distributed via the circulation to the fin, as is considered standard for the complement system. Interestingly, however, local production might contribute as well.

   Overall our new data support our conclusion that the complement system is an important regulator of osteoblast migration in vivo, since the receptors are present in osteoblasts (see also response to the next issue), while systemic and local expression can provide the precursors for injury-induced production of the activated factors that might act as guidance cues.

   To judge whether this osteoblast's migratory response is cell-type specific and cell-autonomous it would be good to know if c5ar1 and c3ar are solely expressed in osteoblasts, or rather broadly within tissue lining the hemirays.

   While we had already shown that c5aR1 is expressed in osteoblasts, we have now added additional RNAscope in situ analysis for c5aR1 showing that the receptor is also expressed in other cell types (new data in Figure 5 – figure supplement 1A). We have also attempted RNAScope for c3aR in fins, which however did not produce specific staining, thus remained inconclusive; we have not added these data to the manuscript. However, we established fluorescent activated cell sorting from bglap:GFP transgenic fins, which gives us an additional tool to analyse to which extent expression is specific to osteoblasts. By qRT-PCR analysis we found that c5aR1 and c3aR are expressed in both GFP+ osteoblasts and other cells that are GFP– (these will mainly represent epidermis and fibroblasts, to a lesser extent endothelial and other cell types). These new data can be found in Figure 5 – figure supplement 1B.

   While our qRT-PCR data and the c5aR1 RNAScope results show that the complement receptors are not specifically expressed in osteoblasts, we do not consider this result to be in conflict with our model that the complement system regulates osteoblast migration. Other cell types migrate after fin amputation as well, which is best described for epidermal cells (Chen et al., Dev Cell 2016, 10.1016/j.devcel.2016.02.017), but likely also occurs for fibroblasts (Poleo et al., DevDyn 2001, doi: 10.1002/dvdy.1152), and it is conceivable that the complement system plays a role in regulating these events as well.

   Reviewer #3 (Public Review):

   Weaknesses:

   1. The major conclusions on osteoblast dedifferentiation and migration are solely based on a bglap:GFP strain, which does not allow a pulse-chase approach in injury responses. Specificity of this strain to osteoblasts is also doubtful because as many as 20% of GFP+ cells are in proliferation. Specificity of bglap:GFP to mature osteoblasts is a major concern. Important caveats associated with this reporter strain are not carefully considered.

   To address these comments, we have performed several additional experiments as described below. In addition, we would like to refer the reviewer to our previous papers, where we have analysed the process of osteoblast dedifferentiation (Knopf et al., Dev Cell 2011, doi: 10.1016/j.devcel.2011.04.014; Geurtzen et al., Development 2014, doi: 10.1242/dev.105817; Mishra et al. Dev Cell 2020, doi: 10.1016/j.devcel.2019.11.016). Using transgenic reporters and immunofluorescence we have shown in these previous papers that osteoblasts in the non-injured fin express Bglap but not the pre-osteoblast marker Runx2 (and are thus by our definition differentiated). We apologize if we failed to explain the logic of our approach in this manuscript, we have restructured the results to clarify these, as indicated below.

   We have also performed the following additional experiments.

   1. To confirm the specificity of the bglap:GFP line for mature osteoblasts, we have performed three experiments:

   a) immunofluorescence against Runx2 on 7 dpa regenerates, at a stage where blastema proliferation at the distal tip of the regenerate produces new osteoblast progenitors, while in more proximal (older) regions osteoblasts have already started to differentiate and new bone matrix has formed. We found that Runx2 is expressed in distal regions in pre-osteoblasts, while bglap:GFP is only expressed in proximal regions in osteoblasts which do not express Runx2. Thus, formation of new bony segment during regenerative growth, bglap:GFP is activated in mature osteoblasts and the population does not include osteoblast precursor cells. These new data are found in Figure 2 – figure supplement 2B.

