JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 16115-24 (1990)
Extrusion of Colonic Epithelial Cells In Vitro DAVID A. BARON AND DONALD H. MILLER Departments of Cell and Molecular Pharmacology (D.A.B.,D.H.M.) and Anatomy and Cell Biology (D.A.B.),Medical University of South Carolina, Charleston, South Carolina 29425
KEY WORDS
Intestine, Mucosal barrier, Tight junction, Freeze-fracture
ABSTRACT
Rat colonic mucosae fixed in situ in Ussing chambers provided a model of the extrusion of absorptive enterocytes and less commonly of goblet and enteroendocrine cells. The cells were lost at extrusion zones midway between crypt mouths. Even in mucosae in which the number of extruding cells was large, epithelial continuity was maintained as evaluated morphologically and electrophysiologically. Beneath points of remaining contact between desquamating cells and the epithelial sheet, microfilaments of the terminal web formed band-like structures linking adjacent junctional complexes. Freeze-fracture replicas disclosed extensive macular regions of tight junction strands in the plasma membranes of desquamating cells. Tight junctions between newly neighboring cells were often irregular and often occurred beneath the terminal web region. Dithiothreitol enhanced cell loss and increased basal epithelial conductance, but histological continuity was maintained and the mucosae continued to respond typically to bradykinin. These observations suggest that during the loss of senescent enterocytes, tight junctions are maintained; old junctional elements are lost, and tight junctions are formed between remaining adjacent cells. This model offers a means to study the synthesis and turnover of tight junctions and the maintenance of the colonic epithelial barrier.
INTRODUCTION The terminal event of intestinal epithelial cell renewal is in principle as important as the mitotic cycle in determining whether the cell population remains constant, expands, or contracts. Yet the mechanism of cell loss in these epithelia has received little attention. Cell extrusion, in addition to being a determinant of epithelial cell number, must play a role in the maintenance of the epithelial barrier to water, ions, macromolecules, and organisms. Similarly, little attention has been paid to the mechanism of reconstitution of the colonic mucosal barrier following cell loss due to senescence or injury (Argenzio et al., 1988). One signal posited for the extrusion of senescent cells is the decreased synthesis or degradation of cell adhesion proteins (Altmann, 1975) in concert with a generalized decrease in enzyme synthesis and nuclear atrophy (Leblond, 1981). These changes occur in “predesquamating” cells concentrated in an extrusion zone defined anatomically by intestinal region (Nordstrom et al., 1968). Not all intestinal epithelia such as the duodenum, however, have a clearly defined extrusion zone (Partridge and Simpson, 1980). The use of mouse aggregation chimeras has shown that extrusion zones in the colon exist between domains demarcated by the narrow cuff of surface epithelial cells surrounding the mouth of the crypts (Schmidt et al., 1985).In the rat 7.3 cells/crypt/hr are produced in the proliferative zone (Sunter et al., 1979); presumably the frequency of cell extrusion over time approximates this rate in the absence of injury or disease. Following acute injury the colonic barrier is rapidly reconstituted, which cannot be accounted for by increased cell proliferation (Craven et al., 1986). Recov-
0 1990 WILEY-LISS, INC.
ery of porcine colon following acute injury has been explained by accelerated cell migration which was evident a t 8 minutes and complete within 2 hours (Argenzio et al., 1988). Rat colonic epithelium damaged by bile salts recovered within 4 hours despite the significant exfoliation of surface cells, and was independent of the subsequent proliferative response (Craven et al., 1986). In contrast to the above injury-induced events, the observation of senescent enterocytes is a relatively rare event in the normal colonic mucosa. Gaps or denudation of the epithelial basal lamina has not been observed in normal animals (Linp, 1983). By scanning electron microscopy cell-sized gaps have been described (Potten and Allen, 1977) but it was not clear if the base of the gaps was defined by the epithelial basal lamina or by cytoplasmic extensions of adjacent cells. The present study was undertaken in an in vitro system in which exfoliation is more frequent or can be enhanced, and in which the electrophysiological response t o secretagogues such as bradykinin can be measured (Cuthbert and Margolius, 1982; Manning et al., 1982). Specifically, this study addresses the fate of tight junctions in the extrusion zone, the fate of senescent non-absorptive enterocytes, and the capacity of the coIonic mucosa to maintain cellular continuity over the epithelial basal lamina.
Received July 31, 1989; accepted in revised form August 30, 1989. David A. Baron, Ph.D., is currently at Searle Research and Development, G.D. Searle and Co.,4901 Searle Parkway, Skokie, IL 60077. Address reprint requests there.
16
D.A. BARON AND D.H. MILLER
Fig. 1. Colonic mucosa. This cross section defined by luminal absorptive cells (top)and muscularis mucosa (bottom) shows moderate cell loss from extrusion zones occurring in areas approximately equidistant from adjacent crypt mouths. Multiple extruding cells are often seen a t these areas. x 320.
MATERIALS AND METHODS
For routine microscopy the tissue was rinsed briefly in distilled water, post-fixed for 1hour in 2% OsO, in the Preparations of colonic mucosae were prepared a s same buffer, and rinsed again in distilled water. The described previously (Baron, et al., 1986). Rat descend- tissue was then stained en bloc with 0.5% uranyl aceing colon, stripped of serosa and major muscle layers, tate in 0.1 M Na-H-maleate buffer (pH 5.0) and dehywas voltage-clamped at a potential difference of zero in drated in graded ethanols followed by propylene oxide. Ussing chambers filled with Krebs-Henseleit solution, The 1-2 mm2 pieces were flat embedded so as to obtain and the resulting short circuit current and mucosal cross sections of the colonic mucosa. Thick sections (1 resistance were monitored. Tissues were subsequently pm) were stained with 1%toluidine blue. Thin sections fixed a t various intervals by the simultaneous addition were stained with lead citrate and saturated uranyl of 50% glutaraldehyde to the mucosal and serosal baths acetate and examined with a n Hitachi HS-8-2 electron to achieve a final concentration of 2%. After 15 min- microscope. utes, the muc0sa.e were minced, fixed continuously Tissues for freeze-fracture were stored in 0.1 M Naovernight in the same fixative, and washed three times cacodylate buffer (pH 7.4) until they were soaked in over 15 minutes in 0.1 M Na-cacodylate buffer (pH 7.4). increasing concentrations of glycerol (5-25%) in the
EXTRUSION OF COLONIC EPITHELIAL CELLS IN VITRO
Fig. 2. Extruding absorptive enterocytes. A This extrusion zone is midway between two goblet cells of adjacent crypt mouths. The epithelium is continuous and tight junctions are present between the newly neighboring cells and the extruding cell. The extruding cell is
17
highly vacuolated and has irregular microvilli along its abluminal borders. x 1,900. B: This freeze-fracture replica shows an extruding cell above the luminal plane. Tight junction strands can be seen in a cytoplasmic vacuole (arrow) and in the plasma membrane. x 14,500.
