Abstract
Abstract
Abstract
Regeneration is a post-embryonic developmental process that ensures complete Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here 46 used to comparatively investigate the main repair phase events after injury as well as the 47 presence and expression of immune system and extracellular matrix (i.e. collagen) 48 molecules using both microscopy and molecular tools.
Abstract
Our results showed that emergency reaction and re-epithelialisation are similar in both 50 echinoderm models, being faster and more effective than in mammals. Moreover, in 51 comparison to the latter, both echinoderms showed delayed and less abundant collagen (brittle stars), were described for the first time during echinoderm regeneration providing 55 promising starting points to investigate the immune system's role in these regeneration 56 models.
Abstract
Overall, the similarities in repair events and timing within the echinoderms and the 58 differences with what has been reported in mammals suggests that effective repair 59 processes in echinoderms play an important role for the subsequent ability to regenerate.
Abstract
Targeted molecular and functional analyses will shed light on the evolution of these abilities 61 in the deuterostomian lineage. 

Among the different echinoderm models, starfishes (Asteroidea) and brittle stars 137 (Ophiuroidea) are becoming valid experimental models to study arm regenerative process 138 (Ben Khadra et al., 2017; Biressi et al., 2010; Czarkwiani et al., 2013 Czarkwiani et al., , 2016 . Nevertheless, 139 in both classes, the cellular/tissue and molecular aspects of the repair phase have never 140 been simultaneously and comparatively investigated and with a multidisciplinary approach.
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Therefore, this research aims to describe and compare the phenomena occurring during the 142 repair phase after traumatic arm amputation using both the brittle star Amphiura filiformis 143 (Ophiuroidea) and the starfish Echinaster sepositus (Asteroidea). Classical histological and 144 ultrastructural methods are employed for the description of the main repair events from a 145 cell/tissue perspective, whereas molecular techniques are used to investigate the 146 involvement of inflammatory/immune responses and the ECM (mainly collagen). Overall, a 147 detailed knowledge on how echinoderms heal severe wounds, and actually regenerate, will 148 possibly shed light on similarities and/or differences with other animals able to regenerate 149 whole lost body parts and, also, with those unable to do it, humans included. 
Light (LM) and transmission electron microscopy (TEM)

