New functional vessels form after spinal cord injury in zebrafish

  1. Ana Ribeiro
  2. Mariana Rebocho da Costa
  3. Carmen de Sena-Tomás
  4. Elsa Charas Rodrigues
  5. Raquel Quitéria
  6. Tiago Maçarico
  7. Susana Constantino Rosa Santos
  8. Leonor Saúde

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Abstract

The vascular system is inefficiently repaired after spinal cord injury in mammals, resulting in secondary tissue damage and immune deregulation that contribute to the limited functional recovery. Unlike mammals, zebrafish can repair the spinal cord and restore motility, but the vascular response to injury has not been investigated. Here we describe the zebrafish spinal cord vasculature, from the body size-dependent vessel ingression during development to the stereotypic vessel organization and barrier specialisation in adulthood. After injury, vessels rapidly regrow into the lesion, preceding the glial bridge and regenerating axons. The initial vascularisation of the injured tissue is done by dysmorphic and leaky vessels. Dysfunctional vessels are later removed, as pericytes are recruited and the blood-spinal cord barrier is re-established. Vascular repair involves an early burst of angiogenesis, likely in response to pro-angiogenic factors detected in the injured spinal cord, including the Vegf pathway. However, the inhibition of the Vegfr2 using genetic and pharmacological methods was not able to efficiently block the formation of new blood vessels, suggesting that other signalling pathways are also involved in this process. This study demonstrates that zebrafish can successfully re-vascularise the spinal tissue, reinforcing the value of this organism as a regenerative model for spinal cord injury.

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    Reply to the reviewers

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

    Spinal cord injury (SCI) is a damage to the spinal cord, that causes temporary or permanent changes in its function. While in mammals the regeneration process are very limited zebrafish are able to repair the spinal cord. Based on the hypothesis, that the vascular response might affect the regeneration capacity, the paper by Ribeiro et al addresses the structure and injury response of the spinal cord vasculature. As the growth of zebrafish larvae and juveniles depends a lot on the individual response to the environment, the authors first established comparable body measurement parameters (other than age) and observed the natural spinal cord vascularization process, starting from 6mm body length of the animals. Using transgenic lines the authors describe the formation and patterning of endothelial cells and pericytes up to 9mm length, when a more developed vascular network was present. They observe the processes of vascular regeneration after a contusion based SCI model at different time points (days post injury (dpi)) and in correlation with glial and axonal regrowth, also observing BSCB barrier integrity, angiogenesis, pericyte recruitment and the dependence on Vegf signaling.

    The study is interesting and novel, vascular structures in the zebrafish adult spinal cord have not been reported yet and neither has the vascular response to SCI. Currently the study remains very descriptive, although the authors tried to add functional data, by inhibiting Vegf signaling.

    Major points for revision: The authors fail to establish whether there is any relationship between spinal cord regeneration and vessel regeneration. While I do very well understand the challenges and limitations the authors should put more effort into functional analyses.

    For example: the authors address EC proliferation as a marker for angiogenesis, but do not analyse whether or how much EC proliferation is required for revascularization and regeneration. Pharmacological inhibition of proliferation should be possible and used. From a vascular point of view it would also be interesting whether there is a differential influence of tip or stalk cell proliferation.

    Although we agree that it would be interesting to inhibit EC proliferation to assess its role in spinal cord regeneration, the use of proliferation inhibiting drugs would likely have a widespread effect on the lesioned spinal cord, since many cell types proliferate in response to injury. Therefore, a pharmacological approach would not allow us to dissect the specific role of endothelial proliferation.

    The same is true for pericyte recruitment: the role of pericytes for the vascular repair or the spinal cord regeneration is not clear. The authors could use use mutants with impaired pericyte development or e.g. nitroreductase mediated ablation of pericytes.

    These experiments have been performed in larvae by Tsata et al. (2021). Although it would be interesting to repeat in adults, we believe that these experiments are beyond the focus of our study.

    The statements regarding the role of Vegf are too bold. The problem lies in the limitations of assessing the efficiency of Vegf inhibition. The heatshock promotor has been shown to induce transcription for up to 4 hours, depending on the efficiency of heatshock. There are no data on the stability of dnVegfaa protein. Likewise the pharmacological inhibition could be far from complete. A full inhibition of Vegf signaling is expected to stop vessel growth or angiogenesis. While it is a sign of good practice, that the authors combined a genetic model with a pharmacological one, both leave the same unresolved issue. However if we believe a very limites requirement of Vegf-signaling, it would be interesting to look for other signaling pathways, like cxcl, IL, or FGF to regulates regenerative angiogenesis.

