Stages leading to the formation of inverted gap junctions between certain basal replacement or interstitial cells in the mid-gut of adult Limultts can be followed by freeze-fracturing. Free, 13-nm EF intramembranous particles first appear to be organized into short linear arrays or small clusters of particles, which then become transformed into anastomosing particulate networks covering a considerable surface area. These subsequently become concentrated into smaller, more nearly circular, macular plaques of EF particles or PF pits. These EF particles, both when free or assembled into macular arrays, possess a central channel or pore. Numerous formed gap junctions are present in Limulus mid-gut, which suggests that cell-to-cell communication is an important feature of the mature tissue. The results show that arthropod tissues can be used to study the development of gap junctions not only in differentiating systems but also in adult tissues during normal cell turnover.

A number of studies have been made on vertebrate tissues to determine the changes in particle distribution leading to the formation of gap junctions; these include investigations on embryonic tissues (Revel, Yip & Chang, 1973; Revel, 1974; Decker & Friend, 1974; Hasty & Hay, 1977), regenerating or maturing systems (Yee, 1972; Benedetti, Dunia & Bloemendal, 1974; Albertini & Anderson, 1974), experimentally stimulated material (Decker, 19766) and, in vitro, on cells in culture (Pinto da Silva & Gilula, 1972; Johnson, Hammer, Sheridan & Revel, 1974; Elias & Friend, 1976).

These experiments have primarily made use of freeze-fracturing, since this technique permits the analysis of any changes in the number, pattern or distribution of the intramembranous particles which comprise the gap junctions. When fully formed in vertebrates, mature gap junctions consist of macular aggregations of PF particles, about 10 nm in diameter, usually packed fairly tightly in hexagonal arrays (Staehelin, 1974). There are some exceptions to this tight packing in mature junctions, such as in mesangial cells (Pricam, Humbert, Perrelet & Orci, 1974), retina (Raviola & Gilula, 1973), mesenteric arteries (Simionescu, Simionescu & Palade, 1976) and frog ventricle (Kensler, Brink & Dewey, 1977), but they are in the minority.

In invertebrate tissues, however, with the exception of those of molluscs and tunicates, whose junctional particles fracture on to the PF (Flower, 1971, 1977; Gilula & Satir, 1971; Lorber & Rayns, 1972, 1977), gap junctions consist of macular arrays of loosely packed EF particles, about 12-13 nm in diameter, often referred to as ‘inverted’ gap junctions (Flower, 1972). Although mature gap junctions have been described in a variety of invertebrate cell types, ranging from tissues such as epithelia in platyhelminthes and annelids (Flower, 1977), molluscs (Flower, 1971, 1977; Gilula & Satir, 1971), Hydra (Hand & Gobel, 1972; Filshie & Flower, 1977), tunicates (Lorber & Rayns, 1972, 1977) and arthropods (Hudspeth & Revel, 1971; Flower, 1972, 1977; Satir & Fong, 1973; Satir & Gilula, 1973; Johnson, Herman & Preus, 1973; Noirot-Timothée & Noirot, 1974; Baerwald, 1975; Gilula, 1975; Skaer, Berridge & Lee, 1975; Dallai, 1975), to the central nervous system of Crustacea and insects (Peracchia, 1973a, b;Skaer & Lane, 1974; Lane, Skaer & Swales, 1975, 1977; Lane & Swales, 1976), few studies have so far been made of their development. Those which have been made include experiments to investigate the effect of hormones on chelicerate arthropods. Very loose gap junctions in the horseshoe crab were interpreted as being in process of formation, as an apparent response to ecdysterone (Johnson, Quick, Johnson & Herman, 1974); this study, however, has been reported in abstract form only. More extensive studies on the development of gap junctions have been carried out on insects in vivo; these investigations on differentiating larval and pupal tissues of the blow-fly Calliphora (Lane & Swales, 1977b, c) have shown that the gap junctions in the perineurium and glia of the CNS form in early larval stages, apparently disaggregate in early pupae and reform in late pupae before the emergence of adult flies. In the moth Manduca sexta, embryonic tissues also display stages in the formation of gap junctions (Lane & Swales, 1977 a). In Calliphora, the stages in the development of the junctions show some similarities to those observed in vertebrates, except that the junctions are inverted, so that the component particles fracture on to the EF not the PF, and the particles are usually of larger diameter than those of vertebrates. In addition, they tend not to display clear ‘formation plaque’ areas (see Decker, 1976a) nor the much larger precursor particles (as in Decker & Friend, 1974, and Revel, 1974), and they do not present linear arrays ordered with the same precision as in vertebrates (see Lane & Swales, 1977b, c). However, the junctional particles apparently change from being scattered, free 13-nm EF particles to small clusters and linear arrays which gradually aggregate to form the mature macular junctions (Lane & Swales, 1977b, c). The present study on the mid-gut of Limulus suggests that stages in the formation of gap junctions may occur normally in adult tissues, presumably as the interstitial or replacement cells move up to take their place in, or make contact with, the mature columnar epithelial cells. A similar system of continuous regeneration occurs in insect gut (Smith, 1968) and, interestingly, has been found to be associated with the presence of continuous junctions (Satir & Gilula, 1973) which also are present in Limulus mid-gut (Lane & Harrison, 1977).

