Zheng 2022

RESEARCH ARTICLE
Regeneration of the larval sea star

nervous system by wounding induced
respecification to the Sox2 lineage
Minyan Zheng†, Olga Zueva, Veronica F Hinman*
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United

States
Abstract The ability to restore lost body parts following traumatic injury is a fascinating area
of biology that challenges current understanding of the ontogeny of differentiation. The origin of
new cells needed to regenerate lost tissue, and whether they are pluripotent or have de- or trans-
differentiated, remains one of the most important open questions . Additionally, it is not known
whether developmental gene regulatory networks are reused or whether regeneration specific
networks are deployed. Echinoderms, including sea stars, have extensive ability for regeneration,
however, the technologies for obtaining transgenic echinoderms are limited and tracking cells
involved in regeneration, and thus identifying the cellular sources and potencies has proven chal-
lenging. In this study, we develop new transgenic tools to follow the fate of populations of cells in
the regenerating larva of the sea star Patiria miniata. We show that the larval serotonergic nervous
system can regenerate following decapitation. Using a BAC-transgenesis approach we show that
expression of the pan ectodermal marker, sox2, is induced in previously sox2 minus cells , even when
cell division is inhibited. sox2+ cells give rise to new sox4+ neural precursors that then proceed
*For correspondence: along an embryonic neurogenesis pathway to reform the anterior nervous systems. sox2+ cells
[email protected] contribute to only neural and ectoderm lineages, indicating that these progenitors maintain their
Present address: †Department normal, embryonic lineage restriction. This indicates that sea star larval regeneration uses a combi-
of Genetics, Harvard Medical nation of existing lineage restricted stem cells, as well as respecification of cells into neural lineages,
School, Boston, United States and at least partial reuse of developmental GRNs to regenerate their nervous system.
Competing interest: The authors
declare that no competing
interests exist. Editor's evaluation
This manuscript presents a careful study of nervous system regeneration in the larval sea star using
Funding: See page 20
new transgenic tools for marking and following cells involved in regeneration. The authors find that
Preprinted: 08 August 2021 these animals can regenerate their nervous system by the re-specification of existing cells, which are
Received: 11 August 2021
induced to express the embryonic neurogenesis program. The experimental approach is robust and
Accepted: 13 January 2022
creative and the data interpretation sound. For its contribution to our understanding of how cells are
Published: 14 January 2022
induced to contribute to specific cell lineages during regeneration, this work will be of interest to
Reviewing Editor: Phillip A the broad community of researchers in regenerative and developmental biology.
Newmark, Morgridge Institute
for Research, United States
‍ ‍Copyright Zheng et al. This

article is distributed under the
terms of the Creative Commons
Introduction
Attribution License, which Regeneration is a fascinating phenomena that challenges the tenet of an irreversible, directional
permits unrestricted use and ontogeny. Decades of research have provided extensive understanding of how embryonic cells acquire
redistribution provided that the their fate through the action of GRNs and the sequential turning on, and off, of regulatory genes.
original author and source are During embryogenesis, these developmental GRNs start from a very particular cell type, the single-
credited. celled zygote, while in regeneration another source of multi-, or toti-potent cells must be the source of
Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 1 of 23

Research article Developmental Biology
cells for regeneration. The source of the cells, their molecular state and how, or even whether, devel-
opmental 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 hemichor-
dates, are an especially important system to study from the standpoint of regeneration. The verte-
brates, 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 ultrastruc-
tural 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 develop-
ment 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 dedifferentia-
tion 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, particu-
larly 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 regen-
erate 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 embryo-
genesis. 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 expres-
sion 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, embry-
onic lineage restriction.
Results
Patiria miniata larvae fully regenerate their nervous system
Sea star bipinnaria larvae have an extensive nervous system (Katow et al., 2009; Murabe et al., 2008;
Nakajima et al., 2004; Carter et al., 2021; Elia et al., 2009; Hinman and Burke, 2018). Figure 1A–B’’
shows the 7-day post fertilization (7dpf) P. miniata larval nervous system labeled via localization of anti-
synaptotagmin B (SynB) and serotonin antibodies. It has been shown that the pan-neuronal marker
SynB labels all neurons in many echinoderm species (Burke et al., 2006; Nakajima et al., 2004). The
neural bodies and axonal tracts are apparent throughout the two ciliary bands, the lip of the mouth,
domains lateral to the mouth that mark the location of the apical ganglia in this stage, and the esoph-
agus (Figure 1A). Serotonin immunoreactivity is concentrated predominantly in the anterior part of
the larvae; in the dorsal ganglia with a subset of cells across the aboral surface (Figure 1B, D’ and E’).
The presence of serotonin, characteristic neural cell morphology including long axonal processes and
lack of markers of cell division, are taken as evidence that these are differentiated cells.
We have shown previously that larvae are able to regenerate their anterior body when they are
bisected below the mouth (Cary et al., 2019b; Figure 1D–I, and Figure 1—figure supplement
1). This bisection, therefore, removes much of the anterior nervous system, including the anterior
ciliary band loop (preoral ciliary band), the dorsal ganglia, as well as the mouth and its associated
neurons. We tested whether these bisected larvae regenerate their nervous system. By one week post
bisection (Figure 1C–C’), we find neural bodies and axonal tracts along the newly formed anterior
ciliary band and complex neural networks lateral to the mouth suggestive of the dorsal ganglia. The
reformed mouth also has associated neurons. We also specifically examined the serotonergic neurons
(Figure 1D’–i). This neural subtype is found in the dorsal ganglia where they are present as clusters of
large cell bodies with long axonal processes (Figure 1D’–e). These cells originally form in the anterior-
most ectoderm of the late gastrula but migrate posteriorly to a location dorsal to the mouth in the
larva (Cheatle Jarvela et al., 2016; Yankura et al., 2013). Serotonergic neurons are also found on the
lower lip of the mouth where they have smaller cell bodies and shorter axonal processes. We therefore

Figure 1. Regeneration of the sea star larval nervous system. (A) A schematic of a 7-day old Patiria miniata larva. (B-B”) Immunostaining with anti-
synaptotagmin B (SynB; red) and anti-serotonin (5-HT; green) shows the larval nervous system organisation. Serotonergic neurons are distributed along
the pre-oral ciliary band, post-oral ciliary bands and around the mouth. Larvae are bisected beneath the lip of the mouth shown by the dotted line (B’’
and C’). (C-C’’) A latero-ventral view of regenerated larva at 7 days post bisection, the nervous system stained with Syn B. As bisection was performed at
the site represented with the white dashed line. Above the dashed line in (C’) is the regenerated anterior tissue with reformed pre-oral ciliary band and
the mouth. (D–I) Schematics of larvae at the normal (uncut) and regenerated stages corresponding to immunostaining with 5-HT images in (D’-I’) and
(d-i). (d-i). The expanded view of the region highlighted with the dashed-line box in (D’-I’). (D’) The serotonergic neurons are located bilaterally in uncut
larvae, on the dorsal side as shown in lateral view in (E’). (d-e). Neural bodies are embedded in the ectoderm, and project long axonal processes typical
of this neural type. (F’-f) Bisection removes the serotonergic neurons. (G’) By 5 dpb, serotonergic neurons are detected at the regeneration leading
edge with emerging neural morphology as shown in (g). (H’) By 7 dpb, regenerated serotonergic neurons with mature neural morphology (h) are located
at the lateral side of the regenerated anterior. (I’-i). Regenerated serotonergic neurons are bilaterally located to reform the dorsal ganglia by 21 dpb.
Arrowheads highlight the serotonergic neurons. Dpf: day-post fertilization; dpb: day-post bisection. DV: dorsal view. LV: lateral view. Scale bar: 50 µm.
The numbers shown in the lower right corner of each image indicate the number of larvae showing a positive IHC signal among the larvae examined.
The online version of this article includes the following figure supplement(s) for figure 1:
Figure supplement 1. Bright-field micrograph showing sea star larvae undergo whole-body regeneration (WBR).
Figure supplement 2. Quantification of serotonergic neurons in larvae shows a stable number of serotonergic neurons over time.
reasoned that serotonergic neural bodies were removed entirely by our manual bisection protocol.
Indeed, when we bisect the larvae across the midline, 40 of 45 stained larvae show no remaining
serotonergic neurons 1 day later, when they were first assayed. Figure 1F’–f shows one such example.
Of the remaining five, only one or two cells were found which, due to their location, are likely those
normally associated with the lower lip of the mouth.
By examining a time series of regeneration, we show that serotonergic neurons are first revealed by
antibody staining at the anterior regenerating leading edge in 8/14 larvae 5 days following bisection
(Figure 1G’–g). By 7 days post bisection (dpb), serotonergic neurons are found in 12/15 regenerating
larvae at the anterior edge (Figure 1H’–h). Three weeks following bisection, the regenerated seroto-
nergic neurons are bilaterally patterned at the dorsal side of the regenerating larvae, resembling the
dorsal ganglia structure found in intact larvae (Figure 1I’–i).

