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# Epithelial-to-mesenchymal plasticity from development to disease: An introduction to the special issue

Hervé Acloque, Jing Yang, Eric Theveneau

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Hervé Acloque, Jing Yang, Eric Theveneau. Epithelial-to-mesenchymal plasticity from development to disease: An introduction to the special issue. Genesis - The Journal of Genetics and Development, 62, 2023, 10.1002/dvg.23581. hal-04427551

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# Epithelial-to-mesenchymal plasticity from development to disease: An introduction to the special issue

The term “Epithelial-Mesenchymal Transformation” was introduced by Elizabeth Hay to define the phenomena that cells can switch from epithelial to mesenchymal states during tissue morphogenesis and homeostasis. The term “transformation” is later replaced by the more accurate word “transition” to better reflect the non-binary nature of the process (see [Hay, 2005] and references therein). EMT is indeed a staple of embryonic development as cells must proliferate and move in three dimensions to form and rearrange tissues and organs at the right place and time. For that, they frequently toggle between relatively cohesive and stable epithelial states and more dynamic and loose mesenchymal arrangements (Nieto et al., 2016). In many cases, and as discussed below, EMT occurs in cells that will eventually migrate. However, one should not systematically associate EMT with migratory behavior. During fibrosis, there are several examples of cells adopting partial E/M phenotypes that do not undertake migration but nonetheless contribute to disease progression via this phenotypic change (see [Liu et al., 2022] for discussion).

Classical examples of EMT include gastrulation and neural crest development. Mesoderm is induced at the interface between ectoderm and endoderm. Initially epithelial, mesoderm progenitor cells undergo a conversion towards mesenchymal states to ingress, migrate and intercalate between ectoderm and endoderm during gastrulation. At the cervical and trunk level, the most axial of these migratory mesodermal cells re-epithelialize into repetitive structures called somites on either sides of the neural tube (Benazeraf & Pourquie, 2013). These somites will then undergo another round of mesenchymalization at their ventral side to form the sclerotome, which later produces vertebrae, and at their dorsal side to produce the dermis and muscle progenitors. Neural crest cells are multipotent stem cells induced at the lateral border of the prospective central nervous system (Gouignard et al., 2018). As neurulation proceeds, neural crest cells emerge from the neuroepithelium by converting into highly migratory mesenchyme cells, many of which will later re-aggregate to form solid structures ranging from condensed connective tissue, such as ganglia of the peripheral nervous system, to epithelial cells of the corneal endothelium of the eye (Dupin et al., 2006). Other examples of EMT during development occurs in the lateral mesoderm, the liver diverticulum, the pancreatic buds or the endocardium (Lim & Thiery, 2012).

Importantly, EMT is not specific to embryonic development but also occurs during various conditions, including wound healing, fibrosis and cancer metastasis (Yang et al., 2020). Molecular and cellular mechanisms controlling EMT are evolutionally conserved due to their

physiological importance. Therefore, these settings provide knowledge databases on how EMT is controlled, what signals may trigger EMT and how cells change their E/M characteristics over time. The range of EMT possibilities and variations is huge. For instance, gastrulation occurs in all animals, except Porifera (sponges) and Placozoa (Lanna, 2015; Martindale, 2005). While the basic principles are conserved, EMT during gastrulation happens at different initial conditions in each species (i.e., number of cells, topology etc.). As for neural crest delamination, there are variations from species to species but also among neural crest subpopulations in each animal. Therefore, by studying the wide diversity of physiological EMTs in multiple experimental models, we can build a catalogue of various possible scenarios for cells to undergo EMT and its reverse transition, MET.

We all hope for the right marker (or set of markers) whose expression could discriminate between cells that have not yet undergone EMT and the ones that are engaged in EMT. Ideally at the earliest possible moment, so that these expressions might have predictive value regarding to what cells will do next. However, the overwhelming diversity of EMTs in physiological settings suggests that the search for the right markers may be a wild goose chase. This is why we think that the previously suggested term “Epithelial-Mesenchymal Plasticity (EMP)” (Haerinck et al., 2023; Yang et al., 2020) should be used to define the framework within which cells’ journeys around E and M status are studied. This makes it possible to think about and address plasticity between epithelial and mesenchymal cell states as a whole without being limited to the simplistic and reductive definitions often linked to the EMT/MET terminologies. The terms EMT and MET are still valid but they represent specific cases of unidirectional changes between E and M that take place at a smaller time scale within the global long-term EMP context. This framework puts less pressure on the use of markers as proof of a cell status and more importance on cell behavior and overall capabilities of cells to interact with one another.

One telling example is that of cadherins. The expressions of these calcium-dependent cell adhesion molecules are often used as definitive markers of epithelial versus mesenchymal identities such that cells expressing E-cadherin (Cadherin-1, CDH1) are considered epithelial while those expressing N-cadherin (Cadherin-2, CDH2) are thought to be mesenchymal. However, neural crest development gives a different perspective on this idea. In Xenopus, pre-migratory cephalic neural crest cells are epithelial and turn into mesenchymal migratory cells via an E to N cadherin switch (Scarpa et al., 2015). However, it should be noted that these cells maintain some
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Epithelial-to-Mesenchymal Plasticity from development to disease: an
introduction to the special issue.

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Hervé Acloque¹, Jing Yang² and Eric Theveneau³*

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1. Université Paris Saclay, INRAE, AgroParisTech, GABI, Domaine de Vilvert, 78350 Jouy en Josas, France

2. Department of Pharmacology and of Pediatrics, Moores Cancer Center, University of California, San Diego, School of Medicine, La Jolla, CA 92093, USA

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3. Molecular, Cellular and Developmental Biology department (MCD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 118 route de Narbonne 31062, Toulouse Cedex 09, France.

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*corresponding author: eric.theveneau@univ-tlse3

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Abstract

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Epithelial-Mesenchymal Transition (EMT) refers to the ability of cells to switch between
epithelial and mesenchymal states, playing critical roles in embryonic development, wound healing,
fibrosis, and cancer metastasis. Here, we discuss some examples that challenge the use of specific
markers to define EMT, noting that their expression may not always correspond to the expected
epithelial or mesenchymal identity. In concordance with recent development in the field, we emphasize
the importance of generalizing the use of the term Epithelial-Mesenchymal Plasticity (EMP), to better
capture the diverse and context-dependent nature of the bidirectional journey that cells can undertake
between the E and M phenotypes. We highlight the usefulness of studying a wide range of physiological
EMT scenarios, stress the value of the dynamic of expression of EMP regulators and advocate,
whenever possible, for more systematic functional assays to assess cellular states.

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Main Text

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The term “Epithelial-Mesenchymal Transformation” was introduced by Elizabeth Hay to define
the phenomena that cells can switch from epithelial to mesenchymal states during tissue
morphogenesis and homeostasis. The term “transformation” is later replaced by the more accurate
word “transition” to better reflect the non-binary nature of the process (see (Hay, 2005) and references
therein). EMT is indeed a staple of embryonic development as cells must proliferate and move in three
dimensions to form and rearrange tissues and organs at the right place and time. For that, they
frequently toggle between relatively cohesive and stable epithelial states and more dynamic and loose
mesenchymal arrangements (Nieto et al., 2016). In many cases, and as discussed below, EMT occurs in
1

2

Epithelial-to-Mesenchymal Plasticity from development to disease: an
introduction to the special issue.

