Model systems for regeneration: Planarians

Abstract

Planarians are a group of flatworms. Some planarian species have remarkable regenerative abilities, which involve abundant pluripotent adult stem cells. This makes these worms a powerful model system for understanding the molecular and evolutionary underpinnings of regeneration. By providing a succinct overview of planarian taxonomy, anatomy, available tools and the molecular orchestration of regeneration, this Primer aims to showcase both the unique assets and the questions that can be addressed with this model system.

Full text

PRIMER
Model systems for regeneration: planarians
Mario Ivankovic
1,
*, Radmila Haneckova
1,2,
*, Albert Thommen
1,3,
*, Markus A. Grohme
1,
*, Miquel Vila-Farre
1,2,
*,
Steffen Werner
4,
* and Jochen C. Rink
1,2,‡
ABSTRACT
Planarians are a group of flatworms. Some planarian species have
remarkable regenerative abilities, which involve abundant pluripotent
adult stem cells. This makes these worms a powerful model system
for understanding the molecular and evolutionary underpinnings of
regeneration. By providing a succinct overview of planarian
taxonomy, anatomy, available tools and the molecular orchestration
of regeneration, this Primer aims to showcase both the unique assets
and the questions that can be addressed with this model system.
KEY WORDS: Flatworms, Planarians, Tricladida, Triploblastic animals
Introduction
Planarians have long been known to possess astonishing
regenerative capabilities. As succinctly stated by John Graham
Dalyell in 1814, planarians ‘…may almost be called immortal under
the edge of the knife’(Dalyell, 1814). For example, if a planarian
worm is chopped into three pieces (Fig. 1A), each of the pieces
regenerates back into a complete and perfectly proportioned animal
within ∼2 weeks. In case of the tail (bottom) piece, this entails de
novo formation of a head complete with brain, eyes and functional
neuronal connections to the pre-existing tissue. Likewise,
regeneration of the head (top) piece necessitates the de novo
specification and formation of the trunk and tail. The central trunk
(middle) piece needs to regenerateboth a head and a tail; the fact that
these always form at the front and rear of the piece, respectively,
indicates that the regeneration process is primed by the polarity of
pre-existing tissues.
Planarians are similarly capable of regenerating tissue along their
medio-lateral (M-L) axis. Worms chopped along the midline
regenerate the missing half of all paired organs (Fig. 1B) and
even thin, lateral slices that have to form the midline de novo are
capable of restoring bilateral symmetry (Fig. 1C). Furthermore,
planarians can restore perfectly proportioned animals from
challenges such as oblique cuts (Fig. 1D), triangular deletions
(Fig. 1E) or cut-out ‘windows’(Fig. 1F). Regeneration also works
over a wide range of sizes. Over 100 years ago, T. H. Morgan
reported the regeneration of a piece that he estimated to correspond
to 1/279th of the donor animal (Morgan, 1898) and later studies
placed the lower size limit at <10,000 cells (Montgomery and
Coward, 1974). However, even though the planarian regeneration
response is extremely robust, the underlying control mechanisms
can be tricked into making ‘mistakes’, as illustrated for example by
the double-headed or double-tailed ‘monsters’that often result from
anteriorlyor posteriorly split animals (Randolph, 1897). Furthermore,
even the ‘almost-immortal’planarians have regenerative ‘weak
spots’: in the model species chosen precisely for their regenerative
powers, the tip of the head in front of the eyes and the pharynx are
incapable of regeneration(Reddien and Sánchez Alvarado, 2004) and
these tissue pieces consequently die if severed from the rest of the
animal. Others amongst the many hundreds of planarian species
worldwide (see below) have anatomically restricted regenerative
abilities (e.g. no head regeneration in the posterior body half) or
seemingly no regeneration at all (Brøndsted, 1969; Vila-Farré and
Rink, 2018).
Overall, planarians therefore offer unusually broad experimental
access to the many unknowns of regeneration. Species with robust
and rapid whole-body regeneration provide a model system for
studying universal aspects of the regeneration response, for example
the mechanisms that signal injury and how they lead to the re-
formation of specific organs or body parts. In addition, the
comparative analyses of species with poor or absent regeneration
provide an opportunity to understand the mechanistic causes of
regeneration defects and likely also the evolutionary dimension of
regeneration, i.e. why some worms regenerate whereas others
cannot. This Primer aims to provide an overview of planarians as a
model system for studying regeneration. We start with a brief
overview of planarian phylogeny, biodiversity and anatomy and of
the currently available tools and techniques. We then discuss the
current knowledge regarding planarian regeneration and its relation
to steady-state tissue dynamics. The Primer ends with a subjective
outlook on how the study of planarians could help address broader
questions about regenerative mechanisms and associated problems.
Planarians as model systems for studying regeneration
Planarian phylogeny and biodiversity
Planarians are a group of worms with a flattened body architecture
that belongs to the aptly named phylum Platyhelminthes ( platy=flat;
Model systems for regeneration
This article is part of a series entitled ‘Model systems for regeneration’.
This series of articles aims to highlight key model systems and species
that are currently being used to study tissue and organ regeneration.
Each article provides background information about the phylogenetic
position of the species, its life-cycle and habitat, the different organs and
tissues that regenerate, and the experimental tools and techniques that
are available for studying these organisms in a regenerative context.
Importantly, these articles also give examples of how the study of these
models has increased our understanding of regenerative mechanisms
more broadly, and how some of the open questions in the field of
regeneration may be answered using these organisms. To see the full
collection as it grows, please visit: https://dev.biologists.org/collection/
regeneration_models
1
Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse
108, 01307 Dresden, Germany.
2
Department of Tissue Dynamics and
Regeneration, Max Planck Institute for Biophysical Chemistry, am Fassberg 11,
37077 Go
ttingen, Germany.
