Renard Emmanuelle
email: emmanuelle.renard@univ-amu.fr

Chapter 5 : Origin and Evolution of epithelial cell types

5.1. Introduction 5.1.1. Epithelial-like structures are not a prerogative of animals 5.1.2. The definition of epithelium in Metazoa 5.1.3. The classification of animal epithelial cell types 5.1.4. The definition of junction types 5.1.5. Cell polarity complexes and cell domains 5.1.6. Do all metazoans have (the same) epithelia? 5.2. The Origin of animal epithelia 5.2.1. Some genes encoding for extracellular matrix proteins predate the emergence of animal epithelia 5.2.2. Integrins: an ancient protein family 5.2.3. Genes encoding for Cell-cell Adhesive proteins predate the emergence of animal epithelia 5.2.4. Do some genes encoding for members of cell polarity complexes predate the emergence of animal epithelia? 5.2.5. Main conclusion: cell adhesion and polarity may have emerged several times during the evolution of Holozoa 5.3. The probably ancestral features of the metazoan epithelium 5.3.1. The composition of the ancestral basement membrane 5.3.2. The ancestral toolkit to achieve cell polarity depends on non-bilaterian relationships 5.3.3. Were adhesive junctions present ancestrally? 5.3.4. Epithelial regulatory signatures 5.4. Conclusions and future challenges

Introduction

Epithelial-like structures are not a prerogative of animals

Multicellularity emerged several times independently during the evolution of life [START_REF] Abedin | Diverse evolutionary paths to cell adhesion[END_REF][START_REF] Hinman | The evolution of gene regulation[END_REF][START_REF] King | The Unicellular Ancestry of Animal Development[END_REF][START_REF] King | Evolution of Key Cell Signaling and Adhesion Protein Families Predates Animal Origins[END_REF][START_REF] Niklas | The Evolutionary-Developmental Origins of Multicellularity[END_REF][START_REF] Parfrey | Multicellularity Arose Several Times in the Evolution of Eukaryotes (Response to DOI 10.1002/Bies.201100187)[END_REF], and so did epithelial-like tissues [START_REF] Dickinson | A Polarized Epithelium Organized by Beta-and Alpha-Catenin Predates Cadherin and Metazoan Origins[END_REF][START_REF] Hynes | The evolution of metazoan extracellular matrix[END_REF][START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Maizel | Plant Biology: The Making of an Epithelium[END_REF][START_REF] Miller | The Evolutionary Origin of Epithelial Cell-Cell Adhesion Mechanisms[END_REF]. In cell biology [START_REF] Lowe | Chapter 3 -Epithelial Cells[END_REF], epithelia are defined generally as a sheet of cells tightly bound together and capable of coordinated movements during morphogenesis. In the different lineages where they emerged, they usually shape and line organs (when present), cavities and free external borders, they control molecule and ion exchanges between the body and the environment and/or between different compartments of the body involved in vital physiological processes.

Because multicellularity and epithelial-like structures emerged several time independently, the molecules involved in their composition and patterning are different [START_REF] Kania | Polar delivery in plants; commonalities and differences to animal epithelial cells[END_REF][START_REF] Reynolds | Epithelial Organization: New Perspective on α-Catenin from an Ancient Source[END_REF]). Nevertheless, very ancient proteins predating multicellularity can play a key role in the establishment of epithelial features [START_REF] Nagawa | RHO GTPase in plants[END_REF] such as Rho/ROP GTPase for polarity because of their conserved involvement in controlling cytoskeleton and vesicular trafficking, and other ancient proteins can have been co-opted independently to perform a quite similar result, such as catenins in Ameobozoa and Metazoa (see section 2).

After this short synopsis, this chapter will focus on the origin and evolution of animal epithelia only.

The definition of epithelium in Metazoa

Epithelia of animals are usually considered as one of the four fundamental tissue types along with connective, nervous and muscular tissues [START_REF] Lowe | Chapter 3 -Epithelial Cells[END_REF][START_REF] Yathish | Cell-Cell Junctions and Epithelial Differentiation[END_REF]. Epithelial tissues cover all the surfaces of the body exposed to the external environment and line organs and body cavities (named in this case: Covering and lining epithelia) hence providing protection and compartmentalization of the body. Epithelia also form much of the glandular tissue of an animal body (in this case they are named glandular epithelia). During embryological development, epithelia are patterned early, after cleavage [START_REF] Tyler | Epithelium-The Primary Building Block for Metazoan Complexity1[END_REF]. Indeed, the organization of cells within tissues is the first sign of cell differentiation during embryogenesis and the epithelial blastoderm is the starting point for morphogenesis during the development of a wide range of metazoans [START_REF] Pozzi | The nature and biology of basement membranes[END_REF]. It is only from epithelium that mesenchyme arises by epithelial-mesenchymal transition (EMT) during gastrulation [START_REF] Tyler | Epithelium-The Primary Building Block for Metazoan Complexity1[END_REF]. In bilaterian adults, epithelial cells derive from all three major embryonic layers (endo-, meso-, and ectoderm): for example in vertebrates, the skin, part of the mouth and nose, and the anus develop from the ectoderm; cells lining the airways and most of the digestive system originate from the endoderm while the epithelium lining vessels derive from the mesoderm. Epithelia are fundamental structures controlling permeability and allowing selective transfer of molecules between animal and its environment and between body compartments. Epithelial tissues also provide protection from physical, chemical, and biological agents and allow for coordinated tissue movements. Consequently, disruptions in epithelial properties cause developmental defects and are responsible for diseases in adult tissues [START_REF] Miller | The Evolutionary Origin of Epithelial Cell-Cell Adhesion Mechanisms[END_REF][START_REF] Royer | Epithelial cell polarity: a major gatekeeper against cancer?[END_REF][START_REF] Sekiguchi | Chapter Four -Basement Membranes in Development and Disease[END_REF].

Currently, as for many other animal morpho-anatomical features, the histological characteristics defining an epithelium were postulated according to what was found in model animals, all pertaining to the bilaterian taxa, such as mammalian epithelial cells and Drosophila epithelial tissues (for review see [START_REF] Tyler | Epithelium-The Primary Building Block for Metazoan Complexity1[END_REF]. From these studies, epithelia have been defined as layers of cells showing coordinated polarity, with differences in structure and function between the apical side facing the external medium (or internal cavities) and a basal side in contact with a basement membrane (BM) made of collagen IV (figure 5.1) (Rodriguez-Boulan et Nelson 1989). In addition, epithelial cells exhibit cell-cell junctions and cell-matrix contacts (figure 5.1) which maintain cohesion between cells during morphogenesis processes and allow the coordination of cell movements by providing a seal between cells [START_REF] Jefferson | Plakins: Goliaths That Link Cell Junctions and the Cytoskeleton[END_REF]. focal Adhesions (FAs), Hemi-Desmosomes (HDs), Gap junctions, Adherens Junctions (AJs); 3) Basement membrane, a dense sheet of extracellular matrix proteins providing support and increasing cohesiveness of cells.

The classification of animal epithelial cell types

Despite this general definition, epithelia can harbor different organizations and structures. In addition to the distinction based on their general role (compartmentalizing versus glandular epithelia), epithelial tissues are identified by both the number of layers (simple, stratified or pseudostratified) and the shape of the cells (squamous, cuboidal or columnar) (summarized in These different kinds of epithelial tissue generally perform different functions (absorption, regulation, excretion, filtration, secretion, protection, detection) according to their body position and their structural organization.

The definition of junction types

Junction types involved in the establishment and patterning of epithelia are usually classified in two major categories: cell-cell junctions and cell-matrix junctions.

In bilaterians, cell-cell junctions include sealing junctions that link cells to form a regulated barrier: these are tight junctions (TJs) in Chordata and septate junctions (SJs) in Protostomia.