3

Hervé Acloque¹, Jing Yang² and Eric Theveneau³*

4

5

6

1. Université Paris Saclay, INRAE, AgroParisTech, GABI, Domaine de Vilvert, 78350 Jouy en Josas, France

2. Department of Pharmacology and of Pediatrics, Moores Cancer Center, University of California, San Diego, School of Medicine, La Jolla, CA 92093, USA

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3. Molecular, Cellular and Developmental Biology department (MCD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 118 route de Narbonne 31062, Toulouse Cedex 09, France.

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*corresponding author: eric.theveneau@univ-tlse3

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**Abstract**

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Epithelial-Mesenchymal Transition (EMT) refers to the ability of cells to switch between
epithelial and mesenchymal states, playing critical roles in embryonic development, wound healing,
fibrosis, and cancer metastasis. Here, we discuss some examples that challenge the use of specific
markers to define EMT, noting that their expression may not always correspond to the expected
epithelial or mesenchymal identity. In concordance with recent development in the field, we emphasize
the importance of generalizing the use of the term Epithelial-Mesenchymal Plasticity (EMP), to better
capture the diverse and context-dependent nature of the bidirectional journey that cells can undertake
between the E and M phenotypes. We highlight the usefulness of studying a wide range of physiological
EMT scenarios, stress the value of the dynamic of expression of EMP regulators and advocate,
whenever possible, for more systematic functional assays to assess cellular states.

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**Main Text**

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The term “Epithelial-Mesenchymal Transformation” was introduced by Elizabeth Hay to define
the phenomena that cells can switch from epithelial to mesenchymal states during tissue
morphogenesis and homeostasis. The term “transformation” is later replaced by the more accurate
word “transition” to better reflect the non-binary nature of the process (see (Hay, 2005) and references
therein). EMT is indeed a staple of embryonic development as cells must proliferate and move in three
dimensions to form and rearrange tissues and organs at the right place and time. For that, they
frequently toggle between relatively cohesive and stable epithelial states and more dynamic and loose
mesenchymal arrangements (Nieto et al., 2016). In many cases, and as discussed below, EMT occurs in
expression of E-cadherin (Huang et al., 2016). During migration of Xenopus neural crest cells, E-cadherin is no longer involved in cell-cell junctions and its loss of function impairs adhesion to fibronectin (Huang et al., 2016). Interestingly, the role of maintaining cell-cell junctions during migration is performed by N-cadherin whose loss-of-function impairs cell-cell adhesion (Theveneau et al., 2010). Therefore, Xenopus neural crest cells display an intermediate E/M phenotype but this is due to the fact that these cells maintain some transient junctions while migrating (via N-cadherin), not because they have some residual level of E-cadherin expression. In other cell types, E-cadherin has been shown to be involved in cell-cell adhesions allowing collective motion (Bazellieres et al., 2015). By contrast, in chicken embryos, cephalic neural crest cells emerge from a neuroepithelium that expresses both E and N-cadherin. These cells become mesenchymal and initiate migration while expressing E and N-cadherin (Dady et al., 2012; Dady & Duband, 2017; Rogers et al., 2018; Theveneau et al., 2007). Whereas, at trunk level, chicken neural crest cells depart from a neuroepithelium that only expresses N-cadherin and their mesenchymalization occurs while maintaining N-cadherin expression (Shoval et al., 2007). These three examples demonstrate that mesenchymalization of neural crest cells can occur concomitantly with an upregulation, a stable expression or a loss of N-cadherin expression at mRNA level and a subsequent complex dynamics at protein level. Thus, the change from E-to-M in neural crest cells cannot be simply attributed to a cadherin switch. Similar observations can be made in other cell types. In gastrulating paraxial mesoderm, N-cadherin is used for epithelialization of the somites from the mesenchymal presomitic mesoderm (Chal et al., 2017). In adults, N-cadherin is endogenously expressed in multiple organs under normal conditions (i.e., liver, testis, adrenal gland, and cardiomyocytes). These different examples highlight the fact that high expression levels of N-cadherin at mRNA level may correspond to very different situations in terms of stability of the N-cadherin protein and cell behavior (i.e., migration, active epithelialization or stable differentiated organ). This is true for the neuroepithelium and migratory neural crest cells. In the former, N-cadherin is involved in stable junctions and maintains the epithelium. In the latter, the N-cadherin protein is cleaved and endocytosed, thus preventing neural crest cells from forming stable cell-cell adhesions (Kuriyama et al., 2014; Shoval et al., 2007). Overall, this indicates that expressions of cadherins by themselves are not indicative of a mesenchymal or epithelial status and that the context in which these expressions occur has to be taken into account.

Another example is that of transcription factors that act upstream of the EMP programs during development such as Snai, Twist, and Zeb family members. Developmental studies highlighted the critical roles that many of these genes play in destabilizing epithelial features by repressing cadherin expressions, upregulating proteases and modifying extracellular matrix. When examining EMP transcription factors in the Human Protein Atlas (Human Protein Atlas proteinatlas.org; [Karlsson et al., 2021; Uhlen et al., 2015]), one cannot help noticing that several of them also have normal expression in certain cell types. One extreme example is that of the specialized epithelial cells of the male gonads, known as Sertoli cells. These cells have an endogenous

expression of Twist1, Zeb1, MMP14, and N-cadherin. By all measures, a cell expressing simultaneously these four proteins would be considered a highly invasive migratory cell by most developmental and cancer biologists. Yet, Sertoli cells are epithelial and non-migratory. Another example is the expression of Snai1 and Snai2 in breast adipocytes, cells with limited migratory potential under normal physiological conditions. The same observation can be made for metalloproteinases whose expression is often used as a sign of invasive behavior. While such enzymes can degrade extracellular matrix, generate tracks for migration and invasion, numerous epithelia express metalloproteinases without displaying signs of EMP. During neural tube development, MMP14 mRNA is strongly detected throughout the entire tissue (Andrieu et al., 2020). However, MMP14-dependent EMP only occurs in the neural crest (Andrieu et al., 2020) while the rest of the neural tube maintains an epithelial organization and a continuous basement membrane. Therefore, while MMPs are involved in invasion, expression of MMPs alone cannot define invasiveness, a trait only assessed via functional assays such as assessing motility, matrix degradation or the ability to intermix with other cells. Finally, in some cell types, E-cadherin is co-expressed with known repressors of its expression under normal circumstances. Such examples include the co-expression of E-cadherin and Snai2 in migratory cephalic neural crest cells in chicken embryos (Rogers et al., 2018). This shows that levels of regulation other than gene expression, post-transcriptionally and post-translationally, are relevant as well.

What do we learn from all of this? First, that the dynamic of expression, taking into consideration the actual protein level, is likely to be more informative than a single measure at the RNA level. Unfortunately, expression analyses at multiple time points in pathological contexts is often technically challenging to achieve and/or initial control expression level in each patient/tissue may not be known. When possible, such analyses may yield seemingly surprising results if only a small set of factors is considered, such as the described reduction of Snai2 expression in malignant prostate cancer compared to normal prostate (Esposito et al., 2015). If taken stricto sensu in the classical EMT framework, such loss of Snai2 expression may be interpreted as an absence of conversion from E to M associated with prostate cancer progression. A more likely situation is that of a progressive change of transcription factor signature as seen in melanoma where the progression from proliferation to invasion is associated with a change from Snai2/Zeb2 to a Twist1/Zeb1 profile (Caramel et al., 2013). Interestingly, such transitions are also observed in neural crest development. In Xenopus cephalic neural crest cells, Snai1/Snai2 are expressed in pre-migratory cells (Aybar et al., 2003). Then, several hours later, Twist expression starts (Hopwood et al., 1989). As migration proceeds, Twist expression is maintained and increased while those of Snai1 and 2 are progressively lost. Functional evidence further indicate that Twist physically interacts with Snail proteins to inhibit their function (Lander et al., 2013). Second, that the context plays a crucial part in interpreting the data. EMP gene expression in cells that do or do not express such genes under normal physiological conditions will not carry the same weight. Third, part of that context may be the subcellular localization of some of the putative EMP regulators
cells that will eventually migrate. However, one should not systematically associate EMT with migratory behavior. During fibrosis, there are several examples of cells adopting partial E/M phenotypes that do not undertake migration but nonetheless contribute to disease progression via this phenotypic change (see (Liu et al., 2022) for discussion).

