J. Cell Set. 84, 1-17 (1986)Printed in Great Britain © The Company of Biologists Limited 1986

TIGHT AND GAP JUNCTIONS IN THE INTESTINALTRACT OF TUNICATES (UROCHORDATA):A FREEZE-FRACTURE STUDY

NANCY J. LANEAFRC Unit of Insect Neurophysiology and Pharmacology, Department of Zoology,University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK

R. DALLAIInstitute of Zoology, University of Siena, Italy

P. BURIGHEL AND G. B. MARTINUCCIDepartment of Biology, University of Padua, Italy

SUMMARYThe intestinal tracts from seven different species of tunicates, some solitary, some colonial, were

studied fine-structurally by freeze-fracture. These urochordates occupy an intermediate positionphylogenetically between the vertebrates and the invertebrates. The various regions of their gutwere isolated for examination and the junctional characteristics of each part investigated. All thespecies examined exhibited unequivocal vertebrate-like belts of tight-junctional networks at theluminal border of their intestinal cells. No septate junctions were observed. The tight junctionsvaried in the number of their component strands and the depth to which they extended basally,some becoming loose and fragmented towards that border. The junctions consisted of ridges orrows of intramembranous particles (IMPs) on the P face, with complementary, but offset, E facegrooves into which IMPs sometimes fractured. Tracer studies show that punctate appositions, thethin-section correlate of these ridge/groove systems, are sites beyond which exogenous moleculesdo not penetrate. These junctions are therefore likely to represent permeability barriers as in thegut tract of higher chordates. Associated with these occluding zonular junctions are intermediatejunctions, which exhibit no identifiable freeze-fracture profile, and macular gap junctions, charac-terized by a reduced intercellular cleft in thin section and by clustered arTays of P face particles infreeze-fractured replicas; these display complementary aggregates of E face pits. The diameters ofthese maculae are rarely very large, but in certain species (for example, dona), they are unusuallysmall. In some tissues, notably those of Diplosoma and Botryllus, they are all of rather similar size,but very numerous. In yet others, such as Molgula, they are polygonal with angular outlines, asmight be indicative of the uncoupled state. In many attributes, these various junctions are moresimilar to those found in the tissues of vertebrates, than to those in the invertebrates, which theadult zooid forms of these lowly chordates resemble anatomically.

INTRODUCTION

The junctional complexes of the digestive tract of mammalian systems have beenextensively studied at the fine-structural level; such epithelia are characterized by aseries of intercellular specializations along their lateral borders, which include zonulaand macula adherens, tight junctions (zonulae occludentes) and gap junctions(Farquhar & Palade, 1963). Although the tissues of the gut of those invertebrates

Key words: gap junctions, tight junctions, freeze-fracture, tunicate gut, ascidian intestine,Urochordata.

2 N. J. Lane and others

examined to date reveal desmosomal contacts and gap junctions, they also possess theso-called septate junctions (Wood, 1959; Noirot-Timothe'e & Noirot, 1980) but notight junctions. Although septate junctions were for some time considered to be theinvertebrate equivalent of the vertebrate tight junctions (Satir & Gilula, 1973; Greenet al. 1979), it has recently become clear that tight junctions proper do exist ininvertebrates, in tissues where permeability barriers are to be found (Lane, 1981a),although they do not generally occur in the intestinal tract of these animals (Lane &Skaer, 1980; Skaer et al. 1980).

The tunicates (Urochordata) (also called ascidians) occupy a unique positionphylogenetically, in that they are deemed to be chordates by virtue of the noto-chord and neural tube that their motile and ephemeral larval form possesses. In theadult, however, after they have undergone a retrogressive metamorphosis, theirnervous system is reduced to a single, invertebrate-like ganglion, housed in a sessile,relatively simplified zooid. In this way they appear to occupy an intermediateposition between the two major divisions of the animal kingdom - invertebrate andvertebrate.