Even though TJs and SJs have the same function they are quite different in morphology and their molecular composition differ. TJs, the most apical junctions in vertebrates, are composed of the transmembrane proteins occludin and claudins that are linked to the actin cytoskeleton through zonula occludens (ZO) proteins (figure 5.1, for recent reviews see [START_REF] Garcia | Cell-Cell Junctions Organize Structural and Signaling Networks To Regulate Epithelial Tissue Homeostasis[END_REF][START_REF] Yathish | Cell-Cell Junctions and Epithelial Differentiation[END_REF][START_REF] Zihni | Tight Junctions: From Simple Barriers to Multifunctional Molecular Gates[END_REF]. SJs are more basal [START_REF] Tyler | Epithelium-The Primary Building Block for Metazoan Complexity1[END_REF]) and contain claudin-like proteins, contactin (Cont), neurexin IV (Nrx IV) and neuroglian (Nrg) among many other components (figure 5.1) (for review see [START_REF] Hall | Septate Junction Proteins Play Essential Roles in Morphogenesis Throughout Embryonic Development in Drosophila[END_REF].

A second type of cell-cell junction, signaling gap junctions allow direct cell-to-cell communication [START_REF] Skerrett | A structural and functional comparison of gap junction channels composed of connexins and innexins[END_REF][START_REF] Yathish | Cell-Cell Junctions and Epithelial Differentiation[END_REF] and are composed in vertebrate and in prostostome lineages of respectively connexin and innexin proteins, two similar but non homologous proteins (figure 5.1). In other words, two distinct lineages found a convergent solution to solve the same problem of intercellular communication [START_REF] Alexopoulos | Evolution of Gap Junctions: The Missing Link?[END_REF][START_REF] Skerrett | A structural and functional comparison of gap junction channels composed of connexins and innexins[END_REF].

The third type of cell-cell junctions are anchoring junctions linking cells between them (adherens junctions (AJs), desmosomes (DSs)). Contrary to DSs (containing the desmosomal transmembrane cadherin Desmogleins and cytosolic desmoplakins, [START_REF] Johnson | Desmosomes: Regulators of Cellular Signaling and Adhesion in Epidermal Health and Disease[END_REF][START_REF] Magie | Cell-Cell Adhesion in the Cnidaria: Insights Into the Evolution of Tissue Morphogenesis[END_REF][START_REF] Yathish | Cell-Cell Junctions and Epithelial Differentiation[END_REF] which are restricted to vertebrates and which provide a link to intermediate filaments through desmoplakin, AJs are present in all bilaterian taxa and comprise classical cadherins which interact to the actin cytoskeleton through α-, β-and δ-catenins (figure 5.1) [START_REF] Garcia | Cell-Cell Junctions Organize Structural and Signaling Networks To Regulate Epithelial Tissue Homeostasis[END_REF][START_REF] Miller | The Evolutionary Origin of Epithelial Cell-Cell Adhesion Mechanisms[END_REF][START_REF] Yathish | Cell-Cell Junctions and Epithelial Differentiation[END_REF].

Finally concerning cell-matrix junctions, they enable the attachment of epithelial cells to the underlying basement membrane. These are focal adhesion (FAs) and hemi-desmosomes (HDs).

As well as cell-cell junctions, cell-matrix junctions are needed to achieve collective migration and coordination of various epithelial morphogenetic processes important during development, [START_REF] Magie | Cell-Cell Adhesion in the Cnidaria: Insights Into the Evolution of Tissue Morphogenesis[END_REF][START_REF] Miller | Analysis of a Vinculin Homolog in a Sponge (Phylum Porifera) Reveals That Vertebrate-like Cell Adhesions Emerged Early in Animal Evolution[END_REF]Walko, Castañón, et Wiche 2015a). While FAs are found in non-epithelial cells types (such as, for example, neurons or fibroblasts [START_REF] Fischer | Filopodia and focal adhesions: An integrated system driving branching morphogenesis in neuronal pathfinding and angiogenesis[END_REF])), hemidesmosomes (HD) are specific to epithelia [START_REF] Walko | Molecular architecture and function of the hemidesmosome[END_REF].

It is usually accepted that variations of types and of organization of junctions have allowed the development and differentiation of tissue types and have played a key role in the evolution of animal body plans [START_REF] Abedin | Diverse evolutionary paths to cell adhesion[END_REF][START_REF] Magie | Cell-Cell Adhesion in the Cnidaria: Insights Into the Evolution of Tissue Morphogenesis[END_REF].

Cell polarity complexes and cell domains

The precise location of the previously defined junctions is one of the numerous demonstrations of cell polarity. This polarity is also obvious by the localization and orientation of other cell features such as the nucleus, cilia or other cell extensions, the cytoskeleton and vesicular trafficking. The cell is generally divided into three domains: the basal domain facing the basement membrane, the apical domain facing the external medium or cavity lumens, and, in between, the lateral domain which is more or less extended depending on the shape of cells (table 5.1, figures 5.1 and 5.2). This polarized structural and ultrastructural organization of an epithelial cell is patterned by at least three protein complexes named polarity complexes, each composed of three interacting proteins (figure 5.2) [START_REF] Assémat | Polarity Complex Proteins[END_REF][START_REF] Bivic | Evolution and Cell Physiology. 4. Why Invent yet Another Protein Complex to Build Junctions in Epithelial Cells?[END_REF]):

-The apical PAR complex is composed of atypical Protein Kinase C (aPKC), the Partition defective 3 (PAR3) and Partition defective 6 (PAR6);

-The apical CRUMBS complex made of CRUMBS (CRB), stardust (Sdt, or MPP5 (Membrane Palmitoylated Protein 5) in mammals also known as PALS1 (protein associated with Lin-7 1) and PALS1-associated tight junction protein (PATJ);

-The lateral SCRIBBLE complex is made with SCRIBBLE (SCRIB), lethal giant larvae (LGL) and Disc large (DLG). These polarity complexes interact and regulate each other and are themselves regulated by signaling pathways in a complex manner [START_REF] Assémat | Polarity Complex Proteins[END_REF][START_REF] Bivic | Evolution and Cell Physiology. 4. Why Invent yet Another Protein Complex to Build Junctions in Epithelial Cells?[END_REF].

Do all metazoans have (the same) epithelia?

There is no doubt that the last common ancestor of all extant animals was multicellular [START_REF] King | Embracing Uncertainty in Reconstructing Early Animal Evolution[END_REF]. And, whatever the variability in timing, relative importance and order of cell mechanisms involved between phyla (and sometimes among species of a same phylum (A.

Ereskovsky 2010), in all metazoans, the patterning of an epithelial or epithelial-like cell sheet is one of the earliest important event occurring during embryogenesis after the acquisition of multicellularity (more often at the blastula stage). The acquisition of such an epithelial level of organization is intimately connected with the acquisition of cellular adhesion and cell-communication toolkits. One of the main questions raised is: did these key cell properties emerge once or several times? In other words, are all animal epithelia and epithelial-like sheets homologous among metazoans? And is it possible to trace back the origin and evolution of all animal epithelial cell types?

