G Model

YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS

Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journal homepage: www.elsevier.com/locate/semcdb

Review

The role of the EGFR signaling pathway in stem cell differentiation

during planarian regeneration and homeostasis
Sara Barberán, Francesc Cebrià ∗
Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona and Institute of Biomedicine of the University of
Barcelona(IBUB), Spain

a r t i c l e i n f o a b s t r a c t

Article history: Cell signaling is essential for cells to adequately respond to their environment. One of the most
Received 12 February 2018 evolutionarily conserved signaling pathways is that of the epidermal growth factor receptor (EGFR).
Received in revised form 7 May 2018 Transmembrane receptors with intracellular tyrosine kinase activity are activated by the binding of their
Accepted 14 May 2018
corresponding ligands. This in turn activates a wide variety of intracellular cascades and induces the up-
Available online xxx
or downregulation of target genes, leading to a specific cellular response. Freshwater planarians are an
excellent model in which to study the role of cell signaling in the context of stem-cell based regeneration.
Keywords:
Owing to the presence of a population of pluripotent stem cells called neoblasts, these animals can regen-
Planarian
Epidermal growth factor receptor erate the entire organism from a tiny piece of the body. Here, we review the current state of knowledge
Signaling of the planarian EGFR pathway. We describe the main components of the pathway and their functions
Regeneration in other animals, and focus in particular on receptors and ligands identified in the planarian Schmidtea
Stem cells mediterranea. Moreover, we summarize current data on the function of some of these components dur-
Cell differentiation ing planarian regeneration and homeostasis. We hypothesize that the EGFR pathway may act as a key
Schmidtea mediterranea regulator of the terminal differentiation of distinct populations of lineage-committed progenitors.
© 2018 Elsevier Ltd. All rights reserved.

Contents

1. Cell signaling in planarians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2. The EGFR signaling pathway: receptors and ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. The EGFR pathway in planarians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. EGF receptors and ligands in S. mediterranea: structure and expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. The role of EGFR signaling in development and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Role of the EGFR pathway during planarian regeneration and homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.1. Smed-egfr-3/Smed-nrg-7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.2. Smed-egfr-5/Smed-egf-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6.3. Smed-egfr-1/Smed-nrg-7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7. EGFR signaling in planarians: a general regulator of neoblast differentiation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Cell signaling in planarians

To better understand a variety of cellular processes, including

∗ Corresponding author at: Department of Genetics, Microbiology and Statis-
proliferation, differentiation, and migration, it is essential to deci-
tics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona,
pher the mechanisms by which single-cell organisms, as well as
Catalunya, Spain. individual cells of complex multicellular animals, respond to phys-
E-mail address: fcebrias@ub.edu (F. Cebrià). ical, chemical, and molecular signals within their environment. A

https://doi.org/10.1016/j.semcdb.2018.05.011
1084-9521/© 2018 Elsevier Ltd. All rights reserved.

Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
2 S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx
small number of signaling pathways are conserved in most animals,
and play pivotal roles during development. These include the Wnt,
TGF␤/BMP, Notch, Hedgehog, JAK/STAT, and RTK (receptor tyro-
sine kinase) pathways. Most of the literature on these pathways
relates to their roles in cell behavior, patterning, and morphogen-
esis during embryogenesis in a variety of animal models [1–4].
However, as recently reported, these pathways have been function-
ally conserved to regulate cell responses in other postembryonic
developmental contexts such as homeostasis and regeneration
[5,6].
Signaling pathways are activated by the binding of a ligand to
a transmembrane receptor. This binding usually triggers an intra-
cellular cascade that influences the expression of a particular set of
genes, the activation or repression of which translates into a spe-
cific cellular response. Remarkably, despite the great variety of cell
types, tissues, and morphologies throughout the animal kingdom,
these few signaling pathways are implicated in numerous cellular
contexts and developmental stages. This suggests a high degree of
flexibility, which appears to be mediated by 5 key features [7]: 1) a
given receptor can activate different intracellular cascades depend-
ing on the tissue in which it is expressed; 2) different responses can
be generated depending on the specific kinetics of ligand-receptor
binding; 3) different signaling pathways can integrate to each other
to regulate the same process; 4) different cell types can express dis-
tinct transcription factors and therefore respond differently to the
activation of a particular signaling pathway; and 5) signal trans-
duction can be compartmentalized within the cell. In addition to
being flexible, signaling pathways must be also sufficiently robust
to ensure that the same response is always generated in a given
context. This robustness appears to be underpinned by the action
of positive and negative regulatory feedback [8].
Most of the aforementioned signaling pathways have been
conserved in planarians, in which they play key roles during
regeneration and homeostasis. Specification of dorsoventral and
anteroposterior polarity is mediated by the BMP pathway [9,10]
and by the Wnt and Hedgehog pathways [11–14], respectively,
while FGFR signaling is implicated in restricting the differentiation
of brain tissues in the head region [15]. EGFR (epidermal growth
factor receptor) signaling, another RTK pathway, plays a variety of
conserved roles throughout animal phylogenies, ranging from stem
cell proliferation and differentiation to cell adhesion and migra-
tion. Moreover, overactivation of the EGFR pathway is observed in
a large proportion of human cancers. Except for a study suggest-
ing a potential pro-mitotic role of EGF in planarian stem cells [16],
little was known until recently about the importance of this sig-
naling pathway during planarian regeneration. However, several
recent studies have shown that EGFR signaling is required for the
differentiation of different cell types [17–21]. Here, we provide a
general overview of this signaling pathway, based on descriptions
of their main components, receptors, and ligands in planarians and
other animal models. In addition, we review current knowledge of
their roles in planarian regeneration and homeostasis.
2. The EGFR signaling pathway: receptors and ligands
Epidermal growth factor receptors (EGFR) belong to subclass I
of the receptor tyrosine kinases (RTK) superfamily. All members
of this subclass share a characteristic structure consisting of an
extracellular domain bound via a transmembrane domain to an
intracellular domain with tyrosine kinase activity [22] (Fig. 1A).
The extracellular domain consists of 4 subdomains defined by their
ability to specifically bind their ligands or by their cysteine rich-
ness. The interaction between these subdomains induces a specific
protein folding essential for ligand binding [23,24]. The intracel-
lular region includes the tyrosine kinase domain as well as the
Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
phosphorylation sites [25]. In vertebrates this family consists of
4 members: ErbB1 (or HER1, EGFR), ErbB2 (or HER2, p185, neu),
ErbB3 (or HER3, p160), and ErbB4 (or HER4) [26]. All share the
structure described above, except for ErbB3, which has very little
tyrosine kinase activity [27,28] (Fig. 1B). Invertebrate model organ-
isms such as Drosophila melanogaster and Caenorhabditis elegans
have a single EGFR (DER and let-23, respectively) [29,30], and their
extracellular domain contains an additional cysteine-rich subdo-
main (Fig. 1B).
Epidermal growth factors (EGF) belong to a family characterized
by the presence of one or more repeats of a domain (EGF domain)
containing 6 conserved cysteine residues. The particular distribu-
tion of the cysteine residues has the potential to establish disulfide
bridges and induce protein folding to enable receptor binding
[31,32]. The EGF structure consists of an extracellular domain, in
which the EGF domain(s) are found, a hydrophobic transmembrane
domain, and a cytoplasmic domain (Fig. 2A). Some members of
this family express additional domains, such as immunoglobulin-,
neuregulin-, or heparin-binding domains. In vertebrates, many lig-
ands can bind specifically to different receptors [33,34]. By contrast,
only one single EGF ligand has been described in C. elegans [35] and
4 in D. melanogaster [29] (Fig. 2B). In other platyhelminthes, such as
Schistosoma mansoni and Echinococcus multilocularis, only one EGFR
has been described [36,37].
In many cases EGF ligands are processed so that they are
secreted as active forms that bind to their receptors, while in oth-
ers EGF ligands remain attached to the membrane and bind to their
receptors in adjacent cells. Upon binding to their ligands, receptors
form homo- or heterodimers and subsequently activate their tyro-
sine kinase domains [38]. The phosphorylated tyrosine residues
serve as binding sites for several signaling proteins involved in
signal transduction [39]. Many different intracellular cascades,
including phospholipase C gamma (PLC␥), JAK/STAT, MAPK/ERK,
and phosphatidylinositol 3 kinase (PI3K), can be activated by EGFR
signaling (see Fig. 3) and subsequently participate in the regulation
of biological processes such as cell proliferation, differentiation,
migration, adhesion, and angiogenesis.
3. The EGFR pathway in planarians
Recent extensive searches of available databases enabled the
identification of a large number of putative homologs of EGF recep-
tors and ligands in the planarian Schmidtea mediterranea [19,20],
adding to the list of previously described receptors and ligands
[17,18,40]. Because the EGF domain found in EGF ligands is a motif
present in a great diversity of proteins it has been difficult to
identify them in planarians. Taking a conservative approach and
searching for candidate genes being full length, with only one
EGF domain and with an structure similar of known EGF ligands,
Barberán and colleagues identified 9 putative EGF ligands in S.
mediterranea [20]. In this study, the orthology of the identified EGF
receptors and ligands was demonstrated by phylogenetic analy-
ses, in 19 different animal lineages (Fig. 4). Those authors proposed
that the last common ancestor of all metazoans had a single EGF
receptor and EGF-type ligand, and that neuregulin-type EGF lig-
ands are likely an innovation of the Bilateria. Moreover, their study
demonstrated that the EGFR pathway evolved via many indepen-
dent expansions of this original complement in multiple lineages
(Fig. 4) [20]. In this respect, the evolutionary history of the EGFR
pathway does not appear to differ significantly from that of other
conserved signaling pathways such as the Wnt, TGF-␤, FGFR, Notch,
and Hedgehog pathways [41,42].
Whereas Barberán and colleagues identified 9 putative EGF
ligands in S. mediterranea [20], Lei and coworkers described 12
putative EGF ligands [19], but provided no phylogenetic support
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx 3

