Z. Zellforsch. 96, 162--172 (1969)

Ultrastructure of the Tube Foot Wall of a Regular Echinoid, Diadema antillarum Philippi*

R. COLEMAN

Department of Zoology, Bedford College, University of London

Received January 6, 1969

Summary. An ultrastructural study of the tube foot wall of a regular echinoid, Diadema antillarum, was made. Ciliated microvillous epithelial cells, their inclusions, amoebocytes, and the arrangement of collagen and muscle in the tube feet are described. The neuropile, together with its vesicular components and possible neurosecretory elements, is considered and discussed as are connective tissue bridges across the lumen. Consideration is given to possible functions for the various components of the tube foot.

Introduction

The activities exhibited by echinoderm tube feet during locomotion are extremely complex and a high degree of coordination is shown by these unique hydraulic organs. The histology of a var ie ty of tube feet from the different classes of echinoderms has been studied (see SMITH, 1937, 1950; NICHOLS, 1962, 1964) bu t these studies have been restricted by the l imita t ions imposed by optical microscopy. Wi th the adven t of the electron microscope and the ref inement of u l t ras t ruc tura l techniques it has become possible to demonst ra te subcellular morphology and to confirm or modify the earlier optical microscopic studies. Suckered echinoid tube feet, which in addi t ion to being organs of locomotion may also be involved in anchorage, respirat ion and possibly have secondary sensory funct ions (NICHOLS, 1961) have been a neglected field of s tudy at the electron microscopic level. One exception is a recent s tudy of the podial wall of the Japanese urchin, Hemicentrotus pulcherrimus (KAwAGUTI, 1964). This present work describes some ul t ras t ruc tura l observations on the wall of suckered tube feet of a tropical urchin, Diadema antillarum. The structure of the sucker is described separately (COLEMAN, 1969).

Material and Methods Tube feet were dissected from fresh Diadema antiUarum PHILIPPI from Madeira and

fixed immediately by immersion in ice-cold 3% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) for 1 hr prior to post-fixation in veronal acetate-buffered 1% osmium tetroxide. Tissues were then dehydrated in graded ethanols, treated with propylene oxide and embedded in Shell Epikote Resin (Epon 812), which was polymerized at 60 ~ C for 24 hrs. Silver sections (ca. 60--90 nm), cut with glass knives on a Cambridge (Huxley) ultramicro- tome, were mounted on uncoated copper grids and doubly stained in uranyl acetate (WATSON, 1958) and lead citrate (REYNOLDS, 1963). Sections were examined in an AEI EM6B electron microscope containing a 25 or 50 ixm objective aperture at 60 kv. Original magnifications varied from 2,500 to 40,000.

* I am indebted to Professor N. MILLOTT for his help and encouragement during the course of this work, to Mr. RAY~OR L. JONES for his expert technical assistance, and to Dr. H. G. VEVERS and the Zoological Society of London.

Ultrastructure of Eehinoid Tube Foot 163

Results

The general construction of the wall of suckered tube feet in Diadema is illustrated diagrammatically in Fig. 1.

Externally the tube feet are covered by an epithelial layer, which is in the form of folds or papillae over most of the tube feet except near the base where papillae are not present. This podial epithelial layer is continuous with the external epithelium overlying the test of the rest of the urchin. The podial epithelium is composed of fairly columnar highly vacuolated cells.

P E

f

/

Fig. 1. Diagram illustrating the general construction of the side wall of Diadema tube feet. C, dense collagen layer; Co, loose connective tissue layer, mainly collagen; CB, connective tissue "bridge"; E, epithelial cells; L, lumen; Mu, muscle cells; N P, neuropile; P, pigment cells

The free surface of the epithelial cells is covered with microvilli, which are branching and pleomorphic, being about 1.8 ~tm long and 0.1 ~zm in diameter (Figs. 2, 3). These microvilli terminate distally in knob-like structures containing an apical electron-dense region. The outer surface of these knob-like endings is coated by a fine matrix of filaments in the form of a web similar to that described for the microvilli of bat intestinal epithelium (FAwCETT, 1966) and those of the pedicellariae of Echinus (COBB, 1968b). Fibrils can be detected running longi- tudinally within the microvilli. In addition to the mierovilti, cilia occur on the surface of a large number of epithelial cells, though usually no more than a single cilium is encountered on each cell. In construction the cilia are of the typical motile variety now described in so many different phyla. The cilia project from the surface of the cells at least 8 ~m and possibly considerably more; they are about 0.3 ~m in diameter and possess a typical 9 outer and 2 inner fibril configura- tion. Two striated rootlets, which may exceed 3 ~m in length, project from the basal body, which is accompanied by an adjacent complementary centriole. The rootlets bear dense transverse striations approximately every 0.06 ~m along

164 R. CoL~MA~:

Figs. 2 and 3

Ultrastructure of Echinoid Tube Foot 165

their length with 3 or 4 less dense striations within each of these periods. These cilia correspond well with those recently described on epithelial cells of Echinus pedieellariae (COBB, 1968b).

