THE ANATOMICAL RECORD 200:253-258 (1981) 7/ Unusually Complex Basement Membranes in the Midgut of Two Decapod Crustaceans, the Stone Crab (Menippe mercenaria) and the Lobster (Homarus americanus) JAN ROBERT FACTOR Smithsonian Institution, Ft. Pierce Bureau, Ft. Pierce, Florida 33450 ABSTRACT UltrastructuraJ studies of the stone crab (Menippe mercenaria) and the lobster [Homarus americanus) demonstrate that the basement membrane of the midgut (intestine) is unusually complex. In both species, the basement membrane is three-layered and has processes that form extensive networks pro- truding into the connective tissue. The possible functional significance of this complex structure is discussed. The basement membrane underlying epithe- lial tissues is generally considered to be a con- tinuous, electron-dense, extracellular sheet which ranges in thickness from 200 to 50,000 A and often appears fibrous or flocculent. When viewed at low magnifications, it has been variously described in textbooks as "amorphous" (Threadgold, 76) or "homogene- ous" with poorly defined inner and outer limits (Fawcett, '66). Closer examination shows that this layer may be a mat or meshwork of fine fil- aments embedded in an amorphous matrix (Bloom and Fawcett, 75). The basement mem- brane (also commonly referred to as the basal lamina or lamina densa) often appears to be separated from the epithelial cells by an inter- vening transparent layer (the lamina rara). The structure, function, and biochemistry of the basement membrane have recently been re- viewed by Kefalides et al. (79). Several elaborations of this basic plan have been reported. The basement membrane of ver- tebrate skin may be connected to the underly- ing connective tissue by anchoring fibrils em- bedded in this tissue (Briggaman et al., 71). Additionally, Fox (76) has shown that larger anchoring fibers may be composed of a varia- ble number of fibrils in the skin of several aquatic vertebrates. Among the invertebrates, several authors have described unusual struc- tural patterns for the basement membrane of the midgut in a variety of insects (Terzakis, '67; Richards and Richards, '68; Gouranton, 70; Holter, 70; Reinhardt and Hecker, 73; Hess and Pinnock, 75; Bayon and Francois, 76). These substructural variations usually in- clude cylinders or grid-like patterns, some- times composed of hexagonal units. Ultrastructural studies of the connective tis- sue layer surrounding the digestive epithelium in two decapod crustaceans, the stone crab Menippe mercenaria (Brachyura: Xanthidae) and the lobster Homarus americanus (Macrura: Nephropidae), demonstrate that the basement membrane of the adult midgut (intestine) is unusually complex. This is the first description of a complex basement membrane in this major group of invertebrates. MATERIALS AND METHODS The stone crabs used in this study were trapped in the Indian River coastal lagoon of Florida and the American lobsters were ob- tained from commerical sources. The digestive tissues of both species were excised from living animals, fixed for light microscopy in seawater Bouin's fluid, and embedded in Paraplast. Sec- tions were stained with Harris's hematoxylin and eosin, Mallory's triple connective tissue stain, 1% alcoholic aniline blue and picric acid- acid fuchsin, and periodic acid-Schiff s reagent (Humason, '62). Tissues were fixed for electron microscopy in 3% glutaraldehydein 0.2M sodi- um cacodylate buffer at pH 7.0. The buffer con- tained 30 mg/ml NaCl and 20 /tg/ml CaCL Fix- ation was carried out at 6?C for 3-6 hours and was followed by 5 washes in solutions of buffer with decreasing concentrations of salts. Speci- Received December 2. 1980; accepted December 19. 1980. 0003-276X/81/2003-0253S02.00 1981 ALAN R LISS. INC. 254 JAN ROBERT FACTOR mens were post-fixed in 1.0% OsO, in buffer for 5 hours, treated with 2% uranyl acetate, dehy- drated in ethanol which was replaced by propy- lene oxide, and embedded in a mixture of Epon 812-Araldite 506. Thin sections were stained with uranyl acetate and lead citrate. OBSERVATIONS The basement membrane of the midgut of the lobster is illustrated in Figures 1-3. When viewed with the light microscope (Fig. 1) it can be seen to be a wavy structure which follows the folding of the simple, columnar epithelium of the midgut and separates it from the under- lying connective tissue. The basement mem- brane stains positively with PAS and aniline blue and is slightly eosinophilic. It varies in thickness from 0.2 to 0.4 /im and can be seen electron-microscopically to be pierced by branching channels (Figs. 2 and 3). The central layer consists of a finely granular, extremely uniform material and is coated on both sides with thin, fibrous, relatively electron-dense outer layers (Fig. 3). In addition, structures which range from roughly circular to elongate in section lie adjacent to the basement mem- brane proper and can be seen to consist of the same finely granular material coated with a similar electron-dense outer layer. Serial sec- tions indicate that the larger of these exten- sions, at least, are processes that extend from the basement membrane. The fibrous outer layer coating the basement membrane and its processes is drawn out into strands which bridge the channels in the membrane and which also interconnect its processes (Fig. 3). The electron-dense strands, together with the