Fig. 1. Structure of EGF receptors. (A) Typical domain structure of EGF receptors. (B) Receptor domain structures in Caenorhabditis elegans, Drosophila melanogaster, and
mammals.

Table 1 obtained from phylogenetic analyses it appears that a significant

Correspondence between the nomenclature used by two different studies to name
expansion of the main components of the EGFR pathway has
the several putative EGF ligands found in S. mediterranea.
occurred in S. mediterranea (Fig. 4). In the platyhelminthes Macros-
Barberán et al. [20] Lei et al. [19] tomum lignano, Prostheceraeus vittatus, Schistosoma mansoni, and
egf-1 ereg Echinochocus multilocularis, 8, 6, 3, and 3 receptors have been iden-
egf-2 tgf-˛ tified, respectively. Orthology analyses of these sequences indicate
egf-3 nrg-9 that these expansions occurred independently in each of these
egf-4 –
groups. The evolution of the ligands appears to have involved both
egf-5 –
egf-6 nrg-1 an expansion and a loss of elements: whereas 9 ligands have been
egf-7 nrg-2 identified in S. mediterranea and P. vittatus, only 1 neuregulin-type
egf-8 – ligand has been identified in S. mansoni and a single ligand of each
egf-9 –
type in M. lignano and E. multilocularis [20].
nrg-1 nrg-5
– nrg-3
– nrg-4
– nrg-6 4. EGF receptors and ligands in S. mediterranea: structure
– nrg-7 and expression
– nrg-8
– nrg-10 S. mediterranea has 6 EGFRs that exhibit the standard domain
architecture, although some contain 1 or 2 additional cysteine
domains. Smed-egfr-6 has a tyrosine kinase domain with reduced
for any of these ligands. As such, further analyses will be necessary activity (Fig. 5) [20]. All EGF ligands in this species for which strong
to determine the exact number of EGF ligands in S. mediterranea phylogenetic support exists contain the characteristic EGF domain,
and whether they are EGF- or neuregulin-like. Currently, the most and Smed-nrg-1 contains an additional immunoglobulin domain
reliable data suggest the existence of at least 8 EGF-type and 1 (Fig. 5) [20]. Analysis of their domains suggests that Smed-egf-
neuregulin-type ligands [20]. Table 1 shows the correspondence 6, Smed-egf-7, and Smed-nrg-1 are synthesized as soluble ligands
between the ligands found in these 2 studies. Based on the results whereas the other EGF ligands are expressed as transmembrane

Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
4 S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

Fig. 2. Structure of EGF ligands. (A) Typical domain structure of the 2 ligand subclasses: EGF-type and neuregulin-type ligands. (B) Ligand domain structures in Caenorhabditis
elegans, Drosophila melanogaster, and mammals.

Fig. 3. Main intracellular signaling cascades activated by the EGFR pathway.

precursors that are subsequently released by the action of proteases
(Fig. 5) [20]. In situ hybridization has demonstrated considerable
variation in the expression patterns observed for both receptors
(Fig. 6) and ligands (Fig. 7). These patterns have been largely corrob-
orated in gene expression analyses using the single-cell sequencing
dataset developed by the Reddien laboratory [43]. The receptors are
expressed in the central nervous system (CNS), gut, pharynx, epi-
dermis, muscle, photoreceptors, protonephridia, and in different
subclasses of neoblasts (Fig. 6; Table 2). Analysis of the expres-
sion of the EGF ligand has also demonstrated significant diversity
of patterns including the gut, CNS, pharynx, muscle, and epidermis
(Fig. 7; Table 2). Although in most cases the expression patterns for
individual genes obtained by in situ hybridization are supported
by corresponding single-cell sequencing data, some discrepancies

Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx 5

Fig. 4. The EGFR pathway in metazoans. Numbers of EGF receptors and ligands (EGF-type and neuregulin-type) across phylogenies. Image from Barberán et al. [20].

Fig. 5. The EGFR pathway in the planarian Schmidtea mediterranea. The number of EGF receptors and ligands, their domain structures, and their possible interactions are
indicated. Defects induced by silencing Smed-egfr-1/Smed-nrg-1, Smed-egfr-3, and Smed-egfr-5/Smed-egf-6 are indicated. See text for more details.

Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
6 S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx

Fig. 6. EGF receptor expression patterns in S. mediterranea. For each of the 6 EGF receptors identified, expression patterns are indicated by a representative whole-mount
in situ hybridization image and analyses at the single-cell sequencing level as obtained from the open database of the Reddien laboratory (https://radiant.wi.mit.edu/app/).
The upper graphs represent the different cell types (left) and neoblast classes (right) and serve as a reference for the specific graphs for each individual EGF receptor.

Table 2
Expression patterns of EGF receptos and ligands in S. mediterranea based on in situ hybridizations and single-cell sequencing data.

Receptors Smed-egfr-1 gut, pharynx, epidermis, photoreceptors, gamma and zeta neoblasts
Smed-egfr-2 gut and gamma neoblasts
Smed-egfr-3 central nervous system and sigma and zeta neoblasts
Smed-egfr-4 central nervous system, photoreceptors, pharynx, muscle, gut and gamma neoblasts
Smed-egfr-5 protonephridia, neurons, muscle
Smed-egfr-6 protonephridia, muscle

Ligands Smed-egf-1 gut

Smed-egf-2 gut
Smed-egf-3 pharynx, parenchyma
Smed-egf-4 central nervous system, pharynx, gut, muscle
Smed-egf-5 pharynx
Smed-egf-6 muscle
Smed-egf-7 central nervous system, pharynx
Smed-egf-8 epidermis, gut, pharynx
Smed-nrg-1 muscle

Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx 7

Fig. 7. EGF ligand expression patterns in S. mediterranea. For each of the 9 EGF ligands identified the corresponding expression patterns are indicated by a representative
whole-mount in situ hybridization image and analyses at the single-cell sequencing level obtained from the open database of the Reddien laboratory (https://radiant.wi.mit.
edu/app/). The bottom right graph represents the different cell types and serve as a reference for the specific graphs for each individual EGF ligand.

Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
8 S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx
are observed, perhaps due to differences in the sensitivity of the 2
methods.
Whereas EGF ligands are expressed in mature differentiated
tissues, several EGF receptors are also expressed in different sub-
classes of neoblasts, the pluripotent stem cells found in adult
planarians [44–46], suggesting an important role of this signaling
pathway in regulating different aspects of neoblast biology, such as
cell proliferation and differentiation, as discussed further below.
Numerous combinations of the different members of the ErbB
family have been identified in vertebrates [38,39]. This variety
of homo- and heterodimers allows sufficient diversity to recog-
nize each of the different ligands and activate specific downstream
pathways to induce distinct molecular and cellular responses. The
formation of heterodimers is essential for ErbB2 and ERbB3 recep-
tors, as ErbB2 alone cannot bind to any of the identified ligands
and ErbB3 contains an intracellular domain with very reduced
tyrosine kinase activity. The presence of a single receptor in C.
elegans and D. melanogaster suggests that EGFR signaling occurs
via homodimerization in those species. S. mediterranea, on the
other hand, possesses at least 6 different receptors with distinct
expression patterns. However, based on current data, it is unclear
whether these receptors exert their functions through the forma-
tion of homo- or heterodimers. The respective expression patterns
of Smed-egfr-1 and Smed-egfr-2 indicate that both are expressed in
the gut and in gamma neoblasts, and could therefore form either
homodimers or heteordimers in them. However, as explained
below, whereas silencing of Smed-egfr-1 results in marked defects
in gut regeneration and maintenance, silencing of Smed-egfr-2 does
not produce an evident phenotype [17,21]. These results suggest
that Smed-egfr-1 could exert its activity in the digestive tissue as
a homodimer. However, it cannot be completely ruled out that
Smed-egfr-1 and Smed-egfr-2 might work as a heterodimer. Fur-
ther experiments specially addressed to uncover the function of
Smed-egfr-2 should help in this direction. Similarly, while both
Smed-egfr-5 and Smed-egfr-6 are expressed in protonephridia, only
silencing of Smed-egfr-5 leads to obvious defects in the excretory
system [18]. In this particular case, it should be noted that Smed-
egfr-6 contains a defective tyrosine kinase domain and therefore its
function needs to be further elucidated [20].
Very little information is currently available regarding the bind-
ing specificity of EGF ligands and their receptors in S. mediterranea.
In the absence of any experimental data demonstrating direct bind-
ing between any of the putative ligands and different EGF receptors,
the only relevant data are those obtained from functional analy-
ses using RNA interference. Given that silencing of Smed-nrg-1 and
Smed-egfr-1 [21] and of Smed-egf-6 and Smed-egfr-5 [18,20] gives
rise to extremely similar phenotypes, it has been proposed that
these may constitute ligand-receptor pairs in planarians (Fig. 5).
5. The role of EGFR signaling in development and
regeneration
As mentioned above, EGFR signaling is involved in regulat-
ing multiple biological processes during development and cancer
progression. Mice carrying mutations in ErbB genes develop mul-
tiorgan failure that leads to premature death. This is characterized
by defects in the normal development of the skin, lungs, pancreas,
digestive system, nervous system, and mammary glands [47–50].
Similarly, mutations in EGF ligands affect the normal development
of the prostate, skin, hair, eyes, and digestive tract [51–53]. In
Drosophila, EGFR signaling is essential for oogenesis [54], dorsoven-
tral patterning [55,56], neurogenesis [57,58], and development of
the eye, epidermis, and tracheal system [59–62].
In mouse, a specific role of this pathway in cell proliferation and
differentiation has been reported, with numerous studies describ-
Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
ing roles in neural and glial precursors [63–66], keratinocytes
[67,68], and intestinal gut cells [69]. In response to brain injury,
astrocytes upregulate the expression of TGF-␣ and ErbB increasing
then their proliferative rate [70]. Also, the administration of EGF to
mice that have suffered ischemia increases significantly the neu-
ronal replacement in the affected region [71]. Another context in
which the EGFR pathway plays an important function is in epi-
dermis homeostasis and hair formation [72], as it regulates the
proliferation and differentiation of the keratinocytes. Thus, EGFR
signaling maintains a basal level of proliferation in the basal lamina
of the epidermis whereas in sub-basal layers this pathway is inhib-
ited in order to promote the differentiation of the keratinocytes
[73,74]. A similar result has been observed in the maintenance of
the mice gut where the EGFR pathway is active in the stem cell
niche of this tissue [75], and is involved in promoting the pro-
liferation of the intestinal stem cells. The overexpression of this
pathway induces an uncontrolled growth of these stem cells and
the expansion of the intestinal niche [69].
Similarly, in invertebrates the EGFR pathway regulates the
proliferation and differentiation of several types of stem cells,
including intestinal [76], nephritic [77], follicular [78], and neu-
ral [54,79,80] stem cells. As an example, in Drosophila, the visceral
muscle around the gut secretes the EGF ligand vein that together
with the other ligands spitz and keren secreted from the enterob-
lasts and enterocytes activate the EGFR pathway in the intestinal
stem cells (ISCs) inducing their proliferation [81]. Although the
EGFR signaling those not seem required for the differentiation of
the ISCs, it is necessary for the maturation of the enterocytes [82]
Moreover, in Drosophila, the EGFR pathway participates in the spec-
ification and differentiation of muscle progenitor cells [83] and
different types of eye cells [84–86]. In the nematode Caenorhab-
ditis elegans, the EGFR pathway is involved in the specification and
differentiation of the cell lineage that will give rise to the vulva
[87,88].
In addition to its contributions to normal development, the role
of the EGFR pathway in cancer has come under particular scrutiny
given its implication in the appearance and progression of sev-
eral types of carcinomas [89–91]. Multiple studies have shown
that EGFRs and their ligands are overexpressed in different types
of human carcinomas, and that this overexpression is an essential
component of the underlying pathogenesis [90,91].
As mentioned above, signaling pathways play fundamental roles
during both embryonic development and regeneration. Studies
have demonstrated the participation of the EGFR pathway in insect
leg regeneration where it is necessary for the re-establishment of
the proximodistal axis [92], cell proliferation and migration during
zebrafish regeneration [93], neural regeneration [94], liver regen-
eration where it plays an important role in triggering the initial
proliferative response [95–97], and gut regeneration [98–100].
6. Role of the EGFR pathway during planarian regeneration
and homeostasis
RNAi-based functional analyses in S. mediterranea have uncov-
ered important roles in regeneration and homeostasis for 3 EGF
receptors and 3 of their putative ligands (Fig. 5) [17–21]. Remark-
ably, the phenotypes obtained are similar between receptor-ligand
pairs, suggesting that they may function as real pairs in vivo,
although further evidence will be required to support this hypoth-
esis.
6.1. Smed-egfr-3/Smed-nrg-7
Smed-egfr-3 is mainly expressed in neoblasts, and weakly
expressed in the CNS and its silencing results in marked defects
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx 9

Fig. 8. Proposed model of the role of the planarian EGFR pathway in the differentiation of lineage-committed progenitors.