same buffer over the course of 1 hour. The square pieces, trimmed to fit the inner diameter of the wells of gold specimen caps and to protrude slightly, were inserted vertically so as to obtain a fracture perpendicular to the plane of the mucosa. The mucosae were frozen by immersion in liquid propane and fractured as sets of three with the cryomicrotome in a Balzer's 400T freeze-fracture-etch device. The specimen-stage temperature was -120°C and the knife temperature was - 196°C. The fractured surfaces were coated with platinum-carbon (20-25 & a t 45" and carbon (200 A) a t 90" for reinforcement. The replicas were cleaned with 25% Clorox for 1 hour, then with 100% Clorox overnight, and finally with 100% chromic acid for 4-6 hours. After washing with distilled water they were picked up on 400 mesh grids.
RESULTS Extrusion of numerous enterocytes from the luminal plane of the colonic mucosa occurred at points approximately midway between the mouths of the crypts (Fig. 1). These cells were highly vacuolated and often occurred in clusters. Cells immediately adjacent to these zones occasionally showed some vacuolization as well, but epithelial continuity appeared to be maintained. Extrusion zones were often recognizable as dimples in the luminal plane. Otherwise the remainder of the muwith the exception Of the lamina appeared propria, which was moderately edematous. The majority of senescent extruding cells were ab-
Fig. 3. Extruding goblet cell. The granules of this cell are intact. There is a n amorphous microfilamentous inclusion in the cytoplasm. The junctional relationships between this goblet cell and the enterocytes beneath are geometrically complex. x 4,000.
18
D.A.BARON AND D.H.MILLER
Fig. 4. Extruding enteroendocrine cell. This cell contains typical secretory granules and is normal in appearance compared with the colonocyte being extruded above it. The basal lamina is covered in this region by squamous extensions of newly adjacent cells terminating in complex tight junctions. x 4,000.
sorptive enterocytes. They often exhibited an irregular brush border on the abluminal aspect of their extruded surface (Fig. 2A). Epithelial continuity was maintained in these regions (Fig. 2A,B). Goblet cells (Fig. 3) and enteroendocrine cells (Fig. 4)were observed as well in the type of extrusion zones typical for absorptive enterocytes. Multiple cell types were occasionally lost from the same zone (Fig. 4).Although there was some evidence of nuclear condensation, goblet and enteroendocrine cells were not vacuolated and otherwise appeared normal. Again, the process of extrusion was consistent with the maintenance of epithelial continuity. In particular, the senescent cells appeared to maintain tight junctions with their neighboring cells during all phases of extrusion (Fig. 5). These tight junctions lost their typical perpendicular orientation to the basal lamina, often running parallel to the basal lamina. Tight junctions between newly neighboring cells frequently showed this type of orientation as well. Points of remaining contact with an extruding cell and its neighbors beneath appeared to be comprised of elements of tight junctions. By freeze-fracture the plasma membrane of an extruded cell showed a focal region of tight junction strands (Fig. 2B). These regions were not typically belt-like, more often occurring in discrete patches. Bands of microfilaments within the terminal web regions were prominent in cells beneath desquamating enterocytes. When these bands terminated in junc-
tional complexes the associated tight junction had a zigzag-like profile (Fig. 6). Pseudopodial cytoplasmic extensions were sometimes seen apical to these bands. No microvilli were present: these regions may actually have consisted of basolateral membrane although they were apically oriented beneath the extruding cell. These bands were also seen beneath extruding enteroendocrine cells (Fig. 7) and goblet cells. The bands often extended from one cell to the next via junctional complexes (Fig. 8). Hemidesmosomes were also observed in this region (Fig. 8). Tight junctions as visualized in freeze-fracture replicas were frequently present on the lateral plasmalemma1 of extruding cells beyond the apical plane of the mucosa (Fig. 9). These structures may be more appropriately termed “macula” occludens, since a common feature was the lack of a belt-like organization. Extensive regions of strands occurred either in vertically oriented domains (Fig. 10) or in circumscribed areas with no obvious orientation (Fig. 11). Less commonly strands were distributed more diffusely (Fig. 121, which may represent a less common cell type. Presumably these junctions (Figs. 9-12) were formed with the remaining neighboring cells beneath. However, regions corresponding to the location of these junctions were only occasionally found in thin sections (Fig. 3); rather, junctional contacts that remained with extruding cells were at their bases (Figs. 5, 6) and not their lateral domains, which often contained the remnants of the brush border (Fig. 2A). Structures identical in appearance to tight junction strands were commonly observed in the membranes of cytoplasmic vacuoles of extruding cells. Tight junctions between newly neighboring cells, so defined during the extrusion of senescent cells, were also non-belt-like. Regions of tight junctions occurred several p.m beneath their normal location as the most apical structure in the junctional complex (Fig. 13). Even in regions of apposition of three cells in which tight junctions normally extend basally, the lateral strands were diffused laterally. As seen in extruding cells, tight junction elements were often identified in cytoplasmic vacuoles. This complex distribution of tight junction strands as defined by freeze fracture corresponded with the complex geometric relationship between newly neighboring and extruding cells apparent in thin sections. Dithiothreitol (1 FM) was used initially as a mucolytic (Horvath et al., 1986). When added to the luminal side bath of the Ussing chamber for 60 minutes it resulted in the accelerated extrusion of colonocytes (Fig. 14). Despite the large numbers of cells lost at each extrusion zone, no instance of denuded epithelial basal lamina was found. In regions of intense desquamation surviving epithelial cells were squamous in profile presumably to cover the greatest possible surface area. Surviving cells in the extrusion zones were more vacuolated than in control preparations. Nevertheless, the anatomy of the extrusion process was qualitatively similar. The addition of dithiothreitol had no effect on the ability of the mucosa to respond to bradykinin M) with an augmented secretion of chloride corresponding to an increase in short circuit current (42.6
EXTRUSION OF COLONIC EPITHELIAL CELLS IN VITRO
19
Fig. 5. Relationships of extruding cell to newly neighboring cells beneath. The junctional planes are mostly parallel to the epithelial basal lamina. Remaining points of contact of the extruded cell and the
remaining epithelium appear to be tight junctions (arrows). Abutting pseudopodial extensions of two neighboring cells do not form a tight junction (*I. x 13,700.