processes of the basement membrane, form an extensive network (approximately 0.4 pm thick) along the connective tissue surface of the basement membrane (Fig. 2). Processes of fibroblasts are commonly associated with this network (Fig. 3). A similar network is only in- termittently present on the surface of the base- ment membrane which faces the epithelium. The basement membrane of the midgut of the stone crab is illustrated in Figures 4-6. It is considerably thicker than that of the lobster (approximately 1.5 /tin) and appears to have two layers when viewed with the light micro- scope (Fig. 4). Its staining characteristics are similar to the basement membrane in the mid- gut of the lobster ? it is strongly PAS-positive, strongly aniline blue-positive, and slightly eo- sinophilic. When observed with the electron microscope, the basement membrane is three- layered and does not appear to have channels (Fig. 5). The central layer has a coarse, reticu- lated appearance and is coated on both sides by outer layers of fibrous material (Fig. 6). The outer layer facing the epithelium is a simple, thin, electron-dense sheet approximately 0.06 urn thick. The outer layer on the connective-tis- sue side of the basement membrane is general- ly thicker (approximately 0.2 /*m) and relative- ly less electron-dense. In addition, extensions of this layer protrude into the connective tis- sue. These extensions are often T-shaped in transverse section and the fibrous material is commonly drawn out into discrete, banded fi- bers at the indistinct boundary between this layer and the general matrix of connective tis- sue (Fig. 6). Extensions of the fibrous layer are especially numerous and prominent where the basement membrane folds toward the lumen of the gut to follow the longitudinal infoldings of the midgut epithelium. In these areas they form a network that extends approximately 1-2 ^m into the connective tissue (Fig. 5). Fi- broblasts are usually associated with this fi- brous outer layer and its extensions (Fig. 6). The basement membranes in the midgut of both lobsters and stone crabs were often separ- ated from the epithelium (Fig. 4), presumably as a result of mechanical damage during prep- aration, but were not separated from the con- nective tissue. Abbreviations BM, Basement membrane BS, Blood sinus CL, Central layer of basement membrane CT, Connective tissue EP, Epithelium F, Fibroblast G, Granulocyte L, Lumen of midgut OL, Outer layer of basement membrane Fig. 1. Basement membrane underlying the midgut epi- thelium of the lobster. Transverse section stained with 1% aniline blue (ale), counterstained with picric acid-acid fuch- sin. x 270, Fig. 2. Basement membrane (lobster) showing channel (arrowhead) and network of processes on connective tissue surface. X 9400. Fig. 3. Portion of the basement membrane (lobster) showing a branching channel, the central layer and fibrous outer layers, processes of the basement membrane (large ar- rowheads), strands which interconnect processes and bridge channels (small arrowheads), processes of fibroblasts, pro- cesses of epithelial cells, and connective tissue, x 46,000. COMPLEX BASEMENT MEMBRANES 255 G --'--.? 2 0 256 JAN ROBERT FACTOR COMPLEX BASEMENT MEMBRANES 257 DISCUSSION The use of the term "basement membrane" to describe the structures in the midgut of the lobster and stone crab may require some justi- fication, particularly in light of the complex na- ture of these structures. "Basement mem- brane" has been used in a variety of ways. Ke- falides et al. (79) point out that the term was coined in 1857 by Robert Todd and William Bowman to describe the extracellular subepi- thelial layer seen with the light microscope in a variety of tissues. Ultrastructural observa- tions have shown that in some instances the basement membrane described from light mi- croscopy is actually a complex of structures that can be distinguished with the electron mi- croscope, and includes the reticular layer, which is now considered to be distinct from the basement membrane. Minor et al. ('76) state, "Microscopically a basement membrane is rec- ognized as a well-defined lamina of extracellu- lar matrix associated with the surface of a vari- ety of cells, including epithelial, mesothelial, endothelial, and muscle cells." Kefalides et al. ('79) use the term to refer to the "dense, amor- phous matrix observed in the electron micro- scope as lying between the lamina lucida and the fibrillar [reticular] layer . . . just below the basal layer of the epidermis," and for the layer underlying the urinary, reproductive, respira- tory, and digestive epithelia. They also use it to describe the extracellular matrix of the glomerular capillary, renal tubule, capsule of the ocular lens, Descemet's membrane of the cornea, Reichert's membrane of the rodent pa- rietal yolk sac, and the layer surrounding Schwann cells, muscle cells, and adipocytes. The term has been applied, then, to extracellu- lar layers with a variety of structural plans ranging from the typical, one-layered, electron- dense lamina with poorly defined inner and outer limits, which underlies many epithelia, to Fig. 4. Basement membrane underlying the midgut epi- thelium of the stone crab. Note artifactual separation of epi- thelium from basement membrane. Transverse section stained with PAS. x 465. Fig. 5. Fold in the basement membrane Istone