3
The Francis Crick Institute, 1 Midland Road, London
NW1 1AT, UK.
4
FOM Institute AMOLF, Department of Systems Biology, Science
Park 104, 1098 XG, Amsterdam, The Netherlands.
*These authors contributed equally to this work
‡
Author for correspondence ( jochen.rink@mpibpc.mpg.de)
J.C.R., 0000-0001-6381-6742
1
© 2019. Published by The Company of Biologists Ltd
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helminthes=worms), within which they form a distinct evolutionary
clade, the order Tricladida (Fig. 2) (Sluys and Riutort, 2018). The
Platyhelminthes in turn group within the superphylum Lophotrochozoa/
Spiralia, along with, for example, leeches, earthworms and snails. The
roundworm Caenorhabditis elegans and the fruit fly Drosophila
melanogaster are only distantly related as they both group within the
Ecdysozoa.
Hundreds (if not thousands) of planarian species exist worldwide in
marine, freshwater or terrestrial habitats and are spread across the three
taxonomic suborders: Maricola, Cavernicola and Continenticola
(Sluys and Riutort, 2018; Vila-Farré and Rink, 2018). The
regenerative abilities of planarian species vary greatly, from robust
whole-body regeneration, as seen in Schmidtea mediterranea or
Dugesia japonica, to anatomically limited regenerative abilities (e.g.
no head regeneration in the posterior body half), as seen in
Dendrocoelum lacteum, or even the reported near-absence of
regeneration in Bdelloura candida and other marine planarians
(Brøndsted, 1969; Vila-Farré and Rink, 2018). S. mediterranea and
D. japonica have been developed into model species precisely for their
robust and rapid whole-body regeneration (Newmark and Sánchez
Alvarado, 2002; Rink, 2018; Saló and Agata, 2012) and the two species
have consequently enjoyed most of the scientific limelight so far.
Anatomy and physiology
In contrast to other flatworm clades, such as tapeworms or flukes,
planarians are non-parasitic. They are triploblastic animals with a
complex internal anatomy (Fig. 3). Organ systems include a true
brain connected to ventral nerve cords and simple eye cups, which
give planarians their characteristic cross-eyed appearance (Cebrià,
2007; Umesono and Agata, 2009). Planarians also possess a highly
branched intestinal system comprising three major branches
(Forsthoefel et al., 2011), which gave rise to the clade designation
Tricladida (tri=three; cladida=branches) (Sluys and Riutort, 2018),
and a protonephridial excretory system with interesting homologies
to the vertebrate kidney (Thi-Kim Vu et al., 2015). Planarians feed
via a muscular pharynx that they extrude through a ventral mouth
opening. The pharynx is the only body opening and also functions
as the anus of the animal. Circulatory and respiratory systems are
absent (Sluys and Riutort, 2018). Neoblasts –the adult stem cells of
planarians (Baguñà, 2012) –reside in the mesenchyme that
surrounds all internal organs. The three-layered body wall
musculature in turn surrounds the mesenchyme like a shell and
provides both mechanical stability and patterning information to the
cells below (Scimone et al., 2017; Witchley et al., 2013).
Planarians generally harbour a hermaphroditic reproductive
system, comprising a pair of ovaries located behind the brain,
testes and yolk glands along the entire anterior-posterior (A-P) axis,
and the copulatory organs in the tail (Newmark et al., 2008; Sluys
and Riutort, 2018). However, asexual reproduction by
parthenogenesis or fission/regeneration is also common amongst
planarians (Vila-Farré and Rink, 2018). Strains that rely on asexual
reproduction by fission often have poorly developed reproductive
0 dpa 3 dpa 14 dpa
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Fig. 1. Planarian regeneration.(A) Images depicting the regeneration of head (top), trunk (middle) and tail (bottom) fragments obtained via amputation of an intact
Schmidtea mediterraneaspecimen (left; red lines indicate approximatecutting planes). Time points in days post-amputation (dpa) are indicated.(B-F) Regeneration
of tissue fragments following amputation/injury as depicted in the cartoons (grey indicates regenerating piece; red lines indicate the cutting plane) at the
indicated dpa. Although intermediate time points are shown for D and E, all fragments eventually regenerate normal body plan proportions.
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organs, as is the case in the clonal laboratory strains of the model
species S. mediterranea and D. japonica (Pongratz et al., 2003;
Vila-Farré and Rink, 2018). Planarian body sizes vary from less
than a millimetre in length to more than one metre in the case of
Bipalium nobile (Kawakatsu et al., 1982). In addition, body shape
and coloration display strong inter-species variation, as do the
number and anatomical placement of eyes or other organ systems
(Sluys and Riutort, 2018).
Experimental accessibility and tools
Planarians are generally cheap and easy to maintain in the laboratory
(Merryman et al., 2018) and a broad and rapidly expanding cell and
molecular biology tool kit is now available. The organism-wide
knockdown of gene function by RNA interference (RNAi) (Rouhana
et al., 2013; Sánchez Alvarado and Newmark, 1999) remains the
workhorse in the field. This technique is often used in combination
with robust in situ hybridization and (immuno)histological staining
protocols (Adell et al., 2018; Forsthoefel et al., 2018; King and
Newmark, 2018; Solana, 2018; Umesono et al., 1997; Winsor and
Sluys, 2018). Simple fluorescence-activated cell sorting protocols
and the identification of specific surface labels allow the isolation and
characterization of neoblasts and other cell types (Hayashi and Agata,
2018; Hayashi et al., 2006; Moritz et al., 2012; Zeng et al., 2018).
Whole-body or regionalized irradiation protocols provide a
convenient means of stem cell ablation (Abnave et al., 2017;
Bardeen and Baetjer, 1904; Guedelhoefer and Sánchez Alvarado,
2012), and single-cell and tissue transplantation protocols permit the
investigation of differentiation potential (Rojo-Laguna and Saló,
2018; Wagner et al., 2011; Wang et al., 2018).