To try to answer this question, the first step is to examine the presence/absence in non-bilaterian taxa of the previously cited three histological criteria defining an animal epithelium (figure 5.3) in order to: 1) decipher whether the term and definition of "epithelium" refers to equivalent structures throughout Metazoa, 2) determine, if epithelia are an ancestral character, which of epithelial features were ancestrally present. [START_REF] Fidler | Collagen IV and Basement Membrane at the Evolutionary Dawn of Metazoan Tissues[END_REF][START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Ledger | Septate junctions in the calcareous sponge Sycon ciliatum[END_REF][START_REF] Leys | Epithelia, an Evolutionary Novelty of Metazoans[END_REF][START_REF] Magie | Cell-Cell Adhesion in the Cnidaria: Insights Into the Evolution of Tissue Morphogenesis[END_REF][START_REF] Moroz | Independent origins of neurons and synapses: insights from ctenophores[END_REF][START_REF] Satterlie | Gap Junctions Suggest Epithelial Conduction within the Comb Plates of the Ctenophore Pleurobrachia Bachei[END_REF][START_REF] Smith | Adherens Junctions Modulate Diffusion between Epithelial Cells in Trichoplax adhaerens[END_REF] the joint presence of the three criteria supposed to define an epithelium in animals is far from being systematically conserved in non-bilaterian phyla. For junctions: "seal." stands for sealing, "adh." stands for adhesive and "comm." For communicating. In this schema, if two symbols are indicated for the same character in one phylum, it means that the observation can be different depending on the species considered. The status "unlikely homologous" means that a type of junction harboring histological features compatible with either sealing, adhesive or communicating properties were described, but that their characteristics suggest that they are from a different nature that what is found typically in other animals (for sponges: (E. D. M. Adams, Goss, et Leys 2010b; Sally P. Leys et Hill 2012b;[START_REF] Leys | Epithelia, an Evolutionary Novelty of Metazoans[END_REF][START_REF] Leys | The Biology of Glass Sponges[END_REF][START_REF] Leys | Epithelia and Integration in Sponges[END_REF]; for ctenophores: [START_REF] Hernandez-Nicaise | Intercellular Junctions in Ctenophore Integument[END_REF]S. L. Tamm et Tamm 1991;[START_REF] Tamm | Novel Bridge of Axon-like Processes of Epithelial Cells in the Aboral Sense Organ of Ctenophores[END_REF]). This schema shows that either many non-bilaterian species do not fit the definition of an epithelium or that the definition postulated decades ago does not depict and fit the diversity really found across animal epithelia.

The first finding is that the organization of epithelial or epithelial-like sheets found in nonbilaterian phyla is much more variableeven in a single phylumthan it is in bilaterians. The second obvious finding is that, among non-bilaterians, only cnidarian epithelia meets the three criteria of polarity, basement membrane and junctions (whatever their type) at the same time [START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Magie | Cell-Cell Adhesion in the Cnidaria: Insights Into the Evolution of Tissue Morphogenesis[END_REF]. In contrast, Placozoa do not meet the accepted definition of an epithelium (according to the very partial data available so far in this phylum)

because of the absence of a basement membrane [START_REF] Fidler | Collagen IV and Basement Membrane at the Evolutionary Dawn of Metazoan Tissues[END_REF][START_REF] Ruthmann | The Ventral Epithelium of Trichoplax Adhaerens (Placozoa): Cytoskeletal Structures, Cell Contacts and Endocytosis[END_REF].

As far as Ctenophora and Porifera are concerned only some of the species have tissues that fully meet the definition of epithelium: this is the case for the Homoscleromorpha class in Porifera [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF][START_REF] Boute | Type IV Collagen in Sponges, the Missing Link in Basement Membrane Ubiquity*[END_REF] (according to the partial data available so far in this phylum) [START_REF] Fidler | Collagen IV and Basement Membrane at the Evolutionary Dawn of Metazoan Tissues[END_REF]. It is therefore very surprising that the presence of a bona fide epithelium was questioned only for sponges.

Indeed, while Porifera were classically excluded from the "Eumetazoa" clade partly because of the absence of "true" epithelia with a basement membrane, and even several years after [START_REF] Boute | Type IV Collagen in Sponges, the Missing Link in Basement Membrane Ubiquity*[END_REF] showed a basement membrane existed in homoscleromorph sponges (A. V. and when such an epithelium originated and evolved: this is the item of the following section.

Tracing back the evolution of epithelial cell types is not only related to the questionings concerning germ layer homology, we have also to consider that the similarity of cell features (here presence of junctions, polarity and basement membrane, as previously mentioned) can result from convergent evolution by co-option of similar molecular actors or that shared proteins can have undergone neofunctionalization. In order to try to tackle this issue, the recent development of comparative whole organism single cell transcriptomic approaches (sc-RNAseq) should enable to define cell type specific core regulatory complex (CoRC) (Arendt 2008;[START_REF] Arendt | The Origin and Evolution of Cell Types[END_REF][START_REF] Marioni | How Single-Cell Genomics Is Changing Evolutionary and Developmental Biology[END_REF]. Thanks to the conservation of regulatory mechanisms, a homologous cell type is expected to remain recognizable across species [START_REF] Arendt | Genes and Homology in Nervous System Evolution: Comparing Gene Functions, Expression Patterns, and Cell Type Molecular Fingerprints[END_REF](Arendt , 2008)). We will also discuss this point in section 2.

5.2.The Origin of animal epithelia

In order to understand the origin of this metazoan synapomorphy, it is useful to study the presence of 'preadapted' molecular tools that were present before the emergence of animals. [START_REF] Fairclough | Premetazoan genome evolution and the regulation of cell differentiation in the choanoflagellate Salpingoeca rosetta[END_REF][START_REF] King | The Genome of the Choanoflagellate Monosiga Brevicollis and the Origin of Metazoans[END_REF][START_REF] Suga | The Capsaspora Genome Reveals a Complex Unicellular Prehistory of Animals[END_REF][START_REF] Williams | The Predicted Secretomes of Monosiga Brevicollis and Capsaspora Owczarzaki, Close Unicellular Relatives of Metazoans, Reveal New Insights into the Evolution of the Metazoan Extracellular Matrix[END_REF]. This means that most of the proteins found in animal ECM and basement membrane (for instance the laminin and fibronectin key components) are metazoan innovations and emerged probably in part by domain shuffling [START_REF] Babonis | Phylogenetic evidence for the modular evolution of metazoan signalling pathways[END_REF]Fahey and Degnan, 2012;[START_REF] Richter | Gene Family Innovation, Conservation and Loss on the Animal Stem Lineage[END_REF][START_REF] Suga | The Capsaspora Genome Reveals a Complex Unicellular Prehistory of Animals[END_REF][START_REF] Williams | The Predicted Secretomes of Monosiga Brevicollis and Capsaspora Owczarzaki, Close Unicellular Relatives of Metazoans, Reveal New Insights into the Evolution of the Metazoan Extracellular Matrix[END_REF]. Interestingly, most recent studies suggest that a canonical type IV collagen could have been already present in the common ancestor of filastereans, choanoflagellates and animals [START_REF] Fidler | The Triple Helix of Collagens -an Ancient Protein Structure That Enabled Animal Multicellularity and Tissue Evolution[END_REF][START_REF] Grau-Bové | Dynamics of Genomic Innovation in the Unicellular Ancestry of Animals[END_REF]) therefore suggesting a more ancient origin of type IV collagen than previously accepted and its neo-functionalization in metazoans, as it has also been suggested for integrin adhesome or cadherins [START_REF] King | The Genome of the Choanoflagellate Monosiga Brevicollis and the Origin of Metazoans[END_REF]Sebé-Pedrós and Ruiz-Trillo, 2010;Sebé-Pedrós et al., 2010). In other words, the characterization of type IV collagen in non-metazoans challenges the previously accepted idea that spongin short chain collagen (SSCC) is ancestral to type IV collagen [START_REF] Aouacheria | Insights into Early Extracellular Matrix Evolution: Spongin Short Chain Collagen-Related Proteins Are Homologous to Basement Membrane Type IV Collagens and Form a Novel Family Widely Distributed in Invertebrates[END_REF]; Sally P. Leys et Riesgo 2012).

Integrins: an ancient protein family

As previously mentioned, (section 1.4), the key proteins shared by focal adhesions (FAs) and According to the presently (scarce) available genomic data, it seems that at least three cadherin families were present in the last common ancestor of Choanozoa (taxa grouping Choanoflagellata and Metazoa): lefftyrins, coherins and hedglings [START_REF] Nichols | Origin of metazoan cadherin diversity and the antiquity of the classical cadherin/β-catenin complex[END_REF]). Among these families some were lost secondarily in bilaterians or earlier. To date, only one cadherin gene was found in the filasterean C. owczarzaki [START_REF] King | The Genome of the Choanoflagellate Monosiga Brevicollis and the Origin of Metazoans[END_REF][START_REF] Nichols | Origin of metazoan cadherin diversity and the antiquity of the classical cadherin/β-catenin complex[END_REF][START_REF] Richter | The Genomic and Cellular Foundations of Animal Origins[END_REF]. It is now therefore obvious that cadherins predate metazoans. But only metazoans have bona fide classical cadherins containing the two domains required for its interaction with catenins (JMD and CBD) [START_REF] Clarke | Characterization of the Cadherin?Catenin Complex of the Sea Anemone Nematostella Vectensis and Implications for the Evolution of Metazoan Cell?Cell Adhesion[END_REF][START_REF] Miller | The Evolutionary Origin of Epithelial Cell-Cell Adhesion Mechanisms[END_REF][START_REF] Murray | Pre-Metazoan Origins and Evolution of the Cadherin Adhesome[END_REF].

