Wilkie IC and Candia Carnevali MD (2023), Morphological and Physiological Aspects of Muta...

ECB-ART-51524

Mar Drugs 2023 Feb 22;213:. doi: 10.3390/md21030138.

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Morphological and Physiological Aspects of Mutable Collagenous Tissue at the Autotomy Plane of the Starfish Asterias rubens L. (Echinodermata, Asteroidea): An Echinoderm Paradigm.

Wilkie IC , Candia Carnevali MD .

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The mutable collagenous tissue (MCT) of echinoderms has the capacity to undergo changes in its tensile properties within a timescale of seconds under the control of the nervous system. All echinoderm autotomy (defensive self-detachment) mechanisms depend on the extreme destabilisation of mutable collagenous structures at the plane of separation. This review illustrates the role of MCT in autotomy by bringing together previously published and new information on the basal arm autotomy plane of the starfish Asterias rubens L. It focuses on the MCT components of breakage zones in the dorsolateral and ambulacral regions of the body wall, and details data on their structural organisation and physiology. Information is also provided on the extrinsic stomach retractor apparatus whose involvement in autotomy has not been previously recognised. We show that the arm autotomy plane of A. rubens is a tractable model system for addressing outstanding problems in MCT biology. It is amenable to in vitro pharmacological investigations using isolated preparations and provides an opportunity for the application of comparative proteomic analysis and other "-omics" methods which are aimed at the molecular profiling of different mechanical states and characterising effector cell functions.