   b) we have refined and expanded our methods and are now able to determine the expression patterns of markers of the osteoblast differentiation status with single cell resolution using RNAScope in situ hybridization. Using this, we can now show that at 1 day post amputation, in segment -2 of the fin stump, which represents a segment equivalent to the non-injured state, since no dedifferentiation occurs here, bglap:GFP+ cells do not express endogenous runx2a. These new data are found in Figure 1 – figure supplement 1A.

   c) Using RNAScope, we can show that cyp26b1, a gene associated with dedifferentiated osteoblasts, is likewise not detected in bglap:GFP+ cells in segment -2 at 1 dpa (new data in Figure 1 – figure supplement 1B).

   Together, these data confirm that the bglap:GFP line is specific for differentiated osteoblasts, and does not label osteoblast progenitors. See the response to issue 2 below for how we describe these new data in the revised version of the manuscript.

   1. Regarding the proliferation of bglap:GFP osteoblasts: In the experiment the reviewer refers to (now Figure 5 – figure supplement 3A), we make use of the persistence of the GFP protein in the bglap:GFP line to detect dedifferentiated osteoblasts. Thus, at the time of analysis, when these GFP+ cells proliferate, they are not differentiated anymore. We can show this as follows:

   Although bglap expression is downregulated during osteoblast dedifferentiation and thus also GFP levels eventually drop in the transgenic line, we can nevertheless use this line to trace osteoblasts, since GFP protein persists for up to three days in cells that shut down endogenous bglap and also bglap:GFP transgene transcription. While we have already shown this previously (Knopf et al., Dev Cell 2011, doi: 10.1016/j.devcel.2011.04.014; Geurtzen et al., Development 2014, doi: 10.1242/dev.105817; Mishra et al. Dev Cell 2020, doi: 10.1016/j.devcel.2019.11.016), we have now also used RNAScope to confirm this. We analysed the expression of GFP on protein and RNA level in the bglap:GFP line. In bglap:GFP fish, in a mature segment in non-injured fins the regions close to the joints are devoid of cells expressing GFP (Figure 1G). Yet after amputation, we observe GFP+ cells in this distal part of segment -1 (Figure 1G, D). RNAscope in situ shows that these GFP+ cells are negative for gfp RNA (new data in Figure 1D). Thus, the observed fluorescence is due to the persistence of the GFP protein and not due to a potential upregulation of the transgene (Figure 1E).

   Importantly, we have now also added data describing the proliferative state of bglap:GFP+ osteoblasts. First, in the non-injured fin, bglap:GFP+ cells are non-proliferative (new data in Figure 5 – figure supplement 2B). After amputation, proliferation can be detected in GFP+ cells at 2 dpa (Figure 5 – figure supplement 2B), and proliferation is restricted to segment -1 and segment 0 (new data in Figure 5 – figure supplement 2C). As we show in Figure 1B, at 2 dpa, dedifferentiation as defined by bglap downregulation is not complete in segment -1, rather here a mixture of cells with different bglap levels are found. We have thus combined EdU labelling with RNAscope against bglap in segment -1 to analyse to which extent bglap and EdU anticorrelate. These data show that EdU is hardly ever incorporated into cells expressing high levels of bglap, while the majority of the proliferating osteoblasts are dedifferentiated, as they express only low levels of bglap (new data in Figure 5 – figure supplement 2D). Together, these data show that mature osteoblasts are non-proliferative, and upon amputation, when they are dedifferentiated, they become proliferative. Thus, the absence of proliferation in bglap:GFP+ cells in the non-injured fin adds to the evidence that this line is specific for mature osteoblasts, but due to the persistence of the GFP protein it can be used to analyse dedifferentiated osteoblasts.