These data therefore show that the anterior serotonergic neurons are removed following anterior
bisection and are reformed in the correct general location and with extensive axonal projections by
21 days of regeneration.
Embryonic neurogenesis pathways re-emerge during regeneration

We next questioned whether the regenerated serotonergic neurons formed using the embryonic
neurogenesis pathway. We have shown that, during normal embryogenesis, the SRY-box TF, sox2,
(formerly soxb1) is expressed broadly throughout the neurogenic ectoderm (Yankura et al., 2013).
Serotonergic neurons form from cells expressing the SRY-box TF, sox4 (formerly soxc) that are first
present and distributed in a ‘salt and pepper’ pattern throughout the ectoderm of the embryonic
blastula (Yankura et al., 2013; Yankura et al., 2010). Two transcription factors, foxq2 and six3, are
expressed in the anterior ectoderm of the early gastrula and are required for the correct progression
of the anteriorly most located sox4+ neural progenitors to become lhx2+ cells. After asymmetric cell
divisions, lhx2+ progenitors in turn give rise to post-mitotic neurons expressing elav (Figure 2A–C).
These post-mitotic neurons will produce serotonin when they mature (Cheatle Jarvela et al., 2016).
Thus we sought to determine whether these genes are re-expressed following bisection. If so, this can
provide the first indication that embryonic neurogenesis is reactivated during regeneration.
Following bisection, we find that foxq2 and six3 are expressed in the anterior ectoderm of the
leading regenerating edge by 3dpb (Figure 2D, and Figure 2—figure supplement 1A- H). It is
important to note therefore, that foxq2 is now expressed in cells that, in the normal larvae, reside
along the middle of the AP axis and would never normally express anterior markers such as foxq2.
We find similar patterns with other regulatory genes that are expressed along the embryonic AP axis,
with wnt3 re-expressed in posterior regenerating larvae (Figure 2—figure supplement 1I-K). This
indicates that the anterior leading edge cells are being respecified, as defined by expressing new sets
of genes, during regeneration. This respecification, at least partially, recapitulates the embryonic axial
state required for neurogenesis.
Sox2 is expressed at low levels throughout the ectoderm of the normal larvae (Yankura et al., 2013;
Figure 2—figure supplement 2A) but within 1 day following bisection, sox2 expression is dramat-
ically up-regulated at the wound site (Figure 2E–e, and Figure 2—figure supplement 2B), when
compared to larval expression levels. We have previously shown that cell division occurs throughout
the normal larva, but then decreases in the early bisected larva and is then predominantly localized to
the wound adjacent anterior at 3dpb (Cary et al., 2019b). Thus, localized sox2 expression precedes
the formation of the proliferative zone but then remains within this anterior region. This regenerative
proliferative zone is readily visualized by staining for 5-Ethynyl-2´-deoxyuridine (EdU), by incubating
larvae for one to six hours in EdU sea water. This costaining shows that sox2 is highly expressed
throughout this ectodermal zone and within some of these proliferating cells at 3dpb (Figure 2F–f,
and Figure 2—figure supplement 2C) and that this remains through to 5dpb (Figure 2G–g, and
Figure 2—figure supplement 2D).
Sox4 expression, a marker of neural precursor cells, is also observed at the wound site within one
day following bisection (Figure 2H–h’, and Figure 2—figure supplement 2F). In 3dpb larvae, sox4
expression is detected at the regeneration leading edge (Figure 2—figure supplement 2G). Fluo-
rescent labels allow for the detection of coexpression of genes and colocalization of gene expression
with EdU staining. The large number of cells at these stages make colocalization difficult to establish
through conventional confocal microscopy and thus we used Imaris Software (Oxford Instruments)
3D visualization analysis to identify overlapping signals with confidence (Imaris visualization is shown
Figure 2h’ and i’ j’, k1, k2). Thus we show that sox4+ cells clearly undergo cell divisions as FISH
staining colocalizes with EdU (Figure 2I–i’). Proliferating sox4+ cells are also detected in the regen-
erating ectodermal/endodermal mouth (Figure 2I). By 5dpb, sox4 expression is concentrated to the
regenerating anterior, again in dividing cells (Figure 2J–j’, and Figure 2—figure supplement 2H).
The expression of lhx2 is also detected by 5-7dpb in dispersed cells at the regenerating leading
edge (Figure 2 K-K2, and Figure 2—figure supplement 2J). Similarly, the expression of post-mitotic
neural marker elav is also restored at the regenerating anterior at 5-7dpb (Figure 2L–l’, and Figure 2—
figure supplement 2L). Two-color FISH shows that, similar to embryogenesis, sox4/lhx2 co-expres-
sion is found in cells located at the lateral sides of the regeneration leading edge (Figure 2K–l’) and
that lhx2 and elav are also co-expressed.

Figure 2. Recapitulation of embryonic neural gene expression during regeneration. (A) In normal development, the embryonic apical pole domain
(APD) gives rise to serotonergic neurons via the APD-associated neurogenesis pathway illustrated in (B). (C–D) Fluorescent in situ hybridization (FISH)
of APD marker gene foxq2 in embryos (C) and in the leading edge of the regenerating larvae at three dpb (D). (E-j’) EdU labeled and FISH (EdU-FISH)
results show that sox2 and sox4 are expressed in proliferative cells at the regeneration leading edge. (E–G) EdU-FISH of gene sox2. (e–g). Expanded
view of the boxed area in (E–G). (E) Upon bisection, sox2+ cells are highly concentrated at the wound site and later in (F–G) at the regeneration leading
edge. Throughout the time-course, sox2+ cells constantly undergo cell cycling (e–g). (H–J) EdU-FISH of sox4. (h–j). Expanded view of the boxed area in
(H–J). (H). Upon bisection, sox4+ cells are detected at the regeneration leading edge and later in (I–J) expanded to the mouth. Throughout the time-
course, sox4+ cells constantly undergo cell division (h–j). (h’-j’) 3D visualization of EdU + sox4+ cells using Imaris software, highlighted in (h–j), indicated
Figure 2 continued on next page

Figure 2 continued
by white arrowheads. This shows clear double detection of EdU and sox4: the EdU + nucleus is surrounded by sox4 signal. (K-i’) Double fluorescent
in situ hybridization (FISH) shows the recapitulation of APD gene expression trajectory. (K) In 5dpb larvae, sox4 and lhx2 are co-expressed in cells at
the lateral regeneration leading edge. (k1’-k2’) show the Imaris 3D reconstructed view of boxed areas k1 and k2. In the highlighted cells (arrowheads),
nucleus labeled with DAPI is surrounded by both lhx2 and sox4 signals, indicating co-expression in the same cell. Scale bar in (c1): 5 µm; (c2): 7 µm.
(L) In 5 dpb larvae, lhx2 and elav are co-expressed in cells at the lateral regeneration leading edge, scale bar: 100 µm. This is amplified in (l). (l’) shows
the 3D reconstructed view of the cell marked by arrowheads in (l). (M) A proposed Model for the regeneration of neurons in sea star larvae. Dpb: day-
post-bisection. Scale bar in (E–K): 50 µm; (L): 100 µm; (e–i,l): 20 µm; (j): 30 µm; (h’-j’,k1 and l’): 5 µm; (k2): 7 µm. Dpf: day-post fertilization; dpb: day-post
bisection; mo, mouth. The numbers shown in the lower right corner of each image indicate the number of larvae showing a positive FISH signal among
the larvae examined.
Figure supplement 1. Whole-mount in situ hybridization (WMISH) results show the reconstruction of the AP body axis.
Figure supplement 2. WMISH of embryonic neurogenic pathway genes during regeneration.
Collectively, these data show that bisection leads to the activation of embryonic neurogenesis
gene expression states. Sox2 and sox4 expressions are induced upon wounding, and sox4+ cells
located at the lateral leading edge give rise to lhx2+ cells. The lhx2+ cells at the leading edge in
turn become post-mitotic neurons expressing elav (Figure 2M). The location of lhx2 expression at
the leading edge, within the zone of foxq2 expression, also recapitulates the spatial localization of
embryonic serotonergic cell specification.
Newly specified sox4+ neural cell lineage is induced by bisection

The gene expression states propose the intriguing model that neural progenitors, that is newly spec-
ified sox4+ cells, arise at the leading edge following bisection, and that these newly specified neural
progenitors will reform the serotonergic neurons by entering the embryonic neurogenesis pathways.
To test this hypothesis, we needed to establish a reporter system that would distinguish new sox4+
cells from existing sox4+ cells and also follow their fate for several days.
To this end, we developed a highly stable, photoconvertible fluorescent reporter. We modified
an approach we have used previously in which eGFP (GFP) was homologously recombined into the
sox4 coding region of a Bacterial Artificial Chromosome (BAC) clone, to replace the coding sequence
of sox4 with that of GFP while leaving the endogenous basal promoter and 5’UTR in place (Buckley
and Ettensohn, 2019). When the sox4:GFP recombinant BAC is injected into embryos, it faithfully
expresses GFP in sox4+ cells, recapitulating endogenous sox4 expression (Cheatle Jarvela et al.,
2016). It should be noted that transgenic DNAs are inherited mosaically and thus any individual larva
expresses the reporter in only a subset of cells. This demonstrates that the approximately 150 Kbp
surrounding the genomic locus of the sox4 coding region, including the endogenous basal promoter,
is sufficient to drive correct expression of the transgenic GFP reporter gene.
We now exchanged GFP for the photoconvertible Kaede sequence. Kaede fluorescent protein
(FPbase ID: Q31FH) stably converts from green to red after exposure to UV light (Ando et al., 2002).
We experimentally measured the stability of Kaede, by injecting in vitro synthesized kaede mRNA into
zygotes and quantifying fluorescence over time. We show that translated Kaede protein is detect-
able above background for at least 7 days in P. miniata embryos and larvae when grown at 15°C
(Figure 3—figure supplement 1, and Figure 3—source data 1). This supports previous work which
shows that GFP is extremely stable in these embryos, likely because they are grown at low tempera-
tures (Arnone et al., 1997).
The sox4:Kaede BAC construct (Figure 3A) was injected into zygotes, and larvae grown to 7dpf
bipinnaria larval stage. The BAC DNA construct is stably inherited by clones of cells and therefore
present mosaically in the cells of the larvae. The larvae were then bisected and immediately photo-
converted by exposure to 405 nm light (Figure 3—figure supplement 1, and Figure 3—source
data 1). Larvae were inspected under fluorescent microscopy to assess photoconversion, and we
confirm that all cells that had expressed sox4 within the previous 7 days now fluoresce at 560 nm
(red). Decapitated larvae were then individually cultured in 24-well dishes so that we could follow indi-
vidual cell populations in each larva (Figure 3C). At 2dpb and 3dpb we observe green-only, red-only,
as well as yellow (green plus red) cells (Figure 3B and D–d). Red cells in the regenerating larvae are
the historically labeled cells that have expressed sox4 at some point prior to bisection but no longer

