Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.

Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immunoelectron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67-positive epithelial cells were significantly decreased in the colonic crypts of periostin-/- mice (approximately 0.6-fold) compared with periostin+/+ mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger ( approximately 1.5-fold) when cultured with fibroblasts derived from periostin+/+ mice or periostin-transfected NIH3T3 cells than with those from periostin-/- mice or periostin-non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.     

Unaltered distribution of laminins, fibronectin, and tenascin in celiac intestinal mucosa.

Extensive remodeling of the extracellular matrix (ECM) occurs in inflammatory tissues. The celiac lesion in the small intestine is characterized by inflammation accompanied by profound morphological alterations. We used immunohistochemistry to determine the distribution of laminin, fibronectin, and tenascin isoforms in small intestinal biopsies of untreated patients with celiac disease. In normal mucosa, the distribution of laminin isoforms defines three epithelial basement membrane (BM) zones. We found that the organization of these zones was maintained in the celiac mucosa. Thus, components of laminin-5 (alpha3 and beta3) were found in the surface epithelial BM, laminin alpha2 chain was found selectively at crypt bottoms, and laminin alpha5 chain was the sole alpha-type chain in middle crypt BMs. Likewise, the distribution of fibronectin and tenascin resembled that of the normal gut. The organization of pericryptal fibroblasts and lamina propria smooth muscle strands, as defined by immunostaining for alpha-smooth muscle actin, also remained unchanged in the celiac mucosa. Unexpectedly, major ECM changes were not detected in the celiac lesion.     

Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium

Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.     

Immunocytochemical localization of thrombomodulin in the aqueous humor passage of the rat eye

 This report describes the distribution and localization of thrombomodulin (TM) in the rat eye by light and electron microscopic immunocytochemistry. In addition to the endothelium of the entire vasculature, TM was found on the non-vascular structures lining the cavities of the posterior and anterior chambers and the limbus. TM was localized on the basal, lateral, and apical plasma membranes of the inner and outer ciliary epithelium, and the posterior iris epithelium in which there was no polarized expression of TM. In the anterior chamber, TM was localized on the luminal surface of the corneal endothelium, but was negative on the anterior border layer of the iris, which is composed of a discontinuous layer of fibroblasts and collagen fibers. Thus, TM was present at sites of cell-to-cell contact. TM was also present on the endothelia of the trabecular meshwork and the Schlemm’s canal in the limbus. TM was localized not only on the luminal plasma membrane, but also on the cytoplasmic giant vacuoles in the endothelial cells of the Schlemm’s canal. These findings extend the importance of anticoagulant mechanisms to the systems of secretion, circulation, and drainage of the aqueous humor. Accepted: 18 March 1997     

Presence of a novel nuclear lamina protein in Raji cells.

In mammalian tissues, the nuclear lamina is composed of the major lamins A, B, and C, and minor lamins D/E. Although lamin B is present in all cell types, lamins A and C are absent from embryonic cells and most undifferentiated cells from hematopoietic lineage. We have investigated the nuclear lamina protein composition of the Raji cell line, lymphoblast-like cells established from a Burkitt lymphoma patient. Lamins A and C were confirmed absent by immunodetection and Northern blot analysis. Besides lamins B and D/E, a protein migrating around 71 kilodaltons was recognized by a serum directed against the nuclear lamina of BHK-21 fibroblasts. Cellular localization by sequential extraction established this 71-kilodalton protein as an exclusive component of the nuclear lamina fraction. These results indicate that the nuclear lamina has a more complex composition than previously thought to be the case for cells devoid of lamins A and C.     