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For Epon resin embedding regenerating samples were fixed in 2% glutaraldehyde in 0.1 M 178 sodium cacodylate (pH about 7.4) with 1.2% (brittle stars) or 1.4% (starfishes) NaCl and 179 washed overnight at 4°C in 0.1 M cacodylate buffer. They were then processed as described 180 by Ben Khadra and co-workers (2015a) with only slight modifications in decalcification step 181 that was performed after osmium tetroxide post-fixation at 4°C for at least 2-3 days using a AxioImager M1 microscope equipped with a Zeiss AxioCamHRc camera.
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For transmission electron microscopy (TEM) the same samples used for semi-thin sections 188 were used to obtain ultra-thin sections (0.07-0.1 µm) which were collected on copper grids, 189 stained with 1% uranyl acetate followed by lead citrate and finally carbon coated with an 190 EMITECH K400X Carbon Coater. Grids were observed and photographed using a Jeol 191 100SX, a Zeiss EFTEM Leo912ab or a PHILIPS CM 10 transmission electron microscope. (NCBI) databases. After cloning a specific fragment by PCR using these primers, Ese-p4h Table S1 ). Furthermore, the conserved domain architecture retrieval tool
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(cDART, NCBI) showed the 2OG-Fe(II) oxygenase superfamily domain is encoded on the
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Ese-p4h isolated fragment. This domain is characteristic of P4H therefore confirming it was 225 the desired collagen biosynthesis enzyme.
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Degenerate primers from Zhang and Cohn (2006) for vertebrate collagen were tested as 227 well (see Table S2 ). Ese-fibrinogen-like (Ese-fib-like) is a gene belonging to the fibrinogen-228 related (FReD) domain superfamily. Using the cDART tool (NCBI) the presence of a FReD 229 domain was confirmed. This is usually present in fibrinogen, a glycoprotein that helps in the 230 formation of blood clotting in vertebrates forming bridges between platelets and being the 231 precursor of fibrin.
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As previously mentioned, actin 1 and ets1/2 were selected as positive controls: specific 233 primers were designed based on the nucleotide sequence of actin 1 (NCBI accession 234 number: KC858258.1, GI: 525327359; see Supplementary Materials), whereas degenerate 235 primers already available in the laboratory were used to clone ets1/2 (see Table S2 ). For 236 actin 1, since the expected product length was shorter than 300 bp, 3'RACE was performed 237 using a mixed cDNA samples from regenerate stages with the FirstChoice ® RLM-RACE Kit (Ambion) according to manufacturer's instructions (see Supplementary Materials and Table S3 ). We cloned a longer fragment that was used to obtain a longer RNA antisense probe for 240 in situ hybridisation (see below). 
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The Afi-p4h (AfiCDS.id43946.tr460) similarly identified as best BLAST hit in the sea urchin 252 genome (EchinoBase; http://www.echinobase.org/Echinobase/) the prolyl-4-hydroxylase 253 alpha-1 subunit precursor (SPU_027669), whereas in the NCBI non-redundant database 254 the Atlantic herring prolyl-4-hydroxylase subunit alpha-1 (Clupea harengus,
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XP_012689665.1). The cDART tool confirmed the presence of a prolyl-4-hydroxylase alpha 256 subunit domain. Therefore, this transcript was considered as prolyl-4-hydroxylase (p4h).
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The Afi-ficolin gene (AfiCDS.id39565.tr647) was isolated from an A. filiformis cDNA pool.
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The clone sequence was analysed using BLAST-X against the sea urchin genome (S.
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purpuratus; EchinoBase) and NCBI non-redundant database and confirmed to belong to the 260 FReD superfamily and to be a closely related gene to the sea urchin Sp-Fic1 (SPU_000045). Table S1 summarises the best BLAST hits of the identified genes in EchinoBase (SPU best 262 BLAST) and in NCBI (NCBI best BLAST) with corresponding scores and E-values. 
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Primer design

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Different design strategies were followed depending on the gene of interest and sequence 266 availability. For specific primers in both species PRIMER3 Software version 0.4.0
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(http://primer3.ut.ee/) was used, optimising the following parameters: max 3' stability was 268 rrush@example.org and max polyX at 3. For brittle stars their specificity was checked performing a
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BLAST to the A. filiformis developmental transcriptome (Dylus et al., 2017) . Degenerate 270 primers were manually designed as described above. Tables S2 and S3 summarises 
297
Chromogenic WMISH was performed with antisense probes as previously described along 298 with positive and negative controls (Czarkwiani et al., 2013) with the following modifications: 299 hybridisation temperature was raised to 50-55°C depending on the probe length and all 300 washes were conducted in 1X MABT (0.1 M maleic acid pH 7.5, 0.15 M NaCl, 0.1% Tween-301 20). Samples were stored in 50% glycerol at 4°C and subsequently observed under a Zeiss
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AxioImager M1 microscope equipped with a Zeiss AxioCam HRc camera.
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After imaging, WMISH samples were embedded in paraffin wax and sectioned in order to 304 better understand the tissue-specific expression patterns. Briefly, samples stored in 50% 305 glycerol were washed in 1x PBS or 1x MABT at room temperature (RT) and decalcified for 1-2 days in 0.5 M EDTA in 1x PBS (pH 8) 
Results