A few earlier studies on tunicates (Cloney, 1972; Lorber & Rayns, 1972; Georges,1979; Green & Bergquist, 1982) revealed that tight junctions were present in thisgroup, at least in the tissues of the heart, tail and epidermis; comparable occludingjunctions have also recently been discovered, however, in certain tissues of insects(Lane & Treherne, 1972; Lane, 1972a, 1978, 1979, 1981a,fe, 1982; Laneetal. 1977)and arachnids (Lane & Chandler, 1980; Lane, 1981c; Lane et al. 1981), arthropodsthat have quite unequivocal septate junctions (Noirot-Timothe'e & Noirot, 1980)in their digestive tracts (Lane & Skaer, 1980). It therefore seemed of interest toexamine the tissues of the intestinal tract of a range of tunicates, to determinewhether they would exhibit tight or septate junctions between their componentepithelial cells. Three different orders, with different genera from each, have beeninvestigated and, although there are subtle distinctions in the organization andcomplexity of their junctions, as well as in the number and size of their associated gapand intermediate junctions, the apical lateral border in all of the species examinedpossesses unequivocal tight-junctional networks. These exhibit the characteristicfeatures of zonulae occludentes as first described in thin sections by Farquhar &Palade (1963) and later in freeze-fracture replicas by Claude & Goodenough (1973).A short preliminary report on part of this work has been published elsewhere (Leeetal. 1985).

MATERIALS AND METHODSThe tissues studied in this investigation were the intestinal tracts of a range of tunicates, both

solitary and colonial. The order Aplousobranchiata was represented by the species Diplosomalisterianum and Clavelina lepadiformis, the order Stolidobranchiata by Molgula socialis, Botryllusschlosseri and Botrylloides leachi, and the order Phlebobranchiata by Ciona intestinalis andAscidiella aspersa. These organisms were collected variously from the .lagoon of Venice (Italy) orfrom Plymouth (England). They were either studied immediately, or maintained in tanks ofcirculating sea water before use.

Junctions in tunicates 3

In most cases the intestinal tract was dissected out into a number of regions: the large pharynx,the relatively short oesophagus, the stomach and the intestine. This last region was in some casesdivided again into three sections, the anterior intestine, the mid-intestine, which in some casesconsisted of a loop, and the distal intestine, or rectum. In the case of the small colonial forms, suchas Diplosoma, Botryllus and Botrylloides, whole zooids isolated from the colony were studied andthe different regions of the gut determined by their morphological differences in the thin sections orreplicas. The tissues were treated with one of a variety of fixatives, of which the most successfulwas 1-5% glutaraldehyde in 0-2M-cacodylate buffer, pH7-4, plus 1-5% NaCl. In some casesphosphate buffer was used instead of cacodylate and 2 % sucrose instead of NaCl^ was added to thefinal fixative solution. The final measured osmolality was slightly hyperosmotic to that of sea water.In some cases, 1 % colloidal lanthanum was added to the cacodylate buffer, using the electron-opaque tracer lanthanum as an extracellular 'negative' stain. Tissues were then embedded in eitherEpon or Araldite for thin-sectioning, or frozen for the preparation of freeze-fracture replicas. Forthe former, tissues were washed, post-fixed in osmium tetroxide, in some cases stained en bloc inuranyl acetate, dehydrated through an ascending series of ethanols, and embedded in epoxy resin.For the latter, the tissues (fragments of gut or pellets of small zooids) were washed and cryo-protected in glycerol at 10 %, 20 % and 30 % in buffer, for varying periods of time, before freezingby plunging into Freon 22 cooled in liquid nitrogen. The material was then mounted in a Balzersfreeze-fracturing device (BAF301 or BA360M models) and fractured at -100 to —115°C and at2xlO~6Torr (1 T o r r ^ 133-3 Pa). The preparations were shadowed with platinum-carbon ortungsten-tantalum and backed with carbon. The tissue was removed with sodium hypochlorite orbiological detergent, the replicas washed in water and mounted on copper grids for examination in aPhilips EM 300, 420 or Hitachi H600, at 60 or 80 kV.

