Unusually Complex Basement Membranes in the Midgut of Two Decapod Crustaceans, the Stone Crab (Menippe Mercenaria) and the Lobster (Homarus Americanus)

THE ANATOMICAL RECORD 200:253-258 (1981)

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- clude cylinders or grid-like patterns, some- lial tissues is generally considered to be a con- times composed of hexagonal units. tinuous, electron-dense, extracellular sheet Ultrastructural studies of the connective tis- which ranges in thickness from 200 to 50,000 sue layer surrounding the digestive epithelium A and often appears fibrous or flocculent. in two decapod crustaceans, the stone crab When viewed at low magnifications, it has Menippe mercenaria (Brachyura: Xanthidae) been variously described in textbooks as and the lobster Homarus americanus "amorphous" (Threadgold, 76) or "homogene- (Macrura: Nephropidae), demonstrate that the ous" with poorly defined inner and outer limits basement membrane of the adult midgut (Fawcett, '66). Closer examination shows that (intestine) is unusually complex. This is the this layer may be a mat or meshwork of fine fil- first description of a complex basement aments embedded in an amorphous matrix membrane in this major group of (Bloom and Fawcett, 75). The basement mem- invertebrates. brane (also commonly referred to as the basal lamina or lamina densa) often appears to be MATERIALS AND METHODS separated from the epithelial cells by an inter- The stone crabs used in this study were vening transparent layer (the lamina rara). The trapped in the Indian River coastal lagoon of structure, function, and biochemistry of the Florida and the American lobsters were ob- basement membrane have recently been re- tained from commerical sources. The digestive viewed by Kefalides et al. (79). tissues of both species were excised from living Several elaborations of this basic plan have animals, fixed for light microscopy in seawater been reported. The basement membrane of ver- Bouin's fluid, and embedded in Paraplast. Sec- tebrate skin may be connected to the underly- tions were stained with Harris's hematoxylin ing connective tissue by anchoring fibrils em- and eosin, Mallory's triple connective tissue bedded in this tissue (Briggaman et al., 71). stain, 1% alcoholic aniline blue and picric acid- Additionally, Fox (76) has shown that larger acid fuchsin, and periodic acid-Schiff s reagent anchoring fibers may be composed of a varia- (Humason, '62). Tissues were fixed for electron ble number of fibrils in the skin of several microscopy in 3% glutaraldehydein 0.2M sodi- aquatic vertebrates. Among the invertebrates, um cacodylate buffer at pH 7.0. The buffer con- several authors have described unusual struc- tained 30 mg/ml NaCl and 20 /tg/ml CaCL Fix- tural patterns for the basement membrane of ation was carried out at 6°C for 3-6 hours and the midgut in a variety of insects (Terzakis, was followed by 5 washes in solutions of buffer '67; Richards and Richards, '68; Gouranton, with decreasing concentrations of salts. Speci- 70; Holter, 70; Reinhardt and Hecker, 73; Hess and Pinnock, 75; Bayon and Francois, 76). These substructural variations usually in- 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 lated appearance and is coated on both sides 5 hours, treated with 2% uranyl acetate, dehy- by outer layers of fibrous material (Fig. 6). The drated in ethanol which was replaced by propy- outer layer facing the epithelium is a simple, lene oxide, and embedded in a mixture of Epon thin, electron-dense sheet approximately 0.06 812-Araldite 506. Thin sections were stained urn thick. The outer layer on the connective-tis- with uranyl acetate and lead citrate. sue side of the basement membrane is generally thicker (approximately 0.2 /*m) and relative- OBSERVATIONS ly less electron-dense. In addition, extensions The basement membrane of the midgut of of this layer protrude into the connective tis- the lobster is illustrated in Figures 1-3. When sue. These extensions are often T-shaped in viewed with the light microscope (Fig. 1) it can transverse section and the fibrous material is be seen to be a wavy structure which follows commonly drawn out into discrete, banded fi- the folding of the simple, columnar epithelium bers at the indistinct boundary between this of the midgut and separates it from the under- layer and the general matrix of connective tis- lying connective tissue. The basement mem- sue (Fig. 6). Extensions of the fibrous layer are brane stains positively with PAS and aniline especially numerous and prominent where the blue and is slightly eosinophilic. It varies in