The recent embracement of next-generation sequencing by the
planarian research community has generated transcriptome
assemblies of model and ‘wild’planarian species (Adamidi et al.,
2011; Blythe et al., 2010; Cantarel et al., 2008; Liu et al., 2013;
Nishimura et al., 2012; Sandmann et al., 2011; Sikes and Newmark,
2013). This has been complemented by rich documentation of the
effects of individual gene knockdowns (Lin and Pearson, 2014;
Reuter et al., 2015; Tu et al., 2015; van Wolfswinkel et al., 2014),
and by gene expression time series during regeneration (Kao et al.,
2013; Stückemann et al., 2017; Wurtzel et al., 2015). More recently,
organism-wide single-cell sequencing atlases have been developed
(Fincher et al., 2018; Plass et al., 2018). Likewise, the very recent
completion of a high-quality S. mediterranea genome assembly
(Grohme et al., 2018) and a D. japonica genome assembly (An
et al., 2018) have made the genome sequences of the two model
species accessible, for instance, to chromatin immunoprecipitation
(ChIP) protocols that can provide insights into gene regulatory
mechanisms (Dattani et al., 2018; Duncan et al., 2015; Zeng et al.,
2013). A number of community resources provide access to these
data and allow the online querying of planarian biology
(see Box 1).
Remaining community challenges include the establishment of
robust transgenesis protocols, further improvement of live-imaging
strategies (Boothe et al., 2017; Shen et al., 2018), and the broad
general adaptation of the tool kit to the non-model species.
Nevertheless, planarians now provide a bona fide model system for
understanding the mechanistic basis of regeneration.
Mechanisms of regeneration in planarians
Neoblasts: the lynchpin of planarian biology
The regenerative powers of planarians derive largely from an
abundant population of unusual adult stem cells, the neoblasts.
Neoblasts are relatively small, round cells (7-12 µm in diameter)
with a high nuclear-cytoplasmic volume ratio that are distributed
throughout the planarian mesenchyme (Fig. 4A). They often
possess filopodia-like extensions and, interestingly, harbour
prominent RNA/protein granules (chromatoid bodies) with
morphological and molecular similarities to the RNA/protein
granules found in the germ cells of many animals (Agata et al.,
2006; Baguñà, 2012; Reddien and Sánchez Alvarado, 2004; Rink,
2018; Tanaka and Reddien, 2011). Neoblast-specific genes include
those encoding conserved chromatoid body components (Rouhana
et al., 2010, 2012, 2014), but also other conserved germ line genes
including homologues of piwi. piwi-1 expression further continues
to be used as generic neoblast marker (Guo et al., 2006; Reddien
et al., 2005b; Shibata et al., 2016).
Neoblasts are unusually abundant in comparison with adult stem
cells in other animals and have been estimated to account for as
much as 20-30% of all cells (Baguñà and Romero, 1981).
Remarkably, the transplantation of a single neoblast into a stem
cell-depleted host is sufficient to restore a complete animal via the
gradual replacement of all host cells by descendants of the
transplanted neoblast (Wagner et al., 2011). This experiment
demonstrates conclusively that the neoblast fraction contains stem
cells (termed clonogenic or cNeoblasts) capable of giving rise to all
adult cell types, which is the functional definition of pluripotency.
Although this abundance of pluripotent stem cells in adult tissues
Tricladida
Cnidaria
Xenoacelomorpha
Cephalocordata
Vertebrata
Echinodermata
Nematoda
Artropoda
Annelida
Platyhelminthes
Hydra
Human
Sea urchin
C. elegans
Drosophila
Tricladida
Hofstenia
Macrostomum
DeuterostomiaEcdysozoa
Spiralia
Catenulida
Macrostomorpha
Polycladida
Rhabdocoela
Cestoda
Digenea
Taenia solium
Schistosoma
mansoni
PlatyhelminthesMetazoans
Leeches
Earthworms
Mollusca
Snail
Amphioxus
Fig. 2. Planarian phylogeny. Left: Simplified phylogenetic relationship
between flatworms (phylum Platyhelminthes) and major taxonomic groups
within the Metazoa. Boxes highlight major clades. Red text denotes well-
known (model) species representatives of specific groups. Right: Simplified
phylogenetic relationship between planarians (order Tricladida) and other
major taxonomic groups within the flatworms. Figure based on Martı

n-Duran
et al. (2018) and Egger et al. (2015).
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may seem highly exotic by comparison with more familiar stem cell
systems, the comparatively recent discovery of piwi-expressing
(likely) somatic stem cells in a broad range of animals (Lai and
Aboobaker, 2018) indicates that planarian neoblasts may not be so
exotic after all, and that they may represent one end of a continuum
of stem cell architectures in animal phylogeny (Rink, 2018). The
mechanistic basis of neoblast pluripotency and of its indefinite
maintenance in adult planarians therefore poses a fascinating
question for future research, also from a comparative point of view.
Neoblasts are not only a source of all adult cell types, but they are
in fact the only source of new cells in planarians (Baguñà, 2012;
Rink, 2018; Tanaka and Reddien, 2011). This stems from the fact
that neoblasts are the only somatic cells that are division-competent
(Forsthoefel et al., 2011; Newmark and Sánchez Alvarado, 2000;
Reddien et al., 2005b). Not surprisingly, therefore, neoblasts are
essential for regeneration, and the depletion of neoblasts by
irradiation completely blocks regeneration. Moreover, the tip of
the head and pharynx, which are the only ‘naturally’neoblast-
devoid tissues, are the only body parts incapable of regeneration
(reviewed by Baguñà, 2012).
