Fundamental aspects of arm repair phase in two echinoderm models

Abstract

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored. Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools. Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models. Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage.

Keywords: Brittle stars; Collagen; Emergency reaction; Immune/inflammatory response; Starfishes; Wound healing.

Fig. 1. Ultrastructure of the brittle star stump (non-regenerating) epidermis.

Light microscopy (LM) and transmission electron microscopy (TEM). A) Semi-thin sagittal section of the aboral epidermis (arrowhead). B) Semi-thin sagittal section of the oral epidermis (arrowhead). C) The aboral epidermis shows the cuboid epidermal cells nested in the skeletal trabeculae and covered by a well-defined cuticle (arrowhead). The subcuticular space hosts numerous bacteria (asterisks) and beneath the epidermis a presumptive pigment cell is visible (arrow). D) In the oral epidermis bacteria are visible in the subcuticular space (asterisk) and the pleats and folds of the basal lamina (arrow) are present immediately beneath the epidermal cells. E) Detail of Fig. D showing the pleats and folds of the basal lamina and the presence of scattered nervous processes (arrowhead). F) The epidermal cells show microvilli branching in the subcuticular space (arrowheads) and a bacterium inside the cell and surrounded by a membrane (asterisk). G) Detail of Fig. F showing the abundant apical Golgi apparatus (arrows). H) Inclusions of different types (arrows), electron-lucent vesicles (asterisks) and abundant RER (arrowhead) are visible in the epidermal cells. I) The basal lamina shows both thin (white arrowhead) and thick (black arrowhead) structure. Thin collagen fibrils are present immediately underneath. J) In the apical portion of the epidermis the apical zonulae (white arrowhead) and subjacent septate junction (black arrowhead) are visible between two adjacent epidermal cells. Hemidesmosomes (arrows) are connecting the epidermal cells with the underlying basal lamina (asterisk) to maintain epidermis integrity. K) In the presumptive pigment cells the spindle-like electron-dense structures (arrowheads) are present both surrounded or not by a thin membrane. L) The aboral epidermis shows a big presumptive pigment cell underneath the epidermis. Spindle-shaped electron-dense structures (asterisks) are spread in the cytoplasm and are present in lower amount also in some epidermal cells. M) A presumptive secretory cell is scattered among epidermal cells showing long microvilli in the subcuticular space apically breaking the cuticle (arrowhead) and compact electron-dense material packed in roundish membrane-bound vesicles (asterisk) in the cytoplasm. Junction complexes connect this cells to the adjacent epidermal cells. Abbreviations and symbols: acc - aboral coelomic cavity; bl - basal lamina; c - collagen fibril; m in A - muscle; m in J - mitochondrion; n in B - radial nerve cord; n in H - nucleus; t - trabecula; asterisk in C, D, F - bacteria; asterisk in J - basal lamina; asterisk in H - electron-lucent vesicle; asterisk in L - spindle-shaped electron-dense structure; asterisk in M - electron-dense granule.

Fig. 2. Main events of A. filiformis repair phase.

Light microscopy (LM). A) Semi-thin parasagittal section showing the downward and upward movements of the aboral shield and of the oral shield respectively (arrows) to help wound closure. The intervertebral muscles involved in the amputation already show rearrangement phenomena (arrowhead). B) Semi-thin sagittal section where the new epithelium covers the whole wound surface (arrow) and the main body cavities (aboral coelomic cavity and radial water canal) are already sealed. C) Semi-thin sagittal section showing that cells (possibly coelomocytes) are clotting in the aboral coelomic cavity lumen in order to seal it and avoid loss of fluid (arrow). Abbreviations and symbols: acc - aboral coelomic cavity; m - muscle; n - radial nerve cord; p - podium; rwc - radial water canal.

Fig. 3. Main events of the A. filiformis repair phase.