Classical examples of EMT include gastrulation and neural crest development. Mesoderm is induced at the interface between ectoderm and endoderm. Initially epithelial, mesoderm progenitor cells undergo a conversion towards mesenchymal states to ingress, migrate and intercalate between ectoderm and endoderm during gastrulation. At the cervical and trunk level, the most axial of these migratory mesodermal cells re-epithelialize into repetitive structures called somites on either sides of the neural tube (Benazeraf & Pourquie, 2013). These somites will then undergo another round of mesenchymalization at their ventral side to form the sclerotome, which later produces vertebrae, and at their dorsal side to produce the dermis and muscle progenitors. Neural crest cells are multipotent stem cells induced at the lateral border of the prospective central nervous system (Gouignard et al., 2018). As neurulation proceeds, neural crest cells emerge from the neuroepithelium by converting into highly migratory mesenchyme cells, many of which will later re-aggregate to form solid structures ranging from condensed connective tissue, such as ganglia of the peripheral nervous system, to epithelial cells of the corneal endothelium of the eye (Dupin et al., 2006). Other examples of EMT during development occurs in the lateral mesoderm, the liver diverticulum, the pancreatic buds or the endocardium (Lim & Thiery, 2012).

Importantly, EMT is not specific to embryonic development but also occurs during various pathogenesis conditions, including wound healing, fibrosis and cancer metastasis (Yang et al., 2020). Molecular and cellular mechanisms controlling EMT are evolutionally conserved due to their physiological importance. Therefore, these settings provide knowledge databases on how EMT is controlled, what signals may trigger EMT and how cells change their E/M characteristics over time. The range of EMT possibilities and variations is huge. For instance, gastrulation occurs in all animals, except Porifera (sponges) and Placozoa (Lanna, 2015; Martindale, 2005). While the basic principles are conserved, EMT during gastrulation happens at different initial conditions in each species (i.e number of cells, topology etc.). As for neural crest delamination, there are variations from species to species but also among neural crest subpopulations in each animal. Therefore, by studying the wide diversity of physiological EMTs in multiple experimental models, we can build a catalogue of various possible scenarios for cells to undergo EMT and its reverse transition, MET.

We all hope for the right marker (or set of markers) whose expression could discriminate between cells that have not yet undergone EMT and the ones that are engaged in EMT. Ideally at the
cells that will eventually migrate. However, one should not systematically associate EMT with migratory behavior. During fibrosis, there are several examples of cells adopting partial E/M phenotypes that do not undertake migration but nonetheless contribute to disease progression via this phenotypic change (see (Liu et al., 2022) for discussion).

Classical examples of EMT include gastrulation and neural crest development. Mesoderm is induced at the interface between ectoderm and endoderm. Initially epithelial, mesoderm progenitor cells undergo a conversion towards mesenchymal states to ingress, migrate and intercalate between ectoderm and endoderm during gastrulation. At the cervical and trunk level, the most axial of these migratory mesodermal cells re-epithelialize into repetitive structures called somites on either sides of the neural tube (Benazeraf & Pourquie, 2013). These somites will then undergo another round of mesenchymalization at their ventral side to form the sclerotome, which later produces vertebrae, and at their dorsal side to produce the dermis and muscle progenitors. Neural crest cells are multipotent stem cells induced at the lateral border of the prospective central nervous system (Gouignard et al., 2018). As neurulation proceeds, neural crest cells emerge from the neuroepithelium by converting into highly migratory mesenchyme cells, many of which will later re-aggregate to form solid structures ranging from condensed connective tissue, such as ganglia of the peripheral nervous system, to epithelial cells of the corneal endothelium of the eye (Dupin et al., 2006). Other examples of EMT during development occurs in the lateral mesoderm, the liver diverticulum, the pancreatic buds or the endocardium (Lim & Thiery, 2012).

Importantly, EMT is not specific to embryonic development but also occurs during various pathogenesis conditions, including wound healing, fibrosis and cancer metastasis (Yang et al., 2020). Molecular and cellular mechanisms controlling EMT are evolutionally conserved due to their physiological importance. Therefore, these settings provide knowledge databases on how EMT is controlled, what signals may trigger EMT and how cells change their E/M characteristics over time. The range of EMT possibilities and variations is huge. For instance, gastrulation occurs in all animals, except Porifera (sponges) and Placozoa (Lanna, 2015; Martindale, 2005). While the basic principles are conserved, EMT during gastrulation happens at different initial conditions in each species (i.e number of cells, topology etc.). As for neural crest delamination, there are variations from species to species but also among neural crest subpopulations in each animal. Therefore, by studying the wide diversity of physiological EMTs in multiple experimental models, we can build a catalogue of various possible scenarios for cells to undergo EMT and its reverse transition, MET.

We all hope for the right marker (or set of markers) whose expression could discriminate between cells that have not yet undergone EMT and the ones that are engaged in EMT. Ideally at the
<figure><img src="image_1.png" /></figure>

themselves. In the case of metalloproteinases, while they obviously
can affect the matrix, they need to be presented at the cell surface or
released extracellularly to do so in the first place. We now know that
many of such proteins have complex subcellular trafficking and can be
kept intracellularly (Jobin et al., 2017), in some cases to promote EMP,
as seen in Xenopus neural crest (Gouignard et al., 2023). Transcription
factors also traffic between the cytosol and preventing entry into the
nucleus can block their function as shown for TWIST1 in response to
different extracellular matrix rigidities (Fattet et al., 2020; Wei
et al., 2015). Fourth, some EMP regulators may have additional func-
tions beyond regulating EMP. Snail for instance can influence cancer
progression without triggering E-cadherin downregulation and EMP
(Paul et al., 2023) and Snai2 is required for normal hematopoiesis
(Pioli & Weis, 2014). Twist1 is known as an important factor for meso-
derm development and differentiation most likely by modulating FGF
and Shh signaling as well as Hand proteins (Qin et al., 2012) but some
of these targets might still be related to EMP. Overexpression of
Sox10 is sufficient to promote partial or complete mesenchymaliza-
tion in the neuroepithelium (McKeown et al., 2005). However, during
normal neural crest development it promotes pigment cell formation
(Aoki et al., 2003) whereas its inhibition does not affect EMT and
migration (Aoki et al., 2003; Honore et al., 2003). Therefore, in some
cases, expression of some EMP genes may be related to cell identity/
lineage rather than cell behavior. These observations require us to
rethink the framework associated with the initial definition of EMT by
proposing a more flexible paradigm, where the plasticity between E
and M states is tissue and context-dependent and cannot be reduced
to a few key markers.