   These data are described on page 14 of the manuscript as follows:

   “In the non-injured fin, bglap:GFP+ osteoblasts are non-proliferative, but upon amputation osteoblasts proliferate at 2 dpa (Figure 5 – figure supplement 2A, B). Proliferation is restricted to segment -1 and segment 0 (Figure 5 – figure supplement 2C), and RNAscope in situ analysis of bglap expression revealed that the majority of EdU+ osteoblasts have strongly downregulated bglap (Figure 5 – figure supplement 2D). Inhibition of C5aR1 with PMX205 had no effect on osteoblast proliferation in segment -1 at 2 dpa (Figure 5 – figure supplement 3A). Furthermore, upregulation of Runx2 was not changed by PMX205 treatment (Figure 5 – figure supplement 3B), and regenerative growth was not affected in fish treated with either W54011, PMX205 or SB290157 (Figure 5 – figure supplement Figure 3C). We conclude that the complement system specifically regulates injury-induced osteoblast migration, but not osteoblast dedifferentiation or proliferation in zebrafish.”

   1. To support our conclusion that osteoblasts migrate, we performed time-lapse imaging using a transgenic line expressing the photoconvertible protein kaede in osteoblasts (entpd5:kaede). Local photoconversion of only the proximal half of a segment allowed us to trace these photoconverted osteoblasts. This revealed that converted cells appear in the distal part of the segment within 1 dpa, which can only be explained by relocation of the cells. These new data can be found in Figure 1F and they are described on page 7 of the revised manuscript as follows: To trace osteoblasts, we used the transgenic line entpd5:kaede (Geurtzen et al., 2014), in which Kaede fluorescence can be converted from green to red by UV light (Ando et al., 2002). We photoconverted osteoblasts in the proximal half of segment -1, while osteoblasts in the distal half remained green (Fig. 1F). At 1 dpa, red osteoblasts were found in the distal half (Fig. 1F), showing that photoconverted osteoblasts had relocated distally.

   1. The authors poorly define dedifferentiation. They use reduced bglap:GFP or bglap mRNA expression as a sole criterion for dedifferentiation. The authors state that NF-kB and retinoic acid can inhibit osteoblast dedifferentiation. However, this simply reflects of the well-described fact that these signals promote osteoblast differentiation.

   We define dedifferentiation as the reversion of a mature cell into an undifferentiated progenitor-like status. This involves the following characteristics: 1) the expression of markers of the differentiated state are downregulated; 2) early lineage markers are re-expressed; 3) the cells become proliferative; and 4) they have the ability to re-differentiate into mature cells. Based in this definition, the downregulation of an osteoblast-specific marker can be used as a read-out for osteoblast dedifferentiation. Bglap is an established marker for mature osteoblasts (Kaneto et al., 2016 doi.org/10.1186/s12881-016-0301-7¸ Yoshioka et al., 2021 doi: 10.1002/jbm4.10496; Kannan et al., 2020 doi: 10.1242/bio.053280; Sojan et al., 2022 doi.org/10.3389/fnut.2022.868805; Valenti et al., 2020 doi.org/10.3390/cells9081911). While we use downregulation of bglap expression as our main read-out for osteoblast dedifferentiation in our experimental interventions (actomyosin inhibition, retinoic acid treatment, complement inhibition), we have expanded our methods to characterize osteoblast dedifferentiation, and have re-arranged our manuscript to show these data in the beginning of the results.

   Already in the previous version of the manuscript we have shown that endogenous bglap is strongly expressed in segment -2, (the segment that does not respond to fin amputation and thus represents the non-injured state), while it is downregulated in a graded manner in segment -1 and segment 0 (the segments where dedifferentiation happens). We have now moved this data to the re-designed Figure 1B. In addition to bglap, we can now show that entpd5, a gene required for bone mineralization, is strongly expressed in osteoblasts of segment -2, while it is massively downregulated in segment -1 and segment 0. These new data can be found in Figure 1C. Thus, entpd5 is another differentiation marker whose loss characterizes osteoblast dedifferentiation. Importantly, we can confirm by RNAScope that the pre-osteoblast marker runx2a is absent in mature segments but is upregulated in segment 0 and segment -1 at 1 dpa (new data in Figure 1 – figure supplement 1A). Similarly, cyp26b1, an enzyme shown to regulate dedifferentiation, is upregulated in segment 0 and segment -1, but not expressed in segment -2. (new data in Figure 1 – figure supplement 1B). Furthermore, we have repeated all experiments where we have previously quantified dedifferentiation upon experimental interventions using downregulation of bglap:GFP (actomyosin inhibition, retinoic acid treatment, complement inhibition). We now can fully confirm the previous conclusions using the more rigorous quantification of dedifferentiation using RNAScope analysis of endogenous bglap levels. We have replaced all bglap:GFP data with the new bglap RNAScope data. These new data are found in Figure 3F, Figure 3 – figure supplement 1A, Figure 4B and Figure 5F.