Any wounding event, even the prick of a needle, activates
neoblast divisions (Baguñà, 1976a; Wenemoser and Reddien,
2010). Wounds involving tissue removal attract the neoblast
progeny by an unknown mechanism and the consequent
accumulation of postmitotic neoblast progeny underneath the
freshly sealed wound gives rise to a blastema –a mass of
differentiating cells in the process of tissue formation. The blastema
first becomes apparent as a thin rim of unpigmented tissue at ∼24 h
post wounding and continues to grow due to high levels of local
neoblast proliferation at its base (Baguñà, 1976b; Newmark and
Sánchez Alvarado, 2000; Wenemoser and Reddien, 2010).
Bromodeoxyuridine pulse-chase experiments have demonstrated
that the blastema is largely composed of the post-mitotic progeny of
wound-induced neoblast divisions (Eisenhoffer et al., 2008),
34 5 6
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Fig. 3. Planarian anatomy. Schematic (top) and microscopic images (bottom) of the major planarian organ systems. (1) Brain (red, Smed-pc2 in situ
hybridization), CNS and pharynx (both green, α-tubulin immunostaining). (2) Intestine (red, Smed-porcupine-A; green, Smed-sufu in situ hybridization). Nuclear
counterstaining (blue, DAPI) reveals the silhouette of the specimen. (3) Protonephridia. Depth-coded confocal maximum projection showing individual
protonephridial units (acetylated-tubulin immunostaining). (4) Pharynx (red, phalloidin staining of muscle actin; green, acetylated-tubulin immunostaining of
cilia; blue, nuclear counterstaining). (5) Neoblasts (red, confocal maximum projection of piwi-1in situ hybridization in the tail area). (6) Body wall musculature
(depth-coded confocal maximum projection of 6G10 immunostaining; for antibody details, see Ross et al., 2015).
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suggesting that the trans-differentiation of existing cells contributes
little, if any, to planarian regeneration (reviewed by Baguñà,
2012). As regenerating pieces often cannot eat until the completion
of regeneration, the blastema cannot possibly rebuild all missing
tissues in their original size. Rather, the blastema appears to
restore the ends of the cardinal body axes (e.g. the ‘head’or ‘tail’
and ‘body edge’), and subsequent restoration of the adjacent
anatomy is accomplished via the dynamic re-modelling of existing
tissues (Agata et al., 2007). In terms of Morgan’s classic
terminology (Morgan, 1901), planarian regeneration therefore
combines epimorphic aspects in the form of de novo tissue
formation until ∼day 5 of regeneration, with the morphallactic
remodelling of existing tissues occurring during the subsequent
∼9 days. However, neoblast divisions are likely instrumental in
both, which limits the utility of Morgan’s terminology with respect
to planarian biology.
Neoblasts are also crucial for the maintenance of planarian
anatomy in the absence of wounding. Continual neoblast divisions
and their resulting progeny continuously replace all differentiated
cell types. The importance of continuous cell turnover is
underscored by the stereotypic deterioration of irradiated (i.e.
neoblast-depleted) animals, which first lose their head, followed by
ventral curling and eventual lysis of the remaining tissues∼30 days
after irradiation (Bardeen and Baetjer, 1904; Reddien et al., 2005a;
Wolff and Dubois, 1948). Moreover, the basal division rate of
neoblasts increases strongly after every meal (Baguñà, 1976a;
Newmark and Sánchez Alvarado, 2000) and the resulting burst of
postmitotic progenitors translates into a growth burst at the
organismal level. Neoblast divisions continue under starvation,
but the basal division rate is insufficient to replace all cells and the
animals consequently shrink owing to a net loss of cells (Baguñà,
1976a; Baguñà and Romero, 1981; González-Estévez et al., 2012;
Thommen et al., 2019).
The pivotal importance of neoblasts as the sole source of new
cells for regeneration and homeostatic tissue dynamics raises the
problem of how to orchestrate the orderly differentiation of all adult
cell types from a single pluripotent stem cell population (Rink,
2018). In contrast to vertebrate adult stem cells, which supply
progeny to comparatively few tissue-specific cell lineages, every
single cNeoblast can give rise to potentially hundreds of adult cell
types that its division progeny must consequently ‘choose’amongst
(Fig. 4B,C). Several recent studies detailing gene expression in
individual neoblasts have provided some glimpses into how this
might occur. Collectively, they demonstrate the onset of lineage
specification within piwi-1-positive and irradiation-sensitive
‘neoblasts’(Hayashi et al., 2010; Molinaro and Pearson, 2016;
van Wolfswinkel et al., 2014; Wurtzel et al., 2015), which therefore
constitute a heterogeneous cell population. The selective expression
of a number of genes, often transcription factors with evolutionarily
conserved roles in lineage specification, define a number of piwi-1-
expressing neoblast subclasses (Fincher et al., 2018; Molinaro
and Pearson, 2016; Plass et al., 2018; van Wolfswinkel et al., 2014;
Wurtzel et al., 2015; Zeng et al., 2018). Moreover, recent results
have suggested that the operationally defined pluripotent cNeoblasts
(Wagner et al., 2011) –the nexus of all planarian cell lineages –fall
within a subclass marked by the cell surface protein Tetraspanin
(Zeng et al., 2018). Planarian cell fate specification is likely,
therefore, a hierarchical process that involves the initial
‘differentiation’of cNeoblasts into a comparatively small number
of lineage-restricted neoblast subclasses (Fincher et al., 2018;
Molinaro and Pearson, 2016; Plass et al., 2018; van Wolfswinkel
et al., 2014), and terminal cell fates emerge during post-mitotic
progenitor differentiation in concert with the migration of these
progenitors to their target organs (Eisenhoffer et al., 2008; Lapan
and Reddien, 2011; Tu et al., 2015; Wurtzel et al., 2017). Important
open questions include the precise differentiation potential of
specific neoblast subclasses, and the extent to which they function
analogously to the transit-amplifying stages observed in vertebrate
stem cell lineages. But above all, there is the question of how to
generate the right cells at the right time and place, or, more
specifically, how to guide differentiating progenitors through the
maze of the planarian cell lineage tree.