crab), sim- ilar to the area in the box in Figure 4, showing the network of extensions of the fibrous layer (arrowheads) into the con- nective tissue. X 5200. Fig. 6. Portion of the basement membrane Istone crab) il- lustrating the central and fibrous outer layers, extensions of the outer layer on the connective tissue surface (large arrow- head), fibers at the edge of the extension (small arrowheads), and processes of fibroblasts. x 22,100. the three-layered glomerular capillary base- ment membrane, in which the inner and outer layers are continuous with the surface coats of the surrounding cells (Latta and Johnston, '76). Although the basement membrane is of- ten described as "amorphous," substructure in the form of oriented fibrils (Jollie, '68; Clark et al., '75) or hexagonal patterns (Jakus, '64) has been reported, in addition to the reports of grid-like patterns in insect basement mem- branes cited above. Biochemically, basement membranes con- sist of a collagenous component, shown in sev- eral instances to be a unique type of collagen molecule (type IV), and a non-collagenous com- ponent, possibly including glycoproteins and glycosaminoglycans (reviewed by Kefalides et al., '79). The basement membranes in the midgut of the lobster and stone crab appear to fit into the broad morphological definition of "basement membrane" as an extracellular, electron-dense matrix forming an extensive layer between the basal surface of the digestive epithelium and the underlying connective tissue. Additional- ly, the strongly positive PAS-reaction, com- mon to many basement membranes, indicates the presence of carbohydrates possibly associ- ated with the non-collagenous component. Al- though they fit the general definition, the base- ment membranes in the midgut of the lobster and stone crab are more complex than the typi- cal basement membrane in several respects: (1) they have three layers ?a thick central layer which is coated on both sides with thin fibrous outer layers; (2) they lack a substantial elec- tron-transparent zone (lamina rara); (3) the cen- tral layer does not appear amorphous in the stone crab, although it does in the lobster; (4) the basement membrane proper (i.e., the base- ment membrane excluding any processes or ex- tensions) has well-defined limits; and (5) exten- sions of the basement membranes protrude in- to the connective tissue. Perhaps the most interesting features pres- ent are the processes of the basement mem- brane of the lobster and the extensions of the outer layer of the basement membrane of the stone crab. Both are variations of the same structural theme and form extensive networks which appear to connect the basement mem- brane and the connective tissue in this region of transition; they may represent complex ver- sions of the anchoring fibrils and fibers found in the skin of vertebrates. Although the mid- gut epithelium was often artifactually separated from the basement membrane, the 258 JAN ROBERT FACTOR basement membrane was never observed to be separated from the surrounding connective tissue. This observation provides indirect evidence for a possible anchoring function of the processes and extensions of the basement membrane. The midgut of arthropods is distinct from the foregut and hindgut both developmental^ and structurally. The foregut (esophagus and stomach) and hindgut (rectum) are lined with a thin layer of cuticle which likely provides structural reinforcement for the underlying tissues and may provide elasticity when the wall of the gut is distended during feeding. The midgut has no such cuticular lining and it is in- teresting to note that this complex basement membrane has been observed only in the mid- gut of the lobster and stone crab; preliminary observations indicate the presence of a typical basement membrane in other regions of the gut. Complexities of the basement membrane in various insects are also reported to be pres- ent in the midgut. It seems plausible that the complex basement membranes in the midgut of the stone crab and the lobster may serve to reinforce the midgut against dilation during feeding, provide elasticity, or evenly distribute the mechanical forces to underlying tissues. The basement membrane in the midgut of the stone crab is unusually thick and the cen- tral layer can be distinguished from the net- work of extensions with the light microscope. This feature suggests it would be especially suitable for experimental studies on the origin and development of the basement membrane using autoradiography at the light microscope level. Demonstrating incontrovertibly that the structures that underlie the digestive epitheli- um in the midgut of the lobster and stone crab are basement membranes must await detailed biochemical characterization beyond the scope of this report, including the demonstration of type IV collagen. Morphological evidence alone, however, suggests that they are some of the more complex versions of the basement membrane yet to be reported. ACKNOWLEDGMENTS Thanks are extended to Dr. John M. Ander- son, Mr. Mark A. Cukierski, Dr. Robert H. Gore, Dr. Mary E. Rice, Ms. Cindy L. Van Dover, Dr. Doreen E. Ashhurst, Dr. Ronald Minor, and Dr. William A. Wimsatt for reading the manuscript and offering valuable com- ments. Ms. Liberta Poolt assisted with collect- ing stone crabs. 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