   Overall, we support our conclusion that osteoblasts dedifferentiate by the loss of the two differentiation markers bglap and entpd5, the upregulation of the pre-osteoblast marker runx2a and the dedifferentiation-associated gene cyp26b1, and the fact that osteoblasts become proliferative. We hope that the reviewer considers this sufficient evidence.

   In mammals, the available literature relatively convincingly concludes that NF-kB signaling negatively regulates osteoblast differentiation (Yao et al., 2014, doi: 10.1002/jbmr.2108; Swarnkar et al., 2014 doi.org/10.1371/journal.pone.0091421, Chang et al., 2009, doi.org/10.1038/nm.1954). Yet in zebrafish osteoblasts, we have previously shown that NF-kB signaling is active in mature osteoblasts and needs to be downregulated for dedifferentiation to occur (Mishra et al., 2020, 10.1016/j.devcel.2019.11.016). Importantly, in our previous work we showed that at least during fin regeneration, NF-kB signalling is not involved in osteoblast differentiation (Mishra et al., 2020, 10.1016/j.devcel.2019.11.016). Specifically, osteoblasts in which Nf-kappaB signaling is enhanced or inhibited differentiate completely normally during the later stages of fin regeneration in the fin regenerate. Hence, our findings with the Nf-kappaB intervention studies done in this manuscript, where we look at osteoblasts in the stump within 1 dpa, cannot be explained by them affecting osteoblast differentiation.

   For retinoic acid signalling, multiple roles in bone development and repair have been described in mammals. For zebrafish osteoblasts, it was shown that during the outgrowth phase of bone regeneration, retinoic acid negatively regulates osteoblast differentiation in the blastema (Blum & Begemann, 2015, 10.1242/dev.120204). Yet importantly, it also negatively controls the dedifferentiation of osteoblasts in the stump right after amputation (Blum & Begemann, 2015, 10.1242/dev.120204). Thus, the effect we observe at the early timepoints we analyse in our intervention studies (retinoic acid treatment) are due to the effect on osteoblast dedifferentiation.

   We have added a short definition of dedifferentiation to the results section (page 6). There it reads as follows:

   “We have previously shown that osteoblasts dedifferentiate in response to fin amputation, that is they revert from a mature, non-proliferative state into an undifferentiated progenitor-like state, which includes loss of bglap expression and upregulation of the pre-osteoblast marker runx2 (Knopf et al., 2011; Geurtzen et al., 2014).”

   In addition, we have restructured the results to describe our use of tools and the new data on page 6 of the revised manuscript as follows:

