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  1. Evaluation Summary:

    This manuscript presents a careful study of nervous system regeneration in the larval sea star using new transgenic tools for marking and following cells involved in regeneration. The authors find that these animals can regenerate their nervous system by the re-specification of existing cells, which are induced to express the embryonic neurogenesis program. The experimental approach is robust and creative and the data interpretation sound. For its contribution to our understanding of how cells are induced to contribute to specific cell lineages during regeneration, this work will be of interest to the broad community of researchers inregenerative and developmental biology.

    (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. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    Overall, this manuscript presents a careful study of sea star larval nervous system regeneration using new transgenic tools for marking and following cells involved in regeneration. The authors provide a nice, well-written introduction to their study in the Abstract and Introduction sections. I do have one major issue with the wording they are using for describing what can be done with the transgenic tools they have developed.

    They mention in the third paragraph of the Introduction that "Only cell tracking can definitively establish the origin and trajectory of cells during regeneration and resolve the debate as to the role of stem cells versus cellular reprogramming in echinoderms." And then in the final paragraph they state that "We establish a novel cell lineage tracking system to determine the cellular origin of these regenerated neurons."

    The system they develop does mark individual sox2 and sox4 expressing cells but I object to it being called a "cell lineage tracking system" as this is a very specific term used for a set of methods that allow for tracing the fate of individual cells and all of their progeny, traditionally through development or with stem cells. In essence cell lineage tracking/tracing provides the identification of ALL progeny of a single cell. According to a Primer on Lineage Tracing by Kretzschmar and Watt (2012)
    "For any lineage tracer, the key features are that it should not change the properties of the marked cell, its progeny, and its neighbors. The label must be passed on to all progeny of the founder cell, should be retained over time, and should never be transferred to unrelated, neighboring cells."

    I strongly believe that the BAC-reporters developed in this manuscript do not fit that definition of a cell lineage tracing/tracking system and new verbiage should be used to describe these tools. These could very simply be referred to as fluorescent BAC-reporters and describe specifically how they are used to mark and follow the fate of cells expressing the Sox2 and Sox4 genes. The only way the language of a cell lineage tracing/tracking system could be used is if they had created a BAC-reporter for a gene that was expressed constitutively throughout a cell lineage as it progresses or if the protein expression (the tracer) was passed along to all progeny of the cell expressing that gene. My understanding is that the gene expression of Sox2 and Sox4 is highly dynamic and thus the label, by definition, is not going to be passed on to all progeny of the founder cell. I do think this is a powerful system, I just object to how the authors have chosen to describe it in the manuscript. Careful rewording can still make the reader aware of the limitations and advantages of this system and will avoid misunderstanding.

    Therefore, all mentions throughout the manuscript of "a lineage tracing system" would need to be removed and replaced with wording that accurately reflects the true nature of these reporters, simply as photoconvertible expression reporters that can show Sox2 or Sox4 expressing cells. This includes text in the Results and Discussion section, e.g. "To our knowledge, this is the first time that any cell lineage tracing studies have been performed in echinoderm regeneration."


    The authors nicely present their larval regeneration system and highlight the timeline of when serotonergic neurons regenerate over a period of 21 days. They then demonstrate that embryonic neurogenesis pathways are recapitulated during larval regeneration. Then, they present results from their photoconvertible expression reporters and demonstrate three populations of cells in decapitated larvae. The green-only sox4+ cells are the most interesting population - these are cells that are induced to express sox4 only after decapitation. Comparing embryogenesis and larval development demonstrated that the wound response in larvae involves specifying new sox4+ cells, something that had ended by 4dpf in normally developing larvae.

    The co-injected double BAC recombinant larvae showed colocalization of sox2+ and sox4+ in regenerating larvae. De novo sox2 expression following bisection together with colocalization with sox4 expression nicely shows that these new sox2+ cells contribute to the neural lineage. Considering that the colocalization appeared to be a rare-ish event (only observed in 4 out of 15 larvae), it would be nice if the authors could comment on why this may be. Is it just a truly rare event to catch or could it have anything to do with the reporters themselves?

    Same question about the sox2+ cells that do not express sox4:Cardinal by 3dpb. Can the authors comment specifically on whether they think there are multiple subpopulations of sox2+ cells and why some get specified to the neural fate while others do not?