RESULTS

The pharynx and oesophagus in these species of tunicates are characterized bycells with many cilia and a few microvilli. The main epithelial cells that occur in thestomach are either the columnar vacuolated absorbing cells or the zymogen cells(Burighel & Milanesi, 1973, 1977); in both cases they, in contrast, exhibit manymicrovilli but no cilia. Ciliated cells may occur, however, in restricted regions of thestomach. In the intestinal tract, mucous cells that exhibit many cilia and fewmicrovilli, and vacuolated cells that possess microvilli and only rarely, cilia, are themain cell types. These and other features can be used in the small colonial forms todetermine which part of the gut is being examined.

The tight junctions are present near the luminal surface of the component cells,seen in thin sections as punctate cell-cell appositions between adjacent cells(Figs 1,3). Associated with their cytoplasmic face may be fibrous material, pre-sumably cytoskeletal (Fig. 1). Below these apical borders occur regions with thereduced intercellular clefts that characterize gap junctions (Fig. 2); in some casesmany of these are found, each in close association with the next (Figs 2, 5). At highermagnification, the tight junctions exhibit a fusion of the two adjacent outer halfmembrane leaflets (Fig. 1), while the gap junctions reveal a 2-4nm cleft betweentheir apposed membranes (Fig. 2). In the specimens incubated with lanthanum, thelanthanum was halted at the punctate tight-junctional appositions (Fig. 3). At thelevel of the gap junctions the tracer penetrates the intercellular clefts, which thenhave a cross-striated appearance when they are slightly obliquely sectioned (Fig. 2).

In some cases, other, more desmosomal-like structures are present near the tightjunctions. These do not resemble septate junctions, which are not present in these

N. J. Lane and others

Junctions in tunicates 5

tissues. They are characterized in thin section by an enhanced density along thelateral cell borders (inset, Fig. 6), which appears to consist of cytoplasmic fibrilscondensed into a feltwork along the side of the membrane. Sometimes, in fortuitoussections, the fibrils can be seen to have a zonular distribution, where they lie as abundle running from one intercellular junction to another. They appear similar tothe zonula adhaerens or intermediate junctions described by Farquhar & Palade(1963). There is no particular organization of intramembrane particles (IMPs) orany other apparent freeze-fracture profile that can be correlated with these contacts(Figs 4, 6, 12, 16) and so it must be presumed that the fibrils do not insert into themembrane as far as its mid-line, the plane of fracture.

Other than this, the freeze-fracture appearance (Fig. 6) of a particular tissue of anygiven species can be closely correlated with thin sections of comparable tissue(Fig. 5), in that a network of tight junctions can be seen to be present in the apicalregion, while macular gap junctions lie beneath. In some cases the population of gapjunctions is very extensive (Fig. 6); in others, they may be much fewer in number(Figs 4, 7) or none may seem to be present at all (Figs 8—11). This appears to varywith the cell type or with the particular area under consideration; in general, theintestine and pharynx seem to have fewer gap junctions than does the stomach, whilethe number in the oesophagus is more variable.

The tight junctions form a circumferential band between the lateral borders at theapices of the cells. In all the regions of the gut examined these junctions feature