    We agree that our data does not allow us assess the level of inhibition of the Vegf pathway. Since we are unable to confirm this at the moment, we will be excluding the Vegf inhibition data and make this a descriptive study.

    Minor issues

    The correlation with spinal cord repair could be stated more clearly throughout the manuscript. For the uninformed reader it is less clear when exactly the spinal cord is functional again.

    We will include in Fig. 3 a plot of the swimming capacity in contusion-injured fish until 90 dpi and will explain in the text how the vascular response correlates with the functional recovery.

    While I find the model in figure 8 very helpful, it gives 5 to 30 days, for the neuronal regeneration. Maybe a more detailed timeline of EC regeneration and remodeling correlating with neuronal repair would help.

    We will update the model in Fig. 8 with a more detailed timeline and a better description of structures important for regeneration (glial bridge, axonal regrowth).

    In line with that in figure 4 it is not clear whether the images of different time points are indeed one individual animal at the different time points or representative animals for the stage (also figure 4 lacks panel labels, in my copy I can see A, K and L, but no other letters).

    We will detail in the figure legend that the images are of different animals that are representative for each stage.

    For understanding the (re)vascularization, the direction of blood flow might be helpful.

    We will perform an additional experiment to characterise the direction of blood flow in uninjured fish. For this we will use juvenile fish with a body size of 7-9 mm, in which we expect to be able to perform live imaging. We will use a lighsheet microscope to image circulating cells in the spinal cord blood vessels in fish with labelled thrombocytes (Tg(-6.0itga2b:EGFP); Lin et al., 2005) and endothelial cells (Tg(kdrl:ras-mCherry)). These transgenic lines are already available in our fish facility. Even though the vascular network has not yet reached its mature stage at these body sizes, we expect to have enough intraspinal vessels to describe the blood flow circuit.

    Especially for the connection between spinal cord regeneration and vessel regeneration. Does blood flow regulate vessel pruning after 14 dpi?

    Although we agree with the reviewer that it would be interesting to understand how blood flow direction is reestablished in repaired vessels and how blood flow levels correlate with vessel remodelling and pruning, this would be difficult to assess in this system. This could be examined using live imaging, but this technique is challenging in adult zebrafish and has only been carried out in more superficial organs than the spinal cord, such as skin (Castranova et al., 2022) and superficial brain structures (Barbosa et al., 2015; Castranova et al., 2021). In addition, SC-injured fish are more sensitive to external conditions and would probably not survive the long-term/repeated anaesthesia required for imaging.

    This analysis could be performed in fixed samples, for example using the the Golgi complex position in relation to the endothelial nuclei as a proxy for blood flow direction (Kwon et al., 2016), however: (1) this would require a new transgenic line (Tg(fli1a: B4GALT1-mCherry)) that would take time to import and establish in the lab; (2) the identification of regressing vessels is not straightforward in fixed samples and is usually studied in very well established vascular models, such as the mouse retina and zebrafish ISVs (Franco et al., 2015).

    For these reasons, we will not address this question by reviewer 1.

    The combined Vegfaa DN and PTK treatment data looks like it could be inhibiting endothelial cell proliferation (Figure7I).However, Supplementary Figure 8B shows endothelial proliferation does not change. Does it mean the number of endothelial cells is same but the volume of endothelial cells decrees?

    We will not be addressing the changes in endothelial density in the presence of dn-vegfaa and PTK787, since we will be removing the figures related to Vegf inhibition.

    There are also some remaining grammatical errors, for example (but NOT limited to) line 133 to 135.

    We will review grammatical errors in the text.

    As a personal interest I think evaluating the role of Notch in the SCI model would also be very interesting, especially with regard to the vasculature, however that might be out of the scope of the manuscript.

    We agree that Notch signalling may be a player during spinal cord revascularisation. However, mutants for dll4 (the Notch ligand involved in angiogenesis) die between 7-14 dpf and cannot be used for this study. In addition, the use of Notch-inhibiting drugs would likely have pleiotropic effects, since the Notch pathway is also involved in other aspects of spinal cord regeneration, namely in the regulation of regenerative neurogenesis (Dias et al., 2012). To our knowledge, tools that allow the endothelial-specific inhibition of the Notch pathway have not been developed, and therefore we will not be able to address this question.

    Reviewer #1 (Significance (Required)):

    The study is partially descriptive, but very novel as the aspects of vascularisation in a spinal cord injury model have not been described before. If the major revisions regarding functionality are addressed fully, I would wholeheartedly recommend publication and expect an interest for a broad audience. The presented images and their analyses are of very high quality, and therefore also enhance the impact of the study.