331
A brief description of the gross morphology of starfish and brittle star arms is re-called in the
332
Supplementary Materials to facilitate the understanding of the subsequent results (Fig. S1 ).
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Since the epidermis plays a key role during the repair phase (see below) and no data is 
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In A. filiformis, the aboral and oral epidermis lines the trabeculae of the skeletal shields ( Fig. 
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1A, B, C). This epithelium is composed of an external cuticle, the epidermal cells and the 339 underlying basal lamina (Fig. 1C, D, F) . The epidermal cells and the subcuticular space house numerous bacteria (Fig. 1C, D, F) . A sub-epithelial nerve plexus is occasionally 341 detectable underneath the basal lamina (Fig. 1E) . The cuboidal epidermal cells present 342 different organelles and inclusions (Fig. 1F , G, H) and are connected to each other by apical 343 junctional complexes and to the underlying basal lamina (Fig. 1I ) and the dermal layer by 344 hemidesmosomes (Fig. 1J ). Secretory cells (granulated cells) are observable (Fig. 1M) spindle-like electron-dense structures are visible in the dermal layer (Fig. 1C, K, L) . These 347 structures, whose specific nature is still unknown, are sometimes present also in the 348 epidermal cells (Fig. 1L) . provided with microvilli and cuticle, is visible within 8 hours p.a. (Fig. 2B, 3A) . It is composed similarly to starfishes (Fig. S2B) , the new epidermis migrates centripetally over the wound.
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The basal membrane becomes visible only at the middle/late repair phase (after 48-72 hours p.a.), initially as a collection of fragmented pleats and folds rather than a continuous and Therefore, the analyses of some genes relevant for the repair phase were performed, as 416 detailed below.
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Immune/inflammatory response-related genes

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The precise regulation of the immune response after injury is a critical factor. Therefore, the Table S1 ). The
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FReD domain was confirmed also using the cDART tool (NCBI). ISH of Ese-fib-like shows 428 a distinct staining in the new epithelium covering the wound area at one week p.a. (Fig. 4A) 
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and in the regenerating radial nerve cord in the ectoneural and hyponeural systems at 72 430 hours p.a. (Fig. 4D ). In the stump area, Ese-fib-like expression is localised in the epidermis 431 ( Fig. 4A, B) , in the coelomic epithelium lining the perivisceral cavity (Fig. 4B ), the papulae 432 ( Fig. 4B, C) , the radial water canal (Fig. 4E ) and the ampullae ( A. filiformis (see Table S1 ). Afi-ficolin is expressed in the dermal lining of the epidermis in this transcript is localised in the radial water canal epithelium (Fig. S3 ).
Collagen biosynthesis enzyme gene

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Collagen is a key protein of the repair phase and its biosynthesis necessarily needs to be 
448
In starfish regenerating tissues Ese-p4h expression is detected in the new epidermis at both 449 72 hours and one week p.a. (Fig. 5) . The signal in the stump tissues is further described in 450 the Supplementary Materials (Fig. S4A-C) and suggests that other epithelial tissues, such 451 as the coelomic lining and the radial nerve cord, may have a role in collagen biosynthesis.
452
In brittle stars, besides the stump tissues (Fig. S4D ), in the regenerative bud Afi-p4h is 453 expressed in the coelomic lining but only after the repair phase is finished ( Fig. S4E-I ). 
460
The information gathered on the different repair events is discussed below. sepositus (Ben Khadra et al., 2015a; Ben Khadra et al., 2017) and from starfishes in general 466 (Mladenov et al., 1989; Candia Carnevali et al., 1993; Moss et al., 1998) , in brittle stars no 467 evident circular constriction of the arm-tip is detectable. This is consistent with the different 468 brittle star arm anatomy (i.e. conspicuous skeletal elements and the absence of a circular 469 muscle layer surrounding the coelomic cavity). Here the apical contraction of the body wall 470 is sufficient for sealing the narrow fluid-filled vessels/cavities (aboral coelomic cavity and 471 radial water canal). In comparison, blood vessel constriction and wound contraction are 472 fundamental events also in mammal wound healing (Pastar et al., 2014; Ibrahim et al., 2015) 473 but while the former is an almost immediate reaction, the latter is delayed comparing to the 474 events happening in both echinoderm models. In humans, skin wound shrinkage slowly starts almost immediately after injury but its main peak of activity occurs around 10 days 476 after the damage (Shultz et al., 2005) , different from echinoderms, where it is visible within 477 1-2 days p.a. (Fig. 2A, C) . The delay observed in mammals might be due to the "time 478 consuming" activation of fibroblasts resident in the injury's neighbourhood which have to 479 leave their quiescent state, migrate towards the wound and be transformed into 480 myofibroblasts, the ultimate responsible of wound contraction (Martin, 1997).
481
Besides constriction, in both echinoderm species, loss of fluid prevention is also mediated 482 by rapid clotting of circulating cells (coelomocytes), a phenomenon analogous to mammalian 483 platelet clot formation (Peacock, 1984; Clark, 1988; Ibrahim et al., 2015) . Noteworthy, in et al., 2014) . In both echinoderms and mammals (Clark et al., 1982; Larjava et al., 499 1993) a well-defined basal lamina is not detectable until after the complete differentiation of 500 epidermal cells, which facilitates their migratory movements.
501
The events occurring after re-epithelialisation slightly differ in the histological organisation 502 between the two echinoderm models. Indeed, the wound area of starfish arm is 
Conclusions