Figure 3. Existing and de novo Sox4+ lineages regenerate serotonergic neurons. (A) The design of Sox4:Kaede BAC construct. (B–C) Design of
photo-conversion experiment. Sea star zygotes are injected with the Sox4-Kaede BAC. Injected embryos are incubated till larvae for bisection. Photo-
conversion is immediately performed on bisected posterior segments. After regenerating for periods of time as shown (dpb), larvae are observed to
examine the Sox4+ lineages. (D) In Sox4-Kaede transgenic regenerating larvae, there are multiple sources of Sox4+ cells at the regeneration leading
edge. Some Sox4+ cells are derived from the yellow, existing Sox4 lineage (marked by asterisks). Some Sox4+ cells are differentiated cells that no
longer express Kaede (arrows). There are also green Sox4+ cells that are newly specified upon decapitation (arrowheads). The boxed areas in the
regeneration leading edge are amplified in (d1), (d2) and (d3). Scale bar in (D): 50 µm. At least 30 regenerating larvae in two independent batches were
examined.
The online version of this article includes the following source data and figure supplement(s) for figure 3:
Source data 1. Quantification of Kaede colors and protein stability.
Figure supplement 1. Stability of Kaede protein in P.miniata during development.
express sox4. For example, these may have been differentiated neurons that no longer express the
progenitor marker sox4. The yellow cells are from existing larval and regenerative sox4+ cell lineages.
That is, these cells expressed sox4 in the intact larvae and continue to express sox4 in their progeny
after wounding. Most interestingly, there are green-only cells. These are cells that have not previously
expressed sox4 but are induced to start expressing sox4 only upon decapitation.
We questioned whether the presence of newly expressing sox4+ cells is induced by wounding, or
whether there is normally a pool of progenitor cells that are set to become sox4+ over the timepoints
we observed. We therefore compared the rate of sox4+ cell specification between embryogenesis and

Figure 4. Appearance of new Sox4+ cells ends at the larval stage but resumes in regeneration. (A) A schematic for the experimental plan to determine
the source of normal larval Sox4+ cell lineage. Sox4:Kaede transgenic embryos were photoconverted at either embryonic stage or larval stage. Sox4
expression was examined three days later. (B) Embryo injected with Sox4-Kaede BAC is converted at 2dpf. Red Kaede localization therefore marks all
labeled Sox4+ cells. (C) By 5dpf there are several newly specified, green Sox4+ cells indicated by arrowheads. However, (D) when converted at 4dpf
(larval stage), only yellow cells and red cells are observed 3 days later as shown in (E), indicating no specification of Sox4+ cell occurs in the larva.
(F) Quantification of Sox4 specification in embryos, larvae and regeneration. The Hedges' g for two comparisons against the embryonic group are
shown in the above Cumming estimation plot. The raw data is plotted on the upper axes. On the lower axes, mean differences are plotted as bootstrap
sampling distributions. Each mean difference is depicted as a dot. Each 95% confidence interval is indicated by the ends of the vertical error bars. Sox4+
cell specification event is close to 0 by 4dpf, significantly decreased compared to the embryonic (2dpf) state. Specification of Sox4+ cells is restored in
regenerating larvae. Scale bar in (B–E): 50 µm. dpf: day-post-fertilization. At least 40 embryos were examined from five independent batches.
The online version of this article includes the following source data for figure 4:
Source data 1. Quantification of sox4 green cells.
larval development. We again injected Sox4:Kaede BAC into zygotes and photoconverted either 2dpf
embryos or 4dpf larvae (Figure 4A). We then quantified the numbers of green, red and yellow cells
3 days later (5dpf or 7dpf) over normal development (i.e. without bisection, Figure 4—source data 1).
We find that there are high numbers of green+ cells in larvae photoconverted at 2dpf (Figure 4B–C),
but almost none in larvae converted at 4dpf (Figure 4D–E).
This is evidence that new sox4+ cells are actively being specified in embryos, but that this speci-
fication has stopped by 4dpf (i.e. larval stage). Comparing the numbers of green (newly expressing)
sox4+ cells during normal development with those counted in regeneration, we see that specification
of new sox4+ cells is highest in embryos, ends by late embryogenesis, and resumes in response to
bisection induced regeneration (Figure 4F, and Figure 4—source data 1). Thus, decapitation induces
a wound response that re-initiates the specification of new sox4+ cells, a process which had stopped
by day four of embryogenesis. These sox4+ cells then reenter into embryonic neurogenesis pathways
(Figure 2).

Sox2+ progenitor cells are specified to Sox4+ cells to regenerate

neurons
We have shown that there arises a new population of sox4+ cells induced by bisection. During normal
embryogenesis, the neural precursor cells are derived from sox2+ ectoderm. Sox2 is expressed
throughout the ectoderm in embryos and therefore broadly marks this germ layer (Yankura et al.,
2013; Yankura et al., 2010). We therefore asked whether these new bisection-induced neural progen-
itors similarly arise from ectodermal lineages.
Our gene expression analysis showed that sox2 is broadly upregulated throughout the anterior
of the regenerating larva (Figure 2E–g) and that new, dividing, sox4+ cells also form in this region
(Figures 2H–j and 3D). We therefore generated a sox2:Kaede BAC. We first tested whether this
BAC recombinant recapitulated normal Sox2 expression in sea stars. After BAC injection, zygotes
started to express Kaede at 24-48hpf in broad patches of ectodermal cells. By larval stage, Kaede
was detected on the ectoderm including the ciliary bands, dorsal ganglia, and ectodermally derived
neurons (Figure 5—figure supplement 1A-C'). This localization pattern recapitulates the embryonic
sox2 expression pattern reported previously (Yankura et al., 2013; Yankura et al., 2010; Figure 2—
figure supplement 2A).
We next generated a sox4:Cardinal BAC recombinant so that we could examine the potential colo-
calization of sox2+ and sox4+ lineages. This is the same BAC recombinant as sox4:Kaede (Figure 3A)
but with the Kaede coding sequencing replaced by Cardinal (FPbase ID: 7EWEM), which fluoresces
in the far-red range (633 nm). These two BAC recombinants were co-injected to identify potential
double labeling of sox2+ cell lineages (green, red, or yellow depending on the assay) and sox4+ cell
lineages. It is important to note that multiple constructs when co-injected into echinoderm zygotes
are then co-integrated, and are therefore inherited in the same cellular lineages (Cheatle Jarvela
et al., 2014). When we examine expression in normal developing larvae, we find that sox4+ cells
overlap completely with sox2+ cells on the ectoderm as is expected in normally developing larvae
(Figure 5—figure supplement 1D-D''), although not all sox2+ cells are sox4+, as sox2+ also produce
non neural ectoderm.
We next photoconverted the double BAC recombinant larvae (i.e. all sox2:Kaede+ cells are
converted to red), and immediately bisected the larvae and allowed them to regenerate for 2–3 days
(Figure 5A). Strikingly, we found new (green+ cells) expression of sox2, indicating that cell that had
not previously expressed sox2 are now expressing the sox2 gene following bisection. Furthermore,
four of 15 observed larvae exhibited cells with colocalized green Kaede (new sox2 expression) and
Cardinal (sox4,far red, pseudo-colored blue) at the regenerating leading edge (Figure 5 B(b-b3))at
3dpb. For example, in the larva shown in Figure 5, there are 3 cells located at the left side of the
regeneration leading edge that contain green Kaede and Cardinal. As the green Kaede reporter
labels de novo sox2 expression, this shows that these new sox2+ cells start to express sox4 and
thereby enter into neurogenesis pathways. We observe only few newly expressing sox2+ and sox4+
cells in this assay, likely as our reporter system is mosaic, and because there are in reality only small
numbers of newly expressing sox2+ cells at this stage. Our whole mount in situ analyses in Figure 2
also suggested that there will be new sox2+ cells, and that these will become sox4+. The Cardinal
reporter can potentially represent both pre-existing and regenerative sox4 expressions. However, it is
important to note that at the time of bisection, all labeled sox4+ cells overlapped with sox2+ lineage
in the imaged larvae. In other words, all Cardinal + cells are also red Kaede+ at the time of photo-
conversion. Thus, the sox4:Cardinal in Figure 5B must be expressed after wounding. This therefore
supports the hypothesis that cells derived from non sox2+ lineages are induced to express sox2 in
regenerating larvae and will contribute to neural progenitors. Indeed when we follow these larvae
through to 7dpb (Figure 5—figure supplement 2A-B) we find fluorescence in the neural projections
which are characteristic of fully formed neurons (Figure 5—figure supplement 2a1-a3).
Bisection induced Sox2+ cells maintain usual embryonic cell restriction