5.2 pA10.6 cm2 in the presence of dithiothreitol, 39.3 7.7 pA10.6 cm2, control). However, basal epithelial conductance increased from 2.5 mS10.6 cm2 to 6.8 mS1 0.6 cm2 after the treatment with dithiothreitol (P < 0.002). DISCUSSION The extrusion zone, the region from which sensecent enterocytes are lost to the gut lumen, is defined anatomically by region. In the duodenum, the extrusion zone is poorly defined and cells may be lost to maturation pressure as columns of cells migrate toward the villus tip (Partridge and Simpson, 1980). In the jejunum and ileum, the extrusion zone is considered to be at the villus tip itself (Leblond, 1981; Nordstrom et al., 1968).The extrusion zone in the colon appears t o be the area of apposition between the circumferential regions surrounding the crypt mouths (Schmidt et al., 1985). The data presented here support this location and its preservation in vitro. During accelerated extrusion due to dithiothreitol, cells continued to be lost primarily from these zones perhaps reflecting their relative metabolic decline and increased susceptibility to injury and death. Cells in a “predesquamating” condition in
the extrusion zone show a decline in the activity of various enzymes with the exception of alkaline phosphatase (Nordstrom et al., 1968). In keeping with the notion of “ailing” cells prior to extrusion (Leblond, 1981) and the decline of enzymatic activity (Nordstrom et al., 19681, a general hypothesis has been forwarded that the synthesis and turnover of proteins responsible for cell-to-cell adhesion are altered in enterocytes destined to be lost (Altmann, 1975; Leblond, 1981). Alternatively, a mechanical signal mediated by the cytoskeleton or structural proteins in the junctional complex has been proposed to initiate the extrusion process (Hudspeth, 1982). Inhibition of DNA or protein synthesis does not prevent cell loss or more importantly the resealing of the epithelial barrier (Craven et al., 1986; Free1 et al., 1983). The maintenance of cell-to-cell adhesion must be affected during cell loss, but the mechanisms of the process of extrusion and resealing are unknown, whether mechanical, biochemical, or most likely, both. The structure of senescent absorptive enterocytes has been described in various regions of the gut (Leblond, 1981). Goblet cells in the extrusion zone have been noted, the inference being that they are extruded
%
20
D.A. BARON AND D.H. MILLER
Fig. 6. Terminal web region beneath extruding cell. A microfilamentous band extends to each junctional complex both of which are distorted in profile. Above the band and below the extruding cell no microvilli are present. x 13,100.
in much the same way as absorptive enterocytes (Merzel and Leblond, 1969). In this report we have described and illustrated the extrusion of goblet cells and enteroendocrine cells. In both cases the morphology of neighboring cells was comparable to those surrounding extruding absorptive enterocytes. Significantly, these cells were usually not vacuolated and otherwise showed little evidence of sensescence. The tight junction between a goblet cell and its neighbors is considered to be a shunt relative to the tight junctions between absorptive enterocytes (Madara et al., 1980). This difference did not, however, reveal itself morphologically. In no case, even during the desquamation of many cells from a n extrusion zone, was denuded epithelial basal lamina found. This supports the conclusion of others (Linp, 1983) and suggests that “holes” visualized by SEM a t the villus tip (Potten and Allen, 1977) may have been in fact lined abluminally by squamous extensions of adjacent cells. These data allow the conclusion that the colon has a considerable capacity to maintain a n intact epithelial barrier. This would not necessarily imply, however, that basal electrophysiological and permeability characteristics are similarly
maintained, though we have now measured these directly. Characteristic of the newly neighboring cells beneath a n extruding cell were bands of microfilaments consistent in size with actin running parallel to the basal lamina. Since these structures were seen in nearequatorial cross sections they were unlikely to represent the perijunctional actomyosin ring or the rootlets of the microvilli. These bands have been illustrated previously (Argenzio et al., 1988) and may be indicative of the involvement of cytoskeletal contractile events in the extrusion process. The fate of tight junctions during and after cell extrusion is unknown. The data presented here show that junctional elements are lost with the senescent cells as they are shed, that new tight junctions are formed between newly adjacent cells prior to cell loss, and that tight junctions appear to be maintained between the dying cell and the cells beneath it until the actual loss of the extruding cell. The architecture of these junctions is consistent with the fact that histological recovery is more rapid than electrophysiological recovery (Argenzio et al., 1988). Freeze-fracture replicas of tight junctions between newly neighboring cells and be-
EXTRUSION OF COLONIC EPITHELIAL CELLS IN VITRO
~web region ~ beneath ~ extruding i enkroendocrine ~ ~ pig, 7. ~ cell. The microfilamentous band in the terminal web region of the cell beneath the extruded cell and the complex geometry of tight junctions in this region are typical of areas containing extruding absorptive cells. x 7,300.
Fig. 8. Terminal web region beneath extruding cell. The microfilamentous bands in each of three cells of the extrusion zone appear to be connected via junctional complexes. A hemidesmosome is present apically. No microvilli are present. This region consists of basolateral membranes. x 7,900.
21
Fig. 9. Tight junction strands on extruding cell. These tight junction strands are in discrete zones which are macular in shape. The brush l borders of the cells beneath end at the stalk-like region of remaining contact with the extruded cell. x 8,900.
Fig. 10. Tight junction strands on extruding cell. These strands are oriented predominantly perpendicular to the mucosal plane rather than parallel as typically occurs away from extrusion zones. x 14,500.
22
D.A. BARON AND D.H. MILLER
Fig. 11. Tight junction strands on extruding cell. An extensive region of strands is situated in a macular area above a cytoplasmic vacuole. Above this region apical membrane and irregularly spaced microvilli are present. x 14,500.
Fig. 12. Tight junction strands on extruding cell. The plasma membrane of this cell contains an extensive region of diffusely arranged tight junction strands. It may be a goblet or enterendocrine cell. x 8,900.