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

    The study by Ribeiro et al. investigates the formation of new blood vessels after spinal cord injury in adult zebrafish. The authors initially characterize the extend of spinal cord vascularization during the development of juvenile zebrafish and investigate the association of pericytes with the newly forming vasculature. They then injure the spinal cord and describe the subsequent regeneration of blood vessels. They perform assays to analyze the functionality of the newly forming blood vessels and show that initially blood vessels are leaky. Through EdU labelling the authors show that endothelial cells proliferate. Pericytes similarly increased in numbers. Lastly, the authors inhibited VEGF signaling, which only mildly affected vascular regeneration.

    Together, this manuscript describes the re-vascularization of the regenerating spinal cord in adult zebrafish and addresses how blood vessels mature during this process through pericyte recruitment and decrease in leakiness. The manuscript provides some interesting initial insights into spinal cord vascularization, but is mainly descriptive and unfortunately remains superficial in this regard, as specified below:

    1. The authors only refer to "blood vessels" without specifying the type of blood vessels they observe (are these veins, arteries, capillaries)? A wealth of markers and transgenic zebrafish lines are available to better characterize spinal cord vessels. This is not necessary in case the authors solely refer to "blood vessels" as they do, but it greatly limits the insights into spinal cord vascularization. For instance, Wild et al. (2017) showed that new vessels apparently sprout from veins in the spinal cord. Is this also true during regeneration?

    We will perform RNA in situ hybridisation using probes for arterial and venous markers. We will assay the expression of arterial markers (dll4, dlc, flt1 and efnb2a) and venous markers (flt4 and ephb4a) in uninjured spinal cord (to characterise vessel identity in homeostasis) and in 3 and 7 dpi spinal cords (to investigate the identify of angiogenic vessels during regeneration).

    1. The authors state that their characterization revealed a "stereotypic organization of blood vessels". However, the organization does not appear to be stereotypic (as I understand this term as looking the same in each fish) at all. Can the authors compare e.g. 3 or 5 wildtype fish and extract features that all fish share and those that differ between fish? This would greatly enhance our understanding of the vascular variability within the wildtype population.

    We will provide an additional figure comparing the spinal cord vasculature in different fish.

    1. The authors show an interesting metameric organization of the vasculature with regions of high vascularization interspersed with sparsely vascularized areas. Are there any morphological landmarks that would precipitate these differences?

    We will acquire light sheet images of adult spinal cords without removing the vertebrae. This will allow us to determine if the metameric organisation is correlated with the vertebral distribution.

    Can the authors check whether they induce a lesion in a highly or poorly vascularized area? This might greatly influence the degree of re-vascularization.

    We always perform the spinal cord injury in the region between neural arches (Dietrich et al., 2021). Once we determine how the vasculature is organised in relation to the vertebrae, we will be able to determine if the lesions are performed in a region of high or low vascularisation.

    1. The same superficial characterization unfortunately also applies to the cell population the authors refer to as "pericytes". Traditionally, pericytes are characterized as being associated with capillaries and sharing a basement membrane with the endothelium. Is this the case here?

    We will further characterise the association between Tg(pdgfrß:citrine)-positive cells and blood vessels using an anti-laminin antibody (#L9393, Sigma) to label the basement membrane. Preliminary results recently acquired indicate that Tg(pdgfrß:citrine)-positive perivascular cells and endothelial cells are both enveloped by the basement membrane, supporting the identity of Tg(pdgfrß:citrine)-positive cells as pericytes. Moreover, pericytes are generally described as solitary mural cells associated with small diameter blood vessels (the type of distribution we observe for Tg(pdgfrß:citrine)-positive cells), whereas vascular smooth muscle cells (vSMCs) form concentric layers around larger blood vessels (a distribution we do not detect with this transgene) (Hellström et al., 1999). For these reasons we believe that this transgene is labelling pericytes. We will explain more clearly in the text how the morphology, localisation and density of Tg(pdgfrß:citrine)-positive cells suggests these cells are pericytes.

    In addition, pdgfrb is hardly specific for pericytes, as it also labels a multitude of other cell types (refer to e.g. Tsata et al. (2021)).

    The different cell types labelled by the pdgfrb reporter line used in the Tsata et al., 2021 paper were identified not by the use of different cell markers, but by their localisation: perivascular cells (the same cell type that we also detect), myoseptal cells (which we would not expect to detect, since we are only analysing the spinal cord tissue and not the adjacent muscle) and floor plate cells (a reporter distribution that the authors show is lost after 3 dpf and is not present in the adult spinal cord). Moreover, the Tsata et al., 2021 paper also includes a supplementary figure (S1, panel N) showing a restricted perivascular pdgfrb:GFP distribution in the wholemount adult spinal cord, in agreement with our characterisation. By their morphology and density, these perivascular cells are likely pericytes, as argued above.