during regeneration. Thus, treated planarians regenerate smaller
blastemas that fail to properly differentiate normal structures and
cell types. Interestingly, neoblast population size and neoblast
mitotic activity do not appear to be significantly affected by Smed-
egfr-3 silencing [17,19]. On the other hand, certain cell types,
especially neural cells, fail to regenerate after Smed-egfr-3 silencing.
Consequently, regeneration of proper cephalic ganglia is generally
impaired in these animals, although other cell types, such as some
marginal gland cells and epidermal cells, appear to differentiate
properly. As described above, EGFR signaling may mediate some of
its functions through the activation of several intracellular path-
ways (Fig. 3). The intracellular cascades activated by different EGF
receptors in planarians are largely unknown. However, it has been
suggested that Smed-egfr-3 may exert its activity through activa-
tion of the extracellular signal-regulated kinase (ERK) pathway.
Two lines of evidence support this relationship. First, ERK inhibition
results in phenotypes similar to those described after Smed-egfr-
3 RNAi [101]. Thus, planarians treated with ERK inhibitors fail to
grow normal blastemas, mainly due to the impairment of proper
neoblast differentiation. Further supporting a relationship between
Smed-egfr-3 and ERK signaling, silencing of Smed-egfr-3 inhibits the
phosphorylation and consequent activation of ERK [102]. Taken
together, these observations suggest that Smed-egfr-3/ERK plays an
important role in neoblast differentiation during planarian regen-
eration.
In addition to this function, Smed-egfr-3 has been shown to reg-
ulate asymmetric cell division in planarians [19]. Neoblasts are
planarian pluripotent stem cells, and the only planarian cells with
proliferative capabilities [46,103]. Irradiation of planarians results
in complete elimination of the neoblast population and loss of the
regenerative abilities, ultimately leading to death due to an inability
to sustain normal homeostatic cell turnover. However, after sub-
lethal irradiation, some neoblasts can survive and can repopulate
the organism to reconstitute the stem cell population [44,104]. It
is in this specific context, after sub-lethal irradiation and neoblast
repopulation, that Smed-egfr-3 participates in cell division. It has
been shown that Smed-egfr-3 is asymmetrically distributed within
the cell membrane of neoblasts [19]. During neoblast repopula-
tion these stem cells divide either symmetrically or asymmetrically
in a ∼1:1 ratio. However, after Smed-egfr-3 RNAi and sub-lethal
irradiation, neoblasts tend to divide symmetrically, resulting in
defects in neoblast proliferation and differentiation [19]. Similar
results are obtained after silencing the putative EGF ligand Smed-
nrg-7 [19]. It remains unclear how Smed-egfr-3 regulates neoblast
division in this particular context, whether Smed-egfr-3 controls
the DNA damage response, and how the balance between sym-
metrical/asymmetrical cell division specifies self-renewal versus
differentiation of the different cell lineages. Nonetheless, these
results support a role of Smed-egfr-3 in neoblast differentiation in
different scenarios, including amputation-driven regeneration and
irradiation-induced repopulation.
6.2. Smed-egfr-5/Smed-egf-6
The planarian excretory system consists of a network of pro-
tonephridia that extends along the lateral margins of the body.
These protonephridia are epithelial tubes with terminal ciliated
flame cells that mediate filtration. Smed-egfr-5 is expressed in

Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
10 S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx
these terminal flame cells and the adjacent proximal tubules [17].
Silencing of Smed-egfr-5 during regeneration results in abnormal
branching of the proximal portion of the new excretory system,
and a significant reduction in the number of flame cells [17]. A sim-
ilar effect is observed in non-regenerating animals, underscoring
the requirement of Smed-egfr-5 for the maintenance of a functional
excretory system. This loss of flame cells results in the forma-
tion of edemas and subsequent lysis of the organism [17]. On
the other hand, silencing of the predicted ligand Smed-egf-6 phe-
nocopies many aspects of Smed-egfr-5 silencing, suggesting that
these 2 proteins may act as ligand and receptor to regulate flame
cell differentiation in planarians [20]. Expression data suggest that
Smed-egf-6 is expressed in muscle cells mainly located along the
dorsoventral border of the animal (Fig. 7). We hypothesize that the
role of Smed-egf-6 in the differentiation of the excretory system
involves its release from these muscle cells and its interaction, in a
paracrine manner, with differentiated excretory cells and/or com-
mitted progenitors of this lineage in order to regulate their final
differentiation.
6.3. Smed-egfr-1/Smed-nrg-7
Silencing of Smed-egfr-1 during both regeneration and homeo-
static cell turnover induces several phenotypes in different tissues
[17,21]. Smed-egfr-1 RNAi planarians regenerate smaller disorga-
nized pharynges and eyes with reduced numbers of eye-pigment
cells, display general hyperproliferation of neoblasts throughout
their bodies, develop abnormal dorsal outgrowths in the pharyn-
geal region, and are incapable of regenerating and maintaining
the digestive system. It remains unclear how these different phe-
notypes relate to one another and whether they are a direct
consequence of the loss of function of Smed-egfr-1 or an indirect
secondary effect.
The best-characterized function of Smed-egfr-1 is its role in
the digestive system [21]. Smed-egfr-1 is mainly expressed in
the mature digestive system and in neoblasts, including gamma
neoblasts, which have been hypothesized to give rise to gut pro-
genitors [105] (Fig. 6). The planarian gut contains 2 main cell types,
phagocytes and secretory goblet cells surrounded by enteric mus-
cle, and has a stereotypical highly branched pattern [106,107].
Thus, from one anterior and 2 posterior main primary branches
secondary, tertiary, and quaternary branches grow. Smed-egfr-1
silencing inhibits regeneration of the digestive system and its main-
tenance during homeostatic cell turnover in uncut animals [21].
In these biological contexts Smed-egfr-1 silencing inhibits the dif-
ferentiation of new digestive cells. In line with the lack of new
differentiated gut cells, these animals show abnormal accumu-
lations of gut progenitors in the mesenchyme surrounding the
digestive system. These gut progenitor cells are defined by the
expression of markers such as hnf4 and gata4/5/6 and are also pos-
itive for Smed-egfr-1. Therefore, the current model suggests that
Smed-egfr-1 plays a key role in the differentiation of gut progenitors
into mature phagocytes and goblet cells. Remarkably, silencing of
the putative EGF ligand Smed-nrg-1 yields a very similar phenotype
to that produced by Smed-egfr-1 RNAi. It has thus been hypothe-
sized that Smed-nrg-1 may regulate the effect of Smed-egfr-1 on gut
progenitor differentiation. Interestingly, Smed-nrg-1 appears to be
expressed in muscle cells (Fig. 7). Recent studies have suggested
that the planarian musculature may act not only as a supportive
skeletal-like tissue but also as a source of so-called “position control
genes” (PCGs) [108,109]. These PCGs include planarian homologues
of factors such as wnt genes, bmp, admp, noggin-like genes, sfrp-1,
nou-darake, notum, and netrin-2, genes with regionalized expres-
sion patterns whose silencing results in defects in polarity and
patterning [108,110]. For example, bmp secreted by dorsal mus-
cle cells appears to regulate the fate of epidermal progenitors by
Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
inhibiting the expression of genes associated with ventral epider-
mal identity [111]. In addition to PCGs, muscle cells may also be a
source of secreted EGF-like ligands that influence the differentia-
tion of nearby neoblasts. Smed-egf-6 and Smed-nrg-1 appear to be
expressed in muscle (Fig. 7) and to play important roles in the differ-
entiation of protonephridia and digestive cells, respectively [20,21].
Moreover, in models such as Drosophila the enteric musculature
surrounding the gut has been shown to secrete the EGF ligand Vein
activates the EGFR pathway in the intestinal stem cells, regulating
their proliferation [81]. Similarly, in mice the EGFR pathway also
regulates the maintenance and proliferation of intestinal stem cells
[112,113,75]. However, in planarians the EGFR pathway appears to
play a more important role in regulating the differentiation of gut
progenitors. Further studies will be required to determine whether
this pathway is also involved in their proliferation.
It should be noted that silencing of Smed-egfr-1 induces
increases both in neoblast proliferation and in apoptosis through-
out the body [17,21]. In planarians, any type of injury results in
rapid upregulation of so-called wound-induced genes [40] and
triggers well-characterized proliferative and apoptotic responses
[114,115]. Recently, it has been shown that defects in the mainte-
nance and regeneration of the epidermis can generate a stress signal
that results in general increases in apoptosis and neoblast prolif-
eration [116]. Taking these findings into account, it is reasonable
to speculate that degeneration of the digestive system follow-
ing Smed-egfr-1 silencing may induce a similar stress signal that
triggers increases in proliferation and apoptosis. Another possible
explanation for the increased proliferation and apoptosis observed
following Smed-egfr-1 silencing is that the digestive system some-
how functions as a niche for the neoblasts. Thus, the lack of integrity
of the gut could negatively affect any role played by this tissue in
regulating neoblast proliferation. Supporting this view, degenera-
tion of the gut induced by Smed-CerS1 silencing completely inhibits
neoblast proliferation [117]. Finally, a third possible explanation
is that apoptotic cells are capable of stimulating cell proliferation
via a process known as apoptosis-induced compensatory prolifer-
ation, as described in Drosophila and hydra [118,119]. In support
of this hypothesis, a planarian homologue of p53 that has been
implicated in compensatory proliferation [120] is upregulated after
Smed-egfr-1 RNAi [17]. Regardless of the underlying mechanisms,
further studies are necessary to better understand the role of the
EGFR signaling pathway in neoblast proliferation.
7. EGFR signaling in planarians: a general regulator of
neoblast differentiation?
Current functional analysis data suggest that the planarian
EGFR pathway is involved in the differentiation of several tissues
such as the CNS, gut, protonephridia, pharynx, and photorecep-
tors. Neoblasts comprise a heterogeneous population of pluripotent
stem cells and a collection of mitotic and postmitoic progenitor
cells committed to the different mature cell fates [44–46,105,121].
However, little is known of the mechanisms that regulate the
commitment of pluripotent stem cells to specialized progenitor lin-
eages or the differentiation of progenitors into their final fates. The
Smed-mex3 gene appears to regulate the appearance of lineage-
committed progenitors from pluripotent neoblasts [122]. Less is
known about how those progenitors terminally differentiate. In the
case of the epidermal lineage, for instance, the zinc-finger tran-
scription factor Smed-egr-5 has been shown to play an important
role in the differentiation of epidermal progenitors [108]. Here, we
have reviewed the role of the planarian EGFR pathway in cell differ-
entiation. Planarians possess numerous EGF receptors and ligands,
with at least 6 receptor homologues and 9 putative ligand homo-
logues expressed in a wide variety of tissues [19,21]. As discussed
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx
above, Smed-egfr-1 and Smed-nrg-1 are required for the differen-
tiation of gut progenitors [21]. Furthermore, evidence suggests a
role for Smed-egfr-3, a gene expressed in neoblasts and in the CNS,
in cell differentiation, particularly that of neural cells [17]. Smed-
egfr-5 and Smed-egf-6 also appear to regulate cell differentiation in
the excretory system [18,20], and Smed-egfr-1 is required for the