We were interested to determine whether these newly specified sox2+ cells, which were not previ-
ously ectodermal, now maintain an ectodermal lineage. We examined at least 10 larvae over three
independent injection batches and found that in all regenerating larvae the newly expressing sox2+
cells contributed to only tissues normally derived from sox2+ lineages (e.g. Figure 5—figure supple-
ment 1E-F''). We observed sox2:Kaede green cells in the outer ectodermal epithelium, the mouth,

Figure 5. Sox4+ cells are specified from Sox2+ cells at the regeneration leading edge. (A) Schematic representation of the experimental design. Two
BACs: sox2:Kaede and sox4:Cardinal were coinjected. Seven days old transgenic larvae were bisected, Kaede protein was photoconverted immediately
following bisection, then regenerated larvae were analysed at 3 days later. (B3) Some sox4+ cells at the regeneration leading edge are made from de
novo sox2+ cells. Boxed area in (B) is amplified in (b). In 3dpb transgenic larvae, sox4+ cells with de novo sox2 expression are located at the lateral side
of the regeneration leading edge (black solid circle). These cells have (b1) newly expressed, regenerative sox2:Kaede and (b3) sox4:Cardinal expression
(both highlighted with white solid circle), (b2) but have no historic sox2:Kaede (white dotted circle). (C). Sox2+ cells at the regeneration leading edge
have multiple sources. (c) Is the amplified view of the boxed area. Apart from the de novo sox2+ cells, there are also yellow, larval sox2 lineage at the
regeneration leading edge (black solid circle). These cells do not enter sox4-mediated pathways. They are labeled with (c1) green and (c2) red Kaede
(white solid circle), (c3) but not Cardinal (white dotted circle). Images B and C are representative of at least 15 observed samples. Scale bar in (B–
C): 50 µm; (b–b3, c–c3): 20 µm; (d-d3). RL, regeneration leading edge; CB, ciliary band. Dpb: day-post-bisection. Sox2: Kaede (red, yellow, green) and
Sox4: Cardinal (far red, false-colored to blue).
Figure supplement 1. Expression pattern of sox2-Kaede BAC.
Figure supplement 2. Sox2+ cell lineage forms the regenerated nervous system.
and ciliary bands. This indicates that once induced these cells will maintain their normal, embryonic
fate potential.
It is worth noting that not all sox2+ cells enter the neurogenesis pathway in regeneration (Figure 5
C(c1-c3)). For example, in the larva shown in Figure 5, there are two cells at the regeneration leading
edge that contain both green and red Kaede, derived from the sox2 lineage. These cells do not
express sox4:Cardinal by 3dpb. This suggests that while larval sox2 cell lineages are restricted to
ectodermal potency, they do not necessarily contribute only sox4+ neural fates.
New sox2 expression is induced in the absence of cell division

Finally, we questioned whether new sox2 expression could arise in the absence of cell division. Stem
cells, by definition, require an asymmetric division to produce a new daughter cell type and main-
tained stem cell. Thus new sox2 expression should not be present when cell division is blocked if
it is solely derived from asymmetric stem cell division. We bathed sea star larvae in Aphidicolin, a
conserved inhibitor of cell division, that is particularly well characterized in echinoderms where it is

known to block DNA polymerase function, nuclear break down and microtubule organizing centers
(Mashanov et al., 2017; Nishioka et al., 1984). We first used a serial dilution test to determine that
the 25 µM of Aphidicolin was the minimum concentration needed to reliably inhibit cell division as
assayed by EdU staining, while cells in DMSO controls divided normally (Figure 6B). We incubated
regenerated larvae in the drug or vehicle for up to 1 week, with daily changes into fresh drug or
vehicle, and analyzed them every 24 hr to test that Aphidicolin consistently blocked cell division over
this time (Figure 6—figure supplement 1). We then microinjected the sox2:Kaede recombinant into
fertilized oocytes, bisected 7dpf larvae, photoconverted and added Aphidicolin or DMSO as a control
in the sea water immediately after post bisection. Bisected transgenic larvae were continuously incu-
bated in drug or control solutions over three days (Figure 6A). The aphidicolin treatment, therefore,
covered the phases of regeneration, and preceded the first observed sox2 and sox4 expression by at
least one day. At least 10 larvae over three independent injections were analyzed. We then assayed for
red, green, or yellow-positive cells (Figure 6C–D”, and Figure 6—source data 1). We show that there
are multiple yellow, green cells along the leading regenerated edge. This confirms that cells which
were not previously expressing sox2 are now able to initiate expression of this gene in the absence of
cell division and are thus not the result of asymmetric stem cell division.
This experiment additionally addresses a concern that green FP only cells may actually be Green
+ Red, but have lost red FP signal through dilution if the precursor cells are highly proliferative. We
thought this unlikely as green+ FP cells are identified prior to extensive regenerative proliferation and
we individually followed larvae to track previously identified Red FP cells. We now however, can defi-
nitely exclude dilution based on proliferation as a cause for loss of Red FP as Green FP cells emerge
in the absence of any division.
Discussion
The ability that some animals have to restore tissues and body parts following traumatic injury chal-
lenges our current understanding of the mechanisms of cell specification and differentiation. Signif-
icant 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 echi-
noderms (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 echi-
noderm 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 echino-
derms, 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

Figure 6. Sox2+ cells fate specification (continued or de novo expression) does not depend on entering into the cell cycle during the early stage (upto
3dpb) of regeneration. (A) Schematic representation of the experimental design. Sox2:Kaede transgenic larvae were bisected and photoconverted.
The first dose of 25 µM aphidicolin or vehicle (0.08% DMSO) were applied after 3hpb, changed every 24 hr until 3dpb then the regenerated larvae
were analyzed. (B-B”) To monitor the effect of aphidicolin on DNA synthesis, we used an EdU cell proliferation assay. 3dpb larvae in aphidicolin, DMSO
or sea water solutions were incubated in 15 µM EdU for 1 hr. (C–D) Sox2-Kaede cell specification by three dpb under aphidicolin (D-D”) or vehicle (C-
C”) treatment. Scale bar in (B-B”), (C-D) is 50 µm. Dpb: day-post-bisection. Hpb: hour-post-bisection. Arrowheads highlight de novo sox2 expression.
The numbers shown in the lower right corner of (B-B’’) indicate the number of larvae analyzed. (C and D) images are representative of at least 15
transgenic samples from two independent batches. A percentage of yellow (re-used) and green (de novo) Kaede expressing cells at the wound site
during vehicle or drug admission are 71.6 and 2.1; 80 and 1.7 correspondent (Figure 6—source data 1).
The online version of this article includes the following source data and figure supplement(s) for figure 6:
Figure 6 continued on next page

Figure 6 continued
Source data 1. Quantification of sox2 cells under drug/vehicle treatment.
Figure supplement 1. Aphidicolin or vehicle treated bisected larvae during one week of regeneration.
axial patterning is a commonly observed early feature in many species undergoing whole body regen-
eration (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 func-
tion 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 consis-
tent with re-entry into an embryonic state. We further show, using FISH and using IMARIS to confi-
dently 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 demon-
strated 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 regener-
ated 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 devel-
oped 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 embry-
onic 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 swim-
ming 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 regen-
eration 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 dediffer-
entiation 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.
Materials and methods

Key resources table
Reagent type (species) or
resource Designation Source or reference Identifiers Additional information
Commercial assay or kit Label IT Nucleic Acid Labeling Kit, DNP Mirus MIR 3825
Commercial assay or kit TSA Plus System Perkin Elmer NEL753001KT
Commercial assay or kit DIG RNA Labeling Kit (SP6/T7) Roche 11175025910
Click-iT Plus EdU Cell Proliferation Kit for

Commercial assay or kit Imaging, Alexa Fluor 488 dye Invitrogen C10637
Thermo-
mMESSAGE mMachine T7 transcription Fisher
Commercial assay or kit kit Scientific AM1344
Sheep polyclonal anti-digoxigenin AP- 11093274910,

Antibody conjugate Roche RRID:AB_2734716 IHC (1:2000)
Sheep polyclonal anti-digoxigenin POD- 1120773391,

Antibody conjugate Roche RRID:AB_514500 IHC (1:2000)
FP1129,
Antibody anti-DNP HRP-conjugate Perkin Elmer RRID:AB_2629439 IHC (1:500)
S5545,
Antibody Rabbit polyclonal anti-serotonin Sigma RRID:AB_477522 IHC (1:250)
Mouse monoclonal DSHB (Nakajima et al.,