    It is also not clear why the transgenic pdgfrb line the authors use only labels cells next to blood vessels. Tsata et al. show a much broader labelling. The authors need to validate their transgenic line using in situ hybridization showing where pdgfrb is being expressed endogenously and how this overlaps with the fluorescent protein expression of the pdgfrb transgenic line.

    We will perform ISH for pdgfrb to confirm if the Tg(pdgfrß:citrine) reporter reproduces the endogenous expression in the uninjured spinal cord and at 3 and 7dpi. The 3-7 dpi period is approximately equivalent to the 1-2 days post-lesion in larvae and, if the non-perivascular pdgfrb:GFP cells observed in the larval spinal cord are present in the adult, we expect to detect them by ISH during this phase of regeneration.

    There are also several transgenic lines available that allow for the distinction between smooth muscle cells and pericytes (e.g. Shih,..., Lawson, Development 2021 and Whitesell,..., Childs, Plos ONE 2014). As for the vasculature, this more detailed characterization is not necessary in case the authors refer to the cells as "cells labelled by the pdgfrb transgene and reside next to endothelial cells". However, this would not be reflective of the level of detail currently present in the field.

    As we explain above, the morphology and density of the pdgfrb:Citrine-positive cells suggests that these cells are pericytes and not smooth muscle cells (SMCs). To confirm this we will compare the expression of pdgfrb with markers of SMCs (i.e, 𝛼-smooth muscle actin and desmin) using immunohistochemistry and/or ISH.

    The reviewer also suggests the characterisation of pericyte subtypes using the lines described by Shih et al., 2021. Although this would be interesting, we do not consider it is essential for our study. It would be very demanding to import the reporter lines and it is not certain that these subtypes are present in the spinal cord.

    1. The authors state that "New blood vessels rapidly attracted pericytes, formed through proliferation and possibly migration of existing pericytes". This statement is not supported by the data, as the authors do not perform lineage tracing of pre-existing pericytes. The authors need to specifically label existing pericytes and then follow whether these pre-labelled cells can be found on newly forming blood vessels. Tsata et al. provide some evidence for this in zebrafish larvae, but they also conclude that pdgfrb expressing tenocytes contribute to new mural cells.

    We will reformulate the sentence to clarify that we detect pericyte proliferation, but pdgfrb-lineage tracing would be needed to provide evidence that existing pericytes contribute to the generation of mural cells associated to new blood vessels. However, we will not perform the lineage tracing experiment for the revision, as we are unable to currently import this line.

    1. The findings that new blood vessel growth only marginally depended on VEGFA signaling is striking. However, it might also point towards an inefficient inhibition of VEGFA signaling. In particular, other publications, for instance Cattin et al. 2015 have shown that inhibiting VEGFA signaling prevents new blood vessel growth during peripheral nerve regeneration in mouse. It will therefore be important that the authors demonstrate that their approach leads to successful inhibition of VEGFA signaling. VEGFAB mutants appear to be homozygous viable and important for spinal cord vascularization (Matsuoka et al., 2017). In addition, heterozygous VEGFAA mutants already have some vascular phenotypes, but are also viable. Can the authors combine these mutants with their inhibitor treatments to achieve a greater reduction in VEGFA signaling?

    Since we are unable to confirm the level of inhibition of the Vegf pathway and we are unable to import the suggested lines at the moment, we will be excluding the Vegf inhibition data.

    Reviewer #2 (Significance (Required)):

    Together, this publication is the first to describe to some extend the regenerating vasculature after spinal cord injury in adult zebrafish. However, both the vascular and regeneration fields are much more advanced than what the authors cover. Both blood vessels and perivascular cells can be characterized in much more detail, as outlined above. Also, studies on nerve regeneration and its dependence on the vasculature, e.g. during peripheral nerve regeneration in mouse have been carried out with a wealth of functional data available. Therefore, the impact of the present study in its current form will be limited. I am an expert on zebrafish blood vessel development.

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

    Summary: Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    Ribeiro et al. described vascular development in the spinal cord from larval to adult stages in zebrafish, and found the dependence of vessel length on body-size. Then, the authors depicted the vascular regeneration process after spinal cord injury (SCI), which includes initial vascularization, angiogenesis, pericyte recruitment, and blood-spinal cord barrier establishment. Although the molecules or signaling pathways that drive the re-vascularization remain unidentified, this study illustrates the cellular processes of spinal cord vascular development and regeneration from the descriptive level, which may facilitate further understandings of mechanisms underlying vascular regeneration in the spinal cord.

