Blowes LM et al. (2017), Body wall structure in the starfish Asterias ru...

ECB-ART-45629

J Anat 2017 Sep 01;2313:325-341. doi: 10.1111/joa.12646.

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Body wall structure in the starfish Asterias rubens.

Blowes LM , Egertová M , Liu Y , Davis GR , Terrill NJ , Gupta HS , Elphick MR .

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The body wall of starfish is composed of magnesium calcite ossicles connected by collagenous tissue and muscles and it exhibits remarkable variability in stiffness, which is attributed to the mechanical mutability of the collagenous component. Using the common European starfish Asterias rubens as an experimental animal, here we have employed a variety of techniques to gain new insights into the structure of the starfish body wall. The structure and organisation of muscular and collagenous components of the body wall were analysed using trichrome staining. The muscle system comprises interossicular muscles as well as muscle strands that connect ossicles with the circular muscle layer of the coelomic lining. The collagenous tissue surrounding the ossicle network contains collagen fibres that form loop-shaped straps that wrap around calcite struts near to the surface of ossicles. The 3D architecture of the calcareous endoskeleton was visualised for the first time using X-ray microtomography, revealing the shapes and interactions of different ossicle types. Furthermore, analysis of the anatomical organisation of the ossicles indicates how changes in body shape may be achieved by local contraction/relaxation of interossicular muscles. Scanning synchrotron small-angle X-ray diffraction (SAXD) scans of the starfish aboral body wall and ambulacrum were used to study the collagenous tissue component at the fibrillar level. Collagen fibrils in aboral body wall were found to exhibit variable degrees of alignment, with high levels of alignment probably corresponding to regions where collagenous tissue is under tension. Collagen fibrils in the ambulacrum had a uniformly low degree of orientation, attributed to macrocrimp of the fibrils and the presence of slanted as well as horizontal fibrils connecting antimeric ambulacral ossicles. Body wall collagen fibril D-period lengths were similar to previously reported mammalian D-periods, but were significantly different between the aboral and ambulacral samples. The overlap/D-period length ratio within fibrils was higher than reported for mammalian tissues. Collectively, the data reported here provide new insights into the anatomy of the body wall in A. rubens and a foundation for further studies investigating the structural basis of the mechanical properties of echinoderm body wall tissue composites.

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Species referenced: Echinodermata
Genes referenced: ado LOC100893907 LOC115925415 LOC593474 LOC594785 nkx1 ros1 srpl