580
In this study the brittle star Amphiura filiformis and the starfish Echinaster sepositus were 581 used as models to describe and compare the repair phase phenomena after arm amputation 582 within echinoderms as well as with mammals' healing events after wound. The main 583 similarities and differences between them are summarised in Table 1 Moreover, over-deposition of collagen (fibrosis) is never detectable. Overall, the 594 temporary loose configuration of the ECM is likely to be more "plastic" than the 
597
It is important to point out that re-epithelialisation, inflammatory/immune system-related 598 genes and ECM fibrillar organisation/deposition during brittle star and starfish repair phases 599 were here deeply described. Furthermore, interesting differences and similarities in repair 600 events and timing within echinoderms and between echinoderms and mammals were 601 highlighted. The comparison between animals able or unable to regenerate after injury 602 suggests that regenerative abilities are mechanistically diverse, from the very first repair 603 events. These differences, contrary to what is assumed, are not just differences in the 604 subsequent re-growth capacities. In the future, perturbation tests aimed to impair/block re-605 epithelialisation, immune response or ECM deposition should be performed to test the 606 hypothesis that specific repair events are strictly necessary to permit an efficient 607 regenerative process. Moreover, our findings show that echinoderms, and starfishes 608 especially, can be considered valid alternative models to study wound healing and 609 regeneration in light of human health future applications (Gurtner et al., 2008) . 
Extended Materials and Methods

Microscopy analyses

Scanning electron microscopy (SEM) of E. sepositus regenerating samples
After sagittal sectioning, the remaining paraffin embedded half-samples of regenerating arms were used for scanning electron microscopy as described in Ben Khadra and coworkers (2015a, b). Briefly, they were washed several times with xylene followed by absolute ethanol and then transferred to a series of solutions of Hexamethyldisilazane (HMDS) in absolute ethanol in different proportions (1:3, 1:1, 3:1, and 100% HMDS). Finally, they were mounted on stubs, covered by a thin layer of pure gold (Sputter Coater Nanotech) and observed under a scanning electron microscope (LEO-1430).
Gene expression analyses

