phylogenetic relationships within the invertebrata in relation to the ...

J. Cell Set. S3. 279-305 (1982) 279Printed in Great Britain © Company of Biologists Limited 1982

PHYLOGENETIC RELATIONSHIPS WITHIN THE

INVERTEBRATA IN RELATION TO THE

STRUCTURE OF SEPTATE JUNCTIONS AND

THE DEVELOPMENT OF 'OCCLUDING'

JUNCTIONAL TYPES

COLIN R. GREEN AND PATRICIA R. BERGQUISTDepartment of Zoology, University of Auckland,Private Bag, Auckland, New Zealand

SUMMARY

The structures of 13 variants of invertebrate septate junction are reviewed on the basis offreeze-fracture, lanthanum tracer and thin-section studies. In addition, a simple type ofoccluding junction in the phylum Porifera, a variation of tight junction in the phylum Tunicateand the vertebrate tight junction are covered. All the junctions considered form a belt aroundthe apical circumference of cells lining a lumen or an exterior surface. The large number ofthese junctions now recognized permits discussion relating to invertebrate classification andsuggested phylogenetic relationships, and to the development of intercellular junctions. Therelationships revealed are discussed under three headings: Coelenterates and lower inverte-brates, Proterostomia (the annelid, molluscan and arthropod lineage) and the Deuterostomia(the echinoderm and chordate lineage).

It is proposed that the pleated septate junction of the lower invertebrates resembles that ofthe hydrozoan rather than anthozoan Coelenterates. This lower invertebrate pleated septatejunction occurs in several lower invertebrate phyla including the Annelida (of the proterostomelineage), but also occurs in the Sipunculoidea, a group supposedly on the deuterostome lineage.The proterostome line includes the molluscs and the arthropods, which have the mollusc-arthropod pleated septate junction. Several variations of the smooth septate junction are alsoseen in Arthropoda. Among the deuterostomes the Chaetognatha have both a paired septatejunction and a pleated junction and are therefore considered to be not very far removed fromthe Sipunculoidea. The echinoderms and hemichordates also have double-septum septatejunctions. In addition however, these two phyla have anastomosing septate junctions thatare very similar, varying only in their final configuration. Of the two, the echinoderm anasto-mosing septate junction most closely resembles the tight junction seen in the tunicates,and the Hemichordata are therefore considered to be a lateral development from the main lineof chordate evolution. The tunicates have a tight junction similar to that seen in vertebrates;it is however more 'leaky' and has distinctive freeze-fracture characteristics.

In the phylum Porifera a form of simple parallel membrane junction appears to serve anoccluding function. This junction has regular intercellular spacing in the absence of any septaand it is suggested that the spacing in septate junctions is probably not dictated by the septa.This interpretation is reasonable particularly when the diversity of septal types in conjunctionwith stable intercellular spacing is considered. Finally, a theory is put forward suggesting thatin evolution a change from the septate to the tight junction could simply involve a modificationof a ' membrane spacing factor', which allows the membranes of adjacent cells to come togetherat intervals, in the normal tight junction pattern.

280 C. R. Green and P. R. Bergquist

INTRODUCTION

Intercellular junctions are specialized regions of contact between the plasma mem-branes of adjacent cells, they provide a structural basis for cellular interactions.Invertebrate septate junctions are intercellular junctions that occur in an apical beltaround cells that line an epithelium. Since the first description of these junctions inHydra by Wood (1959) they have been studied in many invertebrate phyla and arethought by many workers to serve an occluding function (Filshie & Flower, 1977;Green, Bergquist & Bullivant, 1979; Noirot-Timothee & Noirot, 1980; Wood & Kuda,1980) analogous to that of the vertebrate tight junction (see reviews by McNutt,1977; Staehelin, 1974; Staehelin & Hull, 1978). That is, they form a gasket-likebarrier between cells and slow intercellular leakage across an epithelium. Otherworkers, however, consider their role may be a different one; possibly of adhesionor to confer greater rigidity on the intercellular membranes (see reviews by Lane &Skaer, 1980; Noirot-Timothee & Noirot, 1980).

In fact the term septate junction has been used to describe several junctionalstructures in which, in cross-sectional view, the intercellular space is bridged by aseries of septa. Study of these junctions using electron microscopic techniques hasrevealed 13 structural variants to date (Baskin, 1976; Duvert, Gros & Salat, 1980;Filshie & Flower, 1977; Flower & Filshie, 1975; Green, 1980, 19810-^; Green et al.1979; Green & Flower, 1980; Lane & Harrison, 1978; Noirot-Timothee & Noirot,1980; Skaer, Harrison & Lee, 1979; Welsch & Buchheim, 1977; Wood, 1977) andmore may be found as further invertebrate phyla are studied.

The large number of septate junctions now recognized permits some discussion oftheir occurrence in relation to invertebrate classification and suggested phylogeneticrelationships. In conjunction with other data employed in phylogenetic study, forexample morphological, developmental and biochemical, the structure of junctions asseen in electron microscopy may provide some information relevant to classification.

In this communication we review briefly the range of invertebrate septate junctionsand relate these to a variant of the vertebrate tight junction that occurs in the phylumTunicata. Invertebrate classification in relation to the occurrence and structure ofthese junctions is then discussed and a hypothesis proposed for the development oftight junctions from invertebrate precursor junctions.

METHODS AND TECHNIQUES

Three major electron microscope preparatory techniques have provided the results discussedhere. It is often necessary to combine these techniques in order to identify adequately, ordescribe, a particular junction type. Each of the 3 techniques, conventional thin-sectioning,tracer impregnation (negative staining) and freeze-fracture, give information on quite differentaspects of junction structure.

(i) Conventional thin-sectioning: conventional thin-sectioning is particularly useful forstudying the appearance of a junction in cross-section. The septa of septate junctions, forexample, running between cells, and sectioned so that they are parallel to the electron beam,appear as bars in the space between the contributing cell membranes. Tangential views ofjunctions prepared by this technique are usually not very informative because of the lack ofcontrast between the positively stained junctional structures and their background.

Invertebrate septate junctions and phylogeny 281

(ii) Tracer impregnation: heavy-metal tracers have been used very successfully to showjunctional structures in sections cut tangentially to the junction. Tracers, such as lanthanumnitrate, lanthanum hydroxide, horee-radish peroxidase or tannic acid, are used to fill the inter-cellular space with an electron-opaque precipitate. Subsequent thin-sectioning reveals theunstained junctional structures between the membranes in negative contrast. The techniqueeffectively involves surrounding or ' embedding' junctional structures in the tracer material.

(iii) Freeze-fracture: freeze-fracturing reveals the intramembrane component of junctions.When tissue is freeze-fractured the fracture plane passes preferentially down the centre ofbiological membranes. Two faces are therefore revealed, an E face attached to the extracellularspace (the outer half of the membrane) and a P face attached to the cytoplasm of the cell (theinner half of the membrane). The structures of a junction seen by this technique are thereforethose within the membrane of one of the contributing cells. The 2 faces revealed show differentfeatures in each type of junction. Using the freeze-fracture technique it is also possible to studyfixed and unfixed tissue and in many cases substantial differences are seen in junctionalstructures between the 2 types of preparation. Ideally, studies of junctions should involve bothfixed and unfixed tissue samples. A full description of the freeze-fracture technique is given byBullivant (1973).