Fig. 13. Tight junctions between newly neighboring cells in the extrusion zone. Junctional elements are present in the basolateral region of the cell well beneath the terminal web region (arrow). A basal extension of a tight junction between three cells extends many strands laterally (*). x 8,900.
tween these cells and the extruding cell show that they are more typically macular than zonular. As such it is not surprising that the conductance of the paracellular pathway increases as elicited by dithiothreitol-induced accelerated cell loss. The data suggest then that during the time course of cell loss a paracellular shunt develops which is again sealed after histological continuity is achieved. The process of cell loss and epithelial resealing appears to be similar during the normal cell cycle and as a result of epithelial cell damage; however, the number of paracellular shunts is increased during accelarated cell loss. This paracellular defect may become large enough to allow the passage of macromolecules as well as ions. The presence of tight junction strands in the plasma membranes of extruded cells and cytoplasmic vacuoles of the extruded cells as well as in the remaining cells beneath suggests that elements of the tight junction are recycled or degraded in the remaining viable cells and lost with the extruding cells. The persistence of tight junction strands after the apparent dissolution of the tight junction is consistent with the continued presence of strands following EDTA-induced cell dissociation (Madara and Marcial, 1984), antimycin-induced cell dissociation (Meldolesi et al.. 1978). after single cell isolation (Metz et al., 19771, and following prGeolytic enzyme dissociation ofpu~monaryepithelial cells 1987). The use of in vitrosystems such as the one employed here facilitates the simultaneous study
EXTRUSION OF COLONIC EPITHELIAL CELLS IN VITRO
23
Fig. 14. Colonic mucosa. This cross section shows accelerated cell loss relative to normal mucosae (e.g., Fig. 1) from extrusion zones 60 minutes after the luminal addition of dithiothreitol. Despite the increased cell loss the epithelial basal lamina is not exposed. x 320.
of the morphological and electrophysiological manifestations of cell loss in the colon. ACKNOWLEDGMENTS The expert assistance of Januet Mauney, Robert Ashcraft, and Gayle Hoffmeyer is greatly appreciated. These studies were supported in part by the National Institutes of Health Grants HL 29566 and HL 17705. REFERENCES Altmann, G.G. (1975) Morphological effects of a large single dose of cyclohexamide on the intestinal epithelium of the rat. Am. J. Anat., 143:219-239. Argenzio, R.A., Henrikson, C.K., and Liacos, J.A. (1988) Restitution of barrier and transport function of porcine colon after acute mucosal injury. Am. J . Physiol., 255:G62-G71.
Baron, D.A., Miller, D.H., and Margolius, H.S. (1986) Kinins induce rapid structural changes in colon concomitant with chloride secretion. Cell Tissue Res., 246:589-594. Craven, P.A., Pfanstiel, J., Saito, R., and DeRubertis, F.R. (1986) Relationship between loss of rat colonic surface epithelium induced by deoxycholate and initiation of the subsequent proliferative response. Cancer Res., 465754-5759. Cuthbert, A.W., and Margolius, H.S (1982) Kinins stimulate net chloride secretion by the rat colon. Br. J. Pharmacol., 75587-598. Freel, R.W., Hatch, M., Earnest, D.L., and Goldner, A.M. (1983) Role of tight-junctional pathways in bile salt-induced increases in colonic permeability. Am. J. Physiol., 245:G816-G823. Friend, D.S. (1987) Loss of square othogonal arrays from cultured airway epithelial cells. J. Electron Microsc. Tech., 6:237-246. Horvath, P.J., Ferriola, P.C., Weiser, M.M., and Duffey, M.E. (1986) Localization of chloride secretion in rabbit colon: inhibition by anthracene-9-carboxylic acid. Am. J. Physiol., 250:G1854190. Hudspeth, A.J. (1982) The recovery of local transepithelial resistance following single-cell lesions. Exp. Cell Res., 138:331-342.
24
D.A. BARON AND D.H. MILLER
Leblond, C.P. (1981)The life history of cells in renewing systems. Am. J . Anat., 160:113-158. Linp, V.W. (1983) Ultrastrukturelle befunde zur zellextrusion a n den villi intestinales der Maus. Acta Histochem. [Suppl.] (Jena), 27: 195-206. Madara, J.L., and Marcial, M.A. (1984) Structural correlates of intestinal tight-junction permeability. In: Mechanisms of Intestinal Electrolyte Transport and Regulation by Calcium. M. Donowitz and G.W.G. Sharp, eds. Alan R. Liss, Inc., New York, pp. 77-100. Madara, J.L., Trier, J.S. and Neutra, M.R. (1980) Structural changes in the plasma membrane accompanying differentiation of epithelial cells in human and monkey small intestine. Gastroenterology 78: 963-975. Manning, D.C., Snyder, S.H., Kachur, J.F., Miller, R.J., and Field, M. (1982) Bradykinin receptor mediated chloride secretion in intestinal function. Nature, 299256-259. Meldolesi, J., Castiglioni, G., Parma, R., Nassivera, N., and DeCamilli, P. (1978) Ca' +-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs. J. Cell Biol., 79:156-172.
Merzel, J., and Leblond, C.P. (1969) Origin and renewal of goblet cells in the epithelium of the mouse small intestine. Am. J . Anat., 124: 281-306. Metz, J . , Forssman, W.G., and Ho, S. (1977) Exocrine pancreas under experimental conditions. 111. Membrane and cell junctions in isolated acinar cells. Cell Tissue Res., 177:459-474. Nordstrom, C., Dahlqvist, A., and Josefsson, L. (1968) Quantitative determination of enzymes in different parts of the villi and crypts of rat small intestine. Comparison of alkaline phosphatase, disaccharidases and dipeptidases. J . Histochem. Cytochem., 15713-721. Partridge, B.T., and Simpson, L.O. (1980) Duodenal epithelial cell migration and loss in NZB mice. Micron, 11:63-72. Potten, C.S., and Allen, T.D. (1977) Ultrastructure of cell loss in intestinal mucosa. J. Ultrastruct. Res., 60:272-277. Schmidt, G.H., Wilkinson, M.M., and Ponder, B.A.J. (1985) Cell migration pathway in the intestinal epithelium: An in situ marker system using mouse aggregation chimeras. Cell, 40:425-429. Sunter, J.P., Watson, A.J., Wright, N.A., and Appleton, D.R. (1979) Cell proliferation at different sites along the length of the rat colon. Virchows Arch. IS1 32:75-87.