Maintenance of the planarian body plan
Neoblasts situated in the tail or in a tail blastema do not make eye
progenitors (LoCascio et al., 2017), even though they are equally
pluripotent as elsewhere in the animal. This suggests the existence
of patterning processes that instruct location-specific cell fate
choices, analogous to positional information during development.
Indeed, the experimental perturbation of conserved signalling
pathways results in dramatic body plan transformations. Inhibition
of canonical Wnt signalling, for example, causes the appearance of
eyes in the tail by transforming the existing tail into a head, or by re-
programming tail blastemas into head development in regenerating
animals (Fig. 5A, top) (Gurley et al., 2008; Iglesias et al., 2008;
Petersen and Reddien, 2008). Activation of Wnt signalling causes
the opposite phenotype, namely loss of the head and all anterior
structures and transformation of the entire animal into a mass of tail
tissue, or the re-programming of head blastemas into tails in
regenerating animals (Fig. 5A, bottom) (Gurley et al., 2008; Iglesias
et al., 2011; Stückemann et al., 2017). Interference with the BMP
signalling pathway has similarly dramatic consequences, causing
ventralization of animals inclusive of the duplication of the entire
(ventral) nervous system (Gaviño and Reddien, 2011; Molina et al.,
2011; Reddien et al., 2007). Collectively, these phenotypes identify
BMP signalling and canonical Wnt signalling as determinants of
dorsal or posterior tissue identity, respectively.
Consistent with these phenotypes, the respective signals are
constitutively expressed in homeostatic animals. For example,
Smed-bmp4 (which encodes a planarian BMP4 homologue), is
expressed dorsally in a medio-laterally graded manner (Orii et al.,
Box 1. Online communities and further resources
PlanMine (http://planmine.mpi-cbg.de; Brandl et al., 2016; Rozanski
et al., 2019). Queryable repository of flatworm sequence data, provides
interactive tools for functional and comparative gene/transcript analyses.
Has an associated UCSC-based genome browser instance.
SmedGD (http://smedgd.stowers.org; Robb et al., 2015; Robb et al.,
2008). Queryable database for S. mediterranea sequence data.
Planosphere (https://planosphere.stowers.org; Davies et al., 2017).
Interactive access to S. mediterranea cell and developmental biology
datasets.
Genome browser of D. japonica (http://www.planarian.jp/; An et al.,
2018).
Planaria SCS 2015 (https://radiant.wi.mit.edu/app/; Wurtzel et al., 2015).
Single-cell sequencing data of flow-sorted planarian cells.
Planarian digiworm (https://digiworm.wi.mit.edu/; Fincher et al., 2018).
Large-scale single-cell transcriptomic resource for S. mediterranea.
Planaria SC Atlas (https://shiny.mdc-berlin.de/psca/; Plass et al., 2018).
Large-scale single-cell transcriptomic resource for S. mediterranea,
including cellular lineage trees.
Developmental Studies Hybridoma Bank monoclonal antibodies
(http://dshb.biology.uiowa.edu/; Forsthoefel et al., 2014; Ross et al.,
2015). Collection of monoclonal antibodies with validated reactivity
towards planarian epitopes.
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1998; Reddien et al., 2007). In addition, many canonical Wnt
signalling pathway components are expressed in tail-to-head
gradients (Gurley et al., 2010; Petersen and Reddien, 2009;
Reuter et al., 2015; Stückemann et al., 2017; Sureda-Gómez et al.,
2015), which result in a corresponding gradient of canonical Wnt
signalling activity in the tissue (Stückemann et al., 2017). The tail-
deployed Wnt gradient further appears to functionally oppose
independent signalling gradients emanating from the tip of the head
(Stückemann et al., 2017). Although multiple homologues of the
vertebrate FGF pseudoreceptor FGFRL1 (termed nou-darake in
planarians) are involved (Cebrià et al., 2002; Lander and Petersen,
2016; Scimone et al., 2016), multiple aspects of planarian head
identity specification remain to be clarified. Overall, the above
phenotypes and expression patterns now amount to a rudimentary
coordinate system of the planarian body plan to specify positional
information along the A-P, dorso-ventral (D-V) and M-L axes
(Fig. 5B). Moreover, the identity of the primary patterning signals,
together with the prominent theme of signalling gradients in their
deployment, raise many intriguing parallels to embryonic axis
establishment.
Practically all of the aforementioned signals are expressed in the
multi-layered sheet of muscle fibres that lies beneath the planarian
epithelium (see Fig. 3) (Scimone et al., 2017; Witchley et al., 2013).
Importantly, existing muscle fibres can rapidly and dynamically
change the complement of patterning molecules they express; for
example, swapping tail for head gradient genes in the case of tail
piece regeneration (Witchley et al., 2013). A further important
theme that is currently emerging is the functional specialization of
muscle fibre subtypes with regard to the patterning signals they
express (Scimone et al., 2017). A subclass at the tip of the head and
tail, the so-called pole cells, have been strongly implicated in head
and tail fate specification and likely initiate the expression gradients
of the tail and head signals within the body musculature (Blassberg
et al., 2013; Chen et al., 2013; Oderberg et al., 2017; Reuter et al.,
2015; Vogg et al., 2014). Furthermore, the recent discovery that
specific layers of the body wall musculature express axis-specific
signals is intriguing in light of the stereotypic arrangement of the
fibres along the A-P and M-L axes (Scimone et al., 2017). Although
the actual tissue distribution of the muscle-expressed BMP and Wnt
ligands has not yet been determined, their dramatic influence on
planarian anatomy and cell fate choices strongly suggests that at least
some of them can permeate the neoblast-containing mesenchyme
(Witchley et al., 2013). Hence, a conceptual framework is beginning
to emerge whereby the expression patterns of patterning signals in the
body wall musculature translate into location-specific signalling
environments in the mesenchyme that ultimately mediate location-
specific fate choices of neoblast progeny (Reddien, 2018; reviewed
by Rink, 2018).