These observations stress the importance of monitoring expres-
sion of putative EMP regulators across multiple time points and to
perform functional assays to assess the cells' EMP state and potential.
In the context of cell and developmental biology studies multiple
assays can be (and often are) implemented alongside expression ana-
lyses: migration/invasion assays, cell-cell and cell-matrix adhesion
assays, collision assays, matrix remodeling/degradation assays, and so
forth. Given the wealth of information that can be extracted from
such experiments it would be greatly beneficial if the diagnosis/
prognosis workflow in the clinical context could integrate such
approaches as routine procedures from patients' biopsies. Currently,
oncology centers in which there is a functional daily integration of
clinical and research departments are the exception rather than the
norm. There have been promising attempts to harness the power of
classical embryology techniques in the context of oncology via, for
instance, modified migration/invasion assays using patients' cells
grafted in avian embryos (Delloye-Bourgeois et al., 2017; Jarrosson
et al., 2021; Jarrosson et al., 2023). Still very marginal a few years ago,
the use of these chimeras is expanding, both in the academic world
and biotech companies. Organoid development, from embryonic or
adult stem cells, may also help to recapitulate the features and
dynamic of EMP during organogenesis, in physiology and pathologies.
It allows to access and assess processes that may be difficult to
observe and to quantify either in vivo or in vitro in 2D cell culture
systems.

When people discuss EMP from development to diseases, they
usually mean that EMP can be found in a large spectrum of biological
systems from the most physiological (i.e., development, healing) all
the way up to pathological settings (i.e., fibrosis and cancer). This,
however, maintains the various fields of investigation on parallel tra-
jectories with researchers comparing systems, drawing similarities,
and searching for correlations. But we may be ignoring another level
of analysis that could be more relevant. Should we actually follow
cells from development to disease? Thus, considering the life of cells
from development to normal homeostasis to the pathology? Organs
are composed of cells that have a history through which they have
acquired a given gene expression profile, a given morphology and a
given set of interactions with their neighboring cells in their organs
and with adjacent organs. While some organs are formed from cells
that never underwent EMP (epidermis), other went through one
(i.e., ganglia of the peripheral nervous system), two (i.e., dermis, skel-
etal muscle) or three (i.e., cushion mesenchyme of the heart) round
trips between epithelial and mesenchymal states. Should we treat
the variations in gene expression profiles and protein levels differ-
ently when dealing with cells from tissues that experienced one,
two, three or no EMT-MET at all throughout their lives? Or when
considering cells that display different endogenous expression of
genes with EMP potential? Plasticity around E/M states is known to
impact stemness and survival, in addition to migratory and invasive
properties. Are cells durably affected by a chronic exposure to sig-
nals triggering EMP? Genome-scale epigenetic modifications have
been documented during EMP (Malouf et al., 2013; McDonald
et al., 2011). Do they have long-lasting effects in terms of compe-
tence to toggle between E, M and intermediate states? We hope
that future systematic studies could address the long-term effects
(if any) of successive EMT-MET events on cells and their putative
impact on subsequent EM plasticity.

This Editorial only scratches the surface of the complexity of
EMP and the multiple questions it raises. EMP is a rapidly expanding a
field of research and our understanding of its molecular and cellular
implementation as well as its functional relationship with normal and
pathological processes is a work in progress. In this special issue, we
have assembled a collection of reviews and research articles looking
at EMP in a wide range of contexts such as lateral plate mesoderm
and neural crest development, cancer cell dormancy or kidney fibrosis.
We hope that readers will find the content of the Special Issue to be
intellectually stimulating.

ACKNOWLEDGMENTS

Hervé Acloque is an INRAE senior researcher supported by funding
from ANR PluS4PiGs (ANR-19-CE20-0019). Jing Yang has been sup-
ported by grants from NCI (1R01CA262794, 1R01CA174869,
1R01CA206880, and 1R01CA236386) and CDMRP DOD Breast Can-
cer Program BC170283. Eric Theveneau is a research director at the
French National Research Center (CNRS) and supported by grants
from ANR SingleCrest (ANR-21-CE13-0028-02), the Association pour
la Recherche contre le Cancer (ARC, ARCPJA22020060002084), the
Fondation pour la Recherche Médicale (FRM AJE201224).
65 earliest possible moment, so that these expressions might have predictive value regarding to what cells 66 will do next. However, the overwhelming diversity of EMTs in physiological settings suggests that the 67 search for the right markers may be a wild goose chase. This is why we think that the previously 68 suggested term "Epithelial-Mesenchymal Plasticity (EMP)" (Haerinck et al., 2023; Yang et al., 2020) 69 should be used to define the framework within which cells' journeys around E and M status are studied. 70 This makes it possible to think about and address plasticity between epithelial and mesenchymal cell 71 states as a whole without being limited to the simplistic and reductive definitions often linked to the 72 EMT/MET terminologies. The terms EMT and MET are still valid but they represent specific cases of 73 unidirectional changes between E and M that take place at a smaller time scale within the global long- 74 term EMP context. This framework puts less pressure on the use of markers as proof of a cell status 75 and more importance on cell behavior and overall capabilities of cells to interact with one another. 76

76 One telling example is that of cadherins. The expressions of these calcium-dependent cell 77 adhesion molecules are often used as definitive markers of epithelial versus mesenchymal identities 78 such that cells expressing E-cadherin (Cadherin-1, CDH1) are considered epithelial while those 79 expressing N-cadherin (Cadherin-2, CDH2) are thought to be mesenchymal. However, neural crest 80 development gives a different perspective on this idea. In Xenopus, pre-migratory cephalic neural crest 81 cells are epithelial and turn into mesenchymal migratory cells via an E to N cadherin switch (Scarpa et 82 al., 2015). However, it should be noted that these cells maintain some expression of E-cadherin (Huang 83 et al., 2016). During migration of Xenopus neural crest cells, E-cadherin is no longer involved in cell-cell 84 junctions and its loss of function impairs adhesion to fibronectin (Huang et al., 2016). Interestingly, the 85 role of maintaining cell-cell junctions during migration is performed by N-cadherin whose loss-of- 86 function impairs cell-cell adhesion (Theveneau et al., 2010). Therefore, Xenopus neural crest cells 87 display an intermediate E/M phenotype but this is due to the fact that these cells maintain some 88 transient junctions while migrating, not because they have some residual level of E-cadherin 89 expression. In other cell types, E-cadherin has been shown to be involved in cell-cell adhesions allowing 90 collective motion (Bazellieres et al., 2015). By contrast, in chicken embryos, cephalic neural crest cells 91 emerge from a neuroepithelium that expresses both E and N-cadherin. These cells become 92 mesenchymal and initiate migration while expressing E and N-cadherin (Dady et al., 2012; Dady & 93 Duband, 2017; Rogers et al., 2018; Theveneau et al., 2007). Whereas, at trunk level, chicken neural 94 crest cells depart from a neuroepithelium that only expresses N-cadherin and their 95 mesenchymalization occurs while maintaining N-cadherin expression (Shoval et al., 2007). These three 96 examples demonstrate that mesenchymalization of neural crest cells can occur concomitantly with an 97 upregulation, a stable expression or a loss of N-cadherin expression at mRNA level and a subsequent 98 complex dynamics at protein level. Thus, the change from E-to-M in neural crest cells cannot be simply
earliest possible moment, so that these expressions might have predictive value regarding to what cells
will do next. However, the overwhelming diversity of EMTs in physiological settings suggests that the
search for the right markers may be a wild goose chase. This is why we think that the previously
suggested term "Epithelial-Mesenchymal Plasticity (EMP)" (Haerinck et al., 2023; Yang et al., 2020)
should be used to define the framework within which cells' journeys around E and M status are studied.
This makes it possible to think about and address plasticity between epithelial and mesenchymal cell
states as a whole without being limited to the simplistic and reductive definitions often linked to the
EMT/MET terminologies. The terms EMT and MET are still valid but they represent specific cases of
unidirectional changes between E and M that take place at a smaller time scale within the global long-
term EMP context. This framework puts less pressure on the use of markers as proof of a cell status
and more importance on cell behavior and overall capabilities of cells to interact with one another.

