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Morphology and Cell Proliferation of Subepithelial Fibroblasts in Adult Mouse Jejunum
GASTROENTEROLOGY 67:622- 635, 1974
Vol. 67 , )io. i Printed in U.S.A .
Copyright© 1974 by The Willia ms & Wilkins Co.
MORPHOLOGY AND CELL PROLIFERATION OF SUBEPITHELIAL FIBROBLASTS IN ADULT MOUSE JEJUNUM I. Structural features MICHAEL N. MARSH, D.M., M.R.C .P ., AND JERRY
s.
TRIER, M .D.
Departments of Medicine, Harvard Medical School, Peter Bent Brigham Hospital and Boston University School of Medicine, Boston, Massachusetts; and Professorial Medical Unit, Royal South Hants Hospital , Southampton, Hampshire, England
In this report, the organization and the morphology of subepithelial fibroblasts in adult mouse jejunum are described. Toluidine bluestained 1 f./. Epon sections of osmium-fixed tissue were compared with electron micrographs of adjacent thin sections permitting precise identification of mesenchymal cells adjacent to crypt and villous epithelium. In the crypts and along the base of some villi, there was a continuous subepithelial fibroblast sheath 1 to 3 cells thick. This sheath consisted of fibroblasts which appeared spindle-shaped when sectioned due to thin sheath-like cytoplasmic processes which extended toward the periphery from the center of the cells. These cells were closely applied to the epithelial basal lamina and were surrounded by collagen . In contrast, no well developed sheath was present along the middle and distal portions of the villi. Rather, various mesenchymal cell types, including capillary endothelial cells, macrophages, and leukocytes, as well as fibroblasts, were located adjacent to the epithelial lamina. Furthermore, in contrast to the crypt region, collagen was sparse in the lamina of the villi. Thus, an organized subepithelial fibroblast sheath is seen only in the crypt region of mouse jejunum; it is not found throughout the mucosa as in the rabbit and human colon . It has been firmly established that epithelial cell renewal in the small intestinal mucosa is confined normally to the crypts Received January 29, 197 4. Accepted April3, 197 4. Presented in part at the 74th Annual Meeting of the American Gastroenterological Association in New York City, May, 1973. Address requests for reprints to: Dr. Jerry S. Trier, Gastroenterology Division, Department of Medicine, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115 . This investigation was supported by Research Grant AM-17537 from the National Institutes of Health, Bethesda, Maryland . Part of this study was carried out while Dr. Marsh was the recipient of a Traveling Fellowship of the Medical Research Council, Great Britain . Mrs . Jacqueline Beals provided valuable technical assistance.
of Lieberkuhn. 1- 5 Here, undifferentiated crypt cells proliferate and subsequently migrate onto the villi where they differentiate into mature columnar epithelial cells . However, far less is known about cell renewal among mesenchymal cells in the lamina propria of the small intestine. Attention has been drawn to a population of fibroblasts that is closely applied to the basal aspect of small intestinal, 6 gallbladder, 7 and colonic epithelia. 8 • 9 It has been shown that these subepithelial fibroblasts synthesize DNA and divide. 6 • 10 • 11 These fibroblasts have been carefully studied in rabbit colonic mucosa where 3 [ H ]thymidine radioautography indicated that proliferation of subepithelial fibroblasts is confined to the lower crypts, but
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that some newly formed cells subsequently migrate toward the luminal epithelium in synchrony with their adjacent epithelial cells. As these fibroblasts migrate from the lower crypts to surface epithelium, they show morphological evidence of maturation and even senescence when they reach the surface epithelium. Moreover, whereas the fibroblasts form a continuous sheath around the crypts, the sheath is discontinuous beneath the surface epithelium. 9 • 11 In view of these findings, it seemed important to determine whether a similar replicating subepithelial fibroblast sheath exists in the jejunum. Our intent was to examine the morphology of the fibroblast sheath and its relationship to the jejunal epithelium at all levels of the mucosa from crypts to villous tips. We also wished to determine the site of proliferation and subsequent migratory pattern of subepithelial fibroblasts, and to correlate migration of these fibroblasts to migration of epithelial cells in mouse jejunum. 12 ' 14 In this report, we describe our cytological and electron-microscopic findings of the subepithelial fibroblast sheath: in the companion paper which follows, 15 we describe the proliferation and migration of subepithelial fibroblasts as revealed by radioautography.
Methods Adult, random-bred albino mice (Charles River Laboratories, Wilmington, Mass.), weighing 35 to 40 g, were maintained on a diet of Purina chow (Ralston Purina Co., St. Louis, Mo.) and allowed liberal quantities of tap water. Animals were killed by cervical dislocation, and approximately 0.5 ml of chilled chrome-osmium fixative, 16 containing 0.2 Msucrose and 0.9 mM CaC1 2 , was slowly instilled into the lumen of the proximal jejunum. Slices of proximal jejunum were obtained a few minutes later, and then fixed by immersion in the same solution for 1 to 2 hr, transferred for 1 additional hr to 0.2 M phosphate-buffered formalin (pH 7 .2), dehydrated in graded ethanol solutions, embedded in epoxy resin, 17 and oriented for sectioning on aluminum rods, as described previously.' " Sections, 1 /J. in thickness, were cut with glass knives on a Sorvall MTB-2 ultramicrotome, stained with toluidine blue, 19 and examined with the light microscope. To permit precise identification ofsubepithe-
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lial fibroblasts , and at the same time to facilitate the ultrastructural study of the subepithelial fibroblast sheath, thin sections adjacent to selected 1 /J. toluidine blue-stained sections were cut with a diamond knife on an LKB ultramicrotome, mounted on copper grids, stained with uranyl acetate 20 and lead citrate, 21 and examined with a Philips EM-300 electron microscope. Thus, individual cells seen with the light microscope were also examined with the electron microscope at precisely localized levels in the jejunal mucosa. In this way, a detailed, systematic study of the entire subepithelial region of mouse jejunum could be accomplished.
