Regeneration is a fascinating phenomena that challenges the tenet of an irreversible, directional ontogeny. Decades of research have provided extensive understanding of how embryonic cells acquire their fate through the action of GRNs and the sequential turning on, and off, of regulatory genes. During embryogenesis, these developmental GRNs start from a very particular cell type, the single-celled zygote, while in regeneration another source of multi-, or toti-potent cells must be the source of cells for regeneration. The source of the cells, their molecular state and how, or even whether, developmental GRNs are re-established in these cells are open questions. This knowledge, however, is needed as a prerequisite for understanding the molecular mechanisms of regeneration. Furthermore, an understanding of whether any processes are homologous or if there are taxon-specific mechanisms that are related to the ability for extensive or limited regeneration awaits sufficient sampling across the metazoa.

The deuterostomes, that is vertebrates, invertebrate chordates, echinoderms and hemichordates, are an especially important system to study from the standpoint of regeneration. The vertebrates, with some few exceptions, have limited capacity for regeneration that is usually restricted to specific cells, tissues and organs (Rodriguez and Kang, 2020; Tanaka and Reddien, 2011). The emerging evidence points to the use of tissue-specific stem cells and corresponding specific niches as a cellular source for regeneration (Kragl et al., 2009). Echinoderms on the other hand have an impressive ability for extensive whole body regeneration found in most species of the phylum (Ferrario et al., 2020). For this reason, there has been extensive research into the mechanisms of regeneration in many species of echinoderms (Ferrario et al., 2020; Kondo and Akasaka, 2010; Dupont and Thorndyke, 2007; Piovani et al., 2021; Vickery et al., 2001; García-Arrarás et al., 2018; Mashanov and Zueva, 2019; Mashanov et al., 2020). For example, molecular and ultrastructural studies in brittle star arm regeneration point to use of adjacent epithelial cells to differentiate into sclerocytes needed for arm skeleton regeneration, recapitulating, at least in part, a development program (Piovani et al., 2021). Comprehensive studies in the regeneration of radial nerve chords in sea cucumbers also point to the use of radial glial dedifferentiation in nervous system regeneration (Mashanov and Zueva, 2019; Mashanov et al., 2015a). The emerging consensus from these and other studies is that echinoderms rely extensively on wound-adjacent dedifferentiation for regenerating lost tissues (Ferrario et al., 2020; Mashanov and Zueva, 2019). Conversely, expression of stem-cell marker genes have been identified as possible cell sources in some species (Mashanov et al., 2015b; Reinardy et al., 2015). Whether echinoderms and vertebrates use similar cellular sources and mechanisms for regeneration (Zhang et al., 2003; Joven and Simon, 2018) is important for untangling whether these mechanisms are causative of whole-body versus limited regeneration potential. Understanding these processes in naturally regenerating contexts also has important implications for improved translational applications of induced pluripotent stem cells and other stem cell therapies.

To date, however, while there has been a comprehensive series of studies of the molecular and ultrastructural basis of regeneration in many species of echinoderms, there have been no cell lineage tracking studies. 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. Cell tracking, however, is technically challenging, especially in adult echinoderms where there are many cells and cell types, and regeneration proceeds over days or weeks. Transgenic tools are also limited in adult echinoderms. Indeed, the challenges associated with accurately tracking cell lineages have greatly limited the taxonomic distribution of animals for which we have this knowledge across the metazoa.

Here, we sought to fill this important gap by turning to the echinoderm larval regeneration model. Echinoderms, typically for many marine invertebrates, have a biphasic lifestyle, producing a planktonic larval form that exists for up to many months before undergoing dramatic metamorphosis. It has now been well documented that the larval forms are also capable of extensive regeneration (Oulhen et al., 2016; Vickery et al., 2001; Kasahara et al., 2019). We have recently introduced the larval sea star Patiria miniata as a model system for the study of whole body regeneration (Cary et al., 2019b). Sea stars, as typical for echinoderms, have extensive regenerative capacities, a feature which extends across both their adult and larval life phases (Vickery et al., 2001; Vickery et al., 2002; Ben Khadra et al., 2017; Hernroth et al., 2010; Oulhen et al., 2016). Although pentaradial as adults, there are many parallels between echinoderm larval development and early vertebrate development, particularly in the formation of the anterior-posterior axis and development of the nervous system (Angerer et al., 2011; Hinman and Burke, 2018; Range et al., 2013). Therefore, while vertebrates and sea stars have very different regenerative capacities, there are commonalities in their early development. There are also extensive genomic resources (Cary and Hinman, 2017; Cary et al., 2019a, Cary et al., 2018; Cary et al., 2017; Gildor et al., 2019) and detailed developmental GRNs available for P. miniata (Cary et al., 2020) allows comparisons between developmental and regenerative GRNs and clear gene orthology mapping.