THE INVERTEBRATE SEPTATE JUNCTIONS

In cross-sectional views of the different types of septate junction there is littlevariation, virtually all have septa that 9pan a 15-18 nm intercellular space (e.g. seeFig. 1). In tangential views the most obvious differences are in the conformations ofthe septa themselves, whether they are double, single, straight, pleated or anasto-mosing. In addition the widths of septa vary, and some have side projections fromone or both sides, while others have none.

In freeze-fracture studies the shape and size of junctional structures vary and theface to which the structures adhere is also significant. There is, however, some doubtas to the importance that can be placed on the face to which freeze-fracture structuresadhere. Wood (1977) presents results that differ from those obtained by Filshie &Flower (1977) or Green (1980) when viewing the Hydra type septate junction. In addi-tion Skaer et al. (1979) report particles of the smooth junction predominantly on the Eface of one tissue in contrast to the normal situation for that junction where particles arefound on the P face (Flower & Filshie, 1975; Skaer et al. 1979). However, in the workof Dallai (1976) in which he found 2 septate junctional types occurring concurrently,it is noteworthy that one, the smooth septate junction, showed reversed polaritybetween fixed and unfixed tissue while the other, a pleated septate junction, did not.Clearly the reversal or non-reversal of polarity in this case was a feature of thejunction type, not of a membrane or a tissue as a whole. In this communication, there-fore, it is considered that the side of a freeze-fracture to which junctional structuresadhere is a feature of the junction and is thus significant in identifying the junction.

The phylum Porifera. The transient nature of virtually all intercellular interactionsin the sponges makes it difficult to locate cell junctions. Sponge cell junctions areapparently formed only when required for a specific purpose (Green & Bergquist,1978). There is consequently little information available on junction types in thisphylum. In the case of septate junctions, 2 reports are available (Green & Bergquist,1978; Ledger, 1975), both only showing positive-stained cross-sectional views. Whiletrue septate junctions do therefore occur in the phylum Porifera, little is known oftheir detailed structure.

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Invertebrate septate junctions and phytogeny 283

It is of note, however, that other occluding systems are seen in the Porifera in-cluding a 'parallel membrane' junction (Green & Bergquist, 1978). In this case thecontributing cells are between 10 and 20 nm apart depending upon the species, overlong distances, but there is no evidence of septa or any other organized intercellularmodification maintaining the regular intercellular spacing (Fig. 2). This simple junc-tion is possibly important when considering the development of the septate, andultimately the tight junctions, of the vertebrates.

The phylum Coelenterata. Three variations of the septate junction have been foundin the Coelenterata and all three are unique to the phylum. In the class Hydrozoa thereis the well known Hydra type septate junction. It has been fully described in the orderHydrida (Danilova, Rokhlenko & Bodryagina, 1969; Filshie & Flower, 1977; Green,1980; Hand & Gobel, 1972; Wood, 1959, 1977; Wood & Kuda, 1980) but also occursin the orders Calyptoblastea (Leik & Kelly, 1970) and Gymnoblastea (Overton, 1963).In the class Anthozoa, 2 variations occur. One of these, the anthozoan gastrodermal(double septum) septate junction, is apparently restricted to endodermal tissue andthe other, the anthozoan epidermal junction is apparently restricted to epidermaltissue. Both have been described fully by Green & Flower (1980).

(i) The Hydra type septate junction: The Hydra type septate junction appears intangential view to have straight septa 8-12 nm wide. Close inspection however revealsthat each septum has a central backbone about 2 nm wide with lateral projections onboth sides. In true tangential views these projections are difficult to discern, but if thejunction is tilted relative to the thin section about an axis within the plane of thesection at right angles to the line of the septa, the central narrow core and the pro-jections are readily seen. The projections are up to 4 nm long with a 3-4 nm centre-to-centre spacing. In some views in which septa are seen from an acute angle, theycan appear to be made of a series of circular structures in a chain. A representativetangential view of this junction revealed by lanthanum tracer impregnation is shownin Fig. 3.

Fig. 1. A cross-sectional view of an invertebrate septate junction. This micrograph ofa Hydra type septate junction clearly shows septa spanning a 15 nm intercellular space,x 132000.

Fig. 2. A simple parallel membrane junction in the sponge Inflatella belli seen in cross-section. The membranes of the.contributing cells are a constant 10 nm apart butscarcely any intercellular material is seen, x 110000.Fig. 3. A tangential view of the Hydra type septate junction following lanthanumimpregnation. The arrow indicates a region in which the 2 nm wide central backboneand the side projections are clearest, x 245000.Fig. 4. The anthozoan epidermal septate junction seen in tangential view followinglanthanum impregnation. This micrograph is of tentacle epidermis of a sea anemone,x 160000.

Fig. 5. A representative tangential view of the anthozoan double-septum septatejunction from the gut of a sea anemone, x 95000.Fig. 6. The lower invertebrate pleated septate junction seen in tangential view afterlanthanum impregnation. Arrows indicate pegs at the apices of pleats. This micro-graph shows the junction in the gut of an annelid worm, x 90000.

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In cross-section, septa are seen to span a 15 nm intercellular space. Most appear assolid bars between the plasma membranes, but in some instances a modification where2 bars cross the septa at right angles near their midpoint is seen. This feature is notconsistently seen and is not considered to be a useful identifying feature.

Freeze-fracture of the Hydra type septate junction reveals particles of irregular sizeand shape on the E face of both fixed and unfixed tissue (Fig. 18). In fixed tissue theP face can also have these particles on it, but usually in both fixed and unfixed tissue,the P face has a series of grooves or pits. In unfixed tissue the particles of the E facecan appear as short rods lying across the direction of the septa.

(ii) The anthozoan epidermal septate junction: In this junction septa appear intangential views as long wavy structures 3-4 nm wide running between cells. Septahave prominent side projections up to 7 nm long and spaced about 7 nm apart alongthe septa. These projections appear clearest when they are present on one side of thesepta only. In some areas, they may be seen arising from both sides of a septum butin these cases they are less well defined. A representative tangential view of thisjunction following tracer impregnation is seen in Fig. 4.

Freeze-fracture reveals rows of closely spaced particles on the P face of both fixedand unfixed tissues. These particles vary in size, but most are 8-9 nm in diameter.The E face is characterized by a series of shallow grooves but these are very fine anddifficult to see.

(iii) The anthozoan double-septum septate junction: The anthozoan double-septum septate junction is quite distinct. In tangential sections the septa appeardouble, the 2 halves separated by 6-7 nm. In many places lateral projections up to6 nm long and spaced at about 7-5 nm along the septa are seen. Wherever the septaare sectioned in such a way that they are tilted within a section about an axis withinthe plane of the section and along the line of the septa, they appear as single narrowstructures. In such cases the side projections appear to be 8-9 nm long. A repre-sentative tangential view of this junction seen after lanthanum impregnation is shownin Fig. 5.