    Major comments:

    • Are the key conclusions convincing? The descriptions of spinal cord vascularization during development and vascular regeneration after SCI are convincing. However, inhibition of Vegfaa and Vegfr2 is nearly ineffective. The author might not conclude that the Vegfr2 signaling plays any role.

    Since we are unable to confirm the level of inhibition of the Vegf pathway, we will be excluding the Vegf inhibition data.

    Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Major comments:

    1. In Figure 3, the exact injured site on the spinal cord is not clear. Please include a schematic illustration of full spinal cord to show where is the injured site. Are all the injury experiments in this study done at the same site? If not, is there any site difference regarding the regenerative capability.

    We will include a scheme of the injury site in the spinal cord in Fig.3. All the injury were performed in the same position and this will be clarified in the methods.

    1. Figure 2E showed a segmented pattern of spinal cord vasculature. Is this pattern correlated with the position of vertebra?

    We will acquire light sheet images of adult spinal cords without removing the vertebrae. This will allow us to determine if the metameric organisation is correlated with the vertebral distribution.

    1. In Figure 3, during vascular regeneration after SCI, the author only showed partial regeneration at 30 dpi. Why not show the stage of complete regeneration? At that stage, how about the behaviors of the regenerated animals?

    We will add an additional timepoint (90 dpi) to the characterisation of the revascularisation. Moreover, we will include in Fig. 3 a plot of the swimming capacity in contusion-injured fish until 90 dpi and will explain in the text how the vascular response correlates with the functional recovery.

    1. Only EdU data is not sufficient to conclude that new vessels come from proliferation of remaining endothelial cells. For example, these new vessels might come from transdifferentiation of lymphatic vessels, or immune cells, or glial cells, in the meantime proliferate. This could also explain why the inhibition of Vegfr2 signaling is ineffective on new vessel formation. Cre/loxP-mediated lineage tracings need to be performed to exactly identify where these new vessels originate.

    We will clarify in the text that while the detection of endothelial proliferation suggests existing endothelial cells contribute to new vessels, we cannot exclude that other cell types also give rise to endothelial cells. However, regarding the transdifferentiation of immune and glial cells into endothelial cells, to our knowledge few examples have been described in the literature and generally associated with cancers or in* in vitro* conditions (Fernandez Pujol et al., 2000; Li et al., 2011; Soda et al., 2011). For this reason we do not expect this rare process to occur during spinal cord repair.

    A cell type that has been associated with transdifferentiation into ECs are lymphatic cells (Das et al., 2022). However, we have analysed the expression of a lymphatic marker (Tg(lyve1b:DsRed)) and were only able to detect very few lyve1b:DsRed-positive cells before or after injury, suggesting that any possible lymphatic contribution would likely be very limited. We plan to include these data in the revised submission.

    1. To confirm the Tg(hsp70l:dn-vegfaa) did work in this study, the authors need a positive control. For example, the effects on vasculogenesis or angiogenesis during embryonic development after heat shock. If the transgene works, the vascular development at early stages should be blocked (Marín-Juez et al., 2016).

    We will be removing the vegf inhibition data, therefore we will not address this question.

    Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. The suggested experiments are realistic in terms of time and resources.

    • Are the data and the methods presented in such a way that they can be reproduced? In the method, the author should describe how to identify the Tg(hsp70l:dn-vegfaa) in more details, because there is no fluorescence before and after heat shock.

    We will be removing the vegf inhibition data, therefore we will not address this question.

    Are the experiments adequately replicated and statistical analysis adequate? Yes.

    Minor comments:

    • Specific experimental issues that are easily addressable. In Figure 6, from 30 dpi to 90 dpi, the number of pericytes decreased. Did these pericytes undergo apoptosis from 30 dpi on?

    We have not investigated pericyte apoptosis during vessel remodelling. However, this experiment would require the acquisition of long-term samples (between 60 and 90 dpi) and we would prefer not to address this question.

    Are prior studies referenced appropriately? Yes.

    • Are the text and figures clear and accurate? Please clearly labeled the injured region in Figure 6.

    We will identify more clearly the site of the injury in Fig.6.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions? The number of proliferating ECs at 3 dpi is more than those at 5 dpi (Figure 5G). But the number of total EdU+ cells at 3 dpi is less than those at 5 dpi (Figure 5A-D). These data are consistent with Figure S3, which showed ECs were the leading cell type to enter the lesioned site, then were the axons and glial cells at later stages. Please explain and discuss whether the regeneration of other cell types is dependent on the accomplishment of vascular regeneration.