   Using RNAScope in situ hybridization, we can now show that downregulation of bglap occurs in a graded manner and that entpd5 expression is similarly downregulated during dedifferentiation (Figure 1B, C). At 1 day post amputation (1 dpa), expression of entpd5 and bglap remains high in segment -2, but gradually decreases towards the amputation plane and is almost entirely absent from segment 0, with entpd5 downregulation being more pronounced (Figure 1B, C). While RNA expression of these genes is downregulated within hours after injury, GFP or Kaede fluorescent proteins (FPs) expressed in bglap or entpd5 reporter transgenic lines persist for up to three days, even though transgene transcription is shut down rapidly as well (Knopf et al., 2011). We can confirm these earlier findings using the more sensitive RNAScope in situs. In bglap:GFP transgenics at 2 dpa, gfp RNA and GFP protein colocalized to the same cells in segment -2, where osteoblasts do not dedifferentiate (Fig. 1D). In contrast, in the distal segment -1 GFP protein was present, but barely any gfp transcript could be detected (Fig. 1D). Thus, persistence of FPs in reporter lines can be used for short-term tracing of dedifferentiated osteoblasts (Fig. 1E). At 1 dpa, bglap:GFP+ cells upregulated expression of the pre-osteoblast marker runx2a and of cyp26b1, an enzyme involved in retinoic acid signalling (Blum and Begemann, 2015), which regulates dedifferentiation (Figure 1 – figure supplement 1A, B). Both markers were exclusively upregulated in segment -1 and segment 0 at 1 dpa, but were absent in segment -2. Together, these data show that osteoblasts in segment -1 and segment 0 lose expression of mature markers and gain expression of dedifferentiation markers.

   1. The authors do not rigorously demonstrate that mature osteoblasts indeed migrate. What they showed in this study is simply cell shape changes.

   We have the following evidence for osteoblast migration:

   1. bglap:GFP+ cells relocate from the centre of segments towards the amputation plane (after fin amputations) or towards both injuries in the hemiray model. In this revised manuscript we show that transgene expression is not upregulated in these regions, but that GFP fluorescence there must be due to relocation of cells in which GFP protein persists (new data in Figure 1D, E; see also response to “Weaknesses, issue 1” above)

   2. Using the entpd5:kaede transgenic line, which is expressed in mature osteoblasts throughout segments, we have photoconverted only the proximal half of a segment, which allowed us to trace these photoconverted osteoblasts. This revealed that converted cells appear in the distal part of the segment within 1 dpa, which can only be explained by relocation of the cells. These new data can be found in Figure 1F.

   3. Already in the previous version of the manuscript, we have performed live imaging to track single cell behaviour. Using double transgenic fish expressing both GFP and kaede in osteoblasts, we deliberately only partly converted kaedeGreen to kaedeRed, which resulted in different hues for each osteoblast. This distinct colouring facilitates observing single cells. Video 1 shows the directed movement of cell bodies relative to their surroundings within 2 hours (see also Figure 2 – figure supplement 1A).

   4. Osteoblasts display the typical cell shape changes associated with active migration (elongation along the axis of migration, extension of dynamic protrusions), data in Figure 2.

   Together, we think these are convincing data supporting the conclusion that osteoblasts actively migrate.

   1. The hemiray removal model is highly innovative, but this part of the study is not very well connected to the rest of the study.

   We have rephrased the first sentence of the hemiray paragraph to make the connection more perceptible. It now reads as follows:

   In response to fin amputation, all osteoblast injury responses occur directed towards the amputation plane, that is dedifferentiation is more pronounced distally, osteoblasts migrate distal wards and the proliferative pre-osteoblast population forms distally of the amputation plane. We wondered how osteoblasts respond to injuries that occur proximal to their location. To test this, we established a fin ray injury model featuring internal bone defects.

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3. 

   eLife

   Apr 6, 2022

   Evaluation Summary:

   This work is of interest for readers in the field of bone regeneration, and more broadly to readers in the field of tissue repair and regenerative medicine. The authors took advantage of a well-established in vivo model, live imaging, pharmacological inhibition and genetic strategies to dissect the interrelations of key cellular events in zebrafish fin regeneration. The finding of how distinct generic injury responses are differentially regulated, and are functioning independently from each other, is a valuable piece of information for the community.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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4. 

   eLife

   Apr 6, 2022

   Reviewer #1 (Public Review):