Aldosterone Reduces Crypt Colon Permeability during Low-Sodium Adaptation

Fluid and electrolyte absorption by colonic crypts depends on the transport properties of crypt cellular and paracellular routes and of the pericryptal sheath. As a low-Na⁺ diet increases aldosterone and angiotensin II secretion, either hormone could affect absorption. Control and adrenalectomized (ADX) Sprague-Dawley rats were kept at a high-NaCl (HS) diet and then switched to low-NaCl (LS) diet for 3 days. Aldosterone or angiotensin II plasma concentrations were maintained using implanted osmotic mini-pumps. The extracellular Na⁺ concentration in isolated rat distal colonic mucosa was determined by confocal microscopy using a low-affinity Na⁺-sensitive fluorescent dye (Sodium red, and Na⁺-insensitive BODIPY) bound to polystyrene beads. Crypt permeability to FITC-labelled dextran (10 kDa) was monitored by its rate of escape from the crypt lumen into the pericryptal space. Mucosal ion permeability was estimated by transepithelial electrical resistance (TER) and amiloride-sensitive short-circuit current (SCC). The epithelial Na⁺ channel, ENaC, was determined by immunolocalization. LS diet decreased crypt wall permeability to dextran by 10-fold and doubled TER. Following ADX, aldosterone decreased crypt wall dextran permeability, increased TER, increased Na⁺ accumulation in the pericryptal sheath and ENaC expression even in HS. Infusion of angiotensin II to ADX rats did not reverse the effects of aldosterone deprivation. These findings indicate that aldosterone alone is responsible for both the increase in Na⁺ absorption and the decreased paracellular and pericryptal sheath permeability.     

The intestinal proliferon

The intestinal mucosa consists of several rapidly proliferating cell populations. These may be divided according to their function into two classes: (1) parenchyma: or epithelial cells; and (2) supporting cell populations including fibrocytes and vascular endothelial cells. The kinetics of all the cells in the villus are highly co-ordinated. It is postulated that at the pericryptal progenitor region where the cells originate, they are assembled into complex functional units denominated herewith as proliferons. The proliferon consists of four basic elements; parenchyma, connective tissue, blood vessels and nerve fibers. It starts its existence in the pericryptal lamina propria where the proliferating epithelia and fibroblasts receive their own capillary buds and special nerve supply. The displacement of the whole complex towards the villus tip is accompanied by a gradual elongation of the central villus artery. As the proliferon approaches the villus tip its supporting cell populations are catabolized while the epithelium is sloughed off into the lumen.This model has been previously called upon to describe the kinetics of rodent incisor tooth cell populations.     

Isolation of rat intestinal crypt cells

A technique is presented which yields single cells and intact crypts in suspension from unfixed rat intestinal mucosal epithelium. Everted lengths of intestine were digested by 27 mM sodium citrate in phosphate-buffered saline (pH = 7.3) at 37 degrees C. Mucosal cells were dislodged by vibratory stress (hand vortexing) following incubation for prescribed intervals at 37 degrees C in 1.5 mM ethylenediamine tetraacetic acid (EDTA) and 0.5 mM dithiothreitol (dtt). Alkaline phosphatase determinations, phase microscopy, and in vivo and in vitro evaluations of tritiated thymidine ([3H]TdR) incorporation were performed on isolated intestinal cells. Data indicate that cells were sequentially derived from villus tip to crypt base as judged by cellular morphology, alkaline phosphatase activity/mg protein and radioactivity per microgram protein. Upon completion of the intestinal cell isolation assay, scanning electron microscopy of the remaining intestine revealed that approximately 95% of the crypt openings were vacant; the villi were totally denuded; the supporting structures, including the lamina propria, appeared intact. In vitro radiolabelling of intestinal cell fractions enriched with crypts revealed a linear incorporation of [3H]TdR from 0-60 min which was strongly influenced by the presence of foetal calf serum (FCS). Measurements of the compensatory response of the mucosa to resection of 70% of the small bowel indicated that the mucosal cell separation is capable of detecting alterations in crypt cell proliferation. Previously, such alterations were monitored by other methods utilizing microdissection procedures or stathmokinetic agents.     