Antibody 1E11 2004) IHC (1:5)
Jackson
Goat anti-mouse polyclonal Immuno- 115-165-146,
Antibody Cy3 research RRID:AB_2491007 IHC (1:2000)
Jackson
Goat anti-rabbit polyclonal Immuno-
Antibody Cy3 research 115-165-144 IHC (1:2000)
Goat anti-rabbit polyclonal A11008,

Antibody Alexa Fluor 488 Invitrogen RRID:AB_143165 IHC (1:1000)
Recombinant DNA reagent sox2:Kaede BAC This paper http://echinobase.org
Recombinant DNA reagent sox4:Kaede BAC This paper http://echinobase.org
Recombinant DNA reagent sox4:mCardinal BAC This paper http://echinobase.org
Recombinant DNA reagent sox4:GFP BAC This paper http://echinobase.org
Chemical compound, drug Aphidicolin Sigma A0781 25 μM
Fiji
Software, algorithm Fiji (http://fiji.sc) RRID:SCR_002285
Inkscape
(https://inkscape.
Software, algorithm Inkscape org/en/) RRID:SCR_014479
GIMP
(http://www.
Software, algorithm GNU Image Manipulation Program gimp.org) RRID:SCR_003182
Imaris
(http://www.bitplane.
Software, algorithm Imaris com/imaris/imaris) RRID:SCR_007370
Culture and regeneration

Adult Sea star, Patiria miniata, were obtained from the southern coast of California, USA (Pete Halmay
or Marinus Scientific) and housed in artificial sea water (ASW, Instant Ocean, Aquarium Systems)
at 12°C–15°C. Embryos were cultured in ASW and fed Rhodomonas lens ad libitum as previously
described (Cheatle Jarvela and Hinman, 2014). All studies of regenerating larvae were conducted

with larval cultures beginning at 7dpf at which point the larvae were manually bisected stereotypically
through the foregut, midway along the AP body axis as described earlier (Cary et al., 2019b). Regen-
erating larvae were photographed using differential interference contrast (DIC) optics over the course
of regeneration on a Leica DMI4000B microscope at ×100 magnification using Leica Application Suite
software (Leica; Wetzlar, Germany).
Whole-mount in situ hybridization (WMISH)

Embryos, intact and regenerating larvae for WMISH were fixed in 4% paraformaldehyde (PFA) in a
high-salt MOPS-fix buffer (100 mM MOPS pH 7.5, 2 mM MgSO4, 1 mM EGTA, and 0.8 M NaCl) for
90 mintes at room temperature or overnight at 4 °C. Following fixation, embryos were washed in (v/v)
25%, 50%, 75%, and 100% ice-cold 70% ethanol. Fixed embryos and larvae were stored at –20 °C.
WMISH was conducted as described previously (Hinman et al., 2003; Yankura et al., 2010) with the
following modification. Hybridization of sox4 and lhx2 riboprobes (final concentration of 0.2 ng/mL)
was performed at 55 °C for 5 days. Hybridization of elav riboprobes (final concentration of 0.2 ng/mL)
was performed at 58 °C for 5 days. Hybridization of foxq2, wnt3 and six3 riboprobes (final concen-
tration of 0.1 ng/mL) was performed at 58 °C for 5 days. Larvae were imaged on a Leica DMI4000B
inverted light microscope using DIC microscopy at ×100 magnification and the Leica Application
Suite software (Leica; Wetzlar, Germany). Primer sequences used to prepare insitu probes are listed in
Supplementary file 1.
Double fluorescent in situ hybridization (FISH)

Intact and regenerating larvae of P. miniata at the indicated time points were fixed in a solution of
4% paraformaldehyde in MOPS-fix buffer (0.1 M MOPS pH 7.5, 2 mM MgSO4, 1 mM EGTA, and
0.8 M NaCl) for 90 min at room temperature and transferred into 70% ethanol for long-term storage
at − 20 °C. Double FISH experiments were performed as previously described (McCauley et al.,
2013) using digoxigenin-labeled antisense RNA probes (final concentration of 0.1–0.2 ng/mL) and
dinitrophenol-labeled antisense RNA probes (final concentration of 0.1–0.2 ng/mL). Hybridized probes
were detected using anti-DIG-POD antibody (1:1000, Roche, RRID:AB_514500), anti-DNP-HRP anti-
body (1:1000, Perkin Elmer, RRID:AB_2629439) and tyramide signal amplification (Perkin Elmer). A
1:100 dilution of Cy3 or FITC labeled tyramide in an amplification buffer was used to treat larvae
for 7 min at room temperature in the dark. A Cy3- or FITC-labeled tyramide was deposited near the
hybridized probe in a horseradish peroxidase mediated reaction. This allowed for fluorescence detec-
tion of labeled probes. During PBST washes, larvae were incubated for a total of 20 min in solution
with 1:10,000 dilution of 10.9 mM DAPI, followed by PBST washes. Larvae were photographed with
Zeiss 880 Laser Scanning Microscope at ×200 magnification with 405 nm, 488 nm, and 560 nm chan-
nels in Z-stack settings.
EdU-FISH
Labeling and detection of proliferating cells in P. miniata intact and regenerating larvae were performed
using the Click-it Plus EdU 488 Imaging Kit (Life Technologies), with the following modifications.
Larvae were incubated in a 10 μM solution of EdU for 15 min or 6 hr in seawater at 15 °C followed by
immediate fixation in 4% PFA in PBS buffer with 0.1% Triton x-100. Fixation was performed at room
temperature for 90 min. FISH was conducted as described above in the double FISH method with
the following modification. Either FITC- or Cy3-labeled tyramide was used to detect labeled probes.
Immuno-fluorescent (IF) staining

Whole mount IF staining was performed as described elsewhere (Cheatle Jarvela et al., 2016). Briefly
animals were fixed in 4% PFA prepared in PBS (pH 7.4) at room temperature for 15 min. The samples
were post-fixed in ice-cold methanol for 10 min, allowing for setting of larvae by gravity. The samples
were then washed in PBS, permeabilized in PBS/0.5% Triton X-100 for 30 min, then incubated in
0.1 M glycine for 30 min to quench autofluorescence. After another wash in PBS/0.1% Triton X-100 (3
× 15 min), the larvae were blocked using 3% BSA/PBS/0.1% Triton X-100 for 1 hr. The primary anti-
bodies (Supplementary file 2) were applied at 4 °C overnight. After extensive washing in PBS/0.1%
Triton X-100 (6 × 15 min), the samples were incubated in the secondary antibodies (Supplementary
file 2) for 1 hr at room temperature. Unbound antibodies were removed in four changes of PBS /0.1%

Triton X-100 (15 min each) and nuclei were stained in 1:10,000 dilution of 10.9 mM DAPI (Invitrogen)
for 30 min. After the final round of washes (3 × 10 min), the samples were coverslipped in Slow-
Fade antifade medium (Invitrogen). Stacks of optical sections were taken using the Zeiss 880 confocal
laser scanning microscope. Z- projections were generated in the Fiji image processing software
(RRID:SCR_002285) and Imaris (Oxford Instruments, RRID:SCR_007370). Figures were constructed
using Inkscape 1.1 (RRID:SCR_014479) and GIMP 2.10.30 (RRID:SCR_003182).
Generation of BAC-reporters
The sox4- BAC was recombineered with different fluorescent reporters (Supplementary file 3)
following the established protocol (Buckley and Ettensohn, 2019). The EL250 cells and the GFP
recombination cassette were generous gifts from Dr. Buckley. The GFP coding sequence was replaced
with either Kaede coding sequence or mCardinal coding sequence in the recombination cassette.
Using the same protocol, we generated a sox2-Kaede BAC (Supplementary file 3). Embryos injected
with recombineered BACs were observed at 2dpf, 4dpf, and 7dpf to confirm the expression pattern
recapitulating sox2 or sox4.
Microinjection of BACs
BAC-reporters were injected into fertilized eggs at a final concentration of 10 ng/μl as previously
described (Cheatle Jarvela and Hinman, 2014). For double lineage tracing an equivalent amount
of sox2-Kaede BAC and sox4-Cardinal BAC were mixed at a final concentration of 10 ng/μl. Injected
positive embryos were sorted under fluorescent stereo microscope at 24–48 hpf and then were kept
in sea water at 15 °C until future manipulations.
Photo-conversion of Kaede P. miniata cells

Kaede is a photoconvertible fluorescent protein that changes irreversibly from green to red upon
exposure to violet light (Ando et al., 2002). Photo-conversion of the Kaede was performed using an
Andor Revolution XD spinning Disk confocal microscope emitting at 405 nm with 100% laser power
for 60–80 s with snapshots taken every 10 s. After conversion transgenic embryos and larva were
inspected for completion of conversion using the same microscope as quickly as possible to reduce
the stress and transferred back to the plate with ASW.
Live embryos/larva manipulation and live imaging