Previous work has shown that when these sea star larvae are decapitated, they are able to regenerate their anterior structures (Cary et al., 2019b; Oulhen et al., 2016; Vickery et al., 2001). Larvae have an anterior serotonergic nervous system, which we show here is removed by this decapitation. Extensive previous work in P. miniata embryos has shown how these neurons form during embryogenesis. The SRY-box transcription factor (TF), sox2 (formerly soxb1), is expressed broadly throughout the ectoderm by blastula stage (Yankura et al., 2010). Sox4+ (formerly soxc) neural progenitors arise in the ectoderm, and then divide asymmetrically to produce LIM homeodomain TF lhx2+ (formerly lhx2/9) daughter cells, which in turn divide asymmetrically to form differentiated serotonergic neurons (Cheatle Jarvela et al., 2016). This occurs within the anterior ectoderm, which expresses TFs such as six3 and foxq2, and is a wnt ligand negative territory (Cheatle Jarvela et al., 2016; Yankura et al., 2013).

In this study, we took advantage of cell-type-specific markers of differentiated neurons to confirm that the sea star larvae are able to regenerate their anterior nervous system. We establish a novel photoconvertible BAC reporter system to trace populations of cells to determine the cellular origin of these regenerated neurons. This allows us to specifically test whether regenerated neurons arise through embryonic neurogenesis pathways, and to determine the origin and potency of progenitors. Using a small molecule inhibitor of nuclear DNA replication (to question whether new gene expression can arise in the absence of divisions that are a prerequisite of stem cell origin), we show that expression of the putative pluripotency factor sox2 is induced in previously sox2 minus cells at the wound site. As this occurs even in the absence of new cell division, this result suggests that these cells are respecified rather than arising from asymmetric stem cell division. Both the newly induced and existing, sox2+ cells now give rise to new sox4+ neural precursors that then proceed along an embryonic neurogenesis pathway to reform the anterior nervous systems. The sox2+ cells contribute to only neural and ectoderm lineages, indicating that these progenitors maintain their normal, embryonic lineage restriction.

The ability that some animals have to restore tissues and body parts following traumatic injury challenges our current understanding of the mechanisms of cell specification and differentiation. Significant questions remain surrounding the source of new cells needed to regenerate lost tissues and organs, their potencies and whether developmental GRNs are re-used. A further open question is whether any of these processes are shared across taxa, and can explain the differing capacities for regeneration.

Echinoderms are an especially important system for the study of these questions as they are well known for their extensive capacities for regeneration (Ben Khadra et al., 2018; Byrne, 2020). There has, therefore, been extensive research on regeneration of multiple species, tissues and stages in echinoderms (Vickery et al., 2001; Kasahara et al., 2019; García-Arrarás et al., 2018; Mashanov and Zueva, 2019; Piovani et al., 2021). While providing important insight and an emerging consensus on the role of local dedifferentiation, the availability of transgenic tools has precluded the cell lineage analysis needed to definitively identify cell sources and their potencies. The discovery that many echinoderm larvae are able to regenerate (Wolff and Hinman, 2021), opens the possibility to use genetic engineering tools that are available for embryos, to probe regeneration. Additionally, developmental GRNs and the cell specification pathways are extremely well characterized for many species of echinoderms, providing an opportunity to compare developmental to regenerative GRNs of the same larval cell types. In this study, we develop a new BAC-based transgenesis protocol to identify and track the cellular sources of regenerated neurons of the sea star bipinnaria larva, and to compare neurogenesis pathways between development and regeneration.

We show that bipinnaria larvae of the bat star, P. miniata, are able to regenerate their anterior nervous system. Sea star larvae have remarkably complex nervous systems (Carter et al., 2021; Elia et al., 2009) given their small size and presumably simple behavior. We took advantage of the highly specific marker of differentiated serotonergic neurons, which includes immunoreactivity to anti-serotonin antibody (Sigma, S5545), characteristic axonal projections, and location within the larvae, to confirm that these neurons are removed by surgery and reform within 21 days. The removed pre-oral ciliary band and lip of the mouth are also reformed during regeneration.