Results Light-Microscopic Appearance of Fibroblasts and Related Cells In toluidine blue-stained 1 f.L sections of mouse jejunum, subepithelial fibroblasts were identified as spindle-shaped cells with moderately stained cytoplasm (figs. 1 and 4). A few deeply stained granules, presumably lysosomes, were occasionally seen in the fibroblast cytoplasm . Fibroblast nuclei were elongated or ovoid; their margins were smooth or only slightly irregular. Macrophages tended to be more irregular in shape and larger in size than fibroblasts (fig. 6). Nuclei of macro phages lying alongside the epithelium varied in shape, but were frequently fusiform. However, they were usually larger and less densely stained than fibroblast nuclei. The presence of large numbers of densely stained lysosomes and other vacuoles within the macrophage cytoplasm aided their identification, but it was sometimes impossible to differentiate cytoplasm belonging to fibroblasts from that belonging to macrophages with the light microscope alone, unless the cell nucleus was present in the section. Although some endothelial cells were fusiform in appearance, and, therefore, closely resembled the appearance of fibroblasts (fig. 4), the majority exhibited subtle differences. In particular, their nuclear contours were frequently very irregular, and the surrounding cytoplasm was stained densely with toluidine blue (figs. 1 and 5), a feature which often facilitated their differentiation from fibroblasts with the light microscope. Also, the finding of
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FIG. 1. The inset is a 1 J1. toluidine blue-stained light micrograph which shows part of the pericryptal fibroblast sheath, the cytological features of its fibroblasts, and their prominent cytoplasmic processes · (P) . Note densely staining endothelial cell (EC) . The electron micrograph shows an adjacent thin section of the area within box in inset. This illustrates the fine structure of fibroblast nuclei, cytoplasm, and their processes (P) ; EP, epithelium; C, collagen ( x 6000; inset, x 1500).
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red blood cells in a capillary lumen often permitted identification of endothelial cells (figs. 4 and 5). However, in some instances it was virtually impossible to identify individual cells as fibroblasts or endothelial cells with certainty, without obtaining electron micrographs of adjacent thin sections (figs . 4 and 6) , since their light-microscopic features were so similar. Distribution of Subepithelial Fibroblasts Lower crypts. A well defined population of subepithelial fibroblasts was identified around the jejunal crypts (fig. 1). The cell bodies of these fibroblasts were fusiform or ovoid when sectioned, and gave rise to long, tapering cytoplasmic processes. Both cell bodies and their associated processes were closely applied to the basal aspect of the crypt epithelium (fig. 1). When the plane of sectioning was appropriate, some of the thin cytoplasmic processes could be traced along the base of as many as eight adjacent epithelial cells. Overlapping of adjacent fibroblasts was observed often around the basal portions of the crypts, but only ocasionally along the upper crypts. In the intercellular regions between adjacent crypts, collagen fibrils were abundant and were often in contact with fibroblasts and with the epithelial basal lamina. Upper crypts and proximal portions of villi. In this region of the mucosa, a change in the morphological appearance of the subepithelial fibroblast sheath was evident. A fairly well defined sheath could still be identified extending from the upper crypts into the basal portions of some of the villi (fig. 2) . The fibroblasts comprising the sheath at this level of the mucosa were identical to those occupying the lower crypts, both in their fine structure and in their relationship to the epithelial basal lamina . However, in other regions, other types of mesenchymal cells, especially plasma cells , capillary endothelial cells, and occaFIG. 2. In this electron micrograph from the upper CJYpt region, a fibroblast (F) gives rise to a long, sheet-like cytoplasmic process (arrows ) which pa sses closely along the basal lamina of the epithelium.
There is overlap with adjacent fibroblasts (F2, F3) ( x 4,000).
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sional leukocytes and macrophages, now interrupted the fibroblast sheath and were in direct contact with the epithelial basal lamina (fig. 3). Collagen was also less abundant than in the subepithelial spaces between the lower crypt epithelium. This area of the mucosa, therefore, was representative of a transitional zone which, in its lower part, was in continuity with the well defined fibroblast sheath surrounding the bases of the crypts, but which, in the upper part, differed in that the fibroblast sheath was now discontinuous. Middle and distal portions of the uilli. In the middle and the distal portions of the villi a fibroblast sheath was no longer apparent (figs. 4, 5, 6, and 7). Instead, a variety of mesenchymal cells, in addition to fibroblasts, occupied the space just beneath the epithelial basal lamina. A large proportion of these cells were endothelial cells whose attenuated, fenestrated cytoplasm formed an extensive subepithelial capillary network (figs. 4 and 5). Thin processes of fibroblast cytoplasm located directly beneath the basal lamina were interposed between the basal lamina and the endothelial cells, but did not form a complete sheath (figs. 4, 5, and 6). Of other mesenchymal cells besides endothelial cells that abutted on the epithelium, macrophages and plasma cells were most common. In addition, the epithelial lamina together with its associated subepithelial tissues were interrupted where lymphocytes and eosinophils traversed the lamina to enter the intercellular space between epithelial cells, and, less frequently, where pseudopod-like projections of basal epithelial cell cytoplasm penetrated the basal lamina and made direct contact with various mesenchymal cells (fig. 8). At the tips of the villi, the subepithelial spaces were often occupied by capillary endothelial cells (fig. 9). However, in such areas, a few thin fibroblast cytoplasmic processes were seen with the electron microscope to be located between epithelium and the capillary basement membrane (fig. 9). Although epithelial cells were frequently observed in the process of extrusion from the villous tips, extrusion of
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fibroblasts into the lumen from this site was not observed. Electron-Microscopic Features of Sub epithelial Fibroblasts
The electon-microscopic features of subepithelial fibroblasts in the jejunum correspond in general to descriptions of fibroblasts in other tissues. 22 • 23 Typically, sectioned fibroblast nuclei were fusiform and enclosed by inner and outer nuclear membranes . There were dense accumulations of chromatin along the inner nuclear membrane, and several nucleoli were frequently observed (figs. 1 and 2). The perinuclear cytoplasm (fig. 10) contained mitochondria and scattered accumulations of Golgi material. Granular endoplasmic reticulum was abundant. In addition, spirallike polyribosomes, individual free ribosomes , and small cytoplasmic vesicles, spherical in section, were also present throughout the cytoplasm. The elongated cytoplasmic processes of pericryptal fibroblasts were also packed with similar organelles, with the exception of Golgi material (fig. 11) . The fine structure of subepithelial fibroblasts along the outer parts of the villi was similar to that of fibroblasts surrounding the lower crypts, with one major difference. The peripheral cytoplasmic projections of pericryptal fibroblasts were sheath-like extensions (fig. 2). However, the peripheral cytoplasmic processes of fibroblasts in the distal portion of the villi were extremely slender, thin, finger-like structures which were seen, therefore, only where they crossed the plane of sectioning (figs. 4, 5, 6, and 9). They appeared as small fragments of cytoplasm beneath the epithelial and basal lamina, and it was usually difficult to relate a given fragment to its fibroblast cell body. On the other hand, centrally located cell bodies frequently appeared as isolated nuclei surrounded by a thin layer of cytoplasm (figs . 6 and 7).