As far as catenins are concerned, the Amoebozoa Dictyostelium discoideum, possesses catenins (Aardvark and Ddα-catenin), involved in establishing epithelial-like sheets [START_REF] Dickinson | A Polarized Epithelium Organized by Beta-and Alpha-Catenin Predates Cadherin and Metazoan Origins[END_REF][START_REF] Hynes | The evolution of metazoan extracellular matrix[END_REF]. Interestingly, Aardvark-related proteins are also present in choanoflagellates and filastereans [START_REF] Nichols | Origin of metazoan cadherin diversity and the antiquity of the classical cadherin/β-catenin complex[END_REF][START_REF] Suga | The Capsaspora Genome Reveals a Complex Unicellular Prehistory of Animals[END_REF]. Aardvark and Ddαcatenin are considered close relatives of beta-and alpha-catenins because of shared domain features [START_REF] Miller | The Evolutionary Origin of Epithelial Cell-Cell Adhesion Mechanisms[END_REF][START_REF] Miller | Analysis of a Vinculin Homolog in a Sponge (Phylum Porifera) Reveals That Vertebrate-like Cell Adhesions Emerged Early in Animal Evolution[END_REF][START_REF] Richter | Gene Family Innovation, Conservation and Loss on the Animal Stem Lineage[END_REF][START_REF] Suga | The Capsaspora Genome Reveals a Complex Unicellular Prehistory of Animals[END_REF]). As a consequence, it means that the joint presence of cadherins and catenin-related proteins predate the emergence of Metazoa even if they are predicted to not interact. Unfortunately, there is no experimental data so far available to establish the function of these proteins in these organisms [START_REF] Nichols | Origin of metazoan cadherin diversity and the antiquity of the classical cadherin/β-catenin complex[END_REF]. But the recent acquisition of a transfection protocol in choanoflagellates is expected to enable to soon fill this gap [START_REF] Booth | Choanoflagellate Transfection Illuminates Their Cell Biology and the Ancestry of Animal Septins[END_REF].

Concerning genes encoding for proteins involved in other types of junction, none of the components of gap junctions have been characterized in non-metazoans yet [START_REF] Moroz | Independent origins of neurons and synapses: insights from ctenophores[END_REF], while, in contrast, a claudin-like gene was retrieved in Capsaspora and Monosiga genomes [START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF]) (see figure 5).

Do some genes encoding for members of cell polarity complexes predate the emergence of animal epithelia?

Though the typical collar cell of choanoflagellates shows an obvious basal-apical cell polarity, to date the genes encoding for the three metazoan polarity complexes (CRUMBS, SCRIBBLE and PAR, figure 2) remain nearly unexplored. Among the various actors involved in polarity complexes (see section 1.5), only DLG, a member of the SCRIBBLE complex was shown to predate the emergence of the metazoans and has been found in Choanoflagellata, Filasterea and Ichthyosporea [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF][START_REF] Fahey | Origin of Animal Epithelia: Insights from the Sponge Genome: Evolution of Epithelia[END_REF][START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Bivic | Evolution and Cell Physiology. 4. Why Invent yet Another Protein Complex to Build Junctions in Epithelial Cells?[END_REF][START_REF] Murray | Pre-Metazoan Origins and Evolution of the Cadherin Adhesome[END_REF][START_REF] Richter | Gene Family Innovation, Conservation and Loss on the Animal Stem Lineage[END_REF]. For now, without no further contrary evidence, all other components are considered metazoan innovations (figure 5.4). In addition, the key pathways known to regulate these 3 epithelial polarity complexes (among which Wnt and PCP pathways) are also considered as metazoan specificities [START_REF] Babonis | Phylogenetic evidence for the modular evolution of metazoan signalling pathways[END_REF].

If metazoans evolved these specific means to control their epithelial cell polarity, it is obvious that cell polarity is more generally necessary for proper functioning of numerous other cell types such as, for example, neurons in animals, pollen tubes in plants and hyphae in Fungi. The achievement of cell polarity is intimately linked to cytokinesis, orientation of mitotic spindles and asymmetric separation. Some molecular elements controlling cell polarity are known to be highly conserved across eukaryotes including animals, in epithelial and non-epithelial cell types, such as MOB proteins and Rho GTPase [START_REF] Bornens | Cell polarity: having and making sense of direction-on the evolutionary significance of the primary cilium/centrosome organ in Metazoa[END_REF][START_REF] Hoff | Heads, Tails, and Tools: Morphogenesis of a Giant Single-Celled Organism[END_REF][START_REF] Lefèbvre | Secretory Pathway-Dependent Localization of the Saccharomyces Cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites[END_REF][START_REF] Slabodnick | The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis[END_REF].

Main conclusion: cell adhesion and polarity may have emerged several times during the evolution of Holozoa

According to the previously cited results in Choanoflagellata and Filasterea, the proteins involved in cell-cell adhesion and cell-matrix adhesion to achieve aggregative/transient multicellular stages in these organisms are different from what is found in Metazoans.

Nevertheless, key protein domains involved in major cell-cell or cell-matrix protein interactions are highly conserved in holozoans. It means that, during the evolution of Holozoans aggregative/multicellular stages probably emerged thanks to intense domain shuffling of shared building blocks, as Lego® bricks, and duplication-divergence events in already present gene families (e.g. cadherins and catenines). Thanks to these ancestral domains and families, the metazoan lineage evolved specific tools, representing metazoan synapomorphies (such as polarity complexes, classical E-cadherin and signaling pathways), to achieve permanent multicellular level of organization with epithelial features.

The probably ancestral features of the metazoan epithelium

The composition of the ancestral basement membrane

To date, comprehensive biochemical experiments to determine the protein composition of the basement membrane of animals have only been carried out in cnidarians and bilaterians [START_REF] Fidler | A Unique Covalent Bond in Basement Membrane Is a Primordial Innovation for Tissue Evolution[END_REF][START_REF] Fidler | The Triple Helix of Collagens -an Ancient Protein Structure That Enabled Animal Multicellularity and Tissue Evolution[END_REF][START_REF] Halfter | New Concepts in Basement Membrane Biology[END_REF]. As a consequence, only partial biochemical information is available for the other animal lineages, and in particular for sponges and ctenophores which are considered the most basal taxa in the metazoan tree. Most of our present knowledge for these two phyla therefore relies on indirect hypotheses based of transcriptomic and genomic surveys.

A few studies have compared the extracellular matrix (ECM) genes found in the genomes of early-diverging animals such as sponges and cnidarians with that conserved across bilaterians.

These comparisons enabled to identify an ancestral core set of ECM components, receptors and degrading proteases (J. C. [START_REF] Adams | Extracellular Matrix Evolution: An Overview[END_REF]Adams , 2018;;[START_REF] Özbek | The Evolution of Extracellular Matrix[END_REF][START_REF] Tucker | Chapter Eight -Adhesion Networks of Cnidarians: A Postgenomic View[END_REF][START_REF] Williams | The Predicted Secretomes of Monosiga Brevicollis and Capsaspora Owczarzaki, Close Unicellular Relatives of Metazoans, Reveal New Insights into the Evolution of the Metazoan Extracellular Matrix[END_REF]. For what is known from bilaterians and cnidarians, the extracellular matrix is mainly composed of type I collagen, elastin and fibronectins but there is also a great variety of glycoproteins (as laminin), proteoglycans and different types of collagens (except type IV). Its density, fluidity and composition is variable between taxa and between different tissues of the same taxa [START_REF] Fidler | The Triple Helix of Collagens -an Ancient Protein Structure That Enabled Animal Multicellularity and Tissue Evolution[END_REF][START_REF] Hynes | The evolution of metazoan extracellular matrix[END_REF][START_REF] Kular | The Extracellular Matrix: Structure, Composition, Age-Related Differences, Tools for Analysis and Applications for Tissue Engineering[END_REF].