To trace cell fate we kept transgenic embryos and larvae individually in a small petri dish with ASW
at 15 °C. Before imaging the animals were immobilized in 500 mM high-salt sea water for 1–2 min
to remove the cilia, then mounted on the slides. Immediately after imaging, coverslip was carefully
picked up and animals were transferred back to the dish. To increase survival rate the animals were
manipulated as quickly as possible, the different laser power settings were used to minimize tissue
damage in embryonic and larval stages. Regenerated larvae at different time points were imaged with
consistent settings for comparison. Imaging was performed by using the Andor Revolution XD spin-
ning Disk confocal microscope with Andor IQ3 system. Z-projections of stacks of optical sections were
generated in the Fiji image software (RRID:SCR_002285). Figures were constructed using Inkscape
1.1(RRID:SCR_014479) and GIMP 2.10.30 (RRID:SCR_003182).
Establishing Kaede protein stability in embryos

Generation and injection of kaede mRNA
Kaede PCR product was amplified from the plasmid (RRID:Addgene_54726) for generating the
template for capped mRNA synthesis (primers F: 5'- TAA TAC GAC TCA CTA TAG GGG TCG CCA
CCA TGA GTC TGA T –3'; R: 5'-TTG CCG ATT TCG GCC TAT TGG –3') with mMESSAGE mMACHINE
T7 transcription kit (ThermoFischer). Kaede mRNA was injected into the fertilized eggs at a final
concentration of 300 ng/µl. Kaede-positive embryos were sorted under 488 nm fluorescent light at 15
hpf, then were kept in a plate with artificial sea water (ASW) at 15 °C until live imaging. These embryos
were starved prior to imaging to avoid fluorescent background from their algal stomach contents.
Generation of the conversion plot and the conversion rate plot

Photo-conversion was completed manually with Andor Revolution XD spinning Disk confocal micro-
scope as described above. The time-lapse data were analyzed with Fiji. The pixel values (fluorescent

intensity, or FI) of 488 nm and 560 nm channels within selected areas were measured at each time
point. The background FI was subtracted to obtain the net FI which was plotted over time to generate
the photo-conversion plot. The ratio of net FI (560 nm) over net FI (488 nm) was calculated and plotted
over time to generate the conversion rate plot (Figure 3—source data 1).
Kaede stability - the fluorescence intensity curve

Twelve embryos injected with Kaede mRNA were photoconverted at 24 hpf and were imaged everyday
up to 12dpf. Out of the 12 converted embryos, 6 survived the repetitive imaging session. The net FI of
the 488 nm and 560 nm channels were collected and calculated following the steps described above.
The net FI of both channels were plotted over time to generate the fluorescence intensity curve
(Figure 3—source data 1).
Quantification of green Sox4+ cells and statistical analyses

Quantification of green Sox4+ cells was conducted manually in larvae. Unhealthy, abnormal or dying
larvae were not quantified. Quantification data of 5dpf larvae (Kaede converted at 2dpf embryonic
stage, or embryonic conversion) and 7dpf larvae (Kaede converted at 4dpf larval stage, or larval
conversion) were then processed for statistical analyses using the website https://www.estimation-
stats.com/#/. Shared control comparison was performed using Hedge’s g comparison (Ho et al.,
2019). The confidence interval width was 95%. Hedge’s g was chosen because it compares effect
sizes across experiments and is corrected for small-sample bias. The p-value was calculated with the
permutation T test.
The green Sox4+ cells at the regeneration leading edge were quantified manually in seven larvae
at 3dpb using Fiji. The data was compared to the 2dpf embryonic conversion data. The effect size
was Cliff’s delta and the mean of data. The confidence interval width was 95%. Two side p-value was
calculated with the Brunner-Munzel test. The data was compared to the 7dpf larval conversion data.
Delta’s cliff did not apply to these two groups. Instead, we compared the means of the two groups.
The confidence interval width was 95%. Two side p-value was calculated with the Mann Whitney test
(Figure 4—source data 1).
Quantification of Kaede+ cells and determination of colors

Quantification of green, red and yellow Sox4+ cells was conducted manually in Z-stack images. The
cellular area of each colored cell was compared to an adjacent background area with no Kaede
signal to measure FI of 488 nm and 560 nm channels, respectively. The FI in the background area
was subtracted from the FI at each channel within the cellular area to generate the net FI of the
colored cell at 488 nm and 560 nm channels. We then calculated the ratio of net FI (560 nm) over
net FI (488 nm) for each cell. The three colored groups were defined as follows: if the net FI ratio
of 560 nm/488 nm >5, the cell is considered as a red cell; if the ratio is between Y and 5, the cell is
considered as a yellow cell; if the ratio of 560 nm/488 nm< Y, the cell is considered as a green cell.
Inhibition of cell division

To inhibit cell division, we used aphidicolin (Sigma A0781) at a dosage of 8.3 μg/g (25 μM) in sea
water. To prevent drug elimination, we changed Aphidicolin and vehicle (DMSO) every 24 hr, so the
larvae were continuously exposed. The first treatment was done immediately post-injury, followed by
two more drug changes every 24 hr. Control animals received 0.08% DMSO (vehicle) incubation. The
aphidicolin treatment, therefore, covered the phases of regeneration, and preceded the first observed
sox2 and sox4 expression by at least 1 day. These treatment parameters were established after a
series of pilot experiments using two drug concentrations (25 uM, 50 uM), followed by 1 hr incubation
in 15 uM EdU, which was detected by standard protocol (invitrogen C10637). To elucidate sox2+,
sox4+ cells fate without cell division the fertilized eggs were injected with sox2, sox4 Bacs, 1-week-old
larvae were bisected, photoconverted, incubated in aphidicolin solution for 3 days and then imaged.
Sample size of experiments

At least 30–50 embryos and/or larvae in three to five separate trials were analyzed in each in situ and
immunostaining experiment. For quantification of FPs expression in live embryos and/or larvae were
analyzed 7–10 samples from three to five separate matings for each injection of each BAC construct.

Acknowledgements
The authors are grateful to Dr. Buckley for the assistance in BACs recombineering and Dr. Cheryl
Telmer, Dr. Andrew Wolff, and Jonathan Andrade for helpful comments on the manuscript. The work
was supported by NSF. IOS 1557431, NIH 1R24OD023046 and DSF Charitable Foundation.
Additional information
Funding
Funder Grant reference number Author
National Science NSF. IOS 1557431 Veronica Hinman

Foundation
National Institute of NIH 1R24OD023046 Veronica Hinman

General Medical Sciences
DSF Charitable Foundation Veronica Hinman
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Minyan Zheng, Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualiza-
tion, Writing – original draft, Writing – review and editing; Olga Zueva, Conceptualization, Data cura-
tion, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft,
Writing – review and editing; Veronica F Hinman, Conceptualization, Formal analysis, Funding acqui-
sition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original
draft, Writing – review and editing
Author ORCIDs
Minyan Zheng ‍ ‍http://orcid.org/0000-0001-7990-1773
Veronica F Hinman ‍ ‍http://orcid.org/0000-0003-3414-1357
Decision letter and Author response

Decision letter https://doi.org/10.7554/eLife.72983.sa1
Author response https://doi.org/10.7554/eLife.72983.sa2
Additional files
Supplementary files
• Supplementary file 1. Primers for probe amplification.
• Supplementary file 2. List of antibodies.
• Supplementary file 3. List of BAC.
• Transparent reporting form
Data availability
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.
References
Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A. 2002. An optical marker based on the UV-induced
green-to-red photoconversion of a fluorescent protein. PNAS 99:12651–12656. DOI: https://doi.org/10.1073/
pnas.202320599, PMID: 12271129
Angerer LM, Yaguchi S, Angerer RC, Burke RD. 2011. The evolution of nervous system patterning: insights from
sea urchin development. Development 138:3613–3623. DOI: https://doi.org/10.1242/dev.058172, PMID:
21828090