The epithelial cells have intercellular junctions of striking appearance (Figs. 2, 3). At their apical ends there is a " c u p " with an internal electron-dense coating and this leads on to an area, which may exceed 1 txm in length, where the adjacent plasma membranes from the two cells are in intimate contact. This zone probably corresponds to a type of desmosome (macula adherens) found between epithelial cells of other phyla (FAwCETT, 1966). In Diadema these intercellular junctions are septate; "desmosomes" similarly constructed to those found in Diadema are currently considered to be a universal feature of echinoderm epitheha (COBB, 1968b).

The epithelial cells have fairly large regular nuclei, about 4 ~m in diameter, with no indication of any indentations. Many of these nuclei contain one, or occasionally two, extremely conspicuous, electron-dense nncleoli of granular appea- rance; these are homogeneous, rounded and in many instances exceed 3 ~m in diameter.

The mitochondria of the epithehal cells are short and rounded, about 1 ~m in diameter. They are situated in the apical regions of the cells or immediately adjacent to the lateral plasma membrane.

Golgi bodies are also found predominantly in the apical zones of the epithelial cells. They are fairly conspicuous consisting of flattened saccules with dilated ends tha t are apparently in the process of budding off vesicles (Figs. 2, 3). Large numbers of vesicles, which lack any evidence of electron-dense contents, are found in the immediate vicinity of the Golgi bodies. Some cytoplasmic vesicles may have diameters exceeding 0.5 Ezm and occasionally some epithelial cells are found possessing enormous vacuoles with diameters in excess of 2 ~zm.

The background matrix of the epithelial cells is fairly electron-transparent. Occasionally membrane-bound vesicles filled with contents virtually identical to this background matr ix are found external to the apical surface of the epithelial cells. Because of the similarity in electron-densities and general appearance, it is highly likely that the external vesicles have been released from the epithelial cells, possibly as some sort of apocrine secretion, though the function of such secretion remains to be established.

There is little evidence of any rough endoplasmic reticulum occurring in the epithelial cells, though there is a possibility that some of the membranous cytoplasmic components may be agranular reticulum. The virtual absence of rough endoplasmic reticulum coupled with the appearance of the Golgi bodies would suggest tha t these epithelial cells are not very active in terms of protein synthesis or general metabolism.

Droplets, up to 0.75 ~m diameter, bound by a unit membrane and containing material of high electron-density are infrequently encountered in the apical region of some epithelial cells. These droplets may be lysosomal in nature, though this

Fig. 2. Epithelial cells near the base of a tube foot; note nervous elements at base of epithelium external to the basal lamina, f, fibrous elements

Fig. 3. Typical epithelial cells showing vacuolated appearance and characteristic intercellular junctions. G, Golgi body

Figs. 4--6

R. COLeMAn: Ultrastructure of Echinoid Tube Foot 167

remains to be established cytochemically. The function of such organelles within echinoderm epithelial cells also remains to be determined.

Many of the epithelial cells possess groups of cytoplasmic fibrous elements running from the base of the cells towards their apices (/, Fig. 2). The character of these fibrous components is not apparent as no diagnostic banding can be seen.

Immediate ly below the apical surface of many epithelial cells lie membrane- limited vesicles of about 0.75 ~m diameter that contain aggregations of electron- dense granules measuring about 7.5 nm diam. and these appear as though they could be ferritin, the iron-storage protein. Certainly these granules agree in appearance with tha t of ferritin (HAGGIS, 1966) and the vesicles may be the site where iron is concentrated and stored in order to play some, as yet undeter- mined, role in echinoid metabolism. Isolated ferritin-like granules are occasionally found in extra-vesicular sites lying free in the cytoplasmic matrix in the immediate vicinity of the vesicles, though it is possible they may have been displaced during processing.

Mucus-producing cells are extremely rarely encountered in the tube foot wall, but when present occur in the outer epithelial layer. Each mucus cell, which may exceed 15 fzm in diameter, is divided into a series of compartments filled with homogeneous mucoid material tha t differs slightly in electron-density from com- par tment to compartment.

Amoebocytes are found, either singly or in groups between epithelial cells, especially in some papillae. In similar sites some other cells are occasionally encountered that have their cytoplasm packed with unit membrane-limited bodies filled with an extremely electron-dense pigment. These cells may be wandering pigment-transporting amoebocytes.