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	Figure 4. Light microscopy (LM). Histology of dorsolateral body wall. Parasagittal (A,B) and sagittal (C–F) sections stained with Milligan’s trichrome [31]: collagenous tissue is blue, muscle and other cellular tissues are magenta. In all sections, distal end is on left and dorsal side is on top. (A) General view including intact breakage zone (approximate position indicated by arrowheads). Scalebar = 0.2 mm. (B) Intact breakage zone. Scalebar = 0.1 mm. (C) Breakage zone at level of carinal ossicles. Scalebar = 0.1 mm. (D) Attachment area between interossicular muscle and carinal ossicle. Scalebar = 10 µm. (E–G) Autotomising breakage zone (between arrowheads). (E) General view (coelomic layers missing). Double arrows indicate rupturing intercarinal muscle and single arrows rupturing intercarinal ligament. Scalebar = 0.2 mm. (F) Rupturing intercarinal muscle (double arrows) and intercarinal ligament (single arrows). Scalebar = 50 µm. (G) Coelomic side of autotomising body wall. Although inner dermis is disintegrating, adjacent apical muscle and coelothelium are intact. Scalebar = 20 µm. Asterisk, expanded sparsely fibrous region; am, apical muscle; ce, coelothelium; cmb, circular muscle bundle; co, carinal ossicle; do, dorsolateral ossicle; ep, epidermis; id, inner dermis; il, interossicular ligament; im, interossicular muscle; is, intradermal space; od, outer dermis; pa, papula; pe, pedicellaria; sf, sparsely fibrous region; te, tendon.
	Figure 1. A specimen of Asterias rubens undergoing arm autotomy after intracoelomic injection of 0.56 M KCl. (A) General view of whole animal. (B) One arm shows basal constriction at the level of the autotomy plane (arrows) and the adjacent arm is undergoing detachment (arrowheads). (C) After complete detachment of both arms. pc, pyloric caeca.
	Figure 2. Diagram of a transverse section of an arm of Asterias rubens at the level of the autotomy plane. Not to scale. Blue: collagenous tissue; pink: muscle; yellow: ossicles; aao, adambulacral ossicle; am, apical muscle; ao, ambulacral ossicle; at, ampulla of tube-foot; ce, coelothelium; co, carinal ossicle; de, dermis; ep, epidermis; ids, inner dermal sheath; lim, longitudinal interambulacral muscle; me, mesentery; pa, papula; pd, pyloric duct; pe, pedicellaria; ph, perihaemal canal; pvc, perivisceral coelom; rh, radial haemal sinus; rn, radial nerve cord; rs, retractor strand; rw, radial water vascular canal; sp, spine; tf, tube-foot. * Adjacent pairs of ambulacral ossicles are connected by longitudinal interambulacral ligaments (not shown). ** Adjacent adambulacral ossicles are connected by longitudinal interadambulacral ligaments and muscles (not shown).
	Figure 3. Stereomicroscopy (STM). (A) Proximal end of detached arm of A. rubens in frontal view immediately after autotomy. Scalebar = 1 mm. (B) Inner view of dorsal body wall at base of intact A. rubens preserved in 70% ethanol; distal end at bottom. Scalebar = 1 mm. Arrowheads, approximate position of breakage zone; ao, ambulacral ossicle; at, ampulla of tube-foot; dl, dorsolateral body wall; do, dorsolateral ossicles; go, gonad; pc, pyloric caecum; pd, pyloric duct; ps, pyloric stomach; rpd, ruptured end of pyloric duct; rn, radial nerve cord; tf, tube-foot.
	Figure 5. Transmission electron microscopy (TEM). Ultrastructure of dorsolateral body wall: extracellular components. (A) Expanded sparsely fibrous layer (ESFR) of outer dermis: general view. Transverse sections of collagen fibrils appear as electron-lucent spaces. Scalebar = 1 µm. (B) ESFR adjacent to epidermis. Scalebar = 0.2 µm. (C) Inner dermis: general view. Scalebar = 2 µm. (D) Inner dermis. Scalebar = 0.5 µm. (E,F) Tendons of interossicular muscle. (E) General view. Scalebar = 2 µm. (F) Junction between tendon and stereom trabecular coat. Scalebar = 0.5 µm. bl, basal lamina; cf, collagen fibril; ep, epidermis; gf, granulo-filamentous material; im, interossicular muscle; jp, juxtaligamental cell process; mf, microfibril; st, decalcified stereom bar; tc, trabecular coat; te, tendon ((A) adapted by permission of Taylor and Francis Group, LLC, a division of Informa plc., from reference [19], copyright 1990).
	Figure 6. TEM. Proteoglycan histochemistry of dorsolateral body wall. (A–E) ESFR. (A–C) Cupromeronic blue (method of Erlinger et al. [37]). (A) Longitudinal section of collagen fibrils showing electron-dense deposits arranged regularly at each D-period (arrowheads). Scalebar = 0.2 µm. (B) Transverse sections of collagen fibrils with deposits appearing as interfibrillar bridges (arrowhead). Scalebar = 0.2 µm. (C) ESFR adjacent to epidermis, with deposits at basal lamina and in granulo-filamentous areas (arrowheads). Scalebar = 0.2 µm. (D,E) Polyethyleneimine (method of Sauren et al. [36]). (D) Electron-dense granules located on outer surface of collagen fibrils (arrowheads). Scalebar = 0.5 µm. (E) Adjacent to epidermis, basal lamina is labelled (arrowheads) but granulo-filamentous areas are unlabelled. Scalebar = 0.5 µm. (F,G) Inner dermis. (F) Cupromeronic blue: deposits arranged D-periodically on surface of collagen fibrils and forming interfibrillar bridges (arrowheads). Scalebar = 0.2 µm. (G) Polyethyleneimine: electron-dense granules arranged D-periodically. Scalebar = 0.2 µm. bl, basal lamina; cf, collagen fibril; ep, epidermis; gf, granulo-filamentous material.