The present report indicates that arthropod tissues can be used to study the stages in development of gap junctions, not only in maturing systems during growth and differentiation, but also in adult tissues during normal cell turnover or when mature cells begin to make a fresh contact with another cell to establish new intercellular junctions. Earlier studies on the mid-gut of Limulus showed that the gap junctions ordinarily are circular in outline with a fairly loose packing of their component particles (Johnson et al. 1973; Lane & Harrison, 1977), as is typical of mature arthropod gap junctions generally.

The tissue used was the mid-gut of adult specimens of the horseshoe-crab, Limulus polyphemus; hepatopancreas was also examined. The animals were obtained from Woods Hole, Mass., U.S.A, and maintained in large aerated tanks of sea water at around 16 °C. The material was fixed in one of a variety of ways, optimal preservation being obtained with fixation at 4 °C in 0·75% glutaraldehyde in 0·1 M cacodylate buffer, pH 7·4, plus 5% formalin, 1% acrolein, 3 % NaCl and 3·5 % sucrose (Fahrenbach, 1976). Tissues to be embedded were washed, post-osmicated, en bloc stained with uranyl acetate, dehydrated, and embedded in Araldite. Thin sections were stained with lead citrate and uranyl acetate. The tissues for freeze-cleaving were left in fixative for about 0·5 h, washed briefly in several changes of 0·1 M cacodylate buffer, pH 7·4, plus 8% sucrose, and treated with 20% glycerol in the cacodylate buffer rinse for 20–30 min before mounting and rapid freezing in Freon 22 cooled with liquid nitrogen. Unfixed tissues were also treated with buffered 20 % glycerol prior to freezing. Freeze-fracturing took place in a Balzers BA 360M freeze-etching device at about 133 × 10−4 N m-1 (15 × 10−5 torr) at— 100 °C and the fracture face was shadowed with tungsten-tantalum or platinum-carbon and backed with carbon. The replicas were cleaned by brief treatment with concentrated H2SO4 followed by concentrated sodium hypochlorite and then were treated with distilled water washes after rinsing in dimethyl formamide. Replicas were mounted on coated grids and both they and the thin sections were examined in a Philips EM300.

The freeze-fracture micrographs are mounted so that the direction of shadow is from the bottom or side.

The mid-gut of Limulus is lined by a simple columnar epithelium with microvilli on the luminal surface (Fig. 1). The adjacent lateral cell membranes are associated by desmosomes at the very edge of the lumen, and below that, by ‘stacked ‘continuous junctions (see Fig. 1; Lane & Harrison, 1977). In the areas of these zonulae continuas, gap junctions (maculae communicantes) occur; the latter are characterized in thin sections by a reduced intercellular space between the apposed plasma membranes (Fig. 2) and occur singly (Fig. 2A) or in groups (Fig. 2B). The continuous junctions are more conspicious when closer to the apical border of microvilli (Figs. 1, 2A). In the more basal areas of the cells the gap junctions sometimes appear less lengthy, at times almost punctate (Fig. 2 c).