Extrusion of Colonic Epithelial Cells In Vitro DAVID A. BARON AND DONALD H. MILLER Departments of Cell and Molecular Pharmacology (D.A.B.,D.H.M.) and Anatomy and Cell Biology (D.A.B.),Medical University of South Carolina, Charleston, South Carolina 29425
KEY WORDS
Intestine, Mucosal barrier, Tight junction, Freeze-fracture
ABSTRACT
Rat colonic mucosae fixed in situ in Ussing chambers provided a model of the extrusion of absorptive enterocytes and less commonly of goblet and enteroendocrine cells. The cells were lost at extrusion zones midway between crypt mouths. Even in mucosae in which the number of extruding cells was large, epithelial continuity was maintained as evaluated morphologically and electrophysiologically. Beneath points of remaining contact between desquamating cells and the epithelial sheet, microfilaments of the terminal web formed band-like structures linking adjacent junctional complexes. Freeze-fracture replicas disclosed extensive macular regions of tight junction strands in the plasma membranes of desquamating cells. Tight junctions between newly neighboring cells were often irregular and often occurred beneath the terminal web region. Dithiothreitol enhanced cell loss and increased basal epithelial conductance, but histological continuity was maintained and the mucosae continued to respond typically to bradykinin. These observations suggest that during the loss of senescent enterocytes, tight junctions are maintained; old junctional elements are lost, and tight junctions are formed between remaining adjacent cells. This model offers a means to study the synthesis and turnover of tight junctions and the maintenance of the colonic epithelial barrier.
INTRODUCTION The terminal event of intestinal epithelial cell renewal is in principle as important as the mitotic cycle in determining whether the cell population remains constant, expands, or contracts. Yet the mechanism of cell loss in these epithelia has received little attention. Cell extrusion, in addition to being a determinant of epithelial cell number, must play a role in the maintenance of the epithelial barrier to water, ions, macromolecules, and organisms. Similarly, little attention has been paid to the mechanism of reconstitution of the colonic mucosal barrier following cell loss due to senescence or injury (Argenzio et al., 1988). One signal posited for the extrusion of senescent cells is the decreased synthesis or degradation of cell adhesion proteins (Altmann, 1975) in concert with a generalized decrease in enzyme synthesis and nuclear atrophy (Leblond, 1981). These changes occur in “predesquamating” cells concentrated in an extrusion zone defined anatomically by intestinal region (Nordstrom et al., 1968). Not all intestinal epithelia such as the duodenum, however, have a clearly defined extrusion zone (Partridge and Simpson, 1980). The use of mouse aggregation chimeras has shown that extrusion zones in the colon exist between domains demarcated by the narrow cuff of surface epithelial cells surrounding the mouth of the crypts (Schmidt et al., 1985).In the rat 7.3 cells/crypt/hr are produced in the proliferative zone (Sunter et al., 1979); presumably the frequency of cell extrusion over time approximates this rate in the absence of injury or disease. Following acute injury the colonic barrier is rapidly reconstituted, which cannot be accounted for by increased cell proliferation (Craven et al., 1986). Recov-
0 1990 WILEY-LISS, INC.
ery of porcine colon following acute injury has been explained by accelerated cell migration which was evident a t 8 minutes and complete within 2 hours (Argenzio et al., 1988). Rat colonic epithelium damaged by bile salts recovered within 4 hours despite the significant exfoliation of surface cells, and was independent of the subsequent proliferative response (Craven et al., 1986). In contrast to the above injury-induced events, the observation of senescent enterocytes is a relatively rare event in the normal colonic mucosa. Gaps or denudation of the epithelial basal lamina has not been observed in normal animals (Linp, 1983). By scanning electron microscopy cell-sized gaps have been described (Potten and Allen, 1977) but it was not clear if the base of the gaps was defined by the epithelial basal lamina or by cytoplasmic extensions of adjacent cells. The present study was undertaken in an in vitro system in which exfoliation is more frequent or can be enhanced, and in which the electrophysiological response t o secretagogues such as bradykinin can be measured (Cuthbert and Margolius, 1982; Manning et al., 1982). Specifically, this study addresses the fate of tight junctions in the extrusion zone, the fate of senescent non-absorptive enterocytes, and the capacity of the coIonic mucosa to maintain cellular continuity over the epithelial basal lamina.
Received July 31, 1989; accepted in revised form August 30, 1989. David A. Baron, Ph.D., is currently at Searle Research and Development, G.D. Searle and Co.,4901 Searle Parkway, Skokie, IL 60077. Address reprint requests there.
16
D.A. BARON AND D.H. MILLER
Fig. 1. Colonic mucosa. This cross section defined by luminal absorptive cells (top)and muscularis mucosa (bottom) shows moderate cell loss from extrusion zones occurring in areas approximately equidistant from adjacent crypt mouths. Multiple extruding cells are often seen a t these areas. x 320.
MATERIALS AND METHODS
For routine microscopy the tissue was rinsed briefly in distilled water, post-fixed for 1hour in 2% OsO, in the Preparations of colonic mucosae were prepared a s same buffer, and rinsed again in distilled water. The described previously (Baron, et al., 1986). Rat descend- tissue was then stained en bloc with 0.5% uranyl aceing colon, stripped of serosa and major muscle layers, tate in 0.1 M Na-H-maleate buffer (pH 5.0) and dehywas voltage-clamped at a potential difference of zero in drated in graded ethanols followed by propylene oxide. Ussing chambers filled with Krebs-Henseleit solution, The 1-2 mm2 pieces were flat embedded so as to obtain and the resulting short circuit current and mucosal cross sections of the colonic mucosa. Thick sections (1 resistance were monitored. Tissues were subsequently pm) were stained with 1%toluidine blue. Thin sections fixed a t various intervals by the simultaneous addition were stained with lead citrate and saturated uranyl of 50% glutaraldehyde to the mucosal and serosal baths acetate and examined with a n Hitachi HS-8-2 electron to achieve a final concentration of 2%. After 15 min- microscope. utes, the muc0sa.e were minced, fixed continuously Tissues for freeze-fracture were stored in 0.1 M Naovernight in the same fixative, and washed three times cacodylate buffer (pH 7.4) until they were soaked in over 15 minutes in 0.1 M Na-cacodylate buffer (pH 7.4). increasing concentrations of glycerol (5-25%) in the
EXTRUSION OF COLONIC EPITHELIAL CELLS IN VITRO
Fig. 2. Extruding absorptive enterocytes. A This extrusion zone is midway between two goblet cells of adjacent crypt mouths. The epithelium is continuous and tight junctions are present between the newly neighboring cells and the extruding cell. The extruding cell is
17
highly vacuolated and has irregular microvilli along its abluminal borders. x 1,900. B: This freeze-fracture replica shows an extruding cell above the luminal plane. Tight junction strands can be seen in a cytoplasmic vacuole (arrow) and in the plasma membrane. x 14,500.