Discussion In this study, precise identification of the cell types adjacent to the epithelial basal lamina was achieved by examining
FIG. 3. This is a representative electron micrograph of the region of the crypt-villus junction. Here, fibroblast processes (arrows) comprise a fairly substantial but incomplete sheath. Note presence of subepithelial capillary (CAP), and below another capillary profile with an endothelial cell (EC) cut through the plane of its nucleus . Various other mesenchymal cells, e.g., plasma cells (P) and an eosinophil (EO) lie in very close proximity to the epithelial basal lamina ( x 5,000) . 6:27
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FIG. 4. The 1 p. Epon section shown in the inset is from the junction of the proximal and middle one-third of the villus. The fibroblast (cell I), with its densely stained process extending upwards, is easy to spot. The cell beneath (cell 2) might easily be mistakenly identified as a fibroblast by light microscopy. Electron microscopy of an adjacent thin section shows cell2 to be an endothelial cell . Small arrows delineate the capillary lumen. On the opposite side is a larger vessel with a red cell in its lumen. Only a few fragments of fibroblast cytoplasm (arrowheads) are present between this capillary and the epithelium. Thus, there is no well defined sheath at this level of the jejunal mucosa ( x 5,000; inset, x 600).
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FIG. 5. The inset is a light micrograph of the junctional zone between the middle and distal one-third of a villus. Note the extensive capillary network with typical, densely stained endothelial cell nuclei; R, erythrocyte. A detailed view of the right hand network is shown in the adjacent thin section examined by electron microsco py . The three panels, A, B , and C, refer to corresponding areas marked in the inset. Note the absence of a fibroblast sheath ; in fact , hardly any fibroblast cytoplasm is present along t his broad expanse of basal villous epithelium, save for a few thin cell processes (arrows); EC, endothelial cell ( x 5,000; inset, x 800) .
with the electron microscope "thin" sec- became clear that it was occasionally diffitions cut adjacent to selected toluidine cult, and , in a few instances, virtually blue-stained 1 J.L "thick" sections . In this impossible, to identify with certainty, with way, it was possible to evaluate systemati- the light microscope, the nature of every cally the cytology of the entire subepithe- cell lying against the base of the epithelium lial cell · population and correlate light- (figs. 1, 4, and 5). When such difficulties microscopic · with electron-microscopic arose, it usually was the differentiation structural features. Using this approach, it between fibroblasts and endothelial cells
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FIG. 6. A light micrograph of the upper one-third of a villus, except for its tip, is shown in the inset. A large number of cells occupy the lamina propria, but fibroblasts are not obvious. Details of the upper box are shown in the accompanying electron micrograph, and of the lower box are shown in figure 7. Two very densely stained cells in the upper box of the light micrograph are seen to be a fibroblast (F) and an endothelial cell (EC) in the electron micrograph. The remaining lamina propria is largely occupied by a macrophage (M) . This micrograph illustrates well the thin, narrow fibroblast processes which appear as isolated fragments of cytoplasm (arrows) when sectioned. AG, endocrine epithelial cell ( x 4,000; inset, x 400).
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FIG. 7. The lower box outlined in the inset of figure 6 contains an erythrocyte (R) and a fibroblast (F) . The latter has the appearance frequently noted on the villi of a nucleus surrounded by a thin rim of cytoplasm. P, Plasma cell; L , lymphocyte ( x 5,000).
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chymal cell populations and permitted better characterization of light-microscopic features of the specific cell types. Our findings indicate that a well developed fibroblast sheath surrounds the lower half of the crypts in the proximal small intestine of mice. At this level of the mucosa, the fibroblasts are closely applied to the basal aspect of almost all epithelial cells; indeed, there often is considerable overlap between adjacent fibroblasts. This results, in some areas, in a multilayered or shingled sheath of fibroblasts applied to the crypts (fig. 2). At the mouth of the crypts and at the base of the villi, the fibroblast sheath becomes less well defined, and other mesenchymal cell types, such as endothelial cells, macrophages, and plasma cells, are interposed between fibroblasts and make direct contact with the basal surface of epithelial cells. In the villi, the gaps between fibroblasts and their peripheral cell processes increase in size and frequency to such a degree that no well defined fibroblast sheath is present in the distal two-thirds of the villi (fig. 12). A structural feature of individual fibroblasts, which differs with their location in the jejunal mucosa, was the nature of the cytoplasm peripheral to the central perinuclear portion of the cells. Around the crypts the peripheral cytoplasm projected from the fibroblasts in the form of thin, sheetlike extensions which contributed to the continuity of the fibroblast sheath in this region of the mucosa (figs. 1, 2, and 12). In contrast, in the villi, the peripheral cytoplasmic processes were thin, finger-like projections (figs. 4, 9, and 12). The conversion to finger-like from sheet-like peripheral cytoplasmic projections contributed to FIG. 8. This electron micrography shows an epi- the discontinuous nature of the sheath in thelial basal cytoplasmic process (P) interrupting the the more distal regions of the mucosa (fig. continuity of the basal lamina and subepithelial 12). Additionally, collagen was abundant tissues in the middle one-third of the villus . The in the intercellular spaces of the lamina process is in direct contact with cytoplasm of unidenpropria surrounding the crypts, but was tified cell, probably a fibroblast cytoplasmic process. quite sparse in the intercellular spaces of Below is a fibroblast nucleus with its thin rim of the lamina propria of the villi. cytoplasm; collagen is sparse at this level of the Our findings indicate that distinct difmucosa ( x 10,000). ferences exist between the subepithelial (see figs. 4 and 5). Thus, electron micros- fibroblast sheath of the small intestine of copy was particularly valuable in defining the mouse and that described in the colon the nature of these subepithelial mesen- of rabbits and man. 9 • 11 In the small intes-
FIG. 9. This electron micrograph shows a villus cross-sectioned immediately beneath its tip. Widened intercellular spaces (x) are seen between adjacent epithelial cells . The subepithelial space is almost entirely filled by a capillary ; only a few fragments of fibroblast cytoplasm (arrows) are interposed between vascular and epithelial basal laminae (x 6,000).