formation of photoreceptors and the pharynx [17]. But how does
the EGFR pathway regulate cell differentiation in planarians? In
the case of the digestive system it appears to do so not by regulat-
ing the specification of gut progenitors from pluripotent neoblasts,
but by mediating the differentiation of those gut progenitors into
mature cells. Given that the silencing of EGFRs also results in defects
in the production of normal numbers of other differentiated cells,
including neurons, photoreceptors, and protonephridia, it would
be interesting to determine whether EGFRs are also expressed in
the respective specialized progenitors, in which they could play a
role in regulating their final differentiation (Fig. 8). For example,
Smed-egfr-1 and Smed-nrg-1 could regulate the differentiation of
eye progenitors, Smed-egfr-3 of neural progenitors, and Smed-egfr-5
and Smed-egf-6 of protonephridia progenitors.
8. Conclusions
Planarian regeneration is dependent on the differentiation of
pluripotent stem cells, the neoblasts, into all the required cell
types, and the organization of these new cells in time and space
to generate properly patterned tissues, organs, and structures.
Distinct signaling pathways are involved in different aspects of
regeneration, particularly polarity and patterning. One of the most
conserved signaling pathways in animals is the EGFR pathway.
This pathway plays many important roles in stem cell prolifer-
ation and differentiation, adhesion, migration, and angiogenesis.
Moreover, given its overactivation in many human cancers it is
an important target of anti-cancer therapies. Our understanding
of EGFR signaling in planarians has deepened with the recent
identification of many receptors and putative ligands. The great
variety in the expression patterns of receptors and ligands sug-
gests multiple roles of the EGFR pathway in planarians. Based
on functional analyses carried out to date, its main function in
planarians appears to be the regulation of neoblast differentia-
tion. This has been demonstrated in the digestive lineage, where
Smed-egfr-1 and Smed-nrg-1 regulate the final differentiation of
gut progenitors. Given these findings, the aforementioned diver-
sity of expression patterns, and the defects observed in different cell
lineages after silencing of different receptors and ligands, an impor-
tant objective of future research will be to establish whether the
EGFR signaling pathway plays a general role in planarian neoblast
differentiation, with distinct receptors and ligands mediating the
regeneration and maintenance of different cell types. To accomplish
that, and because the large number of receptors and ligands future
experimental approaches should include the search for biochemical
evidence of ligand-receptor pairing as well as for the downstream
pathways and target genes regulated specifically by each of the
different receptors and ligands.
Acknowledgements
This work was supported by grant BFU2015-65704P from Minis-
terio de Economía y Competitividad (Spain) to F.C. We thank Owen
Howard for advice on English style.
References
[1] P.W. Ingham, A.P. McMahon, Hedgehog signaling in animal development:
paradigms and principles, Genes Dev. 15 (2001) 3059–3087.
Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
11
[2] K.M. Cadigan, R. Nusse, Wnt signaling: a common theme in animal
development, Genes Dev. 11 (1997) 3286–3305.
[3] J. Massague, D. Wotton, Transcriptional control by the TGFbeta/Smad
signaling system, EMBO J. 19 (2000) 1745–1754.
[4] J.S. Mumm, R. Kopan, Notch signaling: from the outside in, Dev. Biol. 228
(2000) 151–165.
[5] A. Sánchez-Alvarado, P.A. Tsonis, Bridging the regeneration gap: genetic
insights from diverse animal models, Nat. Rev. Genet. 7 (2006) 873–884.
[6] E.M. Tanaka, P.W. Reddien, The cellular basis for animal regeneration, Dev.
Cell 21 (2011) 172–185.
[7] A. Pires-daSilva, R.J. Sommer, The evolution of signalling pathways in animal
development, Nat. Rev. Genet. 4 (2003) 39–49.
[8] M. Freeman, Feedback control of intercellular signalling in development,
Nature 408 (2000) 313–319.
[9] M.D. Molina, E. Saló, F. Cebrià, The BMP pathway is essential for
re-specification and maintenance of the dorsoventral axis in regenerating
and intact planarians, Dev. Biol. 311 (2007) 79–94.
[10] P.W. Reddien, A.L. Bermange, A.M. Kicza, A. Sánchez-Alvarado, BMP
signaling regulates the dorsal planarian midline and is needed for
asymmetric regeneration, Development 134 (2007) 4043–4051.
[11] J.C. Rink, K.A. Gurley, S.A. Elliott, A. Sánchez-Alvarado, Planarian Hh
signaling regulates regeneration polarity and links Hh pathway evolution to
cilia, Science 326 (2009) 1406–1410.
[12] C.P. Petersen, P.W. Reddien, Smed-betacatenin-1 is required for
anteroposterior blastema polarity in planarian regeneration, Science 319
(2008) 327–330.
[13] M. Iglesias, J.L. Gómez-Skarmeta, E. Saló, T. Adell, Silencing of
Smed-betacatenin1 generates radial-like hypercephalized planarians,
Development 135 (2008) 1215–1221.
[14] K.A. Gurley, J.C. Rink, A. Sánchez-Alvarado, Betacatenin defines head versus
tail identity during planarian regeneration and homeostasis, Science 319
(2008) 323–327.
[15] F. Cebrià, C. Kobayashi, Y. Umesono, M. Nakazawa, K. Mineta, K. Ikeo, T.
Gojobori, M. Itoh, M. Taira, A. Sánchez-Alvarado, K. Agata, FGFR-related gene
nou-darake restricts brain tissues to the head region of planarians, Nature
419 (2002) 620–624.
[16] J. Baguñá, E. Saló, R. Romero, Effects of activators and antagonists of the
neuropeptides substance P and substance K on cell proliferation in
planarians, Int. J. Dev. Biol. 33 (1989) 261–266.
[17] S. Fraguas, S. Barberán, F. Cebrià, EGFR signaling regulates cell proliferation,
differentiation and morphogenesis during planarian regeneration and
homeostasis, Dev. Biol. 354 (2011) 87–101.
[18] J.C. Rink, H.T. Vu, A. Sánchez-Alvarado, The maintenance and regeneration of
the planarian excretory system are regulated by EGFR signaling,
Development 138 (2011) 3769–3780.
[19] K. Lei, H.T. Vu, R.D. Mohan, S.A. McKinney, C.W. Seidel, R. Alexander, K.
Gotting, J.L. Workman, A. Sánchez-Alvarado, Egf signaling directs neoblast
repopulation by regulating asymmetric cell división in planarians, Dev. Cell
38 (2016) 413–429.
[20] S. Barberán, J.M. Martín-Durán, F. Cebrià, Evolution of the EGFR pathway in
metazoa and its diversification in the planarian Schmidtea mediterranea,
Sci. Rep. 6 (2016) 28071.
[21] S. Barberán, S. Fraguas, F. Cebrià, The EGFR signaling pathway controls gut
progenitor differentiation during planarian regeneration and homeostasis,
Development 143 (2016) 2089–2102.
[22] I. Lax, R. Fischer, C. Ng, J. Segre, A. Ullrich, D. Givol, J. Schlessinger,
Noncontiguous regions in the extracelular domain of EGF receptor define
ligand-binding specificity, Cell Regul. 2 (1991) 337–345.
[23] D.L. Cadena, G.N. Gill, Receptor tyrosine kinases, FASEB J. 6 (1992)
2332–2337.
[24] A. Ullrich, J. Schlessinger, Signal transduction by receptors with tyrosine
kinase activity, Cell 61 (1990) 203–212.
[25] B.L. Margolis, I. Lax, R. Kris, M. Dombalagian, A.M. Honegger, R. Howk, D.
Givol, A. Ullrich, J. Schlessinger, All autophosphorylation sites of epidermal
growth factor (EGF) receptor and HER2/neu are located in their
carboxyl-terminal tails. Identification of a novel site in EGF receptor, J. Biol.
Chem. 264 (1989) 10667–10671.
[26] Y. Yarden, M.X. Sliwkowski, Untangling the ErbB signalling network, Nat.
Rev. Mol. Cell Biol. 2 (2001) 127–137.
[27] P.M. Guy, J.V. Platko, L.C. Cantley, R.A. Cerione, K.L. Carraway, Insect
cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity,
Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 8132–8136.
[28] M.P. Steinkamp, S.T. Low-Nam, S. Yang, K.A. Lidke, D.S. Lidke, B.S. Wilson,
erbB3 is an active tyrosine kinase capable of homo- and heterointeractions,
Mol. Cell Biol. 34 (2014) 965–977.
[29] R. Schweitzer, B.Z. Shilo, A thousand and one roles for the Drosophila EGF
receptor, Trends Genet. 13 (1997) 91–96.
[30] R.V. Aroian, M. Koga, J.E. Mendel, Y. Ohshima, P.W. Sternberg, The let-23
gene necessary for Caenorhabditis elegans vulval induction encodes a
tyrosine kinase of the EGF receptor subfamily, Nature 348 (1990) 693–699.
[31] R.C. Harris, E. Chung, R.J. Coffey, EGF receptor ligands, Exp. Cell Res. 284
(2003) 2–13.
[32] J. Massague, A. Pandiella, Membrane-anchored growth factors, Annu. Rev.
Biochem. 62 (1993) 515–541.
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
12 S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx
[33] A.C. Dreux, D.J. Lamb, H. Modjtahedi, G.A. Ferns, The epidermal growth
factor receptors and their family of ligands: their putative role in
atherogenesis, Atherosclerosis 186 (2006) 38–53.