Freeze-fracture replicas of this junction show that the intramembrane particlesapproximate the twin septal arrangement seen in tangential view. On replicas ofunfixed tissue, twin rows of particles are seen on the P face of the junction. These rowsare about 7 nm apart, but the 2 halves of each septum are distinctly different. Onerow has small, 5-6 nm diameter particles, while the other has elongated particles5-6 nm wide and 8-9 nm long. These elongated particles lie at right angles to theline of the septum. The E face has an array of broad shallow grooves, occasionallywith fine cross-striations up to 12 nm long and 7-5 nm apart. Replicas of fixed tissueshow essentially the same features though less clearly. Some distortion appears tooccur during fracturing. The size difference between the twin particle rows is lessevident and in extreme cases the P face particles appear in single broad jumbled rows.The E face has a series of broad shallow grooves.

The lower invertebrate pleated septate junction (The phyla Platyhelminthes, Annelida,Sipunculoidea, Brachiopoda, Nemertina and Bryozoa): The lower invertebratepleated septate junction was first described by Baskin (1976) on the basis of thin-


sectioning work, this study was followed with freeze-fracture work by Welsch &Buchheim (1977). There has been some confusion over defining where this junctionoccurs within the Invertebrata because of its close similarity to a type of pleatedseptate junction which is well known in the molluscs and arthropods. Further, it wasinitially called a polychaete septate junction and later an annelid septate junction.Green (1981a) described this junction fully and differentiated it from the molluscanarthropodan form.

The main features of the lower invertebrate pleated septate junction when seen intangential view are that the septa are pleated and often have side projections fromthe apex of each pleat. The septal backbone is narrow, about 2 nm wide, with theperiodicity of the pleating being between 16 and 22 nm depending upon the angleof a section in relation to the septa. The angle of pleating is about 100-1300. The sideof a pleat is 6-8 nm long with 3-5 nm long projections from the apex of each pleat.Where two or more septa run closely parallel the apices of their pleats often alignand their projections fuse to form hexagonal chambers n - u n m across. A repre-sentative tangential view of this junction following lanthanum impregnation is shownin Fig. 6.

In cross-sectional views septa span a 15-18 nm intercellular space, but the adjacentcell membranes often have a slightly scalloped appearance rather than runningstrictly parallel.

Freeze-fracture replicas of the lower invertebrate pleated septate junction showparticles on the P face of fixed tissue. Particles are irregularly shaped and vary in sizefrom less than 3 nm to about 12 nm wide. They are generally in ragged bands, ratherthan neat rows (Fig. 19). The E face of the junction has a complementary assortmentof pits with the occasional particle (Fig. 19). Replicas of unfixed tissue show particlesare again on the P face but here they are in neat rows and nearly all a standard8—10 nm in diameter; however, some size variation remains. The E face has a seriesof furrows or grooves rather than pits.

The Mollusca and Arthropoda: The molluscs and arthropods both possess the wellknown mollusc-arthropod pleated septate junction (Green, 1981 a; Noirot-Timothee& Noirot, 1980; Staehelin, 1974). In the molluscs this junction is found in all tissues,but in the arthropods it is limited to ectodermal tissues. Arthropod endodermal tissuescharacteristically have a smooth septate junction (Flower & Filshie, 1975; Noirot-Timothee & Noirot, 1980; Skaer et al. 1979). In the divergent groups the Merosto-mata (king crabs) and Pycnogonida (sea spiders), however, variations of the smoothseptate junction pattern are found (Lane & Harrison, 1978; Green, 198 ib).

(i) The mollusc-arthropod pleated septate junction: The mollusc-arthropodpleated septate junction is very similar to the lower invertebrate pleated septatejunction. The septa are narrow (2-3 nm wide) and pleated when seen in tangentialview. The angle of pleating is ico-1300 with a 16-22 nm periodicity. Septa runningclosely parallel can cross-link to form hexagonal chambers, but projections from pleatapices are less obvious than in the lower invertebrate pleated septate junction andoften are not apparent at all. The pleating of this junction appears less pronouncedthan in the lower invertebrate pleated septate junction even though the angle of


pleating and periodicity of pleating in the 2 junctions is the same. The more pro-nounced pleating of the lower invertebrate pleated septate junction is in fact an illusioncreated by the presence of more prominent apex projections. Fig. 7 shows a tangentialview of the mollusc-arthropod pleated septate junction after impregnation withlanthanum.

In cross-section this junction has septa spanning a 15-18 nm intercellular spacewith the contributing membranes often appearing scalloped.

On freeze-fracture replicas of the mollusc-arthropod pleated septate junction, bothfixed and unfixed tissue fractures have particles on the P face. These particles arespaced 16-24 n m aPaft with a 8-12 nm diameter (Fig. 20). The E face has a series offine furrows about 4-6 nm wide. It is difficult to distinguish replicas of this junctionfrom those of unfixed tissue replicas of the lower invertebrate pleated septate junction.

(ii) The smooth septate junction: Tangential views of the arthropod smooth septatejunction reveal broad septa 7-8 nm wide. Septa are an even width, straight-edged andfollow a straight or slightly wavy course between cells. They often have rows of3-5 nm diameter pegs running parallel to the septa. Septa generally appear solid butoccasionally a finer substructure is apparent. A representative tangential view of thisjunction following lanthanum impregnation is given in Fig. 8.

In cross-sectional view, the membranes of the adjacent contributing cells runexactly parallel, separated usually by a 15-18 nm intercellular space; however, aspacing of 11—15 nm has been reported (Noirot-Timothee & Noirot, 1980). The septaappear as clear bars spanning this gap.

Freeze-fracture of the smooth septate junction reveals a series of short rods andparticles on the P face of fixed tissue with a series of prominent grooves on the E face.The E face also has a considerable number of particles. In unfixed tissue the junctionshows a reversal of polarity with the particles on the E face and grooves on the P face.In unfixed tissue the particle separation is more complete, very few particles are seenon the P face. In one instance, however (dipteran Malpighian tubules), Skaer et al.

Figs. 7-12. Representative tangential views of 6 of the invertebrate septate junctionvariations following lanthanum tracer impregnation.

Fig. 7. The mollusc-arthropod pleated septate junction in the hindgut of the crabPetrolisthes elongatus. x 95000.

Fig. 8. The smooth septate junction as seen in the gut of the barnacle Elminiusmodestus. x 105000.

Fig. 9. The merostomatan septate junction in the horseshoe crab Limulus (fromLane & Harrison, 1978). x 220000.

Fig. 10. The pycnogonid septate junction in the Antarctic pycnogonid Ammotheaclausi. x 130000.

Fig. 11. Two views of the chaetognath paired septate junction in the intestine ofSagitta setosa. A. Shows the ' tight' formation of this junction type and B the ' loose'formation, A, X 129000; B, 212500 (from Duvert et al. 1980).

Fig. 12. The hemichordate anastomosing septate junction in Balanoglosms australi-evsis endodermal tissue, x 130000.


(1979) report that they found particles on the E face in both fixed and unfixed tissuereplicas.

