Guatelli S , Ferrario C , Bonasoro F , Anjo SI , Manadas B , Candia Carnevali MD , Varela Coelho A , Sugni M .

POCI-01-0145-FEDER-029311 Fundação para a Ciência e a Tecnologia, POCI-01-0145-FEDER-402-022125 National Mass Spectrometry Network

	Fig. 1. a–e CE of non-regenerating arm. a Semithin longitudinal section of M. glacialis non-regenerating arm tip showing coelomic compartments lined by CE (arrows): perivisceral coelomic cavity (cc), radial water canal (rwc), ampullae (a), tube feet (tf), hyponeural sinus (hs), terminal tube foot (ttf). Scale bar = 400 µm. b Diagrammatic representation of perivisceral CE structure consisting of apical peritoneocytes (p); longitudinally oriented subapical myocytes (lm); common basal lamina (bl); basiepithelial nerve processes (np); and neurosecretory cells (nc). Circular muscle layer (cm) is housed within the dermis (d) and is surrounded by a proper basal lamina (bl). Unusual vesicular cells (vc) are detected close to circular myocytes: they project their flagella into intercellular niches. Note the apocrine secretion(s) of peritoneocyte apical portions. c Semithin longitudinal section of proximal CE showing peritoneocyte layer (p); longitudinal (lm); and circular (cm) muscle layers separated by connective tissue (ct); dermis (d) infiltrated by muscle bundles (asterisk). Scale bar = 10 µm. d TEM micrograph of the distal CE showing peritoneocytes (green); longitudinal muscle layer (blue); vesicular cell layer (red). Note the absence of the circular muscle layer. Scale bar = 2 µm. e Semithin longitudinal section of the distal CE showing peritoneocyte layer releasing blebs (arrow); longitudinal muscle layer; vesicular cell layer (arrowheads). Scale bar = 10 µm. Abbreviations: CE, coelomic epithelium; ct, connective tissue; o, ossicles; rnc, radial nerve cord; TEM, transmission electron microscopy
	Fig. 2. a–k CE of non-regenerating arm. a TEM micrograph of peritoneocytes. Apical junctions among adjacent cells are visible (black arrows) as well as typical flagella (white arrow). Intense apocrine secretion (asterisk) can be observed. Inset: detail of flagellum surrounded by a crown of microvilli. Scale bar A = 2 µm, scale bar inset = 500 nm. b TEM micrograph of peritoneocytes. Secreted cytoplasmic portion of different shapes and sizes (asterisks) are visible in the perivisceral coelomic lumen. Scale bar = 2 µm. c TEM micrograph of peritoneocytes. Phagosomes are visible at the cell base (arrows). Scale bar = 1 µm. d TEM micrograph of secreted peritoneocyte products filled with granular electron-dense material and other small vesicles (arrow). Scale bar = 2 µm. e Semithin longitudinal section of perivisceral CE showing an evident apocrine secretion. Scale bar = 5 µm. f Semithin longitudinal section of radial water canal CE: a massive apical secretion is shown. Scale bar = 10 µm. g TEM micrograph of CE vesicular cell layer. Vesicular cells are characterized by electron-lucent vesicles and flagella (arrow), and are in direct contact with the longitudinal muscle layer (lm). Scale bar = 2 µm. h TEM micrograph of vesicular cell containing small RER cisternae (black asterisks) and a presumptive phagosome (arrow). Note the presence of flagellum (red asterisk). Scale bar = 1 µm. i TEM detail of flagella of vesicular cells (arrowheads) projecting in an intercellular niche. Scale bar = 1 µm. j TEM detail of vesicular cell showing RER cisternae (arrows). Scale bar = 1 µm. k TEM micrograph of vesicular cells containing phagosomes (arrows) and presumptive lipid droplets (asterisks). Scale bar = 2 µm. Abbreviations: cc, coelomic cavity; CE, coelomic epithelium; RER, rough endoplasmic reticulum; rwc, radial water canal; TEM, transmission electron microscopy; vc, “vesicular” cells