	Figure 7. TEM. Ultrastructure of dorsolateral body wall: cellular components of inner dermis. (A–F) Intact body wall. (A) Three types of LDCV-containing JLC processes are present (1–3). Scalebar = 1 µm. (B) Aggregation of cell bodies and processes. Type 1 and 3 JLC processes and a nucleated type 1 juxtaligamental cell body are visible. Another cell body contains many presumptive lipid droplets. Scalebar = 1 µm. (C) Edge of cellular aggregation with possible gliocyte. Scalebar = 1 µm. (D–F) LDCVs of JLCs in sections stained with cupromeronic blue. (D) ESFR. Electron-dense deposits are present at LDCV membranes (arrowheads). Scalebar = 0.5 µm. (E) ESFR. Type 1 LDCV showing paired deposits separated by vesicle membrane (arrowheads). Scalebar = 0.1 µm. (F) Inner dermis. Electron-dense deposits are present at LDCV membranes (arrowhead). Scalebar = 0.2 µm. (G,H) JLC processes in autotomising body wall. (G) Type 1 LDCVs (1) show reduction in electron density, which is not evident in type 2 LDCVs (2). Scalebar = 1 µm. (H) Cluster of type 2 and 3 JLC processes alone. Scalebar = 0.5 µm. cf, collagen fibrils; ci, cilium; ex, vesicle undergoing exocytosis; jb, juxtaligamental cell body; gc, possible gliocyte; gp, possible gliocyte process; li, possible lipid inclusion; nu, nucleus ((G) and (H) adapted by permission of Taylor and Francis Group, LLC, a division of Informa plc., from reference [19], copyright 1990).
	Figure 8. Scanning electron microscopy (SEM). Dorsal views of specimens of A. rubens from which most of dorsolateral body wall has been removed. (A–C) Soft tissues incompletely digested with bleach. (A) General view of one arm and adjacent mouth frame. Arrowheads indicate commonest level of breakage at autotomy (in larger animals). Box delineates margins of isolated preparations of ambulacral body wall used in physiological experiments (see Section 3.2). Scalebar = 1 mm. (B) More magnified view of proximal portion of specimen in A. Arrowheads indicate commonest level of breakage at autotomy. Scalebar = 0.2 mm. (C) Three interambulacral joints showing remnants of collagenous components. Arrows indicate attachment areas of longitudinal interambulacral muscles, which have been completely digested. Scalebar = 0.1 mm. (D,E) Undigested specimen showing location of extrinsic stomach retractor apparatus (ESRA). (D) General view. Scalebar = 1 mm. (E) ESRA associated with one arm. Arrowheads indicate location of autotomy breakage zone. Scalebar = 0.4 mm. ao, ambulacral ossicle; cs, cardiac stomach; do, dorsolateral ossicles; dr, distal retractor strand; es, extrinsic stomach retractor apparatus; ids, inner dermal sheath; lil, longitudinal interambulacral ligament; me, mesentery; mf, mouth frame; no, nodule; pr, proximal retractor strand; tfo, opening for tube-foot; til, transverse interambulacral ligament.
	Figure 9. LM. Histology of ambulacral body wall. Horizontal sections stained with Milligan’s trichrome. In all sections, distal end is at bottom. (A) General view of one side of ambulacral ridge of arm undergoing autotomy, with radial nerve cord included. Arrow indicates breakage plane at which longitudinal ambulacral ligament has ruptured (arrowheads). Adjacent longitudinal interambulacral ligaments are intact. Radial nerve has started to fracture (asterisks). Scalebar = 0.2 mm. (B) Intact longitudinal interambulacral ligament, which is continuous with inner dermal sheath. Scalebar = 0.1 mm. (C) Intact longitudinal interambulacral ligament, which includes acidophilic cell bodies and processes (arrowheads). Scalebar = 20 µm. (D) Ruptured longitudinal interambulacral ligament at autotomising breakage plane, showing fibre disaggregation (arrow). Scalebar = 20 µm. (E–G) Ambulacral ridge of arm of another animal undergoing autotomy. Plane of section at more dorsal level than those shown in (A–D). (E) General view. Arm is undergoing autotomy at level indicated by arrow. Longitudinal interambulacral ligament at breakage plane is undergoing disaggregation. Longitudinal interambulacral muscles have ruptured at breakage plane (asterisk) and at other levels. Scalebar = 0.1 mm. (F) More magnified view of breakage plane shown in (E). Longitudinal interambulacral muscle has ruptured completely; longitudinal interambulacral ligament, inner dermal sheath and coelothelium have also ruptured. Scalebar = 50 µm. (G) Longitudinal interambulacral muscle and adjacent tissues at level two interambulacral joints distal to that shown in (F). Muscle rupture is less advanced than in (F). Longitudinal interambulacral ligament, inner dermal sheath and coelothelium appear to be intact. Scalebar = 50 µm. ao, ambulacral ossicle; at, ampulla; ce, coelothelium; ids, inner dermal sheath; lil, longitudinal interambulacral ligament; lim, longitudinal interambulacral muscle; mf, mouth frame; ph, perihaemal canal; rh, radial haemal sinus; rn, radial nerve cord; tf, tube-foot.
	Figure 10. TEM. Ultrastructure of longitudinal interambulacral ligament. (A) Longitudinal and transverse sections of collagen fibres, each fibre comprising a tightly packed bundle of fibrils. Scalebar = 1 µm. (B) Cellular aggregation. Two types (1, 2) of JLC processes and somata are present. Scalebar = 2 µm. (C) Enlarged detail of JLC components in (B). Scalebar = 1 µm. (D) Cellular aggregation that includes presumptive gliocyte components adjacent to type 2 JLC processes. Scalebar = 2 µm. cf, collagen fibrils; gc, presumptive gliocyte; jb, juxtaligamental cell body; nu, nucleus.