(iii) The merostomatan septate junction: In the horseshoe crab Limulus midgut avariation of the smooth septate junction occurs (Lane & Harrison, 1978). Tangentialviews of this junction reveal broad, 5-7 nm wide septa that are predominantlyarranged in stacks of differing lengths. Septa are relatively short and close examinationshows them to be composed of small subunits. Spaces between septa have stain-occluding pegs about 3-4 nm in diameter, which can appear circular, less regular orhollow. These pegs do not occur in a single row as they do in conjunction with thesmooth septate junction. Fig. 9 shows a representative view of the merostomatanseptate junction when seen tangentially after lanthanum impregnation.

Freeze-fracture of Limulus tissue reveals junctional structures that are less obviousthan those of the arthropod smooth septate junction. In both fixed and unfixedtissues, particles appear on the E face, while the P face junctional structures consistmainly of grooves with the occasional particle still adhering. The particles of the Eface are slightly elliptical with their long axis across the rows. As seen in tangentialintermembrane views, the intermembrane arrangement is in short stacks of septa.

(iv) The pycnogonid septate junction: Within the Pycnogonida a further variation ofthe smooth septate junction has been described by Green (19816). Its main featurewhen seen in tangential view is that it has broad 5-6 nm wide septa that follow a wavycourse between cells. These septa span a 15 nm intercellular space. Close examina-tion shows that septa have a subunit construction often appearing cross-striated oreven to form delicate chain-like structures. Septa often have 2-4 nm diameter pegsin a single row between them. Fig. 10 shows this junction in tangential view followinglanthanum impregnation.

Freeze-fracture replicas of fixed tissues reveal particles predominantly on the E face.Particles vary in width from 3-14 nm wide and are up to 22 nm long. Like those ofthe Limulus septate junction, they have a low profile and normal 8-10 nm diametermembrane particles stand out clearly in comparison. The P face of the junction hasgrooves in most places and these are relatively prominent compared with the size ofthe E face particles. In some regions of the P face, however, it is difficult to discernany obvious structures and in these areas junctional structures appear to consist ofoccasional indistinct particles with small pits between. This junction has not beenstudied in unfixed tissue.

The phylum Chaetognatha: Studies of the intestine of Sagitta setosa (Chaetognatha)by Duvert et al. (1980) have revealed a paired septate junction in this species. Tan-gential views show septa, generally in pairs, about 3 nm wide. These septa run in a' loose' formation or a ' tight' formation and appear pleated with a periodicity of about11-14 nm. In the loose formation septa are spaced, with small rings, short segmentsor a network structure between them. The tight formation, less frequently seen thanthe loose formation, has paired septa running closely together with no other elementsbetween them. The pleating of the septa in these tight pairs is out of phase giving thema twisted appearance. Occasionally, unpaired septa are seen associated with the tightformation. The tight and loose formations are continuous, with septa in the tight


formation flaring apart to form a loose formation. Representative tangential views ofthis junction following lanthanum impregnation are seen in Fig. 11.

From positive stained cross-sections Duvert et al. (1980) report that this junctionhas an intercellular spacing of about 12 nm, slightly less than the usual 15-18 nmseptate junction spacing. Our own measurements of their micrographs, however,indicate a slightly wider result (15-18 nm) and clearly there is a small variance ininterpretation of where the septa start and finish in relation to the outer leaflets of thecontributing plasma membranes.

Freeze-fracturing of fixed chaetognath tissue shows ridges on the P face with 10 nmdiameter particles in rows on the top. Between these ridges no particles are found.On the E face there are furrows, with 10 nm diameter particles in the bottom. In thisseptate junction, therefore, particles are apparently distributed on both faces. Nounfixed chaetognath tissue has been freeze-fractured.

The chaetognath paired septate junction coexists in some areas with a pleatedseptate junction (periodicity approximately 18-5-24 nm) although little informationis as yet available on this second chaetognath junction (Duvert et al. 1980).

The phylum Hemichordata: In the hemichordates 2 further variations of septatejunction have been found. One of these in tangential views appears complicated andsemi-anastomosing, while the second type is a parallel array, double-septum, septatejunction. Both have septa spanning a 15 nm intercellular space and are described byGreen (1981c) as they occur in Balanoglossus australiensis.

(i) The hemichrodate anastomosing septate junction: This junction has been foundin tissues that are of endodermal derivation. In tangential view septa are seen toconsist of a complicated arrangement of circles, parts of circles or short rods. Thesestructures are often linked so that considerable areas of junction can consist of onelarge complex piece of septum. When the septal circles are complete, they usuallyhave a central core that ranges from less than 3 to about 12 nm in diameter. Thecircles themselves have a relatively constant inside diameter of 9-20 nm with 6—10 nmwide walls. Septa often have short bars off the side and have a rough-edged, semi-pleated appearance. Fig. 12 shows a representative tangential view of this junctionfollowing lanthanum impregnation.

Freeze-fracture replicas of the hemichordate anastomosing septate junction in fixedtissue reveal a dense array of particles on the P face (Fig. 21). The particles are aregular size and shape, being rounded and mostly 7-8 nm in diameter. The E faceof the junction has a complementary array of pits with the occasional particle leftadhering. No unfixed tissue of this junction type has been studied.

(ii) The hemichordate double-septum septate junction: The hemichordate double-septum septate junction has been found only in epidermal tissue of Balanoglossus.In tangential views septa 2-5 nm wide are seen to occur in 2 configurations, either aparallel arrangement in which short straight septa are seen in groups of 3-8, or apaired arrangement. Despite the fact that there may be an odd number of septa inthe arrays, they are almost inevitably paired when not in that configuration. Thepaired septa have a slight undulating gap of 3-6 nm between them, though this maybe less where the twin septa have 'pinched' together. In other places the halves of

2Q0 C. R. Green and P. R. Bergquist

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a septal pair can flex apart. Close examination shows that septa, which generally have asolid line appearance, have a substructure and consist of rows of closely apposedparticles. Fig. 13 shows a representative tangential view of this junction followinglanthanum impregnation.

No freeze-fracture replicas have been made of this type of junction.The phylum Echinodermata: The Echinodermata, like the Hemichordata, have

2 types of septate junction. One of these, the echinoderm anastomosing septatejunction, has been described fully by Green et al. (1979) and the other, the echino-derm double-septum septate junction, by Green (1981^). The echinoderm anasto-mosing junction appears to be confined to endodermal tissue, the double-septumjunction to epidermal tissue. Both types have septa spanning a 15 nm intercellularspace.

(i) The echinoderm anastomosing septate junction: When seen in tangential viewthe echinoderm anastomosing septate junction has septa in a configuration that isremarkably similar to that of the vertebrate tight junction (Fig. 24). The anastomosingcompartments are, on average, about 30—35 nm wide and the septa 6—8 nm wide.The septa have a rough-edged appearance and appear semi-pleated in a similarfashion to the septa of the hemichordate anastomosing septate junction. In placesshort branches arise from the septa. Fig. 14 shows a representative tangential view ofthis junction following lanthanum impregnation.

In freeze-fracture of fixed material the junction appears as a pattern of 7-8 nmdiameter particles on the P face (Fig. 22). The particles are rounded in shape and ofeven size. They occur in an anastomosing pattern, although this may not be obviouswithout a prior knowledge of the intermembrane septal pattern. The E face has anoccasional particle left adhering within a fine network of shallow pits and shortgrooves. Freeze-fracture of unfixed tissue reveals an almost identical pattern with

Figs. 13-15. Representative tangential views of 3 invertebrate septate junctionsfollowing lanthanum impregnation.