	Fig. 3. a–h CE of stump in a regenerating sample: 24–48 h p.a. a Semithin longitudinal section of M. glacialis arm tip at 24 h p.a. Note the presence of coelomocyte clots at the level of the wound (asterisk), the thickening of the perivisceral CE immediately close to the amputation site, and the new thin epithelium covering the wound edges (arrow). Scale bar = 400 µm. b Semithin longitudinal section of the regenerating CE at 24 h p.a. showing peritoneocyte layer with massive apical secretion; longitudinal muscle layer (lml); thickening of vesicular cell layer (asterisk) associated to circular muscle layer (cml); connective tissue layer (ct). Scale bar = 10 µm. c TEM micrograph of regenerating CE at 24 h p.a. showing peritoneocytes (arrow); longitudinal muscle layer (lml); circular muscle layer (cml) with first signs of rearrangement; vesicular cells (asterisks). Scale bar = 2 µm. d TEM micrograph showing details of vesicular cell layer. A degenerating nucleus (an) is visible among cell debris (cd); a vesicular cell contains a phagosome (arrow). Scale bar = 2 µm. e Semithin longitudinal section of the regenerating CE at the level of the wound closure. The yellow line indicates the beginning of the looser CE structure with the progressive disappearing of both longitudinal (lml) and circular (cml) muscle layers. Scale bar = 50 µm. f TEM micrograph of circular muscle layer (cml). Fibers undergo rearrangement phenomena indicated by evident modifications of their contractile apparatus (arrows). Flagellated vesicular cells are apparently involved. Yellow line marks the rearrangement beginning in both longitudinal and circular muscle fibers. Scale bar = 5 µm. g TEM micrograph of vesicular cells showing flagella (arrow) projecting into intercellular niches. Big phagosomes are visible (asterisks). Scale bar = 2 µm. (h) TEM detail of a large phagosome in vesicular cell. Scale bar = 1 µm. Abbreviations: a, ampulla; cc, coelomic cavity; CE, coelomic epithelium; o, ossicle; p.a., post amputation; TEM, transmission electron microscopy; vc, “vesicular” cell
	Fig. 4. a–j CE of 1 week p.a. regenerating sample. a Semithin longitudinal section of M. glacialis regenerating arm tip. Re-growing perivisceral coelomic cavity (cc) and radial water canal (rwc) appear to penetrate an area of loose coelomocytes clot (asterisk) at the level of the wound site. Scale bar = 400 µm. b Semithin longitudinal section of the regenerating perivisceral coelomic cavity (arrows). A rearranging ampulla (a) is recognizable. Scale bar = 50 µm. c Semithin longitudinal section of the regenerating radial water canal (rwc) lined by the CE. Note the peritoneocyte apocrine secretion at the very tip of the new regenerating structure. Scale bar = 20 µm. d Semithin longitudinal section of CE at the level of the closure of the wound. Both longitudinal and circular muscle layers are not detectable. Scale bar = 50 µm. e TEM micrograph of vesicular cell layer showing several big phagosomes (asterisks). Scale bar = 2 µm. f TEM micrograph of the new coelomic cavity tip, showing details of peritoneocytes. Apical secretion phenomena are evident (asterisks). Adjacent cells are apically joined by cell junctions (double arrows); numerous myelin figures (arrow) and phagosomes (arrowhead) are present in their cytoplasm. Scale bar = 2 µm. g TEM micrograph of regenerating perivisceral CE (p). A faint basal lamina (arrows) is present under coelothelial cells (p) and separates them from the underlying mesenchymal tissue. Scale bar = 2 µm. h TEM micrograph of regenerating perivisceral CE (p) showing evident cell migration toward the mesenchymal tissue (mt). Triple white arrowheads indicate the site of EMT. Cells have high nucleus/cytoplasm ratio (asterisks). Scale bar = 2 µm. i TEM micrograph of new radial water canal (rwc) CE. Peritoneocytes are releasing apical cytoplasmic portions (asterisks). Scale bar = 5 µm. j TEM micrograph of radial water canal (rwc) CE showing the presence of SLSs (asterisks) in peritoneocytes (p). Scale bar = 2 µm. Abbreviations: CE, coelomic epithelium; cf, collagen fibrils; ct, connective tissue; ml, mesenchymal layer; n, nucleus; o, ossicle; p.a., post amputation; rnc, radial nerve cord; SLS, spindle-like structures; TEM transmission electron microscopy
	Fig. 5. Proteins identified in the M. glacialis CE by LC–MS/MS (a, b). a IDA analysis results; b SWATH analysis results. Each colored circle corresponds to one of the three biological replicates analyzed by LC–MS/MS: in pink, number of proteins identified from A1E1 replicate; in green, number of proteins identified from B1F1 replicate; in blue, number of proteins identified from C1D1 replicate. The total number of proteins identified for each biological replicate is shown between parentheses
	Fig. 6. Gene ontology results from STRAP analysis (a–c). a Biological process; b cellular components; c molecular function

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