same buffer over the course of 1 hour. The square pieces, trimmed to fit the inner diameter of the wells of gold specimen caps and to protrude slightly, were inserted vertically so as to obtain a fracture perpendicular to the plane of the mucosa. The mucosae were frozen by immersion in liquid propane and fractured as sets of three with the cryomicrotome in a Balzer's 400T freeze-fracture-etch device. The specimen-stage temperature was -120°C and the knife temperature was - 196°C. The fractured surfaces were coated with platinum-carbon (20-25 & a t 45" and carbon (200 A) a t 90" for reinforcement. The replicas were cleaned with 25% Clorox for 1 hour, then with 100% Clorox overnight, and finally with 100% chromic acid for 4-6 hours. After washing with distilled water they were picked up on 400 mesh grids.
RESULTS Extrusion of numerous enterocytes from the luminal plane of the colonic mucosa occurred at points approximately midway between the mouths of the crypts (Fig. 1). These cells were highly vacuolated and often occurred in clusters. Cells immediately adjacent to these zones occasionally showed some vacuolization as well, but epithelial continuity appeared to be maintained. Extrusion zones were often recognizable as dimples in the luminal plane. Otherwise the remainder of the muwith the exception Of the lamina appeared propria, which was moderately edematous. The majority of senescent extruding cells were ab-
Fig. 3. Extruding goblet cell. The granules of this cell are intact. There is a n amorphous microfilamentous inclusion in the cytoplasm. The junctional relationships between this goblet cell and the enterocytes beneath are geometrically complex. x 4,000.
18
D.A.BARON AND D.H.MILLER
Fig. 4. Extruding enteroendocrine cell. This cell contains typical secretory granules and is normal in appearance compared with the colonocyte being extruded above it. The basal lamina is covered in this region by squamous extensions of newly adjacent cells terminating in complex tight junctions. x 4,000.
sorptive enterocytes. They often exhibited an irregular brush border on the abluminal aspect of their extruded surface (Fig. 2A). Epithelial continuity was maintained in these regions (Fig. 2A,B). Goblet cells (Fig. 3) and enteroendocrine cells (Fig. 4)were observed as well in the type of extrusion zones typical for absorptive enterocytes. Multiple cell types were occasionally lost from the same zone (Fig. 4).Although there was some evidence of nuclear condensation, goblet and enteroendocrine cells were not vacuolated and otherwise appeared normal. Again, the process of extrusion was consistent with the maintenance of epithelial continuity. In particular, the senescent cells appeared to maintain tight junctions with their neighboring cells during all phases of extrusion (Fig. 5). These tight junctions lost their typical perpendicular orientation to the basal lamina, often running parallel to the basal lamina. Tight junctions between newly neighboring cells frequently showed this type of orientation as well. Points of remaining contact with an extruding cell and its neighbors beneath appeared to be comprised of elements of tight junctions. By freeze-fracture the plasma membrane of an extruded cell showed a focal region of tight junction strands (Fig. 2B). These regions were not typically belt-like, more often occurring in discrete patches. Bands of microfilaments within the terminal web regions were prominent in cells beneath desquamating enterocytes. When these bands terminated in junc-
tional complexes the associated tight junction had a zigzag-like profile (Fig. 6). Pseudopodial cytoplasmic extensions were sometimes seen apical to these bands. No microvilli were present: these regions may actually have consisted of basolateral membrane although they were apically oriented beneath the extruding cell. These bands were also seen beneath extruding enteroendocrine cells (Fig. 7) and goblet cells. The bands often extended from one cell to the next via junctional complexes (Fig. 8). Hemidesmosomes were also observed in this region (Fig. 8). Tight junctions as visualized in freeze-fracture replicas were frequently present on the lateral plasmalemma1 of extruding cells beyond the apical plane of the mucosa (Fig. 9). These structures may be more appropriately termed “macula” occludens, since a common feature was the lack of a belt-like organization. Extensive regions of strands occurred either in vertically oriented domains (Fig. 10) or in circumscribed areas with no obvious orientation (Fig. 11). Less commonly strands were distributed more diffusely (Fig. 121, which may represent a less common cell type. Presumably these junctions (Figs. 9-12) were formed with the remaining neighboring cells beneath. However, regions corresponding to the location of these junctions were only occasionally found in thin sections (Fig. 3); rather, junctional contacts that remained with extruding cells were at their bases (Figs. 5, 6) and not their lateral domains, which often contained the remnants of the brush border (Fig. 2A). Structures identical in appearance to tight junction strands were commonly observed in the membranes of cytoplasmic vacuoles of extruding cells. Tight junctions between newly neighboring cells, so defined during the extrusion of senescent cells, were also non-belt-like. Regions of tight junctions occurred several p.m beneath their normal location as the most apical structure in the junctional complex (Fig. 13). Even in regions of apposition of three cells in which tight junctions normally extend basally, the lateral strands were diffused laterally. As seen in extruding cells, tight junction elements were often identified in cytoplasmic vacuoles. This complex distribution of tight junction strands as defined by freeze fracture corresponded with the complex geometric relationship between newly neighboring and extruding cells apparent in thin sections. Dithiothreitol (1 FM) was used initially as a mucolytic (Horvath et al., 1986). When added to the luminal side bath of the Ussing chamber for 60 minutes it resulted in the accelerated extrusion of colonocytes (Fig. 14). Despite the large numbers of cells lost at each extrusion zone, no instance of denuded epithelial basal lamina was found. In regions of intense desquamation surviving epithelial cells were squamous in profile presumably to cover the greatest possible surface area. Surviving cells in the extrusion zones were more vacuolated than in control preparations. Nevertheless, the anatomy of the extrusion process was qualitatively similar. The addition of dithiothreitol had no effect on the ability of the mucosa to respond to bradykinin M) with an augmented secretion of chloride corresponding to an increase in short circuit current (42.6
EXTRUSION OF COLONIC EPITHELIAL CELLS IN VITRO
19
Fig. 5. Relationships of extruding cell to newly neighboring cells beneath. The junctional planes are mostly parallel to the epithelial basal lamina. Remaining points of contact of the extruded cell and the
remaining epithelium appear to be tight junctions (arrows). Abutting pseudopodial extensions of two neighboring cells do not form a tight junction (*I. x 13,700.