   In this manuscript, Sehring et al. investigate the process of osteoblast migration and dedifferentiation during zebrafish fin regeneration after amputation. In a previous study the authors explored the process of osteoblast dedifferentiation and identified NF-kappaB signaling/retinoid acid as negative regulators of osteoblast dedifferentiation. In the first part of this study, the authors studied osteoblast migration in this model, and show, using approaches combining live imaging, pharmacological inhibition and genetic strategies, that osteoblast dedifferentiation and migration can be uncoupled. While NFkappaB/RA regulates dedifferentiation without affecting migration, migration involves the complement system that is part of the innate immune system, and actomyosin dynamics. In the second part of the study, …

   Reviewer #1 (Public Review):

   In this manuscript, Sehring et al. investigate the process of osteoblast migration and dedifferentiation during zebrafish fin regeneration after amputation. In a previous study the authors explored the process of osteoblast dedifferentiation and identified NF-kappaB signaling/retinoid acid as negative regulators of osteoblast dedifferentiation. In the first part of this study, the authors studied osteoblast migration in this model, and show, using approaches combining live imaging, pharmacological inhibition and genetic strategies, that osteoblast dedifferentiation and migration can be uncoupled. While NFkappaB/RA regulates dedifferentiation without affecting migration, migration involves the complement system that is part of the innate immune system, and actomyosin dynamics. In the second part of the study, the authors established a trauma model by removing two fin ray segments on each side of a segment. They show that regenerative bone formation occurs only distally while the generic injury response (i.e. dedifferentiation and migration) occurs proximally and distally.

   Strengths:

   The data throughout the manuscript is of high quality and the conclusions are in general well supported by the results. The authors built on their previous work that demonstrated the role of NF-KappaB and retinoic acid in osteoblast dedifferentiation in regenerating zebrafish fins. Similar approaches and imaging techniques are employed in this study to describe changes in osteoblast morphology and migration after fin amputation, and to interfere with actomyosin dynamics using inhibitor treatment. Methods used in the previous study to reveal the role of NF-kappaB and RA in osteoblast dedifferentiation are used again here to show that this pathway does not affect migration. The novelty of this study is also to address the role of the complement system in osteoblast cell shape change in migration after amputation.

   The authors describe a new model of hemiray removal to further emphasise their analyses on the process of polarized bone growth distally to the remaining fin segments, and to reveal that the initial steps of cell shape change, and migration occurs symmetrically on both distal and proximal ends of fin segments, independently from the bone regenerative response.

   Weaknesses:

   The data presented in the first part of the study are convincing. However, it is unclear whether each step of cell elongation and alignment, cell migration, cell dedifferentiation and regenerative response, is required for fin regeneration following amputation. As indicated in the discussion, the authors cannot provide evidence for the requirement of migration or dedifferentiation for the overall success of fin regeneration. Such limitations should be more clearly stated.

   In the second part of the study, the term trauma needs to be clarified or reconsidered. A trauma model would imply that healing is impaired. Evidence for a non-healing phenotype is lacking and is expected in support of a trauma model.

   The authors describe the process of fin regeneration that may share common features with bone regeneration in other species. In the absence of direct evidence of common mechanisms between fin regeneration and bone regeneration in other systems, the authors should remain focused on "fin regeneration" in their conclusions rather than referring to "bone regeneration" and "bone formation" in more general terms.

   Read the original source
5. 

   eLife

   Apr 6, 2022

   Reviewer #2 (Public Review):

   The manuscript by Sehring et al. is a continuation of notable contributions by the Weidinger lab towards understanding the cell biology underlying bone regeneration. Again, the authors elegantly apply the toolbox of drug treatments, transgene-mediated modulation of cell signaling and powerful in vivo imaging to dissect how key cellular events, including osteoblast dedifferentiation, proliferation, migration and blastema formation, are interrelated in a well-established injury model at the level of bony fin rays in the zebrafish tail.