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	Figure 1. General anatomy of starfish Asterias rubens. The main image shows a specimen of the starfish A. rubens with one of the rays transversely dissected and with the aboral body wall of two other rays removed to reveal the digestive glands (pyloric caeca, PC) and the ridge of ambulacral ossicle heads (AMOh). Inset (A) shows a close‐up of the transverse cross‐section of the ray with ambulacral ossicles (AMO) and tube feet (TF) on the oral side of the cross‐section and pyloric caeca attached to the inner side of the aboral body wall. The positions of the apical carinal ossicle (CO) and lateral reticular ossicles (ROs) are labelled. Inset (B) shows the outer surface of the aboral body wall centred on the mid‐line of the arm showing the positions of spines (SPs) that are located over the row of carinal ossicles and clusters of papulae (CPa). Inset (C) shows a close‐up of a pedicellaria, a pincer‐shaped defensive organ. Inset (D) shows a close‐up of the inner surface of the aboral body wall with voids in the body wall that are overlain by clusters of papullae (CPa) on the outer body wall surface (see inset C). The position of the longitudinally orientated apical muscle (AM) is outlined. Scale bar (on main figure): 1 cm.
	Figure 2. Trichrome‐stained transverse section of a decalcified ray from Asterias rubens. The voids formed by decalcification of the body wall ossicles show the positions and shapes of the different ossicle types: the ambulacral ossicles (AMO), the adambulacral ossicles (ADO), the marginal ossicles (MO), the reticular ossicles (RO) and the carinal ossicle (CO). The red speckling within the ossicle voids are stained cells, which are located in pores between the calcite struts of the ossicle stereom in vivo. Surrounding the ossicle network can be seen a dense meshwork of collagenous tissue (blue), which forms the bulk of the soft tissue in the body wall. The interossicular muscles that link adjacent ossicles can also be seen. These are most prominent adorally, where there are large muscles that link adjacent ambulacral ossicles (longitudinal supra‐ambulacral muscle, LSM; transverse infra‐ambulacral muscle, TIM; transverse supra‐ambulacral muscle, TSM) or that link ambulacral ossiscles with adambulacral ossicles (inner and outer transverse lateral muscles; TLM; longitudinal lateral muscles, LLM). The smaller interossicular muscles (IOM) linking ossicles of the aboral skeleton are also evident but these are seen more clearly at higher magnification (see Fig. 3). Occupying the coelomic space internal to the body wall can be seen the prominent pair of digestive glands (pyloric caeca, PC), which are connected via mesenteries (*) to the aboral coelomic lining (ACL) of the body wall. The coelomic lining is detached from the body wall in this stained section, which is an artifact probably caused by shrinkage of the body wall dermis during tissue processing. Note that the aboral lining of the coelom is thicker in the midline position due to the presence of the longitudinally oriented apical muscle (AM), which causes aboral flexion of the ray when it contracts in vivo. The prominent V‐shaped radial nerve cord (RNC) can be seen between the two rows of tube feet podia (TF), which are connected to the intracoelomic bulb‐shaped ampullae (AMP) by tubular connections that run between adjacent ambulacral ossicles (as seen here on the right side). Note also other appendages that are associated with the external body wall surface, including spines (SP), pedicellariae (Pe) and papulae (Pa) that overlay voids (V) between the ossicles forming the aboral body wall skeleton. Scale bar: 150 μm.
	Figure 3. Trichrome stained sections of starfish body wall showing ossicles and associated muscles and collagenous tissue. (A) Adjacent reticular ossicles (RO) are linked by an interossicular muscle (IOM) and are embedded within a collagenous tissue meshwork (blue). The calcareous struts of the ossicles appear as voids, due to decalcification of the tissue, and the cellular stroma appears red. (B) High magnification image of adjacent reticular ossicles showing how muscle fibres of an interossicular muscle (IOM) insert between and around strut voids near the surface of each ossicle (white arrows). The wrapping of collagen fibres (blue) around ossicle strut voids (black arrows) can be clearly seen in this image. (C) Muscle strands (arrows) derived from the circular muscle layer (CML) above the apical muscle (AM; longitudinal muscle) extend through the collagenous inner dermis (blue) and insert on the carinal ossicle (CO). (D) Ambulacral ossicle heads (AMOhs) are inter‐connected by transverse supra‐ambulacral muscle (TSM) and longitudinal supra‐ambulacral muscles (LSMs). The ambulacral ossicles (AMOs) are furthermore connected by transverse infra‐ambulacral muscle (TIM). Other abbreviations as in Fig. 2. (E). Collagen fibres (blue) below the transverse supra‐ambulacral muscle (TSM) connect antimeric ambulacral ossicle heads strapping around ossicle struts. (F) The collagen fibres (blue) above the transverse infra‐ambulacral muscle (TIM) have a predominantly transverse horizontal orientation with a macro‐crimp (wavy appearance). Scale bar (3F): 50 μm (A), 25 μm (B), 50 μm (C), 100 μm (D), 12.5 μm (E), 7.8 μm (F).