Transmission electron microscopy (TEM). A) The new epithelium presents cells with an oval/roundish nucleus and well-defined nucleolus. The cuticle is already observable (arrowhead) and numerous bacteria (arrows) are present both underneath the epithelium and in the subcuticular space. B) Detail of an apical junction complex (arrow) between adjacent cells of the new epithelium. C) Detail of bacteria enveloped by a thin membrane. D) New epithelial cells show a well-defined cuticle (arrowhead) and patchy nuclei; several phagosomes are detectable. E) Detail of D on phagosomes. F) The new epidermis presents elongated epidermal cells and a well-defined cuticle. Numerous phagosomes (arrowheads) and mitochondria (asterisk) are visible in both epidermal cells and in the underneath thick layer of cells. G) Different cytotypes are present beneath the new epidermis and create a layer dividing the rearranging/regenerating area from the stump extracellular matrix mainly composed of collagen fibrils. H) The new basal lamina (arrowhead) is visible as pleats and folds beneath the epidermal cells. I) Different cytotypes are observable underneath the new epidermis: cells do not form a syncytium and present abundant RER, phagosomes (arrowhead), spindle-shaped electron-dense structures (arrow) and numerous mitochondria. J) Numerous nervous processes (arrowheads) with mitochondria are visible scattered among the different cytotypes. K) In the regenerating area new epidermal cells present a flat-cubic shape and the rearranging contractile apparatus of several myocytes (arrows) is phagocytised by cells underneath the new epidermis. L) In the rearranging/regenerating area spindle-shaped electron-dense structures (arrowhead) are visible together with myofilaments (asterisk). Myelin figures are present as well (arrow). M) The rearranging contractile apparatus of a myocyte (arrow) inside the phagosome of a cell underneath the new epidermis. Abbreviations and symbols: c - collagen; m - mitochondrion; n - nucleus; RER - rough endoplasmic reticulum; asterisk in F - mitochondria; asterisk in L - myosin filaments.

Fig. 4. Expression pattern of Ese-fib-like on E. sepositus regenerating arms (A-G) and of Afi-ficolin on A. filiformis regenerating arms (H-J).

A) Ese-fib-like is expressed in the new epidermis (orange arrowhead), in the circular coelomic muscles (black arrowhead) and in the epidermis of the stump (arrow). B) In the stump expression is detectable in the coelomic epithelium (arrowhead), in the coelomic lining of the papulae (arrows) but no signal is present in the mucous gland (asterisk). C) Cells in the papulae (possibly coelomocytes) are stained (arrow). D) The regenerating radial nerve cord is stained in both ectoneural (arrowhead) and hyponeural (arrow) systems. E) Ese-fib-like is expressed at the level of the radial water canal epithelium (arrow) of the stump. F) The inner lining of the stump ampullae (arrowhead) expresses this transcript. G) Sagittal section scheme where black boxes indicate corresponding images of this figure to facilitate the understanding of the expression pattern location. H) WMISH sample showing that Afi-ficolin is expressed in the dermal layer below the epidermis (arrowhead). I) Post in situ paraffin section showing the expression of Afi-ficolin in the dermal layer of the regenerative bud (arrowheads). J) Sagittal section scheme showing Afi-ficolin expression pattern in the regenerative bud. Signal is highlighted in violet. Red dotted lines: amputation plane. Abbreviations and symbols: AV - aboral view; c - coelom; ct - connective tissue; e - epidermis; m - muscle; o - ossicle; p - podium; SS - sagittal section; asterisk - mucous gland.

Fig. 5. Expression pattern of Ese-p4h on E. sepositus regenerating arms.

A) In a 72 hours p.a. sample Ese-p4h is expressed in the regenerating epidermis (dotted square) and in the epidermis of the stump (arrowhead). B) Detail of A on the signal in the regenerating epidermis (arrow). C) The new epidermis at one week p.a. shows a signal (arrow). D) Sagittal section scheme where black boxes indicate corresponding images of this figure to facilitate the understanding of the expression pattern location. Red dotted line: amputation plane. Abbreviations: ct - connective tissue; m - muscle; p - podium.

Fig. 6. Main similarities/differences in the events of the repair phase among starfishes (E. sepositus), brittle stars (A. filiformis) and mammals.

See colour legend embedded in the figure.