    As the reviewer points out, our data suggest that endothelial cells display an earlier peak of proliferation than spinal cord cells in general and colonise the lesioned tissue before new axons and glial cells. Although these observations could point to a role for ECs in the regeneration of other cell types, we would need to inhibit vascular repair to assess this possibility, which we were unable to do using Vegf inhibition. In our discussion we already mention some possible roles for ECs in stem cell proliferation, neurogenesis and axonal regrowth, but can expand this discussion if necessary.

    Reviewer #3 (Significance (Required)):

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    Although this study has characterized the development and regeneration of spinal cord vasculature in details, the significance of the advance needs to be improved due to lack of mechanisms. Obviously Vegfa is not essential for the vascular regeneration after SCI. It is better for the authors to identify one or two factors required for this process, in addition to identify cell origins of new vessels. With those, the significance of this study will be improved because the cell origins and required factors will provide potential therapeutic targets after SCI.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    • State what audience might be interested in and influenced by the reported findings. The audience includes people who are interested in vascular development and regeneration, and spinal cord clinicians.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. My field of expertise includes brain vascular regeneration, digestive organ development and regeneration. This study reported spinal cord vascular development and regeneration, which fit my expertise.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    Ribeiro et al. described vascular development in the spinal cord from larval to adult stages in zebrafish, and found the dependence of vessel length on body-size. Then, the authors depicted the vascular regeneration process after spinal cord injury (SCI), which includes initial vascularization, angiogenesis, pericyte recruitment, and blood-spinal cord barrier establishment. Although the molecules or signaling pathways that drive the re-vascularization remain unidentified, this study illustrates the cellular processes of spinal cord vascular development and regeneration from the descriptive level, which may facilitate further understandings of mechanisms underlying vascular regeneration in the spinal cord.

    Major comments:

    • Are the key conclusions convincing?

    The descriptions of spinal cord vascularization during development and vascular regeneration after SCI are convincing. However, inhibition of Vegfaa and Vegfr2 is nearly ineffective. The author might not conclude that the Vegfr2 signaling plays any role.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Major comments:

    1. In Figure 3, the exact injured site on the spinal cord is not clear. Please include a schematic illustration of full spinal cord to show where is the injured site. Are all the injury experiments in this study done at the same site? If not, is there any site difference regarding the regenerative capability.
    2. Figure 2E showed a segmented pattern of spinal cord vasculature. Is this pattern correlated with the position of vertebra?
    3. In Figure 3, during vascular regeneration after SCI, the author only showed partial regeneration at 30 dpi. Why not show the stage of complete regeneration? At that stage, how about the behaviors of the regenerated animals?
    4. Only EdU data is not sufficient to conclude that new vessels come from proliferation of remaining endothelial cells. For example, these new vessels might come from transdifferentiation of lymphatic vessels, or immune cells, or glial cells, in the meantime proliferate. This could also explain why the inhibition of Vegfr2 signaling is ineffective on new vessel formation. Cre/loxP-mediated lineage tracings need to be performed to exactly identify where these new vessels originate.
    5. To confirm the Tg(hsp70l:dn-vegfaa) did work in this study, the authors need a positive control. For example, the effects on vasculogenesis or angiogenesis during embryonic development after heat shock. If the transgene works, the vascular development at early stages should be blocked (Marín-Juez et al., 2016).
    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    The suggested experiments are realistic in terms of time and resources.

    • Are the data and the methods presented in such a way that they can be reproduced?

    In the method, the author should describe how to identify the Tg(hsp70l:dn-vegfaa) in more details, because there is no fluorescence before and after heat shock.

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    In Figure 6, from 30 dpi to 90 dpi, the number of pericytes decreased. Did these pericytes undergo apoptosis from 30 dpi on?

    • Are prior studies referenced appropriately?

    Yes.

    • Are the text and figures clear and accurate?

    Please clearly labeled the injured region in Figure 6.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    The number of proliferating ECs at 3 dpi is more than those at 5 dpi (Figure 5G). But the number of total EdU+ cells at 3 dpi is less than those at 5 dpi (Figure 5A-D). These data are consistent with Figure S3, which showed ECs were the leading cell type to enter the lesioned site, then were the axons and glial cells at later stages. Please explain and discuss whether the regeneration of other cell types is dependent on the accomplishment of vascular regeneration.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    Although this study has characterized the development and regeneration of spinal cord vasculature in details, the significance of the advance needs to be improved due to lack of mechanisms. Obviously Vegfa is not essential for the vascular regeneration after SCI. It is better for the authors to identify one or two factors required for this process, in addition to identify cell origins of new vessels. With those, the significance of this study will be improved because the cell origins and required factors will provide potential therapeutic targets after SCI.

    • Place the work in the context of the existing literature (provide references, where appropriate).
    • State what audience might be interested in and influenced by the reported findings.