Arnone MI, Bogarad LD, Collazo A, Kirchhamer CV, Cameron RA, Rast JP, Gregorians A, Davidson EH. 1997.
Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis
in embryos and larvae. Development 124:4649–4659 PMID: 9409681.,
Ben Khadra Y, Sugni M, Ferrario C, Bonasoro F, Varela Coelho A, Martinez P, Candia Carnevali MD. 2017. An
integrated view of asteroid regeneration: tissues, cells and molecules. Cell and Tissue Research 370:13–28.
DOI: https://doi.org/10.1007/s00441-017-2589-9, PMID: 28331971
Ben Khadra Y, Sugni M, Ferrario C, Bonasoro F, Oliveri P, Martinez P, Candia Carnevali MD. 2018. Regeneration
in Stellate Echinoderms: Crinoidea, Asteroidea and Ophiuroidea. Results and Problems in Cell Differentiation
65:285–320. DOI: https://doi.org/10.1007/978-3-319-92486-1_14, PMID: 30083925
Buckley KM, Ettensohn CA. 2019. Techniques for analyzing gene expression using BAC-based reporter
constructs. Methods in Cell Biology 151:197–218. DOI: https://doi.org/10.1016/bs.mcb.2019.01.004, PMID:
30948008
Burke RD, Osborne L, Wang D, Murabe N, Yaguchi S, Nakajima Y. 2006. Neuron-specific expression of a
synaptotagmin gene in the sea urchin Strongylocentrotus purpuratus. The Journal of Comparative Neurology
496:244–251. DOI: https://doi.org/10.1002/cne.20939, PMID: 16538680
Byrne M. 2020. The Link between Autotomy and CNS Regeneration: Echinoderms as Non-Model Species for
Regenerative Biology. BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology
42:e1900219. DOI: https://doi.org/10.1002/bies.201900219
Carter HF, Thompson JR, Elphick MR, Oliveri P. 2021. The Development and Neuronal Complexity of Bipinnaria
Larvae of the Sea Star Asterias rubens. Integrative and Comparative Biology 61:337–351. DOI: https://doi.org/
10.1093/icb/icab103, PMID: 34048552
Cary GA, Cheatle Jarvela AM, Francolini RD, Hinman VF. 2017. Genome-wide use of high- and low-affinity Tbrain
transcription factor binding sites during echinoderm development. PNAS 114:5854–5861. DOI: https://doi.org/
10.1073/pnas.1610611114, PMID: 28584099
Cary GA, Hinman VF. 2017. Echinoderm development and evolution in the post-genomic era. Developmental
Biology 427:203–211. DOI: https://doi.org/10.1016/j.ydbio.2017.02.003, PMID: 28185788
Cary GA, Cameron RA, Hinman VF. 2018. EchinoBase: Tools for Echinoderm Genome Analyses. Methods in
Molecular Biology 1757:349–369. DOI: https://doi.org/10.1007/978-1-4939-7737-6_12, PMID: 29761464
Cary GA, Cameron RA, Hinman VF. 2019a. Genomic resources for the study of echinoderm development and
evolution. Methods in Cell Biology 151:65–88. DOI: https://doi.org/10.1016/bs.mcb.2018.11.019
Cary GA, Wolff A, Zueva O, Pattinato J, Hinman VF. 2019b. Analysis of sea star larval regeneration reveals
conserved processes of whole-body regeneration across the metazoa. BMC Biology 17:16. DOI: https://doi.
org/10.1186/s12915-019-0633-9, PMID: 30795750
Cary GA, McCauley BS, Zueva O, Pattinato J, Longabaugh W, Hinman V. 2020. Systematic Comparison of
Developmental GRNs Explains How Novelty Is Incorporated in Early Development. [bioRxiv]. DOI: https://doi.
org/10.1101/2020.08.07.231464
Cheatle Jarvela AM, Brubaker L, Vedenko A, Gupta A, Armitage BA, Bulyk ML, Hinman VF. 2014. Modular
evolution of DNA-binding preference of a Tbrain transcription factor provides a mechanism for modifying gene
regulatory networks. Molecular Biology and Evolution 31:2672–2688. DOI: https://doi.org/10.1093/molbev/
msu213, PMID: 25016582
Cheatle Jarvela AM, Hinman V. 2014. A method for microinjection of Patiria miniata zygotes. Journal of
Visualized Experiments e51913:e51913. DOI: https://doi.org/10.3791/51913, PMID: 25226153
Cheatle Jarvela AM, Yankura KA, Hinman VF. 2016. A gene regulatory network for apical organ neurogenesis
and its spatial control in sea star embryos. Development 143:4214–4223. DOI: https://doi.org/10.1242/dev.
134999, PMID: 27707794
Dupont S, Thorndyke M. 2007. Bridging the regeneration gap: insights from echinoderm models. Nature
Reviews Genetics 8:320–329. DOI: https://doi.org/10.1038/nrg1923-c1
Elia L, Selvakumaraswamy P, Byrne M. 2009. Nervous system development in feeding and nonfeeding asteroid
larvae and the early juvenile. The Biological Bulletin 216:322–334. DOI: https://doi.org/10.1086/
BBLv216n3p322, PMID: 19556597
Feng R, Wen J. 2015. Overview of the roles of Sox2 in stem cell and development. Biological Chemistry
396:883–891. DOI: https://doi.org/10.1515/hsz-2014-0317, PMID: 25781683
Ferrario C, Sugni M, Somorjai IML, Ballarin L. 2020. Beyond Adult Stem Cells: Dedifferentiation as a Unifying
Mechanism Underlying Regeneration in Invertebrate Deuterostomes. Frontiers in Cell and Developmental
Biology 8:587320. DOI: https://doi.org/10.3389/fcell.2020.587320, PMID: 33195242
García-Arrarás JE, Lázaro-Peña MI, Díaz-Balzac CA. 2018. Holothurians as a Model System to Study
Regeneration. Results and Problems in Cell Differentiation 65:255–283. DOI: https://doi.org/10.1007/978-3-
319-92486-1_13, PMID: 30083924
Gehrke AR, Neverett E, Luo YJ, Brandt A, Ricci L, Hulett RE, Gompers A, Ruby JG, Rokhsar DS, Reddien PW,
Srivastava M. 2019. Acoel genome reveals the regulatory landscape of whole-body regeneration. Science
363:eaau6173. DOI: https://doi.org/10.1126/science.aau6173, PMID: 30872491
Gerber T, Murawala P, Knapp D, Masselink W, Schuez M, Hermann S, Gac-Santel M, Nowoshilow S, Kageyama J,
Khattak S, Currie JD, Camp JG, Tanaka EM, Treutlein B. 2018. Single-cell analysis uncovers convergence of cell
identities during axolotl limb regeneration. Science 362:eaaq0681. DOI: https://doi.org/10.1126/science.
aaq0681, PMID: 30262634

Gildor T, Cary GA, Lalzar M, Hinman VF, Ben-Tabou de-Leon S. 2019. Developmental transcriptomes of the sea
star, Patiria miniata, illuminate how gene expression changes with evolutionary distance. Scientific Reports
9:16201. DOI: https://doi.org/10.1038/s41598-019-52577-9, PMID: 31700051
Hernroth B, Farahani F, Brunborg G, Dupont S, Dejmek A, Sköld HN. 2010. Possibility of mixed progenitor cells
in sea star arm regeneration. Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution
314:457–468. DOI: https://doi.org/10.1002/jez.b.21352, PMID: 20700890
Hinman VF, Nguyen AT, Davidson EH. 2003. Expression and function of a starfish Otx ortholog, AmOtx: a
conserved role for Otx proteins in endoderm development that predates divergence of the eleutherozoa.
Mechanisms of Development 120:1165–1176. DOI: https://doi.org/10.1016/j.mod.2003.08.002, PMID:
14568105
Hinman VF, Burke RD. 2018. Embryonic neurogenesis in echinoderms. Wiley Interdisciplinary Reviews.
Developmental Biology 7:e316. DOI: https://doi.org/10.1002/wdev.316, PMID: 29470839
Ho J, Tumkaya T, Aryal S, Choi H, Claridge-Chang A. 2019. Moving beyond P values: data analysis with
estimation graphics. Nature Methods 16:565–566. DOI: https://doi.org/10.1038/s41592-019-0470-3, PMID:
31217592
Joven A, Simon A. 2018. Homeostatic and regenerative neurogenesis in salamanders. Progress in Neurobiology
170:81–98. DOI: https://doi.org/10.1016/j.pneurobio.2018.04.006, PMID: 29654836
Kasahara M, Kobayashi C, Yamanaka A, Kitazawa C. 2019. Regeneration of the cell mass in larvae of
temnopleurid sea urchins. Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution
332:245–257. DOI: https://doi.org/10.1002/jez.b.22899, PMID: 31532079
Katow H, Elia L, Byrne M. 2009. Development of nervous systems to metamorphosis in feeding and non-feeding
echinoid larvae, the transition from bilateral to radial symmetry. Development Genes and Evolution 219:67–77.
DOI: https://doi.org/10.1007/s00427-008-0266-4, PMID: 19031082
Kondo M, Akasaka K. 2010. Regeneration in crinoids. Development, Growth & Differentiation 52:57–68. DOI:
https://doi.org/10.1111/j.1440-169X.2009.01159.x, PMID: 20078653
Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, Tanaka EM. 2009. Cells keep a memory of their
tissue origin during axolotl limb regeneration. Nature 460:60–65. DOI: https://doi.org/10.1038/nature08152,
PMID: 19571878
Mashanov VS, Zueva OR, García-Arrarás JE. 2015a. Expression of pluripotency factors in echinoderm
regeneration. Cell and Tissue Research 359:521–536. DOI: https://doi.org/10.1007/s00441-014-2040-4, PMID:
25468557
Mashanov VS, Zueva OR, García-Arrarás JE. 2015b. Heterogeneous generation of new cells in the adult
echinoderm nervous system. Frontiers in Neuroanatomy 9:123. DOI: https://doi.org/10.3389/fnana.2015.
00123, PMID: 26441553
Mashanov VS, Zueva OR, García-Arrarás JE. 2017. Inhibition of cell proliferation does not slow down
echinoderm neural regeneration. Frontiers in Zoology 14:12. DOI: https://doi.org/10.1186/s12983-017-0196-y,
PMID: 28250799
Mashanov V, Zueva O. 2019. Radial Glia in Echinoderms. Developmental Neurobiology 79:396–405. DOI:
https://doi.org/10.1002/dneu.22659, PMID: 30548565
Mashanov V, Akiona J, Khoury M, Ferrier J, Reid R, Machado DJ, Zueva O, Janies D. 2020. Active Notch
signaling is required for arm regeneration in a brittle star. PLOS ONE 15:e0232981. DOI: https://doi.org/10.
1371/journal.pone.0232981, PMID: 32396580
McCauley BS, Akyar E, Filliger L, Hinman VF. 2013. Expression of wnt and frizzled genes during early sea star
development. Gene Expression Patterns 13:437–444. DOI: https://doi.org/10.1016/j.gep.2013.07.007, PMID:
23899422
Murabe N, Hatoyama H, Hase S, Komatsu M, Burke RD, Kaneko H, Nakajima Y. 2008. Neural architecture of the
brachiolaria larva of the starfish, Asterina pectinifera. The Journal of Comparative Neurology 509:271–282.
DOI: https://doi.org/10.1002/cne.21742, PMID: 18473389
Nakajima Y, Kaneko H, Murray G, Burke RD. 2004. Divergent patterns of neural development in larval echinoids
and asteroids. Evolution & Development 6:95–104. DOI: https://doi.org/10.1111/j.1525-142x.2004.04011.x,
PMID: 15009122
Nishioka D, Balczon R, Schatten G. 1984. Relationships between DNA synthesis and mitotic events in fertilized
sea urchin eggs: aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule
organizing centers, but not cycles of microtubule assembly. Cell Biology International Reports 8:337–346. DOI:
https://doi.org/10.1016/0309-1651(84)90161-9, PMID: 6428758
Oulhen N, Heyland A, Carrier TJ, Zazueta-Novoa V, Fresques T, Laird J, Onorato TM, Janies D, Wessel G. 2016.
Regeneration in bipinnaria larvae of the bat star Patiria miniata induces rapid and broad new gene expression.
Mechanisms of Development 142:10–21. DOI: https://doi.org/10.1016/j.mod.2016.08.003, PMID: 27555501
Piovani L, Czarkwiani A, Ferrario C, Sugni M, Oliveri P. 2021. Ultrastructural and molecular analysis of the origin
and differentiation of cells mediating brittle star skeletal regeneration. BMC Biology 19:9. DOI: https://doi.org/
10.1186/s12915-020-00937-7, PMID: 33461552
Range RC, Angerer RC, Angerer LM. 2013. Integration of canonical and noncanonical Wnt signaling pathways
patterns the neuroectoderm along the anterior-posterior axis of sea urchin embryos. PLOS Biology
11:e1001467. DOI: https://doi.org/10.1371/journal.pbio.1001467, PMID: 23335859
Raz AA, Wurtzel O, Reddien PW. 2021. Planarian stem cells specify fate yet retain potency during the cell cycle.
Cell Stem Cell 28:1307-1322.. DOI: https://doi.org/10.1016/j.stem.2021.03.021, PMID: 33882291