We used fluorescent in situ hybridization (FISH) to show that genes involved in embryonic AP axis specification are re- expressed along the axis of bisected larvae. For example, foxq2, six3, and wnt3 are key nodes in the embryonic GRN for axial specification (Cheatle Jarvela et al., 2016; Yankura et al., 2013), and are re-expressed along the AP axis of the posterior regenerating segment. Reestablishing axial patterning is a commonly observed early feature in many species undergoing whole body regeneration (Gehrke et al., 2019; Reddien, 2019), which we now show also occurs in sea star larvae. Sox2 and sox4 expression is similarly induced adjacent to the wound site within one day. This region will form a proliferative epithelial zone by the third day following bisection. In embryos, sox2 is expressed throughout the ectoderm, while sox4+ cells are scattered throughout the ectoderm where they function as precursors for multiple neural types in the larvae (Cheatle Jarvela et al., 2016). This is the first indication that there is a resetting of gene expression states adjacent to the wound and is consistent with a broad respecification of these cells.

One of the most fascinating questions in regeneration is whether animals reuse their embryonic GRNs to regenerate cell types. If different pathways are used, this indicates that there are regeneration-specific GRNs deployed after traumatic wounding. This further suggests that such pathways may independently evolve under different selection pathways than the embryonic pathways. If embryonic GRNS are reused, the challenge is to understand how they are reactivated in non-zygotic cells. The new expression of embryonic axial patterning genes across the bisected larva (Figure 2C–D) is consistent with re-entry into an embryonic state. We further show, using FISH and using IMARIS to confidently assess colocalization of signal, that sox2+ cells overlap with sox4+ cells, that sox4 expression overlaps with lhx2 expression, which in turn overlaps with elav (Figure 2). We have previously demonstrated that these gene products mark the embryonic neurogenesis pathways, and that ectodermal elav expression is a marker of differentiated neurons (Cheatle Jarvela et al., 2016). We have also previously shown that lhx2 is needed for the differentiation of serotonergic neurons (Cheatle Jarvela et al., 2016). This data therefore supports the hypothesis that embryonic serotonergic neurogenesis pathways are reactivated in the induced sox2+ cells, although we are not yet able to show that any of these gene products are functionally required for neurogenesis in the regeneration context.

The presence of sox2+ and sox4+ cells adjacent to the wound site provide the possibility that these are the cellular source of regenerated neurons. Cell tracking however is needed to test whether these cells do indeed form neurons, and to identify the cellular origin of sox2 and sox4 expression. The wound adjacent expression could arise from existing sox2+ or sox4+ cells in the larvae. This would suggest that these cells function as a resident stem cell population of regenerated neurons. Alternatively, they would arise from other, existing, cells, which may either be derived from an additional source of stem cell or through de- or trans-differentiation. We therefore developed a novel transgenic reporter system based on BAC recombineering that takes advantage of highly stable, irreversibly photoconvertible, fluorescence Kaede reporter (Ando et al., 2002). The injected BAC DNA construct is stably inherited in clonal lineages of cells after one to several embryonic cell division and hence the reporter will express mosaically. In the system we developed, cells that are currently expressing the gene of interest will fluoresce green, and may also contain red FP if they have previously expressed the gene prior to photoconversion. Using this reporter system, we show that the sox4 gene is expressed in existing sox4+ cells, and in cells that have not expressed sox4 within the last seven days (Figure 3). We reasoned that new neural precursor cells (i.e. green only sox4+ cells) may be continuously generated as a normal development process and therefore not specifically induced by wounding. We therefore compared the formation of new sox4+ neural precursors in development and regeneration. We find many new sox4+ cells being produced in early embryos (days 2–5 development) as expected, but this has almost entirely stopped by swimming larval phase (days 4–7 development) and resumed upon wounding (Figure 4). The subsidence of sox4+ cell formation in the larval stage is consistent with data showing that the serotonergic postmitotic neuronal number is stable in larvae from day 4 or 5 of development (Figure 1—figure supplement 2). Using the same experimental strategy, but now with sox2 BAC reporter, we show the sox2 gene is also expressed in existing and new cells. Using an additional Cardinal reporter, we show that some, but not all, of these new sox2+ cells become sox4+ (Figure 5), indicating that they can become neural precursors but also other cell types. We followed these reporters through regeneration to show that they are later found in cells with characteristic axonal projections and located in the lower lip of the mouth, and dorso-lateral domains adjacent to the mouth as stereotypic of serotonergic neurons. Thus, we show that these wound-induced sox2 cells do become neurons, other ectodermal cells and mouth endo/ectoderm in a similar lineage potential as the embryonic sox2+ lineage.