5.2 pA10.6 cm2 in the presence of dithiothreitol, 39.3 7.7 pA10.6 cm2, control). However, basal epithelial conductance increased from 2.5 mS10.6 cm2 to 6.8 mS1 0.6 cm2 after the treatment with dithiothreitol (P < 0.002). DISCUSSION The extrusion zone, the region from which sensecent enterocytes are lost to the gut lumen, is defined anatomically by region. In the duodenum, the extrusion zone is poorly defined and cells may be lost to maturation pressure as columns of cells migrate toward the villus tip (Partridge and Simpson, 1980). In the jejunum and ileum, the extrusion zone is considered to be at the villus tip itself (Leblond, 1981; Nordstrom et al., 1968).The extrusion zone in the colon appears t o be the area of apposition between the circumferential regions surrounding the crypt mouths (Schmidt et al., 1985). The data presented here support this location and its preservation in vitro. During accelerated extrusion due to dithiothreitol, cells continued to be lost primarily from these zones perhaps reflecting their relative metabolic decline and increased susceptibility to injury and death. Cells in a “predesquamating” condition in
the extrusion zone show a decline in the activity of various enzymes with the exception of alkaline phosphatase (Nordstrom et al., 1968). In keeping with the notion of “ailing” cells prior to extrusion (Leblond, 1981) and the decline of enzymatic activity (Nordstrom et al., 19681, a general hypothesis has been forwarded that the synthesis and turnover of proteins responsible for cell-to-cell adhesion are altered in enterocytes destined to be lost (Altmann, 1975; Leblond, 1981). Alternatively, a mechanical signal mediated by the cytoskeleton or structural proteins in the junctional complex has been proposed to initiate the extrusion process (Hudspeth, 1982). Inhibition of DNA or protein synthesis does not prevent cell loss or more importantly the resealing of the epithelial barrier (Craven et al., 1986; Free1 et al., 1983). The maintenance of cell-to-cell adhesion must be affected during cell loss, but the mechanisms of the process of extrusion and resealing are unknown, whether mechanical, biochemical, or most likely, both. The structure of senescent absorptive enterocytes has been described in various regions of the gut (Leblond, 1981). Goblet cells in the extrusion zone have been noted, the inference being that they are extruded
%
20
D.A. BARON AND D.H. MILLER
Fig. 6. Terminal web region beneath extruding cell. A microfilamentous band extends to each junctional complex both of which are distorted in profile. Above the band and below the extruding cell no microvilli are present. x 13,100.
in much the same way as absorptive enterocytes (Merzel and Leblond, 1969). In this report we have described and illustrated the extrusion of goblet cells and enteroendocrine cells. In both cases the morphology of neighboring cells was comparable to those surrounding extruding absorptive enterocytes. Significantly, these cells were usually not vacuolated and otherwise showed little evidence of sensescence. The tight junction between a goblet cell and its neighbors is considered to be a shunt relative to the tight junctions between absorptive enterocytes (Madara et al., 1980). This difference did not, however, reveal itself morphologically. In no case, even during the desquamation of many cells from a n extrusion zone, was denuded epithelial basal lamina found. This supports the conclusion of others (Linp, 1983) and suggests that “holes” visualized by SEM a t the villus tip (Potten and Allen, 1977) may have been in fact lined abluminally by squamous extensions of adjacent cells. These data allow the conclusion that the colon has a considerable capacity to maintain a n intact epithelial barrier. This would not necessarily imply, however, that basal electrophysiological and permeability characteristics are similarly
maintained, though we have now measured these directly. Characteristic of the newly neighboring cells beneath a n extruding cell were bands of microfilaments consistent in size with actin running parallel to the basal lamina. Since these structures were seen in nearequatorial cross sections they were unlikely to represent the perijunctional actomyosin ring or the rootlets of the microvilli. These bands have been illustrated previously (Argenzio et al., 1988) and may be indicative of the involvement of cytoskeletal contractile events in the extrusion process. The fate of tight junctions during and after cell extrusion is unknown. The data presented here show that junctional elements are lost with the senescent cells as they are shed, that new tight junctions are formed between newly adjacent cells prior to cell loss, and that tight junctions appear to be maintained between the dying cell and the cells beneath it until the actual loss of the extruding cell. The architecture of these junctions is consistent with the fact that histological recovery is more rapid than electrophysiological recovery (Argenzio et al., 1988). Freeze-fracture replicas of tight junctions between newly neighboring cells and be-
EXTRUSION OF COLONIC EPITHELIAL CELLS IN VITRO
~web region ~ beneath ~ extruding i enkroendocrine ~ ~ pig, 7. ~ cell. The microfilamentous band in the terminal web region of the cell beneath the extruded cell and the complex geometry of tight junctions in this region are typical of areas containing extruding absorptive cells. x 7,300.
Fig. 8. Terminal web region beneath extruding cell. The microfilamentous bands in each of three cells of the extrusion zone appear to be connected via junctional complexes. A hemidesmosome is present apically. No microvilli are present. This region consists of basolateral membranes. x 7,900.
21
Fig. 9. Tight junction strands on extruding cell. These tight junction strands are in discrete zones which are macular in shape. The brush l borders of the cells beneath end at the stalk-like region of remaining contact with the extruded cell. x 8,900.
Fig. 10. Tight junction strands on extruding cell. These strands are oriented predominantly perpendicular to the mucosal plane rather than parallel as typically occurs away from extrusion zones. x 14,500.
22
D.A. BARON AND D.H. MILLER
Fig. 11. Tight junction strands on extruding cell. An extensive region of strands is situated in a macular area above a cytoplasmic vacuole. Above this region apical membrane and irregularly spaced microvilli are present. x 14,500.
Fig. 12. Tight junction strands on extruding cell. The plasma membrane of this cell contains an extensive region of diffusely arranged tight junction strands. It may be a goblet or enterendocrine cell. x 8,900.