Fig. 1. Section through the pharynx of Botryllus, in the region of the cells of thebranchial stigmata. Note the punctate cell to cell appositions forming the tight junctions(arrows). At these points, fusion of the outer half membrane leaflets of the adjacent cellscan be seen. /, lumen. X110 000.Fig. 2. Section through vacuolated cells in the stomach of Botryllus after incubation withlanthanum showing the extensive gap junctions that may occur between adjacent cells.The intercellular cleft has been penetrated by the La3+ and a cross-striated appearance,typical of slightly oblique sections through the connexons of such junctions, can beobserved (arrows). X70000.Fig. 3. Lanthanum infiltration between mucous cells of the stomach of Botryllus. Notethat the progress of the tracer is halted at a punctate tight-junctional apposition (arrow),indicating that these junctions are able to produce a permeability barrier. /, lumen.X56000.Fig. 4. Freeze-fracture replica from the oesophagus of Clavelina revealing the circum-ferential band of tight-junctional ridges forming a network on the E face (EF), just at theluminal surface under the border of microvilli (mv).' The junctional grooves containoccasional particles while below them gap junctions (gj) are to be found. X35 000.Fig. 5. Thin section through adjacent cells in the stomach of Diplosoma. Immediatelybeneath the lumen (/) of the gut, tight junctions (arrows) occur; a series of gap junctions(gj) are found just below these. X22500.Fig. 6. Freeze-fracture replica from the stomach cells of Diplosoma. In correlation withthe thin-section appearance (seen in Fig. 5 and inset), below the lumen (/) are foundintermediate junctions and a slender band of tight junctions (tj) in a network confor-mation. The IMP arrays found in the region above the tight-junctional (tj) belt exhibit noconsistent profile of particle aggregates. Beneath are seen a vast array of gap-junctionalplaques (gj) of a size consistent with the thin-sectioned junctions. X16500. Inset,X80000.

N. J. Lane and others

Figs 7-11. For legends see p. 8

Junctions in tunicates

Figs 12-16. For legends see p. 8

8 N. J. Lane and others

aligned PF particles ranging around 8-12 nm in diameter, arranged in a reticularnetwork (Figs 7-16). In some cases the particles appear fused into ridges on theP face (Fig. 7), but this is not always so (Fig. 10) and may be due to the angle ofshadowing or the response of the junctional components to the cross-linking effectsof glutaraldehyde. Some particles may fracture up onto the EF, where they liein the grooves to be found there (Figs 10, 11). That these E-face grooves arecomplementary to the PF ridges can be seen when the fracture plane is in transitionfrom EF to PF (Fig. 12). The two are in coincidence with one another, except thatthe PF ridges are slightly offset with respect to the EF grooves (small arrows inFig. 12).

The tight-junctional belt varies in depth as it is composed of a variable number ofstrands; it is a fairly broad band in the tissues of the pharynx, oesophagus, stomachand the anterior part of the intestine (Fig. 10). In some cases, however, occasionallyin the stomach (Figs 6, 25) or posterior intestine, the network band is less wide andmay consist of only a few anastomosing strands, at least insofar as the fracturedmembrane face reveals. There seems to be no consistent pattern to this variable. Inother cases, particularly in the stomach (Figs 15, 16), there is a tendency for the basal

Fig. 7. Replica through the intestine of Clavelina, revealing the band of tight-junctional(tj), P face, ridges (arrows), forming anastomoses around the luminal (/) end of the cells.Gap junctions (gj) of a range of sizes lie beneath and there is a reduced intercellular cleftwhere they occur (at arrows). X46000.

Figs 8-11. Replicas of the pharynx, in the area of the branchial stigmata, of Botryllus(Fig. 8), and of Clavelina intestine (Figs 9-11). Note that the tight-junctional belts arefairly extensive networks and consist of P face (PF) particles that are aligned in rows. Thesame tissue (e.g. Figs 7, 10) may exhibit either tight-junctional particle rows or ridges. Itseems that when the angle of shadowing is at right angles to the rows, they appear as solidridges (large arrow in Fig. 10). The complementary E face (EF) grooves may contain afew particles that have fractured into them, presumably from the P face membrane half.Complementary replicas would be required to check if this is so. Both the PF ridges andparticle rows are in register with the EF furrows (arrows in Fig. 10) although they appearto be slightly offset with respect to them. The membranes are pinched in towards eachother in the region of the tight-junctional punctate appositions (arrows in Fig. 11).Fig. 8, X28000; Fig. 9, X36000; Fig. 10, X53 000; Fig. 11, X61500.