In contrast, the composition of the basement membrane seems less variable between taxa (even though slight variation can occur between tissues). Indeed in all cases the main components are laminins and type IV collagen, interconnected with mainly nidogen and perlecan and, sometimes, with additional molecules such as agrin [START_REF] Fidler | Collagen IV and Basement Membrane at the Evolutionary Dawn of Metazoan Tissues[END_REF][START_REF] Fidler | The Triple Helix of Collagens -an Ancient Protein Structure That Enabled Animal Multicellularity and Tissue Evolution[END_REF][START_REF] Medwig | Breaking down barriers: the evolution of cell invasion[END_REF][START_REF] Pozzi | The nature and biology of basement membranes[END_REF][START_REF] Sekiguchi | Chapter Four -Basement Membranes in Development and Disease[END_REF]. According to genomic surveys, only genes encoding for type IV collagen and laminin are present in ctenophores and homoscleromorph sponges. Surprisingly, despite of the absence of the connecting proteins nidogen and perlecan, they are able to build a basement membrane (at least for Beroe ovata and Pleurobrachia pileus) [START_REF] Fidler | Collagen IV and Basement Membrane at the Evolutionary Dawn of Metazoan Tissues[END_REF][START_REF] Fidler | The Triple Helix of Collagens -an Ancient Protein Structure That Enabled Animal Multicellularity and Tissue Evolution[END_REF]. This finding suggests that these two proteins (type IV collagen and laminin) are essential and shared components of BM in animals and therefore constitute the core toolkit already present in the last common ancestor of all extant metazoans. This also means that the other BM components (perlecan and nidogen) emerged more recently during metazoan evolution. Future experiments are needed to understand the differences in the organization and mechanical properties of the BM between cnidarians/bilaterians and ctenophores/homoscleromorph sponges.

These recent findings in ctenophores and sponges also mean that the absences of BM in some ctenophores, such as Mmemiopsis leidyi, and in the placozoan Trichoplax adhaerans or of genes encoding for laminin and type IV collagen (as in demosponges for instance) are secondary losses. Interestingly, in contrast, the calcareous sponge Sycon coactum possesses genes encoding for several BM proteins whereas a BM structure has never been observed in this sponge class (Sally P. [START_REF] Leys | Epithelia, an Evolutionary Novelty of Metazoans[END_REF][START_REF] Riesgo | The Analysis of Eight Transcriptomes from All Poriferan Classes Reveals Surprising Genetic Complexity in Sponges[END_REF]. More in-depth domain analyzes and functional experiments have to be performed to fully understand this apparent discrepancy between gene content and tissue structure (discussed in [START_REF] Renard | Understanding Animal Evolution: The Added Value of Sponge Transcriptomics and Genomics[END_REF].

The ancestral toolkit to achieve cell polarity depends on non-bilaterian relationships

Cell polarity is an ancestral feature of animal epithelia (Figure 5.3). It now remains to establish whether or not the molecular toolkit involved in the establishment of coordinated cell polarity is the same in non-bilaterian animals as in bilaterians. According to recent gene surveys [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF][START_REF] Riesgo | The Analysis of Eight Transcriptomes from All Poriferan Classes Reveals Surprising Genetic Complexity in Sponges[END_REF]), all the genes encoding for the nine proteins involved in PAR, CRUMBS and SCRIBBLE polarity complexes are present in Cnidaria, Placozoa and Porifera. Whereas the whole set of genes needed to build a CRUMBS complex is absent in Ctenophora, as well as the Scribble gene needed to complete the SCRIBBLE complex (table 5.2, figure 5.4).

Table 5.2 Présence (x)/absence(0) in the four non-bilaterian phyla (Porifera, Ctenophora, Placozoa and

Cnidaria) of genes encoding for proteins involved in the three cell polarity complexes, namely the CRUMBS complex, the SCRIBBLE complex and the PAR complex (figure 5.2), according to transcriptomic and genomic surveys [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF][START_REF] Bivic | Evolution and Cell Physiology. 4. Why Invent yet Another Protein Complex to Build Junctions in Epithelial Cells?[END_REF][START_REF] Riesgo | The Analysis of Eight Transcriptomes from All Poriferan Classes Reveals Surprising Genetic Complexity in Sponges[END_REF])

Porifera Ctenophora Placozoa Cnidaria PAR PAR 3 x x x x PAR 6 x x x x aPKC x x x x CRUMBS CRB x 0 x x PAT J x 0 x x PALS1 x 0 x x SCRIBBLE SCRIB x 0 x x LGL x x x x DLG x x x x
Because the relative phylogenetic position of Ctenophora and Porifera is still uncertain [START_REF] Simion | A Large and Consistent Phylogenomic Dataset Supports Sponges as the Sister Group to All Other Animals[END_REF][START_REF] Whelan | Ctenophore Relationships and Their Placement as the Sister Group to All Other Animals[END_REF]reviewed in King and Rokas, 2017) two scenarios are possible:

either the nine polarity genes (DLG predates the emergence of Metazoa) were present in common metazoan ancestor then some were lost secondarily in ctenophores, or only 5 of these genes were ancestral and the other 4 genes would represent innovations acquired later during animal evolution (Figures 5.4 and 5.5). Moreover, whatever the scenario considered there is no experimental evidence so far that neither these proteins are able to form complexes in vivo in Placozoa and Porifera (even if residue and domain analyzes tend to support this hypothesis, see [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF] nor on their function in these phyla. Interestingly, a very recent immunolocalization of the PAR proteins in the ctenophore Mnemiopsis leydii strongly questions the functional conservation of this protein outside the cnidarian-bilaterian lineage (Salinas-Saavedra et Martindale 2019).

Were adhesive junctions present ancestrally?

According to the analyses of the different types of junctions present in bilaterians and cnidarians, it is clear that their common ancestor must have had AJs, gap junctions and SJs, but the origin of these major innovations remains unknown:

5.3.3.1.Communicating junctions

In bilaterians, gap junctions are either composed of innexin (invertebrates) or connexin (vertebrates). Whatever the evolutionary origin of these two proteins (homology vs.

convergence), the gap junction they compose show amazing structural and functional similarities [START_REF] Skerrett | A structural and functional comparison of gap junction channels composed of connexins and innexins[END_REF]. In cnidarians, innexin also composes gap junctions [START_REF] Alexopoulos | Evolution of Gap Junctions: The Missing Link?[END_REF][START_REF] Takaku | Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps[END_REF], it therefore means that innexin-gap-junctions were already present in the last ancestor of bilaterians and cnidarians. In ctenophores, gap junctions have been reported [START_REF] Satterlie | Gap Junctions Suggest Epithelial Conduction within the Comb Plates of the Ctenophore Pleurobrachia Bachei[END_REF] and, interestingly, a gene encoding for innexin has been characterized in Pleurobrachia [START_REF] Moroz | Independent origins of neurons and synapses: insights from ctenophores[END_REF]. Nevertheless, given that innexin can participate to cell-cell communication via the establishment of either gap junctions or hemichannels [START_REF] Güiza | Innexins: Expression, Regulation, and Functions[END_REF], the localization of the innexin protein at the level of gap junction is needed to establish the possible function conservation of innexin in ctenophores. In contrast, until now, neither the structure nor the innexin genes were reported yet in sponges and in placozoans [START_REF] Moroz | Independent origins of neurons and synapses: insights from ctenophores[END_REF][START_REF] Smith | Adherens Junctions Modulate Diffusion between Epithelial Cells in Trichoplax adhaerens[END_REF]. In sponges, a very different type of communicating junction was described in glass sponges (Hexactinellida), named plug junctions, clearly unrelated to gap junctions found in other animals (S. P. [START_REF] Leys | Embryogenesis in the glass sponge Oopsacas minuta: Formation of syncytia by fusion of blastomeres[END_REF][START_REF] Leys | The Biology of Glass Sponges[END_REF]. Therefore, if we assume 1) that the innexin protein is involved in the formation of gap junctions in ctenophores, and 2) that the Ctenophora-first hypothesis is the right one, then, gap junctions were present ancestrally in animals and lost secondarily in sponges and placozoans. Because none of these hypotheses have so far been validated, it seems premature to draw such a conclusion.