FIG. 10. Electron micrograph of perinuclear cytoplasm of a pericryptal fibroblast. Note the abundance of scattered Golgi material (G), dilated sacs of granular endoplasmic reticulum containing a moderately electrondense material, mitochondria, and a probable lysosome (L). Arrowhead points to polyribosome clusters (x 16,000). 63:1
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FIG. 11. Peripheral cytoplasm from a pericryptal fibroblast with distended profiles of endoplasmic reticulum, a mitochondrion, and a few small vesicles ( x 16,000).
VIlli
FIG. 12. Schematic diagram of subepithelial fibroblast sheath, seen en fa ce on left , and in section on right . The vertical lines, X--X , indicate imaginary plane of section perpendicular to basement membrane, and the corresponding profiles are drawn on the right, thus recalling appearances actually observed during this study . Gray stippling, basement membrane ; corrugated lines, collagen fibers.
tine, as in the colon, the fibroblast sheath is continuous around the epithelium where epithelial cell proliferation is most active ; that is, around the base of the crypts. However, in the villi of the small intestine, the sheath becomes so discontinuous that it is no longer an organized, definable structure. In contrast, in the colon, the sheath remains definable at all levels of the colonic mucosa, although it too becomes less complete and somewhat fenestrated beneath the colonic surface epithelium. 9 Moreover, in the small intestine, intercellular collagen is abundant in the crypt region and sparse in the villi, whereas in the colon it is sparse in the crypt region and abundant beneath the surface epithelium. Finally, whereas fibroblasts beneath the surface epithelial cells of the colon contain more lysosome-like structures than those surrounding the crypts, 9 differences in the lysosome content of crypt and villous fibroblasts were not apparent in the small intestine. REFERENCES 1. Bizzozero G: Uber die regeneration der Elemente der schlauchformingen Drusen und des Epithels des Magendarmkanals. Anat Anz 3:781-974, 1888 2. Leblond CP, Walker BE: Renewal of cell popula· tions. Physiol Rev 36:255-276, 1956 3. Leblond CP, Messier P : The constant renewa l of the intestinal epithelium in the albino rat . Anat
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Rec 132:247-260, 1958 4. Lipkin M, Sherlock P, Bell B: Cell proliferation kinetics in the gastrointestinal tract of man. II. Cell renewal in stomach, duodenum, colon and rectum . Gastroenterology 45:721-729, 1963 5. MacDonald WC, Trier JS, Everett NB: Cell proliferation and migration in stomach, duodenum and rectum of man. Gastroenterology 46:405-417, 1964 6. Deane HW: Some electron microscopic observations on the lamina propria of the gut, with comments on the close association of macrophages, plasma cells and eosinophils. Anat Rec 149:453-473, 1964 7. Kaye GI, Wheeler HO, Whitlock RT eta!: Fluid transport in the rabbit gall bladder. A combined physiological and electron microscopic study. J Cell Bioi 30:237-268, 1966 8. Donellan WL: The structure of the colonic mucosa . The epithelium and subepithelial reticulohistiocytic complex. Gastroenterology 49:496514, 1965 9. Kaye GI , Lane N , Pascal RR: Colonic pericryptal fibroblast sheath: replication, migration, and cytodifferentiation of a mesenchymal cell system in adult tissue . II . Fine structural aspects of normal rabbit and human colon . Gastroenterology 54:852- 865, 1968 10. Kaye GI, Maenza RM, Lane N: Cell replication in rabbit gall bladder. An autoradiographic study of epithelial and associated fibroblast renewal in vivo and in vitro. Gastroenterology 51:670-680, 1966 11. Pascal RR, Kaye GI, Lane N: Colonic pericryptal fibroblast sheath: replication , migration and cytodifferentiation of a mesenchymal system in adult tissue. I. Autoradiographic studies of normal rabbit colon. Gastroenterology 54:835- 851 ,
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1968 12. Quastler H, Sherman FG: Cell population kinetics in the intestinal epithelium of the mouse. Exp Cell Res 17:420-438, 1959 13. Lesher S, Fry RJM, Kohn HI: Influ~nce of age on transit time of cells of mouse intestinal epithelium. I. Duodenum. Lab Invest 10:291-300, 1961 14. Hagemann RF, Sigdestad CP, Lesher S: A quantitative description of the intestinal epithelium of the mouse. Am J Anat 129:41-52, 1970 15. Marsh MN, Trier JS : The morphology and cell proliferation of subepithelial fibroblasts in adult mouse jejunum. II. Radioautographic studies. Gastroenterology 67:636-645, 1974 16. Dalton AJ: A chrome-osmium fixative for electron microscopy (abstr.) Anat Rec 121:281, 1955 17. Luft JH: Improvements in epoxy resin embedding methods . J Biophys Biochem Cytol 9:409-414, 1961 18. Trier JS, Lorenzsonn V, Groehler K: Pattern of secretion of Paneth cells of the small intestine of mice. Gastroenterology 53:240-249, 1967 19. Trump BF, Smuckler EA, Benditt EP: A method for staining epoxy sections for light microscopy. J Ultrastruct Res 5:343-348, 1961 20. Watson ML: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475- 478, 1958 21. Reynolds ES: The use oflead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Bioi 17:208-212, 1963 22. Movat HZ , Fernando NVP: Fine structure of connective tissue. I. The fibroblast. Exp Mol Pathol 1:509- 534, 1962 23. Ross R: The connective tissue fiber forming cell . In Treatise on Collagen, vol 2(4) . New York, Academic Press, 1968, p 1- 82
Vol. 67 , )io. i Printed in U.S.A .