    The audience includes people who are interested in vascular development and regeneration, and spinal cord clinicians.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    My field of expertise includes brain vascular regeneration, digestive organ development and regeneration. This study reported spinal cord vascular development and regeneration, which fit my expertise.

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    Referee #2

    Evidence, reproducibility and clarity

    The study by Ribeiro et al. investigates the formation of new blood vessels after spinal cord injury in adult zebrafish. The authors initially characterize the extend of spinal cord vascularization during the development of juvenile zebrafish and investigate the association of pericytes with the newly forming vasculature. They then injure the spinal cord and describe the subsequent regeneration of blood vessels. They perform assays to analyze the functionality of the newly forming blood vessels and show that initially blood vessels are leaky. Through EdU labelling the authors show that endothelial cells proliferate. Pericytes similarly increased in numbers. Lastly, the authors inhibited VEGF signaling, which only mildly affected vascular regeneration.

    Together, this manuscript describes the re-vascularization of the regenerating spinal cord in adult zebrafish and addresses how blood vessels mature during this process through pericyte recruitment and decrease in leakiness. The manuscript provides some interesting initial insights into spinal cord vascularization, but is mainly descriptive and unfortunately remains superficial in this regard, as specified below:

    1. The authors only refer to "blood vessels" without specifying the type of blood vessels they observe (are these veins, arteries, capillaries)? A wealth of markers and transgenic zebrafish lines are available to better characterize spinal cord vessels. This is not necessary in case the authors solely refer to "blood vessels" as they do, but it greatly limits the insights into spinal cord vascularization. For instance, Wild et al. (2017) showed that new vessels apparently sprout from veins in the spinal cord. Is this also true during regeneration?
    2. The authors state that their characterization revealed a "stereotypic organization of blood vessels". However, the organization does not appear to be stereotypic (as I understand this term as looking the same in each fish) at all. Can the authors compare e.g. 3 or 5 wildtype fish and extract features that all fish share and those that differ between fish? This would greatly enhance our understanding of the vascular variability within the wildtype population.
    3. The authors show an interesting metameric organization of the vasculature with regions of high vascularization interspersed with sparsely vascularized areas. Are there any morphological landmarks that would precipitate these differences? Can the authors check whether they induce a lesion in a highly or poorly vascularized area? This might greatly influence the degree of re-vascularization.
    4. The same superficial characterization unfortunately also applies to the cell population the authors refer to as "pericytes". Traditionally, pericytes are characterized as being associated with capillaries and sharing a basement membrane with the endothelium. Is this the case here? In addition, pdgfrb is hardly specific for pericytes, as it also labels a multitude of other cell types (refer to e.g. Tsata et al. (2021)). It is also not clear why the transgenic pdgfrb line the authors use only labels cells next to blood vessels. Tsata et al. show a much broader labelling. The authors need to validate their transgenic line using in situ hybridization showing where pdgfrb is being expressed endogenously and how this overlaps with the fluorescent protein expression of the pdgfrb transgenic line. There are also several transgenic lines available that allow for the distinction between smooth muscle cells and pericytes (e.g. Shih,..., Lawson, Development 2021 and Whitesell,..., Childs, Plos ONE 2014). As for the vasculature, this more detailed characterization is not necessary in case the authors refer to the cells as "cells labelled by the pdgfrb transgene and reside next to endothelial cells". However, this would not be reflective of the level of detail currently present in the field.
    5. The authors state that "New blood vessels rapidly attracted pericytes, formed through proliferation and possibly migration of existing pericytes". This statement is not supported by the data, as the authors do not perform lineage tracing of pre-existing pericytes. The authors need to specifically label existing pericytes and then follow whether these pre-labelled cells can be found on newly forming blood vessels. Tsata et al. provide some evidence for this in zebrafish larvae, but they also conclude that pdgfrb expressing tenocytes contribute to new mural cells.
    6. The findings that new blood vessel growth only marginally depended on VEGFA signaling is striking. However, it might also point towards an inefficient inhibition of VEGFA signaling. In particular, other publications, for instance Cattin et al. 2015 have shown that inhibiting VEGFA signaling prevents new blood vessel growth during peripheral nerve regeneration in mouse. It will therefore be important that the authors demonstrate that their approach leads to successful inhibition of VEGFA signaling. VEGFAB mutants appear to be homozygous viable and important for spinal cord vascularization (Matsuoka et al., 2017). In addition, heterozygous VEGFAA mutants already have some vascular phenotypes, but are also viable. Can the authors combine these mutants with their inhibitor treatments to achieve a greater reduction in VEGFA signaling?