The epithelial cells of the tube feet all lie on a characteristic basal lamina ca. 0.1 ~zm thick, though a gap of about 0.08 ~zm separates this lamina from the cells. The surface of the lamina most distant from the epithelial cells breaks up into a matrix of fine fibrils of a connective tissue component thought to be collagen.

Collagen is the main connective tissue component in the tube foot wall. Compact bundles of collagen fibres (Figs. 4, 5) provide a zone stretching internally from the basal lamina of the outer epithelial cells for 10--15 ~m. The inner region of this collagen zone is much denser and interdigitates with smooth muscle cells at well-defined collageno-muscular junctions (Fig. 6). The cell bodies of the muscle cells are more internally placed, bordering on the coelomic lining, which consists of a single layer of ciliated cells. The coelomic lumen of the tube foot may be packed with a variety of coelomocytes. I t is of interest to note in most Diadema tube feet, if not in all, sheets of connective tissue, which are a single cell deep and appear to be predominantly packed with collagen, tha t span the lumen of the tube feet in a regular undulating manner.

Fig. 4. Collagen bundles in loose connective tissue layer Fig. 5. Cells containing dense-cored vesicles (dg) in spaces between collagen blocks of loose

connective tissue layer Fig. 6. Collageno-muscular junctions in tube foot; note dense-cored vesicles and mitochondria

(mi); Co, collagen; Mu, muscle

12 Z. Zellforsch., Bd. 96

Fig. 7. Neuropile in tube foot

Fig. 8. Nerve elements adjacent to basal lamina (BL) of epithelium; note clear synaptie-like vesicles (V) in axons

R. COLEMAN: Ultrastructure of Echinoid Tube Foot 169

Nervous Elements

Details of the nervous system are difficult to resolve by optical means, but they can clearly be seen with the electron microscope. The nervous system in the wall of the tube foot consists of a nerve felt or neuropile, which is predomi- nantly situated just external to the basal lamina of the epithelial cells (Fig. 2). The felt consists of a large number of axons running mainly in parallel longitudi- nally in the tube foot (Fig. 7). In many places in the tube foot the felt can be identified as such only by the presence of characteristic vesicles in the axons. In particular two main categories of vesicles can be distinguished on the basis of their size and contents. Both types are bound by a single unit membrane.

a) Dense-Cored Vesicles. These are the more abundant type. They occur mainly in a range of diameters from 0.08--0.1 ~m and are characterized by the presence of extremely electron-dense homogeneous cores that fill the vesicle except for a small peripheral clear zone, though in some cases this appears to be absent and the dense contents completely fill the vesicle.

b) Clear Vesicles (v, Fig. 8). These are smaller and average about 0.067 ~m diam. Their contents are not very electron-dense and in appearance they are almost identical to "synaptic vesicles" described in a wide range of nervous elements in many different phyla by many authors.

Neurotubules, typically 20 nm in diam., can be seen passing longitudinally through most of the axons of the nerve felt.

There is no positive evidence as yet for the presence of synapses between axons in the nerve felt in spite of the presence of clusters of synaptic-like vesicles in axonal swellings. In the immediate vicinity of the synaptic clusters within axons small osmiophilic mitochondria, ca. 0.75 • 0.25 ~m, are occasionally found (Fig. 8).

Both dense-cored vesicles and synaptic vesicles are found in the nervous elements external to the basal lamina of the epithelial cells. Deeper in the tube feet between the collagen blocks are found axonal cisternae filled almost entirely of dense-cored vesicles (dg, Fig. 5), which in some cases may be as large as 0.28 ~m in diam. and frequently can be traced along tracts passing between the collagen bundles.

Dense-cored vesicles are also found in accumulations at the collageno-mnscular junctions especially on the muscle side at the sites of interdigitation and they are frequently accompanied by mitochondria. These dense-cored vesicles are of a large variety with diameters up to 0.27 ~m. The dense-cored vesicles thus appear to penetrate most of the elements, if not all, from which the wall of the tube foot is constructed.

Discussion

The epithelial cells of Diadema tube foot lack the thick mucus coating described in the Japanese echinoid, Hemicentrotus pulcherrimus (KAwAGUTI, 1964). This difference may conceivably be adaptive or alternatively it may be that Diadema possesses a mucoid coat in nature, but after maintenance in laboratory aquaria this coating may become physiologically unnecessary and be lost. This latter reason is thought somewhat unlikely as mucus-producing cells are extremely rarely found in the epithelium of Diadema tube feet. I t is possible the outer

170 R. COLEMAN :

"cut ic le" layer described in Echinus (SMITH, 1937) at the optical microscopic level is identical to the mucus layer identifiable in Hemicentrotus at the electron microscopic level; certainly no cuticle is present in either Diadema or Hemicen- trotus. The function of surface mucus in echinoids is still not clear; it may play a protective role against invasive micro-organisms or it may be lubricative or adhesive.