Fig. 13. The hemichordate double-septum septate junction in epidermal tissue ofB. australiensis. The single arrow indicates the parallel septal array of this junctionand the double arrow the double-septal configuration, x 50000.

Fig. 14. The echinoderm anastomosing septate junction as seen in the pyloriccaecum of a starfish, x 120000.

Fig. 15. The echinoderm double-septum septate junction in the tube foot epi-thelium of a starfish, x 140000..Fig. 16. A positively stained cross-sectional view of the tight junction in the tuni-cate Asterocarpa coerulea. The contributing membranes fuse at intervals to form aseries of membrane fusions, x 130000.Fig. 17. A tangential view of the tunicate tight junction following lanthanumimpregnation. This micrograph, from branchial basket tissue of the tunicate A. coeru-lea, shows an anastomosing pattern where membrane fusions have presumablyexcluded the tracer, x 60000.Fig. 18. A freeze-fracture replica of the E face of a Hydra type septate junction inChlorohydra viridissima. x 70000.


particles on the P face and pits on the E face. However, fewer particles are leftadhering to the E face and the pits are more distinct.

(ii) The echinoderm double-9eptum septate junction: In cross-section followinglanthanum impregnation this junction is seen to have double septa. In tangential viewsepta appear straight, but double with each half varying between 2 and 5 nm wide.The 2 halves can be touching or have a gap between them of up to 3 nm. As a resultthe total septal width varies between 77 and 12 nm. Septa generally follow a courseapproximately parallel to each cell apex, but can become less orientated at the lowerend of the junction, that is, the end away from the lumen. Fig. 15 shows this junctionin tangential view following lanthanum impregnation.

Freeze-fracture replicas of this junction are unique (Green, io,8ii). Replicas offixed tissue show a random array of irregularly sized particles in a 0-8-2-5 / t m wideband on the E face. The P face is characterized by a randomly dispersed band ofshallow pits with the occasional particle left adhering. The E face particles are5-20 nm in length, but those left adhering to the P face are 5-15 nm long. In contrast,replicas of unfixed tissue without cryoprotection reveal prominent particles on theE face, this time aligned in rows as might be expected after viewing septa in tangentialthin section views. These particles vary between 6 and 25 nm in length. The P facehas a series of prominent grooves.

The phylum Tunicata: The tunicates do not have a septate junction, but rather avariation of the vertebrate tight junction (Georges, 1979; Green, 1980; Lorber &Rayns, 1972). In this case there is no intermembrane space with septa, but instead aseries of fusion points of the adjacent plasma membranes of the contributing cells areseen in cros9-sectional views (Fig. 16). In tangential views following lanthanumimpregnation, a typical tight junction anastomosing pattern is seen (Green, 1980).Fig. 17 shows a representative view of this pattern.

Freeze-fracture of the junction reveals an anastomosing network of junctionalstructures. The E face of fixed tissue has a series of particles 5-25 nm long (Fig. 23),

Figs. 19-24. Freeze-fracture replicas of 5 septate junction variations and 2 tightjunction variations. All replicas are of glutaraldehyde-fixed tissue.

Fig. 19. The lower invertebrate pleated septate junctions showing both the P and Efaces. This replica is of polychaete gut tissue, x 55000.

Fig. 20. The P face of the mollusc-arthropod pleated septate in the gastropodNerita melanotragus. x 80000.

Fig. 21. The hemichordate anastomosing septate junction in J3. australicnsis tissue.This micrograph shows the P face, x 95 000.

Fig. 22. The P face of the echinoderm anastomosing septate junction in the pyloriccaecum of a starfish, x 95000.

Fig. 23. The tunicate tight junction is Asterocarpa coerulea. This replica of the Eface shows the anastomosing pattern and distinct particle nature of this junction,x 95000.

Fig. 24. The vertebrate tight junction in mouse liver following fixation. The P facehas a series of distinct ridges and the E face a complementary pattern of grooves,x 90000 (micrograph provided by Dr S. Bullivant).

294 C- R- Green and P. R. Bergquist

while the P face is characterized by a complementary network of shallow pits. Unfixedtissue again has an anastomosing pattern of E face particles, but has distinct grooveson the P face. In unfixed tissue the particles are so close together that long stretchesof rough-topped ridges are formed. In one species however, Georges reports that heobserved reversed polarity of these features at least in fixed tissue. In contrast to theseptate junction pattern, changes in face of a freeze-fracture take place along lines offusion as they do in other vertebrate tight junctions.

This junction differs from the usual vertebrate tight junction pattern (Bullivant,1978; Staehelin, 1973) in 3 ways. In most cases the particles are on the E face of bothfixed and unfixed tissue, this stands in contrast to the normal tight junction patternwhere in fixed tissue particles are on the P face. Further, the E face particles arealways discrete and do not form continuous ridges as they do on replicas of the usualfixed tissue vertebrate tight junction (Fig. 24). This latter feature is normally a featureof unfixed tissue for all other tight junctions. Finally, the junction is leaky to lanthanumtracer (Green, 1980) with no pretreatment. Other tight junctions are generallyimpermeable to this tracer, although Goodenough & Revel (1970) were able to getlanthanum into mouse liver tight junctions after pretreating with acetone.

DISCUSSION

The function of the septate junction

The function of the septate junction remains open to discussion. It is not thepurpose of this article to discuss this in any detail as 2 recent reviews of arthropodjunctions by Noirot-Timothee & Noirot (1980) and Lane & Skear (1980) do this.However, within the context of the following discussion on junction relationships anddevelopment we clearly favour an occluding function for the septate junction analogousto that of the vertebrate tight junction. The more obvious major role for the septatejunction must be that of occlusion, when their analogous position to that of the tightjunction, around the apical edge of cells lining an epithelium, is noted in conjunctionwith the fact that there are no other structures present to fulfil this role in the vastmajority of invertebrate tissues.

Readers should, however, note that while this role of an occlusion barrier isfavoured by many workers (Filshie & Flower, 1977; Green et al. 1979; Noirot-Timothee & Noirot, 1980; Wood & Kuda, 1980), others have suggested that septatejunctions may have a different role (Lane & Chandler, 1980; see reviews by Lane &Skaer, 1980; Noirot-Timothee & Noirot, 1980).

^functional relationships

Relationships between the various types of junction are conveniently discussedunder 3 headings: Coelenterates and lower invertebrates, Proterostomia (the annelid,molluscan and arthropod lineage) and the Deuterostomia (the echinoderm andchordate lineage). The deuterostomes and proterostomes are considered to havearisen independently from a common coelomate ancestor (Hyman, 1940). The phylawithin each group and the junction type characteristic of each phylum are indicated


in Table 3. Tables 1 and 2 summarize the main features of the 13 invertebrate septatejunctions, the tunicate tight junction and the usual vertebrate tight junction. Fig. 25summarizes the phylogenetic relationships implied by comparison of junctionalstructures.