3'RACE and degenerate PCR protocols for E. sepositus cDNA amplification
For Ese-actin standard gradient PCR was performed and the transcript was successfully cloned. However, since the PCR product was short (less than 300 bp), 3'RACE was performed using a mixed cDNA samples from regenerate stages with the FirstChoice ® RLM-RACE Kit (Ambion) according to manufacturer's instructions optimising annealing temperature (55°C) and number of cycles (40) in order to obtain a longer product (predicted length ~ 1 kb) and thus a longer RNA antisense probe. Primers used for 3'RACE are listed in Table S3 . Also these PCRs were successful, therefore both PCR products were cloned and used to transcribe RNA antisense probes.
3'RACE and degenerate PCR protocols for E. sepositus cDNA amplification
For Ese-ets1/2 degenerate primers (100 µM; see Table S2 ) were used on a mixed cDNA samples from different regenerative stages as follows: 95°C for 5' followed by 35 cycles of 95°C for 30'', temperature gradient for 30'' and 72°C for 30'' and a final 7' elongation at 72°C.
3'RACE and degenerate PCR protocols for E. sepositus cDNA amplification
The temperatures of the gradient from the highest to the lowest were: 60°C, 54.5°C, 48°C
3'RACE and degenerate PCR protocols for E. sepositus cDNA amplification
and 45°C. The amplification was successful for all of them and all PCR products were pooled, cloned and used to obtain the RNA antisense probe as already described.
3'RACE and degenerate PCR protocols for E. sepositus cDNA amplification
For Ese-p4h, manually designed degenerate primers (100 µM; see Table S2 ) were used with Q5 High-Fidelity DNA Polymerase (New England BioLabs) and Invitrogen reagents and the following protocol: 98°C for 30'', 35 cycles of 98°C for 10'', temperature gradient for 30'' and 72°C for 30'' and a final 2' elongation at 72°C. After purification with NucleoSpin ® gel and PCR clean-up kit (Macherey-Nagel), cloning and RNA antisense probe transcription were performed as already described.
3'RACE and degenerate PCR protocols for E. sepositus cDNA amplification
Collagen-specific degenerate primers (20 µM; see Table S2 ) from Zhang and Cohn (2006) were used on a mixed cDNA samples from different regenerative stages. To optimise the amplification, the following protocol was tested and subsequently modified: 94°C for 1'
3'RACE and degenerate PCR protocols for E. sepositus cDNA amplification
followed by 35 cycles of 94°C for 45'', temperature gradient for 45'' and 68°C for 2' and a final 10' elongation at 68°C. The temperatures of the gradient from the highest to the lowest were: 60°C, 54.5°C, 50.8°C and 45°C. 50.8°C was selected as best amplification temperature and cycles were increased to 40. The PCR product was then purified with NucleoSpin ® gel and PCR clean-up kit (Macherey-Nagel) and cloned as already described. 
Whole mount in situ hybridisation (WMISH)
For each MABT and HB was performed, followed by a wash of 10 minutes with 75% 1X MABT/25%
Whole mount in situ hybridisation (WMISH)
HB. Two washes with 1X MABT were then followed by two washes with 0.1X MABT supplemented with 0.1% Tween-20. All these washes were performed at 50°C-55°C.
Whole mount in situ hybridisation (WMISH)
Samples were incubated with blocking buffer (BB; 5% goat serum in 1X 1X MABT and 50% glycerol was performed and samples were stored in 50% glycerol till observation.
In situ hybridisation (ISH) on paraffin sections
Two different ISH protocols were optimised and performed on starfish paraffin sections.
In situ hybridisation (ISH) on paraffin sections
Moreover, since it was the first time that ISH was performed on E. sepositus sections negative controls were performed as well (see Extended Results).
In situ hybridisation (ISH) on paraffin sections
The first protocol was modified from the WMISH protocol used for A. filiformis samples (see were pre-hybridised in HB for one hour at 45°-55°C. Probes in HB were subsequently added at a final concentration ranging from 0.02 to 1 ng/µl and left for one or five days at 45°-55°C in humid chamber. The following steps (i.e. washes post-hybridisation, blocking, antibody exposure and staining) were performed as described for brittle star samples (see above).
In situ hybridisation (ISH) on paraffin sections
When staining was complete the reaction was stopped with one wash in 1X MABT with 0.5 M EDTA followed by three washes in 1X MABT. Finally, slides were mounted with 50%
In situ hybridisation (ISH) on paraffin sections
glycerol and stored at RT till observation.
In situ hybridisation (ISH) on paraffin sections
The second protocol was described by Etchevers and co-workers (2001) 
Extended results