Fig. 1.

Thin section through the mid-gut of Limulus showing columnar epithelial cells. The apical border consists of microvilli (mv) and the lateral borders between adjacent cells possess desmosomes (d) at the lumen and thereafter continuous junctions (cj) and gap junctions. Note the cistemae of endoplasmic reticulum lying parallel to these junctions and the dense secretory granules, m, multivesicular bodies, × 6800.

Fig. 1.

Thin section through the mid-gut of Limulus showing columnar epithelial cells. The apical border consists of microvilli (mv) and the lateral borders between adjacent cells possess desmosomes (d) at the lumen and thereafter continuous junctions (cj) and gap junctions. Note the cistemae of endoplasmic reticulum lying parallel to these junctions and the dense secretory granules, m, multivesicular bodies, × 6800.

Fig. 2.

Gap junctions as seen in ultrathin sections, between adjacent epithelial cells of Limulus mid-gut.

A, gap junction (g) fairly close to the lumen of the gut, lying between continuous junctions whose faint cross-striations are scarcely visible (arrows), × 115800.

B, gap junctions (at g) farther removed from the gut lumen, × 91 900.

c, gap junctions (g) closer to the basal area of mid-gut cells; several junctions have undulating membranes in between, and there is one punctate apposition (arrow), perhaps where a gap junction is about to form, × 73700.

Fig. 2.

Gap junctions as seen in ultrathin sections, between adjacent epithelial cells of Limulus mid-gut.

A, gap junction (g) fairly close to the lumen of the gut, lying between continuous junctions whose faint cross-striations are scarcely visible (arrows), × 115800.

B, gap junctions (at g) farther removed from the gut lumen, × 91 900.

c, gap junctions (g) closer to the basal area of mid-gut cells; several junctions have undulating membranes in between, and there is one punctate apposition (arrow), perhaps where a gap junction is about to form, × 73700.

In freeze-fracture preparations, although the particles composing the continuous junctions are often of very low profile and barely distinguishable (for example, see Fig. 10, cj in inset), the mature gap junctions are prominent maculae, consisting of round to oval arrays of loosely packed EF particles (Fig. 4). The component particles range from 10 to 15 nm in diameter (most commonly found to be 12-14 nm, and averaging 13 nm), with complementary pits in the PF (Fig. 4).

Fig. 3.

Area near basal region of mid-gut cells showing complex cellular interdigitations. Note degenerating cell (de), presumably to be replaced by another. n, nuclei, ×6000.

Fig. 3.

Area near basal region of mid-gut cells showing complex cellular interdigitations. Note degenerating cell (de), presumably to be replaced by another. n, nuclei, ×6000.

Fig. 4.

Gap junctions between established mid-gut cells near the apical border of columnar epithelial cells. EF (EF) shows macular arrays of 13-nm particles relatively loosely packed, typical of fully formed gap junctions,-while PF (PF) possesses macular aggregations (gp) of complementary pits. Note the short ridge-like array on the PF (arrow) and a complementary groove on the EF (double arrows) x 51000. Inset, higher magnification of junction cleaved to reveal both EF (EF) and PF (PF). Note central depressions in some of the component EF particles (as at arrow), × 92 250.

Figs. 4–11. Freeze-fracture preparations of mid-gut of Limulus to show changes in 13-nm particle distribution that occur apparently when replacement or interstitial cells are establishing contact with other mid-gut cells. The sequence of micrographs in Figs. 4–10 is such as to portray the possible sequence of events that occur during the development of the junctions, working from Fig. 10 through to Fig. 4; hence the one-quarter plate micrographs from Figs. 4-10 are printed at the same magnification, × 51000.