The papillate or folded nature of the epithelium is probably a mechanism for increasing the surface area available for activities such as respiration, surface ionic exchange or excretion. These activities may be aided by surface currents due to the cilia of the epithelial cells. I t is also conceivable these cilia have some sort of sensory role. The epithelial microvilli, with their unusual knob-like endings, may also be sensory, though this also remains to be established. No completely acceptable function has yet been determined for echinoderm microvilli. The microvilli in Diadema seem more branched and more pleomorphic than those in Hemicentrotus. The function of the apparent apocrine secretion by the epithelial cells also remains a mystery, though it may play some part in excretion, lubri- cation or protection and might account for the absence of a mucoid layer in this species.

The amoebocytes in the epithelial layer may act as wandering cells that protect against invasive micro-organisms in a similar manner to phagocytes in vertebrates, or possibly they are involved in excretion or surface "skin digestion" and absorption as has recently been suggested (PEQUIGNAT, 1966).

There is no evidence of axonic processes arising from the base of epithelial cells of Diadema tube feet, though such sensory extensions have been described in the epithelia covering pedicellariae of Echinus (COBB, 1968b).

The collagen in the tube foot of Diadema is virtually identical in ultrastructure with that recently described in Echinus (COBB and LAVERACK, 1966) and in a holothurian (BACCETTI, 1967), it would appear that in all these cases the major repeat period is about 64 nm, which is very similar to mammalian collagen. The thick layer of collagen in the tube foot may serve to maintain the shape of the tube foot lumen and to provide a firm site of a t tachment and elastic counterbody for muscles during contraction (KAWAGUTI, 1964).

Only a single muscle type appears to be present in the tube foot wall in Diadema, namely longitudinal muscle. I t s fine structure has not been studied here. Some ultrastructural features of echinoid muscles (COBB, 1968a; COBB and LAVERACK, 1966) and asteroid muscles (BARGMANN and BEHREZ~S, 1963) have already been reported. The connective tissue "bridges" across the lumen may provide additional structural support to prevent the lumen being too greatly displaced during tube foot extension and contraction.

The recent review of SMITH (1966) on nervous systems of echinoids quite clearly demonstrates how sparse is the available information especially at the ultrastructural level. Axonal dense-cored vesicles of similar size and appearance to those in the nerve felt of Diadema tube feet have been described in hypo- neural tissue of Echinus lantern (COBB and LAVERACK, 1966) and these vesicles are thought to be possibly neurosecretory. They certainly have the ultrastructural appearance of the elementary granules now described in a variety of neuro-

Ultrastructure of Echinoid Tube Foot 171

secretory cells in both ver tebrates and inver tebra tes (BERN, 1966). Moreover granules, supposedly neurosecretory, have been described in nerves of Asterias (BARGMA~N et al., 1962). I t is necessary, however, before labelling a cell as neurosecretory to establish certain s taining and physiological criteria (SCHAlCRER, 1959). The funct ion in Diadema tube feet of neurosecretory material, if these granules prove to be so, remains undetermined. I t is possible they could play some par t in inducing muscular contract ion as they seem to be present at the collageno-muscular junct ions. The site of elaborat ion of these dense-cored vesicles is also unclear a t the present time.

The smaller nervous vesicles (v, Fig. 8) certainly have the size and ul t rastruc- tural appearance of similar vesicles in what have been described as synapses in the hyponeura l ganglion in Echinus (COBB and LAV~RACK, 1966) and in axons of pedicellariae of the same species (COBB, 1968a).

R e f e r e n c e s

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, U. BR. BEHRENS : Ober den Feinbau des Nervensystems des Seesternes (Asterias rubens L.) II. Mitt. Zur Frage des Baues und der Innervation der Ampullen. Z. Zellforsch. 59, 746--770 (1963).

BERn, H. : On the production of hormones by neurones and the role of neurosecretion in neuroendocrine mechanisms. Symp. Soc. exp. Biol. 20, 325--344 (1966).

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- - In: Echinoderms. London: Hutchinson 1962. - - Echinoderms: experimental and ecological. Oeeanogr. mar. biol. Ann. Rev. 2, 393-423

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172 R. COLEMAn: Ultrastructure of Echinoid Tube Foot

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Dr. RAYMOND COLEMAN Depar tment of Zoology Bedford College (University of London) Regent 's Park London N. W. 1, England