Isolation of rat intestinal crypt cells

Abstract. A technique is presented which yields single cells and intact crypts in suspension from unfixed rat intestinal mucosal epithelium. Everted lengths of intestine were digested by 27 mM sodium citrate in phosphate-buffered saline (pH = 7.3) at 37°C. Mucosal cells were dislodged by vibratory stress (hand vortexing) following incubation for prescribed intervals at 37°C in 1.5 mM ethylenediamine tetraacetic acid (EDTA) and 0.5 mM dithiothreitol (dtt). Alkaline phosphatase determinations, phase microscopy, and in vivo and in vitro evaluations of tritiated thymidine ([³H]TdR) incorporation were performed on isolated intestinal cells. Data indicate that cells were sequentially derived from villus tip to crypt base as judged by cellular morphology, alkaline phosphatase activity/mg protein and radioactivity per μg protein. Upon completion of the intestinal cell isolation assay, scanning electron microscopy of the remaining intestine revealed that approximately 95% of the crypt openings were vacant; the villi were totally denuded; the supporting structures, including the lamina propria, appeared intact. In vitro radiolabelling of intestinal cell fractions enriched with crypts revealed a linear incorporation of [³H]TdR from 0–60 min which was strongly influenced by the presence of foetal calf serum (FCS). Measurements of the compensatory response of the mucosa to resection of 70% of the small bowel indicated that the mucosal cell separation is capable of detecting alterations in crypt cell proliferation. Previously, such alterations were monitored by other methods utilizing microdissection procedures or stathmokinetic agents.     

Cytoskeletal protein and mRNA accumulation during brush border formation in adult chicken enterocytes

We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskeletal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunofluorescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to morphological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spectrin, tropomyosin, myosin I, villin or alpha-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskeletal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key protein(s).     

Localization of NK1 receptors and roles of substance-P in subepithelial fibroblasts of rat intestinal villi

Subepithelial fibroblasts of the intestinal villi, which form a contractile cellular network beneath the epithelium, are in close contact with epithelial cells, nerve varicosities, capillaries, smooth muscles and immune cells, and secrete extracellular matrix molecules, growth factors and cytokines, etc. Cultured subepithelial fibroblasts of the rat duodenal villi display various receptors such as endothelins, ATP, substance-P and bradykinin, and release ATP in response to mechanical stimulation. In this study, the presence of functional NK1 receptors (NK1R) was pharmacologically confirmed in primary culture by Ca(2+) measurement, and the effects of substance-P were measured in an acute preparation of epithelium-free duodenal villi from 2- to 3-week-old rats using a two-photon laser microscope. Substance-P elicited an increase in the intracellular Ca(2+) concentration and contraction of the subepithelial fibroblasts in culture and the isolated villi. The localization of NK1R and substance-P in the villi was examined by light and electron microscopic immunohistochemistry. NK1R-like immunoreactivity was intensely localized on the plasma membrane of villous subepithelial fibroblasts in 10-day- to 4-week-old rats and mice and was decreased or absent in adulthood. The pericryptal fibroblasts of the small and large intestine were NK1R immuno-negative. These villous subepithelial fibroblasts form synapse-like structures with both substance-P-immunopositive and -immunonegative nerve varicosities. Here, we propose that the mutual interaction between villous subepithelial fibroblasts and afferent neurons via substance-P and ATP plays important roles in the maturation of the structure and function of the small intestine.     

A nude mouse xenograft model of fetal intestine development and differentiation.

This report describes a novel in vivo model of intestinal differentiation. Fourteen day, undifferentiated fetal rat small intestine, stripped of the major part of its mesenchyme, suspended in a type I collagen gel and then xenografted into a nude mouse, undergoes small intestinal morphogenesis and cytodifferentiation. All four major epithelial lineages, namely Paneth, goblet, columnar and endocrine are present. Double-label nonisotopic in situ hybridization, employing biotinylated and digoxigenin-labelled whole rat DNA and whole mouse DNA probes, was performed to distinguish donor cells from host cell types. The outer longitudinal smooth muscle layer, and the major part of the lamina propria, including pericryptal fibroblasts, are of host mouse origin; the inner circular smooth muscle layer is of donor rat origin. Cells of the muscularis propria and lamina propria acquired smooth muscle alpha-actin, presumably under the influence of the donor endoderm. Furthermore, this xenograft develops a host vascular network, and cells with the morphological appearance of lymphocytes are present within the intestinal epithelium. The production of chemotactic factors by the endoderm is postulated because grafting of collagen gel alone results in a minimal invasion by stromal cells which do not express smooth muscle alpha-actin.     