The existing, sox2+ sox4+ cells that continue to express these genes (i.e. the yellow cells in our assay) likely function as resident ectodermal- and neural- stem cells as we show that they are dividing and contribute to neural and ectodermal tissues. We wanted to investigate whether the new sox2+ cells (green) arose from existing cells or might arise from another, possibly totipotent resident stem cell population. As stem cells must, by definition, divide to produce a daughter that can be directed along a new differentiation pathway we used small molecule inhibitors to block cell division following bisection. We find that new sox2+ cells still emerge in these larvae (Figure 6). This therefore shows that new sox2+ expression does not require the asymmetric division from a stem cell progenitor, and instead, therefore, is the result of respecifying existing cells.

The human and mouse orthologs of the sea star sox2 gene have well-characterized functions in the induction of pluripotency and maintenance of stem cells, especially neurogenic stem cells (Feng and Wen, 2015). In combination with Oct4, Klf4, and c-Myc, sox2 can reprogram human and mouse somatic cells to pluripotent states (Soufi et al., 2015; Takahashi and Yamanaka, 2006). Other models of echinoderm regeneration have implicated, although not demonstrated, a role for orthologs of these pluripotency factors in de-differentiation (Mashanov and Zueva, 2019; Mashanov et al., 2015a). We have also previously shown that Klf4 is expressed at the wound edge following bisection and is thus presumably co-localized with sox2 (Cary et al., 2019b). The new wound-induced expression of sox2, therefore, presents the hypothesis that induction of sox2 expression is the actual cause of the dedifferentiation of cells, and/or may now function to maintain an ectodermal stem cell population. We have not yet formally shown whether new induction of sox2 leads to dedifferentiation (i.e. reprogramming into a progenitor state), or whether there are other types of reprogramming, as we do not know the differentiation state of cells induced to express sox2 or whether these cells now adopt an identical molecular signature to existing sox2 cells. The extent of lineage conversion are unknown and open questions in stem cell biology in any system, and yet is critical knowledge for translational applications of stem cells. The work here provides foundational knowledge in a tractable system to work toward answers to these questions. We have shown that once respecified, sox2+ cells are restricted to become ectoderm, and the lip of mouth ectoderm/endoderm cell types, indicating that they are multi-potent but germline restricted. In embryos, sox2+ cells are also restricted to ectodermal lineages and the lip of the mouth, and therefore, the wound-induced sox2+ cells now enter their normal, embryonic lineage. A subset of these induced cells recapitulate an embryonic neurogenesis pathway to reform serotonergic neurons around the mouth, and the latero-dorsal ganglia.

As new model systems are added to the study of regeneration, they present an opportunity to re-examine our understanding of regeneration and the ways in which cellular reprogramming is induced by wounding. Early concepts around cellular origins of whole body regeneration suggested a dichotomy between use of stem cells and de- or trans-differentiation, although more recent studies suggest a greater complexity, with a range of mechanisms often existing in any given species. 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). Recent work shows that specialized neoblasts can also divide to produce neoblasts that are pluripotent, suggesting a plasticity of their fate (Raz et al., 2021) Species such as Hydra and axolotl, refate differentiated cells either through dedifferentiation or transdifferentiation (Gerber et al., 2018). Although, Hydra also utilizes interstitial cells as a population of stem cells (Siebert et al., 2008). Cellular plasticity also varies widely, from the totipotent planarian neoblasts to lineage restricted progenitors used by vertebrate animals (Kragl et al., 2009; Zhu and Pearson, 2016).Our data indicate that echinoderm larvae induce non-stem cells into ectodermal neural stem cells, possibly through dedifferentiation or other mechanisms of fate conversion, as we see de novo sox2+ cells becoming neurons in regenerating larvae. This is reminiscent of the mechanisms of refating seen in cnidarian models such as Hydra. Additionally, we show that there is a maintained lineage of sox2+ and sox4+ progenitors in bisected larvae, also reforming the nervous system, as we see yellow neurons in regenerating larvae (Figure 5—figure supplement 2A). This at a broad level echoes the use of lineage restricted stem cells seen in vertebrates and other systems. We posit that echinoderms may have a natural capacity to maintain a cellular plasticity that permits induction of new lineage stem-cells, as well as an ability to use existing lineage-restricted stem cells and that this flexibility may underlie their extensive abilities for regeneration.

All data generated in this study are included in the manuscript. Genomic sequence data is provided with accessing numbers and/or links to echinobase.org.

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Author details

1. Minyan Zheng

   Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United States

   Present address

   Department of Genetics, Harvard Medical School, Boston, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7990-1773

2. Olga Zueva

   Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

3. Veronica F Hinman

   Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   vhinman@andrew.cmu.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3414-1357

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