One telling example is that of cadherins. The expressions of these calcium-dependent cell
adhesion molecules are often used as definitive markers of epithelial versus mesenchymal identities
such that cells expressing E-cadherin (Cadherin-1, CDH1) are considered epithelial while those
expressing N-cadherin (Cadherin-2, CDH2) are thought to be mesenchymal. However, neural crest
development gives a different perspective on this idea. In Xenopus, pre-migratory cephalic neural crest
cells are epithelial and turn into mesenchymal migratory cells via an E to N cadherin switch (Scarpa et
al., 2015). However, it should be noted that these cells maintain some expression of E-cadherin (Huang
et al., 2016). During migration of Xenopus neural crest cells, E-cadherin is no longer involved in cell-cell
junctions and its loss of function impairs adhesion to fibronectin (Huang et al., 2016). Interestingly, the
role of maintaining cell-cell junctions during migration is performed by N-cadherin whose loss-of-
function impairs cell-cell adhesion (Theveneau et al., 2010). Therefore, Xenopus neural crest cells
display an intermediate E/M phenotype but this is due to the fact that these cells maintain some
transient junctions while migrating, not because they have some residual level of E-cadherin
expression. In other cell types, E-cadherin has been shown to be involved in cell-cell adhesions allowing
collective motion (Bazellieres et al., 2015). By contrast, in chicken embryos, cephalic neural crest cells
emerge from a neuroepithelium that expresses both E and N-cadherin. These cells become
mesenchymal and initiate migration while expressing E and N-cadherin (Dady et al., 2012; Dady &
Duband, 2017; Rogers et al., 2018; Theveneau et al., 2007). Whereas, at trunk level, chicken neural
crest cells depart from a neuroepithelium that only expresses N-cadherin and their
mesenchymalization occurs while maintaining N-cadherin expression (Shoval et al., 2007). These three
examples demonstrate that mesenchymalization of neural crest cells can occur concomitantly with an
upregulation, a stable expression or a loss of N-cadherin expression at mRNA level and a subsequent
complex dynamics at protein level. Thus, the change from E-to-M in neural crest cells cannot be simply
## FUNDING INFORMATION

Agence Nationale de la Recherche, Grant/Award Numbers: ANR-19-CE20-0019, ANR-21-CE13-0028-02; Association pour la Recherche sur le Cancer, Grant/Award Number: ARCPJA22020060002084; Congressionally Directed Medical Research Programs, Grant/Award Number: BC170283; Fondation pour la Recherche Médicale, Grant/Award Number: FRM AJE201224; National Cancer Institute, Grant/Award Numbers: 1R01CA262794, 1R01CA174869, 1R01CA206880, 1R01CA236386

Hervé Acloque¹

Jing Yang²

Eric Theveneau³

¹INRAE, AgroParisTech, GABI, Université Paris Saclay, Jouy en Josas, France

²Department of Pharmacology and of Pediatrics, Moores Cancer Center, University of California San Diego, School of Medicine, La Jolla, California, USA

³Molecular, Cellular and Developmental Biology Department (MCD), Centre de Biologie Intégrative (CBI), CNRS, UPS, Université de Toulouse, Toulouse, France

## Correspondence

Eric Theveneau, Molecular, Cellular and Developmental Biology Department (MCD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 118 route de Narbonne 31062, Cedex 09, Toulouse, France.

Email: eric.theveneau@univ-tlse3.fr

## ORCID

Hervé Acloque https://orcid.org/0000-0003-4761-1055

Eric Theveneau https://orcid.org/0000-0001-6510-5717

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Aoki, Y., Saint-Germain, N., Gyda, M., Magner-Fink, E., Lee, Y. H., Credidio, C., & Saint-Jeannet, J. P. (2003). Sox10 regulates the development of neural crest-derived melanocytes in xenopus. *Developmental Biology*, 259(1), 19-33.

Aybar, M. J., Nieto, M. A., & Mayor, R. (2003). Snail precedes slug in the genetic cascade required for the specification and migration of the xenopus neural crest. *Development*, 130(3), 483-494.

Bazellières, E., Conte, V., Elosegui-Artola, A., Serra-Picamal, X., Bintanel-Morcillo, M., Roca-Cusachs, P., Muñoz, J. J., Sales-Pardo, M., Guimerà, R., & Trepat, X. (2015). Control of cell-cell forces and collective cell dynamics by the intercellular adheshome. *Nature Cell Biology*, 17(4), 409-420. https://doi.org/10.1038/ncb3135

Benazeraf, B., & Pourquie, O. (2013). Formation and segmentation of the vertebrate body axis. *Annual Review of Cell and Developmental Biology*, 29, 1-26. https://doi.org/10.1146/annurev-cellbio-101011-155703

Caramel, J., Papadogeorgakis, E., Hill, L., Browne, G. J., Richard, G., Wierinckx, A., Saldanha, G., Osborne, J., Hutchinson, P., Tse, G., Lachuer, J., Puisieux, A., Pringle, J. H., Ansieau, S., & Tulchinsky, E.

(2013). A switch in the expression of embryonic EMT-inducers drives the development of malignant melanoma. *Cancer Cell*, 24(4), 466-480. https://doi.org/10.1016/j.ccr.2013.08.018

Chal, J., Guillot, C., & Pourquie, O. (2017). PAPC couples the segmentation clock to somite morphogenesis by regulating N-cadherin-dependent adhesion. *Development*, 144(4), 664-676. https://doi.org/10.1242/dev.143974

Dady, A., Blavet, C., & Duband, J. L. (2012). Timing and kinetics of E- to N-cadherin switch during neurulation in the avian embryo. *Developmental Dynamics*, 241(8), 1333-1349. https://doi.org/10.1002/dvdy.23813

Dady, A., & Duband, J. L. (2017). Cadherin interplay during neural crest segregation from the non-neural ectoderm and neural tube in the early chick embryo. *Developmental Dynamics*, 246(7), 550-565. https://doi.org/10.1002/dvdy.24517

Delloye-Bourgeois, C., Bertin, L., Thoinet, K., Jarrosson, L., Kindbeiter, K., Buffet, T., Tauszig-Delamasure, S., Bozon, M., Marabelle, A., Combaret, V., Bergeron, C., Derrington, E., & Castellani, V. (2017). Microenvironment-driven shift of cohesion/detachment balance within tumors induces a switch toward metastasis in neuroblastoma. *Cancer Cell*, 32(4), 427-443 e428. https://doi.org/10.1016/j.ccell.2017.09.006

Dupin, E., Creuzet, S., & Le Douarin, N. M. (2006). The contribution of the neural crest to the vertebrate body. *Advances in Experimental Medicine and Biology*, 589, 96-119. https://doi.org/10.1007/978-0-387-46954-6_6

Esposito, S., Russo, M. V., Airoldi, I., Tupone, M. G., Sorrentino, C., Barbarito, G., Di Meo, S., & Di Carlo, E. (2015). SNAI2/slug gene is silenced in prostate cancer and regulates neuroendocrine differentiation, metastasis-suppressor and pluripotency gene expression. *Onco target*, 6(19), 17121-17134. https://doi.org/10.18632/oncotarget.2736

Fattet, L., Jung, H. Y., Matsumoto, M. W., Aubol, B. E., Kumar, A., Adams, J. A., Chen, A. C., Sah, R. L., Engler, A. J., Pasquale, E. B., & Yang, J. (2020). Matrix rigidity controls epithelial-mesenchymal plasticity and tumor metastasis via a mechanoresponsive EPHA2/LYN complex. *Developmental Cell*, 54(3), 302-316.e307. https://doi.org/10.1016/j.devcel.2020.05.031

Gouignard, N., Andrieu, C., & Theveneau, E. (2018). Neural crest delamination and migration: Looking forward to the next 150 years. *Genesis*, 56 (6-7), e23107. https://doi.org/10.1002/dvg.23107

Gouignard, N., Bibonne, A., Mata, J. F., Bajanca, F., Berki, B., Barriga, E. H., Saint-Jeannet, J. P., & Theveneau, E. (2023). Paracrine regulation of neural crest EMT by placodal MMP28. *PLoS Biology*, 21(8), e3002261. https://doi.org/10.1371/journal.pbio.3002261

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Hopwood, N. D., Pluck, A., & Gurdon, J. B. (1989). A xenopus mRNA related to drosophila twist is expressed in response to induction in the mesoderm and the neural crest. *Cell*, 59(5), 893-903.