Radiation-induced changes of fibroblasts in the squamous cell carcinoma of the head and neck

The regrowth of mesenchymal tissue (stroma) surrounding the malignant epithelium is an important step in tissue remodelling during and after irradiation. The radiation-induced fibroblastic changes were studied on tissue samples taken before, during and after the radical irradiation of the squamous cell carcinoma of the head and neck. Elongated fibroblasts with large amount of rough endoplasmic reticulum were seen around the tumor epithelium before radiation. The fibrosis increased during irradiation and at the same time the shape of the fibroblasts changed so that they became more triangular and nuclear structures became more prominent together with hyperchromasia. The amount of cell organelles declined although there was a large amount collagen present. Epithelial cells invaded through the basal lamina. In most samples the basal lamina could not be seen at all and the tumor cells were dispersed between stromal elements. On the other hand there were close contacts between epithelial and mesenchymal cells throughout the study in places where the basal lamina was broken, which might indicate epithelio-mesenchymal interaction. Also the connective tissue formed by fibroblasts and collagen might be part of the radiation induced healing and destruction of the tumor cells.     

Evidence for vasopressin production in the human gastrointestinal system.

In the present study we performed immunohistochemical examination of segments from the human gastrointestinal system for the presence of cells containing vasopressin (VP) and vasopressin-associated human neurophysin (VP-HNP). VP immunoreactivity was found in crypt cells of the stomach and small intestine, and in mononuclear cells within the lamina propria and submucosa. VP-HNP was demonstrated in the crypt and lamina propria regions of the small intestine, and was colocalized with vasopressin in crypt cells. This colocalization indicates local vasopressin synthesis by these cells and raises the possibility that they may perform an endocrine or exocrine function in the human gastrointestinal system.     

The effect of increased crypt cell proliferation on the activity and subcellular localization of esterases and alkaline phosphatase in the rat small intestine

Synopsis The activity and ultrastructural localization of alkaline phosphatase and esterase has been studied in normal rat intestine and after the increased crypt cell proliferation that occurs during recovery after 400 rad X-irradiation. Alkaline phosphatase activity is not present in crypt cells of normal intestine, but becomes apparent after the cell has migrated on to the villus. The enzyme is localized in the microvilli, along the lateral cell membranes and in dense bodies. Its activity increases 10 to 15-fold from the base to the tip of the villus. Morphometric analysis of the cell structureswhere this enzyme is localized reveals no marked changes in their relative proportions during crypt cell development.The expansion of the proliferative cell compartment along the whole length of the crypt which occurs during recovery after irradiation (72 hr after 400 rad X-irradiation) results in a marked reduction of alkaline phosphatase activity in the lower 10–15 cell positions at the base of the villus. During subsequent migration of these cells, the activity increases with cell age but normal values are not attained. From a morphometric analysis it was found that the ultrastructural development is similar to that in controls. These results suggest that during cell maturation, normal values for alkaline phosphatase activity are only attained after a 10–12 hr period of maturation in a non-proliferative state and only after the cell has migrated on to the functional villus compartment.In normal intestine, esterase activity shows a 3-fold increase from the bottom to the tip of the crypt and a 3 to 4-fold increase during migration up to the middle of the villus. Enzyme activity is localized in the endoplasmic reticulum, the dense bodies and the perinuclear space. Morphometric analyses reveal a 2 to 3-fold increase in the absolute size of these subcellular compartments during crypt cell differentiation and a 2-fold increase at the crypt-villus junction. The relative sizes increase 1.5-fold during crypt cell differentiation and at the time of transition of the cells on to the villus.Increased crypt cell proliferation after irradiation leads to a marked decrease in esterase activity both in crypts and villi. Morphometric analyses of electron micrographs indicate that these changes in activity are not related to any changes in the subcellular structures in which the enzyme is localized. It appears that the normal development of esterase activity depends both on the functional state of the cell and its localization in the crypt or villus.     