PycnogonidaMerostomata

Brachiopoda

Annelida

Platyhelminthes

Coelenterata Hydrozoa Anthozoa

(The lengths of the linking lines have no significance)

Fig. 25. A diagrammatic representation of the phylogenetic relationships implied inthis discussion.

The Coelenterates and lower invertebrates. In the phylum Coelenterata all 3 septatejunction forms, the Hydra type and the 2 anemone junctions, have straight septa withside projections. However, the triploblastic invertebrate phyla immediately above thecoelenterates in the structural hierarchy, which possess the lower invertebrate pleatedseptate junction, have a junctional configuration that compares more closely to theHydra type than to either of the anemone types. Both the Hydra type junction andthe lower invertebrate pleated septate junction have narrow (2-3 nm wide) centralbackbone septa with fine side projections (compare Figs. 3 and 6) and both haveparticles that are irregular in shape and size as revealed by freeze-fracture of fixedand unfixed tissues (compare Figs. 18 and 19). In contrast the broad septa of the2 anemone septate junctions and their more rounded and regularly shaped freeze-fracture particle appearance is quite distinct. In addition the type of evenly spaced,

Tab

le I

. Su

mm

ary

of t

he fr

eeze

-fra

ctur

e ch

arac

teri

stic

s of

the

I 3

typ

es o

f in

vert

ebra

te s

epta

te ju

nctio

n, t

he t

unic

ate

tight

junc

tion

and

the

vert

ebra

te ti

ght j

unct

ion

Fix

ed t

issu

e U

nfix

ed t

issu

e r

A

\

r-

A

\

P f

ace

E f

ace

P f

ace

E f

ace

Hy

dra

typ

e se

pta

te

junc

tion

Ane

mon

e ep

ithe

lial

se

pta

te ju

ncti

on

Ane

mon

e do

uble

- se

ptat

e ju

ncti

on

Lo

wer

inv

erte

brat

e pl

eate

d se

ptat

e ju

ncti

on

Mol

lusc

-art

hrop

od

plea

ted

sept

ate

junc

tion

Sm

oo

th s

epta

te j

unct

ion

Lir

nulu

s se

ptat

e ju

ncti

on

Pyc

nogo

nid

sept

ate

jun

ctio

n

Mos

tly

groo

ves

or p

its;

so

me

part

icle

s

Row

s of

clo

sely

spa

ced,

ev

enly

siz

ed p

arti

cles

Tw

in o

r b

road

jum

bled

ro

ws

of p

arti

cles

Rag

ged

part

icle

s of

va

ryin

g si

ze a

nd s

hap

e in

ban

ds

rath

er t

han

ro

ws

Ro

un

ded

par

ticl

es o

f ev

en s

ize

and

sha

pe

Mai

nly

part

icle

s o

r ro

ds

Gro

oves

Pro

min

ent

groo

ves

or

indi

stin

ct p

arti

cles

an

d

pits

Mai

nly

part

icle

s of

G

roo

ves

or

pit

s ir

regu

lar

size

an

d s

hap

e;

som

e gr

oove

s o

r p

its

Fin

e sh

allo

w g

roov

es

Bro

ad s

hall

ow g

roov

es

Pit

s of

var

ying

siz

e an

d

shap

e in

ban

ds

Gro

oves

or

pit

s

Mai

nly

groo

ves;

so

me

part

icle

s

Row

s of

par

ticl

es,

oft

en

indi

stin

ct,

or

stac

ks o

f sh

ort

par

ticl

e ro

ws

Indi

stin

ct p

arti

cles

Row

s of

clo

sely

spa

ced

part

icle

s of

eve

n si

ze

Tw

in r

ows

of p

arti

cles

; o

ne

row

of

rou

nd

ed p

ar-

ticl

es,

the

oth

er r

ow w

ith

pa

rtic

les

elon

gate

d at

ri

gh

t an

gles

to

th

e li

ne

of t

he

sep

ta

Eve

nly

size

d an

d s

hap

ed

par

ticl

es;

occa

sion

al

smal

l o

r la

rge

part

icle

s

Eve

nly

size

d an

d s

hap

ed

part

icle

s

Gro

ov

es

No

res

ult

s av

aila

ble

No

res

ult

s av

aila

ble

Mai

nly

par

ticl

es o

f ir

reg

ula

r si

ze a

nd

sh

ape;

so

me

sho

rt r

od

s ly

ing

acro

ss t

he

sep

ta

Fin

e sh

allo

w g

roov

es

Bro

ad s

hall

ow g

roov

es

Dis

tin

ct g

roov

es o

r fu

rro

ws

Gro

ov

es o

r p

its

Clo

sely

ap

po

sed

par

ticl

es

No

res

ults

ava

ilab

le

No

res

ult

s av

aila

ble

Tab

le I

(c

ont.

)

Cha

etog

nath

pai

red

sept

ate

junc

tion

Hem

icho

rdat

e do

uble

- se

ptu

m s

epta

te ju

ncti

on

Hem

icho

rdat

e an

asto

- m

osin

g se

ptat

e ju

ncti

on

Ech

inod

erm

dou

ble-

se

ptu

m s

epta

te ju

ncti

on

Ech

inod

erm

ana

sto-

m

osin

g se

ptat

e ju

ncti

on

Tu

nic

ate

tigh

t ju

ncti

on

Ver

tebr

ate

tigh

t ju

ncti

on

(Sta

ehel

in,

1973

)

Rid

ges

wit

h ro

ws

of

part

icle

s on

top

No

res

ults

ava

ilab

le

Den

se a

rray

of

even

ly

size

d an

d sh

aped

pa

rtic

les

Ran

dom

arr

ay o

f pi

ts i

n

in a

bro

ad b

and

aro

und

cell

s; s

ome

part

icle

s of

va

ryin

g si

ze

Eve

nly

size

d an

d s

hape

d pa

rtic

les

in a

n an

asto

- m

osin

g pa

tter

n

Ana

stom

osin

g ne

twor

k of

sh

allo

w p

its

Ana

stom

osin

g ne

twor

k of

co

ntin

uous

rid

ges

Gro

oves

wit

h ro

ws

of

part

icle

s at

th

e bo

ttom

No

res

ults

ava

ilab

le

Den

se a

rray

of

pits

, oc

casi

onal

par

ticl

e

Wid

e sc

atte

red

arra

y of

ir

regu

larl

y sh

aped

par

- ti

cles

in

a b

road

ban

d ar

ou

nd

cel

ls

Ana

stom

osin

g ne

twor

k of

pi

ts

Ro

un

ded

par

ticl

es a

nd

sh

ort

rod

s in

an

anas

to-

mos

ing

netw

ork

Ana

stom

osin

g ne

twor

k of

gr

oove

s

No

res

ults

ava

ilab

le

No

res

ults

ava

ilab

le

No

res

ults

ava

ilab

le

Pro

min

ent

groo

ves

Eve

nly

size

d an

d s

hape

d pa

rtic

les

in a

n a

nast

o-

mos

ing

netw

ork

Ana

stom

osin

g ne

twor

k of

gr

oove

s

Ana

stom

osin

g ne

twor

k of

gr

oove

s, s

ome

part

icle

s o

r ro

ds

No

res

ults

ava

ilab

le

No

res

ults

ava

ilab

le

No

res

ults

ava

ilab

le

Pro

min

ent

part

icle

s of

va

ryin

g si

ze a

lign

ed i

n

row

s

Ana

stom

osin

g ne

twor

k of

pi

ts

Par

ticl

es o

r sh

ort

rod

s in

an

asto

mos

ing

netw

ork

Ana

stom

osin

g ne

twor

k of

pa

rtic

les


pegged, double septum seen in the anemone double-septum septate junction iscertainly not apparent in any higher invertebrate groups. The main difference be-tween the Hydra type septate junction and the lower invertebrate pleated septatejunction lies in the pleating of the lower invertebrate junction and the fact that these2 junctions fracture with the majority of particles on opposite faces in both fixed andunfixed tissue (usually the E face for the Hydra type, the P face for the lower in-vertebrate pleated septate junction).