The mechanisms and principles by which patterning signals
influence neoblast fate choices are currently an important focus in the
field. In the case of BMP4-mediated D-V patterning, the presumptive
BMP signalling gradient has been shown to cause D-V gene
B Planarians
A
????
Adulthood
Adulthood Development
Potency
Potency
C Vertebrates
piwi-1/nuclei
Fig. 4. The planarian stem cell system. (A) Whole-mount in situ hybridization highlighting the localization of neoblasts, which are visualized using the generic
neoblast marker piwi-1(smedwi-1;yellow), counterstained with DAPI (blue) to highlight nuclei. Scale bar: 500 μm. (B,C) Schematicscontrasting key organizational
features of the planarian and vertebrate stem cell systems. In adult planarians (B), the indefinitely self-renewing and pluripotent cNeoblasts (grey cells, top) likely
give rise to lineage-committed progenitors of currently unknown self-renewal potential (middle) that terminally differentiate into various postmitotic cell types
(bottom). In vertebrates (C), self-renewing pluripotent stem cells (grey cells, top) only ever occur transiently during early developmental stages; in this context,
multiple multipotent stem cells instead persist in adults (middle) and terminally differentiate into various, occasionally mitotically active, cell types (bottom).
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PRIMER Development (2019) 146, dev167684. doi:10.1242/dev.167684
DEVELOPMENT

expression zonation within neoblasts (Wurtzel et al., 2017). Together
with the observation of bmp4(RNAi)-induced ventralization of newly
differentiating neoblast progeny, rather than ‘reprogramming’of
terminally differentiated cells (Wurtzel et al., 2017), this suggests that
the position of a neoblast and the consequent combination of
patterning signals that it is exposed to restricts the lineage choices that
its progeny can make. In the case of A-P-patterning, Wnt signals
induce the expression of Wnt target genes in neoblasts, including
planarian Hox homologues and other transcription factors (Reuter
et al., 2015; Stückemann et al., 2017). In conjunction with the tail-to-
head Wnt signalling gradient, a neoblast situated in the high Wnt
environment of the tail consequently expresses a different
complement of transcription factors than do neoblasts located in the
low Wnt environment of the head (Reuter et al., 2015). Although the
underlying gene regulatory circuits and networks remain to be
elucidated, the conversion of a Wnt signalling gradient into
transcription factor expression gradients in stem cells provides a
working hypothesis that could help explain a key aspect of positional
information in planarians and, more generally, why neoblasts in the
tail do not make eye progenitors.
Regeneration of the planarian body plan
Given the importance of signalling gradients for the maintenance of
planarian steady-state anatomy, restoration of the signal patterns in
regenerating tissue pieces becomes a key prerequisite for successful
regeneration. An interesting observation in this respect is that trunk
fragments, or any other type of tissue fragments, always regenerate
the head and tail along the orientation of the original body axes (e.g.
Fig. 1A). This demonstrates that planarian tissues are intrinsically
polarized and that tissue polarity in turn instructs the direction of
regeneration. Although the mechanistic basis of planarian tissue
polarity remains unclear, the Wnt inhibitor notum has been
identified as a key polarity effector (Petersen and Reddien, 2011).
notum is selectively expressed at anterior-facing wounds within
∼3 h of amputation, and notum(RNAi) animals regenerate a tail
instead of a head, consistent with the necessity and sufficiency of
Wnt inhibition for head formation (as discussed above). The
anterior-specific expression of notum and many other aspects of the
early regeneration response do not require neoblasts (Gurley et al.,
2010; Wenemoser et al., 2012). However, neoblasts are instrumental
in pattern regeneration as they are necessary for pole regeneration.
Pole cells become specified in the wound vicinity and congregate at
the tip of the blastema by day 3 after amputation (Gurleyet al., 2010;
Hayashi et al., 2011; Oderberg et al., 2017; Scimone et al., 2014;
Vásquez-Doorman and Petersen, 2014; Vogg et al., 2014). In the
absence of pole formation, e.g. in pbx(RNAi) animals, small
blastemas form, but they fail to specify head or tail identity and also
do not acquire a midline (Blassberg et al., 2013; Chen et al., 2013;
Scimone et al., 2014). Thus, pole cell regeneration is likely an
essential aspect of pattern regeneration. The fact that the poles mark
the origin of the head and tail gradients, the distal-to-proximal
regeneration of the Wnt gradient out of the blastema (Stückemann
et al., 2017), and the ability of transplanted head tip cells to initiate
head outgrowth (Oderberg et al., 2017) are all consistent with a role
for pole cells as pattern initiators.
Overall, planarian regeneration can thus be envisaged as being
guided by similar conceptual principles as those that govern steady-
state turn-over (Reddien, 2018; reviewed by Rink, 2018). Polarity
cues acting as an additional aspect of tissue-resident positional
information generate a unique signalling environment at the wound
site that encodes wound type and orientation. The signalling
environment, in turn, gates critical lineage choices of arriving
neoblast progeny, including the tightly gated access to the pole cell
lineage and the likely canonical Wnt signalling-dependent choice
between head and tail pole formation. Interaction of pole cells with
the general body wall musculature then re-initiates gradient
formation and thus restores the ‘regeneration’of positional
information in the tissue piece.