   Previous work from the Weidinger lab had already shown that, upon fin amputation, mature osteoblasts along the cut site dedifferentiate to re-adopt a kind of precursor cell status. Under non-injury conditions, this dedifferentiation is inhibited by a signaling cascade involving …

   Reviewer #2 (Public Review):

   The manuscript by Sehring et al. is a continuation of notable contributions by the Weidinger lab towards understanding the cell biology underlying bone regeneration. Again, the authors elegantly apply the toolbox of drug treatments, transgene-mediated modulation of cell signaling and powerful in vivo imaging to dissect how key cellular events, including osteoblast dedifferentiation, proliferation, migration and blastema formation, are interrelated in a well-established injury model at the level of bony fin rays in the zebrafish tail.

   Previous work from the Weidinger lab had already shown that, upon fin amputation, mature osteoblasts along the cut site dedifferentiate to re-adopt a kind of precursor cell status. Under non-injury conditions, this dedifferentiation is inhibited by a signaling cascade involving retinoic acid and NF-κB. The latter becomes inactivated when bone re-growth is required due to injuring events like amputation. The accordingly dedifferentiated osteoblasts migrate into the wound area where they contribute to blastema formation, and finally re-differentiate into active osteoblasts to form new bone structures. In the current manuscript, Sehring and colleagues set out to further elucidate the interdependence and regulation of the distinct underlying mechanisms, with particular focus on the dedifferentiation and pre-osteoblast migration towards the wound. By applying dedicated pharmaceutical interventions to amputated fins, in combination with in vivo imaging for cell tracking, as well as measuring cell shapes and their orientation, the authors provide evidence that the generic injury responses of dedifferentiation and migration are independent processes. While dedifferentiation is under the control of retinoic acid and NF-κB activity, migration is not. The latter rather requires stimuli from the complement system and further depends on actomyosin but not on microtubule dynamics.

   Furthermore, Sehring et al. refer to a recent study by Cao et al. (2021) where a cavity injury model revealed polarity within the wound, with blastema formation occurring only on its distally facing side. Inhibition of calcineurin function could overcome this polarity, leading to blastema formation and wound closure from both sides as shown by Cao and colleagues. Now, by developing their fin ray model further via the removal of bone at two sites separated by an intact hemiray segment, Sehring et al. found that dedifferentiation, pre-osteoblast migration towards the wound, and even cell proliferation within the intact segment showed no proximo-distal polarity. However, blastema formation occurred only on the distal sides of the double-wound. Also under this injury condition, calcineurin inhibition could partly overcome the proximo-distal wound polarity as Sehring and colleagues found. Of note, this treatment enhanced blastema formation even at the distal-facing side of the wound suggesting that calcineurin displays a more general and not directed role for cellular mobility in the context of blastema formation. In sum, the authors conclude that osteoblast dedifferentiation, migration and proliferation of osteogenic cells close to the wound are generic injury responses that are independently regulated. However, the regionally restricted blastema formation depends on additional, so far not identified, regeneration-specific mechanisms.

   These findings by Sehring and colleagues provide new and highly interesting insights into the complex cellular and molecular machinery controlling bone re-growth in a prominent non-mammalian model. Hence, this work will be well noticed by the scientific community.

   The study by Sehring et al. depends on an extensive and thoroughly acquired collection of data points in combination with a robust and rigorous statistical analysis. I see that the authors have spent a lot of effort into this and I am overwhelmed by the number of analyzed data points that again depend on careful measurements at the cellular level in a more or less intact tissue. However, since just a fraction of cells has been chosen to be incorporated into the statistical analysis, there is a certain risk of a biased selection. I think the reader of the paper would appreciate a somewhat clearer picture of how the authors get to their final numbers, starting from the original image data. This appears of particular importance when it comes to determining the elongation of cells and the angular deviations from the proximo-distal axis. In many cases (e.g. Fig.2 A, B, D and E), the reader has to take those numbers without seeing any primary image data. A practicable solution to that issue would be to complement the accompanying Excel sheets of raw data with corresponding image material. This should show an overview of a representative sample for the dedicated experiment, together with some appropriate magnifications of analyzed cells including the axes along which those measurements have been performed. Also, it would be important to state within the methods section of the paper whether the measurements have been done manually using Fiji or whether a certain automated Fiji plug-in has been used for this part of the analysis.