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Jarrosson, L., Costechareyre, C., Gallix, F., Ciré, S., Gay, F., Imbaud, O., Teinturier, R., Marangoni, E., Aguéra, K., Delloye-Bourgeois, C., & Castellani, V. (2021). An avian embryo patient-derived xenograft model for preclinical studies of human breast cancers. *iScience*, 24(12), 103423. https://doi.org/10.1016/j.isci.2021.103423
attributed to a cadherin switch. Similar observations can be made in other cell types. In gastrulating
paraxial mesoderm, N-cadherin is used for epithelialization of the somites from the mesenchymal
presomitic mesoderm (Chal et al., 2017). In adults, N-cadherin is endogenously expressed in multiple
organs under normal conditions (i.e liver, testis, adrenal gland and cardiomyocytes). These different
examples highlight the fact that high expression levels of N-cadherin at mRNA level may correspond to
very different situations in terms of stability of the N-cadherin protein and cell behavior (i.e. migration,
active epithelialization or stable differentiated organ). This is true for the neuroepithelium and
migratory neural crest cells. In the former, N-cadherin is involved in stable junctions and maintains the
epithelium. In the latter, the N-cadherin protein is cleaved and endocytosed, thus preventing neural
crest cells from forming stable cell-cell adhesions (Kuriyama et al., 2014; Shoval et al., 2007). Overall,
this indicates that expressions of cadherins by themselves are not indicative of a mesenchymal or
epithelial status and that the context in which these expressions occur has to be taken into account.

Another example is that of transcription factors that act upstream of the EMP programs during
development such as Snai, Twist, and Zeb family members. Developmental studies highlighted the
critical roles that many of these genes play in destabilizing epithelial features by repressing cadherin
expressions, upregulating proteases and modifying extracellular matrix. When examining EMP
transcription factors in the Human Protein Atlas (Human Protein Atlas proteinatlas.org; (Karlsson et al.,
2021; Uhlen et al., 2015)), one cannot help noticing that several of them also have normal expression
in certain cell types. One extreme example is that of the specialized epithelial cells of the male gonads,
known as Sertoli cells. These cells have an endogenous expression of Twist1, Zeb1, MMP14 and N-
cadherin. By all measures, a cell expressing simultaneously these four proteins would be considered a
highly invasive migratory cell by most developmental and cancer biologists. Yet, Sertoli cells are
epithelial and non-migratory. Another example is the expression of Snai1 and Snai2 in breast
adipocytes, cells with limited migratory potential under normal physiological conditions. The same
observation can be made for metalloproteinases whose expression is often used as a sign of invasive
behavior. While such enzymes can degrade extracellular matrix, generate tracks for migration and
invasion, numerous epithelia express metalloproteinases without displaying signs of EMP. During
neural tube development, MMP14 mRNA is strongly detected throughout the entire tissue (Andrieu et
al., 2020). However, MMP14-dependent EMP only occurs in the neural crest (Andrieu et al., 2020) while
the rest of the neural tube maintains an epithelial organization and a continuous basement membrane.
Therefore, while MMPs are involved in invasion, expression of MMPs alone cannot define invasiveness,
a trait only assessed via functional assays such as assessing motility, matrix degradation or the ability
to intermix with other cells. Finally, in some cell types E-cadherin is co-expressed with known repressors
of its expression under normal circumstances. Such examples include the co-expression of E-cadherin
attributed to a cadherin switch. Similar observations can be made in other cell types. In gastrulating
paraxial mesoderm, N-cadherin is used for epithelialization of the somites from the mesenchymal
presomitic mesoderm (Chal et al., 2017). In adults, N-cadherin is endogenously expressed in multiple
organs under normal conditions (i.e liver, testis, adrenal gland and cardiomyocytes). These different
examples highlight the fact that high expression levels of N-cadherin at mRNA level may correspond to
very different situations in terms of stability of the N-cadherin protein and cell behavior (i.e. migration,
active epithelialization or stable differentiated organ). This is true for the neuroepithelium and
migratory neural crest cells. In the former, N-cadherin is involved in stable junctions and maintains the
epithelium. In the latter, the N-cadherin protein is cleaved and endocytosed, thus preventing neural
crest cells from forming stable cell-cell adhesions (Kuriyama et al., 2014; Shoval et al., 2007). Overall,
this indicates that expressions of cadherins by themselves are not indicative of a mesenchymal or
epithelial status and that the context in which these expressions occur has to be taken into account.

Another example is that of transcription factors that act upstream of the EMP programs during
development such as Snai, Twist, and Zeb family members. Developmental studies highlighted the
critical roles that many of these genes play in destabilizing epithelial features by repressing cadherin
expressions, upregulating proteases and modifying extracellular matrix. When examining EMP
transcription factors in the Human Protein Atlas (Human Protein Atlas proteinatlas.org; (Karlsson et al.,
2021; Uhlen et al., 2015)), one cannot help noticing that several of them also have normal expression
in certain cell types. One extreme example is that of the specialized epithelial cells of the male gonads,
known as Sertoli cells. These cells have an endogenous expression of Twist1, Zeb1, MMP14 and N-
cadherin. By all measures, a cell expressing simultaneously these four proteins would be considered a
highly invasive migratory cell by most developmental and cancer biologists. Yet, Sertoli cells are
epithelial and non-migratory. Another example is the expression of Snai1 and Snai2 in breast
adipocytes, cells with limited migratory potential under normal physiological conditions. The same
observation can be made for metalloproteinases whose expression is often used as a sign of invasive
behavior. While such enzymes can degrade extracellular matrix, generate tracks for migration and
invasion, numerous epithelia express metalloproteinases without displaying signs of EMP. During
neural tube development, MMP14 mRNA is strongly detected throughout the entire tissue (Andrieu et
al., 2020). However, MMP14-dependent EMP only occurs in the neural crest (Andrieu et al., 2020) while
the rest of the neural tube maintains an epithelial organization and a continuous basement membrane.
Therefore, while MMPs are involved in invasion, expression of MMPs alone cannot define invasiveness,
a trait only assessed via functional assays such as assessing motility, matrix degradation or the ability
to intermix with other cells. Finally, in some cell types E-cadherin is co-expressed with known repressors
of its expression under normal circumstances. Such examples include the co-expression of E-cadherin
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and Snai2 in migratory cephalic neural crest cells in chicken embryos (Rogers et al., 2018). This shows that levels of regulation other than gene expression, post-transcriptionally and post-translationally, are relevant as well.