Reddien PW. 2019. The cells of regeneration. Science 365:314–316. DOI: https://doi.org/10.1126/science.
aay3660, PMID: 31346049
Reinardy HC, Emerson CE, Manley JM, Bodnar AG. 2015. Tissue regeneration and biomineralization in sea
urchins: role of Notch signaling and presence of stem cell markers. PLOS ONE 10:e0133860. DOI: https://doi.
org/10.1371/journal.pone.0133860, PMID: 26267358
Rodriguez AM, Kang J. 2020. Regeneration enhancers: Starting a journey to unravel regulatory events in tissue
regeneration. Seminars in Cell & Developmental Biology 97:47–54. DOI: https://doi.org/10.1016/j.semcdb.
2019.04.003, PMID: 30953740
Sánchez Alvarado A. 2006. Planarian regeneration: its end is its beginning. Cell 124:241–245. DOI: https://doi.
org/10.1016/j.cell.2006.01.012, PMID: 16439195
Siebert S, Anton-Erxleben F, Bosch TCG. 2008. Cell type complexity in the basal metazoan Hydra is maintained
by both stem cell based mechanisms and transdifferentiation. Developmental Biology 313:13–24. DOI: https://
doi.org/10.1016/j.ydbio.2007.09.007, PMID: 18029279
Soufi A, Garcia MF, Jaroszewicz A, Osman N, Pellegrini M, Zaret KS. 2015. Pioneer transcription factors target
partial DNA motifs on nucleosomes to initiate reprogramming. Cell 161:555–568. DOI: https://doi.org/10.
1016/j.cell.2015.03.017, PMID: 25892221
Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast
cultures by defined factors. Cell 126:663–676. DOI: https://doi.org/10.1016/j.cell.2006.07.024, PMID:
16904174
Tanaka EM, Reddien PW. 2011. The cellular basis for animal regeneration. Developmental Cell 21:172–185. DOI:
https://doi.org/10.1016/j.devcel.2011.06.016, PMID: 21763617
Vickery MC, Vickery MS, Amsler CD, McClintock JB. 2001. Regeneration in echinoderm larvae. Microscopy
Research and Technique 55:464–473. DOI: https://doi.org/10.1002/jemt.1191, PMID: 11782075
Vickery MS, Vickery MCL, McClintock JB. 2002. Morphogenesis and organogenesis in the regenerating
planktotrophic larvae of asteroids and echinoids. The Biological Bulletin 203:121–133. DOI: https://doi.org/10.
2307/1543381, PMID: 12414562
Wolff A, Hinman V. 2021. The Use of Larval Sea Stars and Sea Urchins in the Discovery of Shared Mechanisms of
Metazoan Whole-Body Regeneration. Genes 12:1063. DOI: https://doi.org/10.3390/genes12071063, PMID:
34356079
Yankura KA, Martik ML, Jennings CK, Hinman VF. 2010. Uncoupling of complex regulatory patterning during
evolution of larval development in echinoderms. BMC Biology 8:143. DOI: https://doi.org/10.1186/1741-7007-
8-143, PMID: 21118544
Yankura KA, Koechlein CS, Cryan AF, Cheatle A, Hinman VF. 2013. Gene regulatory network for neurogenesis in
a sea star embryo connects broad neural specification and localized patterning. PNAS 110:8591–8596. DOI:
https://doi.org/10.1073/pnas.1220903110, PMID: 23650356
Zhang F, Ferretti P, Clarke JDW. 2003. Recruitment of postmitotic neurons into the regenerating spinal cord of
urodeles. Developmental Dynamics 226:341–348. DOI: https://doi.org/10.1002/dvdy.10230, PMID: 12557212
Zhu SJ, Pearson BJ. 2016. Neo)blast from the past: new insights into planarian stem cell lineages. Current
Opinion in Genetics & Development 40:74–80. DOI: https://doi.org/10.1016/j.gde.2016.06.007, PMID:
27379899

Zheng 2022

Uploaded by

Copyright:

Available Formats

Zheng 2022

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Zheng 2022

Uploaded by

Copyright:

Available Formats

RESEARCH ARTICLE

Regeneration of the larval sea star

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

‍ ‍Copyright Zheng et al. This

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  1 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  2 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  3 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  4 of 23

Embryonic neurogenesis pathways re-emerge during regeneration

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  5 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  6 of 23

Newly specified sox4+ neural cell lineage is induced by bisection

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  7 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  8 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  9 of 23

Sox2+ progenitor cells are specified to Sox4+ cells to regenerate

Bisection induced Sox2+ cells maintain usual embryonic cell restriction

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  10 of 23

New sox2 expression is induced in the absence of cell division

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  11 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  12 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  13 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  14 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  15 of 23

Materials and methods

Commercial assay or kit TSA Plus System Perkin Elmer NEL753001KT

Click-­iT Plus EdU Cell Proliferation Kit for

Sheep polyclonal anti-­digoxigenin AP-­ 11093274910,

Sheep polyclonal anti-­digoxigenin POD-­ 1120773391,

Mouse monoclonal DSHB (Nakajima et al.,

Goat anti-­rabbit polyclonal A11008,

Recombinant DNA reagent sox2:Kaede BAC This paper http://echinobase.org

Recombinant DNA reagent sox4:Kaede BAC This paper http://echinobase.org

Recombinant DNA reagent sox4:mCardinal BAC This paper http://echinobase.org

Recombinant DNA reagent sox4:GFP BAC This paper http://echinobase.org

Chemical compound, drug Aphidicolin Sigma A0781 25 μM

Culture and regeneration

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  16 of 23

Whole-mount in situ hybridization (WMISH)

Double fluorescent in situ hybridization (FISH)

Immuno-fluorescent (IF) staining

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  17 of 23

Photo-conversion of Kaede P. miniata cells

Live embryos/larva manipulation and live imaging

Establishing Kaede protein stability in embryos

Generation of the conversion plot and the conversion rate plot

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  18 of 23

Kaede stability - the fluorescence intensity curve

Quantification of green Sox4+ cells and statistical analyses

Quantification of Kaede+ cells and determination of colors

Inhibition of cell division

Sample size of experiments

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  19 of 23

National Science NSF. IOS 1557431 Veronica Hinman

National Institute of NIH 1R24OD023046 Veronica Hinman

DSF Charitable Foundation Veronica Hinman

Decision letter and Author response

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  20 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://​doi.​org/​10.​7554/​eLife.​72983  21 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 1 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 2 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 3 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 4 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 5 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 6 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 7 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 8 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 9 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 10 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 11 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 12 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 13 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 14 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 15 of 23

Click-iT Plus EdU Cell Proliferation Kit for

Sheep polyclonal anti-digoxigenin AP- 11093274910,

Sheep polyclonal anti-digoxigenin POD- 1120773391,

Goat anti-rabbit polyclonal A11008,

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 16 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 17 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 18 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 19 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 20 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 21 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 22 of 23

Zheng et al. eLife 2022;11:e72983. DOI: https://doi.org/10.7554/eLife.72983 23 of 23