[34] D.L. Falls, Neuregulins: functions, forms, and signaling strategies, Exp. Cell
Res. 284 (2003) 14–30.
[35] R.J. Hill, P.W. Sternberg, The gene lin-3 encodes an inductive signal for
vulval development in C. elegans, Nature 358 (1992) 470–476.
[36] H. Ramachandran, P.J. Skelly, C.B. Shoemaker, The Schistosoma mansoni
epidermal growth factor receptor homologue, SER, has tyrosine kinase
activity and is localized in adult muscle, Mol. Biochem. Parasitol. 83 (1996)
1–10.
[37] M. Spiliotis, A. Kroner, K. Brehm, Identification, molecular characterization
and expression of the gene encoding the epidermal growth factor receptor
orthologue from the fox-tapeworm Echinococcus multilocularis, Gene 323
(2003) 57–65.
[38] M.A. Olayioye, R.M. Neve, H.A. Lane, N.E. Hynes, The ErbB signaling network:
receptor heterodimerization in development and cáncer, EMBO J. 19 (2000)
3159–3167.
[39] J. Schlessinger, Cell signaling by receptor tyrosine kinases, Cell 103 (2000)
211–225.
[40] D. Wenemoser, S.W. Lapan, A.W. Wilkinson, G.W. Bell, P.W. Reddien, A
molecular wound response program associated with regeneration initiation
in planarians, Genes Dev. 26 (2012) 988–1002.
[41] M. Adamska, Developmental signalling and emergence of animal
multicellularity, Evol. Transitions Multicell. Life 2 (2015) 425–450.
[42] G.S. Richards, B.M. Degnan, The dawn of developmental signaling in the
metazoa, Cold Spring Harb. Symp. Quant. Biol. 74 (2009) 81–90.
[43] O. Wurtzel, L.E. Cote, A. Poirier, R. Satija, A. Regev, P.W. Reddien, A generic
and cell-type-specific wound response precedes regeneration in planarians,
Dev. Cell 35 (2015) 632–645.
[44] D.E. Wagner, I.E. Wang, P.W. Reddien, Clonogenic neoblasts are pluripotent
adult stem cells that underlie planarian regeneration, Science 332 (2011)
811–816.
[45] M.L. Scimone, K.M. Kravarik, S.W. Lapan, P.W. Reddien, Neoblast
specialization in regeneration of the planarian Schmidtea mediterranea,
Stem Cell Rep. 3 (2014) 339–352.
[46] S.J. Zhu, B.J. Pearson, (Neo)blasts from the past: new insights into planarian
stem cell lineages, Curr. Opin. Genet. Dev. 40 (2016) 74–80.
[47] P.J. Miettinen, J.E. Berger, J. Meneses, Y. Phung, R.A. Pedersen, Z. Werb, R.
Derynck, Epithelial immaturity and multiorgan failure in mice lacking
epidermal growth factor receptor, Nature 376 (1995) 337–341.
[48] M. Sibilia, E.F. Wagner, Strain-dependent epithelial defects in mice lacking
the EGF receptor, Science 269 (1995) 234–238.
[49] D.W. Threadgill, A.A. Dlugosz, L.A. Hansen, T. Tennenbaum, U. Lichti, D. Yee,
C. LaMantia, T. Mourton, K. Herrup, R.C. Harris, et al., Targeted disruption of
mouse EGF receptor: effect of genetic background on mutant phenotype,
Science 269 (1995) 230–234.
[50] M. Sibilia, R. Kroismayr, B.M. Lichtenberger, A. Natarajan, M. Hecking, M.
Holcmann, The epidermal growth factor receptor: from development to
tumorigenesis, Differentiation 75 (2007) 770–787.
[51] B.D. Abbott, T.M. Lin, N.T. Rasmussen, R.M. Albrecht, J.E. Schmid, R.E.
Peterson, Lack of expression of EGF and TGF-alpha in the fetal mouse alters
formation of prostatic epithelial buds and influences the response to TCDD,
Toxicol. Sci. 76 (2003) 427–436.
[52] G.B. Mann, K.J. Fowler, A. Gabriel, E.C. Nice, R.L. Williams, A.R. Dunn, Mice
with a null mutation of the TGF alpha gene have abnormal skin architecture,
wavy hair, and curly whiskers and often develop corneal inflammation, Cell
73 (1993) 249–261.
[53] K.L. Troyer, N.C. Luetteke, M.L. Saxon, T.H. Qiu, C.J. Xian, D.C. Lee, Growth
retardation, duodenal lesions, and aberrant ileum architecture in triple null
mice lacking EGF, amphiregulin, and TGF-alpha, Gastroenterology 121
(2001) 68–78.
[54] A. Sapir, R. Schweitzer, B.Z. Shilo, Sequential activation of the EGF receptor
pathway during Drosophila oogenesis establishes the dorsoventral axis,
Development 125 (1998) 191–200.
[55] Y. Yagi, T. Suzuki, S. Hayashi, Interaction between Drosophila EGF receptor
and vnd determines three dorsoventral domains of the neuroectoderm,
Development 125 (1998) 3625–3633.
[56] A. González-Reyes, H. Elliott, D. St Johnston, Polarization of both major body
axes in Drosophila by gurken-torpedo signalling, Nature 375 (1995)
654–658.
[57] J.B. Skeath, The Drosophila EGF receptor controls the formation and
specification of neuroblasts along the dorsal-ventral axis of the Drosophila
embryo, Development 125 (1998) 3301–3312.
[58] B.J. Rutledge, K. Zhang, E. Bier, Y.N. Jan, N. Perrimon, The Drosophila spitz
gene encodes a putative EGF-like growth factor involved in dorsal-ventral
axis formation and neurogenesis, Genes Dev. 6 (1992) 1503–1517.
[59] A.L. Kolodkin, A.T. Pickup, D.M. Lin, C.S. Goodman, U. Banerjee,
Characterization of star and its interactions with sevenless and EGF receptor
during photoreceptor cell development in Drosophila, Development 120
(1994) 1731–1745.
[60] F.J. Diaz-Benjumea, E. Hafen, The sevenless signalling cassette mediates
Drosophila EGF receptor function during epidermal development,
Development 120 (1994) 569–578.
Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
[61] C. Cela, M. Llimargas, Egfr is essential for maintaining epitelial integrity
during tracheal remodelling in Drosophila, Development 133 (2006)
3115–3125.
[62] M. Llimargas, J. Casanova, EGF signalling regulates cell invagination as well
as cell migration during formation of tracheal system in Drosophila, Dev.
Genes Evol. 209 (1999) 174–179.
[63] C.S. Weickert, M. Blum, Striatal TGF-alpha: postnatal developmental
expression and evidence for a role in the proliferation of subependymal
cells, Brain Res. Dev. Brain Res. 86 (1995) 203–216.
[64] M. Blum, A null mutation in TGF-alpha leads to a reduction in midbrain
dopaminergic neurons in the substantia nigra, Nat. Neurosci. 1 (1998)
374–377.
[65] H.I. Kornblum, S.D. Zurcher, Z. Werb, R. Derynck, K.B. Seroogy, Multiple
trophic actions of heparin-binding epidermal growth factor (HB-EGF) in the
central nervous system, Eur. J. Neurosci. 11 (1999) 3236–3246.
[66] A.J. Kalyani, T. Mujtaba, M.S. Rao, Expression of EGF receptor and FGF
receptor isoforms during neuroepithelial stem cell differentiation, J.
Neurobiol. 38 (1999) 207–224.
[67] K. Hashimoto, S. Higashiyama, H. Asada, E. Hashimura, T. Kobayashi, K. Sudo,
T. Nakagawa, D. Damm, K. Yoshikawa, N. Taniguchi, Heparin-binding
epidermal growth factor-like growth factor is an autocrine growth factor for
human keratinocytes, J. Biol. Chem. 269 (1994) 20060–20066.
[68] M. Piepkorn, M.R. Pittelkow, P.W. Cook, Autocrine regulation of
keratinocytes: the emerging role of heparin-binding, epidermal growth
factor-related growth factors, J. Invest. Dermatol. 111 (1998) 715–721.
[69] V.W. Wong, D.E. Stange, M.E. Page, S. Buczacki, A. Wabik, S. Itami, M. van de
Wetering, R. Poulsom, N.A. Wright, M.W. Trotter, F.M. Watt, D.J. Winton, H.
Clevers, K.B. Jensen, Lrig1 controls intestinal stem-cell homeostasis by
negative regulation of ErbB signalling, Nat. Cell Biol. 14 (2012) 401–408.
[70] T. Yoshida, M. Takeuchi, Establishment of an astrocyte progenitor cell line:
induction of glial fibrillary acidic protein and fibronectin by transforming
growth factor-beta 1, J. Neurosc. Res. 35 (1993) 129–137.
[71] T. Teramoto, J. Qiu, J.C. Plumier, M.A. Moskowitz, EGF amplifies the
replacement of parvalbumin-expressing striatal interneurons after
ischemia, J. Clin. Invest. 111 (2003) 1125–1132.
[72] M.R. Schneider, S. Werner, R. Paus, E. Wolf, Beyond wavy hairs: the
epidermal growth factor receptor and its ligands in skin biology and
pathology, Am. J. Pathol. 173 (2008) 14–24.
[73] X. Wang, D. Bolotin, D.H. Chu, L. Polak, T. Williams, E. Fuchs, AP-2alpha: a
regulator of EGF receptor signaling and proliferation in skin epidermis, J.
Cell Biol. 172 (2006) 409–421.
[74] S. Getsios, C.L. Simpson, S. Kojima, R. Harmon, L.J. Sheu, R.L. Dusek, M.
Cornwell, K.J. Green, Desmoglein 1-dependent suppression of EGFR
signaling promotes epidermal differentiation and morphogenesis, J. Cell
Biol. 185 (2009) 1243–1258.
[75] T. Sato, J.H. Van Es, H.J. Snippert, D.E. Stange, R.G. Vries, M. Van Den Born, N.
Barker, N.F. Shroyer, M. Van De Wetering, H. Clevers, Paneth cells constitute
the niche for Lgr5 stem cells in intestinal crypts, Nature 469 (2011) 415–418.
[76] B. Biteau, H. Jasper, EGF signaling regulates the proliferation of intestinal
stem cells in Drosophila, Development 138 (2011) 1045–1055.
[77] Z. Li, S. Liu, Y. Cai, EGFR/MAPK signaling regulates the proliferation of
Drosophila renal and nephric stem cells, J. Genet. Genomics 42 (2015) 9–20.
[78] A. Castanieto, M.J. Johnston, T.G. Nystul, EGFR signaling promotes
self-renewal through the establishment of cell polarity in Drosophila follicle
stem cells, Elife 3 (2014), http://dx.doi.org/10.7554/eLife.04437.
[79] A. Aguirre, M.E. Rubio, V. Gallo, Notch and EGFR pathway interaction