    Significance

    Together, this publication is the first to describe to some extend the regenerating vasculature after spinal cord injury in adult zebrafish. However, both the vascular and regeneration fields are much more advanced than what the authors cover. Both blood vessels and perivascular cells can be characterized in much more detail, as outlined above. Also, studies on nerve regeneration and its dependence on the vasculature, e.g. during peripheral nerve regeneration in mouse have been carried out with a wealth of functional data available. Therefore, the impact of the present study in its current form will be limited. I am an expert on zebrafish blood vessel development.

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Spinal cord injury (SCI) is a damage to the spinal cord, that causes temporary or permanent changes in its function. While in mammals the regeneration process are very limited zebrafish are able to repair the spinal cord. Based on the hypothesis, that the vascular response might affect the regeneration capacity, the paper by Ribeiro et al addresses the structure and injury response of the spinal cord vasculature. As the growth of zebrafish larvae and juveniles depends a lot on the individual response to the environment, the authors first established comparable body measurement parameters (other than age) and observed the natural spinal cord vascularization process, starting from 6mm body length of the animals. Using transgenic lines the authors describe the formation and patterning of endothelial cells and pericytes up to 9mm length, when a more developed vascular network was present. They observe the processes of vascular regeneration after a contusion based SCI model at different time points (days post injury (dpi)) and in correlation with glial and axonal regrowth, also observing BSCB barrier integrity, angiogenesis, pericyte recruitment and the dependence on Vegf signaling.

    The study is interesting and novel, vascular structures in the zebrafish adult spinal cord have not been reported yet and neither has the vascular response to SCI. Currrently the study remains very descriptive, although the authors tried to add functional data, by inhibiting Vegf signaling.

    Major points for revision: The authors fail to establish whether there is any relationship between spinal cord regeneration and vessel regeneration. While I do very well understand the challenges and limitations the authors should put more effort into functional analyses.

    For example: the authors address EC proliferation as a marker for angiogenesis, but do not analyse whether or how much EC proliferation is required for revascularization and regeneration. Pharmacological inhibition of proliferation should be possible and used. From a vascular point of view it would also be interesting whether there is a differential influence of tip or stalk cell proliferation.

    The same is true for pericyte recruitment: the role of pericytes for the vascular repair or the spinal cord regeneration is not clear. The authors could use use mutants with impaired pericyte development or e.g. nitroreductase mediated ablation of pericytes.

    The statements regarding the role of Vegf are too bold. The problem lies in the limitations of assessing the efficiency of Vegf inhibition. The heatshock promotor has been shown to induce transcription for up to 4 hours, depending on the efficiency of heatshock. There are no data on the stability of dnVegfaa protein. Likewise the pharmacological inhibition could be far from complete. A full inhibition of Vegf signaling is expected to stop vessel growth or angiogenesis. While it is a sign of good practice, that the authors combined a genetic model with a pharmacological one, both leave the same unresolved issue. However if we believe a very limites requirement of Vegf-signaling, it would be interesting to look for other signaling pathways, like cxcl, IL, or FGF to regulates regenerative angiogenesis.

    Minor issues

    The correlation with spinal cord repair could be stated more clearly throughout the manuscript. For the uninformed reader it is less clear when exactly the spinal cord is functional again. While I find the model in figure 8 very helpful, it gives 5 to 30 days, for the neuronal regeneration. Maybe a more detailed timeline of EC regeneration and remodeling correlating with neuronal repair would help. In line with that in figure 4 it is not clear whether the images of different time points are indeed one individual animal at the different time points or representative animals for the stage (also figure 4 lacks panel labels, in my copy I can see A, K and L, but no other letters).

    For understanding the (re)vascularization, the direction of blood flow might be helpful. Especially for the connection between spinal cord regeneration and vessel regeneration. Does blood flow regulate vessel pruning after 14 dpi?

    The combined Vegfaa DN and PTK treatment data looks like it could be inhibiting endothelial cell proliferation (Figure7I).However, Supplementary Figure 8B shows endothelial proliferation does not change. Does it mean the number of endothelial cells is same but the volume of endothelial cells decrees?

    There are also some remaining grammatical errors, for example (but NOT limited to) line 133 to 135.

    As a personal interest I think evaluating the role of Notch in the SCI model would also be very interesting, especially with regard to the vasculature, however that might be out of the scope of the manuscript.

    Significance

    The study is partially descriptive, but very novel as the aspects of vascularisation in a spinal cord injury model have not been described before. If the major revisions regarding functionality are addressed fully, I would wholeheartedly recommend publication and expect an interest for a broad audience. The presented images and their analyses are of very high quality, and therefore also enhance the impact of the study.

  6. Version published to 10.1101/2022.06.09.495446v1 on bioRxiv