   Along the same line, it would strengthen the statement provided by the statistical diagram in Fig.3A if the authors could show images of cells from segment -1 and -2 for all three experimental conditions. In particular, since the depicted segment -1 osteoblasts look rather roundish than elongated (compare with Fig.1 C and D, images and width/length ratio).

   In regards to the biology itself, Sehring and colleagues claim that the complement system is required for injury-induced directed osteoblast migration. To strengthen this point it would be beneficial if the authors could show that the central complement components C3 and C5 are indeed expressed at the amputation site where the dedifferentiated pre-osteoblasts migrate to. It would be interesting to learn about the localization of C3 and C5 expression in the conventional amputation as well as the double-injury condition. Apparently, the RNAscope-based in situ hybridization seems to work quite well in the Weidinger lab.

   To judge whether this osteoblast's migratory response is cell-type specific and cell-autonomous it would be good to know if c5ar1 and c3ar are solely expressed in osteoblasts, or rather broadly within tissue lining the hemirays.

   Despite these two more substantial but manageable criticisms, the manuscript by Sehring et al. is an outstanding piece of work that provides important new findings to the field.

   Read the original source
6. 

   eLife

   Apr 6, 2022

   Reviewer #3 (Public Review):

   This study aims to define mechanisms underlying the response of osteoblasts to injury, using a zebrafish fin rays amputation model and transgenic reporter strains. The authors used a bglap:GFP strain to label and trace osteoblasts in the fin ray segment adjacent to the amputated one (segment -1) with pharmacological (actomyosin, microtubules, complement system, retinoic acid) and genetic interventions (NF-kB).

   Strengths:
   1. This study utilized an elegant zebrafish fin ray amputation model to reproducibly monitor the behaviors of osteoblasts and other related cell types across multiple segments adjacent to the amputation site.
   2. The authors developed an innovative hemiray removal model, which allows them to examine differences in the responses at the proximal vs. distal injury site.
   3. This study performed …

   Reviewer #3 (Public Review):

   This study aims to define mechanisms underlying the response of osteoblasts to injury, using a zebrafish fin rays amputation model and transgenic reporter strains. The authors used a bglap:GFP strain to label and trace osteoblasts in the fin ray segment adjacent to the amputated one (segment -1) with pharmacological (actomyosin, microtubules, complement system, retinoic acid) and genetic interventions (NF-kB).

   Strengths:
   1. This study utilized an elegant zebrafish fin ray amputation model to reproducibly monitor the behaviors of osteoblasts and other related cell types across multiple segments adjacent to the amputation site.
   2. The authors developed an innovative hemiray removal model, which allows them to examine differences in the responses at the proximal vs. distal injury site.
   3. This study performed pharmacological and genetic interventions to define how various signaling pathways affect the osteoblast behaviors in the adjacent segment.
   4. High-quality data.

   Weaknesses:
   1. The major conclusions on osteoblast dedifferentiation and migration are solely based on a bglap:GFP strain, which does not allow a pulse-chase approach in injury responses. Specificity of this strain to osteoblasts is also doubtful because as many as 20% of GFP+ cells are in proliferation. Specificity of bglap:GFP to mature osteoblasts is a major concern. Important caveats associated with this reporter strain are not carefully considered.
   2. The authors poorly define dedifferentiation. They use reduced bglap:GFP or bglap mRNA expression as a sole criterion for dedifferentiation. The authors state that NF-kB and retinoic acid can inhibit osteoblast dedifferentiation. However, this simply reflects of the well-described fact that these signals promote osteoblast differentiation.
   3. The authors do not rigorously demonstrate that mature osteoblasts indeed migrate. What they showed in this study is simply cell shape changes.
   4. The hemiray removal model is highly innovative, but this part of the study is not very well connected to the rest of the study.

   Read the original source
7. 

   Version published to 10.1101/2022.02.22.481466v1 on bioRxiv

   Feb 22, 2022