Extracellular myocilin affects activity of human trabecular meshwork cells

The trabecular meshwork (TM), a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. The protein product has been localized to both intra and extracellular sites, but its function still remains unclear. This study was to determine whether extracellular myocilin presented in the matrix affects adhesion, morphology, and migratory and phagocytic activities of human TM cells in culture. Cell adhesion assays indicated that TM cells, while adhering readily on fibronectin, failed to attach on recombinant myocilin purified from bacterial cultures. Adhesion on fibronectin was also compromised by myocilin in a dose dependent manner. Myocilin in addition triggered TM cells to assume a stellate appearance with broad cell bodies and microspikes. Loss of actin stress fibers and focal adhesions was observed. TM cell migration on fibronectin/myocilin to scratched wounds was reduced compared to fibronectin controls. Myocilin, however, had little impact on phagocytic activities of TM cells. Cell attachment on fibronectin and migration of corneal fibroblasts, a control cell type, were not altered by myocilin. These results demonstrate that extracellular myocilin elicits anti-adhesive and counter-migratory effects on TM cells. Myocilin in the matrix of tissues could be exerting a similar influence on TM cells in vivo, impacting the flexibility and resilience required for maintenance of the normal aqueous outflow.     

In vivo fluorescence measurement of Na+ concentration in the pericryptal space of mouse descending colon

A methodinvolving surgical exposure of the colonic mucosa, fluorescent dyeaddition, and confocal microscopy has been developed for monitoringcolonic crypt function in vivo in mice. Na⁺ concentrationin the extracellular pericryptal space of descending colon was measuredusing a low-affinity Na⁺-sensitive fluorescent indicatorconsisting of an Na⁺-sensitive chromophore (sodium red) andan Na⁺-insensitive chromophore (Bodipy-fl) immobilized on200-nm-diameter polystyrene beads. The Na⁺ indicator beadsaccumulated in the pericryptal spaces surrounding the colonic cryptsafter a 1-h exposure of the colonic luminal surface to the beadsuspension. Na⁺ concentration ([Na⁺]) in thepericryptal space was 491 ± 62 mM (n = 4). Aftera 70-min exposure to amiloride (0.25 mM), pericryptal[Na⁺] was reduced to 152 ± 21 mM. Blockage of thecrypt lumen with mineral oil droplets reduced pericryptal[Na⁺] to 204 ± 44 mM. Exposure of the colonicmucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dyeinto the crypt lumen with a half time of 19.8 ± 1.0 s, whichwas increased to 77.9 ± 6.0 s after amiloride treatment.These results establish an in vivo fluorescence method to measurecolonic crypt function and provide direct evidence for accumulation ofa hypertonic absorbate in the pericryptal space of descending colon.The pericryptal space represents the first example of a hypertonicextracellular compartment in mammals that is not created by acountercurrent amplification mechanism.

     

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine.

Dividing epithelial cells in the mouse small intestine were examined by thin-section electron microscopy with special attention given to the mode of cytokinesis. As the columnar epithelial cells entered mitosis in the crypt, they became rounded, maintaining their junctional complexes with neighboring cells while detaching themselves from the basal lamina. In such rounded cells the mitotic apparatus was formed with its long axis parallel to the luminal surface. Replicated centrioles moved down from the apical region to locate themselves lateral to the nucleus, where they served as the poles of the mitotic spindle. During mitosis the cell retained microvilli on its luminal surface, though the terminal web became much thinner. At telophase the formation of a cleavage furrow proceeded asymmetrically from the basal side alone, and thus the contractile ring which was prominent at the base of the furrow, merged with the terminal web. Eventually, an intercellular bridge with a midbody was formed on the luminal surface. The space in the furrow was occupied by the flattened cytoplasmic processes of the neighboring cells. The tight junction was also seen on the basolateral surface of the intercellular bridge with the underlying neighboring cells. At very late telophase the intercellular bridge was disconnected from the neighboring cells and protruded into the lumen. These observations have led us to propose a mode by which the simple columnar epithelium maintain the tight junctional seal during cell division in the crypt of the small intestinal epithelium.