Figs 12-16. These replicas, taken from preparations of intestinal ciliated cells ofBotrylloides (Fig. 12), stomach of Molgula (Figs 13, 16), oesophagus of Ascidiella(Fig. 14) and stomach of Botrylloides (Fig. 15), all show tight-junctional networks withassociated gap-junctional plaques. Note that the different tissues and species show tightjunctions as particle rows or ridges with no consistent pattern. One feature that doesappear common, is that the networks are continuous where they face the luminal surface(/), while on the other, more basal, border they frequently break up into discontinuousparticle alignments or ridges (see especially Figs 12, IS, 16). In addition, thePF ridges orIMPs are in register with the EF furrows (arrows in Fig. 12) so that the two appear to becomplementary structures. In many cases, these open-ended alignments run into or lienear intramembranous particle (IMP) clusters; the latter sometimes have a rosette-likeappearance (arrowheads, Figs 12, 14, 16) while in others they are more distinctly gap-junctional in character (as in Figs 13, 15). In some cases these display the reducedintercellular cleft characteristic of these junctions (arrows in Fig. 13). Unequivocal gapjunctions (gj in Fig. 13) may be present as plaques beneath the tight-junctional belts, buttheir numbers vary with the species and the tissues under observation. Fig. 12, X51 000;Fig. 13, X35 000; Fig. 14, X54500; Fig. 15, X24000; Fig. 16, X41000.

Junctions in tunicates 9

component of the tight-junctional network to be in looser strands, which branch outfrom the reticulum. These may continue as extensive individual ridges, running inwavy alignments parallel to the longitudinal axis of the cells, separately, with fewanastomoses; this is particularly striking in Ciona (Fig. 17).

The gap junctions, which are found beneath the tight junctions, may lie very closeto them (Figs 6, 15, 18, 19) or may be further down the lateral border. They varyin number from many (Figs 6, 18) to relatively few (Figs 7, 12). They are alwayscomposed of P-face connexons ranging from 8 nm to 13 nm in diameter (Figs 15, 19),which are usually clustered together in close association, commonly in circularplaques (Figs 6, 15, 18, 20, 21). That these are the sites of the gap-junctional reducedintercellular cleft seen in thin sections is evident where the fracture plane cleavesfrom PF to EF (Figs 7, 13, 19, 20, 23). Frequently, fragments of the E-facemembrane remain attached to the maculae, and the EF pits can be seen on theseremnants (Fig. 22); these exhibit the regular connexon packing rather clearly. Inother cases, some of the PF particles may adhere to the EF (Fig. 23) where thejunctions are recognizable as clusters of pits. There are also some irregular gap-junctional plaques, which assume angular, polygonal outlines (Figs 22, 23, 24),particularly in the stomach of Molgula. These shapes may be found on cell bordersclose to those with more normal irregular outlines and in some cases transition formscan be found (Fig. 25). Examination of all the other parts of the gut tract of Molgulareveals that the gap junctions tend not to be polygonal elsewhere.

The size of the gap junctions in the tunicate gut tends to be within the range from0-1 yxn to 0-6/zm (± a few 0-1 nm; see Figs 6, 18, 25); that is, there tend to be noexceptionally large gap junctions. However, in some relatively rare cases they may beextremely small in diameter (Figs 12, 16, 26, 28), giving the impression that they arebecoming fragmented or perhaps, disaggregated (Figs 26, 27). This may also be thecase when small plaques are found near the end of tight-junctional strands (Fig. 28);this is particularly common in Ciona, where normal-sized gap-junctional plaques areremarkably infrequent.