    The final experiment using cell division inhibitor Aphidicolin was very clever and nicely demonstrates that cells that did not previously express sox2 can be induced in the absence of cell division. It would be helpful if the authors could indicate how many larvae showed this pattern as they did for the previous colocalization experiment.


    In the final paragraph of the Discussion, the authors discuss a dichotomy between the use of stem cells versus de- or trans-differentiation in different model systems of regeneration. They describe the planarian system in the following way: "For example, the freshwater planarian, Schmidtea mediterranea, utilizes a population of heterogeneous, pluripotent somatic stem cells, called neoblasts, to proliferate and differentiate to replace body parts (Sánchez Alvarado, 2006)" and contrast this with Hydra and axolotl, saying "Conversely species such as Hydra and axolotl, refate differentiated cells either through dedifferentiation or transdifferentiation (Gerber et al., 2018)." I think this oversimplifies the current understanding of these systems. For example, a recent paper by Raz et al. 2021 (Cell Stem Cell 28(7): 1307-1322.e5) makes the case that Schmidtea mediterranea is capable of having specialized neoblasts undergo fate-switching and "propose a non-hierarchical lineage model for neoblasts, in which a neoblast can specify one of a diverse set of possible fates in the course of a single division and specialized neoblasts can divide to generate neoblasts that can specify different fates." In essence, this could be considered something more flexible and complicated than what the authors described - just using pluripotent neoblasts to proliferate and differentiate to replace body parts. And although Hydra is known to use trans-differentiation during regeneration, this organism also employs stem cells in the process of regeneration. Please see Siebert et al. 2008 (Developmental Biology 313(1): 13-24) for a discussion of how both mechanisms are employed in this regeneration model. Therefore, I think it is an oversimplification to characterize these regeneration models as either using stem cells OR using de- or trans-differentiation. I think in these systems, there is not a simple dichotomy and more flexibility has been demonstrated in how regeneration is accomplished than the authors describe here and the text would need to be revised accordingly.

  3. Reviewer #2 (Public Review):

    Regeneration is a developmental process that occurs in response to injury. Important questions about regeneration include: 1) to what extent are regeneration gene regulatory networks similar to embryonic gene regulatory networks, 2) what is the source of cells used to build new structures during regeneration, and 3) what aspects of regeneration are deeply conserved and which are taxon specific. Towards addressing these questions, the authors have established the sea star larva as a regeneration model. As invertebrate deuterostomes, echinoderms (such as the sea star) have an informative position on the phylogenetic tree; leveraging these organisms to study regeneration will both help answer outstanding questions in the regenerative biology field and will reveal new insights into the evolutionary history of regeneration in vertebrates. In this study, the authors establish a new method for lineage tracing in the sea star larva to determine the source of cells that participate in regenerating the larval serotonergic nervous system after bisection and regeneration of the anterior end. Previous work by this group has established critical transcription factors in the specification of serotonergic neurons during embryogenesis. Here, the authors demonstrate that during regeneration, these same transcription factors are expressed within one day after bisection in cells that previously did not express these genes. Co-expression analysis revealed that embryonic neurogenesis expression states are repeated during regeneration and that injury induces the formation of proliferative sox4+ neural progenitors. To test the hypothesis that sox4+ progenitors newly arise during regeneration, the authors used BAC recombineering to express the photoconvertible protein Kaede under the sox4 regulatory sequences. The fluorescent protein is stable for at least 7 days, making it possible to use this system to determine the history of the sox4-expressing cells present during regeneration. By converting all existing Kaede to red before bisection, the authors were able to demonstrate that sox4+ neural progenitors are newly specified during regeneration. Importantly, they found that this occurs during a time in normal development when sox4+ progenitor cell specification is complete. The authors next use their BAC Kaede lineage tracing system to determine that the sox4+ cells arise from sox2+ progenitors, similar to what is observed during embryogenesis. Finally, they found that new sox2 expression can be induced in the absence of cell division which strongly suggests that the sox2+ progenitors are derived from the respecification of existing cells, rather than from resident stem cells. Overall, this is an interesting and well-executed study that clearly demonstrates that larval nervous system regeneration involves respecification of cells into the neural lineage and that at least a portion of the embryonic GRN is used to regenerate the larval nervous system. Furthermore, this work firmly establishes sea start larval regeneration as an important model for answering outstanding questions in regenerative biology.