	Figure 4. The ray skeleton of Asterias rubens, as revealed by X‐ray microtomography. (A) Low‐magnification overview of the ray skeleton from a top (aboral) view. Along the midline of the aboral skeleton can be seen the row of overlapping carinal ossicles (COs). Either side of the carinal ossicles are a loose meshwork of reticular ossicles (ROs) and through the gaps bounded by rings of reticular ossicles can be seen the two rows of ambulacral ossicles (AMOs) on the oral side of the ray. Note also the numerous spines located external to the ossicle network; three spines located above the carinal ossicles are labelled with arrowheads. (B) Transverse segment of a starfish ray showing the ambulacral skeleton formed by two rows of ambulacral ossicles (AMO), which are supported orally by the cuboid‐shaped adambulacral ossicles (ADO). Lateral to the adambulacral ossicles are the densely packed marginal ossicles (MO). The aboral region of the ray skeleton is formed by a loose meshwork of reticular ossicles (RO) and the single row of carinal ossicles (CO). Spines (SPs) can be seen on the body wall surface. (C) The aboral ray skeleton viewed from its underside, showing the overlapping row of carinal ossicles (COs) along the midline and the loose meshwork of reticular ossicles (ROs) on either side of the carinal ossicles. This image also illustrates how changes in orientation of the carinal and reticular ossicles, mediated in vivo by contraction/relaxation of interossicular muscles, affects skeletal structure. Thus, on the left hand side of the image ossicles form ring‐shaped structures, whereas on the right hand side of the image the ossicles form oblong‐shaped structures. (D) The ambulacral skeleton viewed at high magnification, looking towards the tip of the ray. The image shows how the slender and tightly packed ambulacral ossicles (AMO) are orientated at an angle, leaning away from the tip‐end of the ray. Furthermore, it can be seen that the aboral ‘head’ (AMOh) of each ambulacral ossicle overlaps an adjacent ossicle more proximal to the central disk. The large gaps between the adjacent ‘heads’ of ambulacral ossicles (arrowheads and asterisks) are occupied in vivo by longitudinally and transversely orientated interossicular muscles, respectively (which can also be seen in Figs 2 and 3). Dashed lines show where tubular connections of the tube feet and ampullae are located. (D') Scanning electron micrograph showing the calcite struts and pores of ambulacral ossicle stereom at high magnification. (E) External view of the marginal ossicles of the body wall. At this high magnification it can be seen that overlapping ossicles with appendages (spines, SP and pedicellariae, Pe) are arranged in longitudinally orientated rows and these ossicles are interlinked radially by smaller ossicles without appendages (arrowheads). Scale bars: (A,C) 2 mm; (B,D) 1 mm; (D’) 40 μm; (E) 500 μm.
	Figure 5. Transmission, Iq5col; Iq5col/Iq5min ratio and vector maps of Asterias rubens aboral body wall and ambulacrum. (A1,A2) X‐ray transmission maps of an aboral body wall (A1) and an ambulacrum sample (A2). The contour scale corresponds to darker regions as regions with higher density of the tissue (higher absorption). Areas of high density in blue (A1, *) indicate positions of spines. The insets are close‐ups of micro‐CT images of corresponding body locations. (B1,B2) Intensity of 5th order Bragg peaks mapped across the aboral (B1) and ambulacrum (B2) samples. Red corresponds to the highest amount of collagen. (C1,C2) Ratio of 5th order Bragg peak intensity from collagen fibrils (Iq5col), to the intensity of diffuse SAX scattering (which arises mainly from mineral components of the tissue) (Iq5min) mapped across the aboral (C1) and ambulacral samples (C2). (D1,D2) Vector and circle plots of collagen fibril structure overlapped with a transmission map of the aboral (D1) and ambulacral samples (D2). The orientations of the vectors are parallel to the orientation of the collagen fibrils. The length of the vector is inversely proportional to Δχ0. The degree of fibril orientation increases 1Δχp with the vector length. The scale vector lengths in the bottom left corner correspond to 1Δχp of 0.23, 0.46, 0.70 and 0.93 [Au] or Δχ0 of 5, 2.5, 1.67 and 1.25 degrees. It is noticable that all the vectors in D2 are shorter than the scale vector corresponding to Δχ0 of 5°, in comparison with 8% of vectors in D1 being longer than the scale vector for Δχ0 of 5°.

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