regulates neural stem cell number and self-renewal, Nature 467 (2010)
323–327.
[80] K. Dumstrei, F. Wang, D. Shy, U. Tepass, V. Hartenstein, Interaction between
EGFR signaling and DE-cadherin during nervous system morphogenesis,
Development 129 (2002) 3983–3994.
[81] M.Y. Pasco, R. Loudhaief, A. Gallet, The cellular homeostasis of the gut: what
the Drosophila model points out, Histol. Histopathol. 30 (2015) 277–292.
[82] N. Buchon, N.A. Broderick, T. Kuraishi, B. Lemaitre, Drosophila EGFR
pathway coordinates stem cell proliferation and gut remodeling following
infection, BMC Biol. 8 (2010) 152.
[83] E. Buff, A. Carmena, S. Gisselbrecht, F. Jimenez, A.M. Michelson, Signalling by
the Drosophila epidermal growth factor receptor is required for the
specification and diversification of embryonic muscle progenitors,
Development 125 (1998) 2075–2086.
[84] M. Dominguez, J.D. Wasserman, M. Freeman, Multiple functions of the EGF
receptor in Drosophila eye development, Curr. Biol. 8 (1998) 1039–1048.
[85] M. Freeman, Reiterative use of the EGF receptor triggers differentiation of all
cell types in the Drosophila eye, Cell 87 (1996) 651–660.
[86] N.E. Baker, S.Y. Yu, The EGF receptor defines domains of cell cycle
progression and survival to regulate cell number in the developing
Drosophila eye, Cell 104 (2001) 699–708.
[87] C. Chang, P.W. Sternberg, C. elegans vulval development as a model system
to study the cancer biology of EGFR signaling, Cancer Metastasis Rev. 18
(1999) 203–213.
[88] R.J. Hill, P.W. Sternberg, The gene lin-3 encodes an inductive signal for
vulval development in C. elegans, Nature 358 (1992) 470–476.
[89] N. Normanno, C. Bianco, L. Strizzi, M. Mancino, M.R. Maiello, A. De Luca, F.
Caponigro, D.S. Salomon, The ErbB receptors and their ligands in cancer: an
overview, Curr. Drug Targets 6 (2005) 243–257.
G Model
YSCDB-2584; No. of Pages 13 ARTICLE IN PRESS
S. Barberán, F. Cebrià / Seminars in Cell & Developmental Biology xxx (2018) xxx–xxx
[90] D.S. Salomon, R. Brandt, F. Ciardiello, N. Normanno, Epidermal growth factor
related peptides and their receptors in human malignancies, Crit. Rev.
Oncol. Hematol. 19 (1995) 183–232.
[91] S. Sigismund, D. Avanzato, L. Lanzetti, Emerging functions of the EGFR in
cancer, Mol. Oncol. 12 (2018) 3–20.
[92] T. Nakamura, T. Mito, K. Miyawaki, H. Ohuchi, S. Noji, EGFR signaling is
required for re-establishing the proximodistal axis during distal leg
regeneration in the cricket Gryllus bimaculatus nymph, Dev. Biol. 319
(2008) 46–55.
[93] A. Rojas-Muñoz, S. Rajadhyksha, D. Gilmour, F. Van Bebber, C. Antos, C.
Rodríguez Esteban, C. Nüsslein-Volhard, J.C. Izpisúa-Belmonte, ErbB2 and
ErbB3 regulate amputation-induced proliferation and migration during
vertebrate regeneration, Dev. Biol. 327 (2009) 177–190.
[94] J. Bremer, J. Skinner, M. Granato, A small molecule screen identifies in vivo
modulators of peripheral nerve regeneration in zebrafish, PLoS One 12
(2017), e0178854.
[95] A. Natarajan, B. Wagner, M. Sibilia, The EGF receptor is required for efficient
liver regeneration, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 17081–17086.
[96] T.A. Zimmers, X. Jin, Z. Zhang, Y. Jiang, L.G. Koniaris, Epidermal growth factor
receptor restoration rescues the fatty liver regeneration in mice, Am. J.
Physiol. Endocrinol. Metab. 313 (2017) 440–449.
[97] J. López-Luque, D. Caballero-Díaz, A. Martínez-Palacián, C. Roncero, J.
Moreno-Càceres, M. García-Bravo, E. Grueso, A. Fernández, E. Crosas-Molist,
M. García-Álvaro, A. Addante, E. Bertran, A.M. Valverde, A.
González-Rodríguez, B. Herrera, L. Montoliu, T. Serrano, J.C. Segovia, M.
Fernández, E. Ramos, A. Sánchez, I. Fabregat, Dissecting the role of
epidermal growth factor catalytic activity during liver regeneration and
hepatocarcinogenesis, Hepatology 63 (2016) 604–619.
[98] C. Wang, X. Guo, R. Xi, EGFR and notch signaling respectively regulate
proliferative activity and multiple cell lineage differentiation of Drosphila
gastric stem cells, Cell Res. 24 (2014) 610–627.
[99] J. Xiang, J. Bandura, P. Zhang, Y. Jin, H. Reuter, B.A. Edgar, EGFR-dependent
TOR-independent endocycles support Drosophila gut epithelial
regeneration, Nat. Commun. 8 (2017) 15125.
[100] M. Janeh, D. Osman, Z. Kambris, Damage-induced cell regeneration in the
midgut of Aedes albopictus mosquitoes, Sci. Rep. 7 (2017) 44594.
[101] J. Tasaki, N. Shibata, O. Nishimura, K. Itomi, Y. Tabata, F. Son, N. Suzuki, R.
Araki, M. Abe, K. Agata, Y. Umesono, ERK signaling controls blastema cell
differentiation during planarian regeneration, Development 138 (2011)
2417–2427.
[102] S. Fraguas, Y. Umesono, K. Agata, F. Cebrià, Analyzing pERK activation during
planarian regeneration, Methods Mol. Biol. 1487 (2017) 303–315.
[103] J.C. Rink, Stem cell systems and regeneration in planaria, Dev. Genes Evol.
223 (2013) 67–84.
[104] A. Salvetti, L. Rossi, L. Bonuccelli, A. Lena, C. Pugliesi, G. Rainaldi, M.
Evangelista, V. Gremigni, Adult stem cell plasticity: neoblast repopulation in
non-lethally irradiated planarians, Dev. Biol. 328 (2009) 305–314.
[105] J.C. van Wolfswinkel, D.E. Wagner, P.W. Reddien, Single-cell analysis reveals
functionally distinct classes within the planarian stem cell compartment,
Cell Stem Cell 15 (2014) 326–339.
Please cite this article in press as: S. Barberán, F. Cebrià, The role of the EGFR signaling pathway in stem cell differentiation during
planarian regeneration and homeostasis, Semin Cell Dev Biol (2018), https://doi.org/10.1016/j.semcdb.2018.05.011
13
[106] C. Kobayashi, S. Kobayashi, H. Orii, K. Watanabe, K. Agata, Identification of
two distinct muscles in the planarian Dugesia japonica by their expression
of myosin heavy chain genes, Zool. Sci. 15 (1998) 861–869.
[107] D.J. Forsthoefel, A.E. Park, P.A. Newmark, Stem cell based growth,
regeneration, and remodeling of the planarian intestine, Dev. Biol. 356
(2011) 445–459.
[108] J.N. Witchley, M. Mayer, D.E. Wagner, J.H. Owen, P.W. Reddien, Muscle cells
provide instructions for planarian regeneration, Cell Rep. 4 (2013) 633–641.
[109] M.L. Scimone, L.E. Cote, P.W. Reddien, Orthogonal muscle fibres have
different instructive roles in planarian regeneration, Nature 551 (2017)
623–628.
[110] P.W. Reddien, Constitutive gene expression and the specification of tissue
identity in adult planaria biology, Trends Genet. 27 (2011) 277–285.
[111] O. Wurtzel, I.M. Oderberg, P.W. Reddien, Planarian epidermal stem cells
respond to positional cues to promote cell-type diversity, Dev. Cell 40
(2017) 491–504.
[112] D. Lee, M. Yu, E. Lee, H. Kim, Y. Yang, K. Kim, C. Pannicia, J.M. Kurie, D.W.
Threadgill, Tumor-specific apoptosis caused by deletion of the ERBB3
pseudo-kinase in mouse intestinal epithelium, J. Clin. Invest. 119 (2009)
2702–2713.
[113] R.B. Roberts, L. Min, M.K. Washington, S.J. Olsen, S.H. Settle, R.J. Coffey, D.W.
Threadgill, Importance of epidermal growth factor receptor signaling in
establishment of adenomas and maintenance of carcinomas during
intestinal tumorigenesis, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 1521–1526.
[114] J. Pellettieri, P. Fitzgerald, S. Watanabe, J. Mancuso, D.R. Green, A.
Sánchez-Alvarado, Cell death and tissue remodeling in planarian
regeneration, Dev. Biol. 338 (2010) 76–85.
[115] D. Wenemoser, P.W. Reddien, Planarian regeneration involves distinct stem
cell responses to wounds and tissue absence, Dev. Biol. 344 (2010) 979–991.
[116] K.C. Tu, L.C. Cheng, T.K.H. Vu, J.J. Lange, S.A. McKinney, C.W. Seidel, A.
Sánchez-Alvarado, Egr-5 is a post-mitotic regulator of planarian epidermal
differentiation, eLife 4 (2015), e10501.
[117] D.J. Forsthoefel, N.P. James, D.J. Escobar, J.M. Stary, A.P. Vieira, F.A. Waters,
P.A. Newmark, An RNAi screen reveals intestinal regulators of branching
morphogenesis, differentiation, and stem cell proliferation in planarians,
Dev. Cell 23 (2012) 691–704.
[118] F.A. Martín, A. Perez-Garijo, G. Morata, Apoptosis in Drosophila:
compensatory proliferation and undead cells, Int. J. Dev. Biol. 53 (2009)
1341–1347.
[119] S. Chera, L. Ghila, K. Dobretz, Y. Wenger, C. Bauer, W. Buzgariu, J.C. Martinou,
B. Galliot, Apoptotic cells provide an unexpected source of Wnt3 signaling to
drive hydra head regeneration, Dev. Cell 17 (2009) 279–289.
[120] A. Bergmann, H. Steller, Apoptosis, stem cells, and tissue regeneration, Sci.
Signal. 3 (2010) re8.
[121] A.M. Molinaro, B.J. Pearson, In silico lineage tracing thorugh sinlge cell
transcriptomics identifies a neural stem cell population in planarians,
Genome Biol. 17 (2016) 87.
[122] S.J. Zhu, S.E. Hallows, K.W. Currie, C. Xu, B.J. Pearson, A mex3 homologue is
required for differentiation during planarian stem cell lineage development,
eLife 4 (2015), http://dx.doi.org/10.7554/eLife.07025.