Sealing/Occluding junctions

As far as sealing junctions are concerned, septate junctions are present in Cnidaria. And despite ultrastructural variations observed by electron microscopy and incomplete information about their molecular composition, bilaterian and cnidarian SJs are currently considered to be evolutionary related [START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF]. Again, as for the other structures examined in this chapter, our knowledge from the three other non-bilaterian phyla is too scarce and therefore prevents us from elaborating a clear evolutionary scenario for the origin of SJs. In Placozoa, ladder-like structures reminiscent of the SJs were described (Ruthmann, Behrendt, et Wahl 1986b) but more precise observations are needed to conclude whether or not these structures are bona fide SJs [START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Smith | Adherens Junctions Modulate Diffusion between Epithelial Cells in Trichoplax adhaerens[END_REF]. In ctenophores, no SJs have been reported so far, although "atypical" junctional structures (without septa) have been [START_REF] Leys | Epithelia and Integration in Sponges[END_REF]. Interestingly, despite the absence of unquestionable SJ outside the Cnidaria-Bilateria lineage, genes encoding for core components of SJs are present in placozoans and some of them in sponges and in ctenophores [START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Riesgo | The Analysis of Eight Transcriptomes from All Poriferan Classes Reveals Surprising Genetic Complexity in Sponges[END_REF]): four proteins (Claudin-like, neuroglian, contactin, neurexin IV) considered as major structural components of SJs would have predated the emergence of the Bilateria-Cnidaria lineage (figure 5.5). Considering the incongruences between the different analyzes performed so far in ctenophores and sponges, it is currently impossible to decipher whether these genes were present ancestrally or not [START_REF] Chapman | The Dynamic Genome of Hydra[END_REF][START_REF] Fahey | Origin of Animal Epithelia: Insights from the Sponge Genome: Evolution of Epithelia[END_REF][START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Leys | Epithelia, an Evolutionary Novelty of Metazoans[END_REF][START_REF] Riesgo | The Analysis of Eight Transcriptomes from All Poriferan Classes Reveals Surprising Genetic Complexity in Sponges[END_REF][START_REF] Suga | The Capsaspora Genome Reveals a Complex Unicellular Prehistory of Animals[END_REF].

Thus, much remains to be explored in order to 1) understand how these three non-bilaterian phyla, namely Placozoa, Ctenophora and Porifera, achieve the sealing of their epithelium, 2) establish the roles of retrieved genes, and 3) be able to trace back the origin of bilateriancnidarian septate junctions.

Adhesive junctions

In contrast to the two previous types of junctions, because of the clear description of adherens junctions or adhesive belts in some sponges (Homoscleromorpha and Calcarea), placozoans and ctenophores (A. V. Ruthmann, Behrendt, et Wahl 1986b;[START_REF] Smith | Adherens Junctions Modulate Diffusion between Epithelial Cells in Trichoplax adhaerens[END_REF][START_REF] Smith | Novel Cell Types, Neurosecretory Cells and Body Plan of the Early-Diverging Metazoan, Trichoplax adhaerens[END_REF][START_REF] Tamm | Massive Actin Bundle Couples Macrocilia to Muscles in the Ctenophore Beroë[END_REF], until recently it was taken for granted that adhesive junctions represented the most ancestral type of junctions. Nevertheless recent transcriptomic and genomic surveys [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF][START_REF] Riesgo | The Analysis of Eight Transcriptomes from All Poriferan Classes Reveals Surprising Genetic Complexity in Sponges[END_REF]) and deep analyses of key functional domain and residues [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF]) started casting doubt on this common view. Indeed, whereas only two classes of sponges harbor adherens junctions (Homoscleromorpha and Calcarea), all four sponge classes possess the genes encoding for the four proteins involved in adherens junctions namely E-cadherin, alpha, beta-, and delta-catenins (CCC complex, figure 1).

Moreover, the key domains of the E-cadherin needed to establish the CCC complex (GBM and JMD) are well conserved in three of these classes (while divergent in glass sponges) [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF]; and the first recent biochemical experiments conducted in a demosponge (devoid of adherens junctions) suggest that this canonical complex is formed in vitro [START_REF] Schippers | Evidence of Signaling and Adhesion Roles for β-Catenin in the Sponge Ephydatia Muelleri[END_REF]. Despite the fact that these new data are very exciting and represent an undeniable step forward for sponge biology, they unfortunately fail to explain the functional role of the CCC complex in sponge classes that are devoid of adherens junction-like structures.

It will be important to explore the homology of adherens junctions of homoscleromorph sponges and of cnidarians/bilaterians. Recently, the involvement of vinculin in cell-cell and cell-ECM contacts in Oscarella pearsei has been proposed following its tissue localization by immunolocalization [START_REF] Miller | Analysis of a Vinculin Homolog in a Sponge (Phylum Porifera) Reveals That Vertebrate-like Cell Adhesions Emerged Early in Animal Evolution[END_REF]. This is highly reminiscent of what is known in other animals [START_REF] Carisey | Vinculin, an Adapter Protein in Control of Cell Adhesion Signalling[END_REF][START_REF] Carisey | Vinculin Regulates the Recruitment and Release of Core Focal Adhesion Proteins in a Force-Dependent Manner[END_REF][START_REF] Miller | Analysis of a Vinculin Homolog in a Sponge (Phylum Porifera) Reveals That Vertebrate-like Cell Adhesions Emerged Early in Animal Evolution[END_REF]), but until Ecadherin is not localized in this class of sponge we will not be able to make the link between the structure (AJ) and its protein composition.

The most surprising result of the analyses conducted by [START_REF] Belahbib | New genomic data and analyses challenge the traditional vision of animal epithelium evolution[END_REF] was the high divergence of the key cytoplasmic domains of the E-cadherin of Ctenophores: these divergences prevent to predict a possible interaction of this protein with catenins, and casts doubt on the formation of a CCC complex in this phylum. Again, as for sponges, biochemical and functional experiments are needed to confirm/infirm in vitro and in vivo these in silico predictions. If this is confirmed, and depending on the phylogenetic hypothesis considered, this may challenge the idea that the last common ancestor of animals was already able to form CCC complexes involved in the formation of adhesive junctions. Indeed the possibility of neofunctionalization of cadherins and catenins is well illustrated by the case of the emergence of desmosomes in vertebrates (for review see Green et al., 2020).

Epithelial regulatory signatures

Comparing similar cell types across different species by sc-RNAseq is in progress since recent years. This approach is expected to provide insight into the evolution of cellular lineages (reviewed in [START_REF] Marioni | How Single-Cell Genomics Is Changing Evolutionary and Developmental Biology[END_REF]. The general principle of this approach is that: 1) a cell type is defined by a unique combination of transcription factors, the terminal selectors, that form a core regulatory complex (CoRC) needed to regulate cell type-specific effector genes [START_REF] Arendt | The Origin and Evolution of Cell Types[END_REF][START_REF] Hobert | The molecular and gene regulatory signature of a neuron[END_REF]; 2) evolutionary relatedness between cell types should be visible through the sharing of similarities of CoRCs and effectors.