Fig. 4.

Gap junctions between established mid-gut cells near the apical border of columnar epithelial cells. EF (EF) shows macular arrays of 13-nm particles relatively loosely packed, typical of fully formed gap junctions,-while PF (PF) possesses macular aggregations (gp) of complementary pits. Note the short ridge-like array on the PF (arrow) and a complementary groove on the EF (double arrows) x 51000. Inset, higher magnification of junction cleaved to reveal both EF (EF) and PF (PF). Note central depressions in some of the component EF particles (as at arrow), × 92 250.

Figs. 4–11. Freeze-fracture preparations of mid-gut of Limulus to show changes in 13-nm particle distribution that occur apparently when replacement or interstitial cells are establishing contact with other mid-gut cells. The sequence of micrographs in Figs. 4–10 is such as to portray the possible sequence of events that occur during the development of the junctions, working from Fig. 10 through to Fig. 4; hence the one-quarter plate micrographs from Figs. 4-10 are printed at the same magnification, × 51000.

An examination of cell membranes closer to the basal border reveals a complex system; here interstitial cells are being inserted and replacement cells are differentiating to reinstate the mid-gut cells lost by degeneration (Fig. 3). In such areas the lateral membranes may display, in some freeze-fractured replicas, elongate, irregularly shaped or dispersed gap junctions (Fig. 5). In some cases these EF particle clusters are relatively small, suggesting that coalescence of such small aggregates may have occurred to form the larger irregular plaques. On other membrane faces closer still to the basal area, the intramembranous EF particles sometimes seem to be grouped together, with 13-nm particle-free membrane ridges between them, but the particles themselves are only very loosely associated (Fig. 6). Small clusters of particles, some in the form of linear strands, may be seen in adjacent membranes, aggregated at random (Figs. 7, 8). In all cases, these areas of loose aggregates, when present, are restricted to certain regions of the membrane face; they are bounded by areas of membrane which are free of such large intramembranous particles (see * in Fig. 7, inset, dotted line in Figs. 8, 10). Other deep membrane faces may show linear arrays of particles seemingly only beginning to be arranged in a loose network (Fig. 9); this may nevertheless be very extensive, covering a considerable patch, but distinctly bordered by a relatively particle-free area of membrane face. In regions close to these, small clusters (Fig. 9, inset) or short linear arrays of individual 13-nm particles can be seen lying scattered at random on the EF, sometimes near free 13-nm particles (Fig. 10), but no conspicuously larger particles have been observed. These loose patterns of intramembranous particles have been found close to the basal border in both fixed and unfixed preparations, but they occur only in a proportion of the preparations examined, presumably those where the membrane of a new cell is being inserted and is seeking to establish low-resistance pathways between itself and the other (i.e. mature) mid-gut cells. In certain cases, the free 13-nm particles can be seen lying beside indistinct rows of other particles (Fig. 10, inset) that are part of the stacked continuous junctions which occur between adjacent mid-gut cells in Limulus (see Fig. 2A and Lane & Harrison, 1977). The ‘formation plaque’ area of membrane where the particles aggregate is not characterized by any special freedom from other particles (Figs. 9, 7, inset), although the PF just beside the formation area often possesses many smaller ones, similar to normal intramembranous particles (arrows in Fig. 6, p in Figs. 8, 10). In some cases certain of these form PF ridges with complementary EF grooves (Fig. 4).

Fig. 5.

13-nm EF particle arrays showing irregular outlines of gap junctions during their formation when component particles have apparently begun to become concentrated in particular areas of membrane. Linear strands (at arrows) are still joining some clustered arrays together, x 51000.

Fig. 5.

13-nm EF particle arrays showing irregular outlines of gap junctions during their formation when component particles have apparently begun to become concentrated in particular areas of membrane. Linear strands (at arrows) are still joining some clustered arrays together, x 51000.

Fig. 6.