Fig. 13. Tight junctions between newly neighboring cells in the extrusion zone. Junctional elements are present in the basolateral region of the cell well beneath the terminal web region (arrow). A basal extension of a tight junction between three cells extends many strands laterally (*). x 8,900.
tween these cells and the extruding cell show that they are more typically macular than zonular. As such it is not surprising that the conductance of the paracellular pathway increases as elicited by dithiothreitol-induced accelerated cell loss. The data suggest then that during the time course of cell loss a paracellular shunt develops which is again sealed after histological continuity is achieved. The process of cell loss and epithelial resealing appears to be similar during the normal cell cycle and as a result of epithelial cell damage; however, the number of paracellular shunts is increased during accelarated cell loss. This paracellular defect may become large enough to allow the passage of macromolecules as well as ions. The presence of tight junction strands in the plasma membranes of extruded cells and cytoplasmic vacuoles of the extruded cells as well as in the remaining cells beneath suggests that elements of the tight junction are recycled or degraded in the remaining viable cells and lost with the extruding cells. The persistence of tight junction strands after the apparent dissolution of the tight junction is consistent with the continued presence of strands following EDTA-induced cell dissociation (Madara and Marcial, 1984), antimycin-induced cell dissociation (Meldolesi et al.. 1978). after single cell isolation (Metz et al., 19771, and following prGeolytic enzyme dissociation ofpu~monaryepithelial cells 1987). The use of in vitrosystems such as the one employed here facilitates the simultaneous study
EXTRUSION OF COLONIC EPITHELIAL CELLS IN VITRO
23
Fig. 14. Colonic mucosa. This cross section shows accelerated cell loss relative to normal mucosae (e.g., Fig. 1) from extrusion zones 60 minutes after the luminal addition of dithiothreitol. Despite the increased cell loss the epithelial basal lamina is not exposed. x 320.
of the morphological and electrophysiological manifestations of cell loss in the colon. ACKNOWLEDGMENTS The expert assistance of Januet Mauney, Robert Ashcraft, and Gayle Hoffmeyer is greatly appreciated. These studies were supported in part by the National Institutes of Health Grants HL 29566 and HL 17705. REFERENCES Altmann, G.G. (1975) Morphological effects of a large single dose of cyclohexamide on the intestinal epithelium of the rat. Am. J. Anat., 143:219-239. Argenzio, R.A., Henrikson, C.K., and Liacos, J.A. (1988) Restitution of barrier and transport function of porcine colon after acute mucosal injury. Am. J . Physiol., 255:G62-G71.
Baron, D.A., Miller, D.H., and Margolius, H.S. (1986) Kinins induce rapid structural changes in colon concomitant with chloride secretion. Cell Tissue Res., 246:589-594. Craven, P.A., Pfanstiel, J., Saito, R., and DeRubertis, F.R. (1986) Relationship between loss of rat colonic surface epithelium induced by deoxycholate and initiation of the subsequent proliferative response. Cancer Res., 465754-5759. Cuthbert, A.W., and Margolius, H.S (1982) Kinins stimulate net chloride secretion by the rat colon. Br. J. Pharmacol., 75587-598. Freel, R.W., Hatch, M., Earnest, D.L., and Goldner, A.M. (1983) Role of tight-junctional pathways in bile salt-induced increases in colonic permeability. Am. J. Physiol., 245:G816-G823. Friend, D.S. (1987) Loss of square othogonal arrays from cultured airway epithelial cells. J. Electron Microsc. Tech., 6:237-246. Horvath, P.J., Ferriola, P.C., Weiser, M.M., and Duffey, M.E. (1986) Localization of chloride secretion in rabbit colon: inhibition by anthracene-9-carboxylic acid. Am. J. Physiol., 250:G1854190. Hudspeth, A.J. (1982) The recovery of local transepithelial resistance following single-cell lesions. Exp. Cell Res., 138:331-342.
24
D.A. BARON AND D.H. MILLER
Leblond, C.P. (1981)The life history of cells in renewing systems. Am. J . Anat., 160:113-158. Linp, V.W. (1983) Ultrastrukturelle befunde zur zellextrusion a n den villi intestinales der Maus. Acta Histochem. [Suppl.] (Jena), 27: 195-206. Madara, J.L., and Marcial, M.A. (1984) Structural correlates of intestinal tight-junction permeability. In: Mechanisms of Intestinal Electrolyte Transport and Regulation by Calcium. M. Donowitz and G.W.G. Sharp, eds. Alan R. Liss, Inc., New York, pp. 77-100. Madara, J.L., Trier, J.S. and Neutra, M.R. (1980) Structural changes in the plasma membrane accompanying differentiation of epithelial cells in human and monkey small intestine. Gastroenterology 78: 963-975. Manning, D.C., Snyder, S.H., Kachur, J.F., Miller, R.J., and Field, M. (1982) Bradykinin receptor mediated chloride secretion in intestinal function. Nature, 299256-259. Meldolesi, J., Castiglioni, G., Parma, R., Nassivera, N., and DeCamilli, P. (1978) Ca' +-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs. J. Cell Biol., 79:156-172.
Merzel, J., and Leblond, C.P. (1969) Origin and renewal of goblet cells in the epithelium of the mouse small intestine. Am. J . Anat., 124: 281-306. Metz, J . , Forssman, W.G., and Ho, S. (1977) Exocrine pancreas under experimental conditions. 111. Membrane and cell junctions in isolated acinar cells. Cell Tissue Res., 177:459-474. Nordstrom, C., Dahlqvist, A., and Josefsson, L. (1968) Quantitative determination of enzymes in different parts of the villi and crypts of rat small intestine. Comparison of alkaline phosphatase, disaccharidases and dipeptidases. J . Histochem. Cytochem., 15713-721. Partridge, B.T., and Simpson, L.O. (1980) Duodenal epithelial cell migration and loss in NZB mice. Micron, 11:63-72. Potten, C.S., and Allen, T.D. (1977) Ultrastructure of cell loss in intestinal mucosa. J. Ultrastruct. Res., 60:272-277. Schmidt, G.H., Wilkinson, M.M., and Ponder, B.A.J. (1985) Cell migration pathway in the intestinal epithelium: An in situ marker system using mouse aggregation chimeras. Cell, 40:425-429. Sunter, J.P., Watson, A.J., Wright, N.A., and Appleton, D.R. (1979) Cell proliferation at different sites along the length of the rat colon. Virchows Arch. IS1 32:75-87.