DISCUSSION

The occurrence of tight junctions in the tissues of tunicates was first reportedbetween the caudal epidermal cells of the ascidian Distaplia (Cloney, 1972), and inthe heart of adult Ascidiella (Lorber & Rayns, 1972) from thin sections. Freeze-fracture studies on heart tissue (Lorber & Rayns, 1972), and on the epidermis andpharyngeal epithelium (Georges, 1979) of adult tunicates, revealed strands ofdistinct particles forming belt-like networks at the apical parts of cells; anastomosingpatterns of IMPs were also observed in the tight-junctional belts of Asterocarpa(Green & Bergquist, 1982). Since these junctional structures were observed initiallyonly in the tissues of adults, or between the differentiated tail epidermal cells inlarvae, it was considered that they were characteristic of cells that were completelydifferentiated, and that they were capable of forming a tight barrier between twocompartments (Georges, 1979). In the tissues of the intestinal tract of the range

Figs 17-19. For legends see p. 12

Junctions in tunicates 11

Figs 20—24. For legends see p. 12

12 N. jf. Lane and others

of ascidian genera examined here, the same interpretation seems justified. Theirdistribution, in fact, parallels that typically encountered in vertebrate gut (Claude& Goodenough, 1973) and clearly demonstrates the basic similarity of the tightjunctions found in urochordates and vertebrates. In thin sections, this likeness is alsoapparent as regards the features of the punctate appositions between adjoining cells,with apparent fusion of the two external half membrane leaflets. Fibrous cyto-plasmic material is in some cases associated not only with the zonula adhaerens, butalso with these junctions, as described in earlier studies on tunicates (Cloney, 1972);this too parallels the observations made on vertebrate systems, where cytoskeletalcomponents have been found inserting into tight-junctional contact sites (Bentzel &Hainau, 1979) where they may be involved in modulating their tightness. Regulationof tight-junctional permeability in vertebrate tissues has been considered in detailelsewhere (Schneeberger & Lynch, 1984), but there is very much less relevantinformation available for urochordates and seemingly none on their gut tract.However, during tail resorption in a tunicate larval tadpole (Cloney, 1972), tightjunctions were thought to be involved in the contraction process mediated by cyto-skeletal components. In such circumstances the role of the filaments that are also seento insert into the zonula adhaerens, which may occur near the tight junctions intunicate tissues, is not clear.

The disposition of the tight-junctional strands within the gut cells of tunicates seenin freeze-fracture replicas is as particle alignments or ridges on the P face and E facegrooves, with occasional rows of distinct particles within them. This distribution

Figs 17—19. These replicas represent the rectum of Ciona (Fig. 17) and the oesophagusof both Botryllus (Fig. 18) and Ascidiella (Fig. 19). Note that although in these speciesthe lower border of the tight-junctional network may be relatively intact (Fig. 19), it mayin some cases end in short discontinuities (Fig. 18) or in very extensive ones (Fig. 17),which may, particularly in rectal tissues, run for considerable distances along the lateralborders; this can be seen on both the PF and EF. The gap junctions that lie beneath thetight-junctional belt may be very infrequent, as in Ciona, or very numerous, but variablein diameter (Figs 18, 19). They exhibit the characteristic reduced intercellular cleft(arrows in Fig. 19). Fig. 17, X20600; Fig. 18, X24000; Fig. 19, X48000.

Figs 20—24. Tissues from the stomach of a range of tunicates, revealing the variety ingap-junctional plaque morphology. Figs 20, 21, Clavelina; Fig. 22, Diplosoma; Figs 23,24, Molgula. The plaques range in outline from relatively circular (Clavellina) to slightlypolygonal (Diplosoma) to very regularly polygonal (Molgula). The reduced intercellularcleft at the EF/PF transition is evident (arrows in Figs 20, 23, 24) and in some cases theEF membrane cleaves to the PF junctions (Fig. 22) or the PF particles fracture intothe EF pits (Fig. 23). Fig. 20, X60000; Fig. 21, X28 500; Fig. 22, X41000; Fig. 23,x46 000; Fig. 24, x 36 000.