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What do we learn from all of this? First, that the dynamic of expression, taking into consideration the actual protein level, is likely to be more informative than a single measure at the RNA level. Unfortunately, expression analyses at multiple time points in pathological contexts is often technically challenging to achieve and/or initial control expression level in each patient/tissue may not be known. When possible, such analyses may yield seemingly surprising results if only a small set of factors is considered, such as the described reduction of Snai2 expression in malignant prostate cancer compared to normal prostate (Esposito et al., 2015). If taken *stricto sensu* in the classical EMT framework, such loss of Snai2 expression may be interpreted as an absence of conversion from E to M associated with prostate cancer progression. A more likely situation is that of a progressive change of transcription factor signature as seen in melanoma where the progression from proliferation to invasion is associated with a change from Snai2/Zeb2 to a Twist1/Zeb1 profile (Caramel et al., 2013). Interestingly, such transitions are also observed in neural crest development. In Xenopus cephalic neural crest cells, Snai1/Snai2 are expressed in pre-migratory cells (Aybar et al., 2003). Then, several hours later, Twist expression starts (Hopwood et al., 1989). As migration proceeds, Twist expression is maintained and increased while those of Snai1 and 2 are lost. Functional evidence further indicate that Twist physically interact with Snail proteins to inhibit their function (Lander et al., 2013). Second, that the context plays a crucial part in interpreting the data. EMP gene expression in cells that do or do not express such genes under normal physiological conditions will not carry the same weight. Third, part of that context may be the subcellular localization of some of the putative EMP regulators themselves. In the case of metalloproteinases, while they obviously can affect the matrix, they need to be presented at the cell surface or released extracellularly to do so in the first place. We now know that many of such proteins have complex subcellular trafficking and can be kept intracellularly (Jobin et al., 2017), in some cases to promote EMP, as seen in Xenopus neural crest (Gouignard et al., 2023). Transcription factors also traffic between the cytosol and preventing entry into the nucleus can block their function as shown for TWIST1 in response to different extracellular matrix rigidities (Fattet et al., 2020; Wei et al., 2015). Fourth, some EMP regulators may have additional functions beyond regulating EMP. Snail for instance can influence cancer progression without triggering E-cadherin downregulation and EMP (Paul et al., 2023) and Snai2 is required for normal hematopoiesis (Pioli & Weis, 2014). Twist1 is known as an important factor for mesoderm development and differentiation most likely by modulating FGF and Shh signaling as well as Hand proteins (Qin et al., 2012) but some of these targets might still be related to EMP. Overexpression of Sox10 is sufficient to promote partial or complete mesenchymalization in
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and Snai2 in migratory cephalic neural crest cells in chicken embryos (Rogers et al., 2018). This shows that levels of regulation other than gene expression, post-transcriptionally and post-translationally, are relevant as well.

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What do we learn from all of this? First, that the dynamic of expression, taking into consideration the actual protein level, is likely to be more informative than a single measure at the RNA level. Unfortunately, expression analyses at multiple time points in pathological contexts is often technically challenging to achieve and/or initial control expression level in each patient/tissue may not be known. When possible, such analyses may yield seemingly surprising results if only a small set of factors is considered, such as the described reduction of Snai2 expression in malignant prostate cancer compared to normal prostate (Esposito et al., 2015). If taken *stricto sensu* in the classical EMT framework, such loss of Snai2 expression may be interpreted as an absence of conversion from E to M associated with prostate cancer progression. A more likely situation is that of a progressive change of transcription factor signature as seen in melanoma where the progression from proliferation to invasion is associated with a change from Snai2/Zeb2 to a Twist1/Zeb1 profile (Caramel et al., 2013). Interestingly, such transitions are also observed in neural crest development. In Xenopus cephalic neural crest cells, Snai1/Snai2 are expressed in pre-migratory cells (Aybar et al., 2003). Then, several hours later, Twist expression starts (Hopwood et al., 1989). As migration proceeds, Twist expression is maintained and increased while those of Snai1 and 2 are lost. Functional evidence further indicate that Twist physically interact with Snail proteins to inhibit their function (Lander et al., 2013). Second, that the context plays a crucial part in interpreting the data. EMP gene expression in cells that do or do not express such genes under normal physiological conditions will not carry the same weight. Third, part of that context may be the subcellular localization of some of the putative EMP regulators themselves. In the case of metalloproteinases, while they obviously can affect the matrix, they need to be presented at the cell surface or released extracellularly to do so in the first place. We now know that many of such proteins have complex subcellular trafficking and can be kept intracellularly (Jobin et al., 2017), in some cases to promote EMP, as seen in Xenopus neural crest (Gouignard et al., 2023). Transcription factors also traffic between the cytosol and preventing entry into the nucleus can block their function as shown for TWIST1 in response to different extracellular matrix rigidities (Fattet et al., 2020; Wei et al., 2015). Fourth, some EMP regulators may have additional functions beyond regulating EMP. Snail for instance can influence cancer progression without triggering E-cadherin downregulation and EMP (Paul et al., 2023) and Snai2 is required for normal hematopoiesis (Pioli & Weis, 2014). Twist1 is known as an important factor for mesoderm development and differentiation most likely by modulating FGF and Shh signaling as well as Hand proteins (Qin et al., 2012) but some of these targets might still be related to EMP. Overexpression of Sox10 is sufficient to promote partial or complete mesenchymalization in
167

the neuroepithelium (McKeown et al., 2005). However, during normal neural crest development it
promotes pigment cell formation (Aoki et al., 2003) whereas its inhibition does not affect EMT and
migration (Aoki et al., 2003; Honore et al., 2003). Therefore, in some cases, expression of some EMP
genes may be related to cell identity/lineage rather than cell behavior. These observations require us
to rethink the framework associated with the initial definition of EMT by proposing a more flexible
paradigm, where the plasticity between E and M states is tissue and context-dependent and cannot be
reduced to a few key markers.

168

169

170

171

172

173

174

These observations stress the importance of monitoring expression of putative EMP regulators
across multiple time points and to perform functional assays to assess the cells' EMP state and
potential. In the context of cell and developmental biology studies multiple assays can be (and often
are) implemented alongside expression analyses: migration/invasion assays, cell-cell and cell-matrix
adhesion assays, collision assays, matrix remodeling/degradation assays etc... Given the wealth of
information that can be extracted from such experiments it would be greatly beneficial if the
diagnosis/prognosis workflow in the clinical context could integrate such approaches as routine
procedures from patients' biopsies. Currently, oncology centers in which there is a functional daily
integration of clinical and research departments are the exception rather than the norm. There have
been promising attempts to harness the power of classical embryology techniques in the context of
oncology via, for instance, modified migration/invasion assays using patients' cells grafted in avian
embryos (Delloye-Bourgeois et al., 2017; Jarrosson et al., 2021; Jarrosson et al., 2023). Still very
marginal a few years ago, the use of these chimeras is expanding, both in the academic world and
biotech companies. Organoid development, from embryonic or adult stem cells, may also help to
recapitulate the features and dynamic of EMP during organogenesis, in physiology and pathologies. It
allows to access and assess processes that may be difficult to observe and to quantify either *in vivo* or
*in vitro* in 2D cell culture systems.

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

When people discuss EMP from development to diseases, they usually mean that EMP can be
found in a large spectrum of biological systems from the most physiological (i.e., development, healing)
all the way up to pathological settings (i.e., fibrosis and cancer). This, however, maintains the various
fields of investigation on parallel trajectories with researchers comparing systems, drawing similarities,
and searching for correlations. But we may be ignoring another level of analysis that could be more
relevant. Should we actually follow cells from development to disease? Thus, considering the life of
cells from development to normal homeostasis to the pathology? Organs are composed of cells that
have a history through which they have acquired a given gene expression profile, a given morphology
and a given set of interactions with their neighboring cells in their organs and with adjacent organs.
While some organs are formed from cells that never underwent EMP (epidermis), other went through

192

193

194

195

196

197

198

199

200
167 the neuroepithelium (McKeown et al., 2005). However, during normal neural crest development it
168 promotes pigment cell formation (Aoki et al., 2003) whereas its inhibition does not affect EMT and
169 migration (Aoki et al., 2003; Honore et al., 2003). Therefore, in some cases, expression of some EMP
170 genes may be related to cell identity/lineage rather than cell behavior. These observations require us
171 to rethink the framework associated with the initial definition of EMT by proposing a more flexible
172 paradigm, where the plasticity between E and M states is tissue and context-dependent and cannot be
173 reduced to a few key markers.