basement membrane folds toward the lumen of thickness from 0.2 to 0.4 /im and can be seen the gut to follow the longitudinal infoldings of electron-microscopically to be pierced by the midgut epithelium. In these areas they branching channels (Figs. 2 and 3). The central form a network that extends approximately layer consists of a finely granular, extremely 1-2 ^m into the connective tissue (Fig. 5). Fi- uniform material and is coated on both sides broblasts are usually associated with this fi- with thin, fibrous, relatively electron-dense brous outer layer and its extensions (Fig. 6). outer layers (Fig. 3). In addition, structures The basement membranes in the midgut of which range from roughly circular to elongate both lobsters and stone crabs were often separ- in section lie adjacent to the basement mem- ated from the epithelium (Fig. 4), presumably brane proper and can be seen to consist of the as a result of mechanical damage during prep- same finely granular material coated with a aration, but were not separated from the con- similar electron-dense outer layer. Serial sec- nective tissue. tions indicate that the larger of these extensions, at least, are processes that extend from the basement membrane. The fibrous outer Abbreviations layer coating the basement membrane and its BM, Basement membrane processes is drawn out into strands which bridge the channels in the membrane and BS, Blood sinus which also interconnect its processes (Fig. 3). CL, Central layer of basement membrane The electron-dense strands, together with the processes of the basement membrane, form an CT, Connective tissue extensive network (approximately 0.4 pm EP, Epithelium thick) along the connective tissue surface of F, Fibroblast the basement membrane (Fig. 2). Processes of fibroblasts are commonly associated with this G, Granulocyte network (Fig. 3). A similar network is only in- L, Lumen of midgut termittently present on the surface of the basement membrane which faces the epithelium. OL, Outer layer of basement membrane The basement membrane of the midgut of the stone crab is illustrated in Figures 4-6. It Fig. 1. Basement membrane underlying the midgut epithelium of the lobster. Transverse section stained with 1% is considerably thicker than that of the lobster aniline blue (ale), counterstained with picric acid-acid fuch- (approximately 1.5 /tin) and appears to have sin. x 270, two layers when viewed with the light microscope (Fig. 4). Its staining characteristics are Fig. 2. Basement membrane (lobster) showing channel (arrowhead) and network of processes on connective tissue similar to the basement membrane in the mid- surface. X 9400. gut of the lobster — it is strongly PAS-positive, strongly aniline blue-positive, and slightly eo- Fig. 3. Portion of the basement membrane (lobster) sinophilic. When observed with the electron showing a branching channel, the central layer and fibrous outer layers, processes of the basement membrane (large ar- microscope, the basement membrane is three- rowheads), strands which interconnect processes and bridge layered and does not appear to have channels channels (small arrowheads), processes of fibroblasts, pro- (Fig. 5). The central layer has a coarse, reticu- 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 three-layered glomerular capillary base- The use of the term "basement membrane" to ment membrane, in which the inner and outer describe the structures in the midgut of the layers are continuous with the surface coats of lobster and stone crab may require some justi- the surrounding cells (Latta and Johnston, fication, particularly in light of the complex na- '76). Although the basement membrane is of- ture of these structures. "Basement mem- ten described as "amorphous," substructure in brane" has been used in a variety of ways. Ke- the form of oriented fibrils (Jollie, '68; Clark et falides et al. (79) point out that the term was al., '75) or hexagonal patterns (Jakus, '64) has coined in 1857 by Robert Todd and William been reported, in addition to the reports of Bowman to describe the extracellular subepi- grid-like patterns in insect basement mem- thelial layer seen with the light microscope in a branes cited above. variety of tissues. Ultrastructural observa- Biochemically, basement membranes con- tions have shown that in some instances the sist of a collagenous component, shown in sev- basement membrane described from light mi- eral instances to be a unique type of collagen croscopy is actually a complex of structures molecule (type IV), and a non-collagenous com- that can be distinguished with the electron mi- ponent, possibly