Figs 25—28. Replicas from stomach of Molgula (Fig. 25) and Botryllus (Fig. 26), andrectal tissue of Molgula (Fig. 27) and Ciona (Fig. 28). The semi-polygonal or transitionalforms of gap junctions can be seen equally clearly in both PF and EF of tissues thatpossess them (Fig. 25). The very small gap junctions that characterize some tissues, maybe continuous with the tight-junctional ridges (arrows in Fig. 28) or may be completelyunassociated (Figs 26, 27). Although they are usually close-packed particle aggregates,in some cases they are loosely clustered (Figs 26, 27, airows) so that it may not be clearif certain IMPs are junctional or not. Fig. 25, X45000; Fig. 26, X30500; Fig. 27,X60000; Fig. 28, X44000.

Junctions in tunicates 13

14 N.J. Lane and others

seems very similar in all the ascidians studied here. In an early study on the epi-dermis by Georges (1979), the PF and EF fracture faces revealed slightly differentarrangements, while Green & Bergquist (1982) found alignments of discrete particles(5-25 nm long) that fracture onto the E face in both fixed and unfixed preparations.There seems to be great disparity in particle diameter, with Georges (1979) reportingthe junctional particles in Phallusia to be more variable in diameter (6-5—25 nm)than those of Ciona and Morchellium (12 ± 2nm). The number of rows of particlesand the degree of their anastomosis is not very consistent in the range of speciesexamined here, although there is a general tendency for the junctional networks tobecome looser as they are further removed from the cell's apical border. Whatvariations have been observed in the several segments of the alimentary tract mayreflect a different functional state of the cells. It has been suggested that the tightjunctions of different tissues can vary their geometric pattern according to theflexibility of the plasma membrane in a given region (Hull & Staehelin, 1976). Thismorphological flexibility would enable the junctions to maintain the transepithelialsealing capacity even when the tissue is undergoing osmotic stress or mechanicalstretching.

The studies with tracers suggest that, in the tunicates, these junctions form themorphological basis of permeability barriers, as they do in higher vertebrates(Claude & Goodenough, 1973; van Deurs, 1980), although they may in certain casesbe leaky to lanthanum (Green & Bergquist, 1982). The variability in the dispositionof their IMPs as separate particles or as ridges may relate to leakiness or may be afeature that is determined in part by their response to the cross-linking effects ofglutaraldehyde (van Deurs & Luft, 1979). The angle of shadowing would also beimportant, as has been observed in studies on the IMPs of septate junctions (Kacheret al. 1986). Rapid freezing would be required to determine if these structures arecylindrical in vivo and hence possibly formed from inverted lipid micelles, stabilizedby protein, as has been suggested for vertebrate tight junctions (Kachar & Reese,1982; Pinto da Silva & Kachar, 1982). As found, until recently, with the vertebratetight junctions (Stevenson & Goodenough, 1984), no entirely satisfactory isolationprocedure has been devised for tunicates, so pure preparations of tight junctions areunavailable for biochemical analysis. Certainly the large numbers of strands in thetunicate gut would make them admirable material for such studies. Moreover, in thecase of both the IMPs and ridges of these tight junctions, the PF structures are offsetwith regard to the complementary EF grooves, which suggests that they have theirstructural basis in a double fibril, as has also been observed in vertebrate tissues(Bullivant, 1978). A similar model has been proposed for the arthropod tightjunctions (Lane, 1981a) and these also, after fast freezing, appear as continuouscylinders (Lane, 1984). The Arthropoda are the only major invertebrate group thatpossesses unequivocal tight junctions (Lane, 1981a) and so are the only groupwithout 'backbones' with which the tunicates may be compared. Tight junctions,rather than septate junctions, also seem to form the basis of the permeability barriersobserved in the arthropods, such as in the blood-brain (Lane, 1972a, 1978, 1981a)and blood—testis (Toshimori et al. 1979) barriers. However, the tight-junctional