Copyright© 1974 by The Willia ms & Wilkins Co.
MORPHOLOGY AND CELL PROLIFERATION OF SUBEPITHELIAL FIBROBLASTS IN ADULT MOUSE JEJUNUM I. Structural features MICHAEL N. MARSH, D.M., M.R.C .P ., AND JERRY
s.
TRIER, M .D.
Departments of Medicine, Harvard Medical School, Peter Bent Brigham Hospital and Boston University School of Medicine, Boston, Massachusetts; and Professorial Medical Unit, Royal South Hants Hospital , Southampton, Hampshire, England
In this report, the organization and the morphology of subepithelial fibroblasts in adult mouse jejunum are described. Toluidine bluestained 1 f./. Epon sections of osmium-fixed tissue were compared with electron micrographs of adjacent thin sections permitting precise identification of mesenchymal cells adjacent to crypt and villous epithelium. In the crypts and along the base of some villi, there was a continuous subepithelial fibroblast sheath 1 to 3 cells thick. This sheath consisted of fibroblasts which appeared spindle-shaped when sectioned due to thin sheath-like cytoplasmic processes which extended toward the periphery from the center of the cells. These cells were closely applied to the epithelial basal lamina and were surrounded by collagen . In contrast, no well developed sheath was present along the middle and distal portions of the villi. Rather, various mesenchymal cell types, including capillary endothelial cells, macrophages, and leukocytes, as well as fibroblasts, were located adjacent to the epithelial lamina. Furthermore, in contrast to the crypt region, collagen was sparse in the lamina of the villi. Thus, an organized subepithelial fibroblast sheath is seen only in the crypt region of mouse jejunum; it is not found throughout the mucosa as in the rabbit and human colon . It has been firmly established that epithelial cell renewal in the small intestinal mucosa is confined normally to the crypts Received January 29, 197 4. Accepted April3, 197 4. Presented in part at the 74th Annual Meeting of the American Gastroenterological Association in New York City, May, 1973. Address requests for reprints to: Dr. Jerry S. Trier, Gastroenterology Division, Department of Medicine, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115 . This investigation was supported by Research Grant AM-17537 from the National Institutes of Health, Bethesda, Maryland . Part of this study was carried out while Dr. Marsh was the recipient of a Traveling Fellowship of the Medical Research Council, Great Britain . Mrs . Jacqueline Beals provided valuable technical assistance.
of Lieberkuhn. 1- 5 Here, undifferentiated crypt cells proliferate and subsequently migrate onto the villi where they differentiate into mature columnar epithelial cells . However, far less is known about cell renewal among mesenchymal cells in the lamina propria of the small intestine. Attention has been drawn to a population of fibroblasts that is closely applied to the basal aspect of small intestinal, 6 gallbladder, 7 and colonic epithelia. 8 • 9 It has been shown that these subepithelial fibroblasts synthesize DNA and divide. 6 • 10 • 11 These fibroblasts have been carefully studied in rabbit colonic mucosa where 3 [ H ]thymidine radioautography indicated that proliferation of subepithelial fibroblasts is confined to the lower crypts, but
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that some newly formed cells subsequently migrate toward the luminal epithelium in synchrony with their adjacent epithelial cells. As these fibroblasts migrate from the lower crypts to surface epithelium, they show morphological evidence of maturation and even senescence when they reach the surface epithelium. Moreover, whereas the fibroblasts form a continuous sheath around the crypts, the sheath is discontinuous beneath the surface epithelium. 9 • 11 In view of these findings, it seemed important to determine whether a similar replicating subepithelial fibroblast sheath exists in the jejunum. Our intent was to examine the morphology of the fibroblast sheath and its relationship to the jejunal epithelium at all levels of the mucosa from crypts to villous tips. We also wished to determine the site of proliferation and subsequent migratory pattern of subepithelial fibroblasts, and to correlate migration of these fibroblasts to migration of epithelial cells in mouse jejunum. 12 ' 14 In this report, we describe our cytological and electron-microscopic findings of the subepithelial fibroblast sheath: in the companion paper which follows, 15 we describe the proliferation and migration of subepithelial fibroblasts as revealed by radioautography.
Methods Adult, random-bred albino mice (Charles River Laboratories, Wilmington, Mass.), weighing 35 to 40 g, were maintained on a diet of Purina chow (Ralston Purina Co., St. Louis, Mo.) and allowed liberal quantities of tap water. Animals were killed by cervical dislocation, and approximately 0.5 ml of chilled chrome-osmium fixative, 16 containing 0.2 Msucrose and 0.9 mM CaC1 2 , was slowly instilled into the lumen of the proximal jejunum. Slices of proximal jejunum were obtained a few minutes later, and then fixed by immersion in the same solution for 1 to 2 hr, transferred for 1 additional hr to 0.2 M phosphate-buffered formalin (pH 7 .2), dehydrated in graded ethanol solutions, embedded in epoxy resin, 17 and oriented for sectioning on aluminum rods, as described previously.' " Sections, 1 /J. in thickness, were cut with glass knives on a Sorvall MTB-2 ultramicrotome, stained with toluidine blue, 19 and examined with the light microscope. To permit precise identification ofsubepithe-
623
lial fibroblasts , and at the same time to facilitate the ultrastructural study of the subepithelial fibroblast sheath, thin sections adjacent to selected 1 /J. toluidine blue-stained sections were cut with a diamond knife on an LKB ultramicrotome, mounted on copper grids, stained with uranyl acetate 20 and lead citrate, 21 and examined with a Philips EM-300 electron microscope. Thus, individual cells seen with the light microscope were also examined with the electron microscope at precisely localized levels in the jejunal mucosa. In this way, a detailed, systematic study of the entire subepithelial region of mouse jejunum could be accomplished.