13-nm particle arrays on the EF (EF) and complementary pits in the PF (PF) of extensive pre-junctional arrays that are just beginning to coalesce or become concentrated into separate plaques. Note the other intramembranous particles that occur, especially in the PF (arrows), × 51000.

Fig. 6.

13-nm particle arrays on the EF (EF) and complementary pits in the PF (PF) of extensive pre-junctional arrays that are just beginning to coalesce or become concentrated into separate plaques. Note the other intramembranous particles that occur, especially in the PF (arrows), × 51000.

Fig. 7.

Loose 13-nm particle arrays in the mid-gut EF. Some particles are becoming clustered and some irregular linear arrays occur (arrows) as particles become aligned, × 51000. Inset shows a low-power view of an area with a comparable particle distribution, to illustrate that not all membranes are junction-bearing. ‘Smooth’ regions with only normal intramembranous particles (*) separate the 13-nm particleladen areas where junctions are in the process of forming, × 27900.

Fig. 7.

Loose 13-nm particle arrays in the mid-gut EF. Some particles are becoming clustered and some irregular linear arrays occur (arrows) as particles become aligned, × 51000. Inset shows a low-power view of an area with a comparable particle distribution, to illustrate that not all membranes are junction-bearing. ‘Smooth’ regions with only normal intramembranous particles (*) separate the 13-nm particleladen areas where junctions are in the process of forming, × 27900.

Fig. 8.

Loose clusters and linear arrays of 13-nm particles in mid-gut EF (EF) with complementary PF (PF) pits. Note boundary (dotted line) of junctional area. p, smaller particles on the PF. × 51000.

Fig. 8.

Loose clusters and linear arrays of 13-nm particles in mid-gut EF (EF) with complementary PF (PF) pits. Note boundary (dotted line) of junctional area. p, smaller particles on the PF. × 51000.

Fig. 9.

Short alignments of 13-nm EF particles (EF) with complementary grooves on the PF (PF). Note other smaller intramembranous particles, especially on the PF. Arrow indicates area, lower third of membrane face, where 13-nm particles are clustered more than in the upper part of the membrane. Inset, small clusters of 13-nm EF particles (EF) with PF pits (arrows) which sometimes occur instead of, or along with, the linear arrays, × 38500.

Fig. 9.

Short alignments of 13-nm EF particles (EF) with complementary grooves on the PF (PF). Note other smaller intramembranous particles, especially on the PF. Arrow indicates area, lower third of membrane face, where 13-nm particles are clustered more than in the upper part of the membrane. Inset, small clusters of 13-nm EF particles (EF) with PF pits (arrows) which sometimes occur instead of, or along with, the linear arrays, × 38500.

Fig. 10.

Free 13-nm EF particles on membrane face above an area (starting at the big arrow) where linear arrays are apparently beginning to form (arrows). The PF (PF) shows pits as well as smaller intramembranous particles (p). The dotted lines show the boundary of the presumptive junctional area, × 51000. Inset shows an area with loose, 13-nm particles beginning to line up in short ridges, two particles long. On the same membrane face, to the right, are particles characteristic of Limulus continuous junctions (cj) which also occur between mid-gut cells (see Fig. 2A). × 55100.

Fig. 10.

Free 13-nm EF particles on membrane face above an area (starting at the big arrow) where linear arrays are apparently beginning to form (arrows). The PF (PF) shows pits as well as smaller intramembranous particles (p). The dotted lines show the boundary of the presumptive junctional area, × 51000. Inset shows an area with loose, 13-nm particles beginning to line up in short ridges, two particles long. On the same membrane face, to the right, are particles characteristic of Limulus continuous junctions (cj) which also occur between mid-gut cells (see Fig. 2A). × 55100.

The differing arrangements of 13-nm particles described above suggest the possibility that they could be different stages in the process of gap junction formation. If so, several stages in the formation of these junctions may occur on the same membrane face (as in Fig. 10); particle patterns typical of a particular stage tend to cluster together, often near the next stage in junctional formation (see Figs. 9, 10) as if the tracts of membrane and their component particles had been organized into a series of developmental steps each of which must occur before they move on to the next stage.