Conceptual problems that can be addressed by studying
planarian regeneration
Given that Hydra, salamanders, zebrafish and a range of other
emerging models all vie for the ‘master of regeneration’title
(Galliot, 2012; Gemberling et al., 2013; Holstein et al., 2003;
Tanaka and Reddien, 2011), what is it that planarians can bring to
the table? First, the complete regeneration of a triploblastic body
plan from arbitrary tissue pieces within 2 weeks, and the availability
of a broad range of molecular tools, simply provide convenient
experimental access to the many remaining fundamental challenges
of regeneration –‘fundamental’because they pertain to a general
understanding of the phenomenon of regeneration, and ‘challenges’
because we are so far lacking a mechanistic understanding in any
model system. Second, the many quirks of planarian physiology
offer unique perspectives on a broad range of important problems in
the current biomedical research landscape. Below, we provide a
subjective selection of some such fundamental challenges.
B
A
Anterior-posterior axis Medio-lateral
axis
Dorso-ventral
axis
wnt5slit-1wnt5
wnt
Ndk
?
bmp4
admp
β-Cat1(RNAi)
APC(RNAi)
14 dpa
Regeneration
Intact
14 dpa
Fig. 5. Planarian patterning systems. (A) Morphological consequences of
experimentally perturbing Wnt signalling in intact worms (left) or in regenerating
trunk pieces (right). Inhibition of canonical Wnt signalling via Smed-β-catenin-
1(RNAi) forces head formation, triggering either tail-to-head conversion and the
appearance of multiple ectopic heads (marked by Smed-sfrp-1 via in situ
hybridization) in intact animals, or double-head formation in regenerating trunk
pieces. Activation of Wnt signalling by Smed-APC(RNAi) forces tail formation,
causing either loss of all anterior structures and global posteriorization in intact
animals, or double-tail formation during trunk piece regeneration. (B) Schematic
depicting currently known primary patterning signals and their deployment
along the indicated cardinal body axes. Note that the depicted signalling
gradients are largelyhypothetical extrapolations fromthe expression patterns of
the respective genes, as only the canonical Wnt signalling gradient has been
experimentally demonstrated so far (see text for details).
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DEVELOPMENT

Regeneration specificity: sensing what’s missing
The essence of regeneration is precise reformation of a tissue or
body part that has been damaged or lost. Irrespective of whether we
are considering limb regeneration in axolotls or whole-body
regeneration in planarians or Hydra, the restoration of ‘whole’
from random pieces necessitates a latent ability of tissues to sense
what’s missing, i.e. whether to initiate the regeneration of head, tail,
or lateral tissues in planarians. Although elements of the
mechanisms underlying regeneration specificity have now
emerged in many model systems (Tanaka, 2016; Vogg et al.,
2016; Wehner and Weidinger, 2015), it is probably fair to state that a
mechanistic understanding of ‘sensing what’s missing’has not been
achieved in any system. In this regard, planarians provide a
particularly powerful experimental paradigm, owing to their rapid
rate of regeneration and near-complete experimental freedom over
the shape, size or anatomical origin of the regenerating tissue
fragments examined. Particularly pertinent current questions
regarding planarian regeneration specificity include the cellular
and molecular basis of ‘tissue polarity’in the head/tail decision
(discussed above), or how the animals manage to restrict the general
regeneration response in the case of limited damage to specific
organs (e.g. the pharynx or the eyes; Adler et al., 2014; LoCascio
et al., 2017). Beyond planarians, permutations of the regeneration
specificity challenge include, for example, the injury site-dependent
formation of various limb elements during amphibian limb
regeneration or the position-dependent variation in regeneration
rate during zebrafish fin regeneration (Kujawski et al., 2014;
Tanaka, 2016). Thus, the general conceptual challenge is to
understand how remnants of positional information can encode
and restore the whole structure from arbitrary starting points.
Organization versus self-organization
In sharp contrast to embryogenesis, regeneration does not initiate
from a precisely defined environment (e.g. the fertilized zygote),
but instead is orchestrated from the entirely random remnants of
injuries. A priori, this rules out pre-positioned fate determinants
(e.g. bicoid or nanos in Drosophila zygotes) and, more generally,
the ‘traditional’textbook manifestation of the morphogen gradient
concept with the source as a pre-specified cell fate as organizing
principles. Self-organizing Turing or similar reaction-diffusion
patterns are conceptually very attractive in this sense, as they can
account both for spontaneous pattern emergence and pattern
regeneration (Gierer and Meinhardt, 1972; Turing, 1952). The
anatomical autonomy of the planarian tail gradient and Wnt-mediated
Wnt expression as one of the core mechanisms of planarian
regeneration (Stückemann et al., 2017) are indeed consistent with a
Turing mechanism, yet the instructive role of intrinsic tissue polarity
is suggestive of organized, rather than spontaneous, pattern
emergence. However, it is known that pharmacologically induced
ectopic heads permanently re-programme the polarity of adjacent
tissues, as revealed by subsequent amputations after drug wash-out
(Oviedo et al., 2010). Hence, even though tissuepolaritycan organize
pattern regeneration, pattern regeneration can also, in turn, organize
tissue polarity. At a systems level, regenerative patterning in
planarians is therefore self-organizing. The interplay between tissue
polarityand pattern establishment mirrors the concept of ‘guided self-
organization’in developmental systems and organoids, which
involves channelling the inherently random nature of self-organized
systems into predictable outcomes via the specification of boundary
conditions (Lancaster et al., 2017; Turner et al., 2016; Werner et al.,
2016). Understanding how self-organization can result in the highly
specific and reproducible outcome of planarian regeneration, yet
essentially random tissue architectures in the case of organoid
differentiation (Lancaster and Knoblich, 2014), therefore poses an
interesting challenge for the coming years.