Results Light-Microscopic Appearance of Fibroblasts and Related Cells In toluidine blue-stained 1 f.L sections of mouse jejunum, subepithelial fibroblasts were identified as spindle-shaped cells with moderately stained cytoplasm (figs. 1 and 4). A few deeply stained granules, presumably lysosomes, were occasionally seen in the fibroblast cytoplasm . Fibroblast nuclei were elongated or ovoid; their margins were smooth or only slightly irregular. Macrophages tended to be more irregular in shape and larger in size than fibroblasts (fig. 6). Nuclei of macro phages lying alongside the epithelium varied in shape, but were frequently fusiform. However, they were usually larger and less densely stained than fibroblast nuclei. The presence of large numbers of densely stained lysosomes and other vacuoles within the macrophage cytoplasm aided their identification, but it was sometimes impossible to differentiate cytoplasm belonging to fibroblasts from that belonging to macrophages with the light microscope alone, unless the cell nucleus was present in the section. Although some endothelial cells were fusiform in appearance, and, therefore, closely resembled the appearance of fibroblasts (fig. 4), the majority exhibited subtle differences. In particular, their nuclear contours were frequently very irregular, and the surrounding cytoplasm was stained densely with toluidine blue (figs. 1 and 5), a feature which often facilitated their differentiation from fibroblasts with the light microscope. Also, the finding of
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FIG. 1. The inset is a 1 J1. toluidine blue-stained light micrograph which shows part of the pericryptal fibroblast sheath, the cytological features of its fibroblasts, and their prominent cytoplasmic processes · (P) . Note densely staining endothelial cell (EC) . The electron micrograph shows an adjacent thin section of the area within box in inset. This illustrates the fine structure of fibroblast nuclei, cytoplasm, and their processes (P) ; EP, epithelium; C, collagen ( x 6000; inset, x 1500).
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red blood cells in a capillary lumen often permitted identification of endothelial cells (figs. 4 and 5). However, in some instances it was virtually impossible to identify individual cells as fibroblasts or endothelial cells with certainty, without obtaining electron micrographs of adjacent thin sections (figs . 4 and 6) , since their light-microscopic features were so similar. Distribution of Subepithelial Fibroblasts Lower crypts. A well defined population of subepithelial fibroblasts was identified around the jejunal crypts (fig. 1). The cell bodies of these fibroblasts were fusiform or ovoid when sectioned, and gave rise to long, tapering cytoplasmic processes. Both cell bodies and their associated processes were closely applied to the basal aspect of the crypt epithelium (fig. 1). When the plane of sectioning was appropriate, some of the thin cytoplasmic processes could be traced along the base of as many as eight adjacent epithelial cells. Overlapping of adjacent fibroblasts was observed often around the basal portions of the crypts, but only ocasionally along the upper crypts. In the intercellular regions between adjacent crypts, collagen fibrils were abundant and were often in contact with fibroblasts and with the epithelial basal lamina. Upper crypts and proximal portions of villi. In this region of the mucosa, a change in the morphological appearance of the subepithelial fibroblast sheath was evident. A fairly well defined sheath could still be identified extending from the upper crypts into the basal portions of some of the villi (fig. 2) . The fibroblasts comprising the sheath at this level of the mucosa were identical to those occupying the lower crypts, both in their fine structure and in their relationship to the epithelial basal lamina . However, in other regions, other types of mesenchymal cells, especially plasma cells , capillary endothelial cells, and occaFIG. 2. In this electron micrograph from the upper CJYpt region, a fibroblast (F) gives rise to a long, sheet-like cytoplasmic process (arrows ) which pa sses closely along the basal lamina of the epithelium.
There is overlap with adjacent fibroblasts (F2, F3) ( x 4,000).
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sional leukocytes and macrophages, now interrupted the fibroblast sheath and were in direct contact with the epithelial basal lamina (fig. 3). Collagen was also less abundant than in the subepithelial spaces between the lower crypt epithelium. This area of the mucosa, therefore, was representative of a transitional zone which, in its lower part, was in continuity with the well defined fibroblast sheath surrounding the bases of the crypts, but which, in the upper part, differed in that the fibroblast sheath was now discontinuous. Middle and distal portions of the uilli. In the middle and the distal portions of the villi a fibroblast sheath was no longer apparent (figs. 4, 5, 6, and 7). Instead, a variety of mesenchymal cells, in addition to fibroblasts, occupied the space just beneath the epithelial basal lamina. A large proportion of these cells were endothelial cells whose attenuated, fenestrated cytoplasm formed an extensive subepithelial capillary network (figs. 4 and 5). Thin processes of fibroblast cytoplasm located directly beneath the basal lamina were interposed between the basal lamina and the endothelial cells, but did not form a complete sheath (figs. 4, 5, and 6). Of other mesenchymal cells besides endothelial cells that abutted on the epithelium, macrophages and plasma cells were most common. In addition, the epithelial lamina together with its associated subepithelial tissues were interrupted where lymphocytes and eosinophils traversed the lamina to enter the intercellular space between epithelial cells, and, less frequently, where pseudopod-like projections of basal epithelial cell cytoplasm penetrated the basal lamina and made direct contact with various mesenchymal cells (fig. 8). At the tips of the villi, the subepithelial spaces were often occupied by capillary endothelial cells (fig. 9). However, in such areas, a few thin fibroblast cytoplasmic processes were seen with the electron microscope to be located between epithelium and the capillary basement membrane (fig. 9). Although epithelial cells were frequently observed in the process of extrusion from the villous tips, extrusion of
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fibroblasts into the lumen from this site was not observed. Electron-Microscopic Features of Sub epithelial Fibroblasts
The electon-microscopic features of subepithelial fibroblasts in the jejunum correspond in general to descriptions of fibroblasts in other tissues. 22 • 23 Typically, sectioned fibroblast nuclei were fusiform and enclosed by inner and outer nuclear membranes . There were dense accumulations of chromatin along the inner nuclear membrane, and several nucleoli were frequently observed (figs. 1 and 2). The perinuclear cytoplasm (fig. 10) contained mitochondria and scattered accumulations of Golgi material. Granular endoplasmic reticulum was abundant. In addition, spirallike polyribosomes, individual free ribosomes , and small cytoplasmic vesicles, spherical in section, were also present throughout the cytoplasm. The elongated cytoplasmic processes of pericryptal fibroblasts were also packed with similar organelles, with the exception of Golgi material (fig. 11) . The fine structure of subepithelial fibroblasts along the outer parts of the villi was similar to that of fibroblasts surrounding the lower crypts, with one major difference. The peripheral cytoplasmic projections of pericryptal fibroblasts were sheath-like extensions (fig. 2). However, the peripheral cytoplasmic processes of fibroblasts in the distal portion of the villi were extremely slender, thin, finger-like structures which were seen, therefore, only where they crossed the plane of sectioning (figs. 4, 5, 6, and 9). They appeared as small fragments of cytoplasm beneath the epithelial and basal lamina, and it was usually difficult to relate a given fragment to its fibroblast cell body. On the other hand, centrally located cell bodies frequently appeared as isolated nuclei surrounded by a thin layer of cytoplasm (figs . 6 and 7).