At higher magnifications the free 13-nm particles sometimes display a central depression (Fig. 11) which may represent a channel to be used for cell-to-cell exchanges in formed junctions. This can be found in 13-nm particles in other stages of junction development as well as in the individual particles within the mature junctions (Fig. 4).

Fig. 11.

13-nm EF particles revealing the central channel or pore (arrows), also characteristic of particles comprising the mature gap junctions (see Fig. 4, inset), × 113600.

Fig. 11.

13-nm EF particles revealing the central channel or pore (arrows), also characteristic of particles comprising the mature gap junctions (see Fig. 4, inset), × 113600.

In adult Limulus hepatopancreas, the gap junctions are sometimes very loose, with circular arrays of 13-nm particles surrounding a strikingly particle-free interior (Figs. 12, 13). These junctions may at other times be very closely packed in hepatopancreas and so a structural heterogeneity is to be found in what would appear to be mature gap junctions; these ring-like particle arrays are also typical of such vertebrate junctions as the mature frog nexus (Kensler et al. 1977) and so they do not necessarily represent a stage in the development of the junctions. The circular gap junctions seen in hepatopancreas are quite distinct from the scattered 13-nm particles of the mid-gut, which seem ultimately to aggregate into macular arrays.

Fig. 12.

Freeze-cleave preparations from Limulus hepatopancreas to show the loose, ring-like gap junctions that occur between the lateral borders of its component cells. These have a particle-free membrane area (*) within the 13-nm particle rings, but other intramembranous particles occur in the EF of the membrane around them. Fig. 12, × 61900; Fig. 13, × 62700.

Fig. 12.

Freeze-cleave preparations from Limulus hepatopancreas to show the loose, ring-like gap junctions that occur between the lateral borders of its component cells. These have a particle-free membrane area (*) within the 13-nm particle rings, but other intramembranous particles occur in the EF of the membrane around them. Fig. 12, × 61900; Fig. 13, × 62700.

Fig. 13.

Freeze-cleave preparations from Limulus hepatopancreas to show the loose, ring-like gap junctions that occur between the lateral borders of its component cells. These have a particle-free membrane area (*) within the 13-nm particle rings, but other intramembranous particles occur in the EF of the membrane around them. Fig. 12, × 61900; Fig. 13, × 62700.

Fig. 13.

Freeze-cleave preparations from Limulus hepatopancreas to show the loose, ring-like gap junctions that occur between the lateral borders of its component cells. These have a particle-free membrane area (*) within the 13-nm particle rings, but other intramembranous particles occur in the EF of the membrane around them. Fig. 12, × 61900; Fig. 13, × 62700.

The different patterns of 13-nm particle distribution observed in the basal area of Limulus mid-gut epithelium, i.e. in those regions where epithelial regeneration would occur, suggest that this could be a site of junctional development between the replacement cells and the others. However, it may be that the forming junctions observed here are making connexions between the mid-gut epithelial cells and the interstitial or reserve cells which have been shown to send cytoplasmic extensions into the mid-gut in Limulus (Johnson et al. 1973). Heterocellular gap junctions have been observed between mid-gut and interstitial cells, although only in thin sections, not in replicas; in lanthanum-impregnated tangential sections the subunit packing of these junctions has been reported to be very loose at times (Johnson et al. 1973), as would be expected in gap junctions which were in process of developing.