Size and shape control
The fact that small or large planarian tissue pieces regenerate into
small or large planarians, or the observation that the size of a
regenerating newt new limb is matched to the size of the
regenerating animal (Tanaka and Reddien, 2011), represent
striking manifestations of a fundamental unresolved challenge:
understanding the mechanisms by which biological systems
specify, gauge and restore spatial dimensions. One component
problem of this challenge is to understand how pattern length scales
are matched to tissue size dimensions. Here, the restoration of
planarian body plan proportions from arbitrary starting points
provides a powerful experimental paradigm that can be used to, for
example, probe the mechanistic basis of downscaling the tail Wnt
gradient to the much shorter dimensions of the tail piece (Gurley
et al., 2010; Stückemann et al., 2017). The evident scaling of
planarian patterning gradients is intriguing because diffusion-based
patterning concepts typically imply fixed-length scales that arise
from physicochemical systems parameters (e.g. diffusion and
degradation constants) (Werner et al., 2016). How scalable
systems might achieve the necessary adjustment of reaction rates
to system size remains an important problem not only in
regeneration, but also in development (Aguilar-Hidalgo et al.,
2018; Ben-Zvi et al., 2011; Werner et al., 2015). Here, the
uncoupling of pattern scaling from tissue growth during the early
stages of planarian regeneration promises a uniquely specific model
system to elucidate the underlying mechanisms.
Planarians also offer an additional experimental approach to the
shape and size challenge because of their general lack of a fixed
body size. Planarians grow when fed and literally shrink when
starving due to dynamic and food supply-dependent adjustments of
total organismal cell numbers (Baguñà et al., 1990). In the case of
S. mediterranea, the momentary size of a single worm fluctuates
between ∼0.5 mm and 20 mm in body length or <10,000 to
8,000,000 cells (Thommen et al., 2019). Planarians further display
tremendous inter-species variations in body size, as illustrated by
the giant ‘shoe-sole-sized’planarians of Lake Baikal (Sluys et al.,
1998) or the meter-long land planarians of Japan (Kawakatsu et al.,
1982). Such a broad spectrum of inter- and intra-specific body size
variations provides a further powerful pattern-scaling paradigm, but
in addition it raises many further questions. For example, what
accounts for the recently demonstrated body size-dependent lipid
storage in planarians and the resulting near-universal ¾-law scaling
of metabolic rate with mass (Thommen et al., 2019)? How can
planarians establish centimetre-length signalling gradients, given
that free diffusion is an unlikely signal propagation mechanism at
such length scales? Or, what encodes the maximal body size of a
species in the genome? Overall, planarians thus clearly provide
unique experimental opportunities for probing the mechanistic basis
of size and shape.
The evolution of regeneration: why some animals can,
but others can’t
Finally, this leaves the big question of why Hydra, planarians,
axolotl and zebrafish can all regenerate lost body parts, but, for
example, humans cannot. In the face of ‘survival of the fittest’,
regeneration as the apparent exception rather than the rule seems
deeply counterintuitive. Various hidden ‘costs’of regeneration have
been proposed, such as increased cancer susceptibility,
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PRIMER Development (2019) 146, dev167684. doi:10.1242/dev.167684
DEVELOPMENT

compromised immune system function or the risk of developing
malformations (Alibardi, 2018; Egger, 2008; Godwin and
Rosenthal, 2014), but all evidence so far remains correlative. A
second related challenge is to understand whether the widespread
distribution of regeneration competence in certain phylogenies
reflects ancestral ‘core mechanisms’that were lost in various
lineages or whether regeneration competence evolved de novo in
multiple branches of animal phylogeny (Sánchez Alvarado, 2000;
Slack, 2017). The answer to this question is deeply relevant with
respect to regenerative medicine in humans, as the former
possibility promises a potential ‘unlocking’of latent regenerative
abilities, whereas the latter one would necessitate the conceptually
even more challenging de novo engineering of the trait.
The complete spectrum of regenerative abiliti es across the planarian
taxa, the demonstrated ability to cultivate many of these species in the
lab (Vila-Farré and Rink, 2018), and the existence of well-developed
model species make planarians a uniquelypowerful model systemthat
can be used to probe the evolutionary dynamics of regeneration. As
already demonstrated by the ‘rescue’of head regeneration in
regeneration-deficient planarian species (Liu et al., 2013; Sikes and
Newmark, 2013; Umesono et al., 2013), comparative approaches can
further provide opportunities to diagnose and understand the
mechanistic basis of regeneration defects. Given the rapidly
evolving toolkit in planarians, the identification of potential base
pair changes in enhancer elements or coding sequences as proximate
causes of regeneration defects is now becoming increasingly feasible.
Such a truly mechanistic understanding of regeneration defects could
lead to a better understanding of what it takes to regenerate, and thus,
eventually, to a systems-level understanding of regeneration.
Conclusions
Planarians have developed into a powerful model system for
studying the mechanistic basis of regeneration, and the comparative
analysis of regeneration-deficient planarian species is beginning to
provide access to the evolutionary dimension of the trait. In addition,
the adult pluripotent stem cells of planarians and their uniquely
dynamic tissue architecture expose multiple fascinating phenomena
to experimental scrutiny; for example, the self-organized assembly
of entire organs, the specification of size and shape, and the
maintenance of a dynamic steady state per se. Overall, the seemingly
quirky biology of planarians reminds us that the handful of more or
less haphazardly chosen ‘classical’model species cover but a
fraction of the fascinating complexity and diversity of biological
mechanisms and, consequently, that much remains to be discovered.
Acknowledgements
We thank all other members of the Rink lab for critical discussions and Hanh Thi-Kim
Vu and James Cleland additionally for the contribution of images. We thank our
reviewers for helpful comments.
Competing interests
The authors declare no competing or financial interests.
Funding
This project received funding from the European Research Council (ERC) under the
European Union’s Horizon 2020 research and innovation program( grant agreement
number 649024) and the Max Planck Society (Max-Planck-Gesellschaft). S.W. was
supported by the Netherlands Organisation for Scientific Research (Nederlandse
Organisatie voor Wetenschappelijk Onderzoek; NWO) viathe FOM program no. 161
(14NOISE07).
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