including glycoproteins and croscope, and includes the reticular layer, glycosaminoglycans (reviewed by Kefalides et which is now considered to be distinct from the al., '79). basement membrane. Minor et al. ('76) state, The basement membranes in the midgut of "Microscopically a basement membrane is rec- the lobster and stone crab appear to fit into the ognized as a well-defined lamina of extracellu- broad morphological definition of "basement lar matrix associated with the surface of a vari- membrane" as an extracellular, electron-dense ety of cells, including epithelial, mesothelial, matrix forming an extensive layer between the endothelial, and muscle cells." Kefalides et al. basal surface of the digestive epithelium and ('79) use the term to refer to the "dense, amor- the underlying connective tissue. Additional- phous matrix observed in the electron micro- ly, the strongly positive PAS-reaction, com- scope as lying between the lamina lucida and mon to many basement membranes, indicates the fibrillar [reticular] layer . . . just below the the presence of carbohydrates possibly associ- basal layer of the epidermis," and for the layer ated with the non-collagenous component. Al- underlying the urinary, reproductive, respira- though they fit the general definition, the base- tory, and digestive epithelia. They also use it ment membranes in the midgut of the lobster to describe the extracellular matrix of the and stone crab are more complex than the typi- glomerular capillary, renal tubule, capsule of cal basement membrane in several respects: (1) the ocular lens, Descemet's membrane of the they have three layers —a thick central layer cornea, Reichert's membrane of the rodent pa- which is coated on both sides with thin fibrous rietal yolk sac, and the layer surrounding outer layers; (2) they lack a substantial elec- Schwann cells, muscle cells, and adipocytes. tron-transparent zone (lamina rara); (3) the cen- The term has been applied, then, to extracellu- tral layer does not appear amorphous in the lar layers with a variety of structural plans stone crab, although it does in the lobster; (4) ranging from the typical, one-layered, electron- the basement membrane proper (i.e., the base- dense lamina with poorly defined inner and ment membrane excluding any processes or ex- outer limits, which underlies many epithelia, to tensions) has well-defined limits; and (5) extensions of the basement membranes protrude in- to the connective tissue. Perhaps the most interesting features pres- Fig. 4. Basement membrane underlying the midgut epi- ent are the processes of the basement mem- thelium of the stone crab. Note artifactual separation of epithelium from basement membrane. Transverse section brane of the lobster and the extensions of the stained with PAS. x 465. outer layer of the basement membrane of the stone crab. Both are variations of the same Fig. 5. Fold in the basement membrane Istone crab), sim- structural theme and form extensive networks ilar to the area in the box in Figure 4, showing the network which appear to connect the basement mem- of extensions of the fibrous layer (arrowheads) into the connective tissue. X 5200. brane and the connective tissue in this region of transition; they may represent complex ver- Fig. 6. Portion of the basement membrane Istone crab) il- sions of the anchoring fibrils and fibers found lustrating the central and fibrous outer layers, extensions of in the skin of vertebrates. Although the mid- the outer layer on the connective tissue surface (large arrowhead), fibers at the edge of the extension (small arrowheads), gut epithelium was often artifactually and processes of fibroblasts. x 22,100. separated from the basement membrane, the 258 JAN ROBERT FACTOR basement membrane was never observed to be ing stone crabs. This work was supported by a separated from the surrounding connective Smithsonian Postdoctoral Fellowship to the tissue. This observation provides indirect author. Article No. 22, Studies on Decapod evidence for a possible anchoring function of Crustacea from the Indian River Region of the processes and extensions of the basement Florida. membrane. The midgut of arthropods is distinct from LITERATURE CITED the foregut and hindgut both developmental^ Bayon, C, and J. Francois (1976) infrastructure de la lame and structurally. The foregut (esophagus and basale du mesenteron chez la larve d'Oryctes nasicornis L. (Coleoptera: Scarabaeidae). Int. J. Insect Morphol. Em- stomach) and hindgut (rectum) are lined with a bryol., 5.205-217. thin layer of cuticle which likely provides Bloom, W., and D.W. Fawcett (1975) A Textbook of Histol- structural reinforcement for the underlying ogy. Tenth Edition. W.B. Saunders Co.. Philadelphia. tissues and may provide elasticity when the Briggaman, R.A.. F.C. DaJldorf, and C.E. Wheeler, Jr. 11971) Formation and origin of basal lamina and anchoring fi- wall of the gut is distended during feeding. The brils in adult human skin. J. Cell Biol., 57.384-395. midgut has no such cuticular lining and it is in- Clark, C.C., R.R. Minor, T.R. Brent, and N.A. Kefalides teresting to note that this complex basement (1975) The embryonic rat parietal yolk sac. Changes in the membrane has been observed only in the mid- morphology and composition of its basement membrane during development. Dev. Biol., 46/243-261. gut of the lobster and stone crab; preliminary Fawcett, D.W. (1966) An Atlas of Fine Structure-The Cell. observations indicate the presence of a typical W.B. Saunders Co., Philadelphia. basement membrane in other regions of the Fox, H. (1976) Anchoring fibrils of the basal lamina and gut. Complexities of the basement membrane basement lamella in the skin of aquatic chordates. J. Mi- croscopic Biol., Cell., 23:229-236. in various insects are also reported to be pres- Gouranton, J. (19701 Etude d'une lame basal presentant une ent in the midgut. It seems plausible that the structure d'une type nouveau. J. Microscopie, 9: complex basement membranes in the midgut 1029-1040. of the stone crab and the lobster may serve to Hess, R.T., and D.E. Pinnock (1975) The ultrastructure of a complex basal lamina in the midgut of larvae of Oryctes reinforce the midgut against dilation during rhinoceros L. (Coleoptera, Scarabaeidae). Z. Morphol. feeding, provide elasticity, or evenly distribute Tiere, 80:277-285. the mechanical forces to underlying tissues. Holter, P. (1970) Regular grid-like structures in the midgut The basement membrane in the midgut of epithelial basement membrane of some Coleoptera. Z. Zellforsch.. 770:373-385. the stone crab is unusually thick and the cen- Humason, GL. (1962) Animal Tissue Techniques. W.H. tral layer can be distinguished from the net- Freeman, San Francisco. work of extensions with the light microscope. Jakus, M.A. (1964) Ocular Fine Structure. Little Brown, This feature suggests it would be especially Boston. Jollie, W.P. (1968) Changes in the fine structure of the pari- suitable for experimental studies on the origin etal yolk sac of the rat placenta with increasing gestation- and development of the basement membrane al age. Am. J. Anat., 722:513-532. using autoradiography at the light microscope Kefalides, N.A., R. Alper, and C.C. Clark (1979) Biochemis- level. try and metabolism of basement membranes. Int. Rev. Demonstrating incontrovertibly that the Cytol., 67:167-228. Latta, H, and W.H. Johnston (1976) The glycoprotein inner structures that underlie the digestive epitheli- layer of glomerular capillary basement membrane as a fil- um in the midgut of the lobster and stone crab tration barrier. J. Ultrastruct. Res., 57:65-67. are basement membranes must await detailed Minor, R.R., P.S. Hoch, T.R. Koszalka, R.L. Brent, and N.A. Kefalides (1976) Organ cultures of the embryonic rat pari- biochemical characterization beyond the scope etal yolk sac. I. Morphologic and autoradiographic stud- of this report, including the demonstration of ies of the deposition of the collagen and noncollagen glyco- type IV collagen. Morphological evidence protein components of basement membranes. Dev. Biol., alone, however, suggests that they are some of 4& 344-364. Reinhardt, C, and H. Hecker (1973) Structure and function the more complex versions of the basement of the basal lamina and of the cell junctions in the midgut membrane yet to be reported. epithelium (stomach) of female Aedes aegypti L. (Insecta, Diptera). Acta Trop.. 30:213-236. ACKNOWLEDGMENTS Richards, A.G.. and PA. Richards (1968) Flea Ctenophthal- mus: heterogeneous hexagonally organized layer in the Thanks are extended to Dr. John M. Ander- midgut. Science, 760:423-425. son, Mr. Mark A. Cukierski, Dr. Robert H. Terzakis, J. A. (1967) Substructure in an epithelial basal lam- Gore, Dr. Mary E. Rice, Ms. Cindy L. Van ina (basement membrane). J. Cell. Biol.. 35.273-278. Dover, Dr. Doreen E. Ashhurst, Dr. Ronald Threadgold, L.T. (1976) The Ultrastructure of the Animal Cell. Second Edition. Pergamon Press, Oxford. Minor, and Dr. William A. Wimsatt for reading Todd. R.B., and W. Bowman (1857) The Physiological Anat- the manuscript and offering valuable com- omy and Physiology of Man. Blanchard and Lea, Phila- ments. Ms. Liberta Poolt assisted with collect- delphia.