So far, no study has focused on the evolution of epithelial cell types. Nevertheless a few transcription factors seem to be highly conserved in some epithelial cells across the animal kingdom and may be expected to be part of the CoRC needed to regulate the cell fate of at least some epithelial cell types. This is for example the case of epithelial specific Ets factors (ESE) that regulate epithelial processes, such as epithelial proliferation and differentiation, in different mammalian epithelial cell types, notably by modulating terminal differentiation pathways [START_REF] Feldman | The Epithelial-Specific Ets Factors Occupy a Unique Position in Defining Epithelial Proliferation, Differentiation and Carcinogenesis[END_REF], and that also seem to be specific markers of one of the epithelial layers found in sponges (pinacocytes forming the pinacoderm layer) [START_REF] Sebé-Pedrós | Early Metazoan Cell Type Diversity and the Evolution of Multicellular Gene Regulation[END_REF]). In the same way, the transcription factor Grainyhead that may play a role in regulating domain-specific effector genes for some epithelial cell types [START_REF] Achim | Whole-Body Single-Cell Sequencing Reveals Transcriptional Domains in the Annelid Larval Body[END_REF][START_REF] Boglev | The unique and cooperative roles of the Grainy head-like transcription factors in epidermal development reflect unexpected target gene specificity[END_REF]) has been found recently to be expressed specifically in some epithelial cell types in the ctenophore Mnemiopsys leydii and the choanoderm (formed by choanocytes) of the sponge Amphimedon queenslandica [START_REF] Sebé-Pedrós | Early Metazoan Cell Type Diversity and the Evolution of Multicellular Gene Regulation[END_REF].

Present data are obviously insufficient to decipher evolutionary relationships between epithelial cell types between animal lineages, but ongoing projects are expected to provide clues in the future years. The main difficulties to deal with at such a large evolutionary scale are: 1) establishing the precise orthology and paralogy relationships of all expressed genes [START_REF] Altenhoff | Inferring Orthology and Paralogy[END_REF], 2) acquiring high-quality reference genomes and gene annotation for all invertebrate lineages which is far from being the case [START_REF] Renard | Understanding Animal Evolution: The Added Value of Sponge Transcriptomics and Genomics[END_REF], 3) a better knowledge of cell types especially in non-bilaterian phyla [START_REF] Sebé-Pedrós | Early Metazoan Cell Type Diversity and the Evolution of Multicellular Gene Regulation[END_REF]) and of their ability to transdifferentiate [START_REF] Sogabe | The Ontogeny of Choanocyte Chambers during Metamorphosis in the Demosponge Amphimedon Queenslandica[END_REF].

Conclusions and future challenges

It is now clear that the main features of bilaterian epithelia predate the emergence of the bilaterian lineage: indeed all these features are clearly shared with cnidarians, meaning that the last common ancestor of cnidarians and bilaterians already had an epithelium with cell polarity, basement membrane and 3 types of junctions, and that these features are achieved with the same molecular toolkit. To trace back the origin of these features it is therefore needed to pay attention to the three other non-bilaterian phyla, namely sponges, ctenophores and placozoans.

Unfortunately, the knowledge available on these three phyla mainly relies 1) on classical electron microscopy and 2) on gene surveys. These data describe major discrepancies between gene content and observed histological features (reviewed in [START_REF] Renard | Understanding Animal Evolution: The Added Value of Sponge Transcriptomics and Genomics[END_REF]. The other great lesson learnt in the last years is that even though the basement membrane was probably present ancestrally, it was lost several times during animal evolution. This finding challenges the common dogma on the absolute requirement of the presence of this structure to compose and pattern an epithelium. Indeed, it has been shown in demosponges that the achievement of epithelial properties can be reached without the presence of a basement membrane (E. D. M. Adams, Goss, et Leys 2010a). Nevertheless, the different regeneration processes found in different sponge species [START_REF] Borisenko | Transdifferentiation is a driving force of regeneration in Halisarca dujardini (Demospongiae, Porifera)[END_REF]A. V. Ereskovsky et al. 2015) suggest that the presence or absence of a basement membrane may influence the dynamics of epithelial morphogenetic processes. Much remains to explore from a functional point of view to understand the consequences of BM secondary losses in several species.

The present state of the exciting quest is that biochemical and functional experiments are needed to establish the exact role of the proteins in these three non-bilaterian lineages. Recent papers show that there are promising growing efforts in this direction. Only these proteomic and functional data will enable us to decipher whether the basement membrane and the cell polarity

Figure 5 . 1 :

 51 Figure 5.1: Schematic drawing summarizing the three features defining animal epithelia : 1) Cell polarity (basalapical polarity : cell can harbor different features along this polarity axis, for instance cilia in some cells); 2) Cellcell and cell-matrix junctions found in Bilaterians and their core molecular composition (here only structural components are noted rather than all the components needed for their trafficking and patterning). Left, junction types found in protostomes and non-chordates: Septate Junctions (SJs). Right, junction types found in vertebrates: Desmosomes (DSs), Tight Junctions (TJs). In between and on both sides are junction types found in all bilaterians:

  tissue shaping and wound healing. Both FAs and HDs are highly specialized structures based on interactions between integrins, fibrillar proteins of the extracellular matrix and the internal intermediate filament or actin network (figure 5.1) (De Pascalis et Etienne-Manneville 2017;

Figure 5 . 2 :

 52 Figure 5.2: Localization and protein composition of the three polarity complexes needed to pattern cell polarity in epithelial cells: PAR, CRUMBS and SCRIBBLE according to what was described in Le Bivic (2013).

Figure 5 . 3 :

 53 Figure 5.3: According to histological observations[START_REF] Fidler | Collagen IV and Basement Membrane at the Evolutionary Dawn of Metazoan Tissues[END_REF][START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Ledger | Septate junctions in the calcareous sponge Sycon ciliatum[END_REF] Sally P. Leys

  hemidesmosomes (HDs) are integrins. Until 2010, because genes encoding for integrins were not retrieved in plant, fungi and choanoflagellate genome surveys, integrins were thought to be a metazoan innovation. Thanks to genomic surveys in several unicellular opisthokont species, integrins were shown to be present in the last common ancestor of Filasterea and Metazoa as well as actors of the integrin machinery (Focal Adhesion kinase and non-receptor protein kinase Src)(Sebé-Pedrós and Ruiz-Trillo, 2010; Sebé-Pedrós et al., 2010). In addition, both integrin α and β in these premetazoan taxa possess the amino acid motifs in their cytoplasmic tails involved in interactions with intracellular scaffolding and signaling proteins therefore suggesting that they could interact in a similar way as their metazoan homologs. Though the role of the integrins in unicellular eukaryotes is currently unknown, it has been evidenced very recently that the Capsaspora integrin are able to recruit human talin. Therefore, this result suggests that the regulation of integrins via talin activity is conserved in Holozoa and predates the emergence of Metazoa[START_REF] Baade | Clustering of Integrin β Cytoplasmic Domains Triggers Nascent Adhesion Formation and Reveals a Protozoan Origin of the Integrin-Talin Interaction[END_REF]).On the other hand, to build HD, plectin is required to form bridges between the cytoplasmic keratin intermediate filament network and the integrins. To date, no plectin has been characterized so far in non-metazoan lineages but other linker proteins allowing the binding between integrins and the cytoskeleton are common among unikonts(Sebé-Pedrós et al., 2010).Altogether, integrins predate the emergence of animal and may play an ancestral role in signaling. The role in cell-ECM adhesion might have been coopted in metazoans(Sebé-Pedrós and Ruiz-Trillo, 2010; Sebé-Pedrós et al., 2010).5.2.3. Genes encoding for Cell-cell Adhesive proteins predate the emergence of animal epitheliaIn the section 1.4 we described the types of junctions found in bilaterians and their molecular composition. To date, because actin-based adhesive-like junctions[START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF][START_REF] Magie | Cell-Cell Adhesion in the Cnidaria: Insights Into the Evolution of Tissue Morphogenesis[END_REF][START_REF] Tamm | Massive Actin Bundle Couples Macrocilia to Muscles in the Ctenophore Beroë[END_REF] and adherens junctions (A. V.[START_REF] Ereskovsky | The Homoscleromorph Sponge Oscarellalobularis, a Promising Sponge Model in Evolutionary and Developmental Biology[END_REF][START_REF] Fahey | Origin of Animal Epithelia: Insights from the Sponge Genome: Evolution of Epithelia[END_REF][START_REF] Leys | Epithelia, an Evolutionary Novelty of Metazoans[END_REF][START_REF] Leys | Epithelia and Integration in Sponges[END_REF] were described in Ctenophora and Porifera respectively, it was assumed that adherens junctions (AJs) constitute the ancestral type of junctions of animals. As a consequence, the cadherins and catenins involved in the establishment of AJs (section 1.4) were searched for in priority in unicellular relatives of animals.