Gross morphology of starfish and brittle star non-regenerating arms
Starfish arms (Fig. S1A) are mainly occupied by a spacious perivisceral coelom containing the pyloric caeca and the ampullae, the inner outgrowths of the podia. Two rows of podia, located on the oral surface, run along the whole arm together with the radial water canal and the radial nerve cord. The body wall is mainly occupied by calcitic ossicles and spines embedded in an abundant dermal layer and joined by muscle bundles.
Gross morphology of starfish and brittle star non-regenerating arms
Brittle star arms (Fig. S1B ) are subdivided in repetitive segments, each one mainly occupied by a set of skeletal elements, namely the central vertebra, the inner aboral, oral and lateral shields and the external spines, all embedded in a thin dermal layer. Muscle bundles and ligaments link the adjacent segments. The aboral coelomic cavity (much reduced if compared to that of starfishes), the radial water canal and the radial nerve cord uninterruptedly run along the whole arm length. Differently from starfishes, the digestive tract is not hosted in the arm. 
Main events of E. sepositus repair phase
The main events of the repair phase are here re-called in Fig. S2 . For a detailed description see Ben Khadra and co-workers (2015a, b). n -nucleus; oe -oedematous area; pl -papula lumen; SEM -scanning electron microscopy; TEM -transmission electron microscopy.
Extended ISH results
For both echinoderm species the gene expression patterns in the stump was evaluated as well since its tissues are in close continuity with the regenerating tissues and might therefore be important during the regenerative process. Moreover, although not strictly relevant for the repair phase, in some cases the signal in the advanced regenerative stages of A.
Extended ISH results
filiformis was considered to confirm/validate the results obtained in the early stages and have a more complete overview of the expression pattern of the identified genes throughout regeneration. Thus, expressions of Afi-ficolin, Ese-p4h and Afi-p4h are described below.
Extended ISH results
The analysis of Afi-ficolin expression pattern reveals a strong signal in the stump radial water canal epithelium (Fig. S3 ). This gene is detectable in the early regenerative phase after injury in the stump and in the regenerating tissues (Fig. 4) suggesting that it is likely to be involved in the repair/early regenerative phases, in line with its immunity-related function. In the stump tissues, Ese-p4h is present in the coelomic epithelium lining the perivisceral coelom, the papulae and the radial water canal, in the pyloric caeca and in the ectoneural and hyponeural systems of the radial nerve cord (Fig. S4A-C) .
Extended ISH results
Considering Afi-p4h, in the stump tissues it is localised in the inner coelomic lining of the podia (Fig. S4D) . At stage 2 the blueish staining visible in the new epidermis is background (Fig. S4E ). In the >50% stages at the proximal side a true signal is localised in the aboral coelomic cavity epithelium, whereas blueish staining detectable in the podia and spine epidermis is background staining (Fig. S4F-I ).
Extended ISH results
Overall, considering both experimental models the p4h gene is consistently expressed, even if at different stages, only in the coelomic lining suggesting a possible involvement of these tissue in collagen biosynthesis. filiformis. A) Ese-p4h is expressed in the coelomic lining of the papulae (arrow). B) A signal is visible in the coelomic lining of the perivisceral cavity (arrowhead) and in the pyloric caeca. C) Ese-p4h is expressed in the coelomic lining of the radial water canal (black arrowhead), and in the ectoneural (arrow) and hyponeural (white arrowhead) systems of the radial nerve cord. D) Afi-p4h is expressed in the inner lining of the stump podium (arrow). E) At stage 2 the blueish staining visible in the epidermis is just background due to long staining step. F) Afi-p4h is expressed in the aboral coelomic cavity epithelium (arrowhead). The blueish staining in the epidermis covering spines and podia is background. G) Cross section of F showing the staining in the aboral coelomic cavity epithelium (arrowhead). The blueish staining in the epidermis is background. H) The blueish staining in the spine epidermis is background. I) Cross section scheme showing Afi-p4h expression pattern (violet). Abbreviations: AV -aboral view; CS -cross section; ct -connective tissue; m -muscle; p -
Fig. S5. Expression pattern of Ese-actin on E. sepositus stump tissues and regenerating arms. A)
Ese-actin is expressed in the coelomic cavity epithelium (arrowhead) and in the pyloric caeca (arrow) of the stump. B) The inner lining of the stump papulae (i.e. coelomic epithelium) shows expression of this transcript (arrows). C) Ese-actin is expressed in the inner lining of the stump ampulla (i.e. coelomic epithelium; arrowhead). D) Ese-actin is expressed in the epidermis of the stump podia (arrow) and in the inner coelomic lining (arrowhead). E) Ese-actin is expressed in the epidermis of the stump (arrow). F) Ese-actin shows an expression in the stump radial nerve cord, in particular in the ectoneural (arrowhead) and in the hyponeural systems (arrow). G) Ese-actin is expressed in the new epidermis (arrow). H) The regenerating radial nerve cord (arrow) and radial water canal 
Negative controls in E. sepositus and A. filiformis
Since ISH protocol was performed for the first time on E. sepositus paraffin sections, negative controls were run to evaluate staining specificity. No signal was detected in different tissues (Fig. S8) , therefore signal specificity was confirmed. For negative controls in A. filiformis see Czarkwiani and co-workers (2013). 

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