Table 2. Summary of the main thin-section tangential view featuresof the 13 types of invertebrate septate junction

Hydra type septate junction

Anemone epithelial septate junc-tion

Anemone double-septum septatejunction

Lower invertebrate pleated sep-tate junction

Mollusc-arthropod pleated sep-tate junction

Smooth septate junctionLimulus septate junction

Pycnogonid septate junction

Chaetognath paired septate junc-tion

Hemichordate double-septumseptate junction

Hemichordate anastomosing sep-tate junction

Echinoderm double-septum sep-tate junction

Echinoderm anastomosing sep-tate junction

Narrow, straight backbone septa with fine projectionsfrom both sides

Wide, straight septa with prominent projections,clearest when off one side only

Wide, straight, double septa with component halvesevenly spaced; side projections

Narrow backbone, pleated septa with fine projectionsfrom the apex of each pleat

Narrow backbone, pleated septa

Broad, straight septa, often with pegs between themBroad, straight septa; irregularly shaped pegs betweenthe septa, which often have a substructure appearance

Broad wavey septa with pegs between, striated orchainlike structure

Narrow, double septa, pleated with narrow-spacedand wide-spaced configurations

Narrow, paired septa or stacks of 3 to 8 septa; septaoften have a substructure appearance

Wide, anastomosing septa with rough-edged appearance;anastomosing pattern of circles, parts of circles androds

Straight, double septa of irregular width and withuneven spacing between the component halves

Wide, anastomosing septa with rough-edged appear-ance and short side branches; anastomosing patternlike that of the vertebrate tight junction

The lower invertebrate pleated septate junction is common to many phyla andoccurs in groups that fall into both deuterostome and proterostome lineages. Thephyla that have this type of junction include the Annelida which are on the protero-stome line, but it also includes the Sipunculoidea, which are claimed by some workersto have given rise to the echinoderms (Nichols, 1967), that is to be on the deuterostomeline. Other phyla so far found to have the lower invertebrate pleated septate junctionare the Platyhelminthes, Bryozoa, Nemertina and Brachiopoda (Green, 1981a).

It appears therefore that on the basis of septate junction structure the hydrozoan


Table 3. Invertebrate classification as used in this paper, in relation to thedistribution of septate junction types

— PORIFERA

— COELENTERATA Hydra type septate junction ,Anemone double-septum septate junction |Anemone epithelial septate junction .

— PLATYHELMINTHES

•NEMERTINA

- BRYOZOA Lower invertebrate pleated septate junction

-ANNELIDA

(Deuterostomia)

- SIPUNCULOIDEA

— CHAETOGNATHA

Chaetognath double-septum septatejunction

• ECHINODERMATAEchinoderm double-septum septatejunction

Echinoderm anastomosingseptate junction

(Protero8tomia)

— BRACHIOPODA

— MOLLUSCA

-HEMICHORDATAHemichordate double-septum septate Ijunction

Hemichordate anastomosingseptate junction

-TUNICATA "]Tunicate tight junction 1

L— CHORDATA

Mollusc-arthropod pleatedseptate junction

ARTHROPODA

Smooth septate junction

PYCNOGONIDAPycnogonid

andMEROSTOMATA Limulus

septate junction

"I


coelenterates have more affinity with platyhelminthes than do the Anthozoa. Fromthe platyhelminthes 2 main evolutionary lines diverged. The first of these, the Pro-terostomia, includes the annelids, arthropods and molluscs; the second, the Deutero-stomia, includes the sipunculids, chaetognaths, echinoderms, hemichordates andchordates. The exact relationships of the Bryozoa, Nemertina and Brachiopoda remainquestionable.

The proterostome lineage. Considering the proterostome lineage further, the nextmajor phylum above the annelids on this evolutionary line is the Mollusca, in whicha mollusc-arthropod pleated septate junction is found. This junction, like the lowerinvertebrate pleated septate junction, has a narrow central backbone and is pleated,although side projections are less obvious. The 2 junctions freeze-fracture with themajority of particles on the P face in both fixed and unfixed tissue replicas. The particlesof the mollusc-arthropod septate junction, however, are rounded and regular in sizeand shape, being about 8-10 nm in diameter. The mollusc-arthropod pleated septatejunction also occurs, as its name suggests, in the Arthropoda but in this phylum it isfound only in superficial epithelial tissue. The other junction found in the arthropodsis the smooth septate junction, which is quite distinct from the pleated septatejunction. It has broad straight septa and unfixed tissue most commonly freeze-fractures with the particles on the opposite face to that obtained in fractures of thepleated septate junction. However, the particles, though extended predominantly intoshort rods, are still rounded in shape and about 8-10 nm wide. Two other protero-stome groups, the Merostomata and Pycnogonida, have septate junctions that aresimilar to the smooth septate junction. The septa are straight and broad but have amore obvious substructure and the junctions also differ from the smooth septatejunction in freeze-fracture appearance. Green (19816) also noted that the 'smoothseptate junction' seen by Dallai (1975) in collembolan midgut tissue is very similar toto those seen in the Pycognida and Merostomata. In all 3 of these rather primitivegroups particles are on the E face and appear very indistinct in comparison withparticles of the smooth septate junction. There is however relatively little data avail-able on the collembolan junction.

The deuterostome lineage. Within the Deuterostomia the Chaetognatha are reportedto have both a double-septum septate junction and the pleated septate junction, other-wise characteristic of mollusc and arthropod tissues, occurring in the same tissue(Duvert et al. 1980). However, in this report identification of the pleated septatejunction rests only on thin-section tangential views and it is more than likely that thejunction being viewed is in fact the lower invertebrate pleated septate junction. If thisis the case, the Chaetognatha appear not very far removed from the Sipunculoidea.It is interesting that the Chaetognatha have a paired septate junction as do both thehemichordates and the echinoderms. The septa of the hemichordate double-septumseptate junction are very similar in some views to those of the Chaetognatha in thatthey are similarly paired (compare Figs. 11 and 13). In all of these phyla the doublesepta are made of thin and irregular component parts as compared to the anemonedouble-septum septate junction (compare Figs. 11,13 and 15 with 5). The anastomosingseptate junctions of the Echinodermata and Hemichordata are remarkably similar.