Although it is not possible to be certain, the loose particle arrays observed in freeze-fracture replicas and the punctate gap junctions seen in thin sections of the more basal areas suggest the presence of forming junctions and these, together with the appearance of distinctly mature gap junctions nearer the lumen, and the consistent 13-nm diameter of the particles composing the various arrays, make it tempting to speculate that stages in the formation of gap junctions are being observed. There would appear to be a sequence of stages, commencing with free 13-nm particles, then linear arrays and clusters of particles (possibly identifiable in thin sections as near-punctate membrane appositions), which became arranged in an anastomosing pattern and finally the particles become loosely, then closely associated and concentrated into macular plaques. Rounding up occurs to transform irregular forms into more circular or elliptical ones. As in insects (Lane & Swales, 1977b, c), such a sequence of events would exhibit certain dissimilarities to the situation generally described for vertebrate tissues, in that the particles are inserted in the EF not PF, no large precursor particles are to be found (see Revel, 1974), while the ‘formation plaque’ area is not particularly particle-free (for example, see Decker, 1976a). This last feature differs from the situation observed in Limulus hepatopancreas, where some gap junctions are very loose, and therefore possibly in the act of forming, and tend to be associated with a remarkably smooth inner membrane area (as in Figs. 12, 13). It is possible that the arrays seen in the mid-gut represent junctions that are breaking down, not forming, but given the nature of the tissue and its function, it seems more reasonable that the gap junctions are in the process of maturing, especially as degenerating gaps might here be expected to be disposed of by internalization as occurs in decidual degeneration (Amsterdam, Bratosin & Lindner, 1976).

The anastomosing network-like arrays of particles observed in Limulus mid-gut are more extensive than the linear arrays of intramembranous particles found in developing gap junctions in vertebrates (such as in Decker, 1976a, b’, Decker & Friend, 1974; Benedetti et al. 1974) or in insects such as larval Calliphora (Lane & Swales, 1977b), larval Manduca (Lane & Swales, 1977a) or late pupal Calliphora (Lane & Swales, 1977c). In these insects as well as in vertebrates, the junctional particles seem to line up in relatively short random linear arrays which are scattered about, before coalescing into mature macular aggregation plaques, while in Limulus large networks of particles are distributed over considerable areas of membrane face and then tend to aggregate. The significance of these differences is as yet obscure, but they may reflect variations in such parameters as the response of the membrane particles to the stimulus to cluster, the chemical nature of the intramembranous particles, the make-up of the fluid plasma membrane within which the particles may move translaterally, or differences in the peripheral glycoproteins (see Hasty & Hay, 1977) with which they may be associated.

It is interesting that hormonal induction of gap junctions in horseshoe crabs seemed to produce only irregular or elongated gap junctions with loosely packed component particles (Johnson et al. 1974), not the extensive anastomosing forms observed here. It is possible that the hormone did not actually induce formation, and that what was observed were some of the less closely packed, mature junctions.

It would appear that gap junctions are being formed between interstitial or replacement cells and established cells in order to maintain cell-to-cell communication between adjacent mid-gut cells in Limulus. Presumably the central pore observed in the junctional particles would correspond to the channel utilized in mature junctions when two cells are coupled, for exchange of information or small-molecular-weight materials, as suggested elsewhere in other invertebrate tissues (Perrachia, 19736; Dallai, 1975; Lorber & Rayns, 1977). The channels found in the free 13-nm particles would presumably be modified in the configuration of their protein subunits so as to be effectively closed; at this stage the two cells would not yet be aligned with respect to their 13-nm particles and hence would be uncoupled.

The short PF ridge-like arrays of particles with complementary EF grooves which are encountered in mid-gut membranes bear similarities to those described in insect CNS (Skaer & Lane, 1974; Lane et al. 1975; Lane & Swales, 1976, 1977a, b, c;Wood, Pfenninger & Cohen, 1977) and tracheoles (Lane, Skaer & Swales, 1977; Lane & Swales, 1977c) and it has been suggested that they could possibly be axoglial maculae occludentes in the developing CNS of Calliphora (Lane & Swales, 19776). They have also been seen in insect muscle (Smith & Aldrich, 1971) as well as in other Limulus tissues (Lane, in preparation) and they have been briefly described as ‘scattered segments of tight junctions’ in flea mid-gut (Ito, Vinson & McGuire, 1975). Their function, however, remains obscure.

I should like to thank Mr. W. M. Lee for his kind assistance in preparing the photographic plates. I am also grateful to Mr J. B. Harrison for assisting with the embedding and sectioning of the material studied.

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