Discussion In this study, precise identification of the cell types adjacent to the epithelial basal lamina was achieved by examining
FIG. 3. This is a representative electron micrograph of the region of the crypt-villus junction. Here, fibroblast processes (arrows) comprise a fairly substantial but incomplete sheath. Note presence of subepithelial capillary (CAP), and below another capillary profile with an endothelial cell (EC) cut through the plane of its nucleus . Various other mesenchymal cells, e.g., plasma cells (P) and an eosinophil (EO) lie in very close proximity to the epithelial basal lamina ( x 5,000) . 6:27
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FIG. 4. The 1 p. Epon section shown in the inset is from the junction of the proximal and middle one-third of the villus. The fibroblast (cell I), with its densely stained process extending upwards, is easy to spot. The cell beneath (cell 2) might easily be mistakenly identified as a fibroblast by light microscopy. Electron microscopy of an adjacent thin section shows cell2 to be an endothelial cell . Small arrows delineate the capillary lumen. On the opposite side is a larger vessel with a red cell in its lumen. Only a few fragments of fibroblast cytoplasm (arrowheads) are present between this capillary and the epithelium. Thus, there is no well defined sheath at this level of the jejunal mucosa ( x 5,000; inset, x 600).
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FIG. 5. The inset is a light micrograph of the junctional zone between the middle and distal one-third of a villus. Note the extensive capillary network with typical, densely stained endothelial cell nuclei; R, erythrocyte. A detailed view of the right hand network is shown in the adjacent thin section examined by electron microsco py . The three panels, A, B , and C, refer to corresponding areas marked in the inset. Note the absence of a fibroblast sheath ; in fact , hardly any fibroblast cytoplasm is present along t his broad expanse of basal villous epithelium, save for a few thin cell processes (arrows); EC, endothelial cell ( x 5,000; inset, x 800) .
with the electron microscope "thin" sec- became clear that it was occasionally diffitions cut adjacent to selected toluidine cult, and , in a few instances, virtually blue-stained 1 J.L "thick" sections . In this impossible, to identify with certainty, with way, it was possible to evaluate systemati- the light microscope, the nature of every cally the cytology of the entire subepithe- cell lying against the base of the epithelium lial cell · population and correlate light- (figs. 1, 4, and 5). When such difficulties microscopic · with electron-microscopic arose, it usually was the differentiation structural features. Using this approach, it between fibroblasts and endothelial cells
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FIG. 6. A light micrograph of the upper one-third of a villus, except for its tip, is shown in the inset. A large number of cells occupy the lamina propria, but fibroblasts are not obvious. Details of the upper box are shown in the accompanying electron micrograph, and of the lower box are shown in figure 7. Two very densely stained cells in the upper box of the light micrograph are seen to be a fibroblast (F) and an endothelial cell (EC) in the electron micrograph. The remaining lamina propria is largely occupied by a macrophage (M) . This micrograph illustrates well the thin, narrow fibroblast processes which appear as isolated fragments of cytoplasm (arrows) when sectioned. AG, endocrine epithelial cell ( x 4,000; inset, x 400).
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FIG. 7. The lower box outlined in the inset of figure 6 contains an erythrocyte (R) and a fibroblast (F) . The latter has the appearance frequently noted on the villi of a nucleus surrounded by a thin rim of cytoplasm. P, Plasma cell; L , lymphocyte ( x 5,000).
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chymal cell populations and permitted better characterization of light-microscopic features of the specific cell types. Our findings indicate that a well developed fibroblast sheath surrounds the lower half of the crypts in the proximal small intestine of mice. At this level of the mucosa, the fibroblasts are closely applied to the basal aspect of almost all epithelial cells; indeed, there often is considerable overlap between adjacent fibroblasts. This results, in some areas, in a multilayered or shingled sheath of fibroblasts applied to the crypts (fig. 2). At the mouth of the crypts and at the base of the villi, the fibroblast sheath becomes less well defined, and other mesenchymal cell types, such as endothelial cells, macrophages, and plasma cells, are interposed between fibroblasts and make direct contact with the basal surface of epithelial cells. In the villi, the gaps between fibroblasts and their peripheral cell processes increase in size and frequency to such a degree that no well defined fibroblast sheath is present in the distal two-thirds of the villi (fig. 12). A structural feature of individual fibroblasts, which differs with their location in the jejunal mucosa, was the nature of the cytoplasm peripheral to the central perinuclear portion of the cells. Around the crypts the peripheral cytoplasm projected from the fibroblasts in the form of thin, sheetlike extensions which contributed to the continuity of the fibroblast sheath in this region of the mucosa (figs. 1, 2, and 12). In contrast, in the villi, the peripheral cytoplasmic processes were thin, finger-like projections (figs. 4, 9, and 12). The conversion to finger-like from sheet-like peripheral cytoplasmic projections contributed to FIG. 8. This electron micrography shows an epi- the discontinuous nature of the sheath in thelial basal cytoplasmic process (P) interrupting the the more distal regions of the mucosa (fig. continuity of the basal lamina and subepithelial 12). Additionally, collagen was abundant tissues in the middle one-third of the villus . The in the intercellular spaces of the lamina process is in direct contact with cytoplasm of unidenpropria surrounding the crypts, but was tified cell, probably a fibroblast cytoplasmic process. quite sparse in the intercellular spaces of Below is a fibroblast nucleus with its thin rim of the lamina propria of the villi. cytoplasm; collagen is sparse at this level of the Our findings indicate that distinct difmucosa ( x 10,000). ferences exist between the subepithelial (see figs. 4 and 5). Thus, electron micros- fibroblast sheath of the small intestine of copy was particularly valuable in defining the mouse and that described in the colon the nature of these subepithelial mesen- of rabbits and man. 9 • 11 In the small intes-
FIG. 9. This electron micrograph shows a villus cross-sectioned immediately beneath its tip. Widened intercellular spaces (x) are seen between adjacent epithelial cells . The subepithelial space is almost entirely filled by a capillary ; only a few fragments of fibroblast cytoplasm (arrows) are interposed between vascular and epithelial basal laminae (x 6,000).