174 These observations stress the importance of monitoring expression of putative EMP regulators
175 across multiple time points and to perform functional assays to assess the cells' EMP state and
176 potential. In the context of cell and developmental biology studies multiple assays can be (and often
177 are) implemented alongside expression analyses: migration/invasion assays, cell-cell and cell-matrix
178 adhesion assays, collision assays, matrix remodeling/degradation assays etc... Given the wealth of
179 information that can be extracted from such experiments it would be greatly beneficial if the
180 diagnosis/prognosis workflow in the clinical context could integrate such approaches as routine
181 procedures from patients' biopsies. Currently, oncology centers in which there is a functional daily
182 integration of clinical and research departments are the exception rather than the norm. There have
183 been promising attempts to harness the power of classical embryology techniques in the context of
184 oncology via, for instance, modified migration/invasion assays using patients' cells grafted in avian
185 embryos (Delloye-Bourgeois et al., 2017; Jarrosson et al., 2021; Jarrosson et al., 2023). Still very
186 marginal a few years ago, the use of these chimeras is expanding, both in the academic world and
187 biotech companies. Organoid development, from embryonic or adult stem cells, may also help to
188 recapitulate the features and dynamic of EMP during organogenesis, in physiology and pathologies. It
189 allows to access and assess processes that may be difficult to observe and to quantify either in vivo or
190 in vitro in 2D cell culture systems.

191 When people discuss EMP from development to diseases, they usually mean that EMP can be
192 found in a large spectrum of biological systems from the most physiological (i.e., development, healing)
193 all the way up to pathological settings (i.e., fibrosis and cancer). This, however, maintains the various
194 fields of investigation on parallel trajectories with researchers comparing systems, drawing similarities,
195 and searching for correlations. But we may be ignoring another level of analysis that could be more
196 relevant. Should we actually follow cells from development to disease? Thus, considering the life of
197 cells from development to normal homeostasis to the pathology? Organs are composed of cells that
198 have a history through which they have acquired a given gene expression profile, a given morphology
199 and a given set of interactions with their neighboring cells in their organs and with adjacent organs.
200 While some organs are formed from cells that never underwent EMP (epidermis), other went through
one (i.e. ganglia of the peripheral nervous system), two (i.e. dermis, skeletal muscle) or three (i.e. cushion mesenchyme of the heart) round trips between epithelial and mesenchymal states. Should we treat the variations in gene expression profiles and protein levels differently when dealing with cells from various tissues that experienced one, two, three or no EMT at all throughout their lives? Or when considering cells that display different endogenous expression of genes with EMP potential? Plasticity around E/M states is known to impact stemness and survival, in addition to migratory and invasive properties. Are cells durably affected by a chronic exposure to signals triggering EMP? Genome-scale epigenetic modifications have been documented during EMP (Malouf et al., 2013; McDonald et al., 2011). Do they have long-lasting effects in terms of competence to toggle between E, M and intermediate states? We hope that future systematic studies could address the long term effects (if any) of successive EMT-MET events on cells and there putative impact on subsequent EM plasticity events.

This Editorial only scratches the surface of the complexity of EMP and the multiple questions it raises. EMP is a rapidly expanding a field of research and our understanding of its molecular and cellular implementation as well as its functional relationship with normal and pathological processes is a work in progress. In this special issue, we have assembled a collection of reviews and research articles looking at EMP in a wide range of contexts such as lateral plate mesoderm and neural crest development, cancer cell dormancy or kidney fibrosis. We hope that readers will find the content of the Special Issue to be intellectually stimulating.

220

221 Acknowledgements

H.A. is an INRAE senior researcher supported by funding from ANR PluS4PiGs (ANR-19-CE20-0019). J.Y. has been supported by grants from NCI (1R01CA262794, 1R01CA174869, 1R01CA206880, and 1R01CA236386) and CDMRP DOD Breast Cancer Program BC170283. E.T. is a research director at the French National Research Center (CNRS) and supported by grants from ANR SingleCrest (ANR-21-CE13-0028-02), the Association pour la Recherche contre le Cancer (ARC, ARCPJA22020060002084), the Fondation pour la Recherche Médicale (FRM AJE201224).

228

229 References

Andrieu, C., Montigny, A., Bibonne, A., Despin-Guitard, E., Alfandari, D., & Theveneau, E. (2020).

MMP14 is required for delamination of chick neural crest cells independently of its catalytic

activity. *Development*, **147**(7). https://doi.org/10.1242/dev.183954
one (i.e. ganglia of the peripheral nervous system), two (i.e. dermis, skeletal muscle) or three (i.e. cushion mesenchyme of the heart) round trips between epithelial and mesenchymal states. Should we treat the variations in gene expression profiles and protein levels differently when dealing with cells from various tissues that experienced one, two, three or no EMT at all throughout their lives? Or when considering cells that display different endogenous expression of genes with EMP potential? Plasticity around E/M states is known to impact stemness and survival, in addition to migratory and invasive properties. Are cells durably affected by a chronic exposure to signals triggering EMP? Genome-scale epigenetic modifications have been documented during EMP (Malouf et al., 2013; McDonald et al., 2011). Do they have long-lasting effects in terms of competence to toggle between E, M and intermediate states? We hope that future systematic studies could address the long term effects (if any) of successive EMT-MET events on cells and there putative impact on subsequent EM plasticity events.

This Editorial only scratches the surface of the complexity of EMP and the multiple questions it raises. EMP is a rapidly expanding a field of research and our understanding of its molecular and cellular implementation as well as its functional relationship with normal and pathological processes is a work in progress. In this special issue, we have assembled a collection of reviews and research articles looking at EMP in a wide range of contexts such as lateral plate mesoderm and neural crest development, cancer cell dormancy or kidney fibrosis. We hope that readers will find the content of the Special Issue to be intellectually stimulating.

220

## Acknowledgements

H.A. is an INRAE senior researcher supported by funding from ANR PluS4PiGs (ANR-19-CE20-0019). J.Y. has been supported by grants from NCI (1R01CA262794, 1R01CA174869, 1R01CA206880, and 1R01CA236386) and CDMRP DOD Breast Cancer Program BC170283. E.T. is a research director at the French National Research Center (CNRS) and supported by grants from ANR SingleCrest (ANR-21-CE13-0028-02), the Association pour la Recherche contre le Cancer (ARC, ARCPJA22020060002084), the Fondation pour la Recherche Médicale (FRM AJE201224).

228

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234

(2003). Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. *Dev*

235

Biol, 259(1), 19-33. http://www.ncbi.nlm.nih.gov/pubmed/12812785

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Aybar, M. J., Nieto, M. A., & Mayor, R. (2003). Snail precedes slug in the genetic cascade required for

237

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240

... Trepat, X. (2015). Control of cell-cell forces and collective cell dynamics by the intercellular

241

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Benazeraf, B., & Pourquie, O. (2013). Formation and segmentation of the vertebrate body axis. *Annu*

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(2013). A switch in the expression of embryonic EMT-inducers drives the development of

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247

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248

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249

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250

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251

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