Both have broad septa with rough edges and side branches (compare Figs. 12 and 14)and both have rounded particles revealed on the P face of fixed tissue freeze-fracturereplicas (compare Figs. 21 and 22). The only real difference is in the final configura-tion that the septa take. In this feature the anastomosing network of the echinodermseptate junction appears closer to the tunicate tight junction (compare Fig. 14 with17 and 24) than does the hemichordate junction. On this basis it is not unreasonableto consider the hemichordates as a branch divergent from the main line of deutero-stome evolution. Certainly the occurrence in the Echinodermata of a structure inter-mediate between the invertebrate septate junction and the vertebrate tight junctionreinforces suggestions of a close and possibly direct relationship between this phylumand the phylum Chordata (Nichols, 1967).

The particle structures seen in freeze-fracture replicas of the echinoderm and hemi-chordate anastomosing septate junctions are very similar to those of the mollusc-arthropod pleated septate junction (compare Figs. 21 and 22 with 20). All of thesejunctions fracture with the particles on the same face in both fixed and unfixed tissue(the situation in unfixed hemichordate tissue is yet to be studied) and all have rounded8-10 nm diameter particles. This may be a conservative feature thus reflecting acommon remote ancestry among the phyla that possess the lower invertebrate pleatedseptate junction and consequently have irregularly shaped junctional particles on theP face.

In the tunicates a true tight junction occurs, but it usually fractures with particlesadhering to the E face in fixed and unfixed tissue; the usual tight junction pattern isthat fixed tissue particles adhere to the P face (Staehelin, 1973). In addition, intunicates the junctional structures consist of short rods and particles rather thancontinuous ridges (compare Figs. 23 and 24) and in this feature recall a septatejunction rather than a tight junction. The particles seen on the E face of fixed tissuetunicate replicas are similar to those seen on the P face of echinoderm anastomosingseptate junction replicas though they are closer together (compare Figs. 22 and 23).This junction is also apparently 'leakier' than tight junctions of similar strandcomplexity.

Theory on occluding junction development

On the basis of the above results, a sequence of occluding junction development canbe traced from the lower invertebrates through to the chordate lineage. The lowerinvertebrates have the lower invertebrate pleated septate junction and in the Chaeto-gnatha it is probably this junction that is seen to coexist with the chaetognath pairedseptate junction. The latter is similar to the double-septum septate junctions of theHemichordata and Echinodermata. The echinoderms are of special interest; they havean anastomosing septate junction very similar in pattern to the anastomosing seen intight junctions. However, the echinoderm junction retains a 15 nm intercellularspace, which is not a feature of tight junctions. With regard to this difference inspacing, the Porifera are known to have a simple parallel membrane type junction,which appears to serve an occluding function (Green & Bergquist, 1978; see Fig. 2).The sponge junction has extremely regular intercellular spacing in the absence of any


septa; the intercellular spacing is obviously controlled by some other factor. Thisraises the possibility that the septate junction could be a modification of a simple occlud-ing junction such as is seen in the sponges, and that in such a situation the septa evolvedin response to a need to partition the intercellular space and thus to provide a moreeffective seal. Such a proposal implies that in the septate junction the 15-18 nm inter-cellular space is not dictated by the septa but by a separate spacing factor. When thediversity of septal types is considered and contrasted with the stability of the 15-18 nmintercellular spacing, it is possible to consider such a sequence in the evolution of aseptate junction. The loss of the intercellular cleft, which would be involved in achange from the septate to the tight junction, would then simply involve a modificationof this 'membrane spacing factor'. The membranes of the adjacent cells could thencome together and the intramembrane junctional components protruding from themembrane, previously insertion points of the intercellular septa, could fuse in a tight-junction-like structure. This would involve a change from a long 'in register' joint toa short ' side-by-side' joint which fits the supposed structure of the vertebrate tightjunction (Bullivant, 1978). The appearance of a paniculate structure seen on freeze-fracture replicas of the tunicate tight junction fits this pattern almost exactly. It is onlyin the Chordata that further components are added to the junction to form the morecontinuous, and presumably more efficient, ridges of the usual vertebrate tightjunction.

Tight junctions in invertebrates

Several papers have reported 'tight junctions' as occurring in certain arthropodtissues (see review by Lane & Skaer, 1980). For example, Lane, Skaer & Swales (1977)and Lane (1978) reported a form of tight junction in some insect central nervoussystems and Lane (1979) also reported a similar junction in cockroach rectal pads.Furthermore, Toshimori, Iwashita & Oura (1979) described a type of tight junctionin the cyst envelope of the silkworm testis. All the structures described in these papersare similar, with ridges of closely packed particles on the P face of membranes andgrooves on the E face. They do not, however, form extensive anastomosing arrays andthe ridges are rarely very long; the junction consists of short narrow networks. Noevidence is available to show that a change of face occurs along the lines of fusionduring freeze-fracturing as it does in true tight junctions (Bullivant, 1978). Otherworkers believe that these structures are not true tight junctions and find it difficult tobelieve that they might have an occluding function (Green et al. 1979; Noirot-Timothee & Noirot, 1980; Wood, 1977). However, a recent paper by Lane & Chandler(1980) clearly demonstrates the existence of what appear to be true tight junctions inthe central nervous system of arachnids. It is possible, therefore, in the context of thisarticle, that both the proterostome and deuterostome lineages have evolved parallelforms of the tight junction. It is necessary to stress, however, that the tight-j unction-like structures of the proterostomes only occur in a few specialized arthropodtissues (Lane & Skaer, 1980) and have never been located to date in any otherinvertebrate group lower than the tunicates. This is significant when discussing thesupposed function of invertebrate septate junctions, as Lane & Chandler (1980)


believe that their role is not one of an occlusion barrier as has been suggested bysome other workers in the field (for example, Filshie & Flower, 1977; Flower &Filshie, 1975; Georges, 1989; Green et al. 1979; Lord & DiBona, 1976; Noirot -Timothee & Noirot, 1980; Noirot-Timothee, Smith, Cayer & Noirot, 1978; Wood,1977). They suggest that because tight junctions have been found in arthropod tissues,then the septate junction can no longer be considered the occluding 'invertebrateequivalent of the vertebrate tight junction'. Certainly it would appear that they arecorrect and the septate junction probably cannot be considered the only invertebrateoccluding structure, but Lane & Chandler do not explain what junction type fulfilsthe necessary role of a permeability barrier in invertebrate tissues other than the fewspecialized arthropod tissues. Lane & Chandler also feel that the differences in inter-cellular spacing between the invertebrate septate junction and the vertebrate tightjunction make it unlikely that septate junctions are evolutionary forerunners ofvertebrate tight junctions as was suggested by Green et al. (1979). In this major pointalso they may well prove correct, but we believe that in this communication we haveproposed a theory that could explain how such a change in spacing might occur.Isolation and biochemical comparisons of septate and tight junctions may well proveinformative in resolving this question.

We wish to thank Dr N. J. Lane and Dr M. Duvert for supplying us with photographs oftheir work, the Limulus and Chaetognatha septate junctions, respectively. We also thank Dr S.Bullivant for the vertebrate tight junction photograph. In addition, we thank Dr Bullivant forhis assistance in this work and preparing the manuscript and Mr G. Grayston and Ms G. Ableyfor technical assistance.

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(Received 29 June 1981)

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