FIG. 10. Electron micrograph of perinuclear cytoplasm of a pericryptal fibroblast. Note the abundance of scattered Golgi material (G), dilated sacs of granular endoplasmic reticulum containing a moderately electrondense material, mitochondria, and a probable lysosome (L). Arrowhead points to polyribosome clusters (x 16,000). 63:1
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FIG. 11. Peripheral cytoplasm from a pericryptal fibroblast with distended profiles of endoplasmic reticulum, a mitochondrion, and a few small vesicles ( x 16,000).
VIlli
FIG. 12. Schematic diagram of subepithelial fibroblast sheath, seen en fa ce on left , and in section on right . The vertical lines, X--X , indicate imaginary plane of section perpendicular to basement membrane, and the corresponding profiles are drawn on the right, thus recalling appearances actually observed during this study . Gray stippling, basement membrane ; corrugated lines, collagen fibers.
tine, as in the colon, the fibroblast sheath is continuous around the epithelium where epithelial cell proliferation is most active ; that is, around the base of the crypts. However, in the villi of the small intestine, the sheath becomes so discontinuous that it is no longer an organized, definable structure. In contrast, in the colon, the sheath remains definable at all levels of the colonic mucosa, although it too becomes less complete and somewhat fenestrated beneath the colonic surface epithelium. 9 Moreover, in the small intestine, intercellular collagen is abundant in the crypt region and sparse in the villi, whereas in the colon it is sparse in the crypt region and abundant beneath the surface epithelium. Finally, whereas fibroblasts beneath the surface epithelial cells of the colon contain more lysosome-like structures than those surrounding the crypts, 9 differences in the lysosome content of crypt and villous fibroblasts were not apparent in the small intestine. REFERENCES 1. Bizzozero G: Uber die regeneration der Elemente der schlauchformingen Drusen und des Epithels des Magendarmkanals. Anat Anz 3:781-974, 1888 2. Leblond CP, Walker BE: Renewal of cell popula· tions. Physiol Rev 36:255-276, 1956 3. Leblond CP, Messier P : The constant renewa l of the intestinal epithelium in the albino rat . Anat
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Rec 132:247-260, 1958 4. Lipkin M, Sherlock P, Bell B: Cell proliferation kinetics in the gastrointestinal tract of man. II. Cell renewal in stomach, duodenum, colon and rectum . Gastroenterology 45:721-729, 1963 5. MacDonald WC, Trier JS, Everett NB: Cell proliferation and migration in stomach, duodenum and rectum of man. Gastroenterology 46:405-417, 1964 6. Deane HW: Some electron microscopic observations on the lamina propria of the gut, with comments on the close association of macrophages, plasma cells and eosinophils. Anat Rec 149:453-473, 1964 7. Kaye GI, Wheeler HO, Whitlock RT eta!: Fluid transport in the rabbit gall bladder. A combined physiological and electron microscopic study. J Cell Bioi 30:237-268, 1966 8. Donellan WL: The structure of the colonic mucosa . The epithelium and subepithelial reticulohistiocytic complex. Gastroenterology 49:496514, 1965 9. Kaye GI , Lane N , Pascal RR: Colonic pericryptal fibroblast sheath: replication, migration, and cytodifferentiation of a mesenchymal cell system in adult tissue . II . Fine structural aspects of normal rabbit and human colon . Gastroenterology 54:852- 865, 1968 10. Kaye GI, Maenza RM, Lane N: Cell replication in rabbit gall bladder. An autoradiographic study of epithelial and associated fibroblast renewal in vivo and in vitro. Gastroenterology 51:670-680, 1966 11. Pascal RR, Kaye GI, Lane N: Colonic pericryptal fibroblast sheath: replication , migration and cytodifferentiation of a mesenchymal system in adult tissue. I. Autoradiographic studies of normal rabbit colon. Gastroenterology 54:835- 851 ,
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1968 12. Quastler H, Sherman FG: Cell population kinetics in the intestinal epithelium of the mouse. Exp Cell Res 17:420-438, 1959 13. Lesher S, Fry RJM, Kohn HI: Influ~nce of age on transit time of cells of mouse intestinal epithelium. I. Duodenum. Lab Invest 10:291-300, 1961 14. Hagemann RF, Sigdestad CP, Lesher S: A quantitative description of the intestinal epithelium of the mouse. Am J Anat 129:41-52, 1970 15. Marsh MN, Trier JS : The morphology and cell proliferation of subepithelial fibroblasts in adult mouse jejunum. II. Radioautographic studies. Gastroenterology 67:636-645, 1974 16. Dalton AJ: A chrome-osmium fixative for electron microscopy (abstr.) Anat Rec 121:281, 1955 17. Luft JH: Improvements in epoxy resin embedding methods . J Biophys Biochem Cytol 9:409-414, 1961 18. Trier JS, Lorenzsonn V, Groehler K: Pattern of secretion of Paneth cells of the small intestine of mice. Gastroenterology 53:240-249, 1967 19. Trump BF, Smuckler EA, Benditt EP: A method for staining epoxy sections for light microscopy. J Ultrastruct Res 5:343-348, 1961 20. Watson ML: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475- 478, 1958 21. Reynolds ES: The use oflead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Bioi 17:208-212, 1963 22. Movat HZ , Fernando NVP: Fine structure of connective tissue. I. The fibroblast. Exp Mol Pathol 1:509- 534, 1962 23. Ross R: The connective tissue fiber forming cell . In Treatise on Collagen, vol 2(4) . New York, Academic Press, 1968, p 1- 82