Figure 5 . 4 :

 54 Figure 5.4: Two scenarios for evolution of the three polarity complexes (CRUMBS, PAR and SCRIBBLE complexes) taking into account the uncertainty of phylogenetic relationships at the base of the animal tree (Porifera or Ctenophora as sister group of all other metazoans): either the 3 polarity complexes were already present in the last common ancestor of all extant animals or partially two of them were present.

Figure 5 . 5 :

 55 Figure 5.5: Diagram summarizing the emergence (colored arrows) of genes involved in key epithelial features (junctions, basement membrane and polarity complexes) according to present available data. In the figure, proteins were located in order to remind their function in cnidarians and bilaterians, even though there is no evidence of the functional conservation of these proteins outside the cnidarian-bilaterian lineage. The reader should also note that as far as genes encoding for Neuroglian, Contactin and Neurexin are concerned, their presence in sponges and ctenophores is unclear because of inconsistencies between analyzes : it makes it hard to trace back their evolution, but there is no doubt about their presence in the last common ancestor of Placozoa/Cnidaria and Bilateria.

table 5 .

 5 1 see Lowe and Anderson 2015 for more details).

Table 5 .1 General classification of animal epithelia according to their organization Cell shape Number of cell layers flat cube parallelepiped irregular 1

 5

	Simple squamous	Simple cuboidal	Simple columnar	pseudostratified
	2 or more

Stratified squamous Stratified cuboidal Stratified columnar

  This second solution has the positive consequence of taking into account the obvious conservation of physiological properties (mechanical resistance and occlusion to small molecules) that is found in epithelia of a demosponge (a group lacking basement membranes), a property that appears to be shared with bilaterian epithelia that have occluding junctions (E. D. M.Adams, Goss, et Leys 2010a;[START_REF] Dunn | The Hidden Biology of Sponges and Ctenophores[END_REF][START_REF] Leys | Epithelia, an Evolutionary Novelty of Metazoans[END_REF].Today, according to most authors, there is no longer any doubt on the presence of functional epithelia in the last common ancestor of animals that harbor cell polarity, adhesion properties and basement membrane support[START_REF] Fidler | Collagen IV and Basement Membrane at the Evolutionary Dawn of Metazoan Tissues[END_REF][START_REF] King | Embracing Uncertainty in Reconstructing Early Animal Evolution[END_REF][START_REF] Leys | The Physiology and Molecular Biology of Sponge Tissues[END_REF]

[START_REF] Ereskovsky | The Homoscleromorph Sponge Oscarellalobularis, a Promising Sponge Model in Evolutionary and Developmental Biology[END_REF]

, in contrast the term "epithelium" is currently used for Placozoa

[START_REF] Armon | Ultrafast Epithelial Contractions Provide Insights into Contraction Speed Limits and Tissue Integrity[END_REF][START_REF] Smith | Adherens Junctions Modulate Diffusion between Epithelial Cells in Trichoplax adhaerens[END_REF]

and Ctenophora (Sidney L.

[START_REF] Tamm | Novel Bridge of Axon-like Processes of Epithelial Cells in the Aboral Sense Organ of Ctenophores[END_REF]

. Given such a terminological inconsistency, there are two solutions: 1) either the present definition of epithelium with 3 criteria is kept as it is, meaning that Placozoa, some Ctenophora and most Porifera have to be considered devoid of epithelia; 2) or consider that the epithelium is a synapomorphy of metazoans whatever the presence or absence of basement membrane. 2012a; Sally P.

[START_REF] Leys | Epithelia, an Evolutionary Novelty of Metazoans[END_REF][START_REF] Medwig | Breaking down barriers: the evolution of cell invasion[END_REF]

. We now have to explore how

Some genes encoding for extracellular matrix proteins predate the emergence of animal epithelia

  To do so, even if the data remain scarce, comparative genomics of the closest unicellular relatives of metazoans are needed: choanoflagellates as the sister group of metazoans is of course of particular interest, but other members of Holozoa (such as Filasterea) and Opimoda (including Opisthokonta and Amoebozoa) can be useful[START_REF] Ferrer-Bonet | Capsaspora Owczarzaki[END_REF]

	Olson 2013; Richter et King 2013; Richter et al. 2018; Sebé-Pedrós, Degnan, et Ruiz-Trillo
	2017).
	5.2.1.

In bilaterians and cnidarians, epithelial morphogenesis has been shown to rely strongly on interactions between the epithelial sheet receptors and components of the extracellular matrix (ECM), including the basement membrane when it is present

[START_REF] Aufschnaiter | In Vivo Imaging of Basement Membrane Movement: ECM Patterning Shapes Hydra Polyps[END_REF][START_REF] Dzamba | Extracellular Matrix (ECM) and the Sculpting of Embryonic Tissues[END_REF][START_REF] Fidler | Collagen IV and Basement Membrane at the Evolutionary Dawn of Metazoan Tissues[END_REF][START_REF] Sekiguchi | Chapter Four -Basement Membranes in Development and Disease[END_REF]

.

A few genes encoding for important extracellular matrix proteins or their cellular receptors predate the emergence of Metazoa: indeed, genes encoding for integrins and integrin adhesion machinery are present in unicellular relatives of metazoans (see section 2.2)

[START_REF] Abedin | Diverse evolutionary paths to cell adhesion[END_REF][START_REF] Babonis | Phylogenetic evidence for the modular evolution of metazoan signalling pathways[END_REF] Sebé-Pedrós and Ruiz-Trillo, 2010; Sebé-Pedrós et al., 2010;[START_REF] Suga | The Capsaspora Genome Reveals a Complex Unicellular Prehistory of Animals[END_REF]

. In both Filasterea (C. owczarzaki) and Choanoflagellata (M. brevicolis and Salpingoeca rosetta), several proteins including protein domains that are present in animal ECM proteins (such as for example LAM G, EGF, fibronectin III) are predicted in the genomes of these organisms. But these protein domains are not combined in the same way either in Filasterea and Choanoflagellata, as they are in Metazoa

  [START_REF] Ereskovsky | The Homoscleromorph Sponge Oscarellalobularis, a Promising Sponge Model in Evolutionary and Developmental Biology[END_REF][START_REF] Fahey | Origin of Animal Epithelia: Insights from the Sponge Genome: Evolution of Epithelia[END_REF][START_REF] Ganot | Structural molecular components of septate junctions in cnidarians point to the origin of epithelial junctions in eukaryotes[END_REF]; Sally P. Leys et Riesgo 2012; S. P. Leys, Nichols, et Adams 2009; Magie et Martindale 2008;

Acknowledgements

The authors are grateful to the AM*Idex foundation that funded a first project that enabled to begin the study of epithelia in the sponge Oscarella lobularis (n° ANR-11-IDEX-0001-02) and recently a second international project that now enables us to begin comparative scRNAseq studies to trace back epithelial cell type evolution. The authors also acknowledge the CNRS for the funding of the French-Australian PICS project STraS funding the development of cell staining protocols in order to study epithelia dynamics.

The authors also thank Pr Sally Leys who proofread the text as being a native English speaker.

already present in the last common ancestor of all extant animal relied on the same molecular actors, and to determine which types of junction were present ancestrally.