	Figure 11. LM. Histology of extrinsic stomach retractor apparatus. Transverse (A–C) and horizontal (D–F) sections stained with Halami’s aldehyde fuchsin and light green [59]: collagenous tissue is purple, muscle and other cellular tissues green. (A) General view of dorsal region of one half of ambulacral ridge, including distal retractor strand. Scalebar = 0.1 mm. (B) Distal retractor strand and mesentery. Scalebar = 40 µm. (C) Proximal retractor strand and mesentery. Scalebar = 40 µm. (D) General view of one half of ambulacral ridge, including distal retractor strand. Scalebar = 0.1 mm. (E) Distal retractor strand. Scalebar = 20 µm. (F) Distal mesentery. Scalebar = 20 µm. ao, ambulacral ossicle; ce, coelothelium; ct, collagenous tissue; dr, distal retractor strand; ids, inner dermal sheath; lil, longitudinal interambulacral ligament; lim, longitudinal interambulacral muscle; me, mesentery; mu, muscle; pvc, perivisceral coelom; til, transverse interambulacral ligament; tim, transverse interambulacral muscle.
	Figure 12. STM. Extrinsic stomach retractor apparatus (ESRA) immediately after autotomy. Dorsal view of ambulacral ridge; dorsolateral body wall removed. Distal end is on left. Arrows show location of ambulacral breakage plane. Components of ESRA on only one side are labelled. Arrowheads indicate location of ESRA breakage point. Scalebar = 1 mm. dr, distal retractor strand; ao, ambulacral ossicle; at, ampulla; me, mesentery; pr, proximal retractor strand.
	Figure 13. Mechanical tests. (A) Position of proximal dorsolateral body wall preparations that include autotomy plane (ABW) and distal preparations that lack it (DBW). (B) Diagram (not to scale) of apparatus used in creep tests in which preparations were subjected to constant load. (C) Idealised extension curve of ABW preparation under constant load. (D) Diagram (not to scale) of mechanical testing machine used for force-extension tests in which dorsolateral body wall preparations were stretched at a predetermined rate. (E) Force-extension curve showing measured parameters: ultimate extension (UE), from which ultimate strain was calculated (change in length/initial length), ultimate force (UF) from which ultimate stress was calculated (UF/cross-sectional area), yield force (YF) from which yield stress was calculated (YF/cross-sectional area), and slope of linear portion of curve (a…a1) from which Young’s modulus was calculated (Δstress/Δstrain, i.e., Δ(force/cross-sectional area)/Δ(extension/initial length)) ((D,E) adapted with permission of Springer Nature from reference [15], copyright 2000).
	Figure 14. Extension curves of isolated dorsolateral body wall preparations subjected to constant load (see Figure 13B,C). Horizontal axis: time (min); vertical axis: extension, vertical bars indicating 1 mm; stars indicate rupture. (A) Normal extension curves of ABW preparations: three preparations from one animal. (B) Effect of 100 mM K+: typical response of ABW (upper curve) and DBW (lower curve) preparations. (C) Effect of 10−3 M acetylcholine on ABW preparations: usually acetylcholine caused sustained arrest of extension (lower curve); very rarely, it caused transient contraction (upper curve). (D) Effect of atropine alone: five ABW preparations from one animal which were immersed in either 10−3 M atropine (upper three curves) or sea-water alone (lower two curves) for 5 min before load was applied (adapted with permission of Taylor and Francis Group, LLC, a division of Informa plc., from reference [19], copyright 1990).
	Figure 15. Size-related trends in mechanical properties of dorsolateral body wall preparations as demonstrated by force-extension tests (see Figure 13D,E). Regression lines (all slopes significant) for combined ABW and DBW preparations are included. (A) Yield stress. (B) Ultimate stress. (C) Young’s modulus. Parameters calculated from force-extension curves as explained in Figure 13E. R, centre to arm tip radius (adapted with permission of Springer Nature from reference [15], copyright 2000).
	Figure 16. Effects of glycation on mechanical properties of dorsolateral body wall preparations. (A) Relative change in ultimate force (B) Relative change in Young’s modulus (C) Ultimate strain. DBW preparations were incubated in 0.2 M ribose in marine phosphate buffer (pH 7.6–7.8) at 30 °C; control preparations were incubated in buffer alone. Each data point is mean of six values; standard error bars are shown. ** p < 0.001; * p < 0.05 (one-way ANOVA and Bonferroni post-hoc test). See Figure 13E for explanation of parameters (I.C. Wilkie and J.J. Keane, unpublished data).
	Figure 17. Effect of elevated [K+] and acetylcholine on ambulacral body wall preparations extending under constant load (see Figure 13B,C). Preparations consisted of central ambulacral ridge of basal region of arm and adjacent portion of mouth frame (Figure 8A). (A) Extension curves illustrating range of responses to 100 mM K+; a, transient increase in extension rate; b, sustained increase in extension rate; c, increase in extension rate culminating in rupture (asterisk); d, transient contraction (starting at arrow) then increase in extension rate and rupture. (B) Concentration-dependent effect of elevated [K+] on compliance of ambulacral body wall preparations. Responses, which varied qualitatively (see (A), a–d), were ranked in order of increasing magnitude of compliance change and scored (−1 to +4). Graph shows mean scores (n = 11–13 at each [K+]) and standard errors. (C) Concentration-dependent effect of acetylcholine on extension under constant load of ambulacral body wall preparations. Percentage of preparations (n = 9 or 10 at each [ACh]) exhibiting each type of response: 1, increased extension rate; 2, decreased extension rate; 3, extension arrested; 4, contraction. (I.C. Wilkie and G.V.R. Griffiths, unpublished data).

References [+] :

Ameye, Ultrastructural localization of proteins involved in sea urchin biomineralization. 1999, Pubmed, Echinobase