Junctions in tunicates 15

belts tend to be rather simple in the insects (Lane, 1982) and, although they appearas more extensive networks in the arachnids (Lane & Chandler, 1980; Lane et al.1981), the tight junctions of tunicates are structurally much more complex in theextent of their networks, like those reported in the tissues of vertebrates. We do notfind strict PF particle adherence in tunicate tight junctions but do not consider thatsuch a characteristic, together with a dearth of continuous ridges, would constitutea close relationship to septate junctions phylogenetically, as suggested elsewhere(Green & Bergquist, 1982). Septate junctions, which occur in the intestine ofarthropods (Skaer et al. 1980), are not to be found in the gut of either tunicates orvertebrates.

The gap junctions seen here, as also described in other tunicate tissues (Lorber &Rayns, 1972, 1977; Georges, 1979), are characteristic of those in vertebrate tissue inthat their component particles all fracture onto the P face, leaving complementaryEF pits. This is also the case for some other invertebrate phyla, although not thearthropods, in which the connexons are larger and fracture onto the E face (Lane &Skaer, 1980). The actual si2e of the individual connexon measured here in the gut is,on average, 8—13 nm, in comparison with the 5nm diameter of the particlescomprising them in tunicate epidermal and pharyngeal cells (Georges, 1979). Thejunctions do exhibit, however, great variability in their number, since in some cases(e.g. Diplosoma and Molgula), gap junctions are very numerous, while in dona(as also noted by Georges, 1979), they are relatively infrequent. The size of thejunctional plaques themselves is also somewhat variable, those of dona beingparticularly small. The significance of the polygonal outlines exhibited by some gapjunctions is not clear, but one possibility is that this reflects their state of coupling(see Peracchia & Dulhunty, 1976; Peracchia, 1977), the more closely packed arraystending to be uncoupled. Again, fast freezing without fixation might elucidate thispoint, although earlier studies on such phenomena in both urochordates (Hannaet al. 1981) and mammalian systems (Raviola et al. 1980), as well as crustaceans(van Deurs et al. 1982) and insects (Swales & Lane, 1983), did not support such acontention.

Certainly these organisms are in an interesting phylogenetic position: in thedistribution of tight junctions throughout their gut tract, in the morphologicalfeatures of these and their associated gap junctions, tunicates appear more chordate-like than invertebrate-like. Further, they appear to lack septate junctions, which area feature unique to the latter group. It is therefore intriguing that in certain otherrespects, such as the relative simplicity of their adult nervous system, which lacksa blood—brain barrier (Lane, 19726), as well as any glial ensheathment ormyelination (Lane, unpublished observations), tunicates are more invertebrate-like.Moreover, scalariform junctions, which are related to septate junctions (Lane &Skaer, 1980), have recently been found in the gut of Botryllus (Burighel et al. 1985);the possession of these has hitherto been considered to be a uniquely arthropod-likecharacteristic. In addition, in previous reports the myofibrillar structures of tunicatestriated muscles have been described as being typical of chordates, while theirsarcotubular system is more similar to that of invertebrates (Burighel et al. 1977).

16 N.jf. Lane and others

Further studies on developing tissues, such as the central nervous system, muscle orgut during tunicate retrogressive metamorphosis, may shed light on this dilemma.

We thank Dr Riccardo Brunetti for the selected specimens, Paolo Salvatici and William Lee fortechnical assistance with the production of the freeze-fracture replicas, Valentino Miolo for helpwith sectioning, and Ermanno Malatesta and Vanessa Rule for help with typing the manuscript.We are also indebted to MPI for grants during the course of this research.

REFERENCESBENTZEL, C. J. & HAINAU, B. (1979). Cellular regulation of tight junction permeability in a

